Expression and pharmacological properties of endothelin receptors (ETRs) were investigated in H9c2 cardiomyoblasts. The mechanism of receptor-mediated modulation of intracellular Ca2+ concentration ([Ca2+](i)) was examined by measuring fluorescence increase of Fluo-3-loaded cells with flow cytometry. Binding assays showed that [I-125]endothelin-1 (ET-1) bound to a single class of high affinity binding sites in cardiomyoblast membranes. Endothelin-3 (ET-3) displaced bound [I-125]ET-1 in a biphasic manner, in contrast to an ETB-selective agonist, IRL-1620, that was ineffective. The ETB-selective antagonist, BQ-788, inhibited [(125) I]ET-1 binding in a monophasic manner and with low potency. An ETA-selective antagonist, BQ-123, competed [I-125]ET-I binding in a monophasic manner. This antagonist was found to be 13-fold more potent than BQ-788. Immunoblotting analysis using anti-ETA and -ETB antibodies confirmed a predominant expression of the ETA receptor. ET-1 induced a concentration-dependent increase of Fluo-3 fluorescence in cardiomyoblasts resuspended in buffer containing I mM CaCl2. Treatment of cells with antagonists, PD-145065 and BQ-123, or a phospholipase C-P inhibitor, U-73122, abolished ET-1-mediated increases in fluorescence. The close structural analogue of U-73122, U-73343, caused a minimal effect on the concentration-response curve of ET-1. ET-3 produced no major increase of Fluo-3 fluorescence. Removal of extracellular Ca2+ resulted in a shift to the right of the ET-I concentration-response curve. Both the L-type voltage-operated Ca2+ channel blocker, nifedipine, and the ryanodine receptor inhibitor, dantrolene, reduced the efficacy of ET-1. Two protein kinase C inhibitors reduced both potency and efficacy of ET-I. Our results demonstrate that ETA receptors are expressed and functionally coupled to rise of [Ca2+](i) in H9c2 cardiomyoblasts. ET-1-induced [Ca2+](i) increase is triggered by Ca2+ release from intracellular inositol 1,4,5-trisphosphate-gated stores; plasma membrane Ca2+ channels and ryanodine receptors participate in sustaining the Ca2+ response. Regulation of channel opening by protein kinase C is also involved in the process of [Ca2+](i) increase. (C) 2002 Elsevier Science Inc. All rights reserved.
ETA receptor-mediated Ca2+ mobilisation in H9c2 cardiac cells
COSTA, BARBARA;ZUCCHI, RICCARDO;LUCACCHINI, ANTONIO;MAZZONI, MARIA ROSA
2003-01-01
Abstract
Expression and pharmacological properties of endothelin receptors (ETRs) were investigated in H9c2 cardiomyoblasts. The mechanism of receptor-mediated modulation of intracellular Ca2+ concentration ([Ca2+](i)) was examined by measuring fluorescence increase of Fluo-3-loaded cells with flow cytometry. Binding assays showed that [I-125]endothelin-1 (ET-1) bound to a single class of high affinity binding sites in cardiomyoblast membranes. Endothelin-3 (ET-3) displaced bound [I-125]ET-1 in a biphasic manner, in contrast to an ETB-selective agonist, IRL-1620, that was ineffective. The ETB-selective antagonist, BQ-788, inhibited [(125) I]ET-1 binding in a monophasic manner and with low potency. An ETA-selective antagonist, BQ-123, competed [I-125]ET-I binding in a monophasic manner. This antagonist was found to be 13-fold more potent than BQ-788. Immunoblotting analysis using anti-ETA and -ETB antibodies confirmed a predominant expression of the ETA receptor. ET-1 induced a concentration-dependent increase of Fluo-3 fluorescence in cardiomyoblasts resuspended in buffer containing I mM CaCl2. Treatment of cells with antagonists, PD-145065 and BQ-123, or a phospholipase C-P inhibitor, U-73122, abolished ET-1-mediated increases in fluorescence. The close structural analogue of U-73122, U-73343, caused a minimal effect on the concentration-response curve of ET-1. ET-3 produced no major increase of Fluo-3 fluorescence. Removal of extracellular Ca2+ resulted in a shift to the right of the ET-I concentration-response curve. Both the L-type voltage-operated Ca2+ channel blocker, nifedipine, and the ryanodine receptor inhibitor, dantrolene, reduced the efficacy of ET-1. Two protein kinase C inhibitors reduced both potency and efficacy of ET-I. Our results demonstrate that ETA receptors are expressed and functionally coupled to rise of [Ca2+](i) in H9c2 cardiomyoblasts. ET-1-induced [Ca2+](i) increase is triggered by Ca2+ release from intracellular inositol 1,4,5-trisphosphate-gated stores; plasma membrane Ca2+ channels and ryanodine receptors participate in sustaining the Ca2+ response. Regulation of channel opening by protein kinase C is also involved in the process of [Ca2+](i) increase. (C) 2002 Elsevier Science Inc. All rights reserved.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
