Download Metabolic regulation via intracellular pH - AJP

Metabolic
regulation
via intracellular
pH
BUSA, WILLIAM
B., AND RICHARD
NUCCITELLI.
Metabolic regulation uia
intruceZZuZarpH. Am. J. Physiol. 246(Regulatory Integrative Comp.Physiol.
15): R409-R438,1984.-Despite earlier notions that intracellular pH (pHi)
stimulus-responsecoupling; cellular dormancy; calmodulin; adenosine
3’,5’-cyclic monophosphate;egg and sperm activation; energy charge; cell
cycle; calcium ion
I. INTRODUCTION
Although efforts to determine the relative acidity of
intracellular fluids date back nearly to the beginning of
this century, only within the last eight years have reliable
examples of intracellular pH (pHi) changes accompanying defined metabolic transitions been reported (Table
1). Indeed, principally because interest in pHi long preceded the technical developments necessary to measure
this very subtle parameter, many early and influential
workers were led to conclude from the inadequate data
then available that pHi was a constant, invariant not
only within a single cell type under various physiological
conditions, but also between different cell types. For
example, based on their calorimetric estimates of pHi in
eggs and zygotes of several organisms, the Needhams
(160) remarked in 1926, “The constancy of. . . [intracellular] pH values . . . is striking. Neither ontogenetic
changes in the individual nor phylogenetic changes. . .
lead to any difference.” Even as late as 1961, the Chambers (37) concluded, “All the valid evidence points toward
the fact that no significant pH shifts occur in the cytoplasmic matrix during different functional states.” We
offer these statements not to criticize these workers (or
0363-6119/84 $1.50 Copyright 0 1984 the American Physiological Society
their peers who came to similar conclusions) but to
document the rather widespread bias that pHi cannot be
a general metabolic effector of any significance because
it is presumed to be invariant.
Though it would be difficult to identify a champion of
this “pHi constancy hypothesis” today, its legacy is nonetheless. evident in too many aspects of modern biology.
As an example, biochemists (or their editors?) too often
fail to publish enzyme pH-activity profiles-a standard
component of any enzyme characterization. Clearly such
omissions are justifiable only if pHi is assumed to be a
constant, and thus of no regulatory significance. Similarly, textbook authors routinely present “balanced” biochemical equations that do not take protons explicitly
into account as substrates and products of metabolic
pathways. In at least one instance this failure has given
rise to widespread misunderstanding (Sect. v).
Because comparative data regarding intracellular pH
changes in a variety of organi sms an.d cell types have
only recently begun to appear, earlier review s regarding
pHi (71, 79, 80, 166, 183, 185, 233) have not addressed
the broad question of the generality and scope of pHimediated metabolic regulation. Our goal in the present
review is to provide a comparative review of these most
R409
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
was invariant with time, recent studies have documentedpHi changesof
from 0.1 to 1.6 U during metabolic and developmental transitions in a
variety of cells. Here we review the evidence for pHi-mediated regulation
of gamete activation, cellular dormancy, the cell cycle, and stimulusresponsecoupling. Intracellular Ca2+level changesalso accompany many
of these sametransitions, and mounting evidence suggeststhat pHi and
Ca2+changescan be interdependent, both in their mechanismsand their
effects. Although the significanceof suchinteractions is still largely unclear,
one example-the pronouncedpH dependenceof Ca2+binding by calmodulin-suggests their potential importance in metabolic regulation. Similar
evidence suggeststhat pHi changesalso influence intracellular adenosine
3’,5’-cyclic monophosphatelevels, and vice versa. Finally we show that
changesin adenylate energy charge can significantly alter pHi. In light of
these interactions-and becausepHi, unlike most other effecters, doesnot
require specializedreceptors-we suggestthat pHi functions asa synergistic
messenger,providing a metabolic context within and through which the
actions of other effecters are integrated.
R410
TABLE
W. B. BUSA
1. Summary
of pHi
neutrophils
Nutritional
status
Saccharomyces
cerevisiae
S. cerevisiae
Physarum
polycephalum
(slime mold)
Measurement
Technique
Event
ApHi
Fertilization
Fertilization
Fertilization
Microelectrode
DMO
DMO
+0.4
+0.4
+0.4
Fertilization
Fertilization
Fertilization
NMR
+0.4
Colorimetry
Fertilization
Microelectrode
Motility
Motility
Motility
Motility
initiation
initiation
initiation
initiation
9AA
NMR
9AA and NMR
9AA
Motility
AR
AR
ASR
initiation
Homogenization
9AA
9AA
9AA
Capacitation
Anaerobic-aerobic
and AR
9AA
transition
Germination
Germination
Insulin
stimulation
of glycolysis
Thrombin
stimulation
of aggregation
secretion
Chemotactic
response to complement
synthetic
peptide
Starvation-refeeding
Catabolite
derepression
Starvation-refeeding
DMO,
9AA
factor
or
w
and NMR
NMR
124
108
40,241
41
?a
+0.2/-0.2”
+0.2
+0.6*
+1.1
r+l.O’
+O.l to 0.2
+0.3
DMO
NMR
NMR
Microelectrode
241
32,33,220
88
237
+1.6h
Methylamine
NMR
and
205
109
109
>+l.og
NMR
Ref. No.
244
124
196
116
247
29,30
201
16
153,157
99,210
146,203
+0.4h
+0.4
+0.4
49
49
158
DMO,
5,5-dimethyl2,4oxazolidinedione;
NMR,
nuclear magnetic
resonance;
9AA, 9-aminoacridine;
AR, acrosome
reaction;
ASR, ascidian
sperm reaction.
a pHi has not been reliably
measured,
but seems likely to change, based on evidence presented
in cited references.
b Small,
transient
acidification
precedes permanent
increase
in pHi.
’ Estimate
based on separate
studies of “dry”
semen (NMR)
and sperm in
seawater
(9AA).
d A dose-dependent
alkalinization
has been qualitatively
demonstrated
in response
to “sperm
motility-initiating
factor.”
This pHi increase is not required
for motility
initiation,
however.
e Initial alkalinization
is followed by permanent
reacidification.
*Based
on preliminary
data.
g Intra-acrosomal
pH; semiquantitative
estimate.
h Reversible
transition.
Alkalinization
accompanies
forward
transition as written.
i Estimate
derived from direct measurement
of dormant
spore pHi and “typical”
values for vegetative
cells (see 24).
j See
Table 2.
recent data and a synthesis of our present understanding
of the roles pHi changes play in regulating metabolic
processes.
A. Definitions
Whereas the terms pH and pHi are routinely used
without definition, as though their meanings are inherently obvious, this is hardly the case. It is important,
first, to recognize that international
convention
no
longer regards pH as the negative logarithm of the hydrogen ion activity. This issue has been definitively
discussed by Bates (l7), and we shall simply state that
the modern pH scale is, of necessity, only a relative
measure of the proton’s chemical potential; it is therefore
seldom legitimate to treat the inverse antilog of pH as
the proton activity or concentration,
except perhaps as
a first approximation.
Even more confusion surrounds the use of the term
pHi. Although this term is often implicitly
intended to
refer to cytosolic pH, we note that the majority of standard pHi-determining
techniques (Sect. II) are incapable
of yielding cytosolic pH values entirely uncontaminated
by contributions from other compartments within eucaryotic cells. The worst offenders in this regard are the
weak acid and base distribution techniques (see Ref. 185
for discussion), and microelectrode-determined
pHi probably comes closest to reflecting cytosolic pH.-Recently
developed optical techniques (91,218) may prove to yield
genuine cytosolic pH values; the technique of Heiple and
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
Rabbit
R. NUCCITELLI
changes accompanying metabolic transitions
Cell/Organism
Gamete activation
Echinoderm
egg
Lytechinus
pictus
L. pictus
Strongylocentrotus
purpuratus
S. purpuratus
Arbacia punctulata
Clypeaster
japonicus
Frog egg
Xenopus laevis
Spermatozoon
L. picks
“L. pictus
S. purpuratus
Limulus
polyphemus
(horseshoe
crab)
Rat
L. pictus
S. purpuratus
Ascidia callosa (solitary
ascidian)
Hamster
Arousal
from dormancy
cyst
Artemia
salina (brine
shrimp embryo)
Spore
Bacillus
Pichia pastoris (yeast)
Cell cycle’
Stimulus-response
coupling
Rana pipiens (frog sartorius)
Human
platelets
AND
INVITED
TECHNIQUES
The numerous means employed to estimate pHi have
been comprehensively
reviewed by Roos and Boron
(185), and recently emerging technologies are compared
by Nuccitelli
(166). Space permits us only the briefest
description of the major limitations
and advantages of
the most important
techniques used in the studies reviewed here.
A. Weak Acid or Base Distribution
Because cells are most permeable only to the uncharged form of a conjugate acid-base pair, the Henderson-Hasselbalch
equation permits calculation of pHi if
the extracellular pH (pH,), the pK of the applied weak
acid or base, and its equilibrium
distribution
ratio between cell and medium are known, provided neither
binding, metabolism, nor active transport of the probe
molecule occurs. Because eucaryotic cells are composed
of compartments
of differing pH values, the pHi calculated from weak acid or base distribution
will represent
some weighted average of the compartments
among
which the probe is distributed.
The technique is not
normally applicable to single cells. Its temporal resolution is limited by the time required for equilibration
of
the probe molecule- from seconds to a half hour or more,
depending mainly on the size of the cell.
The radiolabeled weak acid, 5,5-dimethyl2,4oxazolidinedione (DMO) (234), is the most widely used reliable
probe for distribution
studies and employs only technology available to most laboratories. Because the cells are
usually destroyed for analysis, repetitive or continuous
determinations
are not normally possible with DMO.
The radiolabeled weak base, methylamine,
is sometimes
employed when pHi is acidic.
The fluorescent weak base, 9-aminoacridine
(9AA),
has also been employed. Spectrofluorometry
of 9AAcontaining cell suspensions permits continuous, nondestructive monitoring
of pHi (again, limited by the equilibration time), and fluorescence microscopy of these
cells can yield qualitative
information
regarding pHi
compartmentation.
9AA binds to some cell constituents
and therefore yields pHi estimates that are only qualitative, but corrections for binding have recently been introduced (40).
B. pH Microelectrodes
The Thomas-style
(recessed-tip) pH-sensitive
glass
microelectrode permits continuous monitoring of pHi in
single cells with a spatial resolution of about 1 pm and
temporal resolution of 5-30 s. It is perhaps the most
suitable technique for monitoring small or transient pHi
changes in the aqueous cytoplasm, but its application is
limited to fairly large, immobile, and accessible cells.
Recently the fabrication of pH microelectrodes
has
been simplified by the introduction
of an H+-selective
neutral carrier-based resin (7) (Fluka, Switzerland). This
material is easily placed in the tip of a glass microelectrode, producing reliable pH-sensitive
microelectrodes,
which may make the harder-to-construct
Thomas-style
electrode obsolete.
C. In Vivo 3’P-Nuclear
Spectroscopy
Magnetic
Resonance
Because the nuclear magnetic resonance (NMR) frequency (or chemical shift) of endogenous inorganic phosphate (Pi) is pH dependent within the physiological
range, 31P-NMR spectra of intact cells can be used to
monitor pHi. Accurate absolute values of pHi can be
determined only when the intracellular
ionic strength is
known, but the technique is very suitable for detecting
even small changes in pHi. It is the only noninvasive
technique of those discussed here. NMR-determined
pHi
values usually reflect principally cytoplasmic pH, but in
some cases information
regarding other compartments
(e.g., mitochondria)
can be gained as well. Disadvantages
of NMR include its fairlv low temporal resolution, the
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
Taylor (91) is particularly promising, since the pH-sensitive fluorescent probe is conjugated to an impermeant
protein, thus trapping it in the cytosol after microinjection. Finally manipulations
intended to alter cytosolic
pH (e.g., weak acid or base treatment) will affect the pH
of numerous other subcellular compartments,
with net
effects not easily distinguished from that of cytosolic pH
manipulation
alone (Sect. IV). More explicit consideration of these issues in future pHi studies is called for.
With these difficulties in mind, we shall use the term
pHi to refer to the pH of the intracellular
compartment
under consideration, when such a distinction is possible,
or to refer to the experimentally
determined cellular pH,
which will usually be some average of the pH of many
compartments
and/or cells.
A large fraction of the available data concerning the
effect of pHi changes on cellular metabolism derives from
studies in which pHi is artificially
manipulated
in cells
that have not been observed to undergo natural pHi
changes. We shall often distinguish these pHi manipulations from naturally occurring pHi changes by referring
to the latter as physiological pHi changes. Conceivably,
not every physiological
pHi change is physiologically
significant (i.e., involved in regulation of the physiological process it accompanies). We take as the minimum
evidence required to support a claim to physiological
significance the three tests most recently outlined by
Steinhardt (213). 1) The pHi change must be shown to
occur under conditions relevant to both the cell type and
organism under consideration.
2) Treatments
that inhibit the physiological pHi change must inhibit or modify
the response(s) it is thought to regulate. 3) Treatments
that artificially induce pHi changes of similar timing and
magnitude should (in whole or part) elicit the response(s)
the physiological pHi change is thought to regulate. In
applying these tests it is important to bear in mind that
they describe rather idealized conditions. The mounting
evidence that pHi changes interact with other regulators
(Sect. IV) renders pertinent Atkinson’s (8) admonition
that “when a system or process is regulated by several
inputs there can be no one-to-one relationship between
the response and any one input (unless all others are
fixed) .”
II.
R411
OPINION
R412
W. B. BUSA
III. SOME CELLULAR
MAY PLAY A ROLE
PROCESSES
IN
WHICH
PHi
A. Metabolic Activation
1) Egg activation. The eggs of many organisms are
relatively quiescent, as reflected, for example, by their
low respiratory rate, greatly reduced synthetic activity,
and cell cycle arrest. Fertilization
terminates this state,
setting into motion a sequence of events that Epel (58)
has termed “the program of fertilization.”
This sequence
is best understood in the sea urchin egg (see Refs. 58-61
for review) , where fertilization evokes such “early” ’ events
as plasma membrane depolarization,
a transient release
of Ca2+ from intracellular
stores, and (in response to the
Ca2+ transient) exocytosis of the cortical granules. These
are followed by such “late” events (ca. 2 min to 1.5 h
after fertilization)
as hyperpolarization
of the plasma
membrane due to the appearance of new K+ conductance,
formation of microvilli,
dramatic increases in protein
synthesis and messenger RNA (mRNA) polyadenylation,
activation of DNA synthesis, and finally cell division. As
we discuss below, another “late” event is a substantial
increase in pHi, which is in large measure responsible for
the activation of these other events. This discovery provided the first (and still best) evidence for pleiotropic
metabolic regulation via pHi.
EARLY RESULTS. In retrospect, the first hint that pHi
changes might be involved in sea urchin egg activation
arose in the 1900s from the work of Loeb (128), who
observed that sea urchin eggs could be activated by a
sequence of treatments
including
dilute solutions of
NH40H and raised to maturity without undergoing fertilization. Loeb later applied vital staining with neutral
red in an attempt to monitor pHi during fertilization
(127). His incorrect conclusion that the eggs acidify after
fertilization
prevented him from grasping the true significance of his activation experiments, but the ability
R. NUCCITELLI
of NH3 to activate sea urchin eggs has proven crucial to
modern research concerning sea urchin egg pHi.
The next significant attempt to monitor egg pHi came
in 1926, when the Needhams (160) microinjected eggs of
sea urchins, sea stars, annelids, and ascidians with pHsensitive dyes. They invariably observed a pHi of 6.6
both before and after fertilization
(see Sect. I).
MODERN
RESULTS. Nearly 50 yr passed without further progress until Steinhardt and Mazia (215), building
on the work of Loeb, demonstrated that NH3 (in the
form of seawater titrated to pH 9.1 with NH40H) activates one of the “late” events of fertilization, the appearance of new K+ conductance, in unfertilized sea urchin
eggs (see also Ref. 207). pHi was not monitored in this
study but was presumed to alkalinize in response to the
membrane-permeant
weak base, NH3, as Warburg (235)
had previously shown. There followed soon after further
evidence that other late events [e.g., mRNA polyadenylation (239), increased protein synthesis (62), DNA
synthesis (139), and nuclear membrane breakdown and
chromosome condensation (138)] are all elicited by treating these eggs with NHdOH or NH&l. Procaine, another
membrane-permeant
weak base, likewise activat,es DNA
synthesis and chromosome condensation (227). Low concentrations of NH3 are sufficient to stimulate protein
synthesis, but higher levels are required to initiate chromosome condensation, and the increase in protein synthesis is still achieved when the appearance of K+ conductance is blocked, suggesting that NH3 acts separately
on each of these systems rather than on one regulatory
step in a linear pathway (62).
Despite these compelling observations, conclusive evidence for a pHi increase after fertilization was five years
in coming. In 1976, Johnson et al. (112) measured the
pH of egg homogenates and reported a permanent increase from 6.5 to 6.8 within 10 min after fertilization;
with the same techniques, however, Lopo and Vacquier
(132) observed an initial pH increase followed by a
permanent decline. The conflict between these data underscores the unreliability
of the homogenization
technique (Sect. II). The issue was resolved in 1978 by Shen
and Steinhardt (205), who used Thomas-style
pH microelectrodes to record a permanent pHi increase from
6.8 to 7.3 within 5 min after fertilization
of Lytechinus
pictus eggs. Nearly identical results have also been obtained via the DMO technique (log), and 31P-NMR (241)
reveals a pHi increase of similar magnitude (7.1-7.5)
after fertilization
of Strongylocentrotus purpuratus eggs.
In confirmation
of Warburg’s early results with vital
dyes, all these techniques reveal that NH3 alkalinizes
pHi (109, 205, 241).
The mechanism by which pHi is alkalinized after fertilization remains a topic of active debate. Fertilization
is accompanied by the release of proton equivalents into
the medium (the so-called fertilization
acid) (193) with
a time course roughly similar to that of the pHi increase.
Both fertilization
acid release and the pHi increase require external Na’ (as little as 5 mM), and both are
inhibited by amiloride,
a Na+-H+ exchange inhibitor
(112,206). Whereas these facts have been taken to reflect
the involvement of Na+-H+ exchange by some workers
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
very large volume of sample required (ca. 2-5 ml of cells),
the difficulty of providing adequate culture conditions
within the spectrometer, and the requirement
for advanced instrumentation.
It is encouraging that direct comparisons of these three
techniques usually yield closely similar (though seldom
identical) values for pHi under controlled conditions. The
same cannot be said for two earlier (and cruder) techniques, however. The first of these involves homogenization or lysis of cells and direct determination
of the
homogenate or lysate pH. Significant errors are introduced by dilution, disruption of intracellular
compartments, and the rapid acidification
common to disintegrated cells. Unfortunately
this unreliable technique is
still in occasional use. Topical application or microinjection of colored pH indicator dyes such as neutral red has
also been employed and, with but one exception (88), has
failed to detect known pHi changes and even pHi differences between cell types (37, 160). Metachro matic, protein, or salt errors may be involved. With few (and clearly
noted) exceptions, we have limited our discussion to data
collected with one of the three reliable techniques just
discussed.
AND
INVITED
R413
OPINION
rate of protein synthesis in this system is strongly pH
dependent (lo- to 20-fold greater at pH 7.4 than at pH
6.9), and the optimum pH (7.4) equals the pHi of fertilized eggs. In contrast to Brandis and Raff’s in vivo results
with NH3, increasing pH from 6.9 to 7.4 in this cell-free
system stimulates a 2fold increase in elongation rate
(equivalent to the increase observed in vivo after fertilization), whereas the remaining 5 to lo-fold increase in
synthesis rate is presumed to involve pH-sensitive unmasking of maternal mRNA.
OTHER
PHi. In
LATE
EVENTS
THAT
MAY
BE
REGULATED
BY
addition to the processes so far discussed, others
have been suggested to be regulated by the pHi increase
after fertil .ization, although in these remaining cases the
available evidence is less complete. Glucose-6-phosphate
dehydrogenase (GGPDH) changes its subcellular location
on fertilization
(as judged by centrifugation
of egg homogenates) ; it appe ars in the parti culate (pellet) fraction
of unfertili .zed egg homogenates and the supernatant
fraction of fertilized or NHs-activated
egg homogenates
(9). In vitro, GGPDH is released from the particulate
fraction of egg homogenates by increasing pH from 6.7
to 7.2. Similar results have been obtained for eggs of the
surf clam Spissula soZidissima (105). A pHi change after
fertilization
has not been demonstrated
in this egg to
date but has been suggested, based on circumstantial
evidence (104; see also below). NH3 also activates glycogen catabolism in S. purpuratus
eggs in a manner identical to fertilization
(II), but further efforts will be required to determine whether this is a direct effect of the
pHi increase.
Not only the metabolic activity of the egg, but its
structure as well, undergoes changes after fertilization:
during the late phase of activation the surface microvilli
greatly elongate and develop core bundles of laterally
associated actin microfilaments,
presumably from Gactin sequestered in the cortex of the unfertilized egg.
Alkalinization
of isolated cortices from pH 6.5 to 7.5
stimulates extensive actin polymerization,
but not bundling of filaments, and these and other observations
initially suggested a role for pHi in regulating microfilament formation in the zygote (18). Later studies, however, in which intact eggs were alkalinized
with NH3
failed to confirm this (19, 32, 33). Transferring fertilized
eggs to Na+-free seawater (which blocks the pHi increase)
permits microfilament
formation but not bundling (19,
33), suggesting that Ca2+ rather than pHi regulates polymerization
in vivo. The association of these microfilaments into microvillar
core bundles might yet prove
pHi dependent, however, since alkalinization
of zygotes
in Na+-free seawater (either with NH3 or by return to
normal seawater) enables the previously formed microfilaments to bundle normally (15, 30). A 58,000-A& protein, fascin, has been implicated as the agent that cross
links microfilaments
in the sea urchin zygote’s microvilli
(27), but we are unaware of information
regarding the
pH dependence of its interaction with actin.
AN UNSOLVED
PROBLEM.
The pHi increase accompanying sea urchin egg fertilization appears to occur within
the cytosol; thus the activating effect of NH3 is assumed
to be due to cytosolic alkalinization,
and Winkler’s ob-
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
(112), others (206) have challenged this view, suggesting
instead that Na+ plays a secondary, perhaps allosteric,
role and that amiloride inhibition
may be nonspecific in
light of the very high concentrations
required (0.1-I
mM). Competition
between Na+ and amiloride for their
binding site on the exchanger may provide a plausible
explanation
for this relative insensitivity
to amiloride
(185), but it remains difficult to explain Shen and Steinhardt’s (206) observation that a loo-fold decrease in
external Na+ still permi ts normal alkalinization
of PHi9
since Na+-H+ exe hange would presumably be drive n bY
the Na+ electrochemical
potential gradient across the
plasma membrane (see Sect. IIIC~ ). Most recently, Gillies
et al. (83) have presented evidence suggesting that the
fertilization acid is COz, but their results have not proven
reproducible in others’ hands and may have been due to
CO2 contamination
of the “bicarbonate-free”
seawater
they employed (96). With all these difficulties, we feel it
is safe to say that the mechanism of alkalinization
remains to be clearly elucidated.
TARGETS
OF pHi
MEDIATION
DURING
EGG
ACTIVATION.
The molecular targets of the pHi increase after
fertilization
remain for the most part obscure, but some
progress has been made with regard to its role in the
activation of protein synthesis. NH3-activated
protein
synthesis lags behind the activation induced by fertilization and results (as does fertilization-induced
activation) in a sixfold increase in egg polysome content (62).
Grainger et al. (86) attributed the delay in initiation
of
NHs-induced activation to the slower time course of the
NH3 effect on pHi (compared with fertilization)
but
pointed out that this explanation alone could not account
for the markedly slower rate with which NH3-activated
eggs achieve their maximum synthetic rate (60-120 min
vs. 30 min for fertilized eggs). Ruling out the possibility
of toxic effects, they concluded that factor(s) other than
pHi also play a role in regulating the increase in protein
synthetic rate. Nonetheless their observations underlined the importance
of pHi: when the pHi of NHSactivated or fertilized eggs is lowered (by removing NH3
or introducing acetic acid, respectively) protein synthesis
is reinhibited.
