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CARDIOVASCULAR
Departments of Pharmacology (L.I.B., D.R.M., K.K.H., K.L.B.) and Medicine (L.L.C.), Loyola University—Chicago, Maywood, Illinois
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.
Address correspondence to: Dr. Kenneth L. Byron, Dept. of Pharmacology and Experimental Therapeutics, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153. E-mail: kbyron{at}lumc.edu
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