| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
-Adrenoceptor stimulation-mediated negative inotropism and
enhanced Na+/Ca2+ exchange in mouse
ventricle
Department of Pharmacology, Toho University School of Pharmaceutical Sciences, Chiba 274-8510, Japan
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Mechanisms underlying the negative inotropic response to
-adrenoceptor stimulation in adult mouse ventricular myocardium were
studied. In isolated ventricular tissue, phenylephrine (PE), in the
presence of propranolol, decreased contractile force by ~40% of
basal value. The negative inotropic response was similarly observed
under low extracellular Ca2+ concentration
([Ca2+]o) conditions but was significantly
smaller under high-[Ca2+]o conditions and was
not observed under low-[Na+]o conditions. The
negative inotropic response was not affected by nicardipine, ryanodine,
ouabain, or dimethylamiloride (DMA), inhibitors of L-type
Ca2+ channel, Ca2+ release channel,
Na+-K+ pump, or Na+/H+
exchanger, respectively. KB-R7943, an inhibitor of
Na+/Ca2+ exchanger, suppressed the negative
inotropic response mediated by PE. PE reduced the magnitude of postrest
contractions. PE caused a decrease in duration of the late plateau
phase of action potential and a slight increase in resting membrane
potential; time courses of these effects were similar to that of the
negative inotropic effect. In whole cell voltage-clamped myocytes, PE
increased the L-type Ca2+ and
Na+/Ca2+ exchanger currents but had no effect
on the inwardly rectifying K+, transient outward
K+, or Na+-K+-pump currents. These
results suggest that the sustained negative inotropic response to
-adrenoceptor stimulation of adult mouse ventricular myocardium is
mediated by enhancement of Ca2+ efflux through the
Na+/Ca2+ exchanger.
-adrenoceptors; cardiac muscle; contractile force; negative
inotropism; Na+/Ca2+ exchanger; mouse
myocardium
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
SELECTIVE OVEREXPRESSION or knockout of specific genes by transgenic technology provides useful information on myocardial excitation-contraction mechanisms and their regulation by neuronal and hormonal factors. Although the techniques for creating transgenic animals have become routine only in the mouse, basic information on the physiological and pharmacological properties of the wild-type mouse heart is not yet fully available. The action potential of the adult mouse ventricle has a configuration different from many other experimental animal species. It has a rapid repolarization phase and no plateau at depolarized membrane potentials; the action potential duration at 50% repolarization is only 3-5 ms. Tanaka et al. (31) previously reported that contraction of the mouse ventricular myocardium is relatively resistant to Ca2+ channel antagonists while highly sensitive to ryanodine and cyclopiazonic acid, which indicates its high dependence on Ca2+ release from the sarcoplasmic reticulum (SR). The relation of these profiles with the neuronal and hormonal regulation of myocardial contraction in the mouse remains to be investigated.
The sympathetic nervous system is a major regulator of myocardial
function. Although the neurotransmitter norepinephrine increases the
beating rate and contractile force through stimulation of 
-adrenoceptors in most species, including the mouse
(30), -adrenoceptors are also present in myocardial
tissue, and their stimulation is known to affect cardiac rhythm and
contractility through mechanisms different from those of
-adrenoceptor stimulation (7, 9, 33). Mechanisms such
as increase in transsarcolemmal Ca2+ influx through L-type
Ca2+ channel due to inhibition of transient outward
K+ current and increase in Ca2+ sensitivity in
the myofibrils have been postulated to underlie the positive inotropic
effects of -adrenoceptor stimulation. Negative inotropic responses
to -adrenoceptor stimulation have also been reported in the rat
(4, 21, 38) and bullfrog (11) myocardia, but
the details of the mechanisms remain to be clarified.
In the mouse ventricle, Tanaka et al. (29) have found that
-adrenoceptor stimulation produces sustained negative inotropic responses in the adult. Similar negative inotropic effects were observed with endothelin I and angiotensin II (27).
However, the mechanisms for these inotropic responses had not been
clarified. In the present study, we focused on the negative
inotropic effect of -adrenoceptor stimulation on adult mouse
ventricle. We performed contractile force measurements and action
potential recordings on tissue preparations and voltage-clamp analysis
of membrane currents on isolated myocytes to clarify the ionic
mechanisms underlying the negative inotropic effect.
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Measurement of force of contraction and transmembrane potentials in multicellular preparations. Right ventricular free wall strips were rapidly isolated from adult (4-5 wk old, 16-25 g) ddy strain mice anesthetized with ether. The approximate length and width of preparations were 4 and 2 mm, respectively. Preparations were placed horizontally in a 20-ml organ bath containing modified Ringer solution of the following composition (in mM): 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 15 NaHCO3, and 5.5 glucose (pH 7.4 at 36°C). The solution was gassed with 95% CO2-O2 and maintained at 35-36°C. The preparations were driven by a pair of platinum plate electrodes (field stimulation) with rectangular current pulse (1 Hz, 2 ms, 1.5× threshold voltage) generated from an electronic stimulator. Developed tension was recorded isometrically with a force-displacement transducer connected to a minipolygraph. In all contractile force experiments, propranolol (1 µM) was present throughout. The basal contractile force of preparations under control conditions [150 mM extracellular Na+ concentration ([Na+]o), 2 mM extracellular Ca2+ concentration ([Ca2+]o)] was 49.4 ± 4.6 mg. Low-Na+ ([Na+]o = 105 or 60 mM) solutions contained choline chloride instead of NaCl and also 2 µM atropine sulfate. Contractile force in the presence of phenylephrine was measured at 5-10 min after the addition of the drug, when it had reached steady state.
