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AMER. ZOOL.. 19:145-162 (1979).
Inotropism and Contracture of Aplysiid Ventricles as Relatedtothe Action of
Neurohumors on Resting and Action Potentials of Molluscan Hearts
ROBERT B. HILL
ROBERT E. YANTORNO
AND
Department of Zoology,
University of Rhode Island,
Kingston, Rhode Island
02881
Department of Bioengineering,
University of Pennsylvania,
Philadelphia, Pennsylvania
19174
SYNOPSIS The postsynaptic actions of neurohumors on molluscan muscle may be exerted
through control of force as well as by means of excitation and inhibition. The control of
force may appear as potentiation of the response to excitation, as increased inotropism in
spontaneous contractions, or as an increase in tonus. We have directed our attention to the
alterations of force induced in aplysiid ventricles by applied postulated neurohumors. The
results are interpreted in terms of the known effects of neurohumors on resting potential
and action potentials of molluscan hearts. We recorded compound membrane potentials of
aplysiid ventricles extracellularly, using a single sucrose gap apparatus together with a
force-displacement transducer to measure force in contractions or contracture. Aplysia
cahfornica ventricle has a membrane potential of —52.5 ± 9.4 mV. Ventricles of Aplysia
dactylomela or Aplysia californica are depolarized by increased concentrations of external
potassium ion, with an accompanying contracture. After incubation in calcium-free
medium, KC1 contracture-force is directly dependent on calcium ion concentration. A
depolarization of 8.3 ± 2.14 mV in potassium-free medium is blocked by substitution of
lithium for sodium in the medium, suggesting an electrogenic sodium pump. There is a
sustained depolarization in low chloride medium, which suggests a significant chloride
contribution to the resting potential. Ventricles of Dolabella aunculana or Aplysia dactylomela
are depolarized by acetylcholine (ACh). Threshold for depolarization is lower than
threshold for contracture-force. The ventricle of A. dactylomela is depolarized by 5hydroxytryptamine (5HT) with threshold at 10~9 M and a maximum depolarization of 30
mV at 10~4 M. Depolarization by 5HT may induce beating but does not induce
contracture. Ventricles of Aplysia californica are not depolarized by ACh although beating
ventricles are inhibited, and a depolarized ventricle in a tonic contracture may be
hyperpolarized and relaxed by low concentrations. The force of contraction of the
ventricle of Dolabella auricularia is dependent on the duration of the plateau phase of the
cardiac action potential. The plateau is lengthened by 5HT with an accompanying
increase in force of beat, and shortened by ACh, with an accompanying decrease in force
of beat. The action potential in the ventricle of Aplysia californica is not differentiated
into spike and plateau phases, and neither ACh nor 5HT has any marked effect on the
form of the action potential. Nevertheless, isolated ventricles of Dolabella auricularia, Aplysia
dactylomela, and Aplysia californica are all excited by 5-hydroxytryptamine, with a threshold
at about 10~8 M. Both spontaneous beating and excitation induced in A. californica ventricles by 5HT are blocked by lack of the sodium ion, which may be responsible for pacemaker potentials in molluscan hearts.
Original investigations on Aplysia dactylomela were
carried out in Bermuda, and this is contribution N°
797 from the Bermuda Biological 'Station for Research, Inc. We would like to thank J. Clipper and P.
McDonald-Ordzie for their technical assistance during the investigations in Bermuda, supported by
N.I.H. Grant 2 RO1 NS 08352-04 PHY.
We thank Mr. William Par Hayes for assistance in
the original investigations on Aplysia californica, supported by N.S.F. Grant PCM 76-14950, which were
conducted at the Marine Biological Laboratory,
Woods Hole. The original investigations on Dolabella
auricularia were carried out by R. B. Hill, in part, at
the Shimoda Marine Biological Station of Tokyo
145
Kyoiku University, during the tenure of a Visting
Professorship of the Japan Society for the Promotion
of Science, and thanks are extended to host scientist
Professor Arinobu Ebara. Original investigations on
Dolabella auricularia were continued by R. B. Hill at
the Laboratory of Sensory Sciences of the University
of Hawaii, where the original work on Aplysia californica had its inception, and thanks are extended to
Professor Ian M. Cooke for the invitation to work at
the Laboratory of Sensory Sciences. We wish to thank
Dr. Kiyoaki Kuwasawa for much helpful discussion
during the preparation of this paper, and to thank
Professors Michael J. Greenberg and Harold Atwood
for helpful criticism of an earlier draft of this paper.
146
ROBERT B. HILL AND ROBERT E. YANTORNO
The isolated ventricle of Aplysia califomica will continue beating in low calcium solutions,
but the force of beating is directly related to the extracellular concentration of the calcium
ion, which may be responsible for excitation-contraction coupling in molluscan hearts. In
calcium-free medium, action potentials fail after about 8 min. Both sodium and calcium
ions may be involved in the generation of action potentials in molluscan hearts.
RESTING POTENTIAL AND CONTRACTURE
INTRODUCTION
Cardioregulatory neurocrine substances
released by the innervation of molluscan
hearts are believed to include acetylcholine
(ACh) and 5-hydroxytryptamine (5HT).
The evidence for this was reviewed by Hill
and Welsh (1966). Since then, additional
evidence for the identification of ACh and
5HT as cardioregulatory neurohumors has
been provided for a number of molluscan
species (S.-R6zsa and Perenyi, 1966; Phillis, 1966; S.-R6zsa and Zs.-Nagy, 1967;
Taxi and Gautron, 1969; Evanset al., 1971;
Leake et al., 1971; MacKay and Gelperin,
1972) and Welsh (1971) has reviewed the
history of the concept of neurohumoral
regulation of molluscan hearts. In the
neural control of the heart of Aplysia calif ornica, Liebeswar et al. (1974) have identified two heart inhibitor motoneurons as
cholinergic and one heart excitor motoneuron as serotoninergic. Now we wish to
ask whether or not these neurohumoral
transmitters govern cardiac force in aplysiid gastropods through regulation of the
ionic currents which govern membrane
potential difference and the form of cardiac action potentials. An alternative possibility would be direct involvement of
neurohumors in regulation of excitationcontraction coupling.
Bivalvia
Early intracellular recordings from molluscan hearts revealed a resting potential
of up to about 60 mV (Table 1). Irisawa et
al. (1967a) showed that [K+]o must be a
major factor in maintaining the resting
potential in the Mytilm heart, since resting
potential varied linearly with the logarithm
of [K+]o from 28 mM to 186 raM, in otherwise normal artificial sea water (ASW), and
from 4.7 mM to 186 mM in ASW lacking
the other cations (Na, Ca, and Mg). However, the change in membrane potential
for a tenfold increase in [K+]<» was 45 mV,
rather than 58 mV, suggesting that other
ions such as Na+, Ca ++ , and Cl~ also contribute to the resting potential. Nevertheless, the resting potential of myocardial
cells of the Japanese oyster was not appreciably different in Na+-free ASW, or in 45
mM Ca++ ASW, from the value in normal
ASW (Irisawa <tf al., 1968).
Wilkens (1972a) has used the sucrose
gap technique to investigate the role of
chloride and sodium conductances in
maintaining the low resting potential
characteristic of the isolated ventricle of
the ribbed mussel Modiolus demusus, and
the possible role of a rhythmically Huc-
TABLE 1. Resting potentials of molluscan ventricular cardiac muscle.
