Pharmacological and Electrophysiological Characterization of Store-Operated Currents and Capacitative Ca²⁺Entry in Vascular Smooth Muscle Cells

Abstract

Capacitative Ca2ϩ 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 Ca 2ϩ concentration ([Ca 2ϩ ] i ) have demonstrated that depletion of intracellular Ca 2ϩ stores activates CCE. However, there is disagreement in published studies regarding the regulation of this mechanism by the vasoconstrictor hormone [Arg 8 ]-vasopressin (AVP). We have employed electrophysiological methods to characterize the membrane currents activated by store depletion [store-operated current (I SOC )]. Because of different recording conditions, it has not been previously determined whether I SOC 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 I SOC , including its sensitivity to blockade by 2-aminoethoxydiphenylborane, diethylstilbestrol, or micromolar Gd 3ϩ , 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 I SOC 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 I SOC was reduced in both the TRPC1 siRNAand TRPC1 antisense-expressing cells, suggesting that the TRPC1 channel contributes to the I SOC /CCE pathway. Article, publication date, and citation information can be found at

Stimulation of arachidonic acid release by vasopressin in A7r5 vascular smooth muscle cells mediated by Ca2+‐stimulated phospholipase A2

“…A7r5 vascular smooth muscle cells, although their relative roles may differ under different conditions [4][5][6][7][8][9], influenced by both the pattern of expression of TrpC proteins [7,10] and the nature of the stimulus [7]. It is, however, clear that in A7r5 cells, as in many other cells [11][12][13], arachidonic acid (AA) contributes to the coordinate regulation of CCE and NCCE [5,6,14].…”

Differential Protein Kinase C-dependent Modulation of Kv7.4 and Kv7.5 Subunits of Vascular Kv7 Channels

“…As a negative control, we also visualized TRPC6 non-selective cation channels, which are known to be expressed in MASMCs (33), but, as an ion channel of a different class, would not be expected to physically interact with Kv7 channel subunits. Kv7.4, Kv7.5, and TRPC6 could each be visualized individually in MASMCs (Fig.…”

Imbalance and dysfunction of transient receptor potential channels contribute to the pathogenesis of hypertension

“…Over-expression of the human trpc1 gene in rat pulmonary artery enhanced vasoconstriction via store depletion-mediated Ca 2+ influx [33]. One study found that thapsigargin-stimulated store-operated current was reduced in TRPC1 siRNA-or TRPC1 antisense-expressing cells [34]. Ahmmed et al [35] demonstrated that phosphorylation of TRPC1 via PKC alpha was an important determinant of store-operated Ca 2+ entry in human ECs.…”