endothelin-1 and indo-1

The potent vasoconstrictor polypeptide endothelin-1 (ET-1) plays an important pathophysiological role in progression of cardiovascular diseases and elicits prominent effects on myocardial contractility. Although ET-1 produces a positive inotropy in cardiac muscle of most mammalian species, it induces a sustained negative inotropy in mice. This study was performed to gain an insight into the cellular mechanisms underlying the negative inotropy in adult mouse ventricular myocytes.. Cell shortening and Ca(2+) transients were simultaneously recorded from isolated mouse ventricular myocytes loaded with the Ca(2+)-sensitive fluorescent dye indo-1.. ET-1 decreased cell shortening in a concentration-dependent manner (pD(2) value of 10.1). The ET-1-induced decrease in cell shortening was associated with a decrease in Ca(2+) transients. In addition, the Ca(2+) transient/cell-shortening relationship was shifted to the right by ET-1, indicating decreased myofilament Ca(2+) sensitivity. The instantaneous relationship of the rising phase of the Ca(2+) transient and cell shortening was shifted to the right by ET-1. Decreased Ca(2+) transients and cell shortening induced by ET-1 were markedly attenuated by the specific Na(+)/Ca(2+) exchange inhibitor SEA0400.. ET-1-induced negative inotropy in mouse ventricular myocytes was mediated by decreased Ca(2+) transients and myofilament Ca(2+) sensitivity. These data are entirely consistent with the involvement of increased Ca(2+) extrusion via the Na(+)/Ca(2+) exchanger in the ET-1-mediated decrease in Ca(2+) transients. Decreased Ca(2+) sensitivity may be due to retardation of cell shortening in response to a rise in Ca(2+) transients.

Topics: Actin Cytoskeleton; Aniline Compounds; Animals; Calcium; Calcium Channel Blockers; Calcium Signaling; Cell Shape; Dose-Response Relationship, Drug; Endothelin-1; Fluorescent Dyes; Heart Ventricles; Indoles; Mice; Myocardial Contraction; Myocytes, Cardiac; Nicardipine; Phenyl Ethers; Sodium-Calcium Exchanger

Experiments were carried out in isolated canine ventricular trabeculae and acetoxymethylester of indo-1-loaded single myocytes to elucidate the role of protein tyrosine kinase (PTK) in the inotropic effect of endothelin-1 (ET-1) induced by crosstalk with norepinephrine (NE). The PTK inhibitor genistein was used as a pharmacological tool. Genistein but not daidzein inhibited the positive inotropic effect and the increase in Ca(2+) transients induced by ET-1 by crosstalk with NE at low concentrations. Genistein and daidzein antagonized the negative inotropic effect and the decrease in Ca(2+) transients induced by ET-1 by crosstalk with NE at high concentrations, but genistein did not affect the antiadrenergic effect of carbachol. Genistein but not daidzein enhanced the positive inotropic effect and the increase in Ca(2+) transients induced by NE via beta-adrenoceptors, while the enhancing effect of genistein was abolished by the protein tyrosine phosphatase inhibitor vanadate. These findings indicate that genistein (1) induces a positive inotropic effect in association with an increase in Ca(2+) transients, (2) inhibits the positive inotropic effect of ET-1 induced by crosstalk with NE, and (3) enhances the positive inotropic effect of NE induced via beta-adrenoceptors by inhibition of PTK. In addition, genistein inhibits the negative inotropic effect of ET-1 induced by crosstalk with NE through a PTK-unrelated mechanism. PTK may play a crucial role in the receptor-mediated regulation of cardiac contractile function in canine ventricular myocardium.

Topics: Animals; Calcium Signaling; Cardiotonic Agents; Cell Separation; Dogs; Endothelin-1; Enzyme Inhibitors; Genistein; Heart; Heart Ventricles; In Vitro Techniques; Indoles; Myocardial Contraction; Myocytes, Cardiac; Norepinephrine; Protein-Tyrosine Kinases; Receptor Cross-Talk; Receptors, Adrenergic, beta

Angiotensin II (Ang II). endothelin-1 (ET-1) and phenylephrine are receptor agonists that share the signal transduction acting through acceleration of phosphoinositide hydrolysis in the heart. Because the regulation of myocardial contractility induced by these receptor agonists shows a wide range of species-dependent variation among experimental animals, we carried out experiments to elucidate the mechanism of contractile regulation induced by these agents in mice which are employed currently more as transgenic models. Effects of Ang II, ET-1 and phenylephrine on cell shortening and Ca2+ transients were investigated in single ventricular myocytes loaded with indo-1/AM. Ang II (10(-8), 10(-7) M), ET-1 (10(-10), 10(-9) M) and phenylephrine (10(-6), 10(-5) M in the presence of the beta-adrenoceptor antagonist timolol) decreased the cell shortening [Ang II: 58.4+/-9.03 (n = 8), 50.3+/-11.90% (n = 6); ET-1: 48.4+/-8.27, 31.2+/-6.45% (n = 5); phenylephrine: 45.7+/-11.60, 28.7+/-5.89% (n = 5)]. By contrast, the amplitude of Ca2+ transients was not significantly influenced by these agonists. The selective protein kinase C inhibitor chelerythrine at 10(-6) M significantly inhibited the decrease in cell shortening induced by these receptor agonists. These results indicate that Ang II, ET-1 and phenylephrine elicit a negative inotropic effect with insignificant alteration of Ca2+ transients, which may be mainly mediated by activation of protein kinase C in mouse ventricular cardiomyocytes.

