© 1995 American Heart Association, Inc.
Articles |
Negative Chronotropic Effect of Endothelin 1 Mediated Through ETA Receptors in Guinea Pig Atria
From the Division of Chemical Pharmacology and Phytochemistry (K.O.), National Institute of Health Sciences, Tokyo, Japan; the Division of Molecular and Cellular Pharmacology (A.S., K.S., T.S., G.T.), National Children's Medical Research Center, Tokyo, Japan; the Department of Pharmacology (T.M.), Faculty of Medicine, Kyoto (Japan) University; and the Department of Pharmacology (K.E., K.H.), Yamanashi (Japan) Medical University.
Correspondence to Kageyoshi Ono, PhD, Division of Chemical Pharmacology and Phytochemistry, National Institute of Health Sciences, Setagaya-ku, Tokyo 158, Japan.
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Abstract Endothelins exert potent excitatory cardiac effects by acting on specific receptors on myocytes. In this study, we have examined the signal transduction mechanism for the chronotropic effect of endothelins in guinea pig atria. A competition binding of [125I]endothelin 1 ([125I]ET-1) using the recently developed ETA receptor–selective antagonist BQ123 showed the presence of almost equal populations of ETA (44%) and ETB (56%) receptors in the guinea pig right atria. In a concentration-response study, endothelin 3 (ET-3), an agonist with higher affinity to ETB receptors than to ETA receptors, and sarafotoxin S6c (STXS6c), an ETB receptor–selective agonist, increased the rate of spontaneous beating at all concentrations tested (10 pmol/L to 100 nmol/L). In contrast, ET-1, a nonselective agonist, increased the heart rate at lower concentrations (10 pmol/L to 10 nmol/L) but decreased it at higher concentrations (30 to 100 nmol/L). When ET-1 (100 nmol/L) was applied in a single amount, heart rate was strongly increased; however, this increase was followed by a rapid decline in the response. ET-1 (100 nmol/L) but not ET-3 or STXS6c significantly reduced the heart rate when it was raised by isoproterenol (ISO, 300 nmol/L) either in the absence or presence of a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Correspondingly, ET-1 significantly reduced the ISO-induced elevation of cAMP accumulation (19.1±1.7 pmol/mg protein [n=8] and 12.6±1.2 pmol/mg protein [n=7] in the absence and presence of ET-1, respectively; P<.01), which was also observed even in the presence of IBMX. Treatment with BQ123 (1 µmol/L) abolished these inhibitory effects of ET-1 on both the chronotropy and the cAMP accumulation. Also, pretreatment of guinea pigs with pertussis toxin (5 µg/100 g body wt IV) abolished the inhibitory effects of ET-1. These data showed that ETA receptors are involved in an inhibitory cardiac action of endothelins, which is coupled to a pertussis toxin–sensitive G protein/adenylate cyclase inhibition pathway. This ETA receptor–mediated inhibitory action gives new insights into understanding physiological and pathophysiological modulations of cardiac functions by endothelins.
Key Words: endothelin A receptor • negative chronotropic effect • cAMP • pertussis toxin • guinea pig atria
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Introduction |
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The endothelin family, consisting of endothelin 1, 2, and 3 (ET-1, -2, and -3, respectively), has a variety of biological activities in many tissues.1 This diversity in the pharmacological actions of endothelins (ETs) has been attributed to a wide distribution of ET receptors in different cells that involves at least two types of ET receptors, namely ETA and ETB receptors.1 2 3 4 In the heart, the existence of both subtypes of ET receptors has been demonstrated in the rat, chick, and human by using radioligand binding experiments.5 6 7 8 Furthermore, a recent in situ hybridization study has directly demonstrated that both of these two subtypes are coexpressed in rat myocardial cells.9 Functionally, ETs have been shown to have potent positive inotropic and chronotropic effects,5 10 11 12 13 14 15 16 and the activation of phosphatidylinositol response has been implicated to be closely linked to their cardiac actions.15 16 17 18 On the other hand, a recent study has demonstrated that ETs inhibit cAMP production in the heart,18 which could oppose or offset the cardiotonic effect of the peptide. However, very little information is available regarding the physiological roles or the coupling with the ET receptor subtypes of these intracellular pathways in the heart.