Brandis and Raff (26) pointed out that
the increased synthetic rate following fertilization
involves increases in both the peptide elongation rate and
the level of translatable
(i.e., unmasked)
maternal
mRNA and presented evidence that the latter, but not
the former, is accomplished by the pHi increase (but see
below). The second factor postulated by both Grainger
et al. and Brandis and Raff was identified by Winkler
and co-workers (243) as the transient release of Ca2+,
which is an “early” event in the program of fertilization
but not in NH3 activation. When this Ca2+ transient is
simulated by treating NHS-activated
eggs with the Ca2+
ionophore A23187, the resulting rate of protein synthesis
nearly equals that observed in fertilized eggs; in contrast,
increased intracellular
Ca2+ in the absence of a pHi
increase has no activating effect on protein synthesis.
The recent development of a cell-free protein synthesis
system prepared from L. pictus eggs (240, 242) offers the
prospect of significant advances in our understanding of
the molecular target(s) of pHi-mediated
regulation. The
R414
B. BUSA
AND
R. NUCCITELLI
ers; thus the sperm might be expected to win the race to
activate the egg.
Another important
objection to the current pHi hypothesis raised by Jaffe (107) is that, as Zucker et al.
(252) have shown, a transient intracellular
Ca2+ pulse
accompanies NH3 activation of the sea urchin egg, raising
the possibility that this pulse, not intracellular
alkalinization, is responsible for the activating effect of NH3.
The NHB-induced Ca2+ pulse differs from that accompanying fertilization in three important respects: 1) it is
much briefer than the physiological
Ca2+ pulse, 2) its
source appears to be the extracellular Ca2+ pool rather
than an intracellular
store (i.e., its intracellular
localization may differ), and 3) it fails to activate the early
events of fertilization
(e.g., cortical granule exocytosis).
Nevertheless these observations underscore the importance of testing other means of raising pHi-ideally,
without initiating a detectable Ca2+ pulse. In addition to
the high pH, technique already discussed, other means
might include pressure injection of impermeant buffers,
which has recently been shown to activate sand dollar
eggs (see 88), or iontophoresis of OH-. All these alternatives have the advantage of (directly) altering only
cytosolic pH.
HOW
ATION?
GENERAL
ARE
PHI
CHANGES
AFTER
FERTILIZ-
All the direct observations of pHi discussed so
far pertain to eggs of the Pacific sea urchins S. purpuratus and L. pictus. To our knowledge, no such data are
available for the eggs of the Atlantic sea urchin Arbacia
punctulata, but these too release fertilization
acid in the
presence, but not absence, of external Na+ (33, 220) and
are at least partially activated by NH&l
(32, 33). Eggs
of the sea urchin Psammechinus miZZiaris also release
fertilization
acid (44). Three other genera of sea urchins
have been reported not to alkalinize after fertilization as
determined by 31P-NMR (106), but we consider this
latter study to be seriously flawed due to the rather
unphysiological
(anaerobic) conditions employed. The
reliable data presently available therefore suggest that
most, if not all, sea urchin eggs experience a pHi increase
after fertilization.
As recently demonstrated by Hamaguchi (88), eggs of
another echinoderm, the sand dollar Clypeaster japonicus, also alkalinize by about 0.4 U within 3 min after
fertilization.
This pHi increase requires external Na+. In
this unusual study, pHi was monitored by microinjection
of a calorimetric pH indicator, phenol red-a technique
which we have already mentioned was previously unsuccessful in the hands of such careful workers as the
Needhams (160) and Chambers (37). It remains to be
seen whether Hamaguchi’s success was due to a fortuitous choice of indicator dye or organism, but the supporting data he offers strongly substantiate the conclusion that a pHi increase activates the fertilized sand
dollar egg.
It is important to note that pHi changes after fertilization are not without exception: Johnson and Epel (111)
have recently shown that eggs of the sea star Pisaster
ochraceous do not alkalinize
after fertilization,
nor do
they release significant amounts of acid. Similarly, only
negligible acid release follows fertilization
in two other
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
servations on the regulatory effect of pH changes in a
cell-free protein synthesis system from sea urchin eggs
(240, 242) support this notion. Nonetheless a difficulty
remains: NH3 and the other weak bases that elicit the
late events (ethylamine,
nicotine, and procaine) also
permeate and alkalinize
other intracellular
compartments, with largely unknown m.etabolic consequences.
Indeed the possibility ’ remains that all these chemi .cally
related age nts (basic, membrane-permeant
amines) may
activate eggs through some secondary effect indirectly
related to the pHi change they impose, as has recently
been suggested (107). Thus it would be useful to demonstrate activation with agents other than membranepermeant amines to raise PHiOne obvious alternative is to increase pHi by raising
pH,. With DMO, Johnson and Epel(109) showed pHi to
be a roughly linear function of pH, in the sea urchin egg.
While the authors reported pHi val ues as high as 8.2 in
this study, they di .d not report the effect of such treatments in activating the late events of fertilization,
none
of which are apparent to casual observation. Significantly, reports by Steinhardt and Mazia (215) and Shen
(204) do indic ate that eggs trea ted with high pH, sl.owly
hyperpol arize to the memb rane potential typical of activated eggs, presumably due to the appearance of new K+
conductance. In contrast, however, Mazia and Ruby
(139) reported that the activating effect of NH3 on DNA
synthesis and chromosome condensation is not obtained
with seawater at pH, 9, and a recent abstract (110) states
that protein synthesis is not stimulated by “high pH,.”
Since neither of these negative reports presents experimental details or data, however, their observations
should be cautiously interpreted. Future efforts to test
the effects of high pH, must incorporate direct determinations of pHi to ensure that a sufficient period of
treatment is used.
Presently the most compelling evidence for the activating effect of high pH, is the observation by Nishioka
and Epel (165; see also Ref. 59) that fertilized eggs
arrested by transfer to Na+-free seawater (to block the
normal pHi increase) can be stimulated to cleave by
raising pH, to 9. The significance of this result has
perhaps been clouded by the later report of Nishioka and
Cross (164) that eggs fertilized in Na+-free pH 9 seawater
fail to cleave. This observation has been offered as evidence against the regulatory significance of pHi in this
system ( 107), but a more likely explanation concerns the
obvious polyspermy of these zygotes, which interferes
with cleavage (N. L. Cross, personal communication).
Thus the data presently available suggest to us that
elevation of pHi by high-pH, treatment probably activates sea urchin eggs in a fashion similar to NH3 treatment, but a comprehensive analysis of this issue would
be useful. If this supposition proves correct, an interesting question arises. As Jaffe (107) has noted, pH, within
the sea urchin’s natural environment may rise as high as
10 due to photosynthetic
activity. How, then, is premature activation of eggs avoided? Conceivably the relative
slowness of high-pH,-induced
activation [noted by Loeb
(128) and suggested by the data of Refs. 204 and 2151
may be the answer. Sea urchins are synchronized spawn-
W.
INVITED
OPINION
consisting of an initial (effectively instantaneous) acidification of the medium (not obtained when an eq)lal
volume of cell-free seminal fluid is added in place of
“dry” semen) and a slower, progressive acidification inhibited by cyanide and thus presumed to reflect metabolic COZ production. The fast acid release is not inhibited by cyanide, however, but can be prevented by dilution of semen into Na+-free seawater-a treatment that
also inhibits initiation
of flagellar motility. Both acid
release and motility are stimulated when Na+ is added
to the initially Na+-free suspension. In analogy with the
sea urchin egg, the authors interpreted these results to
reflect a Na”-H+ exchange mechanism in the plasma
membrane and supposed its activity to effect a net alkalinization
of pHi.
Although pHi was not determined
in this seminal
study, the authors’ conclusion was lent considerable
weight by their observation that in Na+-free seawater,
motility is reversibly activated either by increasing pH,
to 9, by addition of 10 mM NH&l,
or by the K+-H+
ionophore, nigericin -treatments
expected to elevate
pHi. Semiquantitative
studies of both L. pictus and S.
purpuratus sperm (39, 40, 123-125) have confirmed the
expected effect of these agents on pHi; more importantly,
they have demonstrated that a considerable alkalinization of pHi accompanies dilution of dry semen in Na+containing (but not Na+-free) seawater. For L. pictus,
this involves an increase from pHi 6.4 to 6.9, as determined with methylamine
and 9AA (124), or from 7.2 in
dry semen to 7.6 in Ca2+ -free seawater as determined by
31P-NMR (108). For S. purpuratus sperm in seawater,
both methylamine
and 9AA reveal a pHi of about 7.4
(40), and the 31P-NMR-determined
pHi of dry sperm is
about 7.0 (241). These studies also confirm that, in both
sea urchins, all treatments that elevate pHi also activate
both motility and its concomitant, increased respiration.
Studies of permeabilized
sperm models provide evidence suggesting that these pHi changes have direct
effects on the flagellar motor. In the presence of ATP,
glycerol-permeabilized
S. purpuratus sperm are immotile
at pH 6, while at pH 8 flagellar motility is maximal (98);
in Triton-permeabilized
S. purpuratus sperm, motility is
also maximal at pH 8 and is completely inhibited below
pH 7.4 (85). Similarly Triton-permeabilized
sperm of the
Hawaiian sea urchin Colobocentrotus atratus display decreasing motility as pH is lowered from 8.3 to 7.5 (76),
and the axoneme-bound dynein ATPase from this same
species displays no activity below pH 7, increasing to its
maximum value at pH 9.5 (77).
Dry S. purpuratus sperm display relatively high levels
of free ATP as revealed by 31P-NMR (241); thus an ATPlimited mechanism of motility initiation
seems unlikely.
On the other hand, the potential for Ca2+ and adenosine
3’,5’-cyclic monophosphate (CAMP) mediation of sperm
motility
(69) demands a more careful consideration
of
the interplay between pHi and these other regulatory
factors (Sect. IV).
A similar pattern of motility initiation
in response to
a Na+-H+ exchange-mediated
alkalinization
of pHi in
mammalian sperm has been inferred by Wong et al. (244).
Mature rat spermatozoa are stored in the cauda epi-
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
genera of starfish (174). Thus, even within the single
phylum Echinodermata
we see that while metabolic regulation via pHi at fertilization
may be widespread, it is
not universal.
Direct measurements of pHi in other invertebrate eggs
are unavailable,
but a growing number have been reported to release fertilization
acid. The molluscs present
a picture similar to that seen in echinoderms. Na+dependent acid release accompanies the activation of
Spissula (104) and Barnea (56) eggs, and NH3 partially
activates both of these (5, 55, 56), suggesting the likelihood of a pHi increase similar to that seen in the sea
urchin egg. In contrast, such other molluscs as the mussel
Myths
californianus
and limpet Acmaea incessa do not
release fertilization
acid (173). The last remaining example of fertilization
acid release of which we are aware
occurs in eggs of the echiuran worm Urechis caupo (173).
Since observations of fertilization
acid release provide
only circumstantial
evidence regarding the behavior of
pHi, the results just cited really do little to answer the
important question of whether sea urchin and sand dollar
or starfish eggs are most representative of animal eggs
in general. Thus it is particularly
significant that the
only vertebrate egg so far studied-that
of the frog Xenopus laevis- displays a 0.24 U alkalinization
during activation as determined either by 31P-NMR or pH microelectrode (168, 237). Although the physiological role of
this alkalinization
remains unknown, the occurrence of
substantial pHi changes in organisms as distantly related
as sea urchins and frogs certainly suggests the possibility
that pHi changes may prove to play a significant role in
the eggs of many organisms.
2) Sperm actiuation. As with the metabolic activation
of the egg, our understanding of the role of pHi in sperm
activation is biased toward the echinoderm gamete. Very
recent studies of arthropod, ascidian, and mammalian
sperm suggest both important
similarities
and differences, however. Below, we compare and contrast these
recent results with our more extensive understanding of
sea urchin sperm activation.
Sea urchins are broadcast spawners, and their sperm
exhibit two distinct metabolic activations: the first as
semen is shed into the water column (and spermatozoa
are thus removed from seminal fluid to sea water) and
the second as the spermatozoon comes under the influence of components of the egg jelly. We discuss each of
these events in turn below.
DILUTION
EFFECT. When sea urchin semen free from
seawater (so-called “dry” semen, >101’ cells ml-‘) is
diluted in seawater, sperm respiration increases dramatically (87)-a response termed the respiratory dilution
effect. Flagellar motility is also activated (164), probably
accounting for a considerable fraction of the increase in
metabolic
rate. Rothschild
(188) observed that even
small decreases in the pH, of the diluent seawater profoundly inhibit sperm respiration, but the first compelling evidence of a role for pHi in seawater-induced activation was the observations of Nishioka and Cross (164).
The authors observed that L. pictus or S. purpuratus
sperm diluted into seawater release proton equivalents,
thus acidifying the medium. The acid release is biphasic,
R415
R416
AND R. NUCCITELLI
results is our inability to separate the effects of elevated
pH, from those of its concomitant,
elevated pHi. In a
novel approach to this problem, Tilney et al. (221) applied a variety of protonophores with widely differing
affinities for other cations to dem .onstrate that intracellular alkalinization-even
in the absence of Ca2+-can
alone activate one step of the acrosome reaction, namely,
polymerization
of previously sequestered actin to form
the acrosomal process. Observing that egg jelly stimulates acid release by the sperm, the authors hypothesized
that a pHi increase might mediate the formation of the
acrosomal process. Previous work suggested the target of
this (then undocumented)
pHi increase to be the actinsequestering protein(s) of the sperm, since at pH 6.4 in
vitro these bind actin monomers, preventing their polymerization, but at pH 8 release actin (219).
Using S. purpuratus sperm treated with a variety of
metabolic inhibitors,
Schackmann et al. (197) showed
that the acid release elicited by egg jelly is independent
of respiration and thus is not due simply to CO2 production. Jelly-induced
acid release requires external Na+,
and 22Na+ uptake and H+ efflux have similar time courses
(but do not always display the equivalent stoichiometries
originally reported in Ref. 198; R. Schackmann, personal
communication).
These findings suggested a role for
Na+-H+ exchange in the acrosome reaction, and it was
not long before a Na+-dependent alkalinization
of pHi in
response to egg jelly was observed, using weak base
distribution
techniques. In both L. pictus (124) and S.
purpuratus
(196) this jelly-induced
alkalinization
amounts to an approximately
0.2 U pHi increase. In L.
pictus the increase is transitory, returning over 5-10 min
to its original low value concomitant with cessation of
motility. This reacidification
of acrosome-reacted sperm
is accompanied by a prodigious increase in Ca2+ accumulation and does not occur in Ca2+-free media. Since
mitochondrial
inhibitors
such as antimycin block this
reacidification
(without effect on Na+-dependent
alkalinization), it seems likely to involve mitochondrial
Ca2+H+ exchange (Sect. IvA).
Ohtake (170) reported that a “sperm-activating
substance” is present in crude extracts of sea urchin egg
jelly; it significantly
increased sperm respiration and
motility at low pH, values, but not at physiological pH,
(ca. 8.1). Garbers and co-workers (70, 90) have more
recently purified a glycine-rich polypeptide from these
extracts, which they have named speract. Speract has
the same activating effect on sperm, again at pH, 6.6 but
not at pH, 8 (115). At low pHO speract stimulates Na+
influx and acid efflux from the sperm and requires external Na+ but not Ca2+ (89). Monensin, a Na+-H+ exchange
ionophore, duplicates the activating effects of speract
(89). Its ineffectiveness at normal seawater pH renders
it difficult to assign a physiological role to speract, and
this led Ohtake to suggest that pH within the egg jelly
layer might be significantly lower than that of seawater
due to a Donnan equilibrium
arising from the high density of fixed anions in the jelly. Holland and Cross (95)
have recently measured the pH within the egg jelly coat
with microelectrodes and observed no significant difference from the pH of the bathing medium. However, the
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
didymus and vas deferens in an immotile state; motility
is initiated on dilution with the secretions of the accessory glands during ejaculation. Epididymal sperm remain
immotile when diluted in Na+-free medium but initiate
sustained motility in an otherwise identical Na+-containing medium. Na+-stimu .ated m.otility ini tiation is accompanied by acidification
of the mediu .m, but the relative
contribution
of CO2 is unclear. Millimolar
amiloride inhibits both Na+-stimulated
motility
initiation
and acid
release. As in the sea urchin, NH&l stimulates motility
in Na+-free sperm suspensions. The authors apparently
interpret th .is as a replacement of NH: for Na+, but a
more likely explanation involves alkalinization
of pHi by
NHS. pHi has not been reliably measured in rat spermatozoa, but homogenization
studies suggest a higher pHi
for the Na+-stimulated
sperm (244).
Babcock et al. (10) have very recently measured the
pHi of bovine epididymal sperm, using the pH-dependent
absorbance difference spectrum of intracellularly
generated carboxyfluorescein.
They reported a pHi of 6.6 at
pH, 7.4. Addition of NH&l
raised pHi and stimulated
both respiration and motility.
SPERM ACTIVATION
BY EGG FACTORS. Throughout
much of the animal kingdom,
sperm exhibit distinct
morphological;
metabolic, and behavioral responses in
the presence of eggs. Perhaps the most familiar of these
is the sea urchin acrosome reaction, which involves both
exocytosis of the acrosomal vesicle and polymerization
and bundling of previously sequestered actin to form the
acrosomal process. Certain taxa (e.g., mamm .als) lack an
acrosomal process but do display acrosomal vesicle fusion. Others, such as ascidians, possess neither acrosomal
vesicle nor process; nonetheless ascidian sperm too
changes in response to
undergo complex morphological
externally borne egg factors. Despite th e considerable
variations on a theme represented by the echinoderm,
mammalian,
and ascidian sperm reactions, pHi changes
of regulatory significance may be common to all three.
Some isolated egg factors also stimulate sperm respiration and/or motility. In the case of the sea urchin, one
such factor may act by modulating pHi, whereas in the
horseshoe crab Limulus a similar factor also affects pHi,
but this pHi change appears inconsequential.
SEA URCHIN ACROSOME
REACTION. The sea urchin
sperm acrosome reaction is elicited by egg jelly, a complex
gel of fucose sulfate polymers and other factors that
forms an investment about the egg. Involvement
of Ca2+
in the acrosome reaction (as in 0th .er membrane fusion
events) is well established (see Ref. 60 for review), and a
role for pHi has also recently emerged.
Three decades ago, Dan (46) observed that elevated
pH, elicits the acrosome reaction in the absence of egg
jelly, but only in Ca2+ -containing media. The interactions
of Ca2+, pH,, and egg jelly were studied in greater detail
by Decker et al. (47) and Collins and Epel (43), using
sperm from A. punctulata, L. pictus, and S. purpuratus.
Both groups fou nd that egg jelly effect ively lowers the
pH, optimum of the acrosom .e rea ction: from pH0 8. 5 to
9.5 (depending on the species) in the absence of egg jelly
toward the physiological pH, range (88.2) in its presence. An obvious difficulty in interpreting
these early
W. B. BUSA
INVITED
OPINION
anating from Limdus eggs. A dose-dependent increase
in pHi (as determined with 9AA) accompanies SMImediated motility initiation, but a similar alkalinization
of pHi by NH&l
does not activate motility.
In sharp
contrast with the response of sea urchin sperm, dilution
of Limulus sperm in Na+-free seawater actually activates
motility, despite the fact that the SAA-determined
pHi
is lower in Na+-free seawater than in normal seawater;
adding Na+ back to the Na+-free sperm suspension again
inhibits motility. These results thus strongly suggest that
pHi does not mediate the motility
response to SMI;
nonetheless it is interesting that here, as in the cases
discussed above, sperm pHi i ncrea ses in response to an
egg factor; thus as-yet-unexplored
aspects of Limulus
sperm physiology may be responsive to pHi changes. In
light of this possibility, it would be of interest to determine whether Limdus sperm activated without a pHi
increase (e.g., by SMI treatment in the presence of elevated CO2) are competent to effect normal fertilization.
Finally, since Limulus sperm motility can be activated
even as pHi decreases (i.e., on dilution in Na+-free seawater), it would be of considerable interest to determine
the pH dependence of motility in permeabilized
sperm
models as well as the pH-activity
profile of the flagellar
dynein ATPase, since Clapper and Epel’s observations
imply that these must differ substantially
from those
observed in sea urchin sperm (see above). The results of
such studies, in combination with determinations
of the
primary structures of the horseshoe crab and sea urchin
sperm dyneins, could contribute to our understanding of
the genetic changes required to confer pH sensitivity on
enzymes (Sect. VI).
MAMMALIAN
SPERM CAPACITATION
AND ACROSOME
REACTION. Of necessity, none of the sea urchin sperm
pHi estimates so far discussed have dealt directly with
the pHi of any specific compartment within the cell (e.g.,
the acrosomal granule, mitochondria,
flagellum, nucleus,
or the much-reduced cytosol of the sperm head). Fluorescence microscopy of SAA-accumulating
sperm should,
in principle, reveal the most acidic compartments of the
cell by their greater fluorescence (to the degree that
artifacts due to binding and concentration quenching can
be ruled out). In sea urchin sperm treated with 9AA, the
entire cell, except the midpiece, fluoresces (124). In the
mammalian
(hamster) sperm, however, a strikingly different pattern of fluorescence is observed, as demonstrated by Meizel and Deamer (143): the distinctive
acrosome is intensely fluorescent, while the remainder
of the cell appears dark. This reflects the highly acidic
pHi of the acrosome rather than simple binding of 9AA,
since nigericin, NH&l,
and low pH, all reverse the
acrosomal accumulation of 9AA. By quantifying the uptake of 9AA or ‘*C-methylamine,
the authors estimated
an acrosomal pHi of 55. This rather acid pHi, like that
of chromaffin granules and lysosomes, appears to be
maintained by a proton-translocating
ATPase within the
acrosomal membrane (245, 246).
The mammalian
acrosome contains an inactive (zymogen) protease, proacrosin, the active form of which,
acrosin, is required for the acrosome reaction. Like certain other zymogen proteases, proacrosin is capable of
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
parameter of interest is the pH in the immediate environment of the jelly-activated
sperm. As Holland and
Cross observed, pH within the jelly coat of fertilized eggs
is temporarily
depressed by 0.25 U, presumably due to
fertilization
acid efflux from the egg. Since sea urchin
sperm also release both protons and CO2 during jellyinduced activation (i.e., before fertilization),
it remains
possible that a similar temporary depression of pH, may
occur in their microenvironment;
if so, speract might
serve to prevent this acidification
from inactivating
the
sperm just short of its goal. It may prove feasible to
resolve this issue using inseminated jelly coats loaded
with a fluorescent pH indicator dye.
As witnessed through the eyes of experimental
biologists, then, the short active life of the echinoid sperm
comprises a sequence of pHi changes via Na+-H+ and
Ca2+-H+ antiport activations, possibly interacting in a
complex fashion with Ca2+, CAMP, and membrane potential to establish a metabolic context appropriate to
each point along the sperm’s life journey. To what extent
the sperm itself shares our present point of view is still
debatable; for example, since dry sea urchin semen does
contain some Na+, the lack of motility in semen cannot
be ascribed solely to inhibition
of Na+-H+ exchange, and
other factors such as the high CO2 partial pressure (Pco~)
within the gonad, must help depress pHi (108).
ASCIDIAN
SPERM REACTION. Although they lack an
acrosome reaction, sperm of the ascidians (invertebrates
of the phylum
Chordata)
still undergo remarkable
changes on contact with the chorion layer of the egg: the
single mitochondrion
rounds, swells, migrates down the
flagellum, and is shed from its tip (117). The ascidian
sperm reaction (ASR) is accompanied by Ca2+ uptake
and acid efflux (116-118), but the contribution
of respiratory CO2 to this efflux is unclear. Elevating pH, to 9.5
increases sperm motility,
initiates the ASR in the absence of egg factors (ll7), and induces acid release even
in Ca2+-free seawater (118); the ability of NH3 or other
weak bases to induce the ASR at normal pH, has not
been reported. Preliminary
estimates of pHi suggest that
it increases during the ASR (116), but a detailed report
has not yet appeared. The inhibitor of intracellular
Ca2+
release, N,N-(diethylamino)-octyl-3,4,5trimethoxybenzoate, blocks the ASR induced by high pH, in Ca2+-free
seawater (C. Lambert, personal communication),
suggesting that the pHi increase accompanying the ASR
acts to mobilize intracellular
Ca2+, which may therefore
be the proximate effector of the sperm response.
