The heart and blood vessels express a range of anion currents (e.g. ICl.PKA) and symporter/antiporters (e.g. Cl(-)/HCO3(-) exchanger) that translocate chloride (Cl(-)). They have been proposed to contribute to a variety of physiological processes including cellular excitability, cell volume homeostasis and apoptosis. Additionally there is evidence that Cl(-) currents or transporters may play a role in cardiac pathophysiology. Arrhythmogenesis, the process of cardiac ischaemic preconditioning, and the adaptive remodelling process in myocardial hypertrophy and heart failure have all been linked to such channels or transporters. We have explored the possibility that selective targeting of one or more of these may provide benefit in cardiovascular disease. Existing evidence points to an emerging role of cardiac cell anion channels as potential therapeutic targets, the 'disease-specificity' of which may represent a substantial improvement on current targets. However, the limitations of current techniques hitherto applied (such as developmental compensation in gene-modified animals) and pharmacological agents (which do not at present possess sufficient selectivity for the adequate probing of function) have thus far hindered translation to the introduction of new therapy.

Enteric neural crest-derived cells (ENCCs) migrate from the anterior foregut in a rostrocaudal direction to colonize the entire gastrointestinal tract and to form the enteric nervous system. Genetic approaches have identified many signaling molecules regulating the migration of ENCCs; however, it remains elusive how the activities of the signaling molecules are regulated spatiotemporally during migration. In this study, transgenic mice expressing biosensors based on Förster resonance energy transfer were generated to video the activity changes of the signaling molecules in migrating ENCCs. In an organ culture of embryonic day 11.25 (E11.25) to E13 guts, ENCCs at the rostral wavefront migrated as a cellular chain faster than the following ENCCs that formed a network. The faster-migrating cells at the wavefront exhibited lower protein kinase A (PKA) activity than did the slower-migrating trailing cells. The activities of Rac1 and Cdc42 exhibited an inverse correlation with the PKA activity, and PKA activation decreased the Rac1 activity and migration velocity. PKA activity in ENCCs was correlated positively with the distribution of GDNF and inversely with the distribution of endothelin 3 (ET-3). Accordingly, PKA was activated by GDNF and inhibited by ET-3 in cultured ENCCs. Finally, although the JNK and ERK pathways were previously reported to control the migration of ENCCs, we did not find any correlation of JNK or ERK activity with the migration velocities. These results suggest that external cues regulate the migration of ENCCs by controlling PKA activity, but not ERK or JNK activity, and argue for the importance of live imaging of signaling molecule activities in developing organs.

Endothelin-1 (ET-1) is a locally acting vasoactive peptide that also has profound effects on the contractile properties and growth of the cardiac myocyte. Binding of ET-1 to its transmembrane heptahelical receptors activates G proteins of the G(q) and G(i) classes. Activation of G(q) stimulates hydrolysis of phosphatidylinositol-4,5-bisphosphate, and the diacylglycerol thus formed stimulates protein kinase C. Subsequently, the protein kinase Raf is activated and this leads to activation of the extracellular signal-regulated protein kinase (ERK) subfamily of mitogen-activated protein kinases. Activation of G(i) counteracts β-adrenoceptor-mediated increases in cAMP concentrations. We have attempted to rationalize the established physiological consequences of ET-1 agonism in the cardiac myocyte (that is, on contraction and growth) in terms of activation of these signaling pathways.

Cardiac myocytes express protein kinase A-dependent Cl(-) (Cl(PKA)) channels that are thought to represent cardiac expression of CFTR. In the present study, the 'Smart' patch clamp technique was used to investigate the distribution of Cl(PKA) channels at the cell surface of isolated guinea-pig ventricular myocytes. Imaging the cell surface using scanning ion conductance microscopy allowed the identification of the mouths to t-tubules and lateral z-grooves with a spacing of 1.86 microm. Cell-attached patch clamp recordings were made from specified locations within the imaged area. Perfusion of the cells with an activating cocktail of isoprenaline (5 microM), forskolin (10 microM) and isobutylmethylxanthine (50 microM) activated large, noisy anion-selective currents in which unitary channel currents could not be identified. Currents were recorded both from within z-grooves and in the inter-groove region but not at the mouths of t-tubules. Power spectral and noise analyses indicated the involvement of 13.5pS channels occurring in clusters of >50 channels. Channel activity was lost on excision of the patch from the cell but could be recovered in inside-out excised patches by application of the catalytic subunit of PKA. These results suggest that CFTR Cl(PKA) channels occur in clusters in the sarcolemma of guinea-pig ventricular myocytes; there was no evidence of a heterogeneous distribution of clusters between the z-grooves and the inter-groove region.

The endothelin axis promotes vasoconstriction, suggesting that antagonists of endothelin signaling might be useful in treatment of heart failure. However, promising results from animal trials have not been recapitulated in heart failure patients. Here we review the role of major signaling pathways in the heart that are involved in cell survival initiated by ET-1. These pathways include mitogen-activated protein kinase, phosphatidyl inositol-1,4,5-triphosphate kinase (PI3K-AKT), nuclear factor-kappaB (NF-kappaB), and calcineurin signaling. A better understanding of endothelin-mediated signaling in cardiac cell survival may allow a reevaluation of endothelin receptor antagonists (ETRAs) in the treatment of heart failure.

Endothelin-1 (ET-1) is a peptide hormone produced within the myocardium which may modulate myocardial contractility in a paracrine-autocrine fashion. In the majority of species, ET-1 has a direct positive inotropic effect on the myocardium that involves both increased myofilament Ca(2+) sensitivity and increased Ca(2+) transients. Ca(2+) entry through reverse-mode Na(+)-Ca(2+) exchange, involving both indirect effects via elevation of intracellular [Na(+)] and direct activation of the Na(+)-Ca(2+) exchanger, have been suggested to contribute to the increase in Ca(2+) transients. Conversely, mouse cardiomyocytes show an exclusively negative inotropic response to ET-1. Here, Nishimaru and colleagues present novel evidence that the negative inotropic effect of ET-1 in mouse cardiomyocytes involves both a reduction in myofilament Ca(2+) sensitivity and increased Ca(2+) extrusion, via Na(+)-Ca(2+) exchange. Data obtained using the selective Na(+)-Ca(2+) exchange blocker, SEA0400, suggest that a re-assessment of the role of the exchanger in Ca(2+)-handling by mouse cardiomyocytes may be necessary.

Tumor necrosis factor-alpha (TNF-alpha) affects contractility and ionic currents in the heart. However, the electrophysiological effects, especially on delayed rectifier K currents (IK), have not yet been fully elucidated. We examined the effects of TNF-alpha on IK. Using a voltage-clamp method, IK was measured in guinea pig ventricular myocytes in the basal state and after pharmacological intervention. To specify the site of the action of TNF-alpha, the myocytes were incubated with pertussis toxin or N-oleoylethanolamine, a ceramidase inhibitor, and IK was measured. TNF-alpha suppressed IK when it was enhanced by isoproterenol, histamine or forskolin but not in the basal state or when IK was augmented by an internal application of cyclic AMP. Both pre-incubation with pertussis toxin and N-oleoylethanolamine abolished the inhibitory action of TNF-alpha on isoproterenol-augmented IK. TNF-alpha inhibits IK, mainly IKs, when it is augmented by PKA as a result of the generation of sphingosine.