Species
Crassostrea gigas
Mytilus edulis
Modiolus demissus
Helix pomatia
Method
Bathingfiuid
Value
Range
Reference
microelectrode
microelectrode
microelectrode
microelectrode
sucrose gap
microelectrode
-57mV
-45.5±3.3mV -40.3 to -58.0
—
-43.5±1.3mV
-55.0±8.2mV
-53.8±7.2mV
snail Ringer -50.6±3.4mV
-
Irisawa era/., 1961a.
Irisawa etal., 1968.
Irisawa et al., 1967a.
Wilkens, 1972a.
Wilkens, 1972a.
Kiss and S.-R6zsa,
microelectrode
microelectrode
microelectrode
microelectrode
sucrose gap
hemolymph -65.6±7.0mV
ASW
70mV
hemolymph -45±4mV
- 3 0 to - 7 5
-30 to-75
hemolymph -47±5mV
ASW
-52.5±9.4mV -22 to -74
Nomura, 1963
Nomura, 1965
Kuwasawa, 1967
Kuwasawa, 1967
This paper
ASW
ASW
ASW
ASW
1iy/jO7^
Dolabella auricularia
Aplysia kurodai
Aplysia calif ornica
INOTROPISM OF APLYSIID VENTRICLES
tuating electrogenic sodium pump in
pacemaker activity. The membrane potential of the heart of Modiolus depends
primarily on the distribution of potassium
ion across the membrane, as in Mytilus, but
replacement of chloride with sulphate or
propionate ions shifts the K+ depolarization curve, indicating a contribution of
chloride ions to the resting membrane potential. In chloride-free ASW, the Modiolus
ventricle goes into a sustained but reversible depolarization of 9 to 22 mV. Wilkens
(1972a) concluded that chloride distribution contributes 15-30% of the resting
membrane potential.
In sodium-free ASW, the ventricle of
Modiolus demissus is hyperpolarized to a
degree directly dependent on initial membrane potential, ranging from about 1 mV
at - 6 5 mV to about 12 mV at - 4 5 mV. In
potassium-free ASW, the ventricle of Modiolus demissus depolarizes and an afterhyperpolarization occurs on return to
normal [K+]o. Possibly the electrogenic
sodium pump fails when extracellular
potassium is not available for exchange
with extruded sodium. Depolarization
would follow the entry of sodium in zero
[K~]°, and transient after-hyperpolarization would follow from stimulation of the
pump by the high [Na+], upon return to
NASW (Wilkens, 1972a). This hypothesis
is supported by depolarization of the Modiolus ventricle in ouabain, cyanide, and
reduced temperatures (Wilkens, 1972a).
147
about -72 mV, and then from 5 mM to
200 mM, increased [K+]o depolarizes, with
a linear relation between the membrane
potential and the logarithm of [K+]° from
20 mM to 200 mM. The threshold for
contracture-force is at 25 mM[K+]o, which
corresponds to a membrane potential of
about —50 mV. Potassium contractureforce is lost in zero [Ca++]o, but caffeine
contractures can still be induced.
Aplysia califomica Cooper and Aplysia
dactylomela Rang. We have used a single
sucrose gap apparatus, similar to that described by Berger and Barr (1969), to record gap potential difference between a
region of the isolated ventricle perfused
with isotonic KC1 and a region perfused
with normal artificial sea water. This
method for measuring membrane potential difference with extracellular electrodes
has now been used extensively with molluscan cardiac tissue (Wilkens, 1972a,6;
Wilkens and Greenberg, 1973; Irisawa et
al., 1973; Hill, I974a,b). We find that a
stable resting potential is frequently maintained for many hours with this method,
but there is sometimes a slow steady decline in resting potential. Taking the maximum initial resting potential for each preparation from Aplysia califomica we find an
average value of 52.5 mV (± 9.4 mV S.D.),
with a range of 22 to 74 mV for 38 animals.
These values are well in the range of resting potential values obtained with microelectrode techniques from other molluscan
hearts. With ventricles from Aplysia dactylomela mounted across the single sucrose
Gastropoda
gap, we find that in most experiments depolarization is maintained as long as one
Helix pomatia L. The resting potential in end of the ventricle is superfused with 0.5
ventricular muscle cells is mainly depen- M KCI but there is frequently a rather
dent on [K+]=, but is diminished by the Na steep decline in degree of depolarization
diffusion potential, as shown by a 20% during the first two hours. Subsequently,
hyperpolarization in zero [Na+]=. A 10x there often is a steady level of depolarizaincrease of [Ca++]» hyperpolarizes by 5 tion for a number of hours but sometimes
mV (Kiss and S.-R6zsa, 1973).
there is a further steady decline. ExperiDolahella auriculuria Solander. Increased ments were abandoned if the second level
+
[K ]o produces a reversible long-lasting of depolarization was not quite steady in
depolarization, giving rise to transient value.
contracting force (Nomura, 1965). In zero
LK+]o the membrane depolarized by more
Isolated ventricles of Aplysia dactylomela
than 10 mV, as would be expected if there are depolarized by an increase in [K+]»over
were an electrogenic sodium pump. At 5 the level (9mM) in NASW, with a threshold
mM[K r |. the membrane hyperpolarizes to for force between 25 and 50 mM KCI, and
148
ROBERT B. H I L L AND ROBERT E. YANTORNO
.20-1
CONTROL QAP POJtMTlAl.
CXPCIttUCNTAL SAP POTENTIAL
FO*CE
I '•
Z9
SO
mM
• M KCI
FIG. 1. KCI contracture of the ventricle of Aplysia
dactylomela. An experiment on the relation of
contracture-force to depolarization by superfusion
with KCI. One end of the ventricle was depolarized
with isotonic KCI throughout, providing the relatively
steady control potential difference (PD) shown, while
the other end was superfused sequentially with increasing KCI concentrations. Between runs, the experimental chamber was washed with ASW until PD
returned to the control value.
a maximum force at 250 raM KCI (Fig. 1)
in six trials on six ventricles. Similarly, the
isolated ventricle of Aplysia califomica is
depolarized by increased [K+]°, with force
generated by the few mV depolarization at
double the concentration of ASW (Fig. 2).
We wished to determine whether the potassium contracture is dependent on Ca++
for excitation-contraction (E-C) coupling
(Podolsky, 1965) and therefore perfused
with zero [Ca ++ ]. for increasing periods of
time until contracture force was finally lost
after 34 minutes of successive trials of depolarization by 100 mM KCI. Once force
has been abolished in zero [Ca++]°, it can
be restored by perfusion with medium
containing calcium (Fig. 3).
When the isolated ventricle of Aplysia
califomica is set up in the sucrose gap apparatus, beating becomes labile. Strong
beating may go on for hours, but alternation between beating and quiescent states
occurs easily. This is because the ventricle
is set up as a stretched collapsed tube,
lacking alike the stimuli to beating provided by internal distension (Straub, 1901,
1904), aeration (Hill, 1964), and factors in
the hemolymph (Nomura, 1963; Kuvvasawa, 1967). Thus, many of our observations in the quiescent state of the ventricle
are complicated by the induction of beating. Several authors have suggested that an
I
75
18
100
1
100
I
150
200
K Cl
FIG. 2. KCI contracture of the ventricle of Aplysia
califomica.
electrogenic sodium pump contributes to
the resting potential of molluscan myocardial cells (reviewed above). When the ventricle of Aplysia califomica is perfused with
zero K+ ASW, it is depolarized by several
millivolts (Fig. 4). The depolarization is
generally accompanied by contracture
force, and is frequently accompanied by
the induction of several beats or oscillations on the initial depolarization, as in Figure 4. However, if the exposure to zero
K+ ASW is repeated in several trials, the
induced excitability fades away, trial by
trial, although the induction of contracture
FIG. 3. Dependence of contracture-force on bath
calcium concentration in a ventricle of Aplysia californica which had lost contractility in zero calcium and
developed no force in lO^M CaCI2. Increasing degrees of contracture force develop in 10~3M, 10"2M,
and 10"' CaCl2. Insets: A. lCT'M CaCl2. B. 10"2M
CaCl2. C. 10~'M CaCl2. a. monitor of resting potential
at low gain. b. resting potential at high gain. c. force.
Calibration signals in insets: once per minute and at
change of solutions.
INOTROPISM OF APLYSIID VENTRICLES
149
across the membrane seems to affect ionic
fluxes. For instance, the Na-dependent
calcium efflux is sensitive to the Na gradient across the membrane in the single
muscle fiber preparation from Balanus
nubilis (Ashley et al., 1976), in guinea-pig
auricle (Jundt et al., 1975) and in bovine
papillary muscle (Reuter and Scholz,
1977). Thus, either a drop in [Na+]= or an
FIG. 4. Perfusion of a ventricle of Aplysia californica increase in [Na+]j may lead to contracture
with K-free ASW, between arrows. Force accom- force due to increased [Ca++]j. If the elecpanies depolarization. Repolarization follows immediately after perfusion of the bath with NASW trogenic pumping contributes to the mag(second arrow) and then merges with increasing nitude of the resting potential, stopping of
after-hyperpolarization as spontaneous cardiac action
the pump may be expected to produce the
potentials appear. The resting potential is indicated at degree of depolarization that we see. The
the left. Time signals in this and all subsequent polysodium-potassium exchange pump congraph recordings: 1 sec and 5 sec.
tributes about 10 mV to the resting potential of the Anisodoris giant neurone (Gorforce persists. When normal artificial sea man and Marmor, 1974) which is about the
water (NASW) is readmitted to the muscle amount of depolarization in Figure 5.
compartment, the muscle repolarizes, and Thus, there may be an electrogenic pump
then continues to a hyperpolarization, the component in the resting potential of the
course of which is often hard to distinguish Aplysia ventricle as in some vertebrate
from the increasing diastolic after-hyper- smooth muscles (Casteels et al., 1973) and
cardiac cells (Noma and Irisawa, 1975).
polarizations of induced beating (Fig. 4).
The depolarization that occurs in zero
Smooth muscle is commonly depolarized
[K+]o is reversibly blocked in zero [Na+]° in K-free solution, hyperpolarizes on rewhen the Na+ ot the ASW is replaced by turn to normal-K solution, and that hyLi+ (Fig. 5). This suggests that the de- perpolarization may be suppressed in Napolarization in zero [K+]« results from a free solution (Kuriyama et al., 1975). These
stopping of the sodium-potassium pump, effects have been attributed to modificaand the consequent increase in the in- tion of Na conductance as well as to modtracellular level of Na + (Hodgkin and ification of the electrogenic sodium pump,
Keynes, 1955; Cookeetal., 1974). Now, the but our results with Li support the likeliratio of sodium concentrations that exists hood that we are dealing with an electrogenic sodium pump.
Hyperpolarization on readmission of
sodium after perfusing with potassiumfree solution (Fig. 4) is typical of a preparation with an electrogenic sodiumpotassium pump (den Hertog, 1973). This
potassium-activated after-hyperpolarization indicates extrusion of the excess sodium which enters during the time in potassium-free solution.
During perfusion with potassium-free
56 mV
Free Li A S W
and sodium-free (Li) ASW there may be
influx of calcium and efflux of sodium
FIG. 5. Perfusion of a ventricle of Aplysia californica (Ashley^al., 1976). Possibly, the transient
with K-free ASW (upper trace) followed by perfusion depolarization of the Aplysia ventricle'that
with K-free medium in which sodium has been re- occurs when NASW is readmitted (Fig. 5)
placed by lithium (lower trace). Only potential differ- after perfusion with zero K+ and zero Na +
ence is displayed and the resting potential is indicated
at the left of each record. The ventricle is not de- ASW, may be due to influx of sodium
preceding the reestablishment of the
polarized in Na-free medium.
150
ROBERT B. HILL AND ROBERT E. YANTORNO
sodium-potassium exchange pump.
A so
II I mV
Similar experiments with zero [K+]- were
performed on six Aplysia californica ventriPD
cles. The overall results were that depo+
larization in zero [K ]= was 8.3 mV ±2.14
low Cl
t | Gm
mV (S.D.), n = 43; transient after-hyperpolarization on return to NASW was 1.8
mV ± 1.7 mV (S.D.), n=40; steady hyperpolarization in zero LK+]° and zero [Na+1»
(Li ASW) was '2.03 mV ± 1.5 mV (S.D.),
n=14; and the transient depolarization
II I mV
after Li ASW was 6.3 mV ± 2.4 mV (S.D.),
+
n= 11. Thus, depolarization in zero [K ]° is
quite large and is significant. Over all trials
on all hearts, transient after-hyperpolarGm
ization does not seem significant, but it was
quite marked in certain individual trials. In
eleven trials on one heart, transient afterhyperpolarization was 2.9 mV ± 0.86 (S.D.).
Depolarization by [K.+]° is certainly blocked FIG. 6. Perfusion of a ventricle of Aplysia californica
with low chloride solutions. A. NaCl replaced by
in Li ASW.
sodium sulfate. Calibration: lOmV, lg. B. NaCl reIt has been suggested that an elec- placed by sodium citrate. Calibration: 10mV, lg.
trogenic sodium pump in pacemaker tissues may operate to compensate for a high molluscan hearts which are put into a
sodium conductance, and thus keep the sustained depolarization in zero [Cl~]o (remembrane potential in a physiological viewed above). We wished to investigate
range (Noma and Irisawa, 1974, 1975). chloride dependence of the resting potenEvan a small strip of muscle from the ven- tial of the ventricle of Aplysia californica by
tricle of Aplysia californica has pacemaker means of anion substitution, although we
properties and may beat spontaneously in recognized that anion binding with Ca++ is
the sucrose gap, so we may well be able to a problem in such experiments (Christofobserve properties related to pacemaker fersen and Skibsted, 1975). Perfusion with
functions in our whole ventricle prepara- low chloride solution depolarizes the ventions. We often observed that our labile tricle of Aplysia californica, whether the
preparations would begin beating at the anion substituted for chloride is nitrate,
point where membrane potential returned propionate, sulphate, or citrate (Fig. 6);
to normal after a depolarization in zero and the membrane is also depolarized
[K+]o. Possibly the usual resting potential of when the NaCl of the ASW is replaced with
Aplysia californica heart is the optimum sucrose. Since the anions used have markvalue for pacemaker activity. Aplysia neur- edly different degrees of calcium binding,
ons, which become depolarized in hypoxia, it seems probable that the depolarization
will then hyperpolarize by 5—10 mV with results from a loss of the chloride conoxygenation, which activates the electro- tribution to the resting potential. Another
genie sodium pump (Steffin, 1975). Thus, possibility would be that chloride replaceif there is an electrogenic sodium pump in ment reduces potassium conductance
molluscan cardiac muscle, preparations (Kuriyama et al., 1975), but the effect does
mounted in a sucrose gap apparatus may not seem to depend on the permeability of
be somewhat depolarized by hypoxia, and the anion used.
tend to fail to beat. Quiescent ventricles of
When a spontaneously beating ventricle
Aplysia dactylomela will beat when the bath is of Aplysia californica is depolarized in zero
aerated (Hill, 1964).