The aim of the present study was to examine the signal transduction mechanism of ET-induced cardiac chronotropic effects, with particular emphasis on the roles of each receptor subtype and the cAMP-dependent pathway. Studying chronotropic effects of ETs in the guinea pig atria, we found that ETA receptors mediate an inhibitory component of the peptide-induced chronotropic effect, which negatively modulates the positive chronotropic effects of the peptide itself as well as other neurotransmitters. Also, we have characterized the role of cAMP for this ETA receptor–mediated inhibitory action. The physiological and pathophysiological roles of the ET-induced cardiac inhibitory action are discussed.
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Materials and Methods |
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Materials
ET-1, ET-3, and sarafotoxin S6c (STXS6c) were purchased from Peptide Institute Inc. Cyclo(D-Trp-D-Asp-L-Pro-D-Val-L-Leu) (BQ123) was kindly provided by Banyu Pharmaceutical Co Ltd. (-)-Isoproterenol (+)-bitartrate (ISO), acetylcholine chloride (ACh), (±)-propranolol HCl, phentolamine HCl, atropine sulfate, phenylmethylsulfonyl fluoride (PMSF), thymidine, dithiothreitol, 3-isobutyl-1-methylxanthine (IBMX), and aprotinin were purchased from Sigma Chemical Co. Pertussis toxin (PTX) was obtained from Funakoshi Co Ltd. cAMP measurement kits were purchased from Yamasa Co Ltd. [125I]ET-1 and [32P]NAD were purchased from Amersham Co. ET-1, ET-3, STXS6c, and BQ123 were dissolved in a phosphate-buffered saline (pH 7.4) containing 0.5% bovine serum albumin (BSA). All other chemicals were of reagent grade.
Organ Bath Experiments
Adult male guinea pigs (Hartley) weighing 300 to 400 g were
deeply anesthetized with phenobarbitone (50 mg/kg body wt IP). The
heart was dissected out after a thoracotomy, and the right atrium was
isolated quickly. No special treatment to remove the endocardium was
given. Preparations were suspended with thread at a resting tension of
0.5 g in siliconized glass organ baths containing a Krebs-Ringer
solution (20 mL) of the following composition (mmol/L): NaCl 113, KCl
4.8, CaCl2 2.2, KH2PO4 1.2,
MgSO4 1.2, NaHCO3 25, and
D-glucose 5.5. The solution was maintained at 37°C and
bubbled with 95% O2/5% CO2.
Spontaneous contractions were measured with a force-displacement
transducer (Nihon Kohden TB-611T) connected to an amplifier (Nihon
Kohden AP-601G). The analog data were put into a microcomputer-aided
data acquisition system (Mac Lab, Analog Digital Instruments), and the
heart rate was counted by using an on-line data analysis program
(CHART, Analog Digital Instruments). All preparations
were allowed to equilibrate for at least 1 hour before drug
applications. All organ bath experiments were performed in the presence
of 1 µmol/L phentolamine, 1 µmol/L propranolol, and 1 µmol/L
atropine, unless otherwise noted.
[125I]ET-1 Binding Studies
Membrane fractions were prepared from a batch of 12 hearts from
adult guinea pigs according to the method by Ishikawa et
al,5 with a modification, and were used immediately in the
receptor binding assays. Briefly, the hearts were separated into right
atria, left atria, and ventricles, and materials were homogenized
(Polytron, at 80% of maximum speed, 20 seconds) in an ice-cold buffer
A of the following composition: 10 mmol/L MOPS, 0.25 mol/L sucrose,
0.1% BSA, 0.1 mmol/L PMSF, and 100 U/mL aprotinin (pH 7.2). The
homogenates were centrifuged at 1000g for 10 minutes twice,
and then the supernatant was centrifuged at 35 000g for 30
minutes. The resultant pellet was resuspended in buffer A containing 10
mmol/L MgCl2.