Topics: Alkaloids; Angiotensin II; Animals; Benzophenanthridines; Calcium; Depression, Chemical; Endothelin-1; In Vitro Techniques; Indoles; Male; Mice; Mice, Inbred C57BL; Myocardial Contraction; Myocardium; Phenanthridines; Phenylephrine; Protein Kinase C

We performed experiments to elucidate the cellular mechanism for the biphasic inotropic response to endothelin-1 of single rabbit ventricular myocytes loaded with a fluorescent dye, acetoxymethylester of indo-1. Endothelin-1 at 10 nM elicited a biphasic inotropic effect: a transient decrease in cell shortening and Ca(2+) transients followed by an increase in cell shortening without significant elevation of peak Ca(2+) transients. The selective endothelin ET(A) receptor antagonist FR139317 (2(R)-[2(R)-[2(S)-[(1-hexahydro-1H-azepinyl)]carbonyl]amino-4-methylpentanoyl]amino-3-[3-(1-methyl-1H-indolyl)propionyl]amino-3-(2-pyridyl)propionic acid) at 1 microM abolished the biphasic effect of endothelin-1 on cell shortening and Ca(2+) transients. The selective protein kinase C inhibitor chelerythrine at 1 microM and the tyrosine kinase inhibitor genistein at 5 microM inhibited the endothelin-1-induced increase in cell shortening without significantly affecting Ca(2+) transients and the transient decrease in cell shortening and Ca(2+) transients. The present results indicate that both protein kinase C and tyrosine kinase may contribute to the increase in myofilament Ca(2+) sensitivity induced by endothelin-1, whereas the decrease in Ca(2+) transients induced by endothelin-1 may be mediated by a signalling pathway different from that involved in the increase in cardiac contractility in rabbit ventricular myocytes.

Topics: Actin Cytoskeleton; Alkaloids; Animals; Azepines; Benzophenanthridines; Calcium; Cell Size; Endothelin Receptor Antagonists; Endothelin-1; Enzyme Inhibitors; Genistein; Heart Ventricles; Indoles; Male; Phenanthridines; Protein Kinase C; Protein-Tyrosine Kinases; Rabbits; Receptor, Endothelin A; Spectrometry, Fluorescence

The effect of the nitric oxide (NO) donor sodium nitroprusside (SNP) on both [Ca(2+)](i)and mechanical activity was studied in the rat isolated pulmonary artery (RPA). In freshly isolated myocytes loaded with 1 microM indo-lacetoxymethyl ester for 30 min, short (40-60 s) application of ATP (100 microM) or ET-1 (0.1 microM) induced 3-6 cyclic rises in [Ca(2+)](i)(Ca-oscillations) of decreasing amplitude. Preincubation of cells with SNP (10-250 microM) for 10 min had no effect on the resting [Ca(2+)](i)value, but progressively abolished the oscillations. A similar effect was obtained with 8-bromo-cGMP (100-500 microM). SNP (0.001-100 microM) concentration-dependently relaxed ATP (10 mM, n = 4) and ET-1 (0.1 microM, n = 4)-precontracted RPA. 1H-[1,2,4]oxadiazolol [4,3,-a]quinoxalin-1-one (ODQ, 10 microM), a potent inhibitor of the cytosolic guanylyl cyclase, fully reversed the effect of SNP on ATP-induced [Ca(2+)](i)oscillations as well as on ATP-precontracted RPA. In contrast, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8, 10 microM), a potent inhibitor of cGMP-dependent protein kinase (PKG), did not alter the effect of SNP. Caffeine (5 mM) induced only one transient [Ca(2+)](i)-increase (n = 24), the amplitude of which was altered neither by SNP nor by 8-bromo-cGMP. Our results show that the relaxing effect of NO in RPA is related, at least in part, to its action on the Ca-signalling pathway. NO interacts with inositol trisphosphate pathway without interacting with the ryanodine-sensitive receptor. Finally, the effect of NO involves an increase in cGMP but appears independent of activation of PKG.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine Triphosphate; Animals; Caffeine; Calcium; Cyclic GMP-Dependent Protein Kinases; Endothelin-1; Indoles; Isometric Contraction; Male; Molsidomine; Muscle, Smooth, Vascular; Nitric Oxide Donors; Nitroprusside; Penicillamine; Pulmonary Artery; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; Sarcoplasmic Reticulum; Vasodilation; Vasodilator Agents