[K+]=, beating may stop and depolarization
is
seen to coincide with a diminution in
Chloride ion probably contributes a
significant part of the resting potential of amplitude of action potentials. Either a
INOTROPISM OF APLYSIID VENTRICLES
151
beating is dependent from beat to beat on
influx of extracellular calcium, but force in
KC1 contracture may draw on intracellular
stores of calcium for E-C coupling.
EFFECTS OF NEUROHUMORS ON RESTING
POTENTIAL
FIG. 7. Isolated ventricle of Aplysia calif ornica. At the
arrow perfusion is switched from NASW to zero-Ca
ASW. Force declines gradually until beating abruptly Bivalvia
ceases after 8 minutes, in zero-Ca.
Acetylcholine has been known for a decspontaneously beating ventricle or a ven- ade to have both depolarizing and hypertricle sustained by perfusion with 10~8M polarizing effects on molluscan cardiac
5HT depolarizes slightly and stops beating muscle (Table 2). For instance, acetylreversibly in zero [Na+]° or in both zero choline (ACh) depolarizes myocardial cells
by an increased per[Na+]° and zero [Ca++]«. From those results, of Mytilus, possibly
+
it would be difficult to say whether the ces- meability to Na , but hyperpolarizes myosation of beating is due to the slight de- cardial cells of the oyster heart, possibly by
polarization or to the ionic environment, an increase in permeability to Cl~ (Irisawa
but ventricles oiDolabella (Hill, 19766) and etal, 19676).
Aplysia calif ornica have been observed many In the depolarizing effect (D-response)
times to accommodate and resume beating of acetylcholine on the heart of Mytilus
even at high levels of depolarization. Ven- edulis (Shigeto, 1970) depolarization of
tricles never resume beating while still per- more than 10 mV frequently blocks spikes,
fused with zero [Na+]°.
but the D-response can also initiate spiking
Spontaneous beating (Fig. 7) eventually in a quiescent heart. The D-response +is not
ceases in zero [Ca++]» but the force of con- sensitive to the presence of Cl~ or K , but
tractions declines before beating ceases,
and in some cases mechanical activity is
entirely lost before action potentials cease
to appear rhythmically. Since action potentials stop reversibly in zero [Ca++]« as
-25%well as in zero [Na+]., they probably depend
on both sodium current and on calcium uID
current, in the ventricle of Aplysia californica o - 5 0 % as in other molluscan hearts reviewed UJ
u
above.
o
The isolated superfused ventricle of
++
Helix aspersa is dependent on [Ca ]° for z -75%, (I min.)
force in isometric contractures induced by O
AC stimulation (Burton and MacKay,
-100% - 1
1970) and, similarly, the isolated perfused
ventricle of Busycon canaliculatum is dependent on [Ca++]» for force in maximally
10
driven isometric contractions (Kuwasawa
niM Co
and Hill, 1973). The isolated ventricle of
Aplysia californica will continue beating in FIG. 8. Relationship between force of beating and
low [Ca++]o but the force of beating is re- bathing concentration of calcium, for an isolated
lated to [Ca++]o (Fig. 8). This may be con- ventricle of Aplysia californica. All values represent an
trasted to the need to pieincubate in zero accommodated steady level, reached after a few sec[Ca++]° before a calcium dependence of onds in the given calcium concentration. However,
one minute in zero-calcium, force fell to zero for
potassium contracture could be demon- after
this ventricle although action potentials continued to
strated. Apparently, force in spontaneous appear rhythmically.
tl.
152
ROBERT B. HILL AND ROBERT E. YANTORNO
when Na+ is replaced with Tris (which at- duce a transient hyperpolarization in
tenuates ACh responses in Aplysia neur- ASW (Table 2). In chloride-free sea water
ones; Wilson et al., 1977), the D-response is the effect of 10~7 M 5HT is sometimes
abolished and ACh has only an inhibitory reversed from inhibition with slight hyeffect on spikes, accompanied by relaxa- perpolarization, to excitation with slight
tion. Li+ can substitute for Na+ in the D- depolarization but the usual effect is to increase the threshold concentration at which
response.
During the hyperpolarizing effect (H- 5HT causes inhibition. From these results,
response) of acetylcholine on the heart of Wilkens and Greenberg (1973) concluded
Crassostrea gigas (Shigeto, 1970) there is a that the ACh depolarization probably rereduction of frequency and increase in sults from an increase in sodium conductamplitude of spikes with slight hyper- ance, but that the mechanism of the 5HT
polarization, and then blocking of spikes at inhibition is more complex, possibly inprofound hyperpolarization. The H- volving both an increase in chloride conresponse is abolished in Cl~-free solution, ductance and a suppression of pacemaker
when sulfate or glutamate are substituted potentials.
for Cl~, but not when nitrate or bromide
Although the ventricle of Modiolus deare substituted for Cl". The H-response missus granosissimus is depolarized by ACh
increases in K+-free solution, but it is not (Wilkens and Greenberg, 1973), the ventrisensitive to the presence or absence of Na+. cle of another subspecies, Modiolus demissus
Resting potentials, ionic concentrations, demissus, is inhibited and slightly hyperand equilibrium potentials are very similar polarized by acetylcholine (Irisawa et al.,
for Mytilns and Crassostrea (Table 3). Appli- 1973). These authors compared the effects
cation of current indicated a reversal po- of ACh and 5HT on membrane potential
tential for the ACh response of about —66 and conductance in Modiolus demissus demV in Crassostrea.
missus (hereafter termed M. demissus) and
4
Wilkens and Greenberg (1973) used a Mytilus edulis (Table 2). ACh (10" M) desucrose gap technique to examine the ef- polarizes :lthe Mytilus ventricle, whereas
the
fects of ACh and 5HT on the isolated ven- ACh (10~ M) slightly hyperpolarizes
B
demissus
ventricle.
5HT
(10~
M)
slightly
M.
tricle of Modiolus demissus granosissimus. Perfusion with 10~4 M ACh depolarizes in depolarizes the Mytilus ventricle, but slightly
normal ASW but not in sodium-free ASW. hyperpolarizes the M. demissus ventricle.
Perfusion with low concentrations of 5HT The difference in the effects of 5HT on
has a negatively chronotropic effect on membrane resistance in Mytilus and M. debeating and higher concentrations pro- missus is of interest, because 5HT excites
TABLE 2 . Effects of neurohumors on resting potentials of molluscan ventricular cardiacmuscle.
