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
https://doi.org/10.1161/01.RES.0000070595.10196.CF
Circulation Research. 2003;92:1024-1032
Originally published May 16, 2003

Jump to

Abstract

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 PKCε 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 pHi 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.

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 β-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 β-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

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 ≈3×3 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

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).

Figure1

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).

Figure2

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).

Figure3

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.

Figure4

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 β-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).

Figure5

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 β-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).

Figure6

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 β-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.

Figure7

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 PKCε Translocation

We had examined the subcellular distribution of 4 major PKC isoforms (α, β, δ, and ε) by immunoblotting with the use of isoform-specific antibodies. We found that the dog right ventricle expressed α and ε isoforms, whereas no significant immunoreactivity was detected for β and δ. Because the subcellular localization of the PKCα isoform did not change in response to pharmacological stimuli, we reported data for the PKCε isoform in the present study.

Representative immunoblots of the PKCε 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 PKCε 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).

Figure8

Figure 8. A, Representative immunoblots of the PKCε isoform in canine ventricular trabeculae. Positions of the molecular mass markers (in kDa) are indicated on the left. B, Quantitative group data for PKCε 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

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 β-stimulation by NE. The regulation of contraction induced by ET-1 involves activation of PKC, of a Gs protein-coupled cAMP/PKA pathway, and of a PTX-sensitive Gi 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 β-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 β-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, β-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 (ICa) induced by isoproterenol.14 Because the inhibitory action of ET-1 on the isoproterenol-induced increase in ICa was suppressed by the treatment with PTX in rabbit myocytes, the inhibition of the cAMP-mediated increase in ICa 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 β-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 pHi in single myocytes loaded with the fluorescent pHi 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 α- and β-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 Gi-proteins, PKG, and phosphatases might be highly effective even in the baseline state, counteracting the Gq-/Gs-mediated PIE via suppression of cAMP-mediated signaling and leading to the absence of a PIE of ET-1; ie, ET-1 stimulates both Gq and Gi 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 β-stimulation might be suppressed by the Gi-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 β-stimulation.10,11,39 Although β2-adrenoceptors that are coupled to Gi 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 β-adrenoceptor agonists, including the β2-selective agonist zinterol, exclude the essential role of β2-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 Gi-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 Gi-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 Gi-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 β-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 β-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 Gi-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.

References

  1. ↵
    Minami M, Yasuda H, Yamazaki N, Kojima S, Nishijima H, Matsumura N, Togashi H, Koike Y, Saito H. Plasma norepinephrine concentration and plasma dopamine-β-hydroxylase activity in patients with congestive heart failure. Circulation. 1983; 67: 1324–1329.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Wei CM, Lerman A, Rodeheffer RJ, McGregor CG, Brandt RR, Wright S, Heublein DM, Kao PC, Edwards WD, Burnet JC Jr. Endothelin in human congestive heart failure. Circulation. 1994; 89: 1580–1586.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    McMurray JJ, Ray SG, Abdullah I, Dargie HJ, Morton JJ. Plasma endothelin in chronic heart failure. Circulation. 1992; 85: 1374–1379.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Sakai S, Miyauchi T, Sakurai T, Kasuya T, Ihara M, Yamaguchi I, Goto K, Sugishita Y. Endogenous endothelin-1 participates in the maintenance of cardiac function in rat with congestive heart failure: marked increase in endothelin-1 production in the failing heart. Circulation. 1996; 93: 1214–1222.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Miller WL, Redfield MM, Burnett JC. Integrated cardiac, renal, and endocrine action of endothelin. J Clin Invest. 1989; 83: 317–320.
    OpenUrlCrossRefPubMed
  6. ↵
    Masaki T, Kimura S, Yanagisawa M, Goto K. Molecular and cellular mechanism of endothelin regulation: implication for vascular function. Circulation. 1991; 84: 1457–1468.
    OpenUrlFREE Full Text
  7. ↵
    Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 1988; 332: 411–415.
    OpenUrlCrossRefPubMed
  8. ↵
    Takanashi M, Endoh M. Characterization of the positive inotropic effect of endothelin on mammalian ventricular myocardium. Am J Physiol. 1991; 261: H611–H619.
    OpenUrlPubMed
  9. ↵
    Banyasz T, Magyar J, Kortvely A, Szigeti G, Szigligeti P, Papp Z, Mohacsi A, Kovacs L, Nanasi PP. Different effects of endothelin-1 on calcium and potassium currents in canine ventricular cells. Naunyn Schmiedebergs Arch Pharmacol. 2001; 363: 383–390.
    OpenUrlCrossRefPubMed
  10. ↵
    Zhu Y, Yang HT, Endoh M. Does nitric oxide contribute to the negative chronotropic and inotropic effects of endothelin-1 in the heart? Eur J Pharmacol. 1997; 332: 195–199.
    OpenUrlCrossRefPubMed
  11. ↵
    Zhu Y, Yang HT, Endoh M. Negative chronotropic and inotropic effects of endothelin isopeptides in mammalian cardiac muscle. Am J Physiol. 1997; 273: H119–H127.
    OpenUrlPubMed
  12. ↵
    Watanabe T, Endoh M. Characterization of the endothelin-1 induced regulation of L-type Ca2+ current in rabbit ventricular myocytes. Naunyn Schmiedebergs Arch Pharmacol. 1999; 360: 654–664.
    OpenUrlCrossRefPubMed
  13. ↵
    Chu L, Endoh M. Biphasic inotropic effects of endothelin-1 in the presence of sympathomimetic drugs at different concentrations in canine ventricular myocardium. Jpn J Pharmacol. 2000; 82 (suppl I): 198P.Abstract.
    OpenUrl
  14. ↵
    Watanabe T, Endoh M. Antiadrenergic effects of endothelin-1 on L-type Ca2+ current in canine ventricular myocytes. J Cardiovasc Pharmacol. 2000; 36: 344–350.
    OpenUrlCrossRefPubMed
  15. ↵
    Chu L, Endoh M. Biphasic inotropic response to endothelin-1 in the presence of various concentrations of norepinephrine in dog ventricular myocardium. J Cardiovasc Pharmacol. 2000; 36 (suppl 2): S9–S14.
    OpenUrlCrossRef
  16. ↵
    Endoh M, Chu L, Takahashi R, Norota I. Regulation of Ca2+ signaling by cross talk of endothelin-1 and norepinephrine. Jpn J Pharmacol. 2001; 85 (suppl I): 20P.Abstract.
    OpenUrlCrossRef
  17. ↵
    Chu L, Endoh M. Regulation of Ca2+ signaling induced by endothelin-1 in indo-1 loaded canine ventricular myocytes. Jpn J Pharmacol. 2001; 85 (suppl I): 83P.Abstract.
    OpenUrlCrossRef
  18. ↵
    Takahashi R, Chu L, Endoh M. Dual inotropic responses to endothelin-1 in the presence of high or low concentrations of norepinephrine involve differential regulation of Ca2+ signaling in aequorin-loaded canine right ventricular myocardium. Jpn J Pharmacol. 2001; 85 (suppl I): 81P.Abstract.
    OpenUrl
  19. ↵
    Yang HT, Sakurai K, Sugawara H, Watanabe T, Norota I, Endoh M. Role of Na+/Ca2+ exchange in endothelin-1-induced increases in Ca2+ transient and contractility in rabbit ventricular myocytes: pharmacological analysis with KB-R7943. Br J Pharmacol. 1999; 126: 1785–1795.
    OpenUrlCrossRefPubMed
  20. ↵
    Endoh M. Correlation of cyclic AMP and cyclic GMP levels with changes in contractile force of dog ventricular myocardium during cholinergic antagonism of positive inotropic actions of histamine, glucagon, theophylline and papaverine. Jpn J Pharmacol. 1979; 29: 855–864.
    OpenUrlPubMed
  21. ↵
    Reid JJ, Lieu AT, Rand MJ. Interactions between endothelin-1 and other chronotropic agents in rat isolated atria. Eur J Pharmacol. 1991; 194: 173–181.
    OpenUrlCrossRefPubMed
  22. ↵
    Reid JJ, Wong-Dusting HK, Rand MJ. The effect of endothelin on noradrenergic transmission in rat and guinea-pig atria. Eur J Pharmacol. 1989; 168: 93–96.
    OpenUrlCrossRefPubMed
  23. ↵
    Volkmann R, Bokvist K, Wennmalm A. Endothelin has no positive inotropic effect in guinea-pig atria or papillary muscle. Acta Physiol Scand. 1990; 138: 345–348.
    OpenUrlPubMed
  24. ↵
    Teerlink JR, Loffler BM, Hess P, Maire JP, Clozel M, Clozel JP. Role of endothelin in the maintenance of blood pressure in conscious rats with chronic heart failure: acute effects of the endothelin receptor antagonist Ro 47-0203 (Bosentan). Circulation. 1994; 90: 2510–2518.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Shimoyama H, Sabbah HN, Borzak S, Tanimura M, Shevlygin S, Scicli G, Goldstein S. Short-term hemodynamic effects of endothelin receptor blockade in canines with chronic heart failure. Circulation. 1996; 94: 779–784.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Spinale FG, Walker JD, Mukherjee R, Iannini JP, Keever AT, Gallagher KP. Concomitant endothelin receptor subtype-A blockade during the progression of pacing-induced congestive heart failure in rabbits. Circulation. 1997; 95: 1918–1929.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Pi YQ, Streekumar R, Huang XP, Walker JW. Positive inotropy mediated by diacylglycerol in rat ventricular myocytes. Circ Res. 1997; 81: 92–100.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Wang JX, Paik G, Morgan JP. Endothelin-1 enhances myofilament Ca2+-responsiveness in aequorin-loaded ferret myocardium. Circ Res. 1991; 69: 582–589.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Fujita S, Endoh M. Effects of endothelin-1 on [Ca2+]i-shortening trajectory and Ca2+ sensitivity in rabbit single ventricular cardiomyocytes loaded with indo-1/AM: comparison with the effects of phenylephrine and angiotensin II. J Card Fail. 1996; 2: S45–S57.
    OpenUrlCrossRefPubMed
  30. ↵
    Kelly RA, Eid H, Kramer BK, O’Neill M, Liang BT, Reers M, Smith TW. Endothelin enhances the contractile responsiveness of adult rat ventricular myocytes to calcium by a pertussis toxin-sensitive pathway. J Clin Invest. 1990; 86: 1164–1171.
    OpenUrlCrossRefPubMed
  31. ↵
    Yang HT, Zhu Y, Endoh M. Species-dependent differences in inotropic effects and phosphoinositide hydrolysis induced by endothelin-3 in mammalian ventricular myocardium. Br J Pharmacol. 1997; 120: 1497–1504.
    OpenUrlCrossRefPubMed
  32. ↵
    Pi YQ, Kemnitz KR, Zhang D, Kranias EG, Walker JW. Phosphorylation of troponin I controls cardiac twitch dynamics: evidence from phosphorylation site mutants expressed on a troponin I-null background in mice. Circ Res. 2002; 90: 649–656.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Sato S, Hasson Talukder MA, Sugawara H, Sawada H, Endoh M. Effects of levosimendan on myocardial contractility and Ca2+ transient in aequorin-loaded right-ventricular papillary muscles and indo-1-loaded single ventricular cardiomyocytes of the rabbit. J Mol Cell Cardiol. 1998; 30: 1115–1128.
    OpenUrlCrossRefPubMed
  34. ↵
    Takahashi R, Hasson Talukder MA, Endoh M. Effects of OR-1896, an active metabolite of levosimendan, on contractile force and aequorin light transient in intact rabbit ventricular myocardium. J Cardiovasc Pharmacol. 2000; 36: 118–125.
    OpenUrlCrossRefPubMed
  35. ↵
    Takahashi R, Hasson Talukder MA, Endoh M. Inotropic effects of OR-1896, an active metabolite of levosimendan, on canine ventricular myocardium. Eur J Pharmacol. 2000; 400: 103–112.
    OpenUrlCrossRefPubMed
  36. ↵
    Winegrad S. Cardiac myosin binding protein C. Circ Res. 1999; 84: 1117–1126.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Endoh M, Norota I, Takanashi M, Kasai H. Inotropic effects of staurosporine, NA 0345 and H-7, protein kinase C inhibitors, on rabbit ventricular myocardium: selective inhibition of the positive inotropic effect mediated by α1-adrenoceptor. Jpn J Pharmacol. 1993; 63: 17–26.
    OpenUrlPubMed
  38. ↵
    Hasson Talukder MA, Endoh M. Differential effects of protein kinase C activators and inhibitors on α- and β-adrenoceptor-mediated positive inotropic effect in isolated rabbit papillary muscle. J Cardiovasc Pharmacol Ther. 1997; 2: 159–170.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Walker CA, Ergul A, Zile MR, Zellner JL, Crumbley AJ, Spinale FG. β-Adrenergic and endothelin receptor interaction in dilated human cardiomyopathic myocardium. J Card Fail. 2001; 7: 129–137.
    OpenUrlCrossRefPubMed
  40. ↵
    Xiao RP, Avdonin P, Zhou YY, Cheng H, Akhter SA, Eschenhagen T, Lefkowitz RJ, Koch WJ, Lakatta EG. Coupling of β2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes. Circ Res. 1999; 84: 43–52.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Chu L, Endoh M. Biphasic inotropic effects of endothelin-1 in the presence of sympathomimetic drugs at different concentrations in canine ventricular myocardium. Jpn J Pharmacol. 2000; 82 (suppl I): 198P.Abstract.
    OpenUrl
  42. ↵
    Gupta RC, Neumann J, Boknik P, Watanabe AM. M2-specific muscarinic cholinergic receptor-mediated inhibition of cardiac regulatory protein phosphorylation. Am J Physiol. 1994; 266: H1138–H1144.
    OpenUrlPubMed
  43. ↵
    Narayan P, Mentzer RM Jr, Lasley RD. Phosphatase inhibitor cantharidin blocks adenosine A1 receptor anti-adrenergic effect in rat cardiac myocytes. Am J Physiol. 2000; 278: H1–H7.
    OpenUrl
  44. ↵
    Ahmad Z, Green F, Subuhi HS, Watanabe AM. Autonomic regulation of type 1 protein phosphatase in cardiac muscle. J Biol Chem. 1989; 264: 3859–3863.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    Gupta RC, Neumann J, Watanabe AM. Comparison of adenosine and muscarinic receptor-mediated effects on protein phosphatase inhibitor-1 activity in the heart. J Pharmacol Exp Ther. 1993; 266: 16–22.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    Sakai R, Shen JB, Pappano AJ. Elevated cAMP suppresses muscarinic inhibition of L-type calcium current in guinea pig ventricular myocytes. J Cardiovasc Pharmacol. 1999; 34: 304–315.
    OpenUrlCrossRefPubMed
  47. ↵
    Herzig S, Meier A, Pfeiffer M, Neumann J. Stimulation of protein phosphatases as a mechanism of the muscarinic-receptor-mediated inhibition of the cardiac L-type Ca2+ channels. Pflugers Arch. 1995; 429: 531–538.
    OpenUrlCrossRefPubMed
  48. ↵
    Chu L, Norota I, Ishii K, Endoh M. Inhibitory action of the phosphatase inhibitor cantharidin on the endothelin-1induced and the carbachol-induced negative inotropic effect in the canine ventricular myocardium. J Cardiovasc Pharmacol. 2003; 41 (suppl 1) S89–S92.
    OpenUrlCrossRefPubMed
View Abstract

This Issue

Jump to

Article Tools

Share this Article

  • Email
  • 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 and Masao Endoh
    Circulation Research. 2003;92:1024-1032, originally published May 16, 2003
    https://doi.org/10.1161/01.RES.0000070595.10196.CF
    del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Related Articles

Cited By...

Subjects