ET A receptor-mediated Ca 2+ mobilisation in H9c2 cardiac cells

Abstract

Expression and pharmacological properties of endothelin receptors (ETRs) were investigated in H9c2 cardiomyoblasts. The mechanism of receptor-mediated modulation of intracellular Ca 2+ concentration ([Ca 2+] i) was examined by measuring fluorescence increase of Fluo-3-loaded cells with flow cytometry. Binding assays showed that [ 125 I ]endothelin-1 (ET-1) bound to a single class of high affinity binding sites in cardiomyoblast membranes. Endothelin-3 (ET-3) displaced bound [ 125 I ]ET-1 in a biphasic manner, in contrast to an ET B-selective agonist, IRL-1620, that was ineffective. The ET B-selective antagonist, BQ-788, inhibited [ 125 I ]ET-1 binding in a monophasic manner and with low potency. An ET A-selective antagonist, BQ-123, competed [ 125 I ]ET-1 binding in a monophasic manner. This antagonist was found to be 13-fold more potent than BQ-788. Immunoblotting analysis using anti-ET A and -ET B antibodies confirmed a predominant expression of the ET A receptor. ET-1 induced a concentration-dependent increase of Fluo-3 fluorescence in cardiomyoblasts resuspended in buffer containing 1 mM CaCl 2. Treatment of cells with antagonists, PD-145065 and BQ-123, or a phospholipase C-β 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 Ca 2+ resulted in a shift to the right of the ET-1 concentration–response curve. Both the L-type voltage-operated Ca 2+ 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-1. Our results demonstrate that ET A receptors are expressed and functionally coupled to rise of [Ca 2+] i in H9c2 cardiomyoblasts. ET-1-induced [Ca 2+] i increase is triggered by Ca 2+ release from intracellular inositol 1,4,5-trisphosphate-gated stores; plasma membrane Ca 2+ channels and ryanodine receptors participate in sustaining the Ca 2+ response. Regulation of channel opening by protein kinase C is also involved in the process of [Ca 2+] i increase.