Vol. 277, Issue 4, H1369-H1374, October 1999
EP receptor-mediated inhibition by prostaglandin
E1 of cardiac L-type
Ca2+ current of rabbits
Taku
Yamamoto1,
Yoshizumi
Habuchi1,
Hideo
Tanaka1,
Fumiaki
Suto3,
Junichiro
Morikawa2,
Kei
Kashima2, and
Manabu
Yoshimura1
Departments of 1 Laboratory
Medicine and 2 Internal Medicine
III, Kyoto Prefectural University of Medicine; and
3 Department of Pediatrics,
Children's Research Hospital, Kyoto Prefectural University of
Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-0841, Japan
 |
ABSTRACT |
Prostaglandin
E1
(PGE1) has cardioprotective
effects on the ischemic-reperfused heart. To clarify the mechanisms
underlying the protective action of
PGE1 on myocardium, we examined
the effect of PGE1 on the L-type
Ca2+ current
(ICa) using
single atrial cells from rabbits.
PGE1 did not show a significant
effect on basal
ICa but inhibited
the ICa prestimulated by isoproterenol (Iso, 30 nM). This inhibition was concentration dependent (EC50 = 0.027 µM). Both sulprostone, a specific PGE receptor subtype
(EP1 and
EP3) agonist, and
11-deoxy-PGE1, an
EP3 agonist, inhibited the
Iso-stimulated
ICa, similar to
PGE1. Pretreatment with pertussis
toxin (PTX) abolished the PGE1
inhibition of
ICa. Both the
application of forskolin plus IBMX and intracellular dialysis with
8-bromoadenosine 3',5'-cyclic monophosphate eliminated the
effect of PGE1.
PGE1 did not show any further
inhibition of ICa
when the effect of Iso was almost fully antagonized by acetylcholine. Methylene blue (guanylate cyclase inhibitor), KT-5823 (cGMP-dependent protein kinase inhibitor), and erythro-9-(2-hydroxy-3-nonyl)adenine (type II phosphodiesterase inhibitor) did not significantly change the
inhibitory effect of PGE1. These
findings suggest that 1) PGE1 inhibits Iso-stimulated
ICa by binding to
the EP3 receptor and
2) the PTX-sensitive and
cAMP-dependent pathway is involved in the
PGE1 inhibition of
ICa, but the
nitric oxide-cGMP-dependent pathway is not. The
PGE1-induced antiadrenergic effect
shown in this study may contribute to the
PGE1 protection of myocardium against ischemia.
adenosine 3',5'-cyclic monophosphate; calcium channel; EP3 receptor; cardiac myocytes; myocardial protection
| |
INTRODUCTION |
NUMEROUS STUDIES HAVE SHOWN that members
of the prostaglandin E family, especially prostaglandin
E1
(PGE1), have various effects on
the heart, suggesting that PGE1 is
a local hormone in the heart (17, 29). The dominant prostaglandin in
the heart under physiological conditions is prostacyclin (28). It has
been shown that during myocardial ischemia, other
prostaglandins including PGE1 are
released in the heart (28). The membrane disruption during
ischemia causes accumulation of arachidonic acid and
dihomo-
-linolenic acid, and the aggregation of platelets and
inflammatory cells would promote the metabolism of these acids to form
various prostaglandins (28).
PGE1 is generally regarded as a
cardioprotective agent for ischemia or reperfusion injury (13,
14, 28). Its application to the coronary arteries during experimental
ischemia consistently reduced the size of myocardial infarction
(15, 28). Because PGE1 dilates
coronary arteries and inhibits platelet aggregation (17), it improves
the collateral blood supply to the ischemic myocardium (15). However,
myocardial salvage independent from the blood supply was indicated
(15). Because previous studies have suggested the presence of specific
PGE receptors (EP receptors) on myocardial sarcolemma (14),
PGE1 may cause some cellular responses that diminish the myocardial disruption during ischemia.
Under various experimental conditions,
PGE1 was reported to have positive
inotropic and chronotropic effects (17, 29). These effects were
accompanied by an increase in the intracellular cAMP concentration
(18), whereas the cAMP-mediated positive inotropic and chronotropic
effects seem to contradict the cardioprotective actions of
PGE1. Hohlfeld et al. (14)
recently reported a negative inotropic effect and a decrease in
adenylate cyclase activity by PGE1
in anesthetized pigs during infusion with isoproterenol (Iso). Their
report indicates an antiadrenergic effect of
PGE1, which is favorable for
myocardial protection. However, prostaglandins may affect the coronary
circulation or neurotransmitter release, and these effects may have
affected the above results. In addition, the prostaglandin-induced
change in cAMP concentration in myocardial cells does not correlate
with the functional responses (9, 10, 18, 19). Therefore, whether
PGE1 affects the myocardial cell
functions (especially via the cAMP-dependent pathway) remains to be
resolved. If so, the role of specific sarcolemmal receptors should be
elucidated. To clarify these points, we used enzymatically isolated
single cardiac cells. In myocardial cells, the L-type Ca2+ current
(ICa) has been
used as a probe to examine changes in intracellular cAMP concentration,
and its increase is largely responsible for the inotropic effects and
intracellular Ca2+ overload
induced by 
-adrenoceptor stimulation. In this study, we examined the
responses of ICa
to PGE1 with special reference to
the antiadrenergic effect.
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METHODS |
Cell isolation.
Single atrial cells were isolated as previously reported (7, 33). All
the procedures conformed to the Guide for the Care and
Use of Laboratory Animals published by the National
Research Council (Washington, DC: Natl. Acad. Press, 1996). The heart
was excised from rabbits (2-2.5 kg) after intravenous injection of 40 mg/kg pentobarbital sodium and 500 IU heparin. The excised heart was
mounted on a Langendorff apparatus and retrogradely perfused for 15 min
with Ca2+-free, phosphate-buffered
solution containing (in mM) 136.9 NaCl, 5.4 KCl, 1.0 MgCl2, 0.33 NaH2PO4,
2.24 Na2HPO4,
and 10 glucose (pH 7.4). The solution was then replaced with a solution
containing 0.02 mg/ml collagenase (Yakult, Tokyo) and 0.015 mg/ml
protease (type XIV, Sigma, St. Louis, MO). The temperature was
maintained at 37°C, and the solutions were saturated with 100%
O2. After the first enzyme
treatment, the right atrium was isolated. It was then agitated in a
second enzyme solution that contained 0.8 mg/ml collagenase (type H,
Sigma). Isolated cells were collected by centrifugation at 70 g (3 min) and stored at 4°C in a
stock solution containing (in mM) 75 potassium glutamate, 10 oxalate, 20 KCl, 10 KH2PO4,
1 MgSO4, 20 taurine, 0.5 EGTA, 5 HEPES, 5 NaCl, 5 creatine, and 10 glucose (pH 7.2 adjusted with KOH).
Electrical measurement and data analysis.
The cells showing a rod shape and clear striation during perfusion with
Tyrode solution were used. The Tyrode solution contained (in mM) 142 NaCl, 5.4 KCl, 1.0 NaH2PO4,
1.0 CaCl2, 1.0 MgCl2, 5 HEPES, and 10 glucose (pH
7.4 adjusted by NaOH).
ICa was measured using the perforated-patch method in most of the voltage-clamp experiments. The pipette solution contained (in mM) 140 CsCl, 6 NaCl,
and 5 HEPES (pH 7.2 adjusted with CsOH). The pipette tip was soaked in
the simple pipette solution, and the pipette was backfilled with
amphotericin B-containing solution. Amphotericin B was dissolved in
DMSO at a concentration of 80 mg/ml and diluted in the pipette solution
to make a final concentration of 0.56 mg/ml. Pipettes had a tip
resistance of 1-1.5 M
. After a gigaohm seal was made during
perfusion with Tyrode solution, the perfusate was changed to an
external test solution containing (in mM) 135 NaCl, 10 CsCl, 0.5 CaCl2, 0.5 BaCl2, 1.0 MgCl2, 5 HEPES, and 10 glucose (pH
7.4 adjusted with NaOH). Test pulses were applied from a holding
potential of 
40 to 0 mV for 300 ms at 0.083 Hz. The temperature
during the experiments was 37°C.
In some experiments, cells were incubated with pertussis toxin (PTX) at
a concentration of 1 µg/ml for 5-8 h in the above-described stock solution containing 0.1% bovine serum albumin. In these experiments, the incubation was judged to be successful when
ICa responded
well to Iso (30 nM) and ACh (1 µM) failed to inhibit it. In other
experiments, the membrane-ruptured patch-clamp method was used to
dialyze the cells with 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP). The pipette solution contained (in mM) 105 CsCl, 0.5 CaCl2, 10 EGTA, 5 MgATP,
5 Na2-phosphocreatine, 0.2 Na3GTP, and 5 HEPES (pH 7.2 adjusted with CsOH). The external solution contained (in mM) 135 NaCl,
10 CsCl, 1.8 CaCl2, 0.5 BaCl2, 1.0 MgCl2, 5 HEPES, and 10 glucose (pH
7.4 adjusted with NaOH). 8-BrcAMP was added to the pipette solution at
a concentration of 100 µM. Pipettes had a tip resistance of 2-3
M. A liquid junction potential of 10 mV was corrected. Holding
potential was 70 mV, and test pulses to 0 mV for 300 ms were
applied after a conditioning pulse at 40 mV for 500 ms (7, 33).
The amplifier used was a TM-1000 (ACT ME, Tokyo, Japan) or an Axopatch
1-D (Axon Instrument, Foster, CA). The filtration frequency was 2 kHz.
The current signal was monitored on a digital oscilloscope (Nicolet
310C, Madison, WI) with a sampling time of 0.2 ms. Digitized data were
subsequently analyzed on a computer (NEC 98, Tokyo). ICa was measured
as the difference between the inward peak and the current at the end of
the test pulse. In most experiments, we tested
PGE1 in the presence of Iso at 30 nM. The inhibitory effect of PGE1
is expressed as percent inhibition of the Iso-induced increase in
ICa (33).
To avoid daily bias caused by the cell isolation, no more than two
experiments with the same protocol were performed on one day. The data
are described as means ± SE. Statistical analyses were based on
paired Student's t-test or ANOVA, and
the differences were considered significant when
P values were <0.05.
Materials.
PGE1,
Na2ATP, MgATP,
Na2-phosphocreatine, 8-BrcAMP,
DMSO, amphotericin B, PTX, and erythro-9-(2-hydroxy-3-nonyl)adenine
(EHNA) were purchased from Sigma. KT-5823 and
11-deoxy-PGE1
(11-dPGE1) were purchased from
Calbiochem (La Jolla, CA) and Cayman Chemicals (Ann Arbor, MI),
respectively. Sulprostone and butaprost were gifts from Ono
Pharmaceutical (Osaka, Japan). All other chemicals were from Wako Pure
Chemicals (Osaka). Iso was dissolved in distilled water containing 1 mg/ml ascorbic acid as a 30 µM stock solution. PGE1, sulprostone, and butaprost
were dissolved in ethanol at a concentration of 1 mM. Forskolin and
11-dPGE1 were stocked in ethanol
at 3 mM and 100 µM, respectively. KT-5823 was stocked in DMSO at 2 mM. Methylene blue and IBMX were directly added to each external
solution with sonication.
| |
RESULTS |
Effects of PGE1 on nonstimulated and
stimulated ICa.
We first tested the effects of
PGE1 on basal (nonstimulated)
ICa.
PGE1 at 0.1 (8 cells from 5 rabbits) and 3 (14 cells from 10 rabbits) µM did not change the
amplitude of basal
ICa
significantly. Although in 4 of 14 cells (10 rabbits),
PGE1 at 3 µM caused a slight
inhibition of basal
ICa (Fig.
1A),
the effect was small (
10% of control amplitude). In contrast to the
trivial effect of PGE1 on basal
ICa,
PGE1 showed a distinct inhibition
of ICa when it
was prestimulated by Iso. As shown in Fig.
1B,
PGE1 at 0.1 µM reversibly
inhibited the Iso-stimulated
ICa. Iso at 30 nM
increased ICa by
183.0 ± 18.8%, and 0.1 µM
PGE1 reduced the Iso-induced
increase in ICa
by 25.1 ± 2.8% (14 cells from 11 rabbits). Figure
1C shows the concentration-response
curve for the inhibitory effect of
PGE1 on Iso-stimulated
ICa, which
indicates that PGE1 inhibited
the 30 nM Iso-stimulated
ICa with an
EC50 of 0.027 µM.
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Fig. 1.
Effects of prostaglandin E1
(PGE1) on L-type
Ca2+ current
(ICa) of rabbit
atrial cells. A: weak effect of
PGE1 (3 µM) on basal
ICa.
B:
PGE1 (0.1 µM) inhibition of
ICa prestimulated
by isoproterenol (Iso, 30 nM). Horizontal bars indicate periods of
exposure to PGE1 or Iso. Actual
records obtained at time indicated by arrows
a-c appear in
inset.
C: concentration-dependent inhibition
of Iso-stimulated
ICa by
PGE1. Effect of
PGE1 is expressed as % inhibition
of 30 nM Iso-induced increase in
ICa
(n = 5-16 for each symbol). A
single concentration of PGE1 was
used for 1 cell.
ICa inhibition by
PGE1 was expressed as % decrease
in ICa = Emax/(1 + EC50/[PGE1]),
where Emax and
EC50 represent maximal response
and concentration eliciting half-maximal response, respectively, and
[PGE1] is
PGE1 concentration.
Emax = 30.9%,
and EC50 = 0.027 µM.
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Involvement of EP receptor.
PGE1 induces cell responses by
binding to specific EP receptors (3, 14). To investigate the
involvement of EP receptors in
PGE1 inhibition of Iso-stimulated
ICa, we examined
the effects of synthesized agonists of EP-receptor subtypes
(sulprostone, 11-dPGE1, and
butaprost). Sulprostone is selective for
EP1 and EP3 receptors (3). As shown in
Fig. 2A,
sulprostone (0.1 µM) inhibited Iso-stimulated
ICa by 23.9 ± 1.9% (11 cells from 8 rabbits; see also Fig.
2D) but did not affect the
stimulated ICa
additionally when PGE1 (3 µM)
was applied in advance (6 cells from 5 rabbits; see Fig.
2B). As was the case with
PGE1, sulprostone did not affect basal ICa (7 cells from 4 rabbits; data not shown). A similar ICa inhibition
was observed with 11-dPGE1, an
EP3-receptor agonist (Ref. 3; Fig.
2D). This inhibition was dose
dependent, i.e., 0.01 and 0.1 µM
11-dPGE1 inhibited stimulated
ICa by 11.1 ± 3.4% (8 cells from 6 rabbits) and 24.5 ± 2.4% (9 cells from 6 rabbits), respectively. In contrast, butaprost (selective agonist of
EP2 receptor; Ref. 3) affected
neither basal (6 cells from 3 rabbits; not shown) nor Iso-stimulated (6 cells from 3 rabbits; Fig. 2, C and
D)
ICa. These
results suggest that the inhibitory effect of
PGE1 on the stimulated
ICa is mediated
by the EP3 receptor.
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Fig. 2.
A and
B: effects of sulprostone, an
EP1 and
EP3 agonist, on Iso-stimulated
ICa. Sulprostone
at 0.1 µM inhibited 30 nM Iso-stimulated
ICa by 24%
(A). Sulprostone at 0.1 µM did not
affect Iso (30 nM)-stimulated
ICa in presence
of PGE1 at 3 µM
(B).
C: no effect of 0.1 µM butaprost, an
EP2 agonist, on Iso (30 nM)-stimulated
ICa.
D: inhibition of Iso-stimulated
ICa by
PGE1 (3 µM), sulprostone (0.1 µM), 11-deoxy-PGE1
(11-dPGE1, 0.1 µM), and
butaprost (0.1 µM); n = 16 for
PGE1, 11 for sulprostone, 6 for
PGE1 + sulprostone, 9 for
11-dPGE1, and 6 for butaprost.
* Significant difference with accuracy of 0.001 (ANOVA); NS, not
significant.
|
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Signal transduction involvement.
The finding that PGE1 inhibited
ICa only when
ICa was
stimulated by Iso indicates the involvement of a cAMP-dependent
pathway. In the experiments shown in Fig.
3, the involvement of cAMP was tested. In
Fig. 3A, the cell was perfused with a
solution containing forskolin (3 µM) and IBMX (100 µM). We
previously showed (7, 33) that this treatment abolished the inhibitory
effect of ACh (10 µM) and ATP (30 µM) on
ICa in rabbit
atrioventricular and atrial cells, respectively. Figure
3A shows that
PGE1 also did not affect the
ICa that was
stimulated by forskolin plus IBMX (6 cells from 5 rabbits). We then
tested the effect of PGE1 on cells
that were dialyzed with 8-BrcAMP (100 µM) using the membrane-ruptured patch-clamp method. A sufficient dialysis was confirmed by applying Iso
(30 nM) in all the cells tested.
PGE1 did not affect the
8-BrcAMP-stimulated ICa (Fig.
3B; 10 cells from 6 rabbits). These
findings clearly indicate a role of cAMP in the inhibitory effects of
PGE1.
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Fig. 3.
A: no effect of
PGE1 in a cell stimulated by
forskolin (3 µM) and IBMX (100 µM). Concentrations of
PGE1 and Iso were 0.1 µM and 30 nM, respectively. B: no effect of
PGE1 in an 8-bromoadenosine
3',5'-cyclic monophosphate (8-BrcAMP)-loaded cell. This
experiment was carried out with the ruptured-patch method. Pipette
contained 8-BrcAMP at 100 µM. Iso (30 nM) and
PGE1 (0.1 µM) were perfused
sequentially. C and
D: effects of
PGE1 and ACh (10 µM) on
Iso-stimulated
ICa. In
C,
PGE1 (0.1 µM) did not show any
additional inhibition to ACh.
ICa amplitudes
with reference to basal
ICa are shown by
bar graph (n = 8). In
D,
PGE1 (3 µM) was applied first,
and subsequent application of ACh inhibited
ICa
(n = 6).
* P < 0.05 (paired
t-test).
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In myocardial cells, some biological agents, such as ACh (4, 12, 25),
adenosine (22), ATP (33), angiotensin II (1, 6), and endothelin-1 (27,
30), are known to regulate the intracellular cAMP concentration through
their specific receptors linking with inhibitory GTP-binding
(Gi) protein. Figure
3C shows that the inhibition of the
Iso-stimulated
ICa by
PGE1 was completely abolished in
the presence of a saturating concentration (10 µM) of ACh (8 cells
from 7 rabbits), suggesting that
PGE1 inhibits ICa using a
common pathway with ACh. However, when the effect of Iso
was antagonized by PGE1 at a
maximal effective concentration (3 µM), additional application of ACh
(10 µM) further reduced ICa (Fig.
3D; 6 cells from 5 rabbits). Thus the
receptor-response coupling seems to be weak in the effects of
PGE1 on
ICa. The
Gi involved in the inhibition of
adenylate cyclase has been consistently shown to be sensitive to PTX.
Figure 4A
shows the effect of PGE1 on
ICa in
PTX-treated cells. The PTX treatment completely abolished the effect of
PGE1 (6 cells from 6 rabbits),
suggesting the involvement of a PTX-sensitive pathway.
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Fig. 4.
A: inhibition of
ICa by
PGE1 sensitive to pertussis toxin
(PTX). Cell was preincubated with PTX (see
METHODS). Concentrations of Iso,
PGE1, and ACh were 30 nM, 0.1 µM
and 1 µM, respectively. B:
PGE1 inhibition of Iso (30 nM)-stimulated
ICa in presence
of methylene blue (MB, 10 µM), erythro-9-(2-hydroxy-3-nonyl)adenine
(EHNA, 30 µM), and KT-5823 (1 µM). None of these compounds
significantly changed control effect of
PGE1 at 0.1 µM (ANOVA);
n = 14 for control, 9 for MB, 6 for
EHNA, and 6 for KT-5823.
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In addition to the Gi-mediated
inhibition of adenylate cyclase, alternative mechanisms have been
suggested for the ACh-induced inhibition of
ICa; these
mechanisms involve activation of cGMP-dependent protein kinase (23) or
activation of cGMP-stimulated (type II) phosphodiesterase (8). To test
the involvement of these alternative pathways, we used methylene blue
(a guanylate cyclase inhibitor), KT-5823 (a cGMP-dependent protein
kinase inhibitor), and EHNA (a type II phosphodiesterase inhibitor).
Figure 4B shows that methylene blue (9 cells from 6 rabbits), KT-5823 (6 cells from 4 rabbits), and EHNA (6 cells from 5 rabbits) did not significantly change the inhibitory
effect of PGE1. Accordingly, the
role of the nitric oxide (NO)-cGMP-dependent pathway seems negligible in the inhibitory actions of PGE1
on the cardiac
ICa.
| |
DISCUSSION |
ICa inhibition by
PGE1 and involvement of EP receptor.
We show here that PGE1 (
0.001
µM) consistently inhibited
ICa in single
cardiac cells when
ICa was
prestimulated by Iso. Sulprostone (EP1 and
EP3 agonist) and
11-dPGE1
(EP3 agonist) inhibited the Iso-stimulated
ICa similarly.
Sulprostone did not affect basal ICa, similar to
PGE1, and showed no additional
effect to PGE1. On the other hand,
the EP2 agonist butaprost did not
mimic PGE1. The presence of the
EP3 receptor in the heart has been
suggested in several mammalian species by Northern blot analyses of the receptor proteins (3, 21). A recent study by Hohlfeld et al. (14)
demonstrated the binding of the
EP3-selective agonist M & B-28767
to the sarcolemmal preparation and the expression of the mRNA of the
EP3 receptor in the pig heart,
whereas no significant expression of the
EP1 receptor was observed in the
heart (3, 32). On the basis of these findings, the
PGE1 inhibition of Iso-stimulated
ICa is likely to
be mediated by EP3 receptors on the myocardial sarcolemma.
Involvement of the cAMP-dependent pathway.
The inhibitory effect of PGE1 on
ICa was very weak
in the absence of Iso. When
ICa was maximally
stimulated by forskolin plus IBMX or by dialysis with 8-BrcAMP,
PGE1 did not affect
ICa. The inhibitory effect of PGE1 was also
abolished when the Iso stimulation of
ICa was almost
fully antagonized by ACh. These results indicate that
PGE1 inhibited Iso-stimulated
ICa via the
cAMP-dependent pathway. Numerous investigators have studied the effects
of PGE1 on myocardium with special
reference to inotropic effects and adenylate cyclase (17, 29).
Depending on the experimental conditions or preparations used,
PGE1 produced either a positive or
no inotropic effect (17, 29). Recently, Hohlfeld et al. (14) clearly
showed the PGE1-induced negative
inotropic effect during infusion with Iso in anesthetized pigs. This
last finding is compatible with our present results showing that
PGE1 has antiadrenergic actions.
In addition to the species-dependent differences in the EP
receptor-response coupling, some of the physiological responses of
myocardium to PGE1 in previous
studies may have been modulated by indirect actions such as
neurotransmitter release (17, 29). The direct effects of
PGE1 on myocardium can be examined
most precisely using isolated single cells, as has been shown in this study.
In contrast with the contradictory reports regarding inotropic effects,
PGE1 has consistently enhanced
adenylate cyclase activity in rat (9, 16, 19), guinea pig (20), kitten
(18), and rabbit (2, 10) cardiac preparations. This enhancement was observed using single cells (2) or homogenized preparations (16, 18),
and thus indirect action was not involved in the PGE1-induced stimulation of
adenylate cyclase. These reports indicated that the
PGE1-induced increase in the
intracellular cAMP concentration does not necessarily lead to a
positive inotropic effect because of compartmentation of cAMP and
cAMP-dependent protein kinase (9, 10, 18, 19). Although we did not
measure the cAMP concentration in this study, Buxton and Brunton (2)
previously showed that PGE1
promptly increased cAMP concentration and stimulated soluble
cAMP-dependent protein kinase in isolated rabbit ventricular cells.
Potentiation of adenylate cyclase by
PGE1 without positive inotropic
response was also observed with perfused rabbit hearts (10). In the
present study, PGE1 stimulation of
adenylate cyclase failed to enhance basal
ICa, presumably
because of the compartmentation of cAMP.