MOTILITY
INITIATION
IN LI~MUEUS SPERM. Evidence
for physiologically
significant pHi changes in sperm from
such diverse taxa as the echinoderms,
ascidians, and
mammals clearly raises the possibility that pHi-mediated
regulation of sperm motility
and metabolism
may be
widespread throughout
the animal kingdom; thus the
recent observation by Clapper and Epel (41) that pHi
appears to play no role in motility initiation
in sperm of
the horseshoe crab (Limulus polyphemus) is germane to
the present discussion. Limulus sperm undergo a brief
flurry of motility upon dilution of dry semen in seawater
but quickly become immotile again until they encounter
a so-called sperm motility-initiating
peptide (SMI) em-
R417
R418
AND
R. NUCCITELLI
mediates the partial activation of the sea urchin egg.
BACTERIA.
The first published observation of a large
pHi change accompanying termination
of cryptobiosis
was that of the Setlows (201), who showed that spores of
the bacteria Bacillus cereus and B. megaterium alkalinize
from pHi 6.3 to 7.4 (determined by the methylamine
distribution
technique) when induced to germinate by
heat shock. The low pHi of the dormant spore has been
confirmed by 31P-NMR (16). Because glycolysis is arrested during spore dormancy, the Setlows suggested that
a potential target for pHi-mediated
regulation in this
system might be phosphoglycerate
mutase, since the in
vitro activity of this enzyme at pH 6.3 is but 10% of that
at pH 7.3; however, more recent results (217) have failed
to support this hypothesis. Artificial
alkalinization
of
dormant spore pHi with NH3 [20 mM (NH&Sod;
pH,
8.6, pHi 7.81 not only fails to activate 3-phosphoglycerate
catabolism but also has no other apparent activating
effects, indicating that the pHi increase cannot be the
soZe mediator of arousal from dormancy in the Bacillus
spore. Whereas it is therefore not yet possible to attribute
physiological significance to the very large pHi change
reported by the Setlows, the possibility remains attractive. For example, the depression of pHi, which may be
presumed to accompany sporulation, may be the physiologically significant factor, but this hypothesis may prove
very difficult to test. Alternatively,
alkalinization
may be
necessary but not sufficient to induce arousal from dormancy in this system.
YEAST.
A pHi increase of similar magnitude also appears to accompany germination in spores of a eucaryote,
the yeast Pichia pastoris. This pHi change has not yet
been directly observed but has been inferred (28) from
31P-NMR evidence that the pHi of dormant Pichia spores
is quite acidic (pHi 5.5-6.3) (16), since the pHi of vegetative yeast cells is roughly 1 U higher than this value.
The acidic pHi of the dormant spore has been suggested
to play a role in the inhibition
of glycolysis (16), but no
attempt to test this hypothesis has appeared as of the
time of writing.
PROTOZOONS.
31P-NMR studies of both encysted and
vegetative cells of the protozoon Acanthamoeba castellanii have failed to reveal any difference in 2Hi between
the dormant and metabolically active forms (50), but this
result is difficult to interpret due to the nominally anaerobic conditions employed, which would be expected
to depress preferentially
the pHi of vegetative cells.
CRUSTACEANS.
In the final cryptobiotic system to be
discussed here, the encysted gastrula-stage embryo (or
cyst) of the brine sh.rimp Artemia salina, pHi appears to
be the physiological mediator of the dormancy-metabolism transition. The embryo, enclosed in a semipermeable shell, is released from the maternal ovisac in a profoundly dormant state (hereafter, aerobic dormancy)
which, under proper conditions, may be maintained for
years; under natural conditions aerobic dormancy is terminated by desiccation of the cyst and subsequent rehydration at a permissive temperature and oxygen tension (see Ref. 42 for review). When cysts are rehydrated
in the absence of oxygen, dormancy can continue for at
least 5 mo (anaerobic dormancy) or may be reimposed by
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
autoactivation.
For purified rabbit proacrosin this process is rapid at pH 8 but is greatly inhibited at pH 6,
suggesting that inhibition
of proacrosin autoactivation
may be one physiological role of the acidic acrosomal pHi
(143).
Unlike the sea urchin, mammalian
sperm normally are
not capable of the acrosome reaction (and, hence, fertilization) immediately
after ejaculation;
generally they
first require extended incubation in female genital tract
fluids or, in vitro, in buffers containing serum albumin
or other macromolecular
factors. The still largely undefined biochemical events that enable the acrosome reaction are referred to collectively as capacitation, and it is
important to note that the pHi estimate of Meizel and
Deamer is for noncapacitated sperm. More recent studies
and
of synchronously capacitated sperm by Working
Meizel (247) reveal that acrosome pHi (as estimated
using 9AA) increases by >l U just before the acrosome
reaction. Significantly,
elevating pHi with NHS, protonophores, or inhibitors of the acrosomal H+-ATPase induces the acrosome reaction in capacitated (247) but not
noncapacitated
(245) sperm. In light of these and other
results, the authors proposed (245) that capacitation
and/or the early events of the acrosome reaction involve
both an increase in proton permeability
by the head
membranes and the inhibition
of the acrosomal H+ATPase (247). The result would be a proton efflux from
the acrosome and thus its alkalinization.
This could
initiate or accelerate the activation of proacrosin, and
other effects may remain to be elucidated. Such pHi
mediation of the mammalian
acrosome reaction is also
suggested by the observation (103) that guinea pig sperm
spontaneously undergo the acrosome reaction in a simple
Ca2+-containing salt solution (without the macromolecular factors usually required for in vitro capacitation) at
high (~7.8) but not at lower pH,.
Characterization
of the acrosomal proton-translocating ATPase has documented its pronounced inhibition
by micromolar Ca2’ (246). In light of this, it is tempting
to speculate that Ca2+ accumulation within the reduced
cytoplasm of the sperm head in response to natural
triggers might easily reach levels inhibitory to the acrosomal ATPase, thus initiating
the alkalinization
of the
acrosome (247).
3) Dormancy-metabolism
transitions.
The relative quiescence of sperm and egg we have discussed so far is a
modest form of dormancy, a spectrum of hypometabolic
states in which cells await conditions favoring continued
growth. In its most extreme form, termed cryptobiosis,
cellular do rmancy approaches a truly ametabolic state.
Although perhaps unfamiliar to most biologists, cryptobiosis is far from a biological curiosity: it is common
among bacterial and fungal spores, protozoans, such
micrometazoa
as nematodes and tardigrades, certain
crustacean embryos, and the seeds of many plants. Although it i.s probably fair to state that very little is known
regardi w the principles l regulating WP tobiosis, some
very recent research raises the possibility
that pHi
changes of very large magnitude may be fundamental to
the esta blishment an .d termination
of c:ryptobiotic dormancy, just as the smaller pHi change after fertilization
W. B. BUSA
INVITED
OPINION
further efforts to explore the physiological significance
of the large pHi changes reported in other cryptobiotic
systems is clearly called for. As one of us has previously
pointed out (28), pHi changes of the magnitude observed
in cryptobiotic systems seem certain to have profound
consequences for numerous en .zymes and strut tural proteins; elucidating these effects
11be an importa nt goal
in the future. Fin .ally the resul for Arte mia are important in another sense: they extend the regulatory role for
pHi in development to include events well beyond fertilization and represent the first demonstration
of pHimediated regulation of development in a multicellular
system.
.
.h’
B. Cell Cycle and Proliferation
Measurements of pHi during the cell cycles of a number of cell types have revealed oscillations associated
with nuclear events such as mitosis and DNA synthesis.
In general, these events are correlated with increased
pHi. There is as yet no conclusive evidence that pHi plays
a causal role in cell cycle control, but a number of
intriguing
observations
suggest that imposed pHi
changes can influence the rate of DNA replication or
affect the timing of mitosis. Three lines of evidence
(discussed below) suggest that pHi may influence nuclear
events.
1) PHi changes during the cell cycle. Intracellular
pH
measurements have been made during the cell cycle in
seven different animal species ( Table 2); the results fall
into two categories. In the embryonic systems listed in
Table 2, pHi changes are quite small, and the premitotic
pHi shift is in the acidic direction. In yeast, slime molds,
ciliates, and lymphocytes, on the other hand, fairly large
pHi oscillations (ca. 0.3 U) occur during the cell cycle
(see NOTE ADDED IN PROOF), and an increase in pHi
generally precedes mitosis or DNA synthesis. In Physarum, for example, mitosis occurs either shortly after or
just as a major alkalinization
occurs and can be delayed
by artificially
lowering pHi (158). As Gerson (72) has
pointed out, the DNA polymerases of numerous species
all exhibit very alkaline pH optima and would be expetted to show increasing activity with increasing pHi.
Unfortun .ately, even where alkalinizations
occur, the onset of DNA synthesis is not always coincident with the
pHi increase. In yeast the onset does not come until 45
min after the pHi maximum,
and in Tetrahymena the
onset begins when the pHi is at one of its lowest points
in the cycle. In both Physarum and lymphocytes, however, the fairly close correlation between the timing of
the pHi rise and the time of mitosis or the increase in
DNA synthesis is consistent with the idea that pHi may
be influencing these events. An additional recent observation consistent with this hypothesis is that cyclosporin
A, which blocks T-cell activation and the associated
DNA synthesis, also blocks the early pHi increase (74).
In both sea urchin and frog embryos there is very little
variation in pHi during the cleavage cycle itself. Both
these embryos exhibit a pHi increase of 0.3-0.4 U after
fertilization
(Sect. III/II),
but this is a relatively permanent increase, on which is superimposed a small, cyclic
acidification
associated with the cell cycle. Moreover
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
withdrawing O2 even after an interval of aerobic development. Presumably anaerobic dormancy is an important asset to the brine shrimp, which inhabits hypersaline waters in which the solubility of O2 can become very
low at high temperatures. The true reversibility
of the
dormancy-metabolism
transition in Artemia (unusual in
dormant systems) has been the key to elucidating the
physiological role of pHi in this system.
Using 31P-NMR to determine the mean pHi of the
4,000 cells comprising the embryo, Busa et al. (30) demonstrated that the transition from aerobic development
into anaerobic dormancy in Artemia cysts involves a >l
U acidification of pHi (from 27.9 to 6.3). pHi is effectively
independent of buffer pH, presumably due to the wellestablished impermeability
of the cyst shell to potential
counterions. This is a significant point if the low pHi of
the dormant cyst plays a role in cryptobiosis, since the
dormant embryo may thus maintain an acidic pHi in the
face of environmental
fluctuations at no direct energetic
expense. Finally, when metabolism and development of
anaerobic dormant embryos are reinitiated by oxygenation, pHi returns to 27.9. This is the largest pHi change
known to occur in any cell system under physiological
conditions.
Although direc t measurements of pHi in aerobic dormant cysts (i.e., those not previously desiccated and
rehydrated) are not yet available, recent studies (29)
provide strong circumstantial
evidence that in these as
in anaerobic dormant cysts, pHi is greatly depressed and
further indicate that low pHi is the fundamental regulator of aerobic dormancy. Aerobic dormant cysts incubated in 40 mM NH&l
(pH, 8.5) hatch as well as or
better than conventionally
activated (i.e., desiccated and
rehydrated) controls (33 and 26% hatch, respectively)
and significantly better than untreated aerobic dormant
controls (~5% hatch; P c 0.005 by Student’s t test). 31PNMR studies of anaerobic dormant embryos demonstrate that this level of NH3 alkalinizes pHi by about 0.9
U.
Since, as the results with NH3 indicate, alkalinization
of pHi terminates dormancy, it might be expected that
aerobic dormancy would be induced by artificial acidification of activated (i.e., desiccated and aerobically rehydrated) cysts. Indeed, this is the case: when activated
cysts are incubated under 60% COZ-40% 02, pHi is depressed from the normal value of 27.9 to 6.75, respiration
is reversibly inhibited by ca. 70%, and development is
reversibly arrested, resuming with normal kinetics on
removal of C02. Thus treatment with weak acid depresses pHi and inhibits metabolism and development,
imposing on these embryos a state closely resembling
aerobic dormancy, whereas weak base treatment raises
pHi and terminates true aerobic dormancy. Unlike the
sea urchin egg, which is only abortively activated by NH3
(P to but not including cell division, presumably due to
tie absence of the sperm centriole), the Artemia embryo
is fully activated by NH3 and develops in an apparently
normal fashion to at Least the free-swimming
nauplius
larva stage.
Given this recent demonstration
of pHi-mediated
metabolic regulation of cryptobiotic dormancy in Artemia,
R419
R420
W. B. BUSA
2. Summary
TABLE
Cell
Type
pHi
of pHi
Measuring
Technique
Synchronized
Culture?
Stimulus
to Initiate
Cell Cycle
Progression
+ Glucose
NMR
Yes (starved)
+ Glucose
DMO
Yes (starved)
T. pyriformis
DMO
Physarum
polycephalum
Recessed-tip pH
microelectrode
Initial
P.
polycephalum
L. pictus
(urchin
embryo)
S. purpuratus
Recessed-tip pH
microelectrode
DMO
+0.55 (15 min)
(DNA synthesis)
Refeed
7.55
Yes (heat)
Cool to 28°C
7.4d
Plasmodial
stage is
syncytium;
natural
synchrony
Plasmodial
stage is
syncytium;
natural
synchrony
Synchronous
cleavagee
Refeed (after
starve 9 h)
-0.30
+0.35
-0.35
+0.25
-0.20
-0.15
+0.20
-0.20
+0.25
-0.30
7.00
Refeed (after
starve 13 h)
6.70
Fertilize
laevis
(amphibian
embryo)
Mouse spleen
lymphocytes’
+0.35 (15 min)
(100 min)
(180 min)
(280 min)
(350 min)
(400 min)
(45 min)
(65 min)
(80 min)
(125 min)
(170 min)
+0.40 (3.5 h)
-0.25 (6 h)
+0.05 (7 h)
-0.10 (8 h)
Approx
Onset
Synthesis
Times of
of DNA
or Mitosish
60 min/90 min
Ref.
No.
84
?
100 min/350 min
(DNA synthesis)
81
90 min/l50 min
(DNA synthesis)
81
4 h (mitosis)
158
+0.7 (6.5 h)
-0.20 (9 h)
6.5 h (mitosis)
158
7.4
-0.12 (80 min)
93 min (cleave)
109
DMO
Synchronous
cleavagef
Fertilize
7.6
NS change
110 min (cleave)
109
Recessed-tip pH
microelectrode
Single
blastomere
Fertilize
7.7
-0.03 (56 min)
80 min (cleave)
237
DMO
Yes
Con A
7.18g
(urchin
embryo)
Xenopus
pHi Maximab*”
(approx
time)
Not previously
oxygenated:
6.75
Previously
oxygenated:
6.95
(ciliate)
(slime mold)
pHy
(8 h)
73'
10 h/48 h
(18 h)
(DNA synthesis)
(60 h)
Yes
LPS
Mouse spleen
DMO
(8 h)
73'
7.1Sg
10 h/54 h
lymphocytes’
(18 h)
(DNA synthesis)
+0.12 (76 h)
NMR, nuclear magnetic resonance; DMO, 5,5-dimethyl-2,4-oxazolidinedione;
NS, not significant; Con A, concanavalin A; LPS, lipopolysaca Often referred to as “basal” or “resting” pH, which can be misleading. As used here, this value is that obtained at time 0 (before
charide.
stimulus) or the 1st value given immediately thereafter (as indicated).
b Each value is given with respect to previous value in co1 (or with
c From cited references, as given, to nearest 0.05 pH U.
respect to initial pHi for 1st value in each ~01).
d Earliest ti me point given (ca. 10
e 1st and 2nd cleavages occur 50 min apart. Difference between fastest and slowest embryos is 18 min.
min after cooling).
f 110 min to 1st
g Unstimulated cell population.
cleavage. Difference between fastest and slowest embryos is 10 min.
’ Via incorporation of 3H-thymidine.
'SEE NOTE ADDED
IN PROOF for conflicting observations.
normal cleavage continues to occur in frog embryos when
pHi is lowered as much as 0.3 U with weak acids (126).
This suggests that the lower pHi of the unfertilized egg
may help suppress mitosis but that pHi changes during
the cleavage cycle are not critical for cycle regulation.
One could argue that those cells exhibiting the larger pHi
oscillations during the cell cycle, such as Physarum, all
have dormant stages at low pHi when the cell cycle is
arrested. Thus an increase in pHi may permit entry into
mitosis but probably does not regulate the mitotic cycle
itself.
2) pHi changes may influence DNA replication and
mitosis. Despite the uncertainty
of the regulatory role of
pHi in the cell cycle, there is evidence from other cell
types that pHi may influence nuclear events.
MITOGENIC
EFFECT
OF ALKALINE
PH,. Gerson re-
+0.22
+0.04
+0.35
+0.22
+0.04
viewed the evidence for a mitogenic effect of alkaline
pH, in 1978 (71). When those data are combined with
more recent studies, four strong observations emerge: 1)
Obara et al. (169) observed that 3T3-cells proceed into
the mitotic cycle from a colcimid block faster at pH, 8
than at pH, 6.6; 2) Rubin (191, 192) reported that both
the rates of 14C-thymidine uptake and initiation
of DNA
synthesis increased with pH,; 3) Calothy et al. (31) found
that the number of SV40 virus particles released (which
depends on replication of the viral genome) was 30,000
times greater at pH, 8.4 than at pH, 6.4; and 4) Zetterberg
and Engstrom (250) found that serum-starved (quiescent) 3T3-cells can be stimulated to divide by increasing
pH,. A 2- to lo-min treatment at pH, 8.5-10 causes more
than 80% of the cells to begin DNA synthesis. The only
compound that was found to be required during and
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
Yes (starved)
Tetrahymena
pyriformis
R. NUCCITELLI
changes during the cell cycle
NMR
Saccharomyces
cerevisiae
(yeast)
S. cerevisiae
AND
INVITED
R421
OPINION
(see Refs. 147 and 185 for review), any changes in its
activity are likely to influence pHi. Direct measurements
of pHi in mammalian
fibroblasts
with DMO (48, 199)
and carboxyethyl-carboxyfluorescein
(150) have indicated an 0.2 U pHi increase within 20 min of serum or
mitogen addition. Na+-H+ exchange is directly involved,
since the evoked pHi change can be reversed by reversing
the Na+ gradient across the plasma membrane (150).
One hypothesis
is that growth factors induce a conformational change in the Na+-H+ exchanger, resulting in
an increase in the exchanger’s
affinity for cytoplasmic
H+ (67, 150).
A possible connection between this effect of mitogens
on the Na+-H+ exchanger and the initiation of cellular
transformation
has recently emerged. The most common
mitogen in serum, platelet-derived
growth factor, has
virtually the same amino acid sequence as that predicted
for the putative transforming
protein of simian sarcoma
virus (54, 236). This raises the possibility that one mode
of action of the viral transforming
protein might be to
modify the catalytic properties of the Na+-H+ exchanger.
There has also been at least one report of no pHi
change after mitogen (lectin) treatment
of thymocytes
(184). Therefore lectins might exert their mitogenic effect without the involvement of a pHi change. This raises
the question of whether or not the pHi increase observed
in fibroblasts
on serum addition is necessary for the
mitogen response. It would thus be of interest to observe
the effect on cell division in mitogen-stimulated
fibroblasts of treatments
that counteract
the alkalinizing
effect of mitogens (i.e., weak acids, low pH,, or low Na+).
Recently, Ca2+ and calmodulin have also been implicated in the mitogenic response. Owen and Villereal(l71)
found that the effect of serum in stimulating
Na+ influx
can be mimicked
by the divalent cation ionophore,
A23187. Since serum also increases Ca2+ influx (226), it
has been suggested that this [Ca2+]i increase may be one
step in the mechanism regulating Na+ influx. In keeping
with this, six agents known to bind calmodulin were
found to inhibit both serum- and A23187-stimulated
Na+
influx in a dose-dependent
manner (171). One possible
explanation for this is that serum increases [Ca”‘]i, and
the Ca2+-calmodulin
complex then stimulates the Na+H+ exchanger to increase both Na+ influx and H+ efflux.
This might also apply to egg activation, where a [Ca2+]i
increase somehow leads to a pHi increase (Sect. IIIA~).
EXTRACELLULAR
DENSITY
CULTURES.
pH
CAN
AFFECT
GROWTH
IN
HIGH-
More than 10 yr ago, Ceccarini and
Eagle (34, 35) found that the maximum population density of a fibroblast
cell culture was pH, dependent. In
bicarbonate-buffered
medium, high cell densities rapidly
acidified the medium and induced contact inhibition at
pH 6.9. However, if a nonvolatile organic buffer was used
to hold pH, at a level between 6.9 and 7.8 the culture
continued to grow to reach a density two to four times
greater than those normally observed. Contact inhibition
could be rapidly induced by simply replacing the medium
with one of pH 6.9, and growth could be reinitiated by
switching back to optimal pH. It is most likely that the
cell-induced acidification
of pH, is due to CO2 production, and in that same year Ahmed and Baron (1) showed
that lymphocyte
pHi falls as the CO2 concentration
of
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
immediately after the alkaline treatment was phosphate
(251). Interpretation
of these data requires some knowledge of how pHi varies with pH,. Many cells are able to
maintain a relatively constant pHi over a range of acidic
pH, values, but this ability often declines sharply at
alkaline pH,. Gillies (79) has documented this for six cell
types, and cultured cells are particularly
poor at pHi
regulation above pH, 8. It is therefore not unreasonable
to attribute the above effects of pH, to an alkalinization
of pHi.
MITOGENIC
EFFECT
OF SERUM.
Cellular responses t0
stimulation by serum or specific mitogens can take many
forms, including proliferation
and degranulation.
Such
serum stimulation
has been documented in at least 10
different cell lines, including neuroblastomas
(48, 149),
pheochromocytomas
(23), myoblasts (231), platelets (99),
and hepatocytes, lymphocytes,
and fibroblasts
from four
different species [(151, 177, 232); for reviews see Refs.
114, 148, and 1891. In some cases the specific mitogens
have been identified and include epidermal growth factor,
nerve growth factor, insulin, glucagon, thrombin, vasopressin,
and phytohemagglutinin.
All these stimulate
Na+ influx, and in at least five of these cell lines Na+H+ exchange is known to be stimulated.
Both insulin
and thrombin
have been shown to stimulate
Na’-H+
exchange and elicit a pHi increase in amphibian muscle
and mammalian platelets, respectively
(Sect. IIIC),
and
combined they stimulate Na+-H+ exchange in hamster
fibroblasts
(177). Insulin treatment activates meiosis in
amphibian oocytes (137) and increases Xenopus oocyte
pHi, apparently by activating Na+-H+ exchange (G. Morrill, personal communication).
An increase in Na+ influx detected with 22Na+ (in the
presence of ouabain to inhibit the Na+ pump) is the most
universal
response to serum addition. In many cells,
other means of increasing Na+ influx, such as Na+ ionophore or toxin application, also stimulate cell proliferation, suggesting that Na+ influx is a causal step in the
control mechanism (189,190,212).
In fact, DNA synthesis rates are usually higher if more than one Na+-influx
stimulus is presented, so that the strength of the proliferative effect appears to be roughly proportional
to the
amount of Na+ influx. Moreover
the inhibitor of Na+
influx, amiloride,
usually
blocks
mitogen-stimulated
DNA synthesis in these cells (149). Since amiloride also
inhibits the Na+-H+ electroneutral
exchanger of many
cells (22), it would also prevent any pHi increase resulting
from the activity of this exchanger. Unfortunately
amiloride also directly inhibits protein synthesis, so its effect
cannot be ascribed to the blockage of Na+-H+ exchange
alone (133).
Increases in Na+ influx would be expected to stimulate
Na+-K+-ATPase
activity, as has been observed in most
of these systems. This will tend to maintain a constant
[Na+]i, and in at least one of these cells (the neuroblastoma) a two- to threefold increase in the pump activity
roughly balances the increase in Na+ influx (48). However, no similar mechanism has been found to maintain
a constant [K+]i, which usually increases when the Na+K+-ATPase
is stimulated.