Transmembrane action potentials were recorded with standard glass microelectrodes filled with 3 M KCl (resistance 30-40 M
). The
microelectrode was coupled via Ag-AgCl junction to a microelectrode preamplifier providing capacity compensation (MEZ-7101; Nihon Kohden).
The preparations were placed horizontally in a 20-ml organ bath
containing modified Ringer solution and stimulated through bipolar
platinum electrodes with rectangular current pulse (1 Hz, 1 ms, 1.2×
threshold voltage). Action potentials were displayed on an oscilloscope
and simultaneously digitized at 25 kHz by an analog-to-digital
converter (Analog Pro; Canopus) attached to a personal computer (PC9801
DA2; NEC) for analysis. Propranolol (1 µM) was applied 15 min before
phenylephrine was applied.
Measurement of membrane currents in single ventricular myocytes. Adult male mice were heparinized (50 IU ip) and anesthetized with ether. The hearts were quickly removed and mounted on a Langendorff apparatus and then perfused for 5-10 min at a rate of 1.2-1.5 ml/min with modified Tyrode solution of the following composition (in mM): 143 NaCl, 4 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.33 NaH2PO4, 5.5 glucose, and 5 HEPES (pH adjusted to 7.4 with NaOH). The hearts were continuously perfused for 15 min with nominally Ca2+-free modified Tyrode solution and then enzymatically digested by perfusion of a nominally Ca2+-free modified Tyrode solution containing 0.2 mg/ml collagenase (Yakult, Tokyo, Japan) for ~20-30 min. Thereafter, the collagenase was washed out for 5 min with nominally Ca2+-free modified Tyrode solution and then perfused with modified Kraftbrühe (KB) solution (18). The ventricular tissue was cut into small pieces in the modified KB solution. The cell suspension was filtered through a 200-µm-pore nylon mesh and stored at 4°C in the solution until use. During cell isolation, solutions were maintained at 36°C and were equilibrated with 100% O2.
Membrane currents were recorded in the whole cell configuration (12) with the use of an Axopatch-1D amplifier (Axon Instruments). Data acquisition and analysis were performed with a Compaq Deskpro 386s personal computer and pCLAMP software (Axon Instruments). For Na+-K+-pump current and Na+/Ca2+ exchange current measurements, holding potential was set to
30 mV. Both currents were measured in response
to ramp voltage-clamp pulses. The range of the ramp pulse was between
120 and +60 mV. Ramp pulses were applied at 0.1 Hz, and the speed was
±90 mV/0.75 s. The resistance of filled electrodes ranged from 2 to 3 M. The compositions of external and internal solutions are listed in
Table 1. The free Ca2+
concentrations of the internal solution for
Na+/Ca2+ exchange current measurement were
calculated to be 70 µM by using Fabiato and Fabiato's equations
(8) with the modification by Tsien and Rink
(36). All external solutions contained propranolol (1 µM) to eliminate the -adrenoceptor agonistic effect of
phenylephrine. All voltage-clamp experiments were performed at
35-36°C, and liquid junction potentials were not compensated.
|
Chemicals. The following drugs were used: L-phenylephrine hydrochloride, ryanodine, and atropine sulfate (Wako Junyaku, Osaka, Japan); dl-propranolol hydrochloride, nicardipine hydrochloride, and ouabain octahydrate (Sigma Chemical, St. Louis, MO); and dimethylamiloride (DMA) hydrochloride (RBI, Natick, MA). Nisoldipine hydrochloride was generously supplied by Bayer Japan (Tokyo, Japan). KB-R7943 was generously supplied to us by Kanebo (Osaka, Japan).
Statistical analysis. All values are expressed as means ± SE. The statistical significance of differences between means was evaluated either by one-way analysis of variance or by the paired t-test.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Ionic requirements for inotropic response to -adrenoceptor
stimulation.
Phenylephrine, in the presence of 1 µM propranolol, produced
sustained negative inotropic responses (Fig.
1A,
a). The contractile force reached its minimum at
~2 min after phenylephrine application. In some cases, the negative
inotropic response was preceded by a transient small increase or
followed by a slight gradual recovery of the contractile force. After
10 µM phenylephrine was applied, the contractile force at the peak of
the initial transient positive phase was 103.3 ± 0.5% of the
initial value, that at its minimum was 53.8 ± 2.1%, and that at
the late steady-state phase was 59.6 ± 2.2% (n = 32). -Adrenoceptor stimulation had no effect on the time course of
contraction and relaxation; the time to reach peak contractile force
before and after application of 10 µM phenylephrine was 48 ± 2 and 47 ± 3 ms, respectively, and the time required for 90%
relaxation was 64 ± 3 and 64 ± 3 ms, respectively
(n = 7). The negative inotropic effect of
-adrenoceptor stimulation was concentration dependent, and 10 µM
phenylephrine produced an ~90% maximum response (not shown)
(29). Thus 10 µM phenylephrine was used for
-adrenoceptor stimulation in the following contractile force
experiments.
|
-adrenoceptor stimulation under a
decreased [Ca2+]o of 0.8 mM was not different
from that observed under normal Ca2+ concentration (Fig.