Binding assays were routinely performed in siliconized glass tubes in a
total volume of 250 µL. Each assay tube contained 200 µL of
membrane preparation (
50 µg of protein), 25 µL of
[125I]ET-1 (100 pmol/L), and 25 µL of BQ123 at various
concentrations. The incubations (25°C, 2 hours) were stopped by
diluting with a cold buffer (10 mmol/L Tris-HCl containing 0.5% BSA,
pH 7.4), and the samples were filtered through Whatman GF/C filters
presoaked in 0.1% BSA. After four washes (5 mL each time) with the
same buffer, the radioactivity remaining on the filter was determined
in a gamma radiation counter at an efficiency of 70%. All assays
were conducted in duplicate. The protein concentration was measured by
using the bicinchoninic acid protein assay kit19 (Pierce)
with BSA used as a standard.
Data from the competition binding studies were analyzed by using a nonlinear regression program (LIGAND, Biosoft Co). The presence of one, two, or three different binding sites was assessed by using the F test in the program. The model adopted was that which provided the significantly best fit (P<.05). Since [125I]ET-1 dissociates from its binding sites with an exceedingly low dissociation rate constant,20 Ki values calculated from models that are based on the equilibrium process (such as LIGAND analysis) may not represent the theoretical ones. Therefore, in the present study Ki values calculated by LIGAND were treated as "apparent" Ki.21 22
Measurements of cAMP
Segments of atria were incubated in a Tyrode's solution (pH
7.4) of the following composition (mmol/L): NaCl 135, KCl 5.4,
D-glucose 5.5, HEPES 5.0, CaCl2 1.8,
MgCl2 0.5, and NaH2PO4 0.33,
bubbled with 100% O2 for 1 hour at 37°C before
experimental manipulations began. One set of samples was collected 30
seconds after incubation with ISO (300 nmol/L), at which time the heart
rate usually reaches the maximal elevation. Another set of samples was
collected after the incubation with ISO for 30 seconds and an
additional 4-minute incubation with ET-1 (100 nmol/L) in the presence
of ISO, at which time reduction of heart rate by ET-1 had reached a
steady level. The preparations were quickly frozen in liquid nitrogen
and homogenized in ice-cold 6% trichloroacetic acid. The homogenates
were centrifuged at 2000g for 10 minutes at 4°C, and the
supernatants were extracted five times with 5 vol of water-saturated
ether. cAMP was measured by radioimmunoassay.23 Another
set of experiments was performed with IBMX (10 µmol/L) used as a
pretreatment for 10 minutes and present throughout the drug
treatments.
Treatment With PTX and ADP-Ribosylation
PTX was injected via the penile vein at 5 µg/100 g body wt
under anesthesia with ether, and 60 to 72 hours later, atrial tissues
were collected and assayed. The efficiency of the in vivo treatment
with PTX was assessed both by abolishment of the response to ACh in
organ bath experiments and by in vitro ADP-ribosylation of cardiac
membranes. For the ADP-ribosylation assay, materials were homogenized
(Polytron, at 80% of maximum speed, 20 seconds) in a cold sucrose
buffer (mmol/L: sucrose 250, Tris-HCl 15, MgCl2 1, and
aprotinin 1, pH 7.4). Then the membrane fraction was collected in the
same way as for the binding assay and was used for
[32P]ADP-ribosylation with preactivated PTX, following
the method of Bokoch et al.24 Briefly, membrane
preparations were incubated with [32P]NAD (10 µmol/L,
5000 to 15 000 cpm/pmol) in the presence of 100 mmol/L Tris-HCl (pH
8.0), 10 mmol/L thymidine, 1 mmol/L ATP, 100 µmol/L GTP, 2.5 mmol/L
MgCl2, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, and
50 µg PTX for 30 minutes at 30°C. At the end of the labeling
period, samples were diluted fivefold into Laemmli sample
buffer25 and were incubated to stop the reaction at 99°C
for 1 minute. Sodium dodecyl sulfate–polyacrylamide gel
electrophoresis and autoradiography were carried out by the method of
Schleifer et al.26 Autoradiography revealed a single
32P-labeled band at Mr of
40 000. 32P content was quantified by BAS 2000 (Fuji Co
Ltd).
Statistics
Values are expressed as mean±SEM. A two-way ANOVA with 95%
confidence limits, followed by a Student's t test on
individual sets of data, was performed by using analytical software
STATVIEW 512+ (BrainPower Inc).
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Results |
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Receptor Binding Assay
Previous studies have shown the coexistence of both ETA and ETB receptors in rat hearts.5 9 To our knowledge, however, no information is available about the relative proportion of the ET receptor subtypes in guinea pig hearts. Before examining the chronotropic effect of ET-1, we determined the relative proportion of the ETA and the ETB receptors in the right and left atrial and ventricular membranes in the guinea pig by using a recently developed ETA receptor–selective antagonist, BQ123,27 to compete with [125I]ET-1 binding. The competition curves for BQ123 suggested the presence of two binding sites with different affinities in all these three preparations (Fig 1
). Because of an exceedingly slow dissociation rate of
ET-1, calculated Ki values were treated as
"apparent" Ki (see "Materials and
Methods").20 21 22 The best fit was by a model having a
high-affinity site for BQ123 with an apparent Ki
of 1 nmol/L and an 1000-fold lower affinity site for BQ123 with
an apparent Ki of 6 to 8 µmol/L; these sites
are thus considered to be the ETA receptors and
ETB receptors, respectively (Table). As
summarized in the Table, the relative proportion of these two affinity
sites varied between atrium and ventricle. From the binding study, we
confirmed that ETA receptors can be selectively blocked by
BQ123 at a concentration of 1 µmol/L, which we used in the following
studies.
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Role of ETA Receptors in the Chronotropic Effect
In spontaneously beating right atria, ET-1, ET-3, and STXS6c all
produced positive chronotropic actions. The threshold concentration of
ET-1 for the positive chronotropic effect was 0.1 to 1 nmol/L, and
the maximum increase was obtained at 10 nmol/L. Increasing
concentrations of ET-1 (up to 100 nmol/L), however, produced consistent
reduction in the heart rate (Fig 2A). In contrast, ET-3
and STXS6c produced only positive chronotropic responses in a
concentration-dependent manner, and they showed no inhibitory effects
even at higher concentrations (Fig 2B and 2C). The threshold
concentrations of ET-3 and STXS6c necessary to elicit the chronotropic
responses were 10 to 100 pmol/L.
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, 
, and
) or presence (
and 
) of BQ123 for 10 minutes, and
then ETs or STXS6c was added. By increasing the concentration of the
peptide stepwise, fractional increases in HR were measured. Values are
expressed by regarding the maximal increase in HR in each tissue as
100%: the maximal increase in HR by ET-1, ET-3, and STXS6c was 63±3.0
(n=5), 50±5.7 (n=5), and 32±2.0 (n=5), respectively. Each point
represents mean±SEM obtained from five different experiments.
*P<.05 vs BQ123-untreated group. Note that ET-1 at a high
concentration in the absence of BQ123 produced a significant reduction
in HR (A), whereas ET-3 (B) and STXS6c (C) did not.
Our finding that the inhibitory effect was characteristic only for ET-1
(Fig 2A
) suggested that this inhibitory effect may be mediated by
ETA receptors.1 2 22 This was further
confirmed by examining the effect of the ETA
receptor–selective antagonist BQ123. Treatment with BQ123 (1 µmol/L)
significantly potentiated the positive chronotropic effects of ET-1 at
lower concentrations and abolished the inhibitory effect of the peptide
at higher concentrations (Fig 2A); thus, ET-1 strongly increased the
heart rate even at high concentrations in the presence of BQ123. In
contrast, the concentration-response curve of ET-3 was not affected at
any concentrations examined in the presence of BQ123 (Fig 2B).
Chronotropic Effects of High Concentrations of ET-1
In the course of our concentration-response studies, it was noted
that the ET-1–induced increase in the heart rate at each concentration
gradually declined after reaching a peak response (Fig 2A). In
contrast, the heart rate response by either ET-3 or STXS6c was
sustained at any concentration examined (Fig 2B and 2C). The reduction
in heart rate in the presence of ET-1 was evident especially at high
concentrations. In the following study, therefore, we used relatively
high concentrations of ET-1 to highlight the inhibitory component
involved in the effect of the peptide. We first examined whether a high
concentration of ET-1 by itself causes such an inhibitory chronotropic
response when added as a single amount rather than cumulatively. As
shown in Fig 3A, a single application of ET-1 (100
nmol/L) caused a small initial drop and then a rapid increase in heart
rate. After reaching the peak response, however, a rapid decrease in
heart rate followed. Treatment with BQ123 (1 µmol/L) markedly
inhibited both the initial drop and the later decline in the heart
rate, without significantly affecting the magnitude of the peak
response (Fig 3A and 3B).
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Using the same time course as that used for the chronotropic response,
we measured intracellular cAMP contents after the single application of
ET-1 (100 nmol/L). As shown in Fig 3C, ET-1 did not increase the cAMP
content 2 minutes after the application irrespective of the presence of
BQ123, whereas it markedly elevated the heart rate by this time,
suggesting that the positive chronotropic effect of ET-1 may not depend
on the elevation of cAMP content. BQ123 (1 µmol/L) did not alter the
basal cAMP content. In the presence of BQ123, ET-1 also did not affect
the cAMP content during the course of the 10-minute experiment (Fig 3C). However, the cAMP content measured 10 minutes after the ET-1
application was significantly (P<.01) lower when BQ123 was
absent in the solution than when it was present; the cAMP contents were
4.5±0.3 pmol/mg protein (n=5) and 7.0±0.3 pmol/mg protein (n=5) in
the absence and presence of BQ123, respectively (Fig 3C).
Effects of ET-1 on ISO-Induced Responses
Next, we examined whether ET-1 produces an inhibitory effect on
heart rate when it was elevated in a cAMP-dependent manner by
stimulating ß-adrenergic receptors. Application of ISO (300 nmol/L)
rapidly increased heart rate, and this increase was sustained for more
than 5 minutes (Fig 4A); the spontaneous reduction in
heart rate measured 4.5 minutes after the application of ISO was only
3.1±0.8% of the maximum response (n=5). Addition of ET-1 (100 nmol/L)
30 seconds after the application of ISO, on the other hand,
significantly decreased heart rate (Fig 4); the reduction 4 minutes
after the application of the peptide was 16.4±2.0% of the maximum
response (n=6, P<.01). Pretreatment with BQ123 (1 µmol/L,
10 minutes) abolished this ET-1–induced inhibitory response; the
reduction in heart rate measured 4 minutes after the application of
ET-1 was only 0.5±0.2% (n=4) (Fig 4C). In contrast, neither ET-3 (100
nmol/L, data not shown) nor STXS6c (100 nmol/L) showed this inhibitory
effect (Fig 4B and 4C).
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The inhibitory effect of ET-1 was also observed even in the presence of
IBMX (Fig 5). Application of IBMX (10 µmol/L) by
itself caused a significant elevation of heart rate, and addition of
ISO (300 nmol/L) further increased heart rate to almost maximal values.
In this condition, addition of ET-1 (100 nmol/L) noticeably decreased
heart rate up to 10 minutes (Fig 5B).
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Effect of ET-1 on ISO-Induced cAMP Response
The tissue cAMP content was increased rapidly by ISO (300 nmol/L)
and remained elevated for at least 4.5 minutes (19.1±1.7 pmol/mg
protein, n=8, Fig 6A). When ET-1 (100 nmol/L) was added
30 seconds after the application of ISO, it caused a significant
(P<.01) reduction in the cAMP content; the nucleotide
content measured 4 minutes after ET-1 administration was 12.6±1.2
pmol/mg protein (n=7, Fig 6A). Consistent with the finding on the
chronotropic effect of ET-1 (Fig 4A), pretreatment with BQ123 (1
µmol/L, 10 minutes) abolished the inhibitory effect of ET-1 on the
ISO-stimulated accumulation of cAMP (ISO, 16.3±2.1 pmol/mg protein
[n=5]; ISO+ET-1, 18.1±1.5 pmol/mg protein [n=9]; Fig 6B). BQ123 by
itself did not significantly affect either the basal level (control,
6.2±0.6 pmol/mg protein [n=6]; BQ123 pretreatment, 5.6±0.5 pmol/mg
protein [n=3]) or the ISO-induced elevation of the cAMP content
(control, 19.1±1.7 pmol/mg protein [n=8]; BQ123 pretreatment,
16.3±2.1 pmol/mg protein [n=5]; Fig 6B).
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Furthermore, this inhibitory effect of ET-1 was not affected even under
blockade of phosphodiesterases with IBMX (10 µmol/L); thus, the
tissue cAMP content at 4.5 minutes after application of 300 nmol/L ISO
was significantly (P<.01) lowered by the addition of ET-1
(100 nmol/L) (ISO, 79.8±10.0 pmol/mg protein [n=7]; ISO+ET-1,
45.9±4.8 pmol/mg protein [n=5]; Fig 6C). Pretreatment with BQ123 (1
µmol/L, 10 minutes) abolished the inhibitory effect of ET-1 on the
ISO-stimulated cAMP accumulation (ISO, 77.3±11.7 pmol/mg protein
[n=7]; ISO+ET-1, 77.5±6.7 pmol/mg protein [n=8]; Fig 6D). These
results showed that the ET-1–induced reduction in heart rate was
mediated by the inhibition of cAMP production but not by the
stimulation of cAMP degradation.
Effect of PTX Treatment
We further examined a possible involvement of G protein in the
ET-1–induced reductions in both heart rate and cAMP production by
using PTX. As shown in Fig 7A, treatment with PTX
potentiated the positive chronotropic effects of ET-1 and abolished the
peptide-induced inhibitory chronotropic response observed at higher
concentrations. Also, PTX treatment resulted in almost complete loss of
the ET-1–induced inhibition of the ISO-induced cAMP production (Fig 7B). In constructing concentration-response curves to ET-1, it was
noted that a rapid decline in heart rate following the application of
ET-1 observed in the PTX-untreated atria (Figs 2 and 3) was markedly
lost in the PTX-treated preparations (data not shown). This PTX-induced
change in the heart rate response was quite similar to that observed in
the presence of BQ123.
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We assessed the efficiency of PTX treatment both by confirming the
attenuation of the negative chronotropic effect of ACh (1 µmol/L) and
by measuring the in vitro ADP-ribosylation of the cardiac membrane. In
the PTX-untreated atrium, ACh significantly reduced the basal heart
rate by 36.6±5.7% (n=5), whereas it decreased the heart rate by only
1.5±1.6% (n=5) in the PTX-treated preparation. Electrophoresis on a
polyacrylamide gel of the membrane proteins obtained from PTX-untreated
atria revealed a single band with Mr of
40 000, into which the in vitro ADP-ribosylation procedure
incorporated radiolabeled ADP (Fig 8). This in vitro
ADP-ribosylation of the protein was markedly reduced by 78.8±7.3%
(n=5) in atria obtained from PTX-treated animals.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ETs are now known to exert potent positive inotropic and chronotropic effects in the heart of various species including the human.1 10 11 12 13 14 15 16 17 28 It has been suggested that these cardiac actions of ETs are linked to phosphatidylinositol hydrolysis/Ca2+ signaling15 16 17 29 and cAMP metabolism18 through specific receptors. Recent evidence has shown the presence of subtypes ETA and ETB for ET receptors.1 2 3 4 22 Moreover, it has recently been shown in the rat heart that these two subtypes coexist in single myocytes.9 However, little is known about either the role of each receptor subtype in the cardiac action of ETs or the association of these subtypes with intracellular signal transduction pathways. In the present study, we demonstrated that the cardiotonic effects of ET-1 on guinea pig atria are offset by an inhibitory component. Using ETA receptor–selective antagonist BQ123,27 we found that both ETA and ETB receptors coexist in this preparation, as shown in binding studies, and that the inhibitory effect of ET-1 was mediated mainly through ETA receptors. Furthermore, our data showed that this ETA receptor–mediated inhibitory effect is closely associated with the inhibition of cAMP accumulation and involves the PTX-sensitive G protein, probably Gi. The positive component of the ET-induced chronotropic response, on the other hand, was resistant to BQ123 and did not accompany alterations in the tissue cAMP content, indicating that it is mediated through ETB receptors, which may mainly couple with a cAMP-independent pathway. Taken together, the data showed that ETs exert cardiac actions that are a net result of the complex interactions of these intracellular mechanisms linked to each ET receptor subtype.
Presently, at least two kinds of cDNA clones of ET receptors have been identified. One type, ETA receptors, has a high affinity for ET-1 but a low affinity for ET-3,2 whereas the other type, ETB receptors, has equal affinity for both ET-1 and ET-3.3 4 30 A previous binding study suggested that these two ET receptor subtypes coexist in the rat heart.5 Moreover, the heart is the only organ so far that is known to coexpress both receptor subtypes in a single cell, as revealed by an in situ hybridization study.9 Recently, a cyclic pentapeptide, BQ123, which selectively binds to the ETA receptor and blocks ET-1–induced responses, has been developed27 31 and was established as a potent and highly selective ETA receptor antagonist.22 27 Our binding study showed that guinea pig atria contained almost equal populations of high- and low-affinity sites for BQ123, and their apparent Ki values are in good agreement with those of cloned receptors expressed in cell lines22 ; thus, they are considered to be ETA and probably ETB receptors, respectively. Among these two receptor subtypes, we found that ETA receptors predominantly mediate the ET-1–induced inhibitory responses, since it was induced only by ET-1 but not by ET-3 or STXS6c and abolished by BQ123. The present study was the first to show the negative functional role of ETA receptors in the heart.
ETA receptors are generally known to be linked to an increase in cytosolic free calcium ions in a variety of systems,1 22 which is closely related to the activation of phospholipase C and mediated by PTX-insensitive G proteins.1 18 Also, ETA receptors stimulate the formation of cAMP through the stimulatory G protein, Gs, in Chinese hamster ovary cells stably expressing cloned ETA receptors.32 In clear contrast to these systems, our present study showed that stimulation of ETA receptors inhibited cAMP accumulation in guinea pig atria through activation of PTX-sensitive G proteins. This seemed to be closely related to the ET-1–induced negative chronotropic response. The reduction in cAMP content is mediated through the inhibition of cAMP production but not through facilitation of its breakdown, since pretreatment with IBMX did not affect the ET-1–induced inhibitory response. A similar observation of the ET-1–induced inhibition of adenylate cyclase in a recent report using rat ventricular myocytes18 supports our findings.
The ETA receptor–mediated inhibition of cAMP production
seems to be functionally operating, inhibiting the heart rate raised by
the cAMP-dependent or -independent mechanism. The inhibitory effect of
ET-1 was evident not only when heart rate was raised by stimulating the
ß-adrenoceptor/cAMP pathway but also when heart rate was increased by
ET-1 itself. Consistent with these results, ET-1 decreased not only
ISO-stimulated cAMP production but also the basal cAMP content. On the
other hand, the ET-1–induced potent positive chronotropic effect is
possibly mediated through an ETB receptor–mediated
cAMP-independent mechanism, since it was not sensitive to BQ123
pretreatment and also was not associated with any change in tissue cAMP
content (Fig 3). However, in the later phase of the ET-1–induced heart
rate response, the rapid decline in heart rate seems to be closely
associated with the ETA receptor–mediated reduction in
cAMP content, which was noted in preparations not treated with BQ123
(Fig 3B and 3C). These results indicate that this ETA
receptor–mediated cAMP inhibition may act to lower heart rate whenever
it is raised; hence, this ETA receptor–mediated inhibitory
action seems to be potentially important in assessing the modulatory
role of ETs in the regulation of cardiac chronotropism. The relative
contribution of the ETA receptor–mediated inhibitory
action may explain part, if not all, of the marked species
differences33 34 and developmental changes5
in the cardiac effects of ETs.
The negatively regulatory effect of ETs seems to be important in pathophysiological conditions in particular, since plasma levels of ETs have been noted to be increased in patients with stresses such as cardiac infarction and cardiogenic shock and in patients undergoing surgical procedures, conditions in which an increase in the sympathetic tone is common.1 Therefore, it may be conceivable that ETs play a regulatory role directly on the cardiac muscle when the sympathetic nervous system is activated. In fact, Reid and colleagues33 35 recently observed that ET-1 decreases inotropic and chronotropic responses induced by sympathetic nerve stimulation without significantly affecting the nerve excitation–induced release of norepinephrine, suggesting that ETs modulate the cardiac response at postjunctional sites. Our data have provided more direct evidence that ET-1 modulates cardiac function postjunctionally and showed further that stimulation of the ETA receptor causes a reduction in cAMP production, thereby inhibiting the ß-adrenergic receptor–stimulated increase in heart rate. Hence, this ETA receptor–mediated inhibitory pathway seems to be potentially important for the modulation by ETs of sympathetic regulation of cardiac function in various stressful conditions.
We have recently shown that ET-1 hyperpolarizes the resting membrane potential and shortens the duration of the action potential by stimulating the muscarinic potassium current [IK(ACh)] and inhibiting the L-type calcium current (ICaL) in adult guinea pig atrial myocytes.36 Similar electrophysiological observations on both the ICaL37 and IK(ACh)38 have been reported recently in cultured neonatal cardiac myocytes. Since ICaL in the heart is regulated by intracellular cAMP39 40 and the activation of the heterotrimeric G protein leads to the direct activation of a certain population of ion channels responsible for the regulation of heart rate, such as IK(ACh),41 42 43 44 the ETA-receptor–mediated activation of the Gi/cAMP inhibition pathway that we found in the present study may cause the negative chronotropic effect of ET-1 through these electrophysiological mechanisms. Further studies of other ionic currents important for pacemaker activity, such as the hyperpolarization-activated current45 46 and the T-type calcium current47 in the pacemaker cells, would be required before the ionic mechanisms involved in the cardiac actions of ET-1 are fully understood.
In conclusion, the present study has shown and highlighted an inhibitory component that is involved in the ET-1–induced positive chronotropic effects. This negatively modulatory effect is mainly mediated through the ETA receptor subtype, which is coupled to the PTX-sensitive G protein (probably Gi), leading to the changes in the intracellular second-messenger cAMP. The activation of the ETA receptor/Gi protein pathway seems to lead to the direct and/or indirect modulation of cardiac ion channels. This negatively modulatory effect is particularly evident when the cardiac excitatory state is elevated and may be a potentially important regulatory mechanism in a variety of physiological and pathophysiological settings. Further work on the regulation and "cross-talk" interactions of these ET receptor subtype–mediated intracellular signaling mechanisms should provide valuable insights concerning the physiological regulation of the cardiac functions by ETs.
Note added in proof. After this work was submitted, a short paper by Vogelsang, Broede-Sitz, Schäfer, Zerkowski, and Brodde was published in J Cardiovasc Pharmacol (1994;23:344-347), reporting an ETA receptor–mediated inhibition of adenylate cyclase in human right atrium.
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Acknowledgments |
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This study was supported in part by a research grant for the encouragement of young researchers, Ministry of Science and Education, Japan. The authors thank Japan Human Science Foundation for financial assistance. We also thank Drs Mitsuo Yano and Masaki Ihara of Banyu Pharmaceutical Co Ltd for kindly providing BQ123 and Dr K. Horie and M. Ueda for their technical help in the ADP-ribosylation assay.
Received February 22, 1994; accepted October 21, 1994.
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