ETA receptor-mediated Ca2+ mobilisation in H9c2 cardiac cells.
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 [125I]endothelin-1 (ET-1) bound to a single class of high affinity binding sites in cardiomyoblast membranes. Endothelin-3 (ET-3) displaced bound [125I]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 [125I]ET-1 binding in a monophasic manner and with low potency. An ET(A)-selective antagonist, BQ-123, competed [125I]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 1mM CaCl(2). Treatment of cells with antagonists, PD-145065 and BQ-123, or a phospholipase C-beta 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.
Ceccarelli
Francesca
F
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università di Pisa, Italy.
Scavuzzo
Maria C
MC
Giusti
Laura
L
Bigini
Gianni
G
Costa
Barbara
B
Carnicelli
Vittoria
V
Zucchi
Riccardo
R
Lucacchini
Antonio
A
Mazzoni
Maria R
MR
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000378:metabolism; D015151:Immunoblotting; D007457:Iodine Radioisotopes; D009206:Myocardium / Q000378:metabolism*; D051381:Rats; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000096:biosynthesis
D019332:Endothelin-1; D007457:Iodine Radioisotopes; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium