title: Calcium-sensing receptor modulates extracellular Ca(2+) entry via TRPC-encoded receptor-operated channels in human aortic smooth muscle cells.
abstract: 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 Ca(2+) 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 Ca(2+) ([Ca(2+)](o)) dose-dependently increased free cytosolic Ca(2+) ([Ca(2+)](cyt)) in HASMC, with a half-maximal response (EC(50)) of 0.52 mM and a Hill coefficient of 5.50. CaSR was expressed in HASMC, and the [Ca(2+)](o)-induced [Ca(2+)](cyt) rise was abolished by dominant negative mutants of CaSR. The CaSR-mediated increase in [Ca(2+)](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, Gö6976. Depletion of membrane cholesterol by pretreatment with methyl-β-cyclodextrin markedly decreased CaSR-induced increase in [Ca(2+)](cyt). Blockade of TRPC channels with 2-aminoethoxydiphenyl borate, SKF-96365, or La(3) significantly inhibited [Ca(2+)](o) entry, whereas activation of TRPC6 channels with flufenamic acid potentiated [Ca(2+)](o) entry. Neither cyclopiazonic acid nor caffeine or ionomycin had any effect on [Ca(2+)](cyt) in [Ca(2+)](o)-free solutions. TRPC6 and PKCε mRNA and proteins were detected in HASMC, and [Ca(2+)](o) induced PKCε phosphorylation, which could be prevented by chelerythrine. Our data suggest that CaSR activation mediates [Ca(2+)](o) entry, likely through TRPC6-encoded receptor-operated channels that are regulated by a PLC/PKCε cascade. Our study therefore provides evidence not only for functional expression of CaSR, but also for a novel pathway whereby it regulates [Ca(2+)](o) entry in HASMC.
authors.affiliation: Department of Medicine, University of California San Diego, La Jolla, CA 92093, USA.
authors.forename: Jimmy Y C
authors.initials: JY
authors.lastname: Chow
authors.forename: Christine
authors.initials: C
authors.lastname: Estrema
authors.forename: Tiffany
authors.initials: T
authors.lastname: Orneles
authors.forename: Xiao
authors.initials: X
authors.lastname: Dong
authors.forename: Kim E
authors.initials: KE
authors.lastname: Barrett
authors.forename: Hui
authors.initials: H
authors.lastname: Dong
mesh_terms: D000704:Analysis of Variance; D001011:Aorta / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D021941:Caveolae / Q000378:metabolism; D002478:Cells, Cultured; D004789:Enzyme Activation; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D051744:Protein Kinase C-epsilon / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D044169:Receptors, Calcium-Sensing / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D050052:TRPC Cation Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000074287:TRPC6 Cation Channel; D014162:Transfection; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D002121:Calcium Channel Blockers; D010726:Phosphodiesterase Inhibitors; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D044169:Receptors, Calcium-Sensing; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; C497255:PRKCE protein, human; D051744:Protein Kinase C-epsilon; D010738:Type C Phospholipases; D002118:Calcium