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(Circulation Research. 2003;92:1024.)
© 2003 American Heart Association, Inc.

Cellular Biology

Signal Transduction and Ca²⁺ Signaling in Contractile Regulation Induced by Crosstalk Between Endothelin-1 and Norepinephrine in Dog Ventricular Myocardium

Li Chu, Reiko Takahashi, Ikuo Norota, Takuya Miyamoto, Yasuchika Takeishi, Kuniaki Ishii, Isao Kubota, Masao Endoh

From the Department of Pharmacology (L.C., R.T., I.N., K.I., M.E.) and the First Department of Internal Medicine (T.M., Y.T., I.K.), Yamagata University School of Medicine, Yamagata, Japan.

Correspondence to Masao Endoh, MD, PhD, Department of Pharmacology, Yamagata University School of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585, Japan. E-mail mendou{at}med.id.yamagata-u.ac.jp

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
In certain cardiovascular disorders, such as congestive heart failure and ischemic heart disease, several endogenous regulators, including norepinephrine (NE) and endothelin-1 (ET-1), are released from various types of cell. Because plasma levels of these regulators are elevated, it seems likely that cardiac contraction might be regulated by crosstalk among these endogenous regulators. We studied the regulation of cardiac contractile function by crosstalk between ET-1 and NE and its relationship to Ca²⁺ signaling in canine ventricular myocardium. ET-1 alone did not affect the contractile function. However, in the presence of NE at subthreshold concentrations (0.1 to 1 nmol/L), ET-1 had a positive inotropic effect (PIE). In the presence of NE at higher concentrations (100 to 1000 nmol/L), ET-1 had a negative inotropic effect. ET-1 had a biphasic inotropic effect in the presence of NE at an intermediate concentration (10 nmol/L). The PIE of ET-1 was associated with an increase in myofilament sensitivity to Ca²⁺ ions and a small increase in Ca²⁺ transients, which required the simultaneous activation of protein kinase A (PKA) and PKC. ET-1 elicited translocation of PKC from cytosolic to membranous fraction, which was inhibited by the PKC inhibitor GF 109203X. Whereas the Na⁺-H⁺ exchange inhibitor Hoe 642 suppressed partially the PIE of ET-1, detectable alteration of pH_i did not occur during application of ET-1 and NE. The negative inotropic effect of ET-1 was associated with a pronounced decrease in Ca²⁺ transients, which was mediated by pertussis toxin-sensitive G proteins, activation of protein kinase G, and phosphatases. When the inhibitory pathway was suppressed, ET-1 had a PIE even in the absence of NE. Our results indicate that the myocardial contractility is regulated either positively or negatively by crosstalk between ET-1 and NE through different signaling pathways whose activation depends on the concentration of NE in the dog.

Key Words: endothelin-1 • norepinephrine • myocardial contractility • Ca²⁺ transients • protein kinase C

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
During the course of cardiovascular disorders, such as congestive heart failure and ischemic heart disease, plasma levels of both endothelin-1 (ET-1) and norepinephrine (NE) tend to increase.^1–4 The signal transduction processes that are triggered by the activation of receptors for these endogenous agonists are different, and, thus, it seems likely that crosstalk between ET-1 and NE might play a critical role in the regulation of cardiac function, determining hemodynamic responses to antagonists of ß-adrenoceptors or endothelin receptors under various pathophysiological conditions. The available evidence implies that these endogenous regulators are engaged in crosstalk at different levels of their respective signaling pathways. For example, the positive feedback mechanism seems to exist at the level of the synthesis of NE by which ET-1 increases the plasma concentration of NE,⁵ whereas NE facilitates the expression of mRNA that encodes the prepro-ET-1⁶ and the production of ET-1.⁷

