Calcium-sensing receptor modulates extracellular Ca2+ entry via TRPC-encoded receptor-operated channels in human aortic smooth muscle cells

Chow JY, Estrema C, Orneles T, Dong X, Barrett KE, Dong H. Calcium-sensing receptor modulates extracellular Ca2+ entry via TRPC-encoded receptor-operated channels in human aortic smooth muscle cells. Am J Physiol Cell Physiol 301: C461-C468, 2011. First published May 11, 2011; doi: 10.1152/ajpcell.00389.2010.-Ca-sensing receptor (CaSR), a member of the G protein-coupled receptor family, regulates the synthesis of parathyroid hormone in response to changes in serum Ca2+ concentrations. The functions of CaSR in human vascular smooth muscle cells are largely unknown. Here we sought to study CaSR activation and the underlying molecular mechanisms in human aortic smooth muscle cells (HASMC). Extracellular Ca2+ ([Ca2+](o)) dose-dependently increased free cytosolic Ca2+ ([Ca2+](cyt)) in HASMC, with a half-maximal response (EC50) of 0.52 mM and a Hill coefficient of 5.50. CaSR was expressed in HASMC, and the [Ca2+](o)-induced [Ca2+](cyt) rise was abolished by dominant negative mutants of CaSR. The CaSR-mediated increase in [Ca2+](cyt) was also significantly inhibited by pertussis toxin, the phospholipase C inhibitor U-73122, or the general protein kinase C (PKC) inhibitor chelerythrine, but not by the conventional PKC inhibitor, Go6976. Depletion of membrane cholesterol by pretreatment with methyl-beta-cyclodextrin markedly decreased CaSR-induced increase in [Ca2+] cyt. Blockade of TRPC channels with 2-aminoethoxydiphenyl borate, SKF-96365, or La-3 significantly inhibited [Ca2+](o) entry, whereas activation of TRPC6 channels with flufenamic acid potentiated [Ca2+](o) entry. Neither cyclopiazonic acid nor caffeine or ionomycin had any effect on [Ca2+](cyt) in [Ca2+](o)-free solutions. TRPC6 and PKCe mRNA and proteins were detected in HASMC, and [Ca2+](o) induced PKC epsilon phosphorylation, which could be prevented by chelerythrine. Our data suggest that CaSR activation mediates [Ca2+](o) entry, likely through TRPC6-encoded receptor-operated channels that are regulated by a PLC/PKC epsilon cascade. Our study therefore provides evidence not only for functional expression of CaSR, but also for a novel pathway whereby it regulates [Ca2+](o) entry in HASMC.