Because the Na+-H+ exchanger is one of the two main
components
of the pHi regulation system in most cells
R422
C. Stimulus-Response Coupling
The second messenger hypothesis has been dramatically successful in explaining the action of many hormones that act at the cell surface but influence metabolic
processes well removed from the hormone-binding
site.
In many cases the second messengers involved in hormonal transduction
are Ca2’ and/or cyclic nucleotides,
but in others concerted efforts have failed to clearly
implicate these messengers in the mediation of hormone
action. Insulin is an example of this latter case, and in
at least one cell type insulin now appears to exert some
of its metabolic effects by altering pHi. Similarly platelet
activation by thrombin
and neutrophil
activation by
chemotactic factors both involve pHi changes, the regulatory significance of which is yet unclear.
1) Insulin and glycolysis. A peptide hormone crucial in
the regulation of carbohydrate, fat, and protein metabolism, as well as a mitogen required for normal growth,
insulin has numerous effects on its target cells. Attempts
to ascribe insulin’s actions to its effects on CAMP or
Ca2+ levels within the cell have been largely unsuccessful
(45)
Expanding on earlier observations that insulin stimulates Na+ efflux from a variety of cells, Moore (152)
characterized the kinetics of the Na+ pump in insulinstimulated frog sartorius muscle and suggested on theoretical grounds that one effect of insulin might be to
activate H+ efflux from the cell, thus raising pHi. Later
studies employing both DMO (153) and 31P-NMR (157)
confirmed that insulin elevates frog sartorius pHi by O.l0.2 U. Na+-H+ exchange across the plasma membrane is
AND
R. NUCCITELLI
the most likely mechanism for this, since either amiloride
or reduced [Na’], inhibits the pHi increase normally
evoked by insulin; in contrast, the Na+-K+-ATPase
inhibitor, ouabain, blocks the insulin-activated
Na+ efflux,
but not the pHi increase (156). In elegant experiments,
Moore and co-workers (154) have further confirmed the
role of Na+-H+ exchange by demonstrating
that reversal
of the Na+ activity gradient across the plasma membrane
causes a depression of pHi in response to insulin.
Treatments that block the elevation of pHi in response
to insulin, such as amiloride and low [Na+lO, also inhibit
insulin’s stimulating
effect on anaerobic glycolysis (156).
Further evidence of the regulatory significance of this
pHi change is provided by studies involving artificial
manipulation
of pHi (66). When frog sartorius pHi is
elevated by decreasing PCO~ from 5 to 2.7% in the absence of insulin (raising pHi by 0.16 U), anaerobic glycolytic flux is increased by more than 50%, quite similar
to the increase induced by insulin treatment. Conversely,
increasing PCO~ to 10% acidifies the cell by 0.1 U and
depresses 4 .ycolytic flux by 50%. Because these experiments we re performed in glucose- #free med ium (plus epinephrine to mobilize glycogen as the substrate for glvcolysis) 1,the effects observed do not reflect activ ,ation of
glucose transport, but rather true inc reases in the activ ity
of the glycolytic pathway.
The most likely target for pHi- mediated regulation of
glycolytic flux, as discussed by Moore and co-workers
(66), is the rate-limiting
enzyme phosphofructokinase
(PFK). As demonstrated by Trivedi and Danforth (224),
PFK activity from both frog and mouse muscle displays
a remarkably sharp pH profile in vitro: under a variety
of conditions, an increase in pH of about 0.1 U is sufficient to stimulate PFK from nearly zero to full activity.
Interestingly
the absolute value of pH at which this
activation occurs depends on both the fructose 6-phosphate and AMP concentrations present, with increasing
levels shifting the pH-activity
profile to lower PH. The
pronounced pH-sensitivity
of PFK is observed at physiological (i.e., millimolar)
ATP concentrations, but not
at submillimolar
levels. Both insulin and artificial alkalinization activate PFK in vivo as judged by “crossover
plots” of glycolytic intermediate
levels (66), and pH
strongly regulates PFK activity in a cell-free rat skeletal
muscle system under carefully controlled physiological
conditions (248).
Although Moore’s work (see Ref. 155 for review) provides strong evidence that the glycolytic stimulatory
activity of insulin in frog skeletal muscle is mediated by
pHi with PFK as its target, this may not be the case for
some other tissues. With 31P-NMR, Bailey et al. (12)
have recently shown that normoxic rat heart pHi is
unaffected by insulin treatment. In addition to the species and tissue differences involved, Moore’s and Bailey’s
studies differed in that the former, but not the latter,
involved simultaneous administration
of epinephrine to
mobilize glycogen. It will be important
to resolve the
differences between these studies. Very recently, it has
also been observed that insulin alkalinizes the pHi of
immature frog oocytes (Sect. IIIB).
2) Throm #bin and platelet activation. In keeping with
their role as important mediators of hem .ostasis, platelets
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
the medium increases. Similarly, Gillies and Deamer (82)
found that endogenously produced COa induces stationary phase in Tetrahymena cultures. They also used the
weak acid, DMO, to lower pHi and mimic the effect of
Cog. Further, decreasing the COa concentration
of the
medium leads to increased maximum culture density at
stationary phase. They suggested that accumu lation of
CO2 in the culture leads to cessati .on of growth throu gh
the elimination
of transmembrane
pH gradients. It is
also worth mentioning
here that CO2 reversibly inhibits
meiosis in frog oocytes (20).
CONCLUSION. The last three lines of evidence discussed
strongly suggest that pHi can indeed influence the cell
cycle by inhibiting
mitosis or meiosis at low pH and by
increasing the rate of DNA synthesis at high pH, thus
shortening the mitotic cycle duration. Mitogenic stimulation of fibroblasts evokes a pHi increase, and this pHi
change seems likely to prove physiologically
significant
based on the known ability of CO2 or low pH, to inhibit
serum-stimulated
mitosis and on the ability of high pH,
to stimulate mitosis in serum-starved fibroblasts. It will
be important, however, to demonstrate this explicitly by
showing, using as lingle cell WP%that these experimental
manipulations
do in fact alter p Hi over th .e same range
of values as does serum . We conclude that pHi changes
can, in some instances, turn the cell cycle on or off (as
observed, e.g., in high-density cultures), but pHi oscillations within the cycle are not universal and their function, where they are present, is not yet clear.
W. B. BUSA
INVITED
R423
OPINION
apparently occur roughly simultaneously
(i.e., within seconds after addition).
Platelet PFK and glycolysis are also activated by collagen (4). Although its effect on platelet pHi has not
been reported, it has been observed that collagen differs
from thrombin in that it does not eli .cit plasma membrane
depolarization
(100). It would be of interest to determine
whether collagen increases platelet pHi; if so, this would
provide some evidence for the regulatory significance of
these pHi changes (i.e., in regulating glycolysis). Further,
since collagen is an important component of the extracellular matrix, its involvement
in pHi-mediated
metabolic regulation might help explain how cellular motility,
behavior, and differentiation
are influenced by the composition of the surrounding
extracellular
matrix materials, although this possibility is, at present, merely speculative.
3) Chemotactic factors and neutrophil activation. In
response to numerous chemotactic
stimuli, circulating
polymorphonuclear
leukocytes
(neutrophils)
leave the
vascular system, invade infected tissues, and there help
to suppress bacterial growth via endocytosis and release
of various antibacterial
agents. With the DMO technique, Molski et al. (146) observed a rapid, biphasic
response of neutrophil pHi to the synthetic chemotactic
factor, formyl-Met-Leu-Phe
(FMLP): a small but significant pHi decrease of about 0.05 U occurs during the first
30 s after addition, followed by a slower (30 s to 7 min)
alkalinization
of about 0.2 U. The response to FMLP is
specific and appears to be receptor mediated, since the
structurally
similar but biologically inactive binding antagonist, hoc-Phe-Leu-Phe-Leu-Phe,
inhibits FMLP’s
effects on pHi. The natural chemotactic
agent C5a (a
complement factor) has an effect on pHi similar to that
of FMLP.
Chemotactic
factor-induced
neutrophil activation involves a complex sequence of cation fluxes including Na’
and Ca2+ influx as well as a release of Ca2+ from intracellular stores. The initial pHi depression (but not the
later alkalinization)
in response to FMLP is inhibited in
Ca2+-free medium, suggesting that this acidification may
be secondary to an influx of Ca2+ and its subsequent
exchange for H+ (203). Millimolar amiloride inhibits the
FMLP-induced
Na+ influx and completely abolishes the
secondary alkalinization
of pHi, suggesting that this response, as in other cases reviewed above, is mediated by
Na+-H+ exchange across th .e plasma membrane. In contrast, ouabain has no effect on alkalinization.
No direct evidence is yet available regarding the physiological significance of the pHi changes induced in neutrophils by chemotactic
factors. The observation
that
neutrophil motility is stimulated at acidic pH, (51) suggests that alkalinization
does not mediate motility, since
pHi is a direct function of pH, in this cell (146).
IV. INTERACTION
OF PHi
METABOLIC
REGULATORS
WITH
OTHER
Clearly, physiological
pHi changes do not function in
a “vacuum”
devoid of other regulatory
influences. For
example, the interaction
of pHi and Ca2+ in regulating
the metabolic activation of the sea urchin egg is increas-
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
respond to a variety of compounds
(e.g., thrombin,
collagen, and ADP) by initiating a complex program involving aggregation, secretion of numerous agents (including
5-hydroxytryptamine,
or 5-HT), and shape changes. The
best studied of these stimulants,
thrombin, may be said
to exhibit a h.ormone-like
activity,
since it binds to
external
receptors
but affects intracellular
activities.
Like insulin, thrombin’s
second messenger(s)
remains
for the most part obscure, and besides their specialized
hormonal effects both thrombin and insulin have a general mitogenic action in a variety of cell types (Sect.
II@.
It is significant
then that thrombin
(in platelets)
shares with insulin (in amphibian muscle) the -ability to
increase pHi.
Using the fluorescent
weak base technique, Horne,
Simons, and co-workers
(99, 210) have shown that
thrombin
elicits a rapid (commencing
about 5 s after
addition) dose-dependent
pHi increase in washed platelets. pHi increases by 0.3 U in response to a saturating
dose of thrombin,
or from 7.0 to 7.3 at pH, 7.4. Careful
controls have ruled out the possibility that this apparent
pHi increase is merely an artifact of probe efflux due to
thrombin-induced
secretion. Apparently
coincident with
this pHi increase, membrane potential depolarizes (as
determined
by a similar fluorescence
technique)
(99,
210). Amiloride (10e4M) blocks both the pHi and potential changes (101,210), raising the possibility that thrombin-induced
alkalinization
proceeds via Na+-H+
exchange, but a rigorous test of this hypothesis remains to
be reported. It is interesting
to note, however, that anotherplatelet
activator, ADP, stimulates Na+ influx into
platelets (64) and fails to activate aggregation in Na+free medium (195). It would be interesting
to test the
effect of ADP on platelet pHi.
Presently no direct evidence exists regarding the physiological significance of the pHi change induced in platelets on exposure to thrombin.
5-HT secretion displays a
thrombin
dose-dependence
identical to that of the pHi
response, but these data alone are insufficient
to demonstrate a role for the pHi increase. Curiously, amiloride
(10D3M) at least partially blocks 5-HT secretion induc.ed
by intermediate
levels of thrombin
(and completely
blocks the pHi increase) (210) but has no effect when
saturating levels of thrombin are used (101); thus 5-HT
secretion does not appear to be activated solely by the
pHi increase.
Another platelet response to thrombin is a pronounced
increase in glycolysis of the “anaerobic”
type (i.e., producing lactate) (52). In serum the principal substrate for
this increased glycolytic flux is glucose (hence, stimulation of glucose transport
might be involved),
but in
glucose-free media thrombin
still evokes a pronounced
increase in lactate production,
and studies of intermediate metabolite levels indicate that thrombin’s
effect under these conditions is to activate PFK (52). In light of
Moore’s results on the activation of muscle glycolysis by
insulin (see above) the possible role of thrombin-induced
pHi changes in activating platelet PFK should be carefully considered in future. As is true of PFK from other
sources, the pH-activity
profile of platelet PFK (3) is
consistent
with activation
at elevated pH. Thrombininduced increases in pHi (99) and glycolytic rate (97)
W. B. BUSA
R424
A. Ca2+ and pHi
Most of the metabolic
transitions
involving
pHi
changes reviewed here are-also known to involve altered
Ca2+ fluxes. The transient Ca2+ release in sea urchin eggs
after fertilization
(Sect. IIIAI ) precedes alkalinization
of
pHi and may well be causally related to it, since all
treatments that evoke the former elicit the latter as well.
This relationship
is not yet understood. The acrosome
reaction of sea urchin sperm (Sect. IIIAZ) involves a
pronounced increase in Ca2+ uptake by the spermatozoon
3. Summary of evidence for coupled changes in pHi and pea;’
TABLE
Manipulation
Cell/Organism
pCaf+
Balanus
(barnacle
nubilus
changes
Xenopus
laevis
Chironomus
thummi
(insect salivary gland
cell)
Sheep Purkinje fiber
to artificial
121
Isometric tension
122
100%
0.2 U pCaf+ decrease
Ca2+-NLS
microelectrode”
H+ iontophoresis, 100%
CO2, nigericin + low
pCaz+ decreased
Aequorin, pH
microelectrode
Myofibril bundles
bathed in Ca2+ buffers
16-Cell embryos; no
pCaf+ changes during
cell cycle
e
100%
COZ”
PHO
15% CO2,
decrease
ca. 0.5 U pHi
0.2
U pCaf+ increase
(squid axon)
5% CO2”
pCa”+ increased
Helix
(snail
5% COZ”
0.16
U pCat+ increase
79% coga
0.26
U pCaf+ increase
aspersa
H. aspersa
temporaria
CO2 + carbonic
anhydrase”
(frog
100%
muscle fiber)
Fundulus
heteroclitus
(fish
ofpHi
Whole cells
Loligo forbesi
neuron)
acidification
Ref.
No.
Aequorin, isometric
tension
COS”
COZ, ca. 1.3 U pHi
decrease
(frog
embryo)
in response
Comment
ca. 0.5 U pCa”+
decreaseb and
contraction
Contraction
100%
muscle fiber)
B. nubilus
Measurement
Techniques
(pCaf+ and pHi)
Response
100%
CO2”
embryo)
pHi changes
No pCa”’ change
observedd
in response
to artificial
pCaf+
186
238
Response requires
external Ca2+
13
Ca2+-NLS
microelectrode
Ca2+-NLS
microelectrode
Isometric tension
Skinned
120
Aequorin
Abstract
21
decrease
([Ca2+]i
6
6
increase)
CaC12 pressure injected
0.1-0.3 U pHi decrease
pH microelectrode
142
(2-6 mmol/l cell H20)
Twitch and tonic
Sheep Purkinje fiber
Inhibition of Na+-K+0.1-0.2 U pHi decrease
230
ATPase decreases
tension; pH
pCaF+g
microelectrode
h
Whole rat heart
94
0.3 U pHi decrease
Resting pressure; NMR
Inhibition of Na+-K+ATPase decreases
pCaF+g
a pHi was not determined.
b Estimate based on aequorin luminescence change.
’ Ca2+-selective neutral ligand sensor microelectrode.
d pCaz+ response inferred from aequorin luminescence response; not quantified.
e Authors also report aequorin luminescence
increases (suggesting pCaF+ decreases) in response to alkalinization of pHi. However, positive correlation between pH and aequorin light output
(at ca. constant [Ca”‘]), which they demonstrate, renders this result difficult to interpret.
f Cells equilibrated with 1% Con.
g pCaf+ change
was not quantified.
h pHi decrease was accompanied by marked decrease in free ATP; no attempt was made to assess contribution of net
H. aspersa
ATP
(snail neuron)
Contraction
Ca2+-NLS
microelectrode; pHi
microelectrode
Aequorin
181
hydrolysis
(see Sect. V) to this pHi change.
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
(and its mitochondria),
which appears to be responsible
for the reacidification
of acrosome-reacted
sperm via
mitochondrial
Ca2+-H+ exchange (see below). On the
other hand, it has been suggested that the pHi increase
proposed to accompany the ASR (Sect. IIIAZ) itself
triggers the uptake of Ca2+ in this system.
Interaction between changes in [Ca2+]; and pHi have
also been reported to occur in a variety of cells in response to artificial manipulation
of one or the other of
these parameters (Table 3). Most commonly, these involve artificial acidification of pHi, and, as Table 3 shows,
all three of the possible responses by [ Ca2+]i are observed,
depending on the cell type involved: in barnacle muscle
fiber, Xenopus embryo, and insect salivary gland cells,
increases in [Ca2+]i have been observed in response to
acidification; in mammalian
Purkinje fibers, squid axon,
and snail neuron, [Ca2+]i decreases as pHi is lowered; in
Fund&s
blastomeres,
intracellular
acidification
has
been reported to have no effect on [Ca2+]; (but see below).
All studies to date involving [ Ca2+3; manipulations
(in
ingly apparent (see Ref. 213 for review), and the relative
significance of these two factors has been the subject of
spirited debate (see, e.g., Ref. 107). Whereas resolution
of this issue awaits the collection of more comparative
data, it is possible to discern the general outlines of a
relationship between pHi and two other important regulators of metabolism emerging from studies of a variety
of cells.
Rana
AND R. NUCCITELLI
INVITED
OPINION
chondria from various cell types possess separate pathways across their inner membranes for uptake and release of Ca2+ in exchange for H+, and their operation has
been suggested to be important in pCai regulation (see
Ref. 162 for review). That they may also play a role in
linking pHi and pCai changes in vivo is suggested by the
work of Meech and Thomas involving large Ca2+ injections into Helix neurons (Table 3), since the mitochondrial Ca2+-uptake inhibitor
ruthenium
red blocks the
rapid pHi decrease normally accompanying Ca2+ injection and apparently retards subsequent -removal of Ca2+
from the cytoplasm [as does the mitochondrial
uncoupler
cyanide-m-chlorophenyl
hydrazone (CCCP)]. However,
the reverse reaction ( [Ca2+]i increase in response to acidification of pHi) has not yet been observed in these same
cells; rather, acidification with 5% CO2 (which, unfortunately, will also lower intramitochondrial
pH) increases
the microelectrode-determined
pCai from its basal value
of 6.77 to 6.93, a 30% decrease in [Ca2+]i (6). In contrast
with this in vivo result, isolated rat liver mitochondria
release preloaded Ca2+ in response to small (0.1-0.6 U)
decreases in buffer pH (2, 206) and maintain
higher
steady-state extramitochondrial
[ Ca2+]i levels at low
than at high pH (161)-responses
opposite to that of the
intact Helix neuron reported by Alvarez-Leefmans
et al.
(6). These authors correctly point out that the seeming
conflict between their results and those of Meech and
Thomas may reflect differences in the response of mitochondria heavily loaded with exogenous Ca2+ vs. more
physiologically
loaded organelles.
Mitochondrial
Ca2+-H+ exchange might be involved in
the [ Ca2+]i increases observed in response to acidification
of Xenopus blastomeres (181) and Chironomus salivary
gland cells (186), but this remains untested. Conversely
this mechanism might also be involved in the acidification observed by Vaughan-Jones et al. (230) and Hoerter
et al. (94) in response to elevation of [Ca2+]i in Purkinje
fibers and whole hearts.
The only evidence yet available for the role of mitochondria in [ Ca2+]i-pHi coupling in cells known to
undergo physiological pHi changes is the recent report
of Lee et al. (124; see also Sect. IIIAZ).
By using 9AA to
estimate pHi during the L. pictus sperm acrosome reaction, they observed an initial, transient alkalinization
followed by a large and permanent reacidification
dependent on external Ca2+ and correlated with the rate of
45Ca2’ uptake. A role for mitochondrial
Ca2+-H+ exchange in this reacidification
is suggested by the ability
of the mitochondrial
inhibitor
antimycin to block the
pHi decrease, whereas valinomycin
(which depolarizes
mitochondria,
stimulating
their release of protons) accelerates it in acrosome-reacted sperm but has no effect
on pHi in unreacted sperm. A mixture of the mitochondrial inhibitors
carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), cyanide, and oligomycin also
inhibits the bulk of Ca2+ uptake (198).
2) Plasma membrane G2+ fluxes. The plasma membranes of excitable and nonexcitable cells possess several
mechanisms
of Ca2+ permeability,
including
voltagegated Ca2+ channels, Na+-Ca2+ antiporters, and an ATPdependent Ca2+ pump. In the internally dialyzed squid
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
snail neuron, mammalian
Purkinje
fiber, and whole
heart) have reported pHi decreases in response to an
increase in [Ca2+]i, as is also the case in the acrosomereacted sea urchin sperm. If the inhibition by Ca2+ of the
mammalian
acrosomal H+-ATPase
(Sect. 1~42)
proves
to be physiologically
significant, this would constitute an
example in which increased (cytoplasmic)
Ca2+ evokes
(acrosomal) alkalinization.
Although the diversity of relationships
between pHi
and [Ca”‘]i evident from these data has important
implications, the limitations
of the studies in Table 3 should
be kept in mind. First, they provide only circumstantial
evidence for a physiological link between pHi and Ca2+,
because they involve pHi changes that are nonphysiological in two senses. With the exception of the Xenopus
embryo, none of these cell types is known to undergo pHi
changes under any biologically
meaningful
conditions.
Further the imposed pHi changes (0.5-1.3 U, where determined) are rather large even for most cells that do
undergo physiological pHi changes. Similarly the Ca2+
injections employed by Meech and Thomas (141, 142)
were massive, and the imposed [Ca2+]i changes in the
remaining tw.0 studies of Table 3 were not quantified.
Another difficulty involves the earlier studies listed here,
which used the luminescence of injected aequorin to
detect [ Ca2+]; changes nonquantitatively.
Considerable
difference of opinion exists regarding the pH dependence
of the luminous response of aequorin, even in simple salt
solutions (11, 145, 208). Rose and Rick’s (186) attempt
at in vivo calibration suggests that light output is diminished with decreasing pHi (i.e., enhanced with increasing
pHi), rendering it difficult to interpret their observation
that alkalinization
of Chironomus
salivary gland cells
increases the aequorin luminescence
(not included in
Table 3). Since the lower limit of detection with aequorin
probably lies within the physiological range of [Ca2+]i, it
is also difficult to interpret results such as those of
Bennett et al. (Zl), who reported no change in aequorin
luminescence when Fund&s
blastomeres were acidified
with 100% C02. In favorable preparations such as the
barnacle muscle fiber (121, 122), isometric tension recordings confirm the [ Ca2+]i increase during acidification
also reported by aequorin, but for other cells the use of
Ca 2+ -selective ion-exchange microelectrodes is preferable
wherever possible, since their response is independent of
pH over the physiological range.
Despite the hazards of interpretation
we have just
considered, the studies of Table 3 (particularly
those
employing Ca2+ microelectrodes)
serve to demonstrate
that pHi and pCai can be interrelated under experimental
conditions. Various mechanisms might account for the
diversity of pH;/pCai relationships (direct in some cells,
inverse in others) listed here. The regulation of [Ca2+]i
is complex and not yet fully understood; it involves
regulation of Ca2+ fluxes across the plasma, endoplasmic
and other membranes as well
reticulum, mitochondrial,
as chemical buffering by cytoplasmic constituents and
membranes. Several of these processes either display
pronounced pH dependences or alter pH in a Ca2+dependent manner.
1) Mitochondrial
Ca2+ -H+ exchange. Isolated mito-
R425
R426
3) Ca2+ accumulation and release by sarcoplasmic reticulum. Nakamaru
and Schwartz (159) observed over a
decade ago that the steady-state Ca2+-binding ability of
isolated sarcoplasmic reticulum (SR) vesicles from skeletal and cardiac muscle is markedly pH dependent in
that it decreases as extravesicular pH is increased from
6.3 to 7.5; as a consequence, Ca2+ is released from these
vesicles in response to alkalinization.