Species
Mytilus edulis
Crassostrea gigas
Modiolus demissus
granosissimus
Modiolus demissus
demissus
Modiolus demissus
granosissimus
Modiolus demissus
demissus
Mytilus edulis
[ACh]
D-response
H-response
10- 4 to 10- 6 g/ml
10~4M
2-13mV
14.3mV
—
6
1 0 - t o 10g/ m l
lO"4 M
—
8-9mV
lO-'M
[5HT]
10"6M
_
10"6M
10-6M
Relative
resistance
Incr.2-3x
l-10mV
—
Incr. 18x
Incr.2-7x
Reference
Shigeto, 1970
Wilkens and
Greenberg, 1973
Shigeto, 1970
slight
Decr.8-36x Wilkens and
Greenberg, 1973
5-6mV
Wilkens and
Greenberg, 1973
Decr.2x Wilkens and
Greenberg, 1973
little change Wilkens and
Greenberg, 1973
slight
slight
Relative
conductance
—
INOTROPISM OF APLYSIID VENTRICLES
153
TABLE 3. Ionic concentrations and equilibrium potentials ofmolluscan ventricular cardiac muscle.
Species
E K+
Range
RP
Reference
Mytilus edulis
98.1mM +39.1mV 171.7mM -73.2mV -43.5±0.2mV - 3 9 t o - 5 0 Shigeto, 1970
Crassostrea gigas 83.1mM +42.2mV 140.9mM -68.2mv -45.5±1.6mv -40.3 t o - 5 8 Shigeto, 1970
the Mytilus ventricle but inhibits the M. demissus ventricle. The reversal potential for
5HT hyperpolarization of the M. demissus
ventricle is at about 15 mV higher than the
resting potential (and thus perhaps about
- 7 0 mV). The reversal potential for ACh
hyperpolarization of the M. demissus ventricle is about 3 mV higher than the resting
potential (and thus perhaps about —58
mV). Irisawa et al. (1973) investigated the
ionic dependencies of these hyperpolarizations. The 5HT effect persisted in both
zero [Cr]= and zero [Na+]., but the decrease in membrane resistance was abolished. The ACh hyperpolarization was
transformed to a 15 mV depolarization at
10~4 M in zero [Cl+]°, while the decrease in
membrane resistance caused by ACh was
reduced. The ACh effect persisted in zero
[Na+]o, but the decrease in membrane resistance was abolished. Irisawa et al. (1973)
concluded that ACh acts on the M. demissus
heart by a remarkable increase in Cl~ conductance, while 5HT acts with only a small
effect on membrane conductance.
Gastropoda
Helix pomatia. Kiss and S.-R6zsa (1975)
used a microelectrode technique to record
a biphasic action of 5-hydroxytryptamine
on the resting potential of ventricular
muscle cells of Helix pomatia L. Low concentrations depolarize by a few millivolts
while high concentrations hyperpolarize
by a few millivolts from the resting potential of about - 4 2 mV. At an intermediate
concentration (10~(l M) 5HT has no effect
on resting potential. Low concentrations
(10~9 to 10"K M) decrease the conductivity
of the membrane, but high concentrations
increase conductivity. Hyperpolarization
by 5HT is apparently due to an increase in
Cl~ conductivity, since it is abolished in
chloride-free solution.
Dolabella auricularia Acetylcholine (2 x
10"" M to 2 x 10"' M) depolarizes the
isolated ventricle of Dolabella auricularia in
a preparation in which the entire ventricle
is mounted in a sucrose gap apparatus
(Hill, 1974a). The depolarization induces
contracture force at concentrations greater
than 2 X 10~* M. Contracture in high
concentrations of acetylcholine has been
commonly observed in experiments using
mechanical recording from gastropod
hearts, e.g., Strombus gigas (Hill, 1967), and
it may be supposed that this indicated depolarization. Concentrations of 5-hydroxytryptamine from 10~8 to 10~5 M all
induce a slight depolarization of quiescent
Dolabella ventricles, followed by the onset
of beating (Hill, 19746).
Aplysia dactylomela. Sucrose gap record-
ing from the isolated ventricle of Aplysia
dactylomela revealed depolarization at 8 X
10~(! M ACh, with maximum contracture
force at 8 x 10~5 M (Fig. 9). The relationship between depolarization and contracture force is roughly similar for KG depolarization and ACh depolarization (Fig.
10) with maximum force at about 30 mV
depolarization. Concentrations of 5HT
-O20-J--OV -
CONTROL OAP POTENTIAL
CXPCRIMENTAL OAP POTENTIAL
FORCE
—O—
10-S
M ACH
FIG. 9. ACh contracture of the ventricle of Aptyua
dactylomela. ACh concentrations were increased sequentially, from 10"6M to 10~4M. After each exposure to ACh, the preparation was washed until resting
gap potential difference returned to normal. Similar
results were obtained in 4 trials on 4 ventricles, over a
range of ACh concentrations from 10~7M to 10~4M.
154
ROBERT B. HILL AND ROBERT E. YANTORNO
higher concentrations of ACh (Hill,
19746), we could observe only a slight
transitory hyperpolarization during the
inhibition of a beating ventricle of Aplysia
califomica by 10~7 M ACh (Fig. 12A). However, a quiescent ventricle with a low resting potential (35 mV) was repeatedly and
reversibly hyperpolarized by 10"7 M ACh
(Fig. 12B). The hyperpolarization was
accompanied by a relaxation, but washout
FIG. 10. Contracture force in the ventricle of Aplysia of ACh was followed in about 6 minutes by
dactylomela. A. Relationship between depolarization by a return to resting potential and resting
acetylcholine and contracture force, in 39 runs on
tonus.
three preparations. B. Relationship between deOur observations suggest that the A.
polarization by potassium and contracture force, in
52 runs on five preparations. All data were converted
dactylomela ventricle possesses a D-response
to percentage of the maximum force or depolarizato ACh, while the A. californica ventricle
tion obtained with a given preparation. A negative %
can show an H-response. This H-response
depolarization indicates hyperpolarization and a
strongly resembles the ACh-induced hynegative % force indicates relaxation. Lines indicate
standard error of the estimate.
perpolarization and relaxation of a depolarization-contracture, which was reported by Van der Kloot (1961) for amabove 10 9 M also depolarize the ventricle phibian slow muscle fibers.
of Aplysia dactylomela, to a maximum of 30
mV at 10~4 M (Fig. 11). However, depolariACTION POTENTIAL AND FORCE OF BEAT
zation by 5HT does not induce contracture, although beating is induced at the Bivalvia
higher concentrations.
Intracellularly recorded action potenAplysia californica. We have looked for an
effect of acetylcholine on the ventricle of tials from molluscan hearts may have a
Aplysia califomica similar to that observed typical "cardiac" form, in that a slowly
for the ventricle of Aplysia dactylomela, rising prepotential appears to trigger a spike,
using a single sucrose gap with rubber which gives way to a prolonged plateau
membranes between compartments. Calo- during repolarization. Irisawaila/. (1961«)
mel reference electrodes were used to
measure electrode potential difference and
a force-displacement transducer to record
mechanical contractions. In repeated trials
on 9 preparations, no concentration of
ACh, from 10"9 to 10"2 M, depolarized the
ventricle or induced contracture-force.
