Pharmacological and Electrophysiological Characterization of Store-operated Currents and Capacitative Ca Entry in Vascular Smooth Muscle Cells

Capacitative Ca 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 concentration ([Ca ]i) have demonstrated that depletion of intracellular Ca stores activates CCE. However, there is disagreement in published studies regarding the regulation of this mechanism by the vasoconstrictor hormone [Arg]-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 Gd , were found to parallel the effects of these drugs on thapsigarginor 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 siRNAand TRPC1 antisense-expressing cells, suggesting that the TRPC1 channel contributes to the ISOC/CCE pathway. Vasoconstrictor hormones cause contraction of vascular smooth muscle (VSM) cells by increasing cytosolic free Ca concentration ([Ca ]i), which in turn activates the contractile apparatus of the cells. A well characterized signal transduction pathway leading to elevation of [Ca ]i involves inositol trisphosphate-mediated release of Ca from the sarcoplasmic reticulum and Ca entry via nonselective calcium-permeable cation channels. This pathway may also contribute to the mitogenic actions of vasoactive hormones (Wang et al., 2001). The A7r5 rat aortic smooth muscle cell line is among the most extensively characterized vascular smooth muscle preparations in terms of agonist-stimulated Ca signaling pathways. Using the Ca -sensitive fluorescent indicator, fura-2, the vasoconstrictor hormone [Arg]-vasopressin (AVP) was 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.). 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 Ca entry in rat vascular smooth muscle cells. J Physiol (Lond), in press. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.095067. ABBREVIATIONS: VSM vascular smooth muscle; TRP, transient receptor potential; TRPC, TRP nonselective cation channel; [Ca ]i, cytosolic free Ca 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 Ca entry; CPA, cyclopiazonic acid; ISOC, store-operated current; LOE 908, (R,S)-(3,4-dihydro 6,7-dimethoxy-isoquinoline-1-yl)-2phenyl-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 Ca entry; OAG, 1-oleoyl-2-acetyl-sn-glycerol; RT, reverse transcription; PCR, polymerase chain reaction; siRNA, small interfering RNA; SOCE, store-operated Ca entry; DES, diethylstilbestrol; MOPS, 4-morpholinepropanesulfonic acid; NMDG, N-methyl-Dglucamine; PBS, phosphate-buffered saline. 0022-3565/06/3172-488–499$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 317, No. 2 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 95067/3098817 JPET 317:488–499, 2006 Printed in U.S.A. 488 at A PE T Jornals on Jne 8, 2017 jpet.asjournals.org D ow nladed from found to activate both capacitative (also known as storeoperated) and noncapacitative Ca entry (CCE and NCCE, respectively) in A7r5 cells (Byron and Taylor, 1995; Broad et al., 1999). Several subsequent studies (Moneer and Taylor, 2002; Moneer et al., 2003, 2005) have utilized fura-2 fluorescence measurements to examine the regulation of these Ca entry pathways by AVP, with some disagreement among the published results (Brueggemann et al., 2005). It remains unclear whether CCE contributes to a sustained elevation of [Ca ]i while AVP is present. CCE and NCCE in A7r5 cells probably involve different Ca -permeable channels, because they appear to have different permeabilities to Sr and Mn (Byron and Taylor, 1995) and are differentially sensitive to pharmacological blockers, such as 2-aminoethoxydiphenylborane (2-APB), LOE 908, and Gd (Broad et al., 1999; Iwamuro et al., 1999; Moneer and Taylor, 2002). However, the earlier fura-2 fluorescence studies did not characterize the electrophysiological properties of the CCE pathway, and the channel proteins that are the presumed targets of the pharmacological blockers remain unidentified. A number of earlier electrophysiological studies (Nakajima et al., 1996; Iwasawa et al., 1997; Iwamuro et al., 1999; Jung et al., 2002) failed to detect store-operated currents in A7r5 cells despite evidence from fura-2 fluorescence studies that Ca influx is stimulated following depletion of Ca stores. In a recent study (Brueggemann et al., 2005), we reported conditions under which store-operated currents can be detected in A7r5 cells, but the resulting current differs in its apparent cation selectivity and voltage dependence from ISOC recorded from other vascular smooth muscle preparations. Conditions used for recording isolated currents are generally much different from the ionic conditions used to detect Ca entry using fura-2. These conditions may affect the behavior of the channels and their responses to pharmacological agents. Thus, there is a paucity of information related to the electrophysiological or molecular nature of the CCE pathway in vascular smooth muscle cells and disagreements among different groups regarding the mechanisms regulating its activity. Molecular candidates for the channels involved in capacitative Ca entry pathways may be emerging from the discovery in the mid-1990s of the Drosophila transient receptor potential (TRP) channels and seven canonical mammalian TRP homolog [TRPC1-TRPC7; reviewed by Montell (2005)]. The primary structure of these proteins indicates that they have six membrane-spanning domains reminiscent of the voltage-gated K channel family. In analogy to K channels, the TRP channels are believed to form as tetramers of individual TRP homolog protein subunits. The TRPC proteins are ubiquitously expressed in mammalian tissues, with many cell types, including VSM expressing multiple TRPC homologs. When expressed heterologously, they generally form functional channels that exhibit electrophysiological properties of nonselective cation channels. In the present study, we use electrophysiological techniques to record store-operated currents (ISOC) in A7r5 cells and mesenteric artery smooth muscle cells (MASMC). The pharmacological characteