Download The pH of Spontaneously Beating Cultured Rat Heart Cells Is

676
The pH of Spontaneously Beating Cultured
Rat Heart Cells Is Regulated by an
ATP-Calmodulin-Dependent Na+/H+ Antiport
Peter L. Weissberg, Peter J. Little, Edward J. Cragoe Jr., and Alex Bobik
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We investigated the mechanisms by which spontaneously beating cultured rat ventricular cells
regulate intracellular pH (pH,). Specifically, the relative contributions of the Na + /H + antiport,
Cr/HCO 3 ~ exchange, ATP, and calmodulin-dependent processes in regulating the pH, of cells
loaded with the intracellular fluorescent pH indicator BCECF were investigated. The pH, of
ventricular cells bathed in HEPES-buffered medium averaged 7.30±0.02. Subsequent exposure
of the cells to CO2-HCO3~-buffered medium resulted in intracellular acidification followed by
recovery to pH, levels approximately 0.1 pH units lower than in controls. Recovery was
inhibited by the Na + /H + antiport inhibitor 5-(W-ethyl-W-isopropyl)amiloride (EIPA). The
recovery from intracellular acidification, induced by a 15-mM ammonium chloride prepulse,
was also dependent solely upon activation of the Na + /H + antiport. Recovery was dependent
upon extracellular sodium, was completely inhibited by EIPA, and could be modulated by
changes in extracellular pH (pHJ. At low pH,, values (6.3) the recovery of pH, was greatly
attenuated, while at high pH, (8.0) the recovery process was accelerated. The final pH, to which
the cells recovered was also dependent upon pH,,. Preincubation of the cells with 2deoxy-D-glucose to deplete cellular ATP levels reduced pH, by approximately 0.2 pH units and
greatly unpaired the cells' ability to recover from 15-mM ammonium chloride-induced acid
load. Similarly, preincubation of cells with the calmodulin inhibitors W-7 and trifluoperazine
also impaired their ability to recover from the acid load. The C1~-HCO3~ exchange played no
role in the cells' ability to recover from intracellular acidosis. However, the presence of HCO3~
significantly increased the resistance of myocardial cells to changes in pH, by approximately
doubling their buffer capacity. These results demonstrated that a Na + /H + antiport is the major
pH,-regulating system in spontaneously beating rat ventricular cells. The ability of the Na + /H +
antiport to regulate myocardial pH, is dependent upon the cells' ability to maintain adequate
levels of ATP. The antiport's dependency on ATP, in conjunction with its dependency on
calmodulin, suggests that activation of the antiport in ventricular cells involves phosphorylation
processes. {Circulation Research 1989;64:676-685)
C
hanges in intracellular pH (pHj) have important effects on both the contractile and
electrical properties of the heart. 12 However, little is known about the mechanisms that
regulate myocardial pH;. Myocardial pHj is considerably more alkaline than would be expected from a
passive distribution of protons across the cell mem-
From the Baker Medical Research Institute and Alfred Hospital
Clinical Research Unit, Melbourne, Australia, and Merck Sharp
& Dohme Laboratories (E.J.C.), West Point, Pennsylvania.
Supported by a Grant-in-Aid from the National Heart Foundation of Australia. P.L.W. received a British Medical Research
Council Travelling Fellowship followed by an Alfred Hospital,
Edward Wilson Fellowship.
Address for correspondence: Dr. A. Bobik, Baker Medical
Research Institute, Commercial Road, Prahran, Victoria,
Australia.
Received October 13, 1987; accepted September 2, 1988.
brane. Furthermore, cardiac cells recover rapidly
from an imposed acid load. In cultured chick embryo
heart cells, recovery from acidosis is dependent on
the transmembrane extrusion of protons by the Na+/
H+ antiport.3 Studies on pHj in nonbeating sheep
Purkinje cells by use of microelectrodes are also
consistent with this observation.4-5 However, the
mechanisms involved in pHj regulation in beating
mammalian heart cells are unknown. In particular,
the relative contributions of the Na + /H + antiport,
intracellular HCO3~, and C1"-HCO3~ exchange to
pHj regulation have not been investigated, nor have
the intracellular processes that influence the activity
of these exchange mechanisms.
A variety of different conditions may induce
acidosis in cardiac cells. During myocardial ischemia, for example, the intracellular acidosis is accom-
Weissberg et al
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panied by a pronounced decrease in energy production relative to demand.6 The same condition does
not apply, however, during respiratory acidosis.
These different conditions could profoundly influence the cardiac cells' subsequent ability to recover
from intracellular acidosis. Indeed, in some cell
lines activation of the Na + /H + antiport appears to
be dependent on ATP7 and related phosphorylation
processes.8 In the studies reported here, cultured
spontaneously beating rat ventricular cells containing the intracellular pH-sensitive fluorophore 2,7biscarboxyethy 1- 5(6)-carboxyfluorscein (BCECF)
were used to investigate 1) the relative contributions of the Na + /H + antiport and Cr-HCO 3 "
exchange in the maintenance of pH;; 2) the dependence of Na + /H + antiport activity on cellular ATP
content; and 3) the relative importance of calmodulinand protein kinase C-dependent processes in the
activation of the Na + /H + antiport.
Materials and Methods
Culture of Cardiac Cells
Cardiac cell cultures were prepared from the
hearts of 3- to 5-day-old female Wistar Kyoto rats
by modification of the method previously used for
chick embryo cardiac cultures.9 Briefly, the hearts
were removed from decapitated animals and placed
into cold Hanks' balanced salt solution. The atria,
large vessels, and pericardium were removed, and
the ventricles were cut into small (—1-2 ^.1) pieces.
The tissue was incubated at 37° C in Medium 199
containing 3 mg/ml collagenase. After 15 minutes,
the medium was discarded, and the tissue was
dispersed into single cells by successive tryptic
digestion in a medium consisting of 0.5% trypsin
and 0.01% deoxyribonuclease in calcium-free Dulbecco's medium containing 1 mM MgSO4. The
suspended cells were collected into fetal calf serum
at 4° C and centrifuged at 150g. The cells were then
suspended in Hanks' balanced salt solution containing 2 mM glutamine, 50 mg/l glycine, 12.5 mg/1
hypoxanthine, essential and nonessential amino
acids, 100 IU/ml penicillin, and 7.1 mM NaHCO3
and seeded into 90-mm plastic culture dishes. After
75 minutes at 37° C, the medium containing unattached cardiac cells was removed from the dishes,
leaving behind fibroblasts, endothelial cells, and
smooth muscle cells attached to the plastic dishes.
Bromo-deoxyuridine (0.1 mM final concentration)
was then added to the medium to inhibit proliferation of any remaining noncardiac cells.10 The medium
containing the cardiac cells was then divided into
aliquots and placed in 35-mm culture dishes containing two 24x10 mm sterile glass coverslips and
incubated at 37° C in 5% CO2 in air. After 12-18
hours, the attached cells were washed with fresh
culture medium (containing bromo-deoxyuridine).
This technique produced a monolayer of spontaneously beating cells that were used in experiments
48-72 hours after plating.
Cardiac IntraceUular pH Regulation
677
Measurement of Intracellular pH
The fluorescent pH indicator BCECF was used to
monitor changes in cytoplasmic pH in the cultured
cardiac cells. BCECF has a slow spontaneous leakage rate, and the intensity of its fluorescence is
linearly related to pH from pH 6.5 to 7 . 5 . " l 2
Coverslips to which spontaneously beating cardiac
cells were attached were taken from the culture
medium and washed three times with 2 ml of
physiological salt solution (PSS). The cells were
then incubated for 30 minutes at 37° C in PSS
containing 1 piM of esterified BCECF (BCECFAM). During this time the (membrane permeable)
BCECF-AM entered the cells and was hydrolyzed
to free (membrane impermeable) BCECF by intracellular esterases, resulting in entrapment of the
fluorescent indicator within the cell. At the end of
the incubation, extracellular indicator was removed
by washing the coverslips with PSS at 37° C. Examination of these cells under a fluorescence microscope indicated that BCECF fluorescence was
evenly distributed throughout the cell cytoplasm.
The spontaneous beating of the cell cultures, examined by phase contrast microscopy, was unaffected
by BCECF.
Coverslips with the BCECF-loaded cells were
placed into a vertical holding device that fits into a
standard fluorescence cuvette and permits rapid
exchange of extracellular medium (~20 ml in 10
seconds) or addition of drugs to the medium without
disturbing the cells or their orientation to the excitation beam.13 Fluorescence of the monolayer of
cells bathed in the various salt solutions (see "Materials and Methods" and "Results") was measured
at 37° C in an LS-5 luminescence spectrometer
(Perkin-Elmer, Eden Prairie, Minnesota) with the
excitation wavelengths set at 500 or 440 nm (bandpass 10 nm) and the emission wavelengths set at 530
nm (bandpass 5 nm). Under these conditions BCECF
fluorescence at 500 nm was maximal and dependent
on pH|, while the fluorescence at 440 nm (its isosbestic point) was unaffected by pH,.14 The ratio of
500:440 fluorescence values, corrected for cellular
autofluorescence at these wavelengths, was used to
estimate pHf. Autofluorescence of unloaded cardiac
cells represented < 1 % of total fluorescence. Over
the pH range of 6.40 to 7.80, the regression line
relating the fluorescence ratio (FR) to pH was
described by the equation FR=5.82 [pHJ-34.0
(r=0.99) (Figure 1). Known values for pHj of the
cardiac cells were obtained as previously described
using high-concentration K+ buffers of various pH
containing 7 /xM nigericin.12 This fluorescence
ratio technique gives consistent estimates of pHj
that are not affected by alterations in cell density
or leakage of BCECF and obviates the need to
calibrate each experiment individually.
Measurement of ATP
Cellular contents of ATP were measured fluorometrically via the hexokinase reaction.15 Briefly,
678
Circulation Research
BCECF
Vol 64, No 4, April 1989
Calibration
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Intracellular
7-5
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1. Relation between apparent intracellular pH of
cardiac cells and intracellular BCECF fluorescence intensity ratio. Nigericin and high-potassium buffers of known
pH were used to derive the relation. Each value represents
mean±SEM of four separate experiments. BCECF, 2,7biscarboxyethyl-5(6)-carboxyfluorescein.
FIGURE
the cells were washed thoroughly with ice-cold
normal saline before extraction of the ATP with
ice-cold 0.4 M perchloric acid. After neutralization
of the extracts with potassium carbonate, 100 /tl
aliquots were assayed for ATP content in 2 ml of
buffer containing 100 mM Tris, 5 mM MgCl2, 5 mM
glucose, 10 fiM nicotinamide adenine dinucleotide
phosphate, and 3.5 units glucose 6-phosphate dehydrogenase. Hexokinase (2.8 units) was added to
initiate the reaction, and the changes in fluorescence were monitored in a Perkin-Elmer LS-5 spectrophotofluorometer whose excitation and emission
wavelengths were set at 340 and 450 nm, respectively. Standardization of each sample was achieved
by monitoring the change in fluorescence after
addition of 2 nmol ATP to the reaction cuvette.
Cellular protein was measured according to the
method of Lowry et al.16
Solutions
The PSS used in the study had the following
millimolar composition: NaCI 135, KC1 5, CaCl21.8,
MgSO4 0.8, glucose 5.5, and N-(2-hydroxyethyl)
piperazine-W-2-ethanesulfonic acid 10 (HEPES)
adjusted to pH 7.4 at 37° C. When a PSS of different
pH values was required, the solution was adjusted
with either hydrochloric acid or sodium hydroxide to
the appropriate pH. In PSS buffered with CO2HCO3", the sodium chloride concentration was reduced to 115 mM and 20 mM NaHC0 3 was substi-
tuted for the HEPES. This solution was equilibrated
with 5% CO2 in air to bring its pH to 7.4 at 37° C.
When low sodium concentrations were required, the
sodium chloride was replaced by equimolar Nmethyl-D-glucamine. In the PSS used for depleting
cardiac energy stores, glucose was replaced by 5.5
mM 2-deoxy-D-glucose. The solution used to calibrate
fluorescent signals from the cardiac cells was nominally Na+-free PSS containing 140 mM KC1 and 7 fiM
nigericin. All experiments were carried out at 37° C.
Materials
Cell culture reagents were purchased from Commonwealth Serum Laboratories, Parkville, Australia. Tissue culture plates were from Flow Laboratories, Melbourne, Australia. The BCECF-AM was
from Molecular Probes, Eugene, Oregon. Nigericin;
ouabain; 4-acetamido-4'-isothiocyanostilbene2,2'-disulfonic acid (SITS); l-(5-isoquinolinesulfonyl)2-methylpiperazine dihydrochloride (H-7); trifluoperazine; /V-methyl-D-glucamine; and 2-deoxyD-glucose were from Sigma Chemical, St. Louis,
Missouri. N-(6-Aminohexyl)-5-chloro-1 -naphthalenesulfonamide hydrochloride (W-7) was from Seikagaku America, St. Petersburg, Florida. 5-(N-EthylN-isopropyl)amiloride (EIPA) was synthesized by
Dr. E.J. Cragoe. All other chemicals were of analytical or tissue culture grade and were purchased
from local chemical suppliers.
Statistics
Results are expressed as the mean±SEM.
Statistical significance was evaluated by twotailed Student's t test.
Results
Determinants of Basal Intracellular pH
The basal pHj of spontaneously beating rat
cardiac cells at 37° C in HEPES-buffered PSS (pH
7.4) averaged 7.30±0.02 (n=ll). This basal pH;
was to some extent maintained by the Na + /H +
antiport since the addition of 200 fiM EIPA, a
potent inhibitor of Na + /H + exchange, caused a
gradual time-related fall in pH, of between 0.01
and 0.02 pH( units/min over the ensuing 5 minutes.
However, the contribution of Na + /H + exchange to
the maintenance of normal pHf could be demonstrated more easily when the cells were changed to
a bicarbonate-buffered PSS (Figure 2). When the
extracellular medium was changed from HEPESbuffered PSS (pH 7.4) to the CO2-HC(V-buffered
PSS (pH 7.4), pHj fell rapidly as dissolved carbon
dioxide entered the cells. Intracellular pH then recovered to a new equilibrium about 0.1 pH units
below the original pHj. The pH; under these conditions averaged 7.19±0.04 (n=6). The maintenance
of pHj under these conditions was greatly dependent
on Na + /H + exchange since EIPA (200 /iM) prevented any recovery from the initial (carbon dioxideinduced) fall in pHj (Figure 2).
Weissberg et al Cardiac Intracellular pH Regulation
679
PSS
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2. Role of Na+/H+ antiport in regulation of
intracellular pH in CO2-HCO3'-buffered PSS medium.
Cells equilibrated in HEPES-buffered PSS (pH 7.4) were
perfused with COrHCO{-buffered PSS (pH 7.4). Top
panel depicts changes in intracellular pH; bottom panel
shows effect of inhibition of Na+IH+ exchange with 200
fiM EIPA on intracellular pH. PSS, physiological salt
solution; EIPA, 5(S-ethyl-N-isopropyl)amiloride.
I
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7-2 -
FIGURE
+
Na /H* Antiport and Recovery From
Intracellular Acidosis
To examine the importance of Na + /H + exchange
in regulation of pHj, we compared the rates of
recovery of pH; after acidification by a 15-mM
NH^Cl prepulse in the absence and presence of
bicarbonate. Upon exposure of the cells to 15 mM
NH,C1 in HEPES-buffered PSS, pHj rose rapidly
from 7.30±0.03 to 7.74±0.02 (n=5). Despite the
continual presence of ammonium chloride, pHs gradually returned towards the basal level, attaining a
pH| of 7.38±0.04 5 minutes after the initial exposure
to ammonium chloride. Upon removal of the ammonium chloride by perfusion with HEPES-buffered
PSS (pH 7.4), pHj fell rapidly to 6.67±0.04 (n=5).
In HEPES-buffered PSS (pH 7.4) containing 135
mM Na + , the recovery from acidosis was rapid; the
half-time for recovery averaged 0.625±0.025 minutes (Figure 3). The recovery was prevented by the
Na + /H + exchange inhibitor EIPA.
Recovery from the ammonium chloride-induced
acidosis was also dependent on extracellular sodium
concentration ([Na],,). Reduction of the [Na]0 by
substitution of an equimolar amount of Nmethyl-D-glucamine for sodium in the HEPES-
-
6-8
6-4-1
FIGURE 3. Representative tracings demonstrating effects
of addition and removal of 15 mM NH4C1 on intracellular
pH of cells equilibrated in HEPES-buffered PSS (pH
7.4). Top panel depicts transient fall in intracellular pH
upon perfusion of ammonium chloride-exposed cells with
HEPES-buffered PSS (pH 7.4). Bottom panel shows
effect of 200 yM EIPA on recovery phase of intracellular
pH. PSS, physiological salt solution; EIPA, 5(N-ethyl-Nisopropyl)amiloride.
buffered PSS (pH 7.4) reduced the initial rate of
recovery (Figure 4). In a nominally Na+-free HEPESbuffered PSS (pH 7.4), there was no recovery from
the acidosis. Eadie-Hofstee analysis of the dependence of pHj recovery rate on [Na]0 under these
conditions gave a Km for extracellular sodium of
9.8±1.6 mM and a maximum recovery rate of
0.84±0.09 pH units/min (Figure 4). The dependence on sodium and the sensitivity to EIPA demonstrate that in the absence of bicarbonate, Na + /H +
exchange is wholly capable of mediating complete
recovery from acidosis.
Since a membrane HCO3"-C1" transport mechanism has been shown to exist in mammalian Purkinje fibers,1718 we also examined how it might
influence the cells' ability to recover from acidosis.
680
Circulation Research
Vol 64, No 4, April 1989
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Extracellular sodium (mM)
FIGURE 4. Effects of extracellular Na+ concentration ([Na+]o) on recovery of cells from acidosis. Left panel is a
composite of several tracings in which recovery ofintracellular pHfrom a 15-mM NH4Cl-induced acidosis was measured
in HEPES-buffered PSS (pH 7.4) containing various concentrations of sodium (Nao). Right panel shows relation between
initial rates of recovery measured from above experiments and [Na+]o. PSS, physiological salt solution.
Upon exposure of the cells to 15 mM NH4CI in
CO2-HC<V-buffered PSS (pH 7.4), pH; rose from
7.14±0.04 to 7.48±0.04 (n=6, p<0.0l). This increase
in pHj (0.29±0.01 pH units) was significantly less
than the corresponding rise in HEPES-buffered PSS
(0.44±0.01 pH units, p<0.01 for difference). After 5
minutes pHj had completely returned to basal levels
(pH 7.17±0.04) (Figure 5). Upon perfusion of the
cells with COrHCCV-buffered PSS, pH; rapidly
fell to 6.71 ±0.04, a value similar to that observed
with the HEPES-buffered PSS. However, in the
CO2-HC(V-buffered PSS, the magnitude of the fall
in pHj (0.46±0.03) was significantly less than that in
HEPES-buffered PSS (0.70±0.01, p<0.01 for difference). Recovery from intracellular acidosis was
unaffected by the presence of HC03~ in the medium
and could be completely inhibited by EIPA (Figure
5). Furthermore, the anion exchange inhibitor SITS
(300 fiM) had no inhibitory effect on the recovery
(not shown). These results indicated that Na + /H +
exchange is the major pHj regulating mechanism
responsible for the cells' ability to recover from
acidosis.
Although C1"-HCO3" exchange plays no part in
the recovery from acidosis, the CO2-HCO3~ does
protect the cell from large fluctuations in pH, by
increasing the cell's buffering capacity. The intrinsic buffering capacity (pi)19 of the cells in the
HEPES-buffered PSS (pH 7.4), derived from the
magnitude of the alkalinization (approximately
0.40 pH units) that occurred upon substitution of
HEPES-buffered PSS (pH 7.4) for COrHCCVbuffered PSS (pH 7.4), averaged 20.7 mM/pH. The
total buffering capacity in the HCO3~-buffered PSS,
calculated from the estimated intracellular HC0 3
concentration according to Roos and Boron,19 averaged 41 mM/pH. Thus, the total buffering capacity
of the cell was approximately doubled by the
presence of bicarbonate.
Extracellular pH and Na+/H+ Antiport Activity
Since reductions in extracellular pH (pH,,) are
known to greatly increase the concentration of
acidic metabolites within the heart,2021 the effects
of alterations in pH,, on Na + /H + exchange were also
examined. In these experiments, Na + /H + antiport
activity was assessed by measurement of the rate of
recovery from an NH^Cl-induced acidosis in HEPESbuffered PSS (pH 6.3-8.0). Under these conditions
the fall in pH,, induced by superfusion of the cells
with the different HEPES-PSS buffers, was similar,
averaging 6.66±0.05 (Figure 6). Na + /H + antiport
activity was greatly attenuated when pHo was low
and was enhanced when pHo was elevated. The
initial rates of recovery (pH units per minute) from
this level of acidosis (NaHE) were related to pH,, by
the equation NaHE=0.755[pH o ]-4.63 (n=13,
r=0.93). The final pHs values achieved under these
conditions were also dependent on pH,, (Figure 6).
Dependency of Na+/H+ Exchange on
Metabolic Energy
Previous studies in a number of cell lines have
provided conflicting evidence as to the dependency
of Na + /H + antiport activity on metabolic
energy.7,22.23 Therefore, we assessed the energy
dependency of the antiport in cardiac cells by
studying their ability to recover from an ammonium
Weissberg et al
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FIGURE 5. Tracings depicting effects of addition and
removal of 15 mM NH4Cl on intracellular pH of cells
equilibrated in CO2-HCOf-buffered PSS (pH 7.4). Top
panel demonstrates transient nature of fall in intracellular pH upon perfusion of ammonium chloride-exposed
cells with COrHCOf-buffered PSS (pH 7.4). Bottom
panel shows effect of 200 fiM EIPA on recovery from
acidosis. PSS, physiological salt solution; EIPA, 5(N-ethyl-N-isopropyl)amiloride.
chloride-induced acidosis after 30 minutes of incubation in HEPES-buffered PSS (pH 7.4) containing
5.5 mM 2-deoxy-r>glucose instead of glucose. This
procedure reduced cellular ATP content by 90%,
from 18.4±0.3 nmol/mg protein (n=3) to 1.7±0.1
nmol/mg protein (n=3). Under these conditions the
cells' ability to recover from the 15-mM NH»C1induced acidosis was markedly attenuated (Figure
7). Furthermore, basal pHi in these cells was reduced
by approximately 0.2 pH units, presumably by the
inability of the antiport to extrude protons. To
examine whether this effect was due to a reduction
in the transmembrane sodium gradient via inhibition of the Na-K pump due to ATP depletion, we
examined the effect of 3 mM ouabain on the recovery process. Incubation of the cells for 60 minutes
in ouabain did not affect their subsequent ability to
recover from an acid load (Figure 7).
Calmodulin Dependence of the Na+/H+ Antiport
The inhibition of Na + /H + antiport activity by
2-deoxy-D-glucose-induced ATP depletion raised
the possibility that an ATP-dependent process such
Cardiac IntraceUular pH Regulation
681
as phosphorylation was required for Na + /H + antiport
activation. Therefore, we investigated the role of
calmodulin and other kinase-dependent processes
in activation of the antiport. A 30-minute preincubation of the cells with the calmodulin-selective antagonist W-7 (200 /iM)24-25 completely impaired their
ability to recover from a 15-mM NH4Cl-induced
acid load in HEPES-buffered PSS (pH 7.4) (Figure
8). Lower concentrations caused partial impairment
of recovery. The IC50 for inhibition of recovery by
W-7 was 50 fiM. To test whether this was a direct
(extracellular) effect of W-7 on the Na + /H + antiport,
similar to that observed with EIPA, we also assessed
its acute effects on the ability of cells to recover
from the acid load. When ammonium chloride was
removed from cells not previously exposed to W-7
with PSS containing W-7 (200 /xM), the initial rate
of recovery from acidosis was unimpaired. However, after approximately 2 minutes the rate of
recovery fell dramatically, and pH, gradually commenced to fall (not shown). After 30 minutes of
exposure to 200 ^.M W-7, the fall in pHj averaged
0.50±0.05 pH units. Preincubation of the cells for
30 minutes with the structurally unrelated calmodulin antagonist trifluoperazine (up to 60 /xM)26 also
had similar effects on the ability of cardiac cells to
recover from an acid load (Figure 8). Fifty percent
inhibition by trifluoperazine occurred at 12 /iM; at
60 fiM, the highest concentration used, trifluoperazine inhibited pH, rate of recovery by 82±3%.
These results were consistent with the exertion of
the inhibitory effect of calmodulin antagonists on
the antiport by indirect mechanisms, presumably
inhibition of phosphorylation. In contrast to the
effects of W-7, the related sulfonamide derivative
H-7, known to inhibit protein kinase C, cyclic
AMP, and cyclic GMP-dependent protein kinase
systems at a concentration of 200 /iM,27-28 had only
a marginal effect on the ability of the cells to
recover from the same acid load (Figure 8).
Discussion
We have demonstrated that an ATP-calmodulindependent Na + /H + antiport is the major p H r
regulating mechanism in synchronously beating
mammalian myocardial cells. Recovery from intracellular acidosis is mediated solely by this antiport.
Our finding that preincubation with 2-deoxyr>glucose or the calmodulin antagonists greatly
attenuated activation of the antiport during acidosis
indicated that activation of the antiport is dependent on cellular metabolic energy and may involve
ATP-dependent phosphorylation processes. The ineffectiveness of the sulfonamide derivative H-7, a
potent inhibitor of several protein kinase systems
including those dependent on cyclic AMP, cyclic
GMP, and diacylglycerols,27-28 suggested that regulation of the antiport in cardiac cells is predominantly calmodulin dependent.
It has been generally assumed that Na + /H +
exchange in cells is an ATP-independent process
682
Circulation Research
Vol 64, No 4, April 1989
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Extracellular pH
+
+
FIGURE 6. Effects of extracellular pH on Na /H antiport-mediated recovery from acidosis. Left panel is a composite
of tracings showing recovery of intracellular pH in acidotic (15-mM NH^Cl-induced) cells under conditions of various
extracellular pH. Right panel shows relation between extracellular pH and final intracellular pH achieved by cells in
above experiments. PSS, physiological salt solution; pH0, extracellular pH.
driven by the inward electrochemical gradient for
sodium and that allosteric regulation of the antiport
by intracellular protons is the major mechanism by
which it functions to maintain pH; homeostasis.29
Our results in beating myocardial cells were consistent with this hypothesis. The activity of the
antiport was greatly increased by intracellular acidification. Furthermore, the extent of the increase in
activity was dependent on [Na]0, particularly at
concentrations below 15 mM. However, the inability of the antiport to maintain pHj when the cells
were incubated with 2-deoxy-r>glucose, a procedure that depletes cellular ATP, strongly indicated
that this allosteric regulation by intracellular protons is subject to additional regulating mechanisms
such as phosphorylation. Although our evidence for
involvement of a phosphorylation process in the
regulation of the antiport is indirect and other
mechanisms may contribute to the observed effects,
reports on the properties of this antiport in other
cell lines support a phosphorylation hypothesis.
Recently, phorbol esters, known activators of protein kinase C, have been shown to stimulate Na + /
H+ exchange in fibroblasts,30 lymphocytes,31 and
myoblasts.32 In the latter instance, the effect of the
phorbol esters on the antiport was to shift its pHj
dependence to a more alkaline pHj. The inability of
the myocardial cells to recover from an acid load
after preincubation with 2-deoxy-r>glucose or the
calmodulin antagonists could well have been due to
a shift in- the pHf dependence of the antiport to a
more acidic pHj. Such an effect has recently been
reported for a cultured human cell line.7 This ability
of processes, presumably phosphorylation, to alter
the activity of the antiport in cardiac cells would
represent an important mechanism by which cardiac cells regulate and maintain intracellular pH
during periods of increased metabolic demand. In
this context it is interesting to note that isoproterenol has been recently reported to indirectly stimulate the Na + /K + pump of isolated cardiac myocytes
of rabbits by increasing the electroneutral influx of
sodium.33
Our finding that Cr-HC0 3 ~ exchange in beating
heart cells does not contribute to the recovery from
acidosis was consistent with recent observations in
cardiac Purkinje strands 1718 and cultured chick
embryo cardiac cells.3 Nevertheless, intracellular
HC03~ does contribute significantly to the overall
buffering capacity, thereby increasing the cells'
ability to withstand shifts of pH; to more alkaline
and acidic values. The intracellular buffering capacity of 42 mM/pH unit, observed under these conditions, is similar to that reported for sheep heart
Purkinje fibers34 and other muscle.19
The observation that pH, was consistently approximately 0.1 pH units lower in HCCV-COj-buffered
PSS than in nominally HCO3~-free PSS was consistent with previous observations on myocardial pH.34
This has been attributed to an HCO3"-CO2 shuttle
movement in which the continuous efflux of HC0 3 "
down its electrochemical gradient imposes a constant acid load on the cell by continuously dissociating carbonic acid.33 However, it is still not clear
why the Na + /H + antiport did not return pHj to
control levels. Na + /H + antiport activity in cardiac
cells was not impaired by the presence of CO2HC0 3 " in the incubation medium since initial rates
of recovery were identical to those observed in
HEPES-buflfered medium. Identical initial rates of
Weissberg et al Cardiac Intracellular pH Regulation
• dd load
683
acid load
I
H-7
7. Comparison of effects of preincubation of
cells with 3 mM ouabain and 5.5 mM 2-deoxy-D-glucose
(2-DG) on the cells' subsequent ability to activate Na+l
H+ antiport after a 15-mM NH4Cl-induced acid load.
Arrow indicates time at which ammonium chloride was
removed from medium, thereby inducing acidosis. Intracellular pH recovery was measured in HEPES-buffered
media containing ouabain or 2-deoxy-D-glucose.
FIGURE
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pH, recovery in HEPES-buffered and HCCV-COjbuffered media have also been reported in cardiac
Purkinje fibers despite a higher intracellular buffer
capacity when the fibers are incubated in HCO3~COr-buffered medium.18 Vanheel et al18 attributed
this higher-than-predicted initial recovery rate from
acidosis to a transient lowering of intracellular
bicarbonate concentration during the initial phase
of the ammonium chloride-induced acidosis. A similar mechanism may also have been responsible for
the apparent equalization of pHj recovery rates in
the cultured heart cells. Clearly, this aspect of pHj
control, together with the reasons as to why the
steady-state pH, is lower in cardiac cells incubated
in the HCO3~-CO2-buffered medium, requires further investigation. However, the physiological consequence of this latter effect is that pH, will be lower
in a respiratory acidosis than in an equivalent
metabolic acidosis. Reductions in pH, are known to
induce graded reductions in the transmembrane
cardiac calcium current36 and also reduce the affinity
of myofibrils for calcium.37 The effects of these
reductions due to increasing levels of intracellular
acidosis would explain why the cardiac inotropic
state is depressed more by respiratory acidosis than
by an equivalent metabolic acidosis. An elevation in
myocardial Na + /H + antiport activity in respiratory
failure could also account for the increased toxicity
of digoxin in these subjects.38 Under these conditions, sodium influx via the Na + /H + antiport is
increased in response to intracellular acidosis, and
in the absence of an adequate Na + /K + pump to
extrude the sodium, intracellular sodium concentration increases. This increase in sodium would in
turn stimulate calcium entry via Na+-Ca2+ exchange.
Such an interaction between the Na + /H + antiport,
the Na+-K+ pump, and Na+-Ca2+ exchange has
FIGURE 8. Comparison of effects of preincubation of
cells with calmodulin inhibitors W-7 (200 fj.M) and trifluoperazine (TFP) (60 fjM) and protein kinase C inhibitor
H-7 (200 nM) on their subsequent ability to activate
Na+/H+ antiport after a 15-mM NH^l-induced acid load
in HEPES-buffered PSS (pH.7.4). Arrow indicates time
at which cells were perfused with ammonium chloridefree HEPES-buffered PSS (pH 7.4) to induce acid load.
Intracellular pH recovery was measured in HEPESbuffered media containing H-7, TFP, or W-7. PSS, physiological salt solution.
recently been proposed to play an important role in
the positive inotropic effects of cardiac glycosides.3940 In this context, it is interesting to note
that both the electrocardiographic and inotropic
effects of digoxin can be antagonized by amiloride.4142 These effects were formerly attributed to
the diuretic's potassium-sparing properties. However, partial inhibition of Na + /H + exchange could
also contribute to the antagonistic effect of amiloride.
Intracellular acidosis due to the retention of acidic
metabolites is also an early event in the progress of
ischemia.20 This is accompanied by a fall in extracellular pH. Our results indicated that under these
conditions proton extrusion by the Na + /H + antiport
will be impaired and pH, will fall, thereby depressing myocardial contractile function. After transient
ischemia in which reperfusion occurs before the
depletion of high-energy phosphates such as ATP
and cytidine 5'-triphosphate (CTP), the Na + /H +
antiport would rapidly extrude accumulated protons and restore pHf to normal. During more severe
ischemia in which ATP levels are reduced, restoration of Na + /H + antiport activity under these conditions will depend upon the synthesis of adequate
cellular ATP.
In conclusion, our results demonstrated that an
ATP-calmodulin-dependent Na + /H + antiport in
beating mammalian heart cells is the sole pHregulating system. The antiport is stimulated by
intracellular acidosis and inhibited by extracellular acidosis. The ability of the antiport to respond
to an intracellular acidosis is dependent on adequate high-energy phosphate stores and calmodulin-
684
Circulation Research
Vol 64, No 4, April 1989
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dependent processes. This raises the interesting
possibility that only phosphorylated Na + /H +
antiport proteins are capable of translocating intracellular protons in exchange for extracellular
sodium. It is tempting to speculate that mechanisms that regulate the extent of phosphorylation
and dephosphorylation of Na + /H + antiport proteins maintain intracellular pH within normal limits. Such a mechanism would in essence be capable of responding to an increased acid load by
recruiting additional antiport units activated by
phosphorylation. Furthermore, the involvement
of calmodulin in the antiport's activation process
suggests that calcium may also be an important
determinant of antiport activity. Clearly, further
work will be required to substantiate these hypotheses and understand precisely how the Na + /H +
antiport is involved in pH, regulation of the normal
and impaired myocardial cell.
Acknowledgment
The technical assistance of Miss Annette Grooms
is gratefully acknowledged.
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+
+
KEY WORDS • intracellular pH • Na /H antiport • ATP
heart cells • BCECF • calmodulin
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The pH of spontaneously beating cultured rat heart cells is regulated by an
ATP-calmodulin-dependent Na+/H+ antiport.
P L Weissberg, P J Little, E J Cragoe, Jr and A Bobik
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Circ Res. 1989;64:676-685
doi: 10.1161/01.RES.64.4.676
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