1B). The negative inotropic response to -adrenoceptor stimulation under an increased [Ca2+]o of 5 mM was greatly reduced under such conditions (Fig. 1, A and
B). Although statistically not significant, the negative inotropic response to -adrenoceptor stimulation was reduced under conditions of 105 mM [Na+]o. Under conditions
of 60 mM [Na+]o, the negative inotropic
response was not observed.
Pharmacological properties of inotropic response to
-adrenoceptor stimulation.
Effects of nicardipine and ryanodine on the negative inotropic response
to -adrenoceptor stimulation were examined. Nicardipine only
slightly decreased the basal contractile force, whereas ryanodine produced marked decreases; the contractile force in the presence of 3 µM nicardipine and 10 nM ryanodine was 86 ± 5%
(n = 7) and 19 ± 3% (n = 5) of
the initial value, respectively. Under these conditions, the magnitude
of the negative inotropic response to -adrenoceptor stimulation was
not different from that under control conditions (Fig.
2B). To clarify the role of
Na+-dependent transporters in the negative inotropic
response to -adrenoceptor stimulation, we examined the effects of
ouabain, a Na+-K+ pump inhibitor; DMA, a
Na+/H+ exchange inhibitor; and KB-R7943, a
Na+/Ca2+ exchange inhibitor. The basal
contractile force in the presence of 1 µM ouabain, 30 µM DMA, or 30 µM KB-R7943 was 263 ± 51% (n = 6), 76 ± 4% (n = 7), or 161 ± 19% (n = 6) of the value in the absence of drugs, respectively. The negative
inotropic response to phenylephrine in the presence of 1 µM ouabain
or 30 µM DMA was not different from that under control conditions
(Fig. 2B). In contrast, KB-R7943 significantly inhibited the
phenylephrine-induced negative inotropic effect in a
concentration-dependent manner. In the presence of 30 µM KB-R7943,
phenylephrine-induced negative inotropic response was abolished (Fig.
2, A and C).
|
Effects of -adrenoceptor stimulation on postrest contraction.
Postrest contractions were measured in the absence and presence of
-adrenoceptor stimulation to asses its effect on SR Ca2+
load (Fig. 3). When regular stimulation
was interrupted by a short rest interval, the resumption of regular
stimulation resulted in a larger potentiated contraction. The magnitude
of the postrest contraction increased with the duration of the rest
interval both in the absence and presence of 10
4 M
phenylephrine; the time to half-maximum potentiation was 3.8 ± 0.7 ms and 4.6 ± 0.5 s (n = 9) in the
absence and presence of phenylephrine, respectively. When compared at a
given rest interval, the postrest contraction under -adrenoceptor
stimulation was significantly smaller, indicating reduced SR
Ca2+ load; the postrest contraction after a rest period of
5 s in the absence and presence of phenylephrine was 231.6 ± 19.6% and 145.1 ± 13.0% (n = 9), respectively,
of the contractile force under regular stimulation in the absence of
phenylephrine.
|
Effects of -adrenoceptor stimulation on action potential.
The action potential of the adult mouse had two phases of
repolarization: the rapid repolarization phase, during which the membrane potential rapidly changes from about +30 mV to 60 mV in a
few milliseconds, and the late plateau phase, with a much slower rate
of repolarization (Fig. 4A).
Phenylephrine (30 µM) significantly shortened the late plateau phase
but did not affect the rapid repolarization phase (Fig. 4A;
Table 2). The time course of the decrease in action potential duration
at 70 mV (Fig. 4B) was similar to that of the decrease in
contractile force (Fig. 4D). Phenylephrine produced a
slight, but significant, shift of the resting membrane potential to the
negative direction (Fig. 4C), which also had a time course
similar to that of the decrease in contractile force.
|
|
Effects of -adrenoceptor stimulation on membrane currents.
The L-type Ca2+ current (ICa) was
observed when depolarization pulses were applied under extra- and
intracellular solutions with Cs+ replaced for
K+ (Fig. 5). The peak current
density of ICa at 0 mV in adult mouse ventricular cells was 17.6 ± 6.2 pA/pF (n = 5).
The current was completely blocked by 3 µM nicardipine (not shown).
These properties of ICa in the mouse ventricle
were similar to those in the ventricle of other species such as the
guinea pig (18). Phenylephrine (30 µM) produced a
gradual increase in ICa that reached steady state at 3-7 min after application (Fig. 5). The shape of the current-voltage relationship was not affected by phenylephrine. The
average peak amplitude of ICa at 0 mV in the
presence of 30 µM phenylephrine was 126 ± 8%
(n = 9) of that in its absence.
|
80 mV.
Ito was greatly reduced by 3 mM 4-aminopyridine
(not shown). Phenylephrine affected neither the amplitude nor the time course of the K+ current (Fig.
6, A and B).