Madeira (134) demonstrated that SR actively accumulating
Ca2+ generates
a transmembrane
pH gradient (0.15-0.5 U, inside alkaline) by ejecting proton equivalents. Net proton ejection
during Ca2+ uptake has been confirmed by the elegant
studies of Chiesi and Inesi (38), who postulated a Ca2+H+ antiport system in the SR similar to that observed
by Smallwood et al. (211) in the human erythrocyte
membrane. Recently, Shoshan et al. (209) have shown
that it is the dissipation of this accumulated pH gradient
in skinned muscle fibers (either by alkalinization
of
extrareticular
pH or by addition of protonophores),
rather than the absolute value of extrareticular pH, that
elicits Ca2+ release, and they suggested that the proton
gradient controls a Ca2+ release channel in the SR. Such
an alkalinization-dependent
Ca2+ release from SR might
account for the response of pCai to pHi changes in sheep
Purkinje fibers (Table 3).
Data that at first appear to conflict with the notion
that alkalinization
decreases the Ca2+ loading of SR were
obtained by the Fabiatos (63), who used the magnitude
of caffeine-induced
contractions of skinned cardiac and
skeletal muscle fibers to assess indirectly net Ca2+ accumulation by the SR at pH 6.2-7.4. The authors inter-
AND
R. NUCCITELLI
preted their results to indicate a decreased rate and
extent of Ca2+ accumulation
with decreasing pH. The
conflict between the data of the Fabiatos and those of,
e.g., Nakamaru and Schwartz may be due to the very
different loading pCa levels used (6.0-7.8 in the former
study and ca. 4.3-5.0 in the latter), since the data of the
Fabiatos demonstrate that the pH optimum for Ca2+
loading is depressed at low pCa. In fact, at the lowest
pCa used by these authors (6-O), alkalinization
reduces
the Ca2+ loading of SR, in keeping with the observations
of Nakamaru and Schwartz and others. This is also seen
in the Fabiatos’ observation that a pH increase (6.2-7.4)
will elicit net Ca2+ release from SR loaded at pCa 6.0
(roughly the physiological level for SR loading), but not
from SR loaded at pCa 7.8. Finally, it should be noted
that the Fabiatos’ failure to detect contraction in response to alkalinization
from pH 6.2 to 7 after loading
at pCa 7.75 (Fig. 4F in Ref. 63) was probably due to their
use of 4 mM ethyleneglycol-bis(P-aminoethylether)N,N ‘-tetra-acetate (EGTA) (which prevents contraction
by buffering the [Ca”‘] of the myofilament
space), since
Shoshan et al. (209) observed that even smaller alkalinizations elicit contractions in skinned psoas fibers in the
absence of EGTA under otherwise similar conditions (SR
loaded at pCa 7.0).
It must be emphasized that pH-mediated
Ca2+ release
from SR cannot presently be considered a physiological
mechanism of excitation-contraction
coupling, not least
because pHi changes of the required magnitude have not
been observed to accompany muscle contraction. However, if this mechanism is also common to other forms
of endoplasmic reticulum, it may prove an important
mechanism in the coupling of pHi and Ca2+ changes in
other cells which either undergo natural pHi changes or
in which such changes are experimentally
imposed. For
example, Ca2+ release from the SR in response to dissipation of the transmembrane
pH gradient appears to
provide an explanation for the [Ca2+]i increase observed
by Lea and Ashley in barnacle (121, 122) and frog (120)
muscle fibers treated with Cd,. Whereas their initial
observations (121) seemed to suggest that acidification
of cytoplasmic pH was important,
later studies (122)
employing mechanically skinned fibers specifically ruled
out this mechanism
by employing bathing solutions
equilibrated with either 0 or 100% CO2 at identical pH.
In the absence of any change in the pH of the medium
bathing the contractile apparatus and SR, 100% CO2 still
elicited contraction
and increased aequorin luminescence. In light of Shoshan’s results (209; see above) it
seems possible that this effect is due to the ability of CO2
to cross the SR membrane, lowering intrareticular
pH
and thus discharging the transmembrane
pH gradient.
In keeping with this, membrane-permeant
weak bases
such as NH3 and methylamine
reversibly inhibit Coninduced Ca2+ release in skinned fibers (at constant bath
pH), presumably by counteracting the effect of CO2 on
intrareticular
pH (119). Curiously the protonophores
FCCP and gramicidin
do not elicit Ca2+ release in
skinned barnacle or frog muscle fibers bathed in 0.1 mM
EGTA, suggesting either that the mammalian fibers used
by Shoshan et al. are unique in this respect or that the
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
axon, acidification of pHi by as little as 0.3 U reversibly
inhibits both ATP-dependent
Ca2+ efflux and Na+-Ca2+
exchange, whereas alkalinization
above the physiological
pHi of 7.3 has no effect on ATP-dependent
efflux but
greatly stimulates Na+-Ca2+ exchange (53). Baker and
co-workers observed a similar inhibition
of Na+-Ca2+
exchange in whole axons at acidic pHi (14) and attributed
the decrease in [ Ca2+]i accompanying acidification (Table
3) to the simultaneous but more extensive inhibition
of
Ca2+ influx (13).
In whole barnacle muscle fibers (see below), both
45Ca2+ influx and efflux are inhibited
by acidification
with 100% CO2 (121); in contrast with the behavior of
squid axon, however, acidification
of barnacle fiber pHi
has a greater effect on the efflux component, resulting in
a small net influx of Ca2+ at depressed pHi.
By using inside-out vesicles prepared from human
erythrocytes, Smallwood et al. (211) have very recently
demonstrated
that the plasma membrane Ca2+-Mg2+ATPase is a Ca2+-H+ antiporter
but were unable to
determine whether one or two protons are exchanged for
each Ca2+ transported. Although this is unlikely to h.ave
an effect on erythrocyte pHi (not least because in the
erythrocyte, unlike most other cells, pHi is passively
maintained
via a Donnan equilibrium),
Ca2+-pumping
from other cell types via a similar ATP-dependent
Ca2+H+ exchanger could, in principle, present a cytosolic acid
load. Similarly cytosolic acidification would be expected
to inhibit the exchanger’s activity, as is observed in the
internally dialyzed squid axon (53).
W. B. BUSA
INVITED
R427
OPINION
enzymes, however, since these activities may themselves
be pH dependent (see Ref. 222 for an example concerning
phosphodiesterase).
Nonetheless it might still be expected that in some cases pHi and [Ca2+]i changes might
have interchangeable effects on calmodulin-mediated
responses. A possible example can be found in a recent
paper by Gilkey (78). The Medaka egg cortical granule
reaction can be elicited by microinjection
of Ca2+. With
combined Ca2+-pH buffers as the injectate, Gilkey observed that higher concentrations of Ca2+ were required
at lower pH levels; the median [Ca2+]i required was about
6.5, 2.0, or 0.65 PM at injectate pH of 6.5, 7, or 7.5,
respectively. Based on Tkachuk and Men’shikov’s
&
values, the fraction of calmodulin complexed with Ca2’
is found to be very nearly constant under these three
conditions. Although the Medaka egg cortical granule
reaction has not been shown to involve calmodunn, this
is suggested for the similar reaction of the sea urchin
egg, based on the ability of various calmodulin inhibitors
and calmodulin antibody to block the reaction (175,214).
In the sea urchin egg, however, even extreme alkalinization of pHi with NH3 (which should, according to our
proposal, activate calmodulin even at constant [ Ca2+]i)
does not evoke the cortical granule reaction, apparently
because high levels of NH3 interfere with the attachment
of cortical granules to the inner face of the plasma
membrane (102).
It will be of great interest to determine whether Gilkey’s results can be extended to the sea urchin egg, as well
as to determine
whether other calmodulin-mediated
processes show a similar interdependence
of pHi and
[Ca”‘]i. It would also be useful to confirm Tkachuk and
Men’shikov’s
Kd values at more physiological
ionic
strengths and with a less exotic methodology than those
employed by them, particularly since their & values are
among the lowest reported. Nonetheless the pronoun ted
pH sensitivity of Ca2+ binding by calmodulin
(anecdotally mentioned by other investigators) is clear, as are its
implications
for the interaction
of pHi and [Ca2+]; as
meta bolic regula tors. Because calmodulin is present in
most (if n.ot all) cells and its amino acid composition 1s
very highly conserved, its pH dependence might constitute a universal mechanism of pHi-mediated
(Ca2+-dependent) metabolic regulation in cells experiencing physiological pHi changes.
B. CAMP and PHi
1) Effect of [cAMP]i changes on PHi. If pHi changes
do interact with changes in [Ca2+]i as suggested above, it
would not be surprising to observe interactions between
pHi and [cAMP]i as well, given the intimate interrelations between Ca2+ and CAMP in all cells (see Ref. 178
for review). Boron et al. (25) observed just such an
interaction in internally dialyzed barnacle muscle fibers.
The normal pHi of these cells (7.3) was depressed to a
steady-state value of ca. 6.9 by continuous internal dialysis with an acidic buffer; on addition of 1 PM CAMP
to the dialysate, pHi increased by at least 0.1 U, and
Cl- efflux was stimulated, suggesting that the effect of
CAMP on pHi was due to stimulation
of the
Cl--HCO,
acid extrusion mechanism.
This was con-
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
protonophore-induced
Ca2+ release they observed might
actually have arisen from, for example, mitochondria
(T.
J. Lea, personal communication).
The effect of increasing
extrareticular
pH in skinned barnacle muscle fibers has
not been reported; if a mechanism similar to that proposed by Shoshan et al. for mammalian
muscle is in fact
involved, alkalinization
of extrareticular
pH should also
elicit Ca2+ release.
The possibility that discharging pH gradients (or, perhaps, H+ electrochemical
gradients) across endoplasmic
reticulum membranes may evoke Ca2+ release points out
yet another hazard in interpreting
the data of Table 3.
Membrane-permeant
weak acids such as CO2 not only
depress cytosolic pH, but under some conditions can
also discharge the transmembrane
pH gradients of internally alkaline organelles. Thus COZ-induced Ca2+ release
might be the result of either cytosolic acidification or the
discharge of transmembrane
pH gradients; if the latter
is the case, then alkalinization
of the cytosol (as, e.g.,
during a physiological pHi increase) might evoke a similar Ca2+ release, since cytosolic alkalinization
could also,
in effect, discharge the pH gradient across an internally
alkaline organelle membrane. Thus a Ca2+ release experimentally evoked with CO2 and attributed to acidification
of pHi might actually occur under physiological conditions in response to cytosolic alkabnization!
In light of
this, Shoshan and co-workers’ results need to be confirmed and expanded; it would be particularly
useful to
determine whether protonophores can elicit Ca2+ release
from purified SR vesicles, as opposed to skinned muscle
fibers.
4) Calmodulin.
The ubiquitous Ca2+-binding regulatory protein, calmodulin, mediates the Ca2+ responses of
such important
enzymes as cyclic nucleotide phosphodiesterase and adenylyl cyclase, Ca2+-ATPase, and protein kinase. Its amino acid sequence is among the most
highly conserved of all proteins and features a histidyl
residue at position 107, raising the possibility that Ca2+
binding by calmodulin
might be pH dependent within
the physiological pHi range. This was shown to be the
case by Tkachuk and Men’shikov
(222), who observed
an order of magnitude decrease in the Ca2+ dissociation
constant (&) of calmodulin over the pH range 6.5-7.5
(& ca. 0.25 or 0.02 PM, respectively). They further
showed that such pH changes have no effect on the
binding of the Ca2+-calmodulin
complex to phosphodiesterase. The possible implications
of these results with
regard to metabolic regulation have not been previously
considered, to our knowledge, but strike us as profound.
As an example, at a typical [ Ca2+]i of 0.1 PM and pHi of
7, 50% of the available calmodulin
will be complexed
with Ca2+ (and thus available to regulate enzyme activities), whereas at pHi 7.5 83% is complexed at the same
[ Ca2+]i. Under appropriate conditions, therefore, a pHi
increase of 0.5 U would have the same effect as a fivefold
increase in [Ca2+]i at constant pHi. In other words,
camodulin might more accurately be considered a Ca2+H+-binding protein, and may serve as a sensor of both
[Ca2+]i and pHi changes. This does not necessarily predict an identical correspondence between the effects of
pHi and [Ca2+]i on the activities of calmodulin-regulated
R428
AND
R. NUCCITELLI
cantly elevated or depressed, respectively-precisely
the
result expected based on the pH-activity profiles of Fig.
1. If we recall that increased [cAMP]i appears to decrease
pHi in the rat liver (68), the foundation for a feedbackinhibited cycle may be perceived.
Another-system- that may prove to exhibit the same
response of [cAMP]i to pHi changes as the liver cell is
the norepinephrine-stimulated
rat adipocyte, in which
CAMP accumulation is dramatically depressed at pH, 6.6
compared with that at pH, 7.4 (92). It is an observation
of long standing that, even in whole animals, the CAMPmediated stimulation
of lipolysis and calorigenesis in
response to ,&adrenergic agents is markedly depressed
by acidosis and increased by alkalosis (for review see
Ref. 182). Although direct effects of pH, on the ligandreceptor interaction
or other steps might also explain
this effect, the pH-activity profile of Fig. 1 suggests that
inhibition of adenylyl cyclase during acidosis should also
be considered.
As with the relation between pHi and pCai (Sect. IvA),
it may not be possible to form broad generalizations
regarding the dependence of [cAMPi] on pHi. This is
illustrated by Fig. 2, which shows the pH-activity profiles
of adenylyl cyclase and a cyclic nucleotide phosphodiesterase from the yeast
Skcharomyces
cereuisiae. The
phosphodiesterase profile is similar in form to that of
the rat liver enzyme, except that its maximum occurs at
a pH more alkaline than physiological pHi, thus rendering it sensitive to pH changes within the physiological
range. As in rat liver, cyclase activity is also pH dependent within this range, but its slope is reversed; activity is
maximal at acidic pH and declines rapidly with alkalinization. As Londesborough
(129) has pointed out, the
opposed pH-activity profiles of the cyclase and phosphodiesterase from S. cereuisiae suggest that acidification of
pHi in this system will eZeuate [cAMP]i-the
opposite of
the response predicted for mammalian
liver. This is of
particular interest as S. cereuisiae is known to undergo
physiological pHi changes over the range between about
6.5 and 7.5, depending on such factors as oxygen availaIOO-
I
I
I
6
I
I
I
’
’
1
7
8
PH
FIG. 1. pH-activity profiles of adenylyl cyclase (AC) and cyclic
nucleotide phosphodiesterase (PDE) from rat liver. Activities are normalized to 100% of maximum activity. Dashed line, pHi routinely
reported for rat liver (185). PDE &as assayed in -tris(hydroxymethyl)aminomethane
(tris) and cacodylate buffers; AC was assayed
in N-tris(hydroxymethyl)methyl-Z-aminoethanesulfonic
acid (TES)
buffer. (Redrawn from data of Refs. 113 and 144.)
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
firmed by the ability of 4acetamido-4’-isothiocyanostilbene-2,2’-disulfonic
acid (SITS), an acid extrusion inhibitor, to block effects of CAMP on both pHi and Clefflux. A similar response to exogenous CAMP has also
been observed in mammalian
myocardium.
Respiratory
acidosis (20% COZ) depresses the DMO-determined
pHi
of the isolated, perfused rat heart from 7.1 to 6.8 (65,
180). Addition of either glucagon (65) or norepinephrine
(180) to the perfusate elicits a significant increase in pHi,
to 7.0 or 6.9, respectively. In contrast, neither agent has
an effect on pHi under normocapnic
conditions. The
receptors for both these hormones couple with the
CAMP-generating
enzyme adenylyl cyclase, and millimolar levels of dibutyryl CAMP mimic their effect, increasing pHi by 0.2 U in hypercapnic myocardium but
not in normocapnic controls.
Whereas the preceding examples demonstrate coupling
between CAMP and pHi only in grossly acid-loaded cells,
some evidence also suggests that CAMP may modulate
the normaL pHi of certain cells. In the normoxic rat liver,
exogenous CAMP acidifies pHi from its control value of
7.3 to about 7.2 as determined with DMO (68). The slime
discoideum acidifies its medium in
mold Dictyosteliuni
response to nanomolar pulses of CAMP (a chemotactic
agent in this cell) (136), but pHi was not measured in
this study, nor was the possible role of respiratory CO2
in medium acidification
assessed. Nevertheless this observation is in keeping with the notion that CAMP may
activate acid extrusion in D. discoideum.
2) Effect of PHi changes on [CAMPlie Although
no
direct observations are yet available, several lines of
circumstantial
evidence su.ggest that moderate pHi
changes may have important effects on [cAMP]i in many
systems. Perhaps the most compelling argument concerns the pronounced pH dependence of the enzymes
adenylyl cylase and cyclic nucleotide phosphodiesterase,
which, respectively, generate and hydrolyze CAMP. The
pH-activity profiles for these enzymes from rat liver are
shown in Fig. 1. Around the physiological pHi value of
7.2 (185), small pHi changes (+ 0.2 U) would be predicted
to have little or no effect on phosphodiesterase, which is
maximally activated at this pH, whereas acidification or
alkalinization
would, respectively, decrease or increase
the activity of the cyclase. The net effect would be an
increase in [cAMP]i with increasing pHi.
Responses in keeping with this prediction have been
reported. Using rat liver slices incubated in bicarbonate
buffers at pH, 6.9 and 7.8, Reynolds and Haugaard (179)
observed significant increases in extractable phosphorylase activity (which they assumed to reflect parallel increases in [cAMP]i) at the more alkaline pH,, the response expected if [cAMP]i increases with increasing
pHi. Exogenous CAMP also activates phosphorylase in
this preparation,
and this response is independent
of
pH,, suggesting that the effect of pH on phosphorylase
activation occurs at the level of adenylyl cyclase and/or
phosphodiesterase rather than at the level of, e.g., CAMPdependent protein kinase. More persuasive evidence is
supplied by the whole-animal
studies of Yajima and Ui
(249), who directly assayed CAMP levels in the livers of
alkalotic and acidotic rats and found them to be signifi-
W. B. BUSA
INVITED
R429
OPINION
c.I> 60z a
s
20-
6
7
8
PH
bility, the nature and availability
of the metabolized
carbon source, and the cell’s position in the cell cycle
(49, 84).
In keeping with Londesborough’s
prediction, it has
been reported that S. cereuisiae respond to treatment
with the oxidative phosphorylation
uncouplers CCCP
and dinitrophenol
with a dramatic increase in [cAMP]i
(135, 140, 223). It has been suggested that this response
is due to plasma membrane depolarization,
but another
depolarizing treatment, osmotic shock, fails to produce a
[cAMP]i increase (172). In our view, a more likely explanation involves the well-established ability of these uncouplers to depress pHi (see Ref. 185 for review). Under
ra .ther more physiological conditi .ons, addition of glucose
to suspensio ns of deenergized S. cereuisiae also evokes a
rapid, transient (ca. l-3 min after addition) increase in
[cAMP]i (140, 229), and the high-resolution
31P-NMR
studies-of den Hollander et al. (49) demonstrate a transient pHi decrease from about 6.75 to 6.5 during this
same interval under roughly similar conditions. After
this acidification, pHi rapidly increases to a steady-state
value of about 7.2, and [cAMP]i declines (229).
Londesborough’s
hypothesis also offers an attractive
explanation for the presence of a second, so-called “highK,” cyclic nucleotide phosphodiesterase in S. cereuisiae
(130). This enzyme’s very high K, for CAMP (75 PM at
pH 7) has made its physiological role somewhat unclear,
since at routinely reported [cAMP]i levels (0.01-l PM)
it can account for only a fraction of the total phosphodiesterase activity of the cell; under these conditions the
“low-K,” enzyme of Fig. 2 is most important.
Interestingly the high-K, enzyme’s pH-activity
profile at physiological CAMP levels (not shown in Fig. 2) is the reverse
of that of its low-K, counterpart,
roughly paralleling
that of adenylyl cyclase. Thus, when the cyclase is stimulated (and the low-K, phosphodiesterase
is inhibited)
by a decrease in pHi, [cAMP]i could increase only until
it reaches levels at which the high-K, enzyme can prevent further increases. The high-K,
enzyme might
V. PHi
AND
ENERGY
METABOLISM
On balance, mammalian
whole-a ,nimal metabolism
is
often a proton-producing
process, as re fleeted by the
high rate of secretion of proton equivalents by the kidney.
The same is often assumed to be generally true for
cellular metabolism as well, an .d the apparent ubiquity of
membrane transport mechanisms for extrusion of proton
equivalents from cells might at first sight seem to confirm
that the principal challenge faced by pHi regulation is to
relieve t ‘he cell of excess protons. Such an excess could
arise by two di fferent mechanisms: 1) passive diffusion
of protons across the plasma membrane down their electrochemical gradient (i.e., into the cell) and 2) net production of proton equivalents by metabolism. The first
of these is an inevitable consequence of the fact that
nearly all cells maintain a pHi higher than that which
would obtain from a simple Donnan equilibrium
with the
extracellular
medium (see 185). In light of this, the
existence of “bailing” mechanisms for proton extrusion
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
FIG. 2. pH-activity
profiles
of adenylyl
cyclase (AC) and “low-K,”
cyclic nucleotide
phosphodiesterase
(PDE)
from yeast Saccharomyces
cereuisicze. Activities
are normalized
to 100% of maximum
activity.
Dashed lines, extremes
of mean pHi values under various
conditions
reported
by den Hollander
et al. (49). PDE and AC were assayed in N2-hydroxyethylpiperazine-N’-2
ethanesulfonic
acid (HEPES)
and piperazine-N-N’-bis(ethanesulfonic
acid) (PIPES)
buffers.
(Redrawn
from data of Refs. 129 and 131 and J. Londesborough,
personal
communication.)
thereby function as a “safety valve,” preventing uncontrolled elevations of [cAMP]i at low pHi (130). In keeping
with this is the extreme pH dependence of this enzyme’s
K, for CAMP-roughly
350 PM at pH 8, decreasing to
about 20 PM at pH 6.
Aside from the brief discussions of Londesborough
cited above, it appears that a physiological role for the
pronounced pH dependence of the adenylyl cyclase and/
or phosphodiesterase(s)
of many organisms has not been
seriously considered to date. Admittedly pH-activity profiles can differ, sometimes dramatically,
depending on
the conditions under which they are determined,
and
comparison of data sets collected under nonstandardized
conditions is fraught with hazards. Further the cyclase
pH-activity profiles presented here were determined under rather nonphysiological
conditions (in the presence
of Mn2+); however, the adenylyl cyclase from Neurospora
recently characterized by Rosenberg and Pall (187) demonstrates an even steeper pH dependence than that of
Fig. 2 in the absence of Mn2+. We believe that the
predictive power of the hypothesis that CAMP and pHi
changes may couple, as reflected in the preceding examples, is quite intriguing and demands further testing. It
would be interesting,
for instance,
to determine
[cAMP]i under aerobic and anaerobic conditions in both
catabolite-repressed
and -derepressed cultures of S. cereuisiae, since the results of den Hollander
et al. (49)
demonstrate that pHi can vary considerably under these
conditions. Any such experiments must also take into
account the known role of Ca2+ in [cAMP]i regulation
(178), particularly
in light of the apparent interactions
of pHi and pCai discussed in section IVA. Rather than
merely representing an annoyance, however, this other
interaction suggests the possibility that H+, Ca2+, and
CAMP might all function interdependently
as synergistic
messengers, with pHi providing
a metabokc context
within which, e.g., a hormonal stimulus might have
rather different consequences at two different pHi levels,
as appears to be the case in the effect of acidosis on
adrenergic stimulation
in some systems.
R430
W. B. BUSA
seems inevitable. Regarding the role of metabolism in
producing protons, however, considerable confusion exists in the literature; references to “the acidifying effect
of metabolism” are common but are largely unsupported.
This state of confusion was apparently first appreciated by Gevers (75), who pointed out that lactic acid
accumulation is not the source of the intracellular
acidification observed in numerous cells during anaerobic
glycolysis, contrary to statements to be found in most
modern biochemistry and physiology texts. This at first
counterintuitive
assertion can be confirmed simply by
careful expression of the individual reactions of glycolysis, balanced with respect to mass and charge at (for the
moment) pH > 8 (75)
glucose + MgATP2glucose 6-phosphate2-
+ H+
+ fructose 6-phosphate2-
fructose 6-phosphate2-
+ MgATP2-
+ fructose diphosphate4fructose diphosphate42 triose phosphate2-
+ MgADP-
+ MgADPl-
--) 2 triose phosphate2-
+ 2 NAD-
+ 2 diphosphoglycerate42 diphosphoglycerate4-
+ H+
+ 2P”-
+ 2 NADH2-
+ 2 H+
+ 2 MgADP-
+ 2 phosphoglycerate3-
+ 2 MgATP2-
2 phosphoglycerate3---) 2 phosphoenolpyruvate32 phosphoenolpyruvate3-
+ 2 MgADP-
+ 2 H+ ---) 2 pyruvate2 pyruvate-
+ 2 H20
+ 2 MgATP2-
+ 2 NADH2+ 2 H+ + 2 lactate-
(2)
(3)
(4)
(5)
(6)
(7
(8
0
Net reaction:
glucose + 2 MgADP-
+ 2 PF- + 2 lactate+ 2 MgATP2-
+ 2 H20
(10)
As written, the protons produced by the reactions of
(Eq. 3), and
hexokinase (Eq. l), phosphofructokinase
glyceraldehyde-3-phosphate
dehydrogenase (Eq. 5) are
consumed by the reactions of pyruvate kinase (Eq. 8)
and lactate dehydrogenase (Eq. 9). The net products of
anaerobic glycolysis (Eq. 10) are thus two lactate- and
two MgATP2-; no net production of proton equivalents
occurs. Nevertheless it is a well-established
fact that
anaerobic glycolysis (e.g., in the ischemic myocardium)
is associated with intracellular
acidification. To account
for this, Gevers drew attention to the familiar equation
for ATP hydrolysis (also at pH > 8)
MgATP2-
+ H20 ---) MgADP-
+ PF- + H+
(11)
Because hydrolysis of one ATP produces one proton
at this pH, the net reaction of glycolysis plus hydrolysis
of the ATP produced (sum of Eqs. 10 and 11) is
+ 2 H+
(12)
so that ATP hydrolysis (Eq. 11 ), not lactate accumulation
(Eq. lo), is the dominant source of the intracellular
acid
load (Eq. L2) accompanying anaerobiosis.
This important concept, quite contrary to the general
misconception
that lactic acid is the end product of
anaerobic glycolysis, aroused surprisingly little interest
for some 6 yr. It was recently rediscovered and lent
quantitative
rigor by Hochachka and Mommsen
(93),
who took into account the pH dependence of the stoichiometries of Eqs. 10 and 11, arising from the fact that
MgATP, MgADP, and Pi all have pKs near physiological
pHi. If we take this into account, the reaction of Eq. 10
actually produces about 0.4 H+ per glucose consumed at
“typical” values of pH (here, 7.4) and free [Mg2+] (4.4
mM) due to the differing pKs of MgATP and its precursors, MgADP and Pi, whereas Eq. 11 produces only 0.8
H+ per ATP hydrolyzed
{MgATPj
+ H20 + {MgADP)
+ {Pi) + 0.8 H+
(13)
where the terms in braces represent the sum of all ionic
species at the specified pH and free [Mg2+] (see 93). The
net effect of these adjustments, however, is that Eq. 12
still yields the same result-precisely
two H+ are generated per glucose consumed, at pH 7.4 as at pH 8, or at
any other pH within the physiological range (93).
If we adopt the approach of Gevers and of Hochachka
and Mommsen, we can similarly show that steady-state
aerobic energy metabolism has no net effect on pHi. The
summary equation for aerobic ATP synthesis (glycolysis
to pyruvate plus the tricarboxylic acid cycle plus oxidative phosphorylation)
at pH 7.4 and 4.4 mM free Mg2+
is
glucose + {38 MgADP}
+ 2 NAD-
R. NUCCITELLI
+ {38 Pi) + 6 02
+ 30.4 H+ -+ 138 MgATP)
+ 6 CO2 + 44 H20
(14)
Equation 14 demonstrates that at pH 7.4, respiration will
consume 0.8 H+ per ATP generated. Since hydrolysis of
each ATP thus generated will yield 0.8 H+ at this pH
(Eq. 13), respiration plus ATP hydrolysis will neither
produce nor consume protons. As with anaerobic glycolysis, this effect can be shown to be pH independent: at
any pHi within the physiological range, aerobic energy
metabolism
will have no net effect on pHi, provided
respiration and ATP hydrolysis are strictly coupled.
Experimental
data supporting our treatment of respiration are available. Tightly coupled mammalian
heart
mitochondria
oxidizing pyruvate alkalinize their incubation medium, in keeping with the reaction
pyruvate + {15 MgADPj
+ {15 Pi) + 2.5 02
+ 13.0 H+ + (15 MgATP)
+ 3 CO2 + 17 Hz0
(15)
When the uncoupler, dinitrophenol,
is added after exhaustion of the available ADP (i.e., during state 4 respiration) the mitochondrial
ATPase is stimulated, ATP
formed during state 3 respiration is hydrolyzed, and the
pH of the medium returns to approximately
its original
value-that
is, protons are consumed by respiration (Eq.
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
(1)
+ glucose 6-phosphate2-
glucose --) 2 lactate-
AND
INVITED
R431
OPINION
A. Dormancy-Metabolism
Embryos
Transition
of Artemia
Anaerobic dormant Artemia embryos (Sect. IId3)
possess very low levels of nucleotide triphosphates and diphosphates (NTP and NDP; see Ref. 30 for examples
and references). During activation of aerobic metabolism
and development, NTP levels increase to the millimolar
concentrations typical of most cells and, roughly simultaneously, pHi increases by >l U. From the biochemical
data of Stocco et al. (216) one can estimate an NTP
increase of about 3 mmol/l
cell water and an NDP
increase of about 1 mmol/l
during the anaerobic-toaerobic transition.
From the data of Busa and Crowe
(29), the intrinsic buffer power of the dormant embryo
is about 32 mmol per pH unit per liter between pHi 6.8
and 7.3. If we assume, as a first approximation,
that all
the NTP and NDP arise from nucleotide monophosphates (NMP) via the adenylate kinase reaction and
oxidative phosphorylation,
and an average H+ consumption of 0.8 H+ per NDP (or 1.6 H+ per NTP) produced
from NMP, activation of respiration can account for at
most about 0.2 U of the X-U pHi increase.
B. Starvation
and Refeeding of Yeast
The pHi of starved S. cerevisiae cells increases by about
0.5 U after refeeding with glucose (Sect. IvB), and starved
cells have very low ATP (15, 49) and ADP (49) levels,
the former increasing by nearly 4 mM on refeeding (49).
At pHi 6.8, fermentation of glucose will consume roughly
0.4 H+ per ATP formed from ADP (93) or 0.8 H+ per
ATP produced from AMP (if we assume the involvement
of adenylate kinase), thus yielding a net consumption of
slightly more than 3 mM H+ by the increase in ATP
after refeeding. If we arbitrarily assume a buffer power
of about 30 mmol per pH unit per liter in these cells, 0.1
U of the 0.5-U pHi increase could be accounted for by
this mechanism. Since activation of the plasma membrane proton pump has also been suggested to account
for some or all of this alkalinization
(49), it would be of
interest to observe pHi during refeeding of starved cells
when the pump is inhibited (e.g., by vanadate; see 194).
In conclusion, the temporary uncoupling of ATP synthesis and hydrolysis seen in two different metabolic
activation events can account for an important fraction,
but by no means all, of the pHi increase accompanying
activation. The magnitude of the changes in ATP levels
in these examples is unusually large; the smaller fluctuations observed under most circumstances in vegetative
cells would, by themselves, have less significant effects
on pHi. However, we have limited our analysis to only a
few aspects of a single (albeit, very high flux) pathway,
the ATP cycle. A number of other important metabolic
pathways either produce or consume protons (e.g., see
75); detailed analysis of the “proton balance” of cellular
metabolism under various conditions will therefore require more sophisticated modeling. Nevertheless the preliminary
analysis offered here suggests an interesting
correlation. In the examples discussed above, cells in a
temporary state of positive energy balance (reflected by
increasing ATP levels) necessarily tend to alkalinize, and
the reverse applies as well-a negative energy balance
can result in a decrease in pHi. Similarly the transition
from aerobic metabolism
(which neither produces nor
consumes protons in the steady state) to anaerobic glycolysis (which produces one H+ per ATP cycled) both
reduces pHi and potentially can lead to negative energy
balance, due to the lower efficiency of anaerobic catabolism of glucose and the accumulation
of end products.
To the extent to which this generalization
also applies
to other cell types under various conditions, pHi could
thus function as a sort of “null meter,” conveying information regarding changes in cellular energy balance to
molecules (such as calmodulin,
see Sect. IVA) that are
not otherwise in direct communication
with the adenylate pool. As we have suggested elsewhere in this review,
pHi might thus provide a metabohc context within which
metabolic pathways and effecters function appropriately.
vI PERSPEC1[‘IVE
l
As we have shown, intracellular
pH changes ranging
from about 0.1 to 1.6 U ([H+]i changes of from 0.25- to
4O-fold) are a fact of life in cells of such diverse organisms
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
15 in this instance) and regenerated in roughly equal
numbers by ATP hydrolysis (Eq. 13) (228). In vivo the
pHi of freshly prepared, aerobic barnacle muscle fibers
is unaffected by inhibition
of the cells’ acid extrusion
mechanism (24), and the pHi of respiring Neurospora is
largely unaffected by inhibition
of its plasma membrane
proton pump (194) . Thus whatever roles membrane
transport processes for extrusion of proton equivalents
serve, they do not appear necessary to counter an acidifying effect of aerobic energy metabolism.
As noted, our analysis of aerobic metabolism
so far
has assumed strict one-to-one coupling between ATP
synthesis and hydrolysis (i.e., a stable ATP-to-ADP
ratio). During certain metabolic transitions such as some
of those we have reviewed here, however, the ATP-toADP ratio (or similar expressions such as the phosphorylation
potential
or adenylate energy charge) can
undergo pronounced changes as either ATP synthesis or
hydrolysis rates are altered. During such “non-steadystate” conditions, when the reactions of Eqs. 13 and 14
temporarily
proceed at different rates, aerobic energy
metabolism could have either an acidifying or alkalinizing effect on pHi, depending on whether the ATP-toADP ratio declines or increases, respecti .vely. It is i.nteresting to note that metabolic activation events are routinely associated with increasing pHi, whereas inactivations, such as initiation
of dormancy, involve pHi decreases (167). Although membrane transport of proton
equivalents appears to be involved in some of these cases,
such as sea urchin egg fertilization
(where ATP does not
increase during activation; Ref. 57), it is important
to
consider the extent to which transitions in energy metabolism during activation might affect pHi in those
systems that do show pronounced changes in ATP levels.
Below we consider two instances in which existing data
regarding pHi and “energy charge” changes during metabolic transitions permit such an assessment.
R432
AND R. NUCCITELLI
proton-to
consider perhaps the most unique feature
that suits it to this role. Because all life is based on
aqueous chemistry and because water spontaneously ionizes, protons cannot be excluded from the intracellular
milieu; their activity must therefore be regulated. It
seemssafe to assumethat, j ust as it does today, from the
very beginnings of life on earth biochemistry has involved weakly ionized compounds and has relied heavily
on acid catalysis; regulation of pHi therefore provided a
powerful means of regulating the metabolism of early
cells without requirini the evolution of special receptor
moZecuZes.Conceivably pH sensitivity can be conferred
on enzymes and structural proteins by single point mutations: for example, mutation of the DNA codon for
arginine to that coding for histidine (by replacing a single
cytosine base with thymine) will replace an amino acid
residue with a pKa outside the physiological pHi range
with one of pKa about equal to pHi. If this residue is so
located as to alter significantly the conformational, binding, or catalytic plroperties of the protein, depending on
its ionization state, pHi-sensitivity will be conferred.
In their landmark 1966 paper, Trivedi and Danforth
(224) observed that “it would be surprising if physiological regulation of enzyme activity by pH were not a
widespread and perhaps primitive method of control.”
Although much obviously remains to be learned, we
believe that the data collected by many workers over the
past decade and reviewed here strongly substantiate this
notion.
NOTE ADDED IN PROOF
Recently, Deutsch et al. (C. Deutsch, J. S. Taylor, and M. Price. J.
CeZZBiol. In press.) have failed to observe alkalinization
of human
peripheral lymphocyte pHi in response to the mitogens succinyl-Con A
and phytohemagglutinin,
using both DMO and “F-NMR to monitor
PHi*
We thank Drs. N. L. Cross, D. W. Deamer, D. Epel, S. J. O’Dell, P.
Setlow, and N. M. Scherer for helpful discussions. W. B. Busa is
indebted to Dr. J. H. Crowe for support and encouragement during the
early stages of writing. Thanks are also due to the numerous workers
who shared with us prepublication copies of their work and who
reviewed draft versions of this manuscript.
REFERENCES
S. A., AND D. N. BARON. Intracellular pH measured in
isolated normal human leukocytes, by using 5,5-dimethyl-&&oxazolidinedione under conditions of varying PCO~ and bicarbonate. Clin. Sci. 40: 482-495, 1971.
2. AKERMAN,
K. E. 0. Effect of pH and Ca2+ on the retention of
Ca2+ by rat liver mitochondria. Arch. Biochem. Biophys. 189: 2561. AHMED,
262,1978.
3. AKKERMAN,
J. W. N., G. GORTER, J. J. SIXMA, AND G. E. J.
STAAL. Human platelet 6-phosphofructokinase:
purification, ki-
netic parameters and the influence of sulfate ions on enzyme
activity. Biochim. Biophys. Acta 370: 102-112, 1974.
J. W. N., G. GORTER, J. J. SIXMA, AND G. E. J.
4. AKKERMAN,
STAAL. Variations in levels of glycolytic intermediates in human
platelets during platelet-collagen interaction. Biochim. Biophys.
Actu 421: 296-307,1976.
5. ALLEN, R. D. Fertilization
and artificial activation in the egg of
the surf-clam, SpissuZu solidissima. BioZ. Bull. 105: 213-239, 1953.
6. ALVAREZ-LEEFMANS,
F. J., T. J. RINK, AND R. Y. TSIEN. Free
calcium ions in neurones of Helix aspersa measured with ionselective micro-electrodes. J. Physiol. London 315: 531-548, 1981.
D., F. LANTER, R. A. STEINER, P. SCHULTHESS,
Y.
7. AMMANN,
SHIJO, AND W. SIMON. Neutral carrier based hydrogen ion selective microelectrode for extra- and intracellular studies. Anal.
Chem. 53: 2267-2269,198l.
8. ATKINSON,
D. E. CeZZuZar Energy Metabolism
and Its Regulation.
New York: Academic, 1977.
9. AUNE, T. M., AND D. EPEL. Increased intracellular pH shifts the
subcellular location of GGPDH (Abstract). J. CeZZ BioZ. 79: 164a,
1978.
10. BABCOCK, D. F., G. A. RUFO, JR., AND H. A. LARDY. Potassiumdependent increases in cytosolic pH stimulate metabolism and
motility of mammalian sperm. Proc. N&Z. Acad. Sci. USA 80:
1327-1331,1983.
11. BAGINSKY,
R. M. On the timing
and activation of glycogen
catabolism in fertilized sea urchin eggs. J. Exp. ZooZ. 216: 201-
203,198l.
12. BAILEY, I. A., G. K. RADDA,
A. L. SEYMOUR, AND S. R. WILLIAMS.
The effects of insulin on myocardial metabolism and acidosis in
normoxia and ischaemia: a 31P-NMR study. Biochim.
Biophys.
Acta 720: 17-27,
1982.
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
as bacteria, yeast, slime molds, protozoons, echinoderms,
chelicerates, crustaceans, fish, amphibians, and mammals. The “pHi constancy hypothesis” is therefore untenable, and the unfortunate practices engendered by its
earlier acceptance (Sect. I) should be avoided in future.
Clearly many (perhaps all?) pronounced metabolic activations or inactivations involve modest-to-large pHi
changes, and in at least four cell systems (sea urchin egg
and sperm, frog muscle, and Artemia embryo) such
changes have been convincingly shown to play a regulatory role. The next important question to be answered is
that of the role pHi may play in events that do not
involve substantial changes in metabolic rate. Does the
fairly extreme nature of all the examples of metabolic
transitions accompanying pHi changes we have reviewed
indicate that pHi is of use only in extreme (hence, comparatively rare) cases, or does it merely arise from a
“sampling error,” due to the fact that extreme transitions
are most readily studied? On this question hangs the
larger issue of the overall scope for pHi-mediated metabolic regulation.
To our minds, the most important insight arising from
this review concerns the intimate and manifold nature
of the potential interactions between pHi and the central
regulators and processes of cellular metabolism. The
interaction between pHi and [Ca2+]; in activating calmodulin may prove to be of considerable significance
wherever moderate-to-large physiological pHi changes
occur, and the remaining evidence for pHi-pCai interactions discussed in Section IVA suggests that the overall
relationship between these factors can be very complex.
Similar but less well-documented interactions between
pHi and CAMP (Sect. IVB) may prove important too and
deserve further investigation. Finally the apparent potential of pHi to communicate information regarding
cellular energy baZance to enzymes and structures that
may share no other common effector (Sect. V) further
emphasizes the integrative character of pHi, precisely the
characteristic required of a central effector of metabolism.
In closing, it is useful to consider why organic evolution
has “chosen” to confer regulatory status on the hydrated
W. B. BUSA
INVITED
OPINION
36. CHAMBERS,
E. L. Na is essential for activation
of the inseminated
sea urchin egg. J. Exp. ZooZ. 197: 149-153,1976.
37. CHAMBERS,
R., AND E. L. CHAMBERS.
Explorations
into the
Nature
of the Living Cell. Cambridge,
MA: Harvard
Univ. Press,
1961.
38. CHIESI, M., AND G. INESI. Adenosine
5’-triphosphate
dependent
fluxes of manganese
and hydrogen
ions in sarcoplasmic
reticulum
vesicles. Biochemistry
19: 2912-2918,
1980.
39. CHRISTEN,
R., R. W. SCHACKMANN,
AND B. M. SHAPIRO. COUpling of sperm respiration,
motility,
and intracellular
pH (pHi) in
Strongylocentrotus
purpuratus
(Abstract).
J. Cell BioZ. 91: 174a,
1981.
40. CHRISTEN,
R., R. W. SCHACKMANN,
AND B. M. SHAPIRO. Elevation of the intracellular
pH activates
respiration
and motility
of sperm of the sea urchin. J. BioZ. Chem. 257: 14881-14890,1982.
41. CLAPPER, D. L., AND D. EPEL. Sperm motility
in the horseshoe
crab. IV. Extracellular
ions and intracellular
pH are not mediators
of motility
initiation.
Gamete Res. 6: 327-342,
1982.
42. CLEGG, J. S., AND F. P. CONTE. A review
of the cellular
and
developmental
biology
of Artemia.
In: The Brine Shrimp
Artermia, edited by G. Persoone,
P. Sorgeloos,
0. Roels, and E. Jaspers.
Wetteren,
Belgium:
Universa,
1980, p. 11-54.
43. COLLINS,
F., AND D. EPEL. The role of calcium
ions in the
acrosome reaction
of sea urchin sperm. Exp. Cell Res. 106: 211222,1977.
44. CUTHBERT,
A., AND A. W. CUTHBERT.
Fertilization
acid production in Psammechinus
eggs under pH clamp conditions,
and the
effect of some pyrazine
derivatives.
Exp. Cell Res. 114: 409-415,
1978.
45. CZECH, M. P. Molecular
basis of insulin
action. Annu.
Rev.
Biochem.
46: 359-384,1977.
46. DAN, J. C. Studies on the acrosome.
I. Reaction
to egg water and
other stimuli.
BioZ. Bull. 103: 54-66, 1952.
47. DECKER, G. L., G. B. JOSEPH, AND W. J. LENNARZ.
A study of
factors involved
in induction
of the acrosome
reaction
in sperm
of the sea urchin, Arbaciapunctulata.
Dev. BioZ. 53: 115-125,1976.
48. DELAAT, S. W., J. BOONSTRA,
W. H. MOOLENAAR,
C. L. MUMMERY, P. T. VAN DER SAAG, AND E. J. J. VAN ZOELEN.
Cation
transport
and growth
control
in neuroblastoma
cells in culture.
In: Membranes
In Growth
and Development,
edited by J. F.
Hoffmann,
G. H. Giebisch,
and L. Bolis. New York: Liss, 1981, p.
211-236.
49. DEN HOLLANDER,
J. A., K. UGURBIL,
T. R. BROWN, AND R. G.
SHULMAN.
Phosphorus-31
nuclear magnetic
resonance
studies of
the effect of oxygen
upon glycolysis
in yeast. Biochemistry
20:
5871-5880,198l.
50. DESLAURIERS,
R., R. A. BYRD, H. C JARRELL, AND I. C. P. SMITH.
31P NMR studies of vegetative
and encysted cells of Acanthamoeba
castellanii.
Eur. J. Biochem.
111: 369-375,
1980.
51. DESTEFANO,
M. J., M. A. DZIEZANOWSKI,
AND M. RABINOVITCH.
Migration
of polymorphonuclear
neutrophils
under agarose is
enhanced
by media of low pH or osmolarity.
J. Cell BioZ. 79: 89a,
1978.
52. DETWILLER,
T. C. Control
of energy metabolism
in platelets.
The
effects of thrombin
and cyanide on glycolysis.
Biochim.
Biophys.
Actu. 256: 163-174,
1972.
53. DIPOLO, R., AND L. BEAUGE. The effect of pH on Ca2+ extrusion
mechanisms
in dialyzed
squid axons. Biochim.
Biophys. Actu 688:
237-245,1982.
54. DOOLITTLE,
R. F., M. W. HUNKAPILLER,
L. E. HOOD, S. G.
DEVARE, K. C. ROBBINS,
S. A. AARONSON,
AND H. N. ANTONIADES. Simian sarcoma virus one gene, v-sis, is derived from the
gene (or genes) encoding a platelet-derived
growth factor. Science
221: 275-277,1983.
55. DUBE, F., AND P. GUERRIER.
Activation
of Barnes candidcz (Mollusca, Pelecypoda)
oocytes by sperm or KCl, but not by NH&l,
requires a calcium influx. Dev. BioZ. 92: 408-417,
1982.
56. DUBE, F., AND P. GUERRIER.
Acid release during activation
of
Barnea
candida
(Mollusca,
Pelecypoda)
oocytes.
Dev. Growth
Differ. 24: 163-171,
1982.
57. EPEL, D. Does ADP regulate respiration
following
fertilization
of
sea urchin eggs? Exp. CeZZ Res. 58: 312-319,
1969,
58. EPEL, D. The program
of and mechanisms
of fertilization
in the
echinoderm
egg. Am. ZooZ. 15: 507-522,1975.
59. EPEL, D. Intracellular
pH and activation
of the sea urchin egg at
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
13. BAKER, P. F., AND P. HONERJAGER.
Influence
of carbon dioxide
on level of ionized calcium
in squid axons. Nature
London
273:
160-161,1978.
14. BAKER, P. F., AND P. A. MCNAUGHTON.
Selective
inhibition
of
the Ca-dependent
Na efflux from intact squid axons by a fall in
intracellular
pH. J. Physiol. London 269: 78P-79P,
1977.
15. BALL, W. J., AND D. E. ATKINSON.
Adenylate
energy charge in
Saccharomyces
cerevisiae during starvatiqn.
J. Bacterial.
121: 975982,1975.
16. BARTON,
J. K., J. A. DEN HOLLANDER,
T. M. LEE, A. MACLAUGHLIN,
AND R. G. SHULMAN.
Measurement
of the internal
pH of yeast spores by 31P nuclear magnetic
resonance.
Proc. N&Z.
Acad. Sci. USA 77: 2470-2473,198O.
17. BATES, R. G. The modern
meaning
of pH. CRC Crit. Rev. Anal.
Chem. 10: 246-278,198l.
18. BEGG, D. A., AND L. I. REBHUN. pH regulates
the polymerization
of actin in the sea urchin
egg cortex.
J. Cell BioZ. 83: 241-248,
1979.
19. BEGG, D. A., L. I. REBHUN,
AND H. HYATT. Structural
organization of actin in the sea urchin egg cortex:
microvillar
elongation
in the absence of microfilament
bundle formation.
J. Cell BioZ. 93:
24-32,1982.
20. BELLE, R., J. BOYER, AND R. OZON. Carbon
dioxide
reversibly
inhibits
meiosis of Xenopus
Zaevis oocyte and the appearance
of
the maturation
promoting
factor. Dev. BioZ. 90: 3X-319,
1982.
21. BENNETT,
M. V. L., J. E. BROWN,
A. L. HARRIS,
AND D. C.
SPRAY. Electrotonic
junctions
between
Fundulus
blastomeres:
reversible
block by low intracellular
pH. BioZ. Bull. 155: 428-429,
1978.
22. BENOS, D. J. Amiloride:
a molecular
probe of sodium transport
in tissues and cells. Am. J. Physiol. 242 (Cell Physiol.
11): Cl31Cl45,1982.
23. BOONSTRA,
J., P. T. VAN DER SAAG, W. H. MOOLENAAR,
AND S.
W. DELAAT. Rapid effects of nerve growth
factor on the Na+,K+
pump in rat pheochromocytoma
cells. Exp. Cell Res. 131: 452-455,
1981.
24. BORON, W. F., W. C. MCCORMICK,
AND A. Roos. pH regulation
in barnacle
muscle fibers: dependence
on intracellular
and extracellular
pH. Am. J. Physiol.
237 (Cell Physiol.
6): Cl85-Cl93,
1979.
25. BORON, W. F., J. M. RUSSELL, M. S. BRODWICK,
D. W. KEIFER,
AND A. ROOS. Influence
of cyclic AMP on intracellular
pH regulation and chloride
fluxes in barnacle
muscle fibers. Nature
London 276: 511-513,1978.
26. BRANDIS, J. W., AND R. A. RAFF. Elevation
of protein
synthesis
is a complex
response
to fertilization.
Nature
London
278: 467469,1979.
27. BRYANT, J., AND R. E. KANE. Separation
and interaction
of the
major components
of sea urchin actin gel. J. Mol. BioZ. 125: 207224,1978.
28. BUSA, W. B. Cellular
dormancy
and the scope of pHi-mediated
metabolic
regulation.
In: IntraceZZuZar
pH: Its Measurment,
Regulation, and Utilization
in Cellular Functions,
edited by R. Nuccitelli and D. W. Deamer.
New York: Liss, 1982, p. 417-426.
29. BUSA, W. B., AND J. H. CROWE.
Intracellular
pH regulates
transitions
between
dormancy
and development
of brine shrimp
(Artemiu
salina) embryos.
Science 221: 366-368,
1983.
30. BUSA, W. B., J. H CROWE, AND G. B. MATSON.
Intracellular
pH
and the metabolic
status of dormant
and developing
Artemia
embryos.
Arch. Biochem.
Biophys.
216: 711-718,1982.
31. CALOTHY,
G., C. M. CROCE, V. DEFENDI,
H. KOPROWSKI,
AND
H. EAGLE. Effect of environmental
pH on rescue of simian virus
40. Proc. NatZ. Acad. Sci. USA 70: 366-368,
1973.
32. CARRON,
C. P., AND F. J. LONGO.
Relation
of intracellular
pH
and pronuclear
development
in the sea urchin, Arbaciapunctulatu.
Dev. BioZ. 79: 478-487,
1980.
33. CARRON,’ C. P., AND F. J. LONGO.
Relation
of cytoplasmic
alkalinization
to microvillar
elongation
and microfilament
formation
in the sea urchin egg. Dev. BioZ. 89: 128-137,1982.
34. CECCARINI,
C., AND H. EAGLE. Induction
and reversal
of contact
inhibition
of growth
by pH modification.
Nature
New BioZ. 233:
271--273,197l.
35. CECCARINI,
C., AND H. EAGLE. pH as a determinant
of cellular
growth
and contact
inhibition.
Proc. NatZ. Acad. Sci. USA 68:
229-233,197l.
R433
R434
W. B. BUSA
fertilization.
In: Cell Reproduction:
In Honor
of Daniel
Mazia,
edited by E. R. Dirkson,
D. M. Prescott,
and C. F. Fox. New York:
Academic,
1978, p. 367-378.
60. EPEL, D. Mechanisms
of activation
of sperm and egg during
fertilization
of sea urchin gametes. Curr. Top. Deu. Biol. 12: 185-
84.
C87-C93,1982.
67. FRELIN,,
C., P. VIGNE,
sensitive
87.
88.
89.
Na/H
antiport
L. CLANCY. The effect
the myocardial
cell pH.
91.
B. WALSH, AND R. D.
of insulin on glycolysis
242: (Cell Physiol. 11):
AND M. LAZDUNSKI.
The amiloridein 3T3 fibroblasts.
J. Biol. Chem. 258:
6272-6276,1983.
68. FRIEDMANN,
N. Effects
of glucagon and cyclic AMP on ion fluxes
in the perfused
liver. Biochim.
Biophys. Acta 274: 214-225, 1972.
69. GARBERS, D. L., AND G. S. KOPF. The regulation
of spermatozoa
by calcium and cyclic nucleotides.
Adv. Cyclic Nucleotide
Res. 13:
251-306,198O.
70. GARBERS, D. L., H. D. WATKINS,
AND K.
synthesis
S. MISONO.
The amino
of speract and of speract
J. R. HANSBROUGH,
A. SMITH,
acid sequence
and chemical
analogues.
J. Biol. Chem. 257:
2734-2737,1982.
71. GERSON, D. F. Intracellular
pH and the mitotic cycle in Physarum
and mammalian
cells. In: CeZZ Cycle Controls, edited by I. Cameron,
G. Padilla,
and A. Zimmerman.
New York: Academic,
1978, p.
105-131.
72. GERSON, D. F. The relation
between
intracellular
pH and DNA
synthesis
rate in proliferating
lymphocytes.
In: IntracelZuZar
pH:
Its Measurement,
Regulation
and Utilization
in Cellular Functions,
edited by R. Nuccitelli
and D. W. Deamer.
New York: Liss, 1982,
p. 375-383.
73. GERSON, D. F., H. KIEFER, AND W. EUFE. Intracellular
pH of
mitogen-stimulated
lymphocytes.
Science 216: 1009-1010, 1982.
74. GERSON, D. F., M. KOPONEN, AND R. LOOR. Cyclosporin
inhibits
the early rise of pHi permissive
for T cell activation
by concanavalin A. Eur. J. Immunol.
In press.
75. GEVERS, W. Generation
of protons
by metabolic
processes
in
1977.
heart cells. J. Mol. Cell. Cardiol. 9: 867-874,
76. GIBBONS, B. H., AND I. R. GIBBONS. Flagellar
movement
and
adenosine
triphosphatase
activity
in sea urchin sperm extracted
with Trition
X-100. J. Cell Biol. 54: 75-97, 1972.
77. GIBBONS, I. R., AND E. FRONK. Some properties
of bound and
soluble dynein
from sea urchin
sperm flagella.
J. CeZZ Biol. 54:
365-381,1972.
78. GILKEY, J. C. The roles of calcium
and pH in activation
of eggs
of the medaka fish, Oryzias latipes. J. CeZZ Biol. 97: 669-678,1983.
79. GILLIES,
R. J. Intracellular
pH and growth control in eukaryotic
cells. In: The Transformed
Cell, edited by I. Cameron
and T. B.
Poole. New York: Academic,
1981, p. 347-395.
80. GILLIES,
R. J., AND D. W. DEAMER.
Intracellular
pH: methods
19: 63-87, 1979.
and applications.
Curr. Top. Bioenerg.
81. GILLIES, R. J., AND D. W. DEAMER. Intracellular
pH changes
during the cell cycle in Tetrahymena.
J. Cell Physiol. 100: 23-32,
induces
86.
90.
R. A., N. C. GONZALEZ,
AND R.
of dibutyryl
cyclic AMP and glucagon on
Respir. Physiol. 32: 213-223, 1978.
66. FIDELMAN,
M. L., S. H. SEEHOLZER,
K.
MOORE.
Intracellular
pH mediates
action
in frog skeletal
muscle. Am. J. Physiol.
1979.
82. GILLIES,
85.
R. J., AND D. W. DEAMER. Endogenously
stationary
phase in Tetrahymena
cultures.
105:221-225,198O.
produced
COz
J. Cell. Physiol.
92.
R. J., M. P. ROSENBURG,
AND D. W. DEAMER.
Carbon
dioxide efflux accompanies
release of fertilization
acid from sea
urchin eggs. J. CelZ. Physiol. 108: 115-122, 1981.
GILLIES,
R. J., K. UGURBIL,
J. A. DEN HOLLANDER,
AND R. G.
SHULMAN.
31P NMR
studies of intracellular
pH and phosphate
metabolism
during cell division cycle of Saccharomyces
cereuisiae.
Proc. Natl. Acad. Sci. USA 78: 2125-2129, 1981.
GOLDSTEIN,
S. F. Starting
transients
in sea urchin sperm flagella.
J. Cell Biol. 80: 61-68, 1979.
GRAINGER,
J. L., M. M. WINKLER,
S. S. SHEN, AND R. A.
STEINHARDT.
Intracellular
pH controls protein
synthesis
rate in
the sea urchin egg and early embryo.
Deu. BioZ. 68: 396-406,1979.
GRAY, J. The effect of dilution
on the activity
of spermatozoa.
J.
Exp. Biol. 5: 337-344, 1928.
HAMAGUCHI,
M. S. The role of intracellular
pH in fertilization
of
sand dollar eggs analyzed
by microinjection
method. Deu. Growth
Differ. 24: 443-451, 1982.
HANSBROUGH,
J. R., AND D. L. GARBERS.
Sodium-dependent
activation
of sea urchin spermatozoa
by speract and monensin.
J.
Biol. Chem. 256: 2235-2241,198l.
HANSBROUGH,
J. R., AND D. GARBERS. Speract. Purification
and
characterization
of a peptide associated
with eggs that activates
spermatozoa.
J. Biol. Chem. 256: 1447-1452,198l.
HEIPLE,
J. M., AND D. L. TAYLOR.
An optical
technique
for
measurement
of intracellular
pH in single living cells. In: Intracellular pH: Its Measurement,
Regulation
and Utilization
in Cellular Functions,
edited by R. Nuccitelli
and D. W. Deamer.
New
York: Liss, 1982, p. 21-54.
HJEMDAHL,
P., AND B. B. FREDHOLM.
Direct antilipolytic
effect
of acidosis in isolated
rat adipocytes.
Acta Physiol.
Stand. 101:
294-301,1977.
93. HOCHACHKA,
P. W., AND T. P. MOMMSEN.
Protons and anaerobiosis. Science 219: 1391-1397,
1983.
94. HOERTER,
J. A., M. V. MICELI,
D. G. RENLUND,
G. GERSTENBLITH, E. G. LAKATTA, AND W. E. JACOBUS. Intracellular
calcium
alters high energy phosphates
and intracellular
pH. A 31P NMR
study of the isolated
rat heart (Abstract).
Biophys.
J. 41: 249a,
1983.
95. HOLLAND,
L. Z., AND N. L. CROSS. The pH within the jelly coat
of sea urchin eggs. Deu. Biol. 99: 258-260,1983.
96. HOLLAND,
L. Z., AND M. GOULD-SOMERO.
Fertilization
acid of
sea urchin eggs: evidence
that it is H+, not COS. Deu. Biol. 92:
549-552,1982.
97. HOLMSEN,
H. Platelet
energy metabolism
in relation
to function.
In: Platelets
and Thrombosis,
edited by D. C. B. Mills and F. I.
Pareti. London:
Academic,
1977, p. 45-62.
98. HOLWILL,
M. E. J. Kinetic
studies of the flagellar
movement
of
1969.
sea urchin spermatozoa.
J. Exp. Biol. 50: 203-222,
99. HORNE, W. C., N. E. NORMAN,
D. B. SCHWARTZ,
AND E. R.
SIMONS. Changes in cytoplasmic
pH and in membrane
potential
in thrombin-stimulated
human
platelets.
Eur. J. Biochem.
120:
295-302,198l.
100. HORNE, W. C., AND E. R. SIMONS.
Probes of transmembrane
potentials
in platelets:
changes in cyanine
dye fluorescence
in
response to aggregation
stimuli.
Blood 51: 741-749, 1978.
101. HORNE, W. C., AND E. R. SIMONS. Effects of amiloride
on the
response
of human platelets
to bovine thrombin.
Thromb.
Res.
13:599-607,1978.
102. HYLANDER,
B. L., AND R. G. SUMMERS.
The effect of local
anesthetics
and ammonia
on cortical
granule-plasma
membrane
attachment
in the sea urchin egg. Dev. Biol. 86: l-11, 1981.
103. HYNE, R. V., AND D. L. GARBERS. Requirement
of serum factors
for capacitation
and the acrosome reaction
of guinea pig spermatozoa in buffered
medium
below pH 7.8. BioZ. Reprod.
24: 257-
266,1981.
104. II, I., AND L. I. REBHUN.
Acid release follows
activation
of surf
clam (Spissula solidissima)
eggs. Dev. Biol. 72: 195-200, 1979.
105. II, I., AND L. I. REBHUN. Release of glucose-6-phosphate
dehydrogenase from cortex of SpissuZa eggs at fertilization
and its recombination
after meiosis. Deu. Biol. 91: 171-182, 1982.
106. INOUE, H., AND T. YOSHI~KA.
Measurement
of intracellular
pH
105: 461-468,
in sea urchin
eggs by 31P-NMR.
J. Cell. Physiol.
1980.
107. JAFFE, L. F. The role of calcium
activating
explosions,
eggs. In: BioZog-y of Fertilization,
waves and pulses in
edited bv C. B. Metz
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
1977.
65. FENTON,
R. NUCCITELLI
83. GILLIES,
245,1978.
61. EPEL, D. Experimental
analysis of the role of intracellular
calcium
in the activation
of the sea urchin egg at fertilization.
In: The Cell
Surface, Mediator
of Developmental
Processes, edited by S. Subtelney and N. Wessells. New York: Academic,
1980, p. 169-185.
62. EPEL, D., R. A. STEINHARDT,
T. HUMPHREYS,
AND D. MAZIA.
An analysis
of the partial
metabolic
derepression
of sea urchin
eggs by ammonia:
the existence
of independent
pathways.
Deu.
Biok 40: 245-255, 1974.
63. FARIATO, A., AND F. FABIATO. Effects of pH on the myofilaments
and the sarcoplasmic
reticulum
of skinned
cells from cardiac and
skeletal muscle. J. Physiol. London 276: 233-255, 1978.
64. FEINBERG, H., W. C. SANDLER, M. SCORER, G. C. LEBRETON, B.
GROSSMAN,
AND G. V. R. BORN. Movement
of sodium into human
platelets
induced by ADP. Biochim.
Biophys.
Acta 470: 317-324,
AND
INVITED
R435
OPINION
133.
134.
135.
136.
137.
138.
139.
140.
141.
142.
143.
144.
145.
146.
147.
148.
149.
150.
151.
152.
153.
154.
155.
pH of sea urchin eggs after fertilization.
Nature London 269: 590592,1977.
LUBIN, M., F. CAHN, AND B. A. COUTERMARSH.
Amiloride,
protein synthesis,
and activation
of quiescent
cells. J. Cell. Physiol.
113: 247-251,1982.
MADEIRA,
V. M. C. Proton movements
across the membranes
of
sarcoplasmic
reticulum
during the uptake of calcium
ions. Arch.
Biochem. Biophys.
200: 319-325,198O.
MAHLER,
H. R., P. K. JAYNES, J. P. MCDONOUGH,
AND D. K.
HANSON.
Catabolite
repression
in yeast: mediation
by CAMP.
Curr. Top. Cell. Regul. 18: 455-474,
1981.
MALCHOW,
D., V. NANJUNDIAH,
B. WURSTER, F. ECKSTEIN, AND
G. GERISCH.
Cyclic AMP-induced
pH changes in Dictyostelium
discoideum
and their control by calcium.
Biochim.
Biophys.
Acta
538: 473-480,1978.
MALLER,
J. L., AND J. W. KOONTZ.
A study of the induction
of
cell division in amphibian
oocytes by insulin. Deu. BioZ. 85: 309316,198l.
MAZIA, D. Chromosome
cycles turned on in unfertilized
sea urchin
eggs exposed to NHdOH.
Proc. NatZ. Acad. Sci. USA 71: 690-693,
1974.
MAZIA, D., AND A. RUBY. DNA synthesis turned on in unfertilized
sea urchin eggs by treatment
with NH,OH.
Exp. Cell Res. 85: 167172,1974.
MAZON,
M. J., J. M. GANCEDO,
AND C. GANCEDO.
Phosphorylation and inactivation
of yeast fructose-bisphosphatase
in viuo by
glucose and by proton ionophores.
Eur. J. Biochem. 127: 605-608,
1982.
MEECH, R. W., AND R. C. THOMAS. The effect of calcium injection
on the intracellular
sodium and pH of snail neurones.
J. Physiol.
London 265: 867-879,1977.
MEECH, R. W., AND R. C. THOMAS.
Effect of measured
calcium
chloride injections
on the membrane
potential
and internal
pH of
snail neurones.
J. Physiol. London
298: ill-129,198O.
MEIZEL,
S., AND D. W. DEAMER. The pH of the hamster
sperm
acrosome. J. Histochem.
Cytochem.
26: 98-105,1978.
MENAHAN,
L. A., K. D. HEPP, AND 0. WIELAND.
Liver 3’,5’nucleotide
phosphodiesterase
and its activity
in rat livers perfused
with insulin. Eur. J. Biochem. 8: 435-443,
1969.
MOISESCU,
D. G., C. C. ASHLEY, AND A. K. CAMPBELL.
Comparative aspects of the calcium-sensitive
photoproteins
aequorin
and
obelin. Biochim.
Biophys. Acta 396: 133-140,
1975.
MOLSKI,
T. F. P., P. H. NACCACHE,
M. VOLPI, L. M. WOLPERT,
AND R. I. SHA’AFI. Specific modulation
of the intracellular
pH of
rabbit neutrophils
by chemotactic
factors. Biochem. Biophys. Res.
Commun.
94: 508-514,198O.
MOODY,
W. J. Intracellular
pH regulation:
a summary
of the
proposed
mechanisms
and meeting
discussion.
In: Intracellular
pH: Its Measurement,
Regulation
and Utilization
in Cellular Functions, edited by R. Nuccitelli
and D. W. Deamer.
New York: Liss,
1982, p. 283-289.
MOOLENAAR,
W. H. Functional
aspects of plasma
membrane
studies: growth regulation
by ionic fluxes. In: Cell Biological
Asand B. A.
pects of Disease, edited by W. T. Daems, E. H. Burger,
Afzelius.
The Hague: Leiden Univ. Press, 1981, p. 27-36.
MOOLENAAR,
W. H., C. L. MUMMERY,
P. T. VAN DER SAAG, AND
S. W. DELATT. Rapid ionic events and the initiation
of growth in
serum-stimulated
neuroblastoma
cells. Cell 23: 789-798,
1981.
MOOLENAR,
W. H., R. Y. TSIEN, P. T. VAN DER SAAG, AND S.
W. DELAAT. Na/H exchange
and cytoplasmic
pH in the action of
growth
factors in human
fibroblasts.
Nature
London
304: 645648,1983.
MOOLENAAR,
W. H., Y. YARDEN, S. W. DELAAT, AND J. SCHLESSINGER. Epidermal
growth
factor induces electrically
silent Na+
influx in human fibroblasts.
J. BioZ. Chem. 257: 8502-8506,
1982.
MOORE,
R. D. Effect of insulin
upon the sodium pump in frog
skeletal muscle. J. Physiol. London 232: 23-45, 1973.
MOORE,
R. D. Elevation
of intracellular
pH by insulin
in frog
skeletal muscle. Biochem.
Biophys.
Res. Commun.
91: 900-904,
1979.
MOORE, R. D. Stimulation
Na:H exchange by insulin. Biophys. J.
33: 203-210,198l.
MOORE,
R. D., M. L. FIDELMAN,
J. C. HANSEN, AND J. N. OTIS.
The role of intracellular
pH in insulin action. In: IntraceZZuZarpH:
Its Measurement,
Regulation,
and Utilization
in CeZZuZar Func-
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
and A. Monroy.
New York: Academic.
In press.
108. JOHNSON,
C. H., D. L. CLAPPER, M. M. WINKLER,
H. C. LEE,
AND D. EPEL. A volatile
inhibitor
immobilizes
sea urchin sperm
in semen by depressing
the intracellular
pH. Deu. Biol 98: 493501,1983.
109. JOHNSON,
C. H., AND D. EPEL. Intracellular
pH of sea urchin
eggs measured
by the dimethyloxazolidinedione
(DMO)
method.
J. Cell Biol. 89: 284-291,
1981.
110. JOHNSON,
C. H., AND D. EPEL. Anomalies
in the pHi hypothesis
of protein
synthesis
activation
in sea urchin eggs (Abstract).
J.
Cell BioZ. 91: 180a, 1981.
111. JOHNSON,
C. H., AND D. EPEL. Starfish
oocyte maturation
and
fertilization:
intracellular
pH is not involved
in activation.
Deu.
BioZ. 92: 461-469,
1982.
112. JOHNSON,
J. D., D. EPEL, AND M. PAUL. Intracellular
pH and
activation
of sea urchin eggs after fertilization.
Nature
London
262: 661-664,1976.
113. JOHNSON,
R. A. Changes
in pH sensitivity
of adenylate
cyclase
specifically
induced
by fluoride
and vanadate.
Arch. Biochem.
Biophys.
218: 68-76,1982.
114. KAPLAN,
J. G. Membrane
cation transport
and the control
of
proliferation.
Annu. Rev. Physiol. 40: 19-41, 1978.
115. KOPF, G. S., D. J. TUBB, AND D. L. GARBERS. Activation
of sperm
respiration
by a low molecular
weight egg factor and by 8-bromoguanosine-3’,5’-monophosphate.
J. BioZ. Chem. 254: 8554-8560,
1979.
116. LAMBERT,
C. C. The ascidian
sperm reaction:
evidence
for Cland HC03involvement
in acid release. Dev. BioZ. 91: 257-262,
1982.
117. LAMBERT,
C. C., AND D. EPEL. Calcium-mediated
mitochondrial
movement
in ascidian
sperm during
fertilization.
Deu. BioZ. 69:
296-304,1979.
118. LAMBERT,
C. C., AND G. LAMBERT.
The ascidian
sperm reaction:
Ca2’ uptake in relation
to H+ efflux. Deu. BioZ. 88: 312-317,
1981.
119. LEA, T. J. Weak bases inhibit
the release of calcium
from the
crustacean
sarcoplasmic
reticulum
induced by CO2 and caffeine.
J. Physiol. London
330: 52P-53P,
1982.
120. LEA, T. J. C02/HCO;-induced
Ca2+ release from frog sarcoplasmic
reticulum
and the effects of weak bases. J. Physiol. London
334:
39P-4OP,
1982.
121. LEA, T. J., AND C. C. ASHLEY. Increase
in free Ca2+ in muscle
after exposure
to Con. Nature
London 275: 236-238,1978.
122. LEA, T. J., AND C. C. ASHLEY. Carbon dioxide or bicarbonate
ions
release Ca2+ from internal
stores in crustacean
myofibrillar
bundles. J. Membr.
BioZ. 61: 115-125,
1981.
123. LEE, H. C., J. G. FORTE, AND D. EPEL. The use of fluorescent
amines for the measurement
of pHi: applications
in liposomes,
gastric microsomes,
and sea urchin gametes. In: Intracellular
pH:
Its Measurement,
Regulation,
and Utilization
in Cellular
Functions, edited by R. Nuccitelli
and D. W. Deamer.
New York: Liss,
1982, p. 135-160.
124. LEE, H. C., C. JOHNSON,
AND D. EPEL. Changes in internal
pH
associated
with initiation
of motility
and acrosome
reaction of sea
urchin sperm. Dev. BioZ. 95: 31-45,1983.
125. LEE, H. C., S. SCHULINDER,
C. JOHNSON,
AND D. EPEL. Sperm
motility
initiation:
changes in intracellular
pH, Ca2’, and membrane potential
(Abstract).
J. Cell BioZ. 87: 39a, 1980.
126. LEE, S. C., AND R. A. STEINHARDT.
Observations
on intracellular
pH during cleavage of eggs of Xenopus
Zaeuis. J. Cell BioZ. 91: 414419,198l.
127. LOEB, J. Weitere
Beobachtungen
uber den Einfluss
der Befruchtung und der Zahl der Zellkerne
auf die Saurebildung
im Ei.
Biochem.
2. 2: 34-42,1906.
128. LOEB, J. Artificial
Parthenogenesis
and Fertilization.
Chicago, IL:
Univ. of Chicago Press, 1913.
129. LONDESBOROUGH,
J. C. Characterization
of an adenosine
3’,5’monophosphate
phosphodiesterase
from baker’s yeast. Biochem.
J. 163: 467-476,
1977.
130. LONDESBOROUGH,
J. Activity
of cyclic-AMP
phosphodiesterase
in permeabilised
cells of baker’s yeast. Eur. J. Biochem.
126: 631637,1982.
131. LONDESBOROUGH,
J. C., AND T. NURMINEN.
A manganese-dependent
adenyl cyclase in baker’s yeast, Saccharomyces
cereuisiae.
Acta Chem. Stand. 26: 3396-3398,1972.
132. LOPO, A., AND V. D. VACQUIER.
The rise and fall of intracellular
R436
156.
157.
158.
159.
160.
162.
163.
164.
165.
166.
167.
168.
169.
170.
171.
172.
173.
174.
175.
176.
177.
178.
179.
180.
181.
182.
183.
184.
185.
186.
187.
188.
189.
190.
191.
192.
193.
194.
195.
196.
AND R. NUCCITELLI
blasts: coupling to ribosomal protein S6 phosphorylation. Proc.
Natl. Acad. Sci. USA 79: 3935-3939,
1982.
RASMUSSEN,
H. Calcium and CAMP as Synarchic
Messengers.
New York: Wiley, 1981.
REYNOLDS,
R. C., AND N. HAUGAARD. The effect of variations of
pH upon the activation of phosphorylase by epinephrine in perfused contracting heart, liver slices, and skeletal muscle. J. Pharm.
Exp. Ther. 156:417-425,1967.
RIEGLE, K. M., AND R. L. CLANCY. Effect of norepinephrine on
myocardial intracellular hydrogen ion concentration. Am. J. Physiol. 229: 344-349,
1975.
RINK, T. J., R. Y. TSIEN, AND A. E. WARNER.
Free calcium in
Xenopus
embryos measured with ion-selective microelectrodes.
NatureLondon
283:658-660,198O.
ROBISON, G. A., R. W. BUTCHER, AND E. W. SUTHERLAND.
Cyclic
AMP. Annu. Rev. Biochem. 37: 149-174,1968.
ROBSON, J. S., J. M. BONE, AND A. T. LAMBIE. Intracellular pH.
Adv. Clin. Chem. 11:213-275,1968.
ROGERS, J., T. R. HESKETH,
G. A. SMITH, AND J. C. METCALFE.
Intracellular pH of stimulated thymocytes measured with a new
fluorescent indicator. J. Biol. Chem. 258: 5994-5997,
1983.
Roos, A., AND W. F. BORON. Intracellular pH. Physiol. Reu. 61:
296-433,1981.
ROSE, B., AND R. RICK. Intracellular
pH, intracellular free Ca,
and junctional cell-cell coupling. J. Membr.
Biol. 44: 377-415,
1978.
ROSENBERG,
G. B., AND M. L. PALL. Characterization of an ATPMg-dependent guanine nucleotide-stimulated
adenylate cyclase
from Neurospora
crassa. Arch. Biochem.
Biophys.
221: 243-253,
1983.
ROTHSCHILD,
L. The physiology of sea urchin spermatozoa. Action of pH, dinitrophenol, dinitrophenol + versene, and usnic acid
on O2 uptake. J. Exp. BioZ. 33: 155-173, 1956.
ROZENGURT,
E. Stimulation of DNA synthesis in quiescent cultured cells: exogenous agents, internal signals and early events.
Curr. Top. Cell. Regul. 17: 59-88, 1980.
ROZENGURT,
E., T. D. GELEHRTER, A. LEGG, AND P. PETTICAN.
Melittin stimulates Na entry, Na-K pump activity and DNA
synthesis in quiescent cultures of mouse cells. Cell 23: 781-788,
1981.
RUBIN, H. The regulation of animal cell multiplication.
J. Cell
Biol. 51: 686-702, 1971.
RUBIN, H. pH, serum and Zn2+ in the regulation of DNA synthesis
in cultures of chick embryo cells. J. CeZZ. Physiol. 82: 231-238,
1973.
RUNNSTROM,
J. Zur Kenntnis der Stoffwechselvorgange bei der
Entwicklungserregung
des Seeigeleies. Biochem. 2. 258: 257-279,
1933.
SANDERS, D., AND C. L. SLAYMAN.
Control of intracellular pH.
Predominant role of oxidative metabolism, not proton transport,
in the eukaryotic microorganism Neurospora.
J. Gen. Physiol. 80:
377-402,1982.
SANDLER, W. C., G. C. LE BRETON, AND H. FEINBERG.
Sodium
involvement in platelet aggregation (Abstract). Federation
Proc.
35: 755, 1976.
SCHACKMANN,
R. W., R. CHRISTEN, AND B. M. SHAPIRO. Membrane potential depolarization and increased intracellular pH
accompany the acrosome reaction of sea urchin sperm. Proc. Nutl.
Acad. Sci. USA
78: 6066-6070,198l.
197. SCHACKMANN,
R. W., E. M. EDDY, AND B. M. SHAPIRO. The
acrosome reaction of Strongylocentrotus
purpurutus
sperm: ion
movements and requirements. Deu. BioZ. 65: 483-495, 1978.
198. SCHACKMANN,
R. W., AND B. M. SHAPIRO. A partial sequence of
ionic changes associated with the acrosome reaction of Strongylocentrotus
purpuratus.
Dev. Biol. 81: 145-154, 1981.
S., AND E. ROZENGURT.
Na/H antiport in Swiss
199. SCHULINDER,
3T3 cells: mitogenic stimulation leads to cytoplasmic alkalinization. Proc. Natl. Acad. Sci. USA 79: 7778-7782,
1982.
200. SETLOW, P., AND A. KORNBERG. Biochemical studies of bacterial
sporulation and germination. XXII. Energy metabolism in early
stages of germination of BacilZus meguterium
spores. J. BioZ.
Chem.
245: 3637-3644,197O.
201. SETLOW, B., AND P. SETLOW. Measurements of the pH within
dormant and germinated bacterial spores. Proc. N&Z. Acad. Sci.
USA
77: 2474-2476,198O.
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
161.
tions, edited by R. Nuccitelli and D. W. Deamer. New York: Liss,
1982, p. 385-416.
MOORE,
R. D., M. L. FIDELMAN,
AND S. H. SEEHOLZER.
Correlation between insulin action upon glycolysis and change in intracellular pH. Biochem. Biophys. Res. Commun. 91: 905-910, 1979.
MOORE, R. D., AND R. D. GUPTA. Effect of insulin on intracellular
pH as observed by 31P NMR spectroscopy. Int. J. Quunt. Chem.
Quant. Biol. Symp. 7: 83-92, 1980.
MORISAWA,
M., AND R. A. STEINHARDT.
Changes in intracellular
plusmodium
during the cell cycle and in response
pH of Physarum
to starvation. Exp. Cell Res. 140: 341-351, 1982.
NAKAMARU,
Y., AND A. SCHWARTZ. Possible control of intracellular calcium metabolism by [H+]: sarcoplasmic reticulum of
skeletal and cardiac muscle. Biochem. Biophys. Res. Commun. 41:
830-836,197O.
NEEDHAM,
J., AND D. M. NEEDHAM.
The hydrogen ion concentration and the oxidation-reduction
potential of the cell interior:
a microinjection study. Proc. R. Sot. London Ser. B 98: 259-286,
1926.
NICHOLLS,
D. G. The regulation of extramitochondrial
free calcium ion concentration by rat liver mitochondria. Biochem. J.
176:463-474,1978.
NICHOLLS,
D. G., AND M. CROMPTON.
Mitochondrial
calcium
transport. FEBS Lett. 111: 261-268,198O.
NISHIMURA,
M., T. ITO, AND B. CHANCE. Studies on bacterial
photophosphorylation.
III. A sensitive and rapid method of determination of photophosphorylation.
Biochim.
Biophys.
Actu 59:
177-182,1962.
NISHIOKA,
D., AND N. CROSS. The role of external sodium in sea
urchin fertilization. In: Cell Reproduction:
In Honor
of Daniel
Maxiu,
edited by E. R. Dirkson, D. M. Prescott, and C. F. Fox.
New York: Academic, 1978, p. 403-413.
NISHIOKA,
D., AND D. EPEL. Intracellular pH and activation of
sea urchin eggs at fertilization (Abstract). J. Cell BioZ. 75: 40a,
1977.
NUCCITELLI,
R. Intracellular pH measurement techniques: their
advantages and limitations. In: IntracellularpH:
Its Measurement,
Regulation
and Utilization
in Cellular
Functions,
edited by R.
Nuccitelli, and D. W. Deamer. New York: Liss, 1982, p. 161-169.
NUCCITELLI,
R., AND J. M. HEIPLE. Summary of the evidence
and discussion concerning the involvement of pHi in the control
of cellular functions. In: Intracellular
pH: Its Measurement,
Regulation, and Utilization
in Cellular Functions,
edited by R. Nuccitelli and D. W. Deamer. New York: Liss, 1982, p. 567-586.
NUCCITELLI,
R., D. J. WEBB, S. T. LAGIER, AND G. B. MATSON.
31P NMR reveals increased intracellular pH after fertilization in
Xenopus eggs. Proc. Natl. Acad. Sci. USA 78: 4421-4425, 1981.
OBARA, Y., H. YOSHIDA,
L. S. CHAI, H. WEINFELD,
AND A. A.
SANDBERG.
Contrast between the environmental pH dependencies of prophasing and nuclear membrane formation in interphase-metaphase cells. J. Cell BioZ. 58: 608-617, 1973.
OHTAKE, H. Respiratory behavior of sea urchin spermatozoa. I.
Effect of pH and egg water on respiratory rate. J. Exp. Zool. 198:
303-311,1976.
OWEN, N. E., AND M. L. VILLEREAL.
Evidence for a role of
calmodulin in serum stimulation of Na+ influx in human fibroblasts. Proc. Natl. Acad. Sci. USA 79: 3537-3541, 1982.
PALL, M. L. Adenosine 3’,5’-phosphate
in fungi. Microbial.
Reu.
45:462-480,1981.
PAUL, M. Release of acid and changes in light scattering properties
following fertilization of Urechis caupo eggs. Dev. BioZ. 43: 299312,1975.
PEAUCELLIER,
G., AND M. DOREE. Acid release at activation and
fertilization of starfish oocytes. Deu. Growth Differ. 23: 287-296,
1981.
PICARD, A., AND M. DOREE. Intracellular microinjection of anticalmodulin drugs does not inhibit the cortical reaction induced
by fertilization, ionophore A 23187 or injection of calcium buffers
in sea urchin eggs. Deu. Growth Differ. 24: 155-162, 1982.
POOLE, B., AND S. OHUMA.
Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages. J. Cell BioZ. 90:
665-669,1981.
POUYSSEGUR,
J., J. C. CHAMBARD,
A. FRANCHI, S. PARIS, AND
E. VAN OBBERGHEN-SCHILLING.
Growth factor activation of an
amiloride-sensitive Na+/H+ exchange system in quiescent fibro-
W. B. BUSA
INVITED
R437
OPINION
Res. Commun.
53: 490-494,1973.
SHOSHAN,
V., D. H. MACLENNAN,
AND D. S. WOOD. A proton
gradient controls a calcium-release channel in sarcoplasmic reticulum. Proc. Natl. Acad. Sci. USA 78: 4828-4832,198l.
210. SIMONS, E. R., D. B. SCHWARTZ, AND N. E. NORMAN. Stimulus
response coupling in human platelets: thrombin-induced changes
pH, Its Measurement,
Regulation,
and
in pHi. In: Intracellular
edited by R. Nuccitelli and D.
UtiZization
in Cellular Functions,
W. Deamer. New York: Liss, 1982, p. 463-482.
211. SMALLWOOD,
J., D. M. WAISMAN,
D. LAFRENIERE,
AND H. RASMUSSEN. The erythrocyte calcium pump catalyzes a Ca:nH exchange. J. BioZ. Chem. 258:11092-11097,1983.
212. SMITH, J. B., AND E. ROZENGURT.
Serum stimulates the Na+,K+
pump in quiescent fibroblasts by increasing Na+ entry. Proc. N&Z.
209.
Acad.
Sci. USA
213. STEINHARDT,
214.
215.
216.
217.
218.
219.
220.
221.
222.
223.
224.
75: 5560-5564,1978.
R. A. Ionic logic in activation of the cell cycle. In:
Ions, Proliferation
and Cancer. New York: Academic. In press.
STEINHARDT,
R. A., AND J. M. ALDERTON.
Calmodulin confers
calcium sensitivity on secretory exocytosis. Nature London 295:
154-155,1982.
STEINHARDT,
R. A., AND D. MAZIA. Development of K-conductance and membrane potentials in unfertilized sea urchin eggs
after exposure to NHIOH. Nature London 241: 400-401,1973.
STOCCO, D. M., P. C. BEERS, AND A. H. WARNER.
Effects of
anoxia on nucleotide metabolism in encysted embryos of the brine
shrimp. Dev. Biol. 27: 479-493, 1972.
SWERDLOW,
B. M., B. SETLOW, AND P. SETLOW. Levels of H+
and other monovalent cations in dormant and germinating spores
of BaciZZus megaterium.
J. Bacterial.
148: 20-29, 1981.
THOMAS,
J. A., R. M. BUSCHBAUM,
A. ZIMNIAK, AND E. RACKER.
Intracellular pH measurements in Ehrlich ascites tumor cells
utilizing spectroscopic probes generated in situ. Biochemistry
18:
2210-2218,1979.
TILNEY,
L. G. The polymerization of actin. III. Aggregates of
nonfilamentous actin and its associated proteins: a storage form
of actin. J. Cell Biol. 69: 73-89, 1976.
TILNEY, L. J., AND L. A. JAFFE. Actin, microvilli, and the fertilization cone of sea urchin eggs. J. Cell Biol. 87: 771-782, 1980.
TILNEY,
L. G., D. P. KIEHART,
C. SARDET, AND M. TILNEY.
Polymerization of actin. IV. Role of Ca++ and H+ in the assembly
of actin and in membrane fusion in the acrosomal reaction of
echinoderm sperm. J. Cell Biol. 77: 536-550, 1978.
TKACHUK,
V. A., AND M. Y. MEN’SHIKOV.
Effect of pH on calcium
binding properties of calmodulin and its interaction with the Cadependent form of cyclic nucleotide phosphodiesterase. Biokhimia. 46: 779-788, 1981. (English translation)
TREVILLYAN,
J. M., AND M. L. PALL. Control of cyclic adenosine
3’,5’ -monophosphate levels by depolarizing agents in fungi. J.
Bacterial.
138: 397-403, 1979.
TRIVEDI, B., AND W. H. DANFORTH.
Effect of pH on the kinetics
of frog muscle phosphofructokinase. J. Biol. Chem. 241: 4110-
4114,1966.
225. TSOKOS, J., T. F. CORNWELL, AND G. VLASUK. Ca2+ efflux from
liver mitochondria induced by a decrease in extramitochondrial
119:297-300,198O.
pH. FEBSLett.
AND F. ZORG226. TUPPER, J. T., M. DEL Rosso, B. HAZELTON,
NIOTTI. Serum-stimulated changes in calcium transport and distribution in mouse 3T3 cells and their modification by dibutyryl
cyclic AMP. J. Cell. Physiol. 95: 71-84, 1978.
227. VACQUIER, V. D., AND B. BRANDRIFF.
DNA synthesis in unfertilized sea urchin eggs can be turned on and turned off by the
addition and removal of procaine hydrochloride. Dev. Biol. 47:
12-31,1975.
228. VAGHY, P. L. Role of mitochondrial oxidative phosphorylation in
the maintenance of intracellular pH. J. Mol. Cell. Cardiol. 11:
933-940,1979.
229. VAN DER PLAAT, J. B. Cyclic 3’,5’-adenosine monophosphate
stimulates trehalose degradation in baker’s yeast. Biochem. Biophys.
Res. Commun.
56: 580-587,1974.
230. VAUGHAN-JONES,
R. D., W. J. LEDERER, AND D. A. EISNER. Ca2’
ions can affect intracellular pH in mammalian cardiac muscle.
NatureLondon
301:522-524,1983.
231. VIGNE, P., C. FRELIN, AND M. LAZDUNSKI.
The amiloride-sensitive Na+/H+ exchange system in skeletal muscle cells in culture.
J. Biol.
Chem.
257: 9394-9400,1982.
232. VILLEREAL,
M. L. Sodium fluxes in human fibroblasts: effect of
serum, Ca2+ and amiloride. J. CeZZ. Physiol. 107: 359-369, 1981.
W. J., AND R. G. BATES. Intracellular pH. Physiol.
233. WADDELL,
Rev. 49: 285-329,1969.
234. WADDELL, W. J., AND T. C. BUTLER. Calculation of intracellular
pH from the distribution of 5,5-dimethyl-2,4-oxazolidinedione
(DMO). J. Clin. Invest. 38: 720-729, 1959.
235. WARBURG, 0. Uber die oxidationen in lebeden Zellen nach Versuchen am Seeigelei. Hoppe-Seyler’s
2. Physiol.
Chem. 66: 305340,1910.
236. WATERFIELD,
M. D., G. T. SCRACE, N. WHITTLE, P. STROOBANT,
A. JOHNSSON, A. WASTESON, B. WESTERMARK,
C.-H. HELDIN,
J. S. HUANG, AND T. F. DEUEL. Platelet-derived growth factor is
structurally related to the putative transforming protein p28sis of
Simian sarcoma virus. Nature London 304: 35-39,1983.
237. WEBB, D. J., AND R. NUCCITELLI.
Intracellular pH changes accompany the activation of development in frog eggs: comparison
of pH microelectrode and 31P-NMR measurements. In: Intracellular pH, Its Measurement,
Regulation,
and Utilization
in Cellular
edited by R. Nuccitelli and D. W. Deamer. New York:
Functions,
Liss, 1982, p. 293-324.
238. WEINGART, R., P. HESS, AND W. R. REBER. Influence of intracellular pH on cell-to-cell coupling in sheep Purkinje fibers. In:
Normal
and Abnormal
Conduction
in the Heart, edited by A. P.
de Carvalho, B. F. Hoffman, and M. Lieberman. New York:
Futura, 1982, p. 73-84.
239. WILT, F. H., AND D. MAZIA. The stimulation of cytoplasmic
polyadenylation in sea urchin eggs by ammonia. Dev. Biol. 37:
422-424,1974.
240. WINKLER,
M. M. Regulation of protein synthesis in sea urchin
eggs by intracellular pH. In: Intracellular
pH: Its Measurement,
Regulation,
and Utilization
in Cellulur
Functions,
edited by R.
Nuccitelli and D. W. Deamer. New York: Liss, 1982, p. 325-340.
241. WINKLER, M. M., G. B. MATSON, J. W. B. HERSHEY, AND E. M.
BRADBURY.
31P-NMR study of the activation of the sea urchin
egg.Exp.
CeZZRes. 139: 217-222,1982.
242. WINKLER, M. M., AND R. A. STEINHARDT. Activation of protein
synthesis in a sea urchin cell-free system. Dev. Biol. 84: 432-439,
1981.
M. M., R. A. STEINHARDT,
J. L. GRAINGER, AND L.
243. WINKLER,
MINNING.
Dual ionic controls for the activation of protein synthesis at fertilization. Nature London 287: 558-560, 1980.
244. WONG, P. Y. D., W. M. LEE, AND A. Y. F. TSANG. The effects of
extracellular sodium on acid release and motility initiation in rat
caudal epididymal spermatozoa in vitro. Exp. CeZZ Res. 131: 97104,1981.
245. WORKING,
P. K., AND S. MEIZEL.
Evidence that an ATPase
functions in the maintenance of the acidic pH of the hamster
sperm acrosome. J. Biol. Chem. 256: 4708-4711,198l.
246. WORKING,
P., AND S. MEIZEL. Preliminary characterization of a
M$+-ATPase in hamster sperm head membranes. Biochem. Bio-
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017
202. SETLOW, B., L. K. SHAY, J. C. VARY, AND P. SETLOW. Production
of large amounts of acetate during germination of Bacillus megaterium
spores in the absence of exogenous carbon sources. J.
Bacterial.
132: 744-746, 1977.
203. SHA’AFI, R. I., P. H. NACCACHE, T. F. P. MOLSKI, AND M. VOLPI.
Chemotactic stimuli-induced changes in the pHi of rabbit neutrophils. In: Intracellular
pH: Its Measurement,
Regulation
and Utilization in CeZZuZur Functions,
edited by R. Nuccitelli and D. W.
Deamer. New York: Liss, 1982, p. 513-525.
204. SHEN, S. S. The effect of extracellular ions on pHi in sea urchin
eggs. In: Intracellular
pH, Its Measurement,
Regulation,
and Utiedited by R. Nuccitelli and D.
lization in Intracellular
Functions,
W. Deamer. New York: Liss, 1982, p. 269-282.
205. SHEN, S. S., AND R. A. STEINHARDT.
Direct measurement of
intracellular pH during metabolic derepression of the sea urchin
egg. Nature
London
272: 253-254,1978.
206. SHEN, S. S., AND R. A. STEINHARDT.
Intracellular pH and the
sodium requirement at fertilization. Nature London 282: 87-89,
1979.
207. SHEN, S. S., AND R. A. STEINHARDT.
Intracellular pH controls
the development of new potassium conductance after fertilization
of the sea urchin egg. Exp. CeZZ Res. 125: 55-61, 1980.
208. SHIMOMURA,
O., AND F. H. JOHNSON. Further data on the specificity of aequorin luminescence to calcium. Biochem. Biophys.
R438
phys.
Res. Commun.
104: 1060-1065,1982.
P. K., AND S. MEIZEL.
Correlation
of increased
intraacrosomal
pH with the hamster
sperm acrosome
reaction.
J.
hp. Zool. 227: 97-107, 1983.
248. WV, T. L., AND E. J. DAVIS. Regulation
of glycolytic
flux in an
energetically
controlled
cell-free
system:
the effects of adenine
nucleotide
ratios,
inorganic
phosphate,
pH, and citrate.
Arch.
Biochem. Biophys.
209: 85-99, 1981.
249. YAJIMA, M., AND M. UI. Hydrocortisone
restoration
of the pHdependent
metabolic
responses to catecholamines.
Am. J. Physiol.
247. WORKING,
W. B. BUSA
AND
R. NUCCITELLI
228: 1053-1059,1975.
250. ZETTERBERG, A., AND W. ENGSTROM.
pH on quiescent,
serum-starved
Mitogenic
effect of alkaline
cells. Proc. NutZ. Acad. Sci. USA
78:4334-4338,198l.
251. ZETTERBERG,
A., W. ENGSTROM,
AND 0. LARSSON.
Growthactivation
of resting cells: induction
of balanced
and imbalanced
growth. Ann. NY Acad. Sci. 397: 130-147,
1982.
252. ZUCKER, R. S., R. A. STEINHARDT, AND M. M. WINKLER. Intracellular
Ca release and the mechanisms
of parthenogenetic
activation of the sea urchin egg. Deu. Biol. 65: 285-295,
1978.
William B. Busa and Richard Nuccitelli
Department of Zoology, University of California,
Davis, California 95616
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on May 3, 2017