A cAMP-dependent pathway was apparently involved in the
PGE1-induced inhibition of
ICa. This finding
is most plausibly explained by assuming that
PGE1 decreased the intracellular
cAMP concentration that was elevated in advance by Iso, similarly to
ACh. However, biochemical studies have demonstrated that
PGE1 did not inhibit the
Iso-stimulated adenylate cyclase and cAMP-dependent protein kinase in
the particulate fraction (2, 9, 10). It may be that the EP
receptor-stimulated pathway preferably decreased the cAMP concentration
in a space near the membrane Ca2+
channel, so that the biochemical measurements using cell homogenate failed to depict this effect of
PGE1. Besides,
PGE1 may inhibit the
phosphorylated Ca2+ channels
downstream of the cAMP-dependent protein kinase. Although the
cGMP-dependent protein kinase can inhibit the stimulated
ICa, presumably
by phosphorylating the channels (8, 23), the results shown in Fig.
4B clearly indicate that the
cGMP-dependent protein kinase was not involved. Phosphatase may also
contribute to the muscarinic inhibition of
ICa (11).
However, PGE1 did not attenuate ICa when the
channels were phosphorylated in the presence of a sufficient amount of
cAMP (Fig. 3B). Thus the
phosphatase-mediated mechanism plays a small role, if present, in the
PGE1 inhibition of
ICa.
PTX-sensitive inhibition of
ICa.
Hohlfeld et al. (14) reported that
PGE1 stimulated GTPase activity,
and guanosine
5'-O-(3-thiotriphosphate)
inhibited the binding of
[3H]PGE1
to the sarcolemmal preparation, indicating the coupling of
EP3 receptor to G proteins. We
also showed that the pathway involved in the
PGE1 inhibition of
ICa is sensitive
to PTX. It is well known that the PTX-sensitive
Gi is involved in the muscarinic regulation of ICa
in myocardium (5, 12, 25). In addition to the inhibition of adenylate
cyclase (5, 7, 12), the Gi-mediated activation of NO
synthesis has been proposed to contribute to the antiadrenergic effects
of muscarinic agonists by activating cGMP-stimulated phosphodiesterase
or the cGMP-dependent protein kinase. (8, 23). However, other reports
have shown that the inhibitors of the NO-cGMP pathway did not attenuate
the effects of ACh or that NO donors did not mimic muscarinic
stimulation (7, 24, 31, 34). As described above, our present results clearly suggest that the NO-cGMP pathway is not involved in
PGE1 effects on
ICa.
Previous reports including our own have suggested that various
nonmuscarinic receptors are also coupled with
Gi and that their stimulation
inhibits ICa via
the PTX-sensitive and cAMP-dependent pathway. These receptors include
A1 (adenosine; Ref. 22),
P2 (ATP; Ref. 33),
AT1 (angiotensin II; Refs. 1, 6),
and ETA (endothelin-1; Refs, 27, 30) receptors. The physiological role of
Gi activation via these receptors
has not been clarified. One possibility is that its activation serves
as a protective mechanism against excessive contraction of myocardium,
Ca2+ overload under some
pathological conditions, or excessive adrenoceptor stimulation. It must
be noted that PGE1 is released in
the heart only under conditions in which a sufficient amount of
arachidonic and dihomo--linolenic acids are accumulated, i.e.,
ischemia (28). Therefore, the antiadrenergic effects of
PGE1 on
ICa observed in
this study could contribute to the
PGE1-induced myocardial protection
during myocardial ischemia. The ATP-regulated
K+ channel
(KATP) is also suggested to be
involved in the PGE1 protection of
ischemic myocardium in rabbits (13), and the
PGE1-induced activation of
KATP was shown in rat coronary
artery cells (26). Further studies, including studies on the synthesis
of specific EP3 receptor
antagonists, are necessary to determine the physiological and
pathophysiological roles of the PTX-sensitive pathway activated by
PGE1.
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FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: Y. Habuchi,
Dept. of Laboratory Medicine, Kyoto Prefectural Univ. of Medicine,
Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-0841, Japan (E-mail:
yhabuchi{at}koto.kpu-m.ac.jp).
Received 5 December 1998; accepted in final form 26 May 1999.
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REFERENCES |
1.
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