Ito and the inward rectifying K+
current were observed on depolarization and hyperpolarization, respectively, from a holding potential of 40 mV. Phenylephrine had no
effect on the current-voltage relationship (Fig. 6C). The peak outward current density on depolarization to +50 mV in the absence
and presence of phenylephrine was 49.5 ± 6.1 and 48.1 ± 6.6 pA/pF, respectively, and the inward current density at the end of a
300-ms hyperpolarizing pulse to 120 mV was 17.0 ± 3.4 and 16.8 ± 2.8 pA/pF, respectively (n = 5).
|
80 mV and
0.26 ± 0.05 pA/pF at 0 mV in the absence of phenylephrine. Five
minutes after phenylephrine was applied, the current density of
Ip was 0.13 ± 0.01 pA/pF at 80 mV and
0.26 ± 0.07 pA/pF at 0 mV (n = 4, respectively).
|
30 ± 2 mV,
n = 8) was considerably more positive than the
calculated value of the equilibrium potential of
Iex (Eex = 60 mV
for 70 nM internal free Ca2+) under our experimental
conditions, this could be explained by the slow kinetics of
Ca2+ chelating by EGTA (6). A similar
phenomenon has been observed in the guinea pig ventricular myocytes
(5). Phenylephrine, at 10 and 30 µM, enhanced
Iex; the response was more rapid and quick at 30 µM. We used 30 µM phenylephrine to analyze changes in the small
Iex accurately. The current density of
Iex at 80 mV was 0.98 ± 0.21 pA/pF in
the absence, and 1.12 ± 0.20 pA/pF in the presence, of
phenylephrine (increased to 118 ± 13%). Current density at +20
mV was 0.53 ± 0.09 pA/pF in the absence, and 0.81 ± 0.25 pA/pF in the presence, of phenylephrine (increased to 143 ± 22%,
n = 4, respectively).
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The objective of the present study was to clarify the mechanisms
underlying the negative inotropic effects of -adrenoceptor stimulation in mouse ventricle. We performed contractile force measurements, action potential recordings, and voltage-clamp analysis of membrane currents and obtained data suggesting that -adrenoceptor stimulation decreases contractile force by enhancement of
transsarcolemmal Ca2+ efflux through the
Na+/Ca2+ exchanger and the resulting decrease
in the amount of Ca2+ released from the SR.
After the rapid repolarization phase of the adult mouse and rat action
potential, a slow repolarization phase, or a late plateau phase, is
present at membrane potentials more negative than about 40 mV. In
both mouse and rat ventricular myocytes, the late plateau was shown to
be shortened by intracellular dialysis with EGTA and also after the
application of ryanodine, which decreases the amount of
Ca2+ released from the SR on stimulation (24).
The late plateau was reported to be prolonged in
Na+/Ca2+ exchanger-overexpressed mice
(41). Thus the late plateau is considered to be the result
of inward current carried by the Na+/Ca2+
exchanger when it pumps out the Ca2+ released from the SR.
In the present study, we observed that the duration of the late plateau
of the mouse ventricular action potential is shortened after
-adrenoceptor-stimulation (Fig. 4, A and B).
The time course of the decrease in action potential duration at 70 mV
was similar to the time course of the negative inotropic effect. This
indicates that -adrenoceptor stimulation somehow decreases the
amount of Ca2+ released from the SR. This was further
supported by our present result showing that
-adrenoceptor-stimulation reduced the magnitude of postrest
contractions, which has been used as an index of SR Ca2+
load in various preparations, including the mouse ventricle
(14). The negative inotropic effect of -adrenoceptor
stimulation was not affected by ryanodine, which inhibits
Ca2+ release from myocardial SR (Fig. 2B).
Inhibition of Ca2+ uptake into the SR by cyclopiazonic acid
was previously shown to result in decreased contractile force and
slower muscle relaxation of the mouse ventricle (31). In
the case of -adrenoceptor stimulation, the decrease in contractile
force was not accompanied by a decrease in the rate of myocardial
relaxation. These results suggest that the decrease in the amount of
Ca2+ release from the SR is not due to direct inhibitory
effects on SR function but is indirectly caused by other mechanisms
such as decreased transsarcolemmal Ca2+ influx or increased efflux.
The density of ICa in mouse ventricular
myocardium was comparable to that of other experimental animal species.
However, the extremely large outward K+ current quickly
repolarizes the membrane within only a few milliseconds after the
initial rapid depolarization (3). Thus only a limited amount of Ca2+ influx occurs during the short action
potential in the mouse ventricle, resulting in the low sensitivity to
Ca2+ channel antagonists of its contraction
(31). The negative inotropic effect of -adrenoceptor
stimulation was not affected by the Ca2+ channel antagonist
nicardipine, suggesting that the effect was not mediated by decrease in
Ca2+ current (Fig. 2B). In fact, in isolated
myocytes, -adrenoceptor stimulation rather produced an increase in
Ca2+ current amplitude (Fig. 5). In other species, in which
-adrenoceptor stimulation produces positive inotropic effects,
either no change (13, 16, 32, 35) or an increase
(22, 23, 42) in Ca2+ current amplitude has
been reported. The positive inotropic effect of -adrenoceptor
stimulation has been attributed to inhibition of K+
currents, leading to prolongation of the action potential duration and
the resulting indirect increase in Ca2+ influx through the
Ca2+ channel (10, 35). In the mouse, the
slight increase in Ca2+ current (Fig. 5) does not produce
positive inotropic effects, probably because of the limited importance
of the Ca2+ current itself in excitation-contraction
coupling. -Adrenoceptor stimulation had no effects on the outward
K+ current amplitude, which is the determinant of action
potential duration (Fig. 6). In fact, the rapid repolarization of the
mouse ventricular action potential was not affected (Fig. 4). Thus
effects on Ca2+ influx through the L-type Ca2+
channel, either direct or indirect, could not be the mechanism for the
negative inotropic effect of -adrenoceptor stimulation.