The endothelin (ET) system consists of 3 ET isopeptides, several isoforms of activating peptidases, and 2 G-protein-coupled receptors, ETA and ETB, that are linked to multiple signaling pathways. In the cardiovascular system, the components of the ET family are expressed in several tissues, notably the vascular endothelium, smooth muscle cells, and cardiomyocytes. There is general agreement that ETs play important physiological roles in the regulation of normal cardiovascular function, and excessive generation of ET isopeptides has been linked to major cardiovascular pathologies, including hypertension and heart failure. However, several recent clinical trials with ET receptor antagonists were disappointing. In the present review, the authors take the stance that ETs are mainly and foremost essential regulators of cardiovascular function, hence that antagonizing normal ET actions, even in patients, will potentially do more harm than good. To support this notion, we describe the predominant roles of ETs in blood vessels, which are (indirect) vasodilatation and ET clearance from plasma and interstitial spaces, against the background of the subcellular mechanisms mediating these effects. Furthermore, important roles of ETs in regulating and adapting heart functions to different needs are addressed, including recent progress in understanding the effects of ETs on diastolic function, adaptations to changes in preload, and the interactions between endocardial-derived ET-1 and myocardial pump function. Finally, the potential dangers (and gains) resulting from the suppression of excessive generation or activity of ETs occurring in some cardiovascular pathological states, such as hypertension, myocardial ischemia, and heart failure, are discussed.

The enteric nervous system (ENS) in vertebrates is derived mainly from vagal neural crest cells that enter the foregut and colonize the entire wall of the gastrointestinal tract. Failure to completely colonize the gut results in the absence of enteric ganglia (Hirschsprung's disease). Two signaling systems mediated by RET and EDNRB have been identified as critical players in enteric neurogenesis. We demonstrate that interaction between these signaling pathways controls ENS development throughout the intestine. Activation of EDNRB specifically enhances the effect of RET signaling on the proliferation of uncommitted ENS progenitors. In addition, we reveal novel antagonistic roles of these pathways on the migration of ENS progenitors. Protein kinase A is a key component of the molecular mechanisms that integrate signaling by the two receptors. Our data provide strong evidence that the coordinate and balanced interaction between receptor tyrosine kinases and G protein-coupled receptors controls the development of the nervous system in mammals.

Endothelin-1 (ET-1) exerts many biological effects in airways, including bronchoconstriction, airway mucus secretion, cell proliferation, and inflammation. We investigated the effect of ET-1 on Na absorption and Cl secretion in human bronchial epithelial cells. Addition of 10(-7) M ET-1 had no effect on the inhibition of the short circuit current (Isc) induced by amiloride, a Na channel blocker. Addition of 10(-7) M ET-1 to the apical bath in the presence of amiloride increased Isc in cultured human bronchial epithelial cells studied in Ussing chambers. No effect was observed when ET-1 was added to basolateral bath, indicating that the involved ET-1 receptors are likely present only in the apical membrane of the cells. Use of Cl-free solutions and bumetanide reduced the ET-1-induced increases in Isc, indicating that ET-1 stimulates Cl secretion. The ET-1-induced increase in Isc was prevented by exposure to the ETB receptor antagonist BQ-788 but not to the ETA receptor antagonist BQ-123. ET-1 did not raise intracellular Ca levels, but increased the intracellular concentration of cAMP. These findings indicate that ET-1 is a Cl secretagogue in human airways and acts presumably through apically located ETB receptors and activation of the cAMP pathway.

The purpose of this study was to test the hypothesis that tumor necrosis factor-alpha (TNF-alpha) rapidly antagonizes the beta-adrenergic responses of the chloride current and to clarify the intracellular mechanisms responsible for the anti-adrenergic action. The whole-cell patch-clamp technique was used to monitor the anti-adrenergic effects of TNF-alpha on the cAMP-dependent chloride current (I(Cl)) recorded from isolated guinea-pig ventricular myocytes. Ramp pulses (+/-120 mV; dv/dt = +/-0.4 V/s) were applied from the holding potential of -40 mV. TNF-alpha rapidly (<15 min) inhibited the isoproterenol (Iso, 0.1 micromol/L)-induced I(Cl) in a concentration-dependent manner (30-1,000 U/ml, IC (50) = 144 U/ml, n=30). The inhibitory action of TNF-alpha was also observed when I(Cl) had been previously stimulated by 1 micromol/L forskolin (n=5). Prior exposure of myocytes to 5 microg/ml pertussis toxin (PTX) hardly affected the anti-adrenergic action of TNF-alpha (n=4). However, when I(Cl) was induced by both 8-bromo-cAMP (100 micromol/L) and isobutylmethylxanthine (0.1 mmol/L), TNF-alpha (1,000 U/ml) failed to decrease I(Cl) amplitude (n=5). Prior exposure of myocytes to 5 mg/ml pertussis toxin (PTX) hardly affected the anti-adrenergic action of TNF-alpha (n=4). Furthermore, despite of the presence of nitro-L-arginine methyl ester (0.1 mmol/L), a nitric oxide synthase (NOS) inhibitor, TNF-alpha reversed the Iso-induced increase in I(Cl) (n=5). These results suggest that TNF-alpha rapidly antagonizes the beta-adrenergic responses of I(Cl) by reducing cAMP concentration. This anti-adrenergic action is mediated by neither the PTX-sensitive G proteins regulatory pathway nor constitutive NOS activation.

Endothelin-1 (ET-1) and nitric oxide (NO) exert opposite effects in the cardiovascular system, and there is evidence that the NO counters the potential deleterious effects of ET-1. We investigated whether NO affects the increased mRNA expression of ET-1 and endothelin receptors induced by (i) 30 min of ischemia with or without 30 min reperfusion in myocytes from isolated rat hearts or (ii) ischemic conditions (acidosis or hypoxia) in cultured rat neonatal ventricular myocytes. Ischemia with or without reperfusion produced more than a twofold increase in mRNA expression of ET-1 as well as the ET(A) and ET(B) receptor (P < 0.05), although these effects were completely blocked by the NO donor 3-morpholinosydnonimine (SIN-1; 1 microM). To assess the possible factors regulating ET expression, myocytes were exposed to acidosis (pH 6.8-6.2) or to hypoxic conditions in an anaerobic chamber for 24 h in the presence or absence of SIN-1. At all acidic pHs, ET-1 and ET(A) receptor mRNA expression was significantly (P < 0.05) elevated approximately threefold, although the magnitude of elevation was independent of the degree of acidosis. These effects were completely prevented by SIN-1. ET(B) receptor expression was unaffected by acidosis. Hypoxia increased ET-1 as well as ET(A) and ET(B) receptor expression threefold (P < 0.05), although this was unaffected by SIN-1. Our results demonstrate that myocardial ischemia and reperfusion upregulate the ET system, which is inhibited by NO. Although increased expression of the ET system can be mimicked by both acidosis and hypoxia, only the effects of the former are NO sensitive. NO may serve an endogenous inhibitory factor which regulates the expression of the ET system under pathological conditions.

Experimental work during the past 15 years has demonstrated that endothelial cells in the heart play an obligatory role in regulating and maintaining cardiac function, in particular, at the endocardium and in the myocardial capillaries where endothelial cells directly interact with adjacent cardiomyocytes. The emerging field of targeted gene manipulation has led to the contention that cardiac endothelial-cardiomyocytal interaction is a prerequisite for normal cardiac development and growth. Some of the molecular mechanisms and cellular signals governing this interaction, such as neuregulin, vascular endothelial growth factor, and angiopoietin, continue to maintain phenotype and survival of cardiomyocytes in the adult heart. Cardiac endothelial cells, like vascular endothelial cells, also express and release a variety of auto- and paracrine agents, such as nitric oxide, endothelin, prostaglandin I(2), and angiotensin II, which directly influence cardiac metabolism, growth, contractile performance, and rhythmicity of the adult heart. The synthesis, secretion, and, most importantly, the activities of these endothelium-derived substances in the heart are closely linked, interrelated, and interactive. It may therefore be simplistic to try and define their properties independently from one another. Moreover, in relation specifically to the endocardial endothelium, an active transendothelial physicochemical gradient for various ions, or blood-heart barrier, has been demonstrated. Linkage of this blood-heart barrier to the various other endothelium-mediated signaling pathways or to the putative vascular endothelium-derived hyperpolarizing factors remains to be determined. At the early stages of cardiac failure, all major cardiovascular risk factors may cause cardiac endothelial activation as an adaptive response often followed by cardiac endothelial dysfunction. Because of the interdependency of all endothelial signaling pathways, activation or disturbance of any will necessarily affect the others leading to a disturbance of their normal balance, leading to further progression of cardiac failure.

The role of ET(A) endothelin receptor (ET(A)R) in the regulation of the delayed rectifier potassium current (I(K)) was examined in guinea pig atrial myocytes. Application of ET-1 (10 nM) together with an ET(B)-receptor-selective antagonist, BQ-788 (300 nM), significantly increased the voltage-dependent activation of I(K) without affecting its half-activation voltage or the slope factor, while it suppressed the calcium current (I(CaL)) and displaced the time-independent background current to the outward direction. The data suggests that the augmentation of I(K) contributes to the ET(A)-receptor-mediated shortening of action potential duration, and hence to the negative inotropic response, in atria.

In excitable cells, receptor-induced Ca(2+) release from intracellular stores is usually accompanied by sustained depolarization of cells and facilitated voltage-gated Ca(2+) influx (VGCI). In quiescent pituitary lactotrophs, however, endothelin-1 (ET-1) induced rapid Ca(2+) release without triggering Ca(2+) influx. Furthermore, in spontaneously firing and depolarized lactotrophs, the Ca(2+)-mobilizing action of ET-1 was followed by inhibition of spontaneous VGCI caused by prolonged cell hyperpolarization and abolition of action potential-driven Ca(2+) influx. Agonist-induced depolarization of cells and enhancement of VGCI upon Ca(2+) mobilization was established in both quiescent and firing lactotrophs treated overnight with pertussis toxin (PTX). Activation of adenylyl cyclase by forskolin and addition of cell-permeable 8-bromo-cAMP did not affect ET-1-induced sustained inhibition of VGCI, suggesting that the cAMP-protein kinase A signaling pathway does not mediate the inhibitory action of ET-1 on VGCI. Consistent with the role of PTX-sensitive K(+) channels in ET-1-induced hyperpolarization of control cells, but not PTX-treated cells, ET-1 decreased the cell input resistance and activated a 5 mM Cs(+)-sensitive K(+) current. In the presence of Cs(+), ET-1 stimulated VGCI in a manner comparable with that observed in PTX-treated cells, whereas E-4031, a specific blocker of ether-a-go-go-related gene-like K(+) channels, was ineffective. Similar effects of PTX and Cs(+) were also observed in GH(3) immortalized cells transiently expressing ET(A) receptors. These results indicate that signaling of ET(A) receptors through the G(i/o) pathway in lactotrophs and the subsequent activation of inward rectifier K(+) channels provide an effective and adenylyl cyclase-independent mechanism for a prolonged uncoupling of Ca(2+) mobilization and influx pathways.

Chronic heart failure (CHF) is characterized by impaired left ventricular function, increased peripheral and pulmonary vascular resistance, reduced exercise tolerance, and dyspnea. Despite considerable progress in the treatment of CHF, especially in targeting activated neurohumoral systems, mortality in these patients remains high. Therefore, new treatment approaches are warranted. Endothelin-1 (ET-1) plasma levels are elevated in CHF and correlate with both hemodynamic severity and symptoms. Plasma levels of ET-1 are strong independent predictors of mortality in CHF. Combined ET(A/B) selective ET(A) receptor antagonists have been evaluated in patients with CHF showing impressive hemodynamic improvements. These results indicate that ET receptor antagonists indeed have a potential to improve hemodynamics, symptoms, and potentially prognosis in patients with CHF, which still carries a high mortality.

1. Electrophysiological effects of endothelin-1 (ET-1) were studied in rabbit sinoatrial node (SAN) using conventional microelectrode and whole-cell voltage and current recordings. 2. In rabbit SAN, RT-PCR detected ET(A) endothelin receptor mRNA. ET-1 (100 nM) increased the cycle length of action potentials (APs) from 305 +/- 15 to 388 +/- 25 ms; this effect was antagonised by the ET(A) receptor-selective antagonist BQ-123 (1 microM). ET-1 increased AP duration (APD50) by 22%, depolarised the maximum diastolic potential (MDP) from -59 +/- 1 to -53 +/- 2 mV, shifted the take-off potential by +5 mV and decreased the pacemaker potential (PMP) slope by 15%. Under exactly the same experimental conditions, ET-1 caused a positive chronotropic effect in guinea-pig SAN with a decrease of 13% in APD50, a shift of -4 mV in the take-off potential and an increase of 8% in the PMP slope. 3. Rabbit SAN exhibited two major cell types, distinguished both by their appearances and by their electrophysiological responses to ET-1. Whereas the spontaneous pacing rate and the PMP slope were similarly decreased by ET-1 (10 nM) in both cell types, ET-1 depolarised MDP from -67 +/- 1 to -62 +/- 4 mV in spindle-shaped cells but hyperpolarised it from -73 +/- 1 to -81 +/- 3 mV in rod-shaped cells. ET-1 decreased APD50 by 8 and 52% and shifted the take-off potential by +5 and -9 mV in spindle- and rod-shaped cells, respectively. 4. ET-1 decreased the high-threshold calcium current (I(CaL)) by about 50% in both cell types, without affecting its voltage dependence, and decreased the delayed rectifier K+ current (I(K)) with significant shifts (of +4.7 and +14.0 mV in spindle- and rod-shaped cells, respectively) in its voltage dependence. It was exclusively in rod-shaped cells that ET-1 activated a sizeable amount of time-independent inward-rectifying current. 5. The hyperpolarisation-activated current (I(f)), observed exclusively in spindle-shaped cells, was significantly increased by ET-1 at membrane potentials between -74.7 and -84.7 mV whereas it was significantly decreased at more negative potentials. ET-1 significantly decreased the slope of the current-voltage (I-V) relation of the I(f) tail without changing its half-maximum voltage. 6. The overall negative chronotropic influence of ET-1 on the whole rabbit SAN is interpreted as resulting from the integration of its different actions on spindle- and rod-shaped SAN cells through electrotonic interaction.

The cardiac Na+-Ca2+ exchanger participates in Ca homeostasis, and Na+-Ca2+ exchanger-mediated ionic current (I(NaCa)) also contributes to the regulation of cardiac action potential duration. Moreover, I(NaCa) can contribute to arrhythmogenesis under conditions of cellular Ca overload. Although it has been shown that the peptide hormone endothelin-1 (ET-1) can phosphorylate the cardiac Na+-Ca2+ exchanger via protein kinase C (PKC), little is known about the effect of ET-1 on I(NaCa). In order to examine the effects of ET-1 on I(NaCa), whole-cell patch clamp measurements were made at 378C from guinea-pig isolated ventricular myocytes. With major interfering currents inhibited, I(NaCa) was measured as the current sensitive to nickel (Ni; 10mM) during a descending voltage ramp. ET-1 (10 nM) significantly increased I(NaCa) ( approximately 2-fold at -100 mV). Application of a PKC activator (PMA; 1mM: phorbol 12-myristate 13-acetate), mimicked the effect of ET-1. In contrast, the PKC inhibitor chelerythrine (CLT, 1mM) abolished the stimulatory effect of ET-1. An inactive phorbol ester, 4-alpha-phorbol-12,13-didecanoate (4a-PDD, 1mM) had no effect on I(NaCa). Collectively, these data indicate that ET-1 activated I(NaCa) through a PKC-dependent pathway. In additional experiments, isoprenaline (ISO; which has also been reported to activate I(NaCa) ) was applied. The increase in I(NaCa) density with ISO (1mM) was similar to that induced by ET-1 (10nM). When I(NaCa) was pre-stimulated by ET-1, application of ISO elicited no further increase in current and vice versa. ISO also had no additional effect on I(NaCa) when the cells were pretreated with PMA. Application of CLT did not alter the response of I(NaCa) to ISO. We conclude that ET-1 stimulated ventricular I(NaCa) via a PKC-dependent mechanism under our recording conditions. Concentrations of ET-1 and ISO that stimulated I(NaCa) to similar extents when applied separately were not additive when co-applied. The lack of synergy between the stimulatory effects of ET-1 and ISO may be important in protecting the heart from the potentially deleterious consequences of excessive stimulation of I(NaCa).

Increases in the expression of endothelin-1 (ET-1) in cardiac myocytes play a critical role in the development of heart failure in vivo. Whereas norepinephrine (NE) is a potent inducer of ET-1 expression in cardiac myocytes, the signaling pathways that link NE to inducible cardiac ET-1 expression are unknown. Adrenergic stimulation results in an increase in intracellular calcium levels, which in turn activates calcineurin. Here, we have shown that stimulation with NE markedly increased the expression of the ET-1 gene in primary cardiac myocytes from neonatal rats. This increase was severely attenuated by a beta-adrenergic antagonist, metoprolol, but not by an alpha-adrenergic antagonist, prazosin. Consistent with these data, the beta-adrenergic agonist isoproterenol (ISO) activated the rat ET-1 promoter activity to an extent that was similar to NE. The ISO-stimulated increase in promoter activity was significantly inhibited by a Ca(2+)-antagonist, nifedipine, and an immunosuppressant, cyclosporin A, which blocks calcineurin. Mutation analysis indicated that the GATA4 binding site is required for ISO-responsive ET-1 transcription. Stimulation with ISO enhanced the interaction between NFATc and GATA4 in cardiac myocytes. Consistent with this interaction, overexpression of GATA4 and NFATc synergistically activated the ET-1 promoter. These findings demonstrate that NE-stimulated ET-1 expression in cardiac myocytes is mediated predominantly via a beta-adrenergic pathway, and that calcium-activated calcineurin-GATA4 plays a role in this process.

It is still controversial whether the cAMP-activated Cl(-) current (I(Cl,cAMP)) is expressed in human cardiomyocytes. The whole-cell configuration of the voltage-clamp technique was used to examine in detail the I(Cl,cAMP) in single human atrial and ventricular myocytes. Human cardiomyocytes were enzymatically isolated from atrial or ventricular specimens obtained from open-heart surgery or cardiac transplantation, respectively. Isoproterenol (1 microM) or forskolin (10 microM) was used to activate the cAMP second-messenger system. The isoproterenol- or forskolin-induced Cl(-) current was elicited in 12 of 54 atrial myocytes but was completely absent from ventricular myocytes. The isoproterenol-induced Cl(-) current in atrial myocytes was time-independent and had a reversal potential close to zero. Endothelin-1 (30 nM) inhibited the isoproterenol-induced Cl(-) current by 75+/-6% (n=4). This inhibitory effect of endothelin-1 was attenuated by pretreating atrial myocytes with the endothelin ET(A) receptor antagonist, BQ485, but not with the ET(B) receptor antagonist, BQ-788. The results provide evidence that the I(Cl,cAMP) exists in human atria, but not ventricle, and is inhibited by endothelin-1.

The endothelin system consists of two G-protein-coupled receptors, three peptide ligands, and two activating peptidases. Its pharmacological complexity is reflected by the diverse expression pattern of endothelin system components, which have a variety of physiological and pathophysiological roles. In the vessels, the endothelin system has a basal vasoconstricting role and participates in the development of diseases such as hypertension, atherosclerosis, and vasospasm after subarachnoid hemorrhage. In the heart, the endothelin system affects inotropy and chronotropy, and it mediates cardiac hypertrophy and remodeling in congestive heart failure. In the lungs, the endothelin system regulates the tone of airways and blood vessels, and it is involved in the development of pulmonary hypertension. In the kidney, it controls water and sodium excretion and acid-base balance, and it participates in acute and chronic renal failure. In the brain, the endothelin system modulates cardiorespiratory centers and the release of hormones. More advanced functional analysis of the endothelin system awaits not only additional pharmacological studies using highly specific endothelin antagonists but also the generation of genetically altered rodent models with conditional loss-of-function and gain-of-function manipulations.

Protein kinase A (PKA) is an important effector enzyme commonly activated by cAMP. The present study focuses on our finding that the vasoactive peptide endothelin-1 (ET1), whose signaling is not coupled to cAMP production, stimulates PKA in two independent cellular models. Using an in vivo assay for PKA activity, we found that ET1 stimulated PKA in HeLa cells overexpressing ET1 receptors and in aortic smooth muscle cells expressing endogenous levels of ET1 receptors. In these cell models, ET1 did not stimulate cAMP production, indicating a novel mechanism for PKA activation. The ET1-induced activation of PKA was found to be dependent on the degradation of inhibitor of kappaB, which was previously reported to bind and inhibit PKA. ET1 potently stimulated the nuclear factor-kappaB pathway, and this effect was inhibited by overexpression of the inhibitor of kappaB dominant negative mutant (IkappaBalpham) and by treatment with the proteasome inhibitor MG-132. Importantly, IkappaBalpham and MG-132 had similar inhibitory effects on ET1-induced activation of PKA without affecting G(s)-mediated activation of PKA or ET1-induced phosphorylation of mitogen-activated protein kinase. Finally, another vasoactive peptide, angiotensin II, also stimulated PKA in a cAMP-independent manner in aortic smooth muscle cells. These findings suggest that cAMP-independent activation of PKA might be a general response to vasoactive peptides.

Endothelins are a family of biologically active peptides that are critical for development and function of neural crest-derived and cardiovascular cells. These effects are mediated by two G-protein-coupled receptors and involve transcriptional regulation of growth-responsive and/or tissue-specific genes. We have used the cardiac ANF promoter, which represents the best-studied tissue-specific endothelin target, to elucidate the nuclear pathways responsible for the transcriptional effects of endothelins. We found that cardiac-specific response to endothelin 1 (ET-1) requires the combined action of the serum response factor (SRF) and the tissue-restricted GATA proteins which bind over their adjacent sites, within a 30-bp ET-1 response element. We show that SRF and GATA proteins form a novel ternary complex reminiscent of the well-characterized SRF-ternary complex factor interaction required for transcriptional induction of c-fos in response to growth factors. In transient cotransfections, GATA factors and SRF synergistically activate atrial natriuretic factor and other ET-1-inducible promoters that contain both GATA and SRF binding sites. Thus, GATA factors may represent a new class of tissue-specific SRF accessory factors that account for muscle- and other cell-specific SRF actions.

Adult cardiac myocytes are terminally differentiated cells that are no longer able to divide. Accumulating data support the idea that apoptosis in these cells is involved in the transition from cardiac compensation to decompensated heart failure. Since a number of neurohormonal factors are activated in this state, these factors may be involved in the positive and negative regulation of apoptosis in cardiac myocytes. beta1-Adrenergic receptor and angiotensin type 1 receptor pathways, nitric oxide and natriuretic peptides are involved in the induction of apoptosis in these cells, while alpha1- and beta2-adrenergic receptor and endothelin-1 type A receptor pathways and gp130-related cytokines are antiapoptotic. The myocardial protection of the latter is mediated, at least in part, through mitogen-activated protein kinase-dependent pathways, compatible with the findings in other cell types. In contrast, signaling pathways leading to apoptosis in cardiac myocytes are distinct from those in other cell types. The cAMP/PKA pathway induces apoptosis in cardiac myocytes and blocks apoptosis in other cell types. The p300 protein, a coactivator of p53, mediates apoptosis in fibroblasts but appears to play a protective role in differentiated cardiac myocytes. The inhibition of myocardial cell apoptosis in heart failure may be achieved by directly blocking apoptosis signaling pathways or by modulating neurohormonal factors involved in their regulation. These may provide novel therapeutic strategies in some forms of heart failure.

The purpose of this study was to investigate the regulation of beta-adrenergic agonist-induced apoptosis by endothelin-1 (ET-1) in cardiac myocytes.

Numerous hormonal factors including norepinephrine and ET-1 are activated in patients with heart failure. These factors may be involved in the positive and negative regulation of myocardial cell apoptosis observed in failing hearts. Recently, it has been shown that norepinephrine can induce myocardial cell apoptosis via a beta-adrenergic receptor-dependent pathway.

Primary cardiac myocytes were prepared from neonatal rats. These cells were stimulated with the beta-adrenergic agonist isoproterenol (ISO) in the presence or absence of ET-1.

The administration of 10(-7) mol/liter of ET-1 completely blocked Iso-induced apoptosis. An endothelin type A receptor antagonist, FR139317, negated the inhibitory effect of ET-1 on apoptosis, while the endothelin type B receptor antagonist BQ788 did not show such a negation. Endothelin-1 also inhibited apoptosis induced by a membrane-permeable cAMP analogue (8-Br-cAMP), which bypassed Gi. The effect of ET-1 was neutralized by an MEK-1-specific inhibitor (PD098059), a phosphatidylinositol 3'-kinase inhibitor (wortmannin) and its downstream pp70 S6-kinase inhibitor, rapamycin.

These findings suggest that ET-1 represents a protective factor against myocardial cell apoptosis in heart failure and that this effect is mediated mainly through endothelin type A receptor-dependent pathways involving multiple downstream signalings in cardiac myocytes.

The goal of this study was to determine the comparative effects of angiotensin II type 1 (AT(1)) receptor inhibition alone, endothelin-1 (ET) receptor blockade alone, and combined receptor blockade on left ventricular (LV) function, contractility, and neurohormonal system activity in a model of congestive heart failure (CHF).

Pigs were randomly assigned to each of 5 groups: (1) rapid atrial pacing (240 bpm) for 3 weeks (n=9), (2) concomitant AT(1) receptor blockade (valsartan, 3 mg/kg per day) and rapid pacing (n=8), (3) concomitant ET receptor blockade (bosentan, 50 mg/kg BID) and rapid pacing (n=8), (4) concomitant combined AT(1) and ET receptor inhibition and rapid pacing (n=8), and (5) sham-operated control (n=9). LV stroke volume was reduced from the control value after rapid pacing, was unchanged with either AT(1) or ET receptor blockade alone, but was improved with combination treatment. LV peak wall stress was reduced in both groups with ET receptor blockade compared with the rapid pacing group. Plasma norepinephrine levels were increased by >3-fold after rapid pacing, remained increased in the monotherapy groups, but were reduced after combination treatment. LV myocyte velocity of shortening was reduced after rapid pacing-induced CHF, remained reduced after AT(1) receptor blockade, increased after ET receptor blockade (compared with rapid pacing-induced CHF values), and returned to within control values after combined blockade.

Combined AT(1) and the ET receptor blockade in this model of CHF improved LV pump function, and contributory factors included the effects of LV loading conditions, neurohormonal system activity, and myocardial contractile performance. Thus, combined receptor blockade may provide a useful combinatorial therapeutic approach in CHF.

The expression of endothelin-1 (ET-1) in cardiac myocytes is markedly induced during the development of heart failure in vivo and by stimulation with the alpha(1)-adrenergic agonist phenylephrine in culture. Although recent studies have suggested a role for cardiac-specific zinc finger GATA factors in the transcriptional pathways that modulate cardiac hypertrophy, it is unknown whether these factors are also involved in cardiac ET-1 transcription and if so, how these factors are modulated during this process. Using transient transfection assays in primary cardiac myocytes from neonatal rats, we show here that the GATA element in the rat ET-1 promoter was required for phenylephrine-stimulated ET-1 transcription. Cardiac GATA-4 bound the ET-1 GATA element and activated the ET-1 promoter in a sequence-specific manner. Stimulation by phenylephrine caused serine phosphorylation of GATA-4 and increased its ability to bind the ET-1 GATA element. Inhibition of the extracellularly responsive kinase cascade with PD098059 blocked the phenylephrine-induced increase in the DNA binding ability and the phosphorylation of GATA-4. These findings demonstrate that serine phosphorylation of GATA-4 is involved in alpha(1)-adrenergic agonist-responsive transcription of the ET-1 gene in cardiac myocytes and that extracellularly responsive kinase 1/2 activation plays a role upstream of GATA-4.

Endothelins (ETs) are a family of peptide hormones that act on G protein-coupled ET(A) and ET(B) receptors. ETs exert inotropic and chronotropic actions in the heart. Myocardial ischemia is associated with increased plasma levels of ET and cell swelling. We examined the effect of ETs on dog atrial swelling-induced chloride current (I(Cl,swell)). Whole-cell patch clamp was used; 10 nM ET-1 or ET-2 increased I(Cl,swell) by approximately twofold. ET-2 had no effect if I(Cl,swell) activation was prevented by hypertonic superfusate. Outward ET-2-induced current was blocked by 150 microM DIDS more effectively than inward current. Overnight pretreatment with phorbol 12-myristate 13-acetate (1.6 microM), pertussis toxin (100 ng/ml), or dialysis of the cell with 300 microM 2'-deoxyadenosine 3'-monophosphate, a P-site inhibitor of adenylyl cyclase, did not diminish the effect of ET-2. The effect of ET-2 was blocked by an ET(A1)- (BQ123), but not an ET(B)-selective (BQ788) antagonist. ET-2-induced currents were inhibited approximately 70% by PD 98059 (30 microM), a selective MAPK kinase (MEK) blocker. PD 98059 did not affect basal whole cell current or I(Cl,swell) before exposure to ET-2. The data suggest that MEK activity is not required for activation of atrial I(Cl,swell) but that ET-2 stimulates I(Cl,swell) by a MEK-dependent pathway.

Anion transport proteins in mammalian cells participate in a wide variety of cell and intracellular organelle functions, including regulation of electrical activity, pH, volume, and the transport of osmolites and metabolites, and may even play a role in the control of immunological responses, cell migration, cell proliferation, and differentiation. Although significant progress over the past decade has been achieved in understanding electrogenic and electroneutral anion transport proteins in sarcolemmal and intracellular membranes, information on the molecular nature and physiological significance of many of these proteins, especially in the heart, is incomplete. Functional and molecular studies presently suggest that four primary types of sarcolemmal anion channels are expressed in cardiac cells: channels regulated by protein kinase A (PKA), protein kinase C, and purinergic receptors (I(Cl.PKA)); channels regulated by changes in cell volume (I(Cl.vol)); channels activated by intracellular Ca(2+) (I(Cl.Ca)); and inwardly rectifying anion channels (I(Cl.ir)). In most animal species, I(Cl.PKA) is due to expression of a cardiac isoform of the epithelial cystic fibrosis transmembrane conductance regulator Cl(-) channel. New molecular candidates responsible for I(Cl.vol), I(Cl.Ca), and I(Cl.ir) (ClC-3, CLCA1, and ClC-2, respectively) have recently been identified and are presently being evaluated. Two isoforms of the band 3 anion exchange protein, originally characterized in erythrocytes, are responsible for Cl(-)/HCO(3)(-) exchange, and at least two members of a large vertebrate family of electroneutral cotransporters (ENCC1 and ENCC3) are responsible for Na(+)-dependent Cl(-) cotransport in heart. A 223-amino acid protein in the outer mitochondrial membrane of most eukaryotic cells comprises a voltage-dependent anion channel. The molecular entities responsible for other types of electroneutral anion exchange or Cl(-) conductances in intracellular membranes of the sarcoplasmic reticulum or nucleus are unknown. Evidence of cardiac expression of up to five additional members of the ClC gene family suggest a rich new variety of molecular candidates that may underlie existing or novel Cl(-) channel subtypes in sarcolemmal and intracellular membranes. The application of modern molecular biological and genetic approaches to the study of anion transport proteins during the next decade holds exciting promise for eventually revealing the actual physiological, pathophysiological, and clinical significance of these unique transport processes in cardiac and other mammalian cells.

The expression and coupling of endothelin (ET) receptors were studied in rat pituitary somatotrophs. These cells exhibited periods of spontaneous action potential firing that generated high-amplitude fluctuations in cytosolic calcium concentration ([Ca(2+)](i)). The message and the specific binding sites for ET(A), but not ET(B), receptors were found in mixed pituitary cells and in highly purified somatotrophs. The activation of these receptors by ET-1 led to an increase in inositol 1,4,5-trisphosphate production and the associated rise in [Ca(2+)](i) and growth hormone (GH) secretion. The Ca(2+)-mobilizing action of ET-1 lasted for 2-3 min and was followed by an inhibition of action potential-driven Ca(2+) influx and GH secretion to below the basal levels. As in somatostatin-treated cells, the ET-1-induced inhibition of spontaneous electrical activity and Ca(2+) influx was accompanied by the inhibition of adenylyl cyclase and by the stimulation of inward rectifier potassium current. In contrast to somatostatin, ET-1 did not inhibit voltage-gated Ca(2+) channels. During prolonged agonist stimulation a gradual recovery of Ca(2+) influx and GH secretion occurred. In somatotrophs treated with pertussis toxin overnight, the ET-1-induced Ca(2+)-mobilizing phase was preserved, but it was followed immediately by facilitated Ca(2+) influx and GH secretion. Both somatostatin- and ET-1-induced inhibitions of adenylyl cyclase activity were abolished in pertussis toxin-treated cells. These results indicate that the transient cross-coupling of Ca(2+)-mobilizing ET(A) receptors to the G(i)/G(o) pathway in somatotrophs provides an effective mechanism to change the rhythm of [Ca(2+)](i) signaling and GH secretion during continuous agonist stimulation.

The aim of this review is to provide basic information on the electrophysiological changes during acute ischemia and reperfusion from the level of ion channels up to the level of multicellular preparations. After an introduction, section II provides a general description of the ion channels and electrogenic transporters present in the heart, more specifically in the plasma membrane, in intracellular organelles of the sarcoplasmic reticulum and mitochondria, and in the gap junctions. The description is restricted to activation and permeation characterisitics, while modulation is incorporated in section III. This section (ischemic syndromes) describes the biochemical (lipids, radicals, hormones, neurotransmitters, metabolites) and ion concentration changes, the mechanisms involved, and the effect on channels and cells. Section IV (electrical changes and arrhythmias) is subdivided in two parts, with first a description of the electrical changes at the cellular and multicellular level, followed by an analysis of arrhythmias during ischemia and reperfusion. The last short section suggests possible developments in the study of ischemia-related phenomena.

Although it is well known that progesterone alters uterine contractility and plays an important role in maintenance of pregnancy, the biochemical mechanisms by which progesterone alters uterine contractility in human gestation are less clear. In this investigation we sought to identify progesterone-induced adaptations in human myometrial smooth muscle cells that may alter Ca2+ signaling in response to contractile agents. Cells were treated with vehicle or the progesterone analog medroxyprogesterone acetate (MPA) for 5 days, and intracellular free Ca2+ concentration ([Ca2+]i) was quantified after treatment with oxytocin (OX) or endothelin (ET)-1. OX- and ET-1-induced increases in [Ca2+]i were significantly attenuated in cells pretreated with MPA in a dose-dependent manner. Progesterone receptor antagonists prevented the attenuated Ca2+ transients induced by MPA. ETA and ETB receptor subtypes were expressed in myometrial cells, and treatment with MPA resulted in significant downregulation of ETA and ETB receptor binding. MPA did not alter ionomycin-stimulated increases in [Ca2+]i and had no effect on inositol trisphosphate-dependent or -independent release of Ca2+ from internal Ca2+ stores. We conclude that adaptations of Ca2+ homeostasis in myometrial cells during pregnancy may include progesterone-induced modification of receptor-mediated increases in [Ca2+]i.

1. Desensitization of ET(A) endothelin receptor (ET(A)R) was compared between the rat and guinea-pig with regard to negative chronotropic response (NC) in the right atria (RA). 2. ET-1 (100 nM) produced distinct NC in the presence of BQ788 (300 nM), and positive chronotropic response (PC) in the presence of BQ123 (1 microM) in both species, showing that ETAR and ET(B) endothelin receptor (ET(B)R) mediate NC and PC, respectively. 3. Repetitive applications of ET-1 (50 nM) desensitized PC, and the second application only induced a strong NC in both species. Later applications of ET-1 produced virtually no response in the rat RA, whereas they produced BQ123-sensitive NCs repetitively in guinea-pig RA, exhibiting marked species difference in desensitization of ETAR-mediated NC. 4. Pretreatment with staurosporine (100 nM) prevented desensitization of ET(A)R in the rat RA altogether. However, phorbol 12-myristate 13-acetate (PMA, 300 nM) failed to induce, but rather hampered, desensitization of ET(A)R. 5. Partial amino acid sequencing of ET(A)Rs, spanning from the 2nd through the 4th intracellular loops, revealed that all the potential Ser/Thr phosphorylation sites, including a protein kinase C (PKC) site, are conserved among guinea-pigs, rats, rabbits, bovines and humans. 6. In guinea pig RA, pretreatment with okadaic acid (1 microg ml(-1)) and PMA did not facilitate desensitization of ET(A)R whereas these agents successfully desensitized ETAR during combined stimulation of beta-adrenoceptor and ET(A)R by isoproterenol (300 nM) and ET-1 (100 nM). 7. These results suggest that species differences in desensitization of ET(A)R are not caused by differences in the site(s) of, but caused by differences in the environment for phosphorylation of the receptor. Desensitization of ET(A)R appears to require phosphorylation of the receptor by PKC as well as a kinase stimulated by beta-adrenoceptor activation.

Ion and fluid transport across the biliary epithelium contributes to bile secretion. Since endothelin (ET)-1 affects ion transport activities and is released by human gallbladder- derived biliary epithelial cells in primary culture, we examined the expression of ET peptides and ET receptors and the influence of ET-1 on ion transport in this epithelium ex vivo. In freshly isolated gallbladder epithelial cells, preproET-1, -2, and -3 mRNAs were detected by reverse transcription PCR and ET-1 isopeptide was identified by chromatography. The cells also displayed ET receptor mRNAs and high-affinity binding sites for ET-1, mostly of the ETB type. Electrogenic anion secretion across intact gallbladder mucosa was stimulated by forskolin, secretin, and exogenous ATP, as assessed by short-circuit current (Isc) increases in Ussing-type chambers. ET-1 inhibited forskolin- and secretin-induced changes in Isc, without affecting baseline Isc or ATP-induced changes. Accordingly, ET-1 significantly reduced the accumulation of intracellular cAMP elicited by forskolin and secretin in the epithelial cells, and this effect was abolished by pertussis toxin. This is the first evidence that ET-1 is synthesized and inhibits, via a Gi protein-coupled receptor, cAMP-dependent anion secretion in human gallbladder epithelium, indicating a role in the control of bile secretion by an autocrine/paracrine mechanism.

Endothelin (ET) isopeptides, ET-1, ET-2 and ET-3, elicit a positive inotropic effect (PIE) in association with a negative lusitropic effect, essentially with identical efficacies and potencies in the isolated rabbit papillary muscle, but with different concentration-dependent properties. Pharmacological analysis indicates that the PIE of ET-1 is mediated by an ETA2 subtype that is less sensitive to BQ-123 and FR139317, whereas the PIE of ET-3 is mediated by an ETA1 subtype that is highly sensitive to these ETA antagonists. ETs increased the amplitude of intracellular Ca2+ transient (CaT) in indo-1 loaded rabbit ventricular myocytes, but the increase was much smaller than that produced by elevation of [Ca2+]o or isoproterenol for a given extent of PIE, an indication of increased myofibrillar Ca2+ sensitivity. ETs stimulate phosphoinositide (PI) hydrolysis, which leads to production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Evidence for the role of IP3-induced Ca2+ release in cardiac E-C coupling is tenuous. Generation of IP3 induced by ET-1 was transient and returned to the baseline level when the PIE reached an elevated steady level. Protein kinase C (PKC) that is activated by DAG and also via other pathways triggered by ETs stimulates Na+-H+ exchanger to lead to an increased [Na+]i and alkalinization. The former may contribute to an increase in the amplitude of CaT through Na+-Ca2+ exchanger, and the latter, to an increase in myofibrillar Ca2+ sensitivity. A number of PKC inhibitors, such as staurosporine, H-7, calphostin C and chelerythrine, consistently and selectively inhibited the PIE of ET-3 without affecting the PIE of isoproterenol and Bay k 8644. The maximum inhibition was 20-30% of the total response. A Na+-H+ exchange inhibitor, [5-(N-ethyl-N-isopropyl) amiloride (EIPA)] or a Ca2+ antagonist, verapamil, could not completely inhibit the PIE of ET-3, but the combination of both inhibitors totally abolished the PIE of ET-3. These findings indicate that activation of PKC and subsequent activation of Na+-H+ exchanger and/or L-type Ca2+ channels may play a crucial role in the cardiac action of ET isopeptides in the rabbit ventricular myocardium.

The effects of angiotensin II (Ang II) on ATP-sensitive K+ channels (K(ATP)) were investigated in ventricular myocytes enzymatically isolated from adult guinea pig heart.

In the whole-cell and cell-attached configurations (including open-cell-attached mode) of the patch-clamp technique, K(ATP) currents (I(KATP)) were activated through metabolic poisoning by the use of inhibitors of both glycolytic and oxidative ATP productions at 37 degrees C. In the whole-cell mode, I(KATP) were reversibly suppressed by increasing extracellular glucose and Ang II (1 nmol/L). In the cell-attached mode, Ang II concentration-dependently inhibited single K(ATP) activities with an IC50 value of 3.2+/-0.5 pmol/L (Hill coefficient=1.3+/-0.3). CV11974 (100 nmol/L), an angiotensin 1 (AT1) receptor-selective antagonist, blocked the inhibitory action of Ang II. Preincubation of myocytes with pertussis toxin (5 microg/mL for > 120 min at 37 degrees C) virtually prevented subsequent Ang II action. The inhibitory effect of Ang II was also abolished in the open-cell-attached mode (achieved by a prior perfusion of streptolysin-O, 0.08 U/mL). In this mode, through tiny membrane holes, the intracellular ATP concentration can be controlled by bathing extracellular solutions containing a known ATP concentration.

The inhibitory actions of Ang II on K(ATP) appear to be mediated by an increase in the subsarcolemmal ATP concentration that results from the inhibition of adenylate cyclase activities via AT1 receptors/PTX-sensitive G proteins.

The effect of endothelin-1 on the phosphorylation of alpha1b-adrenoreceptors, transfected into rat-1 fibroblasts, was studied. Basal alpha1b-adrenoreceptor phosphorylation was markedly increased by endothelin-1, norepinephrine, and phorbol esters. The effect of endothelin-1 was dose dependent (EC50 approximately 1 nM), reached its maximum 5 min after stimulation, and was inhibited by BQ-123, an antagonist selective for ETA receptors. Endothelin-1-induced alpha1b-adrenoreceptor phosphorylation was attenuated by staurosporine or genistein and essentially abolished when both inhibitors were used together. The effect of norepinephrine was not modified by either staurosporine or genistein alone, and it was only partially inhibited when both were used together. These data suggest the participation of protein kinase C and tyrosine kinase(s) in endothelin-1-induced receptor phosphorylation. However, phosphoaminoacid analysis revealed the presence of phosphoserine and traces of phosphothreonine, but not of phosphotyrosine, suggesting that the putative tyrosine kinase(s), activated by endothelin, could act in a step previous to receptor phosphorylation. The effect of endothelin-1 on alpha1b-adrenoreceptor phosphorylation was not mediated through pertussis toxin-sensitive G proteins. Calcium mobilization induced by norepinephrine was diminished by endothelin-1. Norepinephrine and endothelin-1 increased [35S]GTPgammaS binding to control membranes. The effect of norepinephrine was abolished in membranes obtained from cells pretreated with endothelin-1. Interestingly, genistein plus staurosporine inhibited this effect of the endothelial peptide. Endothelin-1 did not induce alpha1b-adrenoreceptor internalization. Our data indicate that activation of ETA receptors by endothelin-1 induces alpha1b-adrenoreceptor phosphorylation and alters G protein coupling.

Effects of endothelins (ETs) on the acetylcholine receptor-operated K+ current (I(KACh)) were examined in isolated guinea pig atrial cells using patch-clamp techniques. ET-1 or ET-3 produced a transient activation of I(KACh) in atrial cells held at -40 mV. When I(KACh) was preactivated by 1 microM carbachol, however, both ETs produced a transient potentiation followed by a sustained inhibition of the current. When I(KACh) was maximally activated by 10 microM carbachol or 100 microM adenosine, these ETs produced only a sustained inhibition of the I(KACh). Their inhibitory effects on the preactivated I(KACh) were concentration dependent, and the half-maximal effective concentrations were 314 pM for ET-1 and 1.13 nM for ET-3. The inhibitory effect of ET-1 was antagonized by BQ-485, a specific ET(A) receptor antagonist, but not by BQ-788, a specific ET(B) receptor antagonist, indicating that the ET-1 effect is mediated by ET(A) receptors. On the other hand, the inhibitory effect of ET-3 was antagonized by BQ-788 and more effectively by BQ-485, suggesting the involvement of "atypical" ET receptors. Both ETs partly reversed the carbachol-induced shortening of the action potential recorded in the current-clamp mode. Inhibitory effects of ET-1 and ET-3 on the preactivated I(KACh) may contribute to the positive inotropic and chronotropic effects of ETs in atrial tissues.

N omega-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, was used to examine whether nitric oxide was involved in the negative chronotropic and inotropic effects of endothelin-1 in the presence of isoprenaline in mammalian heart. In isolated rabbit right atria, endothelin-1 elicited a negative chronotropic effect in the presence of isoprenaline, which was associated with a decrease in the isoprenaline-induced accumulation of cyclic AMP. On the other hand, in the dog ventricular trabeculae, the negative inotropic effect of endothelin-1 was not accompanied by a significant reduction in the isoprenaline-induced accumulation of cyclic AMP. N omega-nitro-L-arginine methyl ester affected neither the negative chronotropic effect nor the negative inotropic effect of endothelin-1. The effects of endothelin-1 on the isoprenaline-induced cyclic AMP accumulation were not influenced by N omega-nitro-L-arginine methyl ester either. These results indicate that the negative chronotropic and inotropic effects of endothelin-1 in the presence of isoprenaline in mammalian cardiac muscle do not involve the nitric oxide-mediated signaling pathway.

Endothelin-1 (ET-1) is a 21-amino acid peptide hormone released from myocardial and endothelial cells, whose receptors (both ETA and ETB are expressed in the myocardium. We report here that ET-1 inhibits the cardiac delayed rectifier K+ current (IK) via a pertussis toxin (PTX)-sensitive mechanism. Ventricular myocytes enzymatically isolated from guinea pig hearts were voltage-clamped by the conventional whole-cell and nystatin-perforated patch technique (intrapipette and extrapipette K+ concentrations, 150 and 5.4 mmol/L, respectively) in the presence of nifedipine (2 mumol/L). Amplitudes of tail and steady state (2-second pulse) currents were measured as IK. ET-1 suppressed the basal IK by 20.9 +/- 2.3% in a concentration-dependent manner, with an IC50 of 1.1 +/- 0.3 nmol/L (n = 19), although it did not suppress the basal IK using the nystatin method. E-4031 (5 mumol/L), a blocker of the rapid component of IK (IKr), did not prevent the inhibitory action of ET-1. ET-1 reduced by 63.4 +/- 6.5% the slow component of IK (IKs) that had been enhanced to approximately 2-fold by isoproterenol (ISO, 20 nmol/L). The action was concentration dependent, with an IC50 of 0.7 +/- 0.4 nmol/L (n = 22), and was also observed using the nystatin method. The effect of ET-1 appeared to be mediated by an ETA receptor, because it was prevented by FR139317, an ETA-selective antagonist (1 mumol/L, n = 4), and sarafotoxin s6c, an ETB-selective agonist (100 nmol/L, n = 4), could not inhibit the ISO-enhanced IK. ET-1 antagonized IKs enhanced by histamine (250 nmol/L, n = 7) and forskolin (500 nmol/L, n = 7) but did not inhibit IKs enhanced by the internal application of cAMP (100 mumol/L, n = 6). Preincubation of myocytes with PTX (5 micrograms/mL for > 60 minutes at 36 degrees C) completely abolished the inhibitory action of ET-1 on the ISO-enhanced IKs (n = 4). Thus, nanomolar ET-1 inhibits IKs via the ETA receptor/PTX-sensitive G protein/PKA pathway.

Plasma levels of endothelin-1 (ET-1) are increased in patients and animals with severe congestive heart failure (CHF). It remains unknown, however, whether ET-1 plays a direct and contributory role in the progression of CHF. Accordingly, the present project tested the hypothesis that chronic blockade of the ETA receptor would have direct and beneficial effects on left ventricular (LV) and myocyte function in a model of CHF.

Global LV and isolated myocyte function were examined in rabbits in the following groups (12 per group): chronic rapid ventricular pacing (RVP; 400 bpm, 3 weeks), RVP and concomitant administration of the selective ETA receptor antagonist (PD 156707 24 mg/d), and sham controls. LV fractional shortening decreased after RVP (17 +/- 5 versus 42 +/- 3%) and end-diastolic dimension increased (2.36 +/- 0.44 versus 1.24 +/- 0.18 cm) compared with controls (P < .05). With RVP plus ETA blockade, LV fractional shortening was increased (33 +/- 6%) and end-diastolic dimension decreased (2.02 +/- 0.30 cm) compared with RVP-only values (P < .05). Plasma norepinephrine and endothelin increased twofold in the RVP group. In the RVP plus ETA blockade group, plasma endothelin increased threefold compared with RVP values. Isolated myocyte shortening velocity declined after RVP (42 +/- 13 versus 72 +/- 10 microns/s, P < .05) compared with controls but was normalized with RVP plus ETA blockade (77 +/- 16 microns/s). Myocyte inotropic response to extracellular Ca2+, beta-receptor stimulation, and ET-1 was reduced in the RVP group and returned to control levels with RVP and concomitant ETA receptor blockade.

The results from this study suggest that chronically elevated ET-1 levels and subsequent activation of the ETA receptor play a direct and contributory role in the progression of the CHF process. Thus, specific ETA receptor blockade may provide a new and useful therapeutic modality in the setting of CHF.

Angiotensin II receptors are reported to be abundant in the guinea pig ventricle; their coupling to adenylate cyclase in the heart, however, remains controversial. Therefore, we investigated the effect of angiotensin II on Cl- conductance activated by cAMP-dependent protein kinase.

After minimizing the contribution of other ionic currents, exposure of single guinea pig ventricular cells to isoproterenol (40 to 50 nmol/L; 36 degrees C) elicited a typical protein kinase A-dependent Cl- conductance. Subsequent application of angiotensin II reduced the isoproterenol-induced conductance with an IC50 of 0.24 +/- 0.08 nmol/L. Angiotensin II also inhibited the Cl- currents, which were activated through stimulation of adenylate cyclase by forskolin and histamine receptors. CV-11974 (1 mumol/L), an antagonist selective for the angiotensin type 1 receptor, prevented the effect of angiotensin II. Angiotensin II did not inhibit the current that had been persistently activated by intracellular GTP gamma S (100 mumol/L), a nonhydrolyzable guanine nucleotide, plus isoproterenol. In addition, prior incubation of myocytes with pertussis toxin prevented the angiotensin II inhibitory action. Cl- conductance, when activated directly by intracellular dialysis with cAMP (1 mmol/L), was not affected by angiotensin II. Radioimmunologic measurement of cellular cAMP in the dissociated myocytes showed that angiotensin II inhibited the isoproterenol-induced increase of cAMP.

Angiotensin II receptors negatively couple to adenylate cyclase via pertussis toxin-sensitive G proteins, thereby inhibiting cardiac protein kinase A-dependent Cl- conductance.

With the advent of the first generation of both selective and nonselective endothelin antagonists being a relatively recent event, the manifold therapeutic potentials of these compounds are only now being explored clinically. Undoubtedly, numerous clinical utilities for these compounds will soon be realized.

In most mammalian species, activation of myocardial endothelin as well as alpha1-adrenergic and angiotensin receptors leads to an increase in contractile function and myocardial cell hypertrophy, in association with acceleration of PI hydrolysis and with resultant production of IP3 and diacylglycerol. Therefore, these receptors may share a common intracellular signal transduction process in cardiac regulation. Although the pathophysiological relevance of endothelin- and angiotensin-mediated signal transduction has been postulated to play a key role in the progress of congestive heart failure, the details of the regulation are still controversial. We carried out experiments to further study the regulation induced by activation of these receptors. In spite of a wide range of species-dependent variation among mammals in the induction of the cardiotonic effect via these receptors, there is an excellent correlation between the extent of acceleration of PI hydrolysis and the positive inotropic effect (associated with a negative lusitropic effect) of the respective receptor agonists under most experimental conditions in rabbit ventricular myocardium. In isolated rabbit ventricular cardiomyocytes loaded with indo-1/AM, activation of these receptors elicited a very similar changes in the relationship between [Ca2+]i and cell shortening: the [Ca2+]i-shortening trajectory was shifted mainly upwards and the relationship of peak shortening vs peak [Ca2+]i was shifted to the left, an indication that the PIE of these agonists is consistently associated with an increase in [Ca2+]i and in the sensitivity of myofilaments to Ca2+ ions under the same experimental condition. Pieces of evidence in biochemical and pharmacological analyses imply that the products of PI hydrolysis, namely diacylglycerol and subsequent activation of protein kinase C, might play a crucial role in the regulation of cardiac function that is induced upon activation of endothelin, angiotensin and alpha-adrenergic receptors in the rabbit ventricular myocardium.

Cardiac endothelial cells, regardless of whether they are from endocardial or from coronary (micro)vascular origin, directly modulate performance of the subjacent cardiomyocytes, resulting in control of the onset of ventricular relaxation and rapid filling of the heart. This review summarizes major features of the morphology, embryology, and comparative physiology of cardiac endothelial cells as well as the experimental observations on how cardiac endothelial cells affect the mechanical performance of the heart. As for the underlying mechanisms of the interaction between cardiac endothelial cells and cardiomyocytes, two working hypotheses have been postulated over the past years; (1) interaction mediated through a trans-endothelial physicochemical gradient for various ions (active blood-heart barrier), and (2) interaction mediated through the release by the cardiac endothelial cells of various cardioactive substances, eg, nitric oxide, endothelin, and prostacyclin. These two mechanisms may act in concert or in parallel.

Endothelial cells within the heart release a number of substances that modulate myocardial contractile function. These agents include nitric oxide, endothelin, prostanoids, adenylpurines, and other substances that have so far been characterized only in bioassay studies. A notable feature of many of these agents is that they influence contractile behavior predominantly by modifying cardiac myofilament properties rather than altering cytosolic Ca2+ transients. A consequence of this subcellular action is often a disproportionate effect on myocardial relaxation and diastolic tone. The paracrine modulation of cardiac myocyte function by endothelial cell factors is likely to be an important mechanism contributing to the overall regulation of cardiac contractile function, both physiologically and in pathological states.