ET-1 has a positive inotropic effect (PIE) in ventricular myocardium of most mammals, but it has no inotropic effect on canine ventricular myocardium.^8,9 By contrast, ET-1 has a negative inotropic effect (NIE) in the presence of catecholamines and antagonizes the ß-adrenoceptor-mediated facilitatory regulation of contractile function in several mammalian species, including the dog.^10–14 Regulation of myocardial contractility induced by crosstalk between ET-1 and NE has not been studied in detail. It has been reported that the acceleration of the hydrolysis of phosphoinositide and the subsequent generation of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol might be responsible for the PIE of ET-1 in certain species,⁸ but the subcellular mechanism involved in the NIE of ET-1 has not been fully elucidated. We designed the present study in an attempt to characterize the contractile regulation induced by crosstalk between ET-1 and NE over a wide range of concentration and its relationship to the regulation of Ca²⁺ signaling in canine ventricular myocardium. We examined subcellular mechanisms responsible for such regulation using selective inhibitors of protein kinases and other types of enzyme. ET-1 had a PIE and NIE, depending on the concentration of NE present before administration of ET-1. The effects were mediated by an increase in the myofilament sensitivity to Ca²⁺ or a decrease in Ca²⁺ transients, which were induced by activation of different signaling pathways in dog ventricular myocardium. Preliminary accounts of this study have been published elsewhere.^13,15–18

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
All manipulations of animals were performed in accordance with the Guide for Animal Experimentation, Yamagata University School of Medicine, and Japanese Governmental Law (No. 105). Approval for all experiments with animals was obtained from the Committee for Animal Experimentation, Yamagata University School of Medicine, before the experiments, and the study was also carried out in accordance with the Helsinki Declaration. Mongrel dogs (7 to 10 kg) of both sexes were used in these experiments, which were performed as described previously.^11,14,19

Isolation and Treatment of Canine Ventricular Trabeculae
The heart was excised, beating was ceased in cold Tyrode solution (7°C) bubbled with 95% CO₂ and 5% O₂, and 2 to 4 thin trabeculae carneae of the right ventricular wall (<1 mm in diameter) were isolated and mounted in 20-mL organ baths that contained Krebs-Henseleit solution.^8,11 The ventricular trabeculae were stimulated electrically with square-wave pulses of 5-ms duration and a voltage that was 20% above the threshold (0.4 V) at a frequency of 0.5 Hz. The average length of muscle preparations was 7.39±0.52 mm, and the average cross-sectional area was 1.43±0.19 mm² (n=165, from a total of 73 dogs).

ET-1 was administered at a single concentration to each muscle preparation. Selective inhibitors were administered 20 to 30 minutes before the addition of ET-1 and were present in the organ bath throughout respective experiments. Pertussis toxin (PTX) at 0.5 µg/mL was allowed to act for 10 hours before experiments were started.

Preparation and Analysis of Canine Ventricular Myocytes
A portion of the free wall of the left ventricle that is supplied via a branch of the left anterior descending artery was excised. The artery was cannulated and perfused with Tyrode’s solution that contained 1.0 mg/mL collagenase and 0.1 mg/mL protease via a recirculating system for 15 to 25 minutes at room temperature (24°C). Then the muscle was perfused with Tyrode’s solution that contained 0.2 mmol/L CaCl₂ and cut into small pieces 3x3 mm² with a scalpel. The resultant cells in suspension were rinsed several times with Tyrode’s solution that contained gradually increasing concentrations of Ca²⁺ up to 1.8 mmol/L.

Procedures used for loading of indo-1, superfusion of myocytes, measurements of fluorescence, and cell length are presented in detail in online data supplement, available at http://www.circresaha.org.

Subcellular Localization of Protein Kinase C Isoforms
The subcellular fractionation procedures, antibodies, and Western blotting techniques are described in the online data supplement.

Statistical Analysis
Experimental values are presented as mean±SE. Significant differences between mean values were estimated by a repeated-measures ANOVA or by Student’s t test with analytic software STATVIEW J-4.5 (Abacus Concepts). P<0.05 was judged to indicate a significant difference.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Influence of NE on the Inotropic Effects of ET-1
Endothelin-1 at 1 to 100 nmol/L did not, by itself, affect the peak twitch force. However, in the presence of NE at the subthreshold concentration of 1 nmol/L, ET-1 at 10 and 100 nmol/L had a definite PIE in association with negative lusitropic and clinotropic (the effect on time to peak tension) effects (data not shown), and these effects of ET-1 were concentration-dependent (Figures 1A and 1B). The EC₅₀ value for ET-1 in the presence of NE at 1 nmol/L was 34.0±5.90 nmol/L (determined in 37 preparations from 14 dogs, including data presented in Reference 15).

