Abstract
Capacitative Ca2+ entry (CCE) in vascular smooth muscle cells contributes to vasoconstrictor and mitogenic effects of vasoactive hormones. In A7r5 rat aortic smooth muscle cells, measurements of cytosolic free Ca2+ concentration ([Ca2+]i) have demonstrated that depletion of intracellular Ca2+ stores activates CCE. However, there is disagreement in published studies regarding the regulation of this mechanism by the vasoconstrictor hormone [Arg8]-vasopressin (AVP). We have employed electrophysiological methods to characterize the membrane currents activated by store depletion [store-operated current (ISOC)]. Because of different recording conditions, it has not been previously determined whether ISOC corresponds to CCE measured using fura-2; nor has the channel protein responsible for CCE been identified. In the present study, the pharmacological characteristics of ISOC, including its sensitivity to blockade by 2-aminoethoxydiphenylborane, diethylstilbestrol, or micromolar Gd3+, were found to parallel the effects of these drugs on thapsigargin- or AVP-activated CCE measured under identical external ionic conditions using fura-2. Thapsigargin-stimulated ISOC was also measured in freshly isolated rat mesenteric artery smooth muscle cells (MASMC). Members of the transient receptor potential (TRP) family of nonselective cation channels, TRPC1, TRPC4, and TRPC6, were detected by reverse transcription-polymerase chain reaction and Western blot in both A7r5 cells and MASMC. TRPC1 expression was reduced in a stable A7r5 cell line expressing a small interfering RNA (siRNA) or by infection of A7r5 cells with an adenovirus expressing a TRPC1 antisense nucleotide sequence. Thapsigargin-stimulated ISOC was reduced in both the TRPC1 siRNA- and TRPC1 antisense-expressing cells, suggesting that the TRPC1 channel contributes to the ISOC/CCE pathway.
Footnotes
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This work was supported by the John and Marian Falk Trust for Medical Research and the National Heart, Lung, and Blood Institute, National Institutes of Health Grant R01HL70670 (to K.L.B.).
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A preliminary account of this work has been presented and accepted for publication as an abstract: Brueggemann LI, Markun DR, Cribbs LL, and Byron KL (2006) Electrophysiological and pharmacological characterization of store-operated currents and capacitative Ca2+ entry in rat vascular smooth muscle cells. J Physiol (Lond), in press.
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doi:10.1124/jpet.105.095067.
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ABBREVIATIONS: VSM vascular smooth muscle; TRP, transient receptor potential; TRPC, TRP nonselective cation channel; [Ca2+]i, cytosolic free Ca2+ concentration; 2-APB, 2-aminoethoxydiphenylborane; AS-TRPC1-GFP, adenoviral vector for expression of antisense TRPC1 DNA and coexpression of green fluorescent protein; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; BSA, bovine serum albumin; CCE, capacitative Ca2+ entry; CPA, cyclopiazonic acid; ISOC, store-operated current; LOE 908, (R,S)-(3,4-dihydro 6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide; MASMC, mesenteric artery smooth muscle cell(s); m.o.i., multiplicity of infection; NCCE, noncapacitative Ca2+ entry; OAG, 1-oleoyl-2-acetyl-sn-glycerol; RT, reverse transcription; PCR, polymerase chain reaction; siRNA, small interfering RNA; SOCE, store-operated Ca2+ entry; DES, diethylstilbestrol; MOPS, 4-morpholinepropanesulfonic acid; NMDG, N-methyl-d-glucamine; PBS, phosphate-buffered saline.
- Received September 1, 2005.
- Accepted January 12, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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