Evidently, the ventricle of Aplysia (Varria) I
dactylomela Rang, 1828 possesses a depolarizing response to acetylcholine (Dresponse) that may be lacking in the ventricle of Aplysia (Neaplysia) califomica Cooper, J863. We repeatedly tried to observe a
membrane depolarization or hyperpolarization component, in the well-known ACh
inhibition of beating Aplysia ventricles (reviewed in Hill, 1964, 1967), in A. califor- FIG. 11. Depolarization of the ventricle of Aplysia
nica. Although there is a membrane de- dactylomela by 5-hydroxytryptamine. Abscissa: Molar
polarization which accompanies inhibition concentration of 5HT. Ordinate: Depolarization in
mV. Symbols indicate trials on three isolated ventriof the ventricle of Dolabella auricularia by cles.
Resting potentials: 61-67 mV.
JO
50
70
% Orpoisnetsn
INOTROPISM OF APLYSIID VENTRICLES
PD
10 mV
JACh|
iGm
PD
10 mV
IGm
JACh
FIG. 12. A. Inhibition by ACh (10"7M) of a beating
ventricle of Aplysia californica. B. Hyperpolarization
by ACh (10"7M) in a quiescent, partly depolarized
and contracted ventricle of Aplysia californica. Hyperpolarization is accompanied by relaxation.
recorded action potentials of this typical
pattern from myocardial cells olCrassostrea
gigas. Spike height ranged from 62 to 23
mV (mean 53.3 ± 10 mV). Since the resting potential was around 57 mV, these
action potentials were sometimes overshooting. C. gigas myocardial cells sometimes beat with another type of action
potential, showing the same time and
amplitude relations for prepotential and
spike but lacking the plateau phase. It thus
appeared that the delayed repolarization
was labile.
When both force and action potential
(suction electrode) were recorded from the
heart of C. gigas, a relation between duration of action potential and force of contraction became apparent (Irisawa et ui,
1961/;). When a current pulse, applied to
the surface of the ventricle, terminated the
action potential at the beginning of the
plateau phase, force was markedly re-
155
duced, but a similar termination late in the
plateau phase did not affect force in the
ensuing beat. Thus, the action potential of
the oyster myocardium may be differentiated into spike and plateau components, and the plateau component may
offer a site for modulation of cardiac force.
Irisawa and Kobayashi (1964) enquired
into the ionic mechanisms of the spike and
plateau phases of the oyster heart, using
both Hexibly mounted microelectrodes and
suction electrodes for recording action
potentials. They found that spontaneous
action potentials depended on [Na+]°, but
that Ba++ or Sr ++ could maintain spontaneous activity in Na+-free sucrose solution, suggesting that although Na+ plays
the major role in the initiation of the action
potential, the ionic mechanism cannot be
very specific. The relation between the
duration of the action potential and force
of contraction was reinforced by observations on oyster hearts beating spontaneously in a medium consisting of 48 mM
BaCl2 in isotonic sucrose solution. The
duration of the action potential steadily
increased in the BaCI2 solution and this
was accompanied by a steady increase in
the duration of beats, superimposed on a
slowly developing contracture.
In contrast to the oyster heart, the heart
oi'Mytilu.s edulis will beat for more than 3 ruin Na+-free solution (Irisawa et ai, 1967a)
but after about 20 minutes in zero calcium
perfusate, spontaneous beating of the isolated Mytilus heart suddenly stops, suggesting that the heart of Mytilus depends
on calcium for spontaneity, even as the
oyster heart depends on sodium. The intracellularly recorded action potential of
the heart of Mytilus has an amplitude of
23.3 ± 3.7 mV (S.D.), range 16.5 to 29.0 mV,
and is thus not overshooting, in NASW.
However, a 10x increase in LCa++]o to 90
mM leads to an increase in the amplitude
of the action potentials, in Na+-free solution, to 43.4 m V ± 2.8 (S.D.). Compared to
the action potential in NASW, this leads to
a spike of remarkably increased amplitude
and shortened duration, with a slight overshoot, indicating that calcium can participate in the initial depolarization phase of
the spike. Permeability to sodium in the
156
ROBERT B. HILL AND ROBERT E. YANTORNO
resting state is thought to be very high in
the Mytilus heart, contributing to the low
resting potential. Since the action potential
can function in the extremely small
amount of Na+ still present in Na+-free
solution, and increases so markedly in high
Ca ++ , it is very probable that the spike of
the Mytilus cardiac action potential is at
least partially a calcium spike. In Crassostrea
gigas (Irisawa et al., 1968) the rhythm of
spike potentials, in sodium-free solution, is
blocked by 10 mM manganese, while the
amplitude and rate of rise of the spikes
increases with [Ca ++ ]. from 0.25 mM to 10
mM, suggesting a calcium spike in Ostrea as
well as in Mytilus. The roles of Na+ and
Ca ++ in the action potentials of bivalve
hearts have been summarized by Irisawa^
al., (1969), who point out that the sensitivity of the action potential to [Na+]« depends
on the resting potential level. Thus, when
the membrane is hyperpolarized, the action potentials vanish in zero [Na+J° and
increase in amplitude in high LNa+]°; but
when the membrane is at a control level,
the action potentials are insensitive to
[Na+]o. They conclude that bivalve cardiac
muscle depends on an intricate interaction
between both Na+ and Ca ++ and the membrane, resulting in the generation of the
spike phase of the action potential.
sults from a transient increase in sodium
current which is regulated by calcium" and
thus increases in calcium-free solution.
The persistent spike has a reduced rate of
rise in zero [Na+]° which indicates that,
when present, sodium current, as well as
calcium current, contributes to the spike.
Gastropoda
Helix pomatia. The action potentials of
ventricular cells of Helix pomatia are abolished in zero [Na+]° (Kiss and S.-R6zsa,
1973), when Tris is substituted for Na+, or
in zero [Ca ++ ]. when sucrose or Na+ are
substituted for Ca ++ . Li+ supports action
potentials when substituted for Na+. Thus,
both Na+ and Ca ++ probably participate in
the generation of the Helix action potential.