View larger version (33K): [in this window] [in a new window]   Figure 1. Inotropic effects of ET-1 in the absence and presence of NE at different concentrations in isolated canine ventricular trabeculae. A and B, PIE of 10 and 100 nmol/L ET-1 in the presence of 1 nmol/L NE. C and D, Inotropic effects of 10 nmol/L ET-1 and their dependence on the concentration of NE applied before ET-1 (C refers to control; no NE applied before ET-1). A, Actual tracings. B, C, and D, Summary of data. Values are mean±SE. Where SE is not shown, it is smaller than the symbol. Numbers in parentheses indicate the numbers of preparations examined. ***P<0.001 vs the force before the addition of ET-1.

NE had a concentration-dependent PIE at 10, 100, and 1000 nmol/L equivalent to 11.5±4.1% (n=5), 80.4±8.4% (n=5), and 248.3±27.4% (n=8) of the basal force, respectively, and the threshold concentration and EC₅₀ value were 3 nmol/L and 0.87±0.09 µmol/L, respectively. When the NE concentration before the administration of ET-1 was increased, the PIE of ET-1 was converted to a NIE, depending on the concentration of NE. In the presence of 10 nmol/L NE, 10 nmol/L ET-1 induced a biphasic inotropic response (ie, a transient NIE followed by a long-lasting PIE); in the presence of NE at higher concentrations (≥100 nmol/L), ET-1 had a definite NIE (Figure 1C). In the presence of NE at 1000 nmol/L, the NIE of ET-1 was markedly reduced, and ET-1 did not have any inotropic effect in the presence of 10 µmol/L NE (Figure 1D).

Regulation of Ca²⁺ Signaling by Crosstalk Between ET-1 and NE
In canine ventricular myocytes, neither ET-1 (10 nmol/L) nor NE (0.1 and 1 nmol/L) by itself affected the cell shortening and Ca²⁺ transients (Figure 2A). When 10 nmol/L ET-1 was administered in the presence of 0.1 nmol/L NE, ET-1 induced an increase in cell shortening (Figures 2A and 2B, bottom) in association with a small increase in Ca²⁺ transients (Figure 2B, top).

View larger version (24K): [in this window] [in a new window]   Figure 2. Increases in cell shortening and in the ratio of indo-1 fluorescence at 405 and 500 nm in response to ET-1 in the presence of NE at a subthreshold concentration in isolated canine ventricular myocytes. A, Actual tracings of the effects of 10 nmol/L ET-1 in the absence (top) and presence (bottom) of 0.1 nmol/L NE. B, Individual signals recorded at times a through c in A (bottom tracings). Individual tracings were obtained by averaging of 5 successive signals. Top, indo-1 fluorescence ratio; bottom, cell shortening. C, Relationship between the maximum ratio of indo-1 fluorescence and maximum shortening in response to ET-1 in the presence of 0.1 nmol/L NE compared with the effects of an increase in [Ca2+]o to 3.6 mmol/L. Basal indicates basal value ([Ca2+]o=1.8 mmol/L) before administration of drugs or elevation of [Ca2+]o. Numbers in parentheses indicate the numbers of cells; vertical and horizontal bars indicate SEM.

The increase in cell shortening induced by 10 nmol/L ET-1 was equivalent to that produced by an increase in extracellular Ca²⁺ concentration ([Ca²⁺]_o) to 3.6 mmol/L. However, the increase in Ca²⁺ transients induced by ET-1 was significantly (P<0.05) smaller than that induced by [Ca²⁺]_o of 3.6 mmol/L (Figure 2C), an indication that the increase in cell shortening induced by ET-1 was attributable, at least in part, to an increase in the myofilament sensitivity to Ca²⁺.

NE at 100 nmol/L induced a pronounced increase in the maximum cell shortening together with a remarkable increase in Ca²⁺ transients. ET-1 markedly decreased the NE-induced increase in cell shortening and Ca²⁺ transients (Figures 3A and 3B). The NE-induced increases in cell shortening and Ca²⁺ transients were inhibited by ET-1 to an essentially similar extent (Figure 3C), and the relationship between the amplitudes of cell shortening and Ca²⁺ transients that was observed with NE alone was unaffected by ET-1 (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 3. Decreases in cell shortening and indo-1 fluorescence ratio in response to ET-1 in the presence of 100 nmol/L NE in isolated canine ventricular myocytes. A, Actual tracings of the effects of 10 nmol/L ET-1 in the presence of 100 nmol/L NE. B, Individual signals recorded at times a through c in A. Individual tracings were obtained by averaging of 5 successive signals. Top, indo-1 fluorescence ratio; bottom, cell shortening. C, Summary of data in A and B. Basal indicates baseline Ca2+ transients and cell shortening before administration of drugs. Numbers in parentheses indicate numbers of cells. ***P<0.001 vs 100 nmol/L NE alone.

In the presence of NE at an intermediate concentration of 10 nmol/L, ET-1 had a biphasic effect, inducing a transient decrease in cell shortening that was associated with a decrease in Ca²⁺ transients, followed by a long-lasting increase in cell shortening that was associated with a statistically insignificant alteration of Ca²⁺ transients (Figures 4A and 4B). Summary of these data are presented in Figure 4C. Our findings indicate that, in the presence of 10 nmol/L NE, the inotropic response to ET-1 involves a combination of facilitatory and inhibitory effects.

View larger version (21K): [in this window] [in a new window]   Figure 4. Biphasic effects of 10 nmol/L ET-1 in the presence of 10 nmol/L NE in isolated canine ventricular myocytes. A, Actual tracings of the effects of ET-1 at 10 nmol/L in the presence of 10 nmol/L NE. B, Individual signals recorded at times a through d in A. Individual tracings were obtained by averaging of 5 successive signals. Top, indo-1 fluorescence ratio; bottom, cell shortening. C, Summary of data in A and B. Basal indicates baseline Ca2+ transients and cell shortening before administration of drugs. Numbers in parentheses indicate numbers of cells. *P<0.05; ***P<0.001 vs 10 nmol/L NE alone.

Similar results were obtained in aequorin-loaded canine right ventricular trabeculae (see the online data supplement).

Signal Transduction Pathway for the ET-1-Induced PIE
Figure 5 shows the effects of selective inhibitors of cAMP-mediated and protein kinase C-mediated (PKC-mediated) pathways on the PIE of ET-1 in the presence of a subthreshold concentration of 1 nmol/L NE. The PIE of ET-1 was abolished by treatment of trabeculae with timolol (1 µmol/L), which blocks ß-adrenoceptors, and with H-89 (1 µmol/L), an inhibitor of protein kinase A (PKA; Figure 5A). These inhibitors, at the concentrations used, did not affect the PIE induced by an elevation of [Ca²⁺]_o (data not shown).

View larger version (33K): [in this window] [in a new window]   Figure 5. Effects of inhibitors of PKA-mediated and PKC-mediated pathways on the PIE of ET-1 in isolated canine ventricular myocardium. A, Effects of treatment with timolol (1 µmol/L), H-89 (1 µmol/L), staurosporine (STS; 10 nmol/L), H-7 (10 µmol/L), and neomycin (NEO; 10 µmol/L) individually on the PIE of ET-1 in the presence of 1 nmol/L NE. ***P<0.001 vs 1 nmol/L NE alone. B, Effects of carbachol (CCh; 0.1 µmol/L) on the PIE of ET-1 in the presence of 1 nmol/L NE. Numbers in parentheses indicate numbers of preparations examined. ***P<0.001 vs 1 nmol/L NE plus 10 nmol/L ET-1.

The inhibitors of PKC, staurosporine (10 nmol/L) and H-7 (10 µmol/L), and an inhibitor of phospholipase C (PLC), neomycin (10 µmol/L), abolished the PIE of ET-1 in the presence of 1 nmol/L NE (Figure 5A). These selective inhibitors, at the concentrations used, did not affect the PIE of NE that was induced by activation of ß-adrenoceptors (data not shown). Carbachol (0.1 µmol/L), which selectively inhibits the cAMP-mediated PIE,²⁰ also reversed the PIE of ET-1 (Figure 5B). These results indicate that the PIE of ET-1 in the presence of NE requires the simultaneous activation of PKA and PKC signaling pathways.

Signal Transduction Pathway for the ET-1-Induced NIE
At concentrations that completely inhibited the PIE of 10 nmol/L ET-1, the inhibitors of PKC and PLC did not suppress but, in fact, enhanced the NIE of ET-1 that was induced in the presence of 100 nmol/L NE (Figure 6A).

View larger version (36K): [in this window] [in a new window]   Figure 6. Effects of inhibitors of PKC and Gi pathways on the NIE of ET-1 in isolated canine ventricular myocardium. A, Effects of treatment with staurosporine (STS; 10 nmol/L), H-7 (10 µmol/L), and neomycin (NEO; 10 µmol/L) individually on the NIE of ET-1 in the presence of 100 nmol/L NE. B, Effects of prior treatment with PTX (0.5 µg/mL), LY83583 (LY; 10 µmol/L), KT5823 (KT; 0.3 µmol/L), and cantharidin (Cant; 10 µmol/L) individually on the NIE of ET-1. The force of contraction before the addition of ET-1 was taken as 100% for each preparation, and changes in the force recorded 20 minutes after the application of ET-1 are expressed as a percentage relative to the maximum force. Numbers in columns indicate numbers of preparations examined. ***P<0.001 vs 100 nmol/L NE alone.

Prior treatment with PTX (0.5 µg/mL) and the treatment with LY83583 (10 µmol/L), an inhibitor of guanylyl cyclase (GC), with KT5823 (0.3 µmol/L), an inhibitor of cGMP-dependent protein kinase (PKG), or with cantharidin (10 µmol/L), an inhibitor of protein phosphatase (PP), almost completely suppressed the NIE of ET-1 in the presence of 100 nmol/L NE (Figure 6B). These selective inhibitors had no effects on the basal force and on the PIE of NE mediated by ß-adrenoceptors (data not shown).

Unmasking of the PIE of ET-1 by Suppression of Inhibitory Pathways
After the pretreatment of trabeculae with PTX (Figure 7A) and the treatment with KT5823 (Figure 7B) or with cantharidin (Figure 7C), ET-1 (10 nmol/L) had a PIE even in the absence of NE.

View larger version (18K): [in this window] [in a new window]   Figure 7. Effects of prior treatment with PTX (0.5 µg/mL) and treatment with KT5823 (KT; 0.3 µmol/L) and cantharidin (Cant; 30 µmol/L) individually on the inotropic effects of ET-1 in the absence of NE in isolated canine ventricular trabeculae. The basal force before the addition of ET-1 was taken as 100% for each preparation, and changes in the force recorded after the application of ET-1 are expressed as a percentage relative to the basal force. Numbers in parentheses indicate numbers of preparations. *P<0.05, **P<0.01, ***P<0.001 vs 10 nmol/L ET-1 alone.

Influence of GF 109203X and Hoe 642 on PKC Translocation
We had examined the subcellular distribution of 4 major PKC isoforms (, ß, , and ) by immunoblotting with the use of isoform-specific antibodies. We found that the dog right ventricle expressed and isoforms, whereas no significant immunoreactivity was detected for ß and . Because the subcellular localization of the PKC isoform did not change in response to pharmacological stimuli, we reported data for the PKC isoform in the present study.

Representative immunoblots of the PKC isoform are shown in Figure 8A. The membrane-associated immunoreactivity was markedly increased in response to phorbol dibutyrate and ET-1+NE. The translocation of PKC to the membranous fraction by ET-1+NE was completely blocked by GF 109203X (a selective PKC inhibitor, 1 µmol/L) but not by Hoe 642 (a Na⁺/H⁺ inhibitor, 1 µmol/L) (Figure 8B). Under the same experimental condition, Hoe 642 suppressed significantly (P<0.05) the PIE induced by combination of ET-1 with NE by 40% (control, 151±5.6%; Hoe 642, 132±4.8%; n=4 each).

View larger version (16K): [in this window] [in a new window]   Figure 8. A, Representative immunoblots of the PKC isoform in canine ventricular trabeculae. Positions of the molecular mass markers (in kDa) are indicated on the left. B, Quantitative group data for PKC translocation. Data are reported as mean±SEM and were obtained from 5 separate experiments. *P<0.01 vs control, #P<0.01 vs ET-1+NE. Details in the experimental procedures are described in the online data supplement.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we demonstrated that the extent and quality of the inotropic effects of ET-1 in canine ventricular myocardium are determined by the concentration of simultaneously applied NE, with the functional outcome of coupling subsequent to stimulation of ET receptors being dependent, apparently, on the extent of ß-stimulation by NE. The regulation of contraction induced by ET-1 involves activation of PKC, of a G_s protein-coupled cAMP/PKA pathway, and of a PTX-sensitive G_i protein-coupled cGMP/PKG/PP pathway.

By itself, ET-1 did not induce any inotropic response, but it had a definite PIE in the presence of NE at 0.1 to 1 nmol/L, which did not significantly affect the basal force. The PIE of ET-1 was converted to a NIE when the concentration of NE was increased. ET-1 had a prominent NIE in the presence of NE at 100 nmol/L and higher, and these observations are essentially consistent with previous reports that ET-1 attenuated the PIE and positive chronotropic effect of ß-stimulation in the dog,¹¹ rat,²¹ and guinea pig.²² Our results indicate that in the presence of NE, positive and negative inotropic responses compete with one another and that, in the presence of NE at high concentrations, the PIE of ET-1 is completely replaced by a NIE. This crosstalk between ET-1 and NE might contribute significantly to the variable inotropic responses to ET-1; in previous studies, ET-1 had no effect,^8,9,23 a PIE,^4,8 and a NIE,^24–26 including a time-dependent component to these phenomena.²⁷

Differential Regulation of Ca²⁺ Signaling by ET-1
The PIE of ET-1 in the presence of a threshold concentration of NE was associated with a small increase in Ca²⁺ transients. This increase was significantly smaller than that induced by an increase in [Ca²⁺]_o that elicited a PIE equivalent to that of ET-1 (Figure 2). These observations imply that the PIE of ET-1 is associated definitively with an increase in the myofilament sensitivity to Ca²⁺, being consistent with previous findings with ET-1 in other species.^19,28–30 Because ET-1 did not have a PIE in the presence of inotropic interventions, such as dihydroouabain or an increase in [Ca²⁺]_o,¹³ ET-1 might require a weak ß-stimulation for induction of its PIE. This synergistic action of ET-1 and NE indicates that there is a critical difference in the regulation between the dog and other mammals.^8,19,29–31 In mice, ß-stimulation and ET-1 regulate cardiac contractility in opposite directions in part through phosphorylation of troponin I on distinct sites,³² indicating that the phosphorylation of contractile proteins plays a crucial role in the regulation, the role of which could not be determined in the present study.

In contrast to the PIE, the NIE of ET-1 was accompanied by a pronounced decrease in Ca²⁺ transients (Figure 3), which may play a key role in the NIE of ET-1. We investigated in dog ventricular myocytes that ET-1 inhibited significantly the increase in L-type Ca²⁺ current (I_Ca) induced by isoproterenol.¹⁴ Because the inhibitory action of ET-1 on the isoproterenol-induced increase in I_Ca was suppressed by the treatment with PTX in rabbit myocytes, the inhibition of the cAMP-mediated increase in I_Ca via the PTX-sensitive inhibitory pathway activated by ET-1 might contribute, to some extent, to the ET-1-induced decrease in Ca²⁺ transients. Involvement of effects on other processes, such as PKA and SR Ca²⁺ release, however, is not excluded.

Present observations with indo-1-loaded myocytes are consistent with findings obtained with aequorin-loaded dog ventricular trabeculae (online data supplement).¹⁸

Subcellular Mechanisms for the PIE of ET-1
Neomycin, staurosporine, and H-7, at a concentration that did not affect the PIE of NE mediated by ß-adrenoceptors, abolished the PIE of ET-1. Furthermore, timolol and H-89 completely suppressed the PIE of ET-1 (Figure 5A), whereas carbachol reversed the PIE of ET-1 (Figure 5B). These observations together imply that the ET-1-induced PIE and increase in the myofilament sensitivity to Ca²⁺ require the simultaneous activation of PKC and PKA.

Although the Na⁺-H⁺ exchange inhibitor Hoe 642 inhibited partially the PIE of ET-1, we could not detect an appreciable alteration of pH_i in single myocytes loaded with the fluorescent pH_i probe SNARF-1 (online data supplement).

We found recently that the Ca²⁺-sensitizing actions of OR-1896 and levosimendan were abolished by carbachol,^33–35 an indication that a Ca²⁺-sensitizing mechanism might exist that requires accumulation of cAMP. Although myosin-binding protein C, which is phosphorylated via a cAMP/PKA signaling pathway, might be a candidate for the source of increased sensitivity to Ca²⁺,³⁶ the target proteins responsible for such a cAMP-mediated increase in Ca²⁺-sensitivity remain to be identified.

The selectivity of staurosporine and H-7 was checked by comparing the concentration-dependent effect of these agents on the - and ß-mediated PIE,^37,38 which is discussed in detail in online data supplement.

Treatment with cantharidin and with KT5823 and pretreatment with PTX unmasked the PIE of ET-1 even in the absence of NE. These findings indicate that the signaling process that leads to activation of G_i-proteins, PKG, and phosphatases might be highly effective even in the baseline state, counteracting the G_q-/G_s-mediated PIE via suppression of cAMP-mediated signaling and leading to the absence of a PIE of ET-1; ie, ET-1 stimulates both G_q and G_i proteins, accounting for the activation of facilitative and inhibitory (Figure 7).

Subcellular Mechanisms for the NIE of ET-1
The selective inhibitors that abolished the PIE enhanced the NIE of ET-1 (Figure 6), an indication that the NIE of ET-1 is mediated by signaling processes that are different from those involved in the induction of the PIE of ET-1. Because the NIE of ET-1 was almost completely inhibited by pretreatment with PTX, the accumulation of cAMP induced by ß-stimulation might be suppressed by the G_i-mediated deactivation of adenylyl cyclase. However, this scenario is unlikely, because the NIE of ET-1 is not accompanied by a significant reduction in the accumulation of cAMP that is mediated by ß-stimulation.^10,11,39 Although ß₂-adrenoceptors that are coupled to G_i proteins are activated by NE⁴⁰ and could come into play in the NIE of ET-1 in the presence of high concentrations of NE, the findings with ET-1 in the presence of various ß-adrenoceptor agonists, including the ß₂-selective agonist zinterol, exclude the essential role of ß₂-subtype in the NIE of ET-1⁴¹ (online data supplement).

Both LY83583, an inhibitor of GC, and KT5823, an inhibitor of PKG, suppressed the NIE of ET-1, an indication that G_i-coupled cGMP/PKG signaling might be responsible for the inhibitory action of ET-1, whereas nitric oxide is not involved in such regulation.^10,11 It has been reported that G_i-coupled receptors, such as muscarinic M₂⁴² and adenosine A₁ receptors,⁴³ counteract the effect of activation of PKA, in part via the activation of PP: the muscarinic agonists acetylcholine and carbachol inhibit protein phosphatase inhibitor-1 (PPI-1) through G_i-mediated stimulation of the PP activity with resultant dephosphorylation of a variety of functional proteins that are phosphorylated by PKA.^44–47 The muscarinic inhibition could occur without a concomitant decrease in the cAMP content or in the PKA activity.^45,46 Our observation that an inhibitor of PP, cantharidin, abolished the NIE of ET-1 is consistent with the results of previous investigations described above. In this context, it is noteworthy that the NIE of ET-1 is more susceptible to cantharidin than the NIE of carbachol and the PIE of NE mediated by ß-adrenoceptors,⁴⁸ an indication that the differential effect of cantharidin on various signaling processes is exerted in dog ventricular myocardium.

In summary, ET-1 has a positive effect, biphasic effect, or NIE, depending on the extent of ß-stimulation in canine ventricular myocardium. The PIE of ET-1 might be attributable to activation of the PLC/PKC pathway, requiring simultaneous activation of the process that is mediated by cAMP. By contrast, the NIE of ET-1 can be ascribed to activation of phosphatases via G_i-coupled cGMP/PKG signaling. The PIE was associated with an increase in the myofilament sensitivity to Ca²⁺, whereas the NIE was attributable to a decrease in Ca²⁺ transients. The crosstalk between ET-1 and NE might play a crucial role in the regulation of myocardial contractility under pathophysiological conditions that are associated with elevated plasma levels of endogenous regulators.

	Acknowledgments

 
This work was supported in part by Grants-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by a Research Grant for Cardiovascular Disease (11-1) from the Ministry of Health and Welfare, Japan.

	Footnotes

 
This manuscript was sent to Richard A. Walsh, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Original received February 18, 2002; resubmission received February 14, 2003; revised resubmission received March 24, 2003; accepted March 27, 2003.

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