title: TRPC1 and STIM1 mediate capacitative Ca2+ entry in mouse pulmonary arterial smooth muscle cells.
abstract: Previous studies in pulmonary arterial smooth muscle cells (PASMCs) showed that the TRPC1 channel mediates capacitative Ca(2+) entry (CCE), but the molecular signal(s) that activate TRPC1 in PASMCs remains unknown. The aim of the present study was to determine if TRPC1 mediates CCE through activation of STIM1 protein in mouse PASMCs. In primary cultured mouse PASMCs loaded with fura-2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular Ca(2+) concentration ([Ca(2+)](i)). The transient but not the sustained rise in [Ca(2+)](i) was partially inhibited by nifedipine. In addition, CPA increased the rate of Mn(2+) quench of fura-2 fluorescence that was inhibited by SKF 96365, Ni(2+), La(3+) and Gd(3+), exhibiting pharmacological properties characteristic of CCE. The nifedipine-insensitive sustained rise in [Ca(2+)](i) and the increase in Mn(2+) quench of fura-2 fluorescence caused by CPA were both inhibited in cells pretreated with antibody raised against an extracellular epitope of TRPC1. Moreover, STIM1 siRNA reduced the rise in [Ca(2+)](i) and Mn(2+) quench of fura-2 fluorescence caused by CPA, whereas overexpression of STIM1 resulted in a marked increase in these responses. RT-PCR revealed TRPC1 and STIM1 mRNAs, and Western blot analysis identified TRPC1 and STIM1 proteins in mouse PASMCs. Furthermore, TRPC1 was found to co-immunoprecipitate with STIM1, and the precipitation level of TRPC1 was increased in cells subjected to store depletion. Taken together, store depletion causes activation of voltage-operated Ca(2+) entry and CCE. These data provide direct evidence that CCE is mediated by TRPC1 channel through activation of STIM1 in mouse PASMCs.
authors.affiliation: Department of Pharmacology, University of Nevada School of Medicine, Reno, NV 89557, USA. lcng@medicine.nevada.edu
authors.forename: Lih Chyuan
authors.initials: LC
authors.lastname: Ng
authors.forename: Mary D
authors.initials: MD
authors.lastname: McCormack
authors.forename: Judith A
authors.initials: JA
authors.lastname: Airey
authors.forename: Cherie A
authors.initials: CA
authors.lastname: Singer
authors.forename: Phillip S
authors.initials: PS
authors.lastname: Keller
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Shen
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Hume