Dolabella auricularia. Dolabella provides a
preparation in which spontaneous myocardial action potentials are always preceded by a prepotential (slow diastolic depolarization), usually followed by an action
potential with both spike and plateau (Nomura, 1963). During a single action potential, membrane potential may cycle from
— 56 mV to —7 mV (Kuwasawa and Matsui,
1970) and thus action potentials do not
overshoot. Nomura noticed that the duraIn zero |_Na+]°, the plateau is lost from tion of action potentials was shorter than
the cardiac action potential of Modiolus normal in mechanically inactive preparadem.Ls.sus, leaving a simple spike (Wilkens, tions, and that there was a linear relation
1972a). This change does not seem to between the magnitude of the resting poaffect force of heartbeat. In zero [Ca+ + Jo, tential and the amplitude of the action
the spike of the Modiolus cardiac action potential. This suggested that the long espotential becomes progressively reduced tablished relationship between stretch and
until it is lost, after about 90 minutes, but force in aplysiid ventricles (Straub, 1901,
the amplitude of the plateau increases 1904; Matsui, 1945, 1961) might result
gradually until it exceeds that of the spike. from an effect of stretch on resting potenFinally, the plateau wave-form persists tial level and on the form of the action poafter the spike is lost, but before that tential. That is, resting potential may conhappens, the spike and plateau wave- trol the rate of repolarization and thereby
forms become disassociated so that a spike control the duration of the action potenor burst of spikes may precede a "plateau." tial. Nomura (1963) investigated the effect
Thus in sodium-free sea water, spikes and of stretch and found that while even a
beating persist after the plateau is lost, but large stretch has very little effect on resting
in calcium-free sea water plateau-forms potential, or on the amplitude of action
persist after spikes and mechanical activity potentials, even a small stretch increases
are lost. Wilkens (1972/>) therefore, con- the rate of depolarization during the presiders that the spike of the action potential potential and prolongs the plateau of the
is calcium-dependent, but the plateau "re- action potential. A rathei slight increase in
INOTROPISM OF APLYSIID VENTRICLES
157
plateau duration may be accompanied by a repolarization. Similarly, Wilkens and
large increase in contractile force, and Greenberg (1973) found that perfusion
there is a linear relation between increase with ACh of the isolated ventricle of Moin passive resting tension, increase in diolus demissus granosissimus led to action poplateau duration, and increase in active tentials lacking the plateau phase and acmechanical force. Nomura concluded that companied by contractions of decreased
plateau duration is the factor which is force.
closely related to mechanical force of contractions.
Gastropoda
Gastropod cardiac muscle resembles
frog myocardium in the relatively small
Helix pomatia. The characteristic effect of
size of the cells, the lack of a transverse (t) 5HT on the ventricle of Helix pomatia L.
tubular system, and the sparseness of or- (Kiss and S.-R6zsa, 1972) is to prolong the
ganized sarcoplasmic reticulum (Kuwa- repolarizing phase (plateau) of the action
sawa, this symposium; Sanger, this sym- potential, even at the threshold concentraposium) as well as in dependence on ex- tion of 10~10 M. This characteristic plateau
ternal sources of calcium for E-C coupling formation is blocked in zero [Na+]°, even
(Kuwasawa and Hill, 1973). Membrane though 5HT will restart action potentials
control of force has been reviewed for frog in Helix ventricles that have stopped beatand mammalian ventricles by Morad and ing in zero [Na+]° (Kiss and S.-R6zsa,
Goldman (1973). Voltage clamp tech- 1975). Thus in Helix as in Modiolus, Na+
niques which permit the action potential to current may maintain the level of debe treated as a controlled variable, sepa- polarization during the plateau induced by
rated from the effects of other interven- 5HT. In zero rCA + + k 5HT (1(T5 M)
tions, provide evidence that in the frog the evokes local oscillation of membrane poduration of the ventricular AP may well tential, without a sharp spike. This may
serve as a "gate" for the entry of extracel- suggest that, in the Helix heart, Na+ is
lular calcium as activator of E-C coupling. sufficient for pacemaker activity, but Ca ++
In vertebrate myocardia, increased is required for spike generation.
+
[K ]o leads to a dramatic shortening of the
Dolabella auricularia. Nomura concluded,
duration of the cardiac action potential in 1963, that the duration of the plateau
(reviewed by Noble, 1975). Similarly, 10 phase of the cardiac action potential is
mV depolarization of ventricular fibers of
Dolabella in 50 mM KC1 causes a reduction
in amplitude and duration of action potential, which is correlated with a decrease 1 2 0 in force of contractions (Nomura, 1965). It gjooshould be illuminating to ascertain whether
or not inward-going rectification, attributed to some K+ channels of vertebrate
cardiac muscle, can be demonstrated in
cardiac muscle oi Dolabella.
EFFECTS OF NEUROHUMORS ON ACTION
POTENTIALS
Bivalvia
Irisawa et at. (1961«) found that application of acetylcholine (\0~A ) to a Cmssostren
gigcv, heart led to a loss of the plateau phase
of the action potential. This left a spike of
undiminished amplitude, but with quick
FIG. 13. Modulation of action potential and force of
contraction in the ventricle of Dolabella auricularia by
5-hydroxytryptamine. Plateau height and force have
been plotted against concentration of 5HT, from
10""M to 10"'M. Plateau height has been calculated
from the second hump of the action potential, when
one was discernible, or from depolarization at 0.2 of
the spike-to-spike interval, when a second hump did
not appear. The inset diagrams represent the form of
the action potential at 10"7M, 10-5M, 10":lM 5HT
concentrations.
158
ROBERT B. H I L L AND ROBERT E. YANTORNO
2 Gms
S 125—i
£
z
o
10 —\
«J 7.5 —
u.
o
0 —I
I
I
I
I
I
I
I
I
1
40% 50% 60% 70% 80% 90% 100% 110% 120%
PLATEAU HEIGHT AS % OF SPIKE
FIG. 14. Relation between plateau height and force
of contraction in the ventricle of Dolabella auricularia.
closely related to the force of the accompanying beat. This has been supported by
observations on the effects of 5HT on action potentials recorded across a sucrose
gap from the ventricle of Dolabella (Hill,
1974«). Concentrations of 5HT from 10~7
M to 10"' M prolong the cardiac action
potential and, over the entire range of
concentrations, this effect is correlated
with increased force of beating (Fig. 13).
There is a direct relationship between
plateau height and force of contraction
(Fig. 14), but no relation between spike
height and force of contraction in the
Dolabella ventricle (Hill, 1974a). Increased
force and plateau duration may be maintained by continuous perfusion of the
Dolabella ventricle with 5HT. Under those
conditions, addition of ACh (2 x 10~7 M)
to the perfusion medium accelerates the
rate of repolarization and a reduction in
force accompanies the shortened plateau
duration.
The length of the plateau phase of cardiac action potentials may govern the entry
of calcium into the myoplasm, from the
extracellular compartment, as the major
source of activator in E-C coupling (reviewed by Morad and Goldman, 1973;
Fozzard, 1977). This provides for control
of the development and maintenance of
force by membrane potential. Apparently,
this mode of control is also possible in
the differentiation into spike-and-plateau
phases that can be observed in the action
potentials of some molluscan hearts (reviewed above). We wished to determine
whether or not perfusion with 5HT would
induce a differentiation of the action potential, but in 14 preparations perfused
repeatedly with concentrations of 5HT
from 10~9 M to 10~! M we never observed
differentiation into spike and plateau
phases (Fig. 15). In addition, the Dolabella
wave form is highly labile, depending on
such factors as stretch and 5HT concentration (reviewed above), while that of
Aplysia californica is stable, varying only
very slightly in response to drastic changes
in conditions of stretch or 5HT perfusion.
Aplysia and Dolabella are quite closely
related, since they both belong to the tectibranch gastropod family Aplysiidae, al-
Aplysio
californica
Dolabella
auricularia
FIG. 15. The action potential of the ventricle of
Aplysia californica lacks the differentiation, into spike
and plateau phases, which may often be observed in
the action potential ot the ventricle of Dolabella auricularia. In this figure, the wave form of action
potentials in the ventricle of Aplysia californica, beating
in 10~6M 5HT, may be compared to the wave form of
action potentials from the ventricle of Dolabella auDolabella.
ricularia, beating in 10"'M 5HT. The action potential
10~3M 5HT is shown for Dolabella as an example of
Aply.sia californica. When we began to re- at
the clear differentiation into spike and plateau phases
cord action potentials in the sucrose gap which is observed over a wide range of concentrations
from ventricles of Aplysia calif ornica we ob- of 5HT, (Fig. 13). Calibration: upper, 1 second and
served a rather simple wave form, without 5mV; lower, 10 second and lOmV.
INOTROPISM OF APLYSIID VENTRICLES
159
that is very similar to the induction of
beating by 5HT (Fig. 17A), frequently be52m"v
ginning with a depolarization of about 5
mV, measured in the sucrose gap. Amino5HT
pyridine-inducfd beating is inhibited by
1 Gm
zero [Na+]o (Fig. 17B) and eventually
zero Na
illlll |
stops. The sodium-sensitive induction of
beating by 4AP may be considered further
FIG. 16. Effect of perfusion of the ventricle of evidence implicating sodium in the initiaAplysia californica with KT'M 5HT in NASW (A), and tion of automaticity in molluscan hearts,
in zero-NA ASW (B). Calibration: lOmV, lgm. Rest- although it remains to be seen whether or
ing potential indicated.
not the action of 4AP on the ventricle of
Aplysia californica also affects potassium
though Aplysia is in the subfamily Aplysiinae, conductance and calcium release (Harvey
while Dolabella belongs in the subfamily and Marshall, 1977).
Dolabellinae (Eales, 1960; Marcus, 1965).
In Aplysia californica, 5HT has a posiIt is thus rather surprising to find such a tively inotropic effect and ACh has a negamarked difference in the wave form of tively inotropic effect on the isolated ventheir cardiac action potentials.
tricle, very much as in Dolabella auricularia,
The isolated ventricles of Aplysia dac- but these effects are not mediated by any
tylomela and Aplysia fasciata are excited by obvious modulation of the form of the
5-hydroxytryptamine, with a threshold for action potential. Acetylcholine reduces
positively inotropic and chronotropic ef- force of contraction in ventricles of Aplysia
fects at 10-* M (Hill, 1958, 1964). The californica beating in 5HT solution, but
ventricle of Aplysia californica is similarly without apparent change in the form of
excited and the induced level of cardiac the action potential. Thus, in these aplysiid
activity may be maintained through several
hours of perfusion with solutions of 5hydroxytryptamine in natural or artificial
II mV
sea water. Frequently, we observed an inhibitory after-effect to washout of per- 52 mV
NAP I
fused 5HT. This has been reported for
other molluscan hearts (Kuwasawa and
Hill, 1973). Beating induced by 5HT (lO""
|lGm
M) is abolished in zero [Na+|» (Fig. 16), an
effect consistent with evidence (reviewed
above) that the pacemaker mechanism of
other molluscan hearts is primarily dependent on Na+ current.
Potassium currents are suppressed by
II mV
aminopyridines in the membrane of the M mV
I zero N a
4AP
giant axon of the squid, while sodium currents remain unaffected (Yeh et al., 1976).
The block of K current is relieved at
higher depolarizations (Yeh et al., 1976).
flllGm
The slow potential oscillations which are
induced in the squid giant axon membrane FIG. 17. A. Effect of perfusion of the ventricle of
by 4-aminopyridine, are set up by an in- Aplysia californica with lmM 4-aminopyridine, becrease in permeability to Na + , accom- tween arrows. B. Perfusion with lmM 4-aminopanied by a depolarization of 6 - 8 mV pyridine in ASW switched at the second arrow to perfusion with lmM 4-aminopyridine in zero-Na ASW.
(Golenhofen and Mandrek, 1977). Beating 4AP-induced beating stops in zero-Na, but strong
is induced in the ventricle of Aplysia califor- beating returns upon washout in ASW, as at the third
nica by 4-aminopyridine (4AP) in a way arrow.
A
111 mV
160
ROBERT B. HILL AND ROBERT E. YANTORNO
ventricles, lacking a plateau on the action
potential, there is not an obvious link between the negative inotropic effect of
acetylcholine and the duration of the cardiac action potential. Possibly the site of
action of the inotropic effects of the
neurohumors involves synthesis of cAMP
(Rapp and Berridge, 1977) which Koester
et al. have shown to be sensitive to 5HT
(this symposium).
It is unlikely that 4AP acts on presynaptic cholinergic nerve terminals (Moritoki et
al., 1978) in the ventricle of Aplysia californica, since ACh release would be inhibitory.
A relationship between plateau duration
and force of contraction is evident in a
number of molluscan species with a spikeand-plateau form of cardiac action potential (Irisawa et al., 1961a,Z>; Nomura, 1963;
Hill and Irisawa, 1966; Hill, 1974a J?). The
ventricle of Dolabella auricularia, for example, possesses a cardiac action potential
of a classical "cardiac" type, reminiscent of
cellular action potentials from vertebrate
ventricular muscle. For Dolabella, plateau
duration may be the point of intervention
at which the postulated cardiac neurotransmitters, ACh and 5HT, modulate
inotropism.
Sodium flux during the plateau phase of
the action potential in vertebrate cardiac
CONCLUSIONS
muscle may govern calcium influx, and
The resting potentials of molluscan thus govern force of contraction (c/.,
ventricular myocardia appear to be bal- Beeler, 1977; McClellan and Winegrad,
anced between the predominance of trans- 1977; Wendt and Langer, 1977). In turn,
membrane potassium ion distribution and increased [Ca++]i may increase outward
several other factors which together result potassium flux and thus provide a feedin a rather low value of resting potential back mechanism for control of the dura(Irisawa et al., 1967a; Irisawa et al., 1968; tion of the action potential (BassingWilkens, 1972a; Kiss and S.-R6zsa, 1973; thwaithee<a/., 1976; Isenberg, 1977).
Nomura, 1965). Primarily, there appears
For molluscan hearts, there is evidence
to be a leakiness to sodium ion which re- presented and reviewed here, that impliduces the value of the resting potential, but cates sodium current in pacemaker activthis is counteracted by a chloride contribu- ity, and both sodium and calcium in the
tion and by an electrogenic sodium-potas- action potential. Wilkens (19726) has
sium pump which may function to keep shown that the plateau phase of the ventrithe membrane potential value at the level cle of Modiolus demissiis is dependent on
where pacemaker activity appears. Pace- sodium, and both Nomura (1963) and Hill
maker activity appears to be sodium de- (1974a) have shown that plateau duration
pendent, but the action potential may in- is directly related to force in the ventricle
volve both sodium and calcium current. of Dolabella auricularia. Possibly sodium
Ventricles of the aplysiids Dolabella au- flux during the plateau phase governs calricularia, Aplysia dactylomela, and Aplysia cium influx in gastropod as well as vertecalifornica all appear to conform to this brate myocardia. This is supported by the
general pattern.
evidence presented here that links [Ca + t ].
Contracture-force is elicited by KC1 de- to both contracture-force and beat-to-beat
polarization of the ventricle of all three force in gastropod hearts.
aplysiids, and depolarization by either KC1
or ACh is related to force in a similar way
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