The major mechanism for transsarcolemmal Ca2+ efflux in
myocardial cells is the Na+/Ca2+ exchanger. In
the present study, the negative inotropic effect of -adrenoceptor
stimulation was reduced or abolished by both increased extracellular
Ca2+ and decreased extracellular Na+ conditions
(Fig. 1), which shift the equilibrium potential of the exchanger to
negative direction and inhibit Ca2+ efflux through the
Na+/Ca2+ exchanger. The increase in contractile
force by high Ca2+ or low Na+ itself could not
explain the abolishment of -adrenoceptor-induced negative inotropism
because it was observed after treatment with ouabain, which also
increased the contractile force (Fig. 2). The negative inotropic effect
of -adrenoceptor stimulation was not observed in the presence of
KB-R7943. The compound is reported to be an inhibitor of the
Na+/Ca2+ exchanger (20, 39), and
the IC50 of the compound for Iex in
ventricular myocytes was ~1 µM (20). In the present
study, KB-R7943 increased basal contractile force and inhibited the
phenylephrine-induced negative inotropic response at 3 µM, suggesting
the involvement of enhanced Na+/Ca2+ exchange
in the response (Fig. 2C). Although there is a report that
KB-R7943 has inhibitory effects on cardiac Na+ current,
ICa, and inward rectifier K+ current
at higher concentrations with IC50 values of 14, 8, and 7 µM, respectively (39), these effects are unlikely to
interfere with the phenylephrine-induced negative inotropic response,
which was shown not to be mediated by changes in
ICa, K+, and Na+
currents (Figs. 1, 2, and 4).
Because these results from contractile force experiments strongly
suggested that the -adrenoceptor stimulation-induced negative inotropic response is mediated by an increase in
Na+/Ca2+ exchanger activity, we performed
voltage-clamp experiments in isolated cardiomyocytes. We obtained
evidence that -adrenoceptor stimulation indeed increases
Iex (Fig. 8). The effect was not voltage
dependent; the exchanger current was increased for both inward and
outward direction. This means that not only Ca2+ efflux but
also Ca2+ influx through the
Na+/Ca2+ exchanger can possibly be enhanced by
-adrenoceptor stimulation. However, the myocardial membrane
potential is considered to be more negative than the equilibrium
potential of the Na+/Ca2+ exchanger for most of
the period in the cardiac cycle, resulting in net Ca2+
efflux. This would be prominent in the mouse ventricular myocardium, which has an extremely short action potential lacking a plateau phase
at depolarized membrane potentials (Fig. 4). Thus enhancement of the
Na+/Ca2+ exchanger by -adrenoceptor
stimulation would result in enhancement of transsarcolemmal
Ca2+ efflux, leading to an eventual decrease in the amount
of Ca2+ released from the SR and a decrease in contractile
force. Decrease in SR Ca2+ load under -adrenoceptor
stimulation was confirmed by reduction of the magnitude of postrest
contractions (Fig. 3). Phenylephrine was reported to stimulate
Na+/Ca2+ exchange in the presence of GTP in
sarcolemmal vesicles of rat ventricular myocardia (1).
-Adrenoceptor stimulation-induced shortening of the action potential
plateau (Fig. 4) might appear contradictory with the enhancement of
Na+/Ca2+ exchanger (Fig. 8). However, these two
phenomena are not necessarily contradictory because the inward current
(Ca2+ efflux) through the Na+/Ca2+
exchanger is affected by the intracellular Ca2+
concentration. Na+/Ca2+ exchanger activity,
enhanced by either pharmacological interventions or transgenic
technology, can result in prolongation of action potential duration
only when the intracellular Ca2+ is maintained. In
Na+/Ca2+ exchanger-overexpressed mice, in which
the SR Ca2+ load was unchanged from the wild type, action
potential duration was longer than that in the wild type
(41). In normal mouse ventricle in the present study,
Iex was enhanced by -adrenoceptor stimulation
under voltage-clamp conditions in which the intracellular Ca2+ concentration was maintained constant (Fig. 8).
However, this only means that Na+/Ca2+
exchanger activity in the presence of -adrenoceptor stimulation is
higher than in its absence when compared under the same
Ca2+ concentration. In the intact ventricular myocyte,
-adrenoceptor stimulation-induced enhancement of the
Na+/Ca2+ exchanger resulted in reduced SR
Ca2+ load (Fig. 3) and reduced contractile force. Reduced
Ca2+ supply to the Na+/Ca2+
exchanger during the repolarization phase caused reduction in the
inward current (Ca2+ efflux and Na+ influx)
through the Na+/Ca2+ exchanger, and thus the
action potential duration was shortened. A previous finding in mouse
ventricle indicating that ryanodine, which was reported to inhibit
Ca2+ release from the SR with no effect on the
Na+/Ca2+ exchanger, markedly shortened APD
(31) also supports this view.
There are reports suggesting effects of -adrenergic stimulation on
mechanisms that might affect intracellular Na+
concentration. In canine Purkinje fiber and rat myocardium,
-adrenoceptor stimulation was reported to enhance
Na+-K+-pump activity (28, 37, 40).
There are also reports suggesting that the -adrenoceptor-induced
inotropic responses are mediated by changes in
Na+/H+ exchanger activity and/or intracellular
pH (17, 32). However, in the present study on mouse
ventricular myocardium, negative inotropic effects of -adrenoceptor
stimulation were observed in the presence of the
Na+-K+-pump inhibitor ouabain and in the
presence of the Na+/H+ exchange inhibitor DMA
(Fig. 2). DMA, at the concentration in this study, has little effect on
other channels and transporters (26). Furthermore,
enhancement of the Na+/Ca2+ exchanger by
-adrenergic stimulation was observed under whole cell voltage-clamp
conditions in which intracellular Na+ and H+
concentrations were maintained constant. Thus -adrenoceptor stimulation-induced inotropism in the mouse ventricle was not mediated
by changes in the activities of the Na+-K+
pump or Na+/H+ exchanger. Furthermore,
-adrenoceptor stimulation did not affect the maximum rate of
rise of the action potential, excluding effects on the Na+
current. There are other mechanisms that might be involved in the
-adrenoceptor-mediated negative inotropism such as changes in the
function of the SR and changes in the Ca2+ sensitivity of
the contractile proteins. These possibilities remain to be investigated.
-Adrenoceptor stimulation produced a slight but significant shift in
the resting membrane potential to the negative direction with a time
course similar to that of the negative inotropic effect (Fig. 4,
A and C). Results from voltage-clamp experiments
exclude changes in K+ currents (Fig. 5) or
Ip (Fig. 7) as mechanisms for hyperpolarization. Also, in the rat ventricle, Tohse et al. (34) observed a
phenylephrine-induced hyperpolarization that was not affected by
ouabain or elevated extracellular K+ concentration. The
-adrenoceptor stimulation-induced enhancement of
Na+/Ca2+ exchanger in the mouse ventricle also
could not explain the hyperpolarization because the
Na+/Ca2+ exchanger current flows in the inward
direction at the resting membrane potential range. Thus the ionic
mechanism and the role of the hyperpolarization induced by
-adrenoceptor stimulation are not clear at present.
The mouse heart has characteristic excitation-contraction coupling
properties and a higher beating rate compared with that of other
experimental animal species. The sympathetic nervous system was
reported to have dominant influence on the heart in the mouse
(2). Increased -adrenoceptor stimulation results in
increased transsarcolemmal Ca2+ influx through
increased frequency of myocardial excitation and cAMP-mediated
enhancement of ICa. Simultaneous enhancement of Na+/Ca2+ exchanger activity through
-adrenoceptors under such conditions would increase transsarcolemmal
Ca2+ efflux and partially balance the increase in
transsarcolemmal Ca2+ influx. In fact, we have observed in
isolated mouse ventricular tissue that the increase in contractile
force by the sympathetic neurotransmitter norepinephrine was enhanced
by the -adrenoceptor antagonist WB-4101 (29). Thus
-adrenoceptor stimulation-induced enhancement of
Na+/Ca2+ exchanger activity appears to be a
mechanism to prevent overactivity of the mouse myocardium under
increased sympathetic nerve activity.
| |
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: H. Tanaka, Dept. of Pharmacology, Toho Univ. School of Pharmaceutical Sciences, Miyama 2-2-1 Funabashi, Chiba 274-8510, Japan (E-mail: htanaka{at}phar.toho-u.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 November 1999; accepted in final form 28 July 2000.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Ballard, A,
and
Schaffer S.
Stimulation of the Na+/Ca2+ exchanger by phenylephrine, angiotensin II and endothelin I.
J Mol Cell Cardiol
28:
11-17,
1996[ISI][Medline].
2.
Barth, E,
Stämmler G,
Speiser B,
and
Schaper J.
Ultrastructural quantitation of mitochondria and myofilaments in cardiac muscle from 10 different animal species including man.
J Mol Cell Cardiol
24:
669-681,
1992[ISI][Medline].
3.
Benndorf, K,
and
Nilius B.
Properties of an early outward current in single cells of the mouse ventricle.
Gen Physiol Biophys
7:
449-466,
1988[ISI][Medline].
4.
Capogrossi, MC,
Kachadorian A,
Gambassi G,
Squrgeon HA,
and
Lakatta EG.
Ca2+ dependence of -adrenergic effects on the contractile properties and homeostasis of cardiac myocytes.
Circ Res
69:
540-550,
1991
5.
Coetzee, WA,
Ichikawa H,
and
Hearse DJ.
Oxidant stress inhibits Na-Ca-exchange current in cardiac myocytes: mediation by sulfhydryl groups?
Am J Physiol Heart Circ Physiol
266:
H909-H919,
1994
6.
Ehara, T,
Matsuoka S,
and
Noma A.
Measurement of reversal potential of Na+-Ca2+ exchange current in single guinea-pig ventricular cells.
J Physiol (Lond)
410:
227-249,
1989
7.
Endoh, M.
Signal transduction of myocardial 1-adrenoceptors: regulation of ion channels, intracellular calcium, and force of contraction
a review.
J Appl Cardiol
6:
379-399,
1991.
8.
Fabiato, A,
and
Fabiato F.
Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells.
J Physiol (Paris)
75:
463-505,
1979[Medline].
9.
Fedida, D,
Braun AP,
and
Giles WR.
1-Adrenoceptors in myocardium: functional aspects and transmembrane signaling mechanisms.
Physiol Rev
73:
469-487,
1993
10.
Fedida, D,
Shimoni Y,
and
Giles WR.
-Adrenergic modulation of the transient outward current in rabbit atrial myocytes.
J Physiol (Lond)
423:
257-277,
1990
11.
Goto, M,
Sun C,
Yatani A,
Urata M,
and
Fujino T.
Antagonistic action of - and -agonists on the bullfrog atrium.
Jpn J Physiol
30:
751-765,
1980[ISI][Medline].
12.
Hamil, OP,
Marty A,
Neher E,
Sakmann B,
and
Sigworth FJ.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch
391:
85-100,
1981[ISI][Medline].
13.
Hartmann, HA,
Mazzocca NJ,
Kleiman RB,
and
Houser SR.
Effects of phenylephrine on calcium current and contractility of feline ventricular myocytes.
Am J Physiol Heart Circ Physiol
255:
H1173-H1180,
1988
14.
He, H,
Giordano FJ,
Hilal-Dandan R,
Choi DJ,
Rockman HA,
McDonough PM,
Bluhm WF,
Meyer M,
Sayen MR,
Swanson E,
and
Dillmann WH.
Overexpression of the rat sarcoplasmic reticulum Ca ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation.
J Clin Invest
100:
380-389,
1997[ISI][Medline].
15.
Hermans, AN,
Glitsch HG,
and
Verdonck F.
The effect of cardiac glycosides on the Na+ pump current-voltage relationship of isolated rat and guinea-pig heart cells.
J Physiol (Lond)
481:
270-291,
1994.
16.
Hescheler, J,
Nawrath H,
Tang M,
and
Trautwein W.
Adrenoceptor-mediated changes of excitation and contraction in ventricular heart muscle from guinea-pigs and rabbits.
J Physiol (Lond)
397:
657-670,
1988
17.
Iwakura, K,
Hori M,
Watanabe Y,
Kitabatake A,
Cragoe EJ,
Yoshida H,
and
Kamada T.
1-Adrenoceptor stimulation increases intracellular pH and Ca2+ in cardiomyocytes through Na+/H+ and Na+/Ca2+ exchange.
Eur J Pharmacol
186:
29-40,
1990[ISI][Medline].
18.
Kato, Y,
Masumiya H,
Agata N,
Tanaka H,
and
Shigenobu K.
Developmental changes in action potential and membrane currents in fetal, neonatal and adult guinea-pig ventricular myocytes.
J Mol Cell Cardiol
28:
1515-1522,
1996[ISI][Medline].
19.
Kimura, J,
Miyamae S,
and
Noma A.
Identification of sodium-calcium exchange current in single ventricular cells of guinea-pig.
J Physiol (Lond)
384:
199-222,
1987
20.
Kimura, J,
Watano T,
Kawahara M,
Sakai E,
and
Yatabe J.
Direction-independent block of bi-directional Na+/Ca2+ exchange current by KB-R7943 in guinea-pig cardiac myocytes.
Br J Pharmacol
128:
969-974,
1999[ISI][Medline].
21.
Kissling, G,
Blickle B,
Ross C,
Pascht U,
and
Gulbins E.
1-Adrenoceptor-mediated negative inotropy of adrenaline in rat myocardium.
J Physiol (Lond)
499:
195-205,
1997[ISI][Medline].
22.
Liu, QY,
Karpinski E,
and
Pang PKT
The L-type calcium channel current is increased by alpha-1 adrenoceptor activation in neonatal rat ventricular cells.
J Pharmacol Exp Ther
271:
935-943,
1994
23.
Liu, SJ,
and
Kennedy RH.
-Adrenergic activation of L-type Ca current in rat ventricular myocytes: perforated patch-clamp recordings.
Am J Physiol Heart Circ Physiol
274:
H2203-H2207,
1998
24.
Mitchel, MR,
Powell T,
Terrar DA,
and
Twist VW.
The effects of ryanodine, EGTA and low sodium on action potentials in rat and guinea-pig ventricular myocytes: evidence for two inward currents during the plateau.
Br J Pharmacol
81:
543-550,
1984[ISI][Medline].
25.
Nakao, M,
and
Gadsby DC.
[Na] and [K] dependence of the Na/K pump current-voltage relationship in guinea-pig ventricular myocytes.
J Gen Physiol
94:
539-565,
1989
26.
Rüsch, A,
Kros CJ,
and
Richardson GP.
Block of amiloride and its derivatives of mechano-electrical transduction in outer hair cells of mouse cochlear cultures.
J Physiol (Lond)
474:
75-86,
1994
27.
Sekine, T,
Kusano H,
Nishimaru K,
Tanaka Y,
Tanaka H,
and
Shigenobu K.
Developmental conversion of inotropism by endothelin-I and angiotensin-II from positive to negative in mice.
Eur J Pharmacol
374:
411-415,
1999[ISI][Medline].
28.
Shah, A,
Cohen IS,
and
Rosen MR.
Stimulation of cardiac alpha receptors increases Na/K pump current and decreases gK via a pertussis toxin-sensitive pathway.
Biophys J
54:
219-225,
1988
29.
Tanaka, H,
Manita S,
Matsuda T,
Adachi M,
and
Shigenobu K.
Sustained negative inotropism mediated by alpha-adrenoceptors in adult mouse myocardia: developmental conversion from positive response in the neonate.
Br J Pharmacol
114:
673-677,
1995[ISI][Medline].
30.
Tanaka, H,
Manita S,
and
Shigenobu K.
Increased sensitivity of neonatal mouse myocardia to autonomic transmitters.
J Auton Pharmacol
14:
123-128,
1994[ISI][Medline].
31.
Tanaka, H,
Sekine T,
Nishimaru K,
and
Shigenobu K.
Role of sarcoplasmic reticulum in myocardial contraction of neonatal and adult mice.
Comp Biochem Physiol A Mol Integr Physiol
120:
431-438,
1998[Medline].
32.
Terzic, A,
Pucéat M,
Clement O,
Scamps F,
and
Vassort G.
1-Adrenergic effects on intracellular pH and calcium and on myofilaments in single rat cardiac cells.
J Physiol (Lond)
447:
275-292,
1992
33.
Terzic, A,
Pucéat M,
Vassort G,
and
Vogel S.
Cardiac 1-adrenoceptors: an overview.
Pharmacol Rev
45:
147-175,
1993[ISI][Medline].
34.
Tohse, N,
Hattori Y,
Nakaya H,
and
Kanno M.
Effects of -adrenoceptor stimulation on electrophysiological properties and mechanics in rat papillary muscle.
Gen Pharmacol
18:
539-546,
1987[ISI][Medline].
35.
Tohse, N,
Nakaya H,
Hattori Y,
Endoh M,
and
Kanno M.
Inhibitory effect mediated by -adrenoceptors on transient outward current in isolated rat ventricular cells.
Pflügers Arch
415:
575-581,
1990[ISI][Medline].
36.
Tsien, RY,
and
Rink TJ.
Neutral carrier ion-selective microelectrodes for measurement of intracellular free calcium.
Biochim Biophys Acta
599:
623-638,
1980[Medline].
37.
Wang, Y,
Gao J,
Mathias RT,
Cohen IS,
Sun X,
and
Baldo GJ.
-Adrenergic effects on Na+-K+ pump current in guinea-pig ventricular myocytes.
J Physiol (Lond)
509:
117-128,
1998
38.
Watanabe, AM,
Hathaway DR,
Besch HRJ,
Farmer BB,
and
Harris RA.
-Adrenergic reduction of cyclic adenosine monophosphate concentrations in rat myocardium.
Circ Res
40:
596-602,
1977
39.
Watano, T,
Kimura J,
Morita T,
and
Nakanishi H.
A novel antagonist, no. 7943, of the Na+/Ca2+ exchange current in guinea-pig cardiac ventricular cells.
Br J Pharmacol
119:
556-563,
1996.
40.
Williamson, AP,
Kennedy RH,
Seifen E,
Lindemann JP,
and
Stimers JR.
1b-Adrenoceptor-mediated stimulation of Na-K pump current in adult rat ventricular myocytes.
Am J Physiol Heart Circ Physiol
264:
H1315-H1318,
1993
41.
Yao, A,
Su Z,
Nonaka A,
Zubair I,
Lu L,
Philipson KD,
Bridge JHB,
and
Barry WH.
Effects of overexpression of the Na+-Ca2+ exchanger on [Ca2+]i transients in murine ventricular myocytes.
Circ Res
82:
657-665,
1998
42.
Zhang, S,
Hiraoka M,
and
Hirano Y.
Effects of 1-adrenergic stimulation on L-type Ca2+ current in rat ventricular myocytes.
J Mol Cell Cardiol
30:
1955-1965,
1998[ISI][Medline].
This article has been cited by other articles:
|
|
 |
|
  G.-Y. Wang, D. T. McCloskey, S. Turcato, P. M. Swigart, P. C. Simpson, and A. J. Baker Contrasting inotropic responses to {alpha}1-adrenergic receptor stimulation in left versus right ventricular myocardium Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H2013 - H2017. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. B. Roman, P. H. Goldspink, E. Spaite, D. Urboniene, R. McKinney, D. L. Geenen, R. J. Solaro, and P. M. Buttrick Inhibition of PKC phosphorylation of cTnI improves cardiac performance in vivo Am J Physiol Heart Circ Physiol, June 1, 2004; 286(6): H2089 - H2095. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A Ross, B. R Rorabaugh, D. Chalothorn, J. Yun, P. J Gonzalez-Cabrera, D. F McCune, M. T Piascik, and D. M Perez The {alpha}1B-adrenergic receptor decreases the inotropic response in the mouse Langendorff heart model Cardiovasc Res, December 1, 2003; 60(3): 598 - 607. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Turnbull, D. T. McCloskey, T. D. O'Connell, P. C. Simpson, and A. J. Baker alpha 1-Adrenergic receptor responses in alpha 1AB-AR knockout mouse hearts suggest the presence of alpha 1D-AR Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1104 - H1109. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Reuter, S. A. Henderson, T. Han, T. Matsuda, A. Baba, R. S. Ross, J. I. Goldhaber, and K. D. Philipson Knockout Mice for Pharmacological Screening: Testing the Specificity of Na+-Ca2+ Exchange Inhibitors Circ. Res., July 26, 2002; 91(2): 90 - 92. [Abstract] [Full Text] [PDF]
|
|
) and presence (
) of 100 µM
PE expressed as a percentage of the regular contractile force under
1-Hz stimulation in the absence of PE. All experiments were performed
in the presence of 1 µM propranolol. Values represent means ± SE from 9 experiments. Values of all data recorded in the presence of
PE were significantly smaller (P < 0.05) than values
of corresponding data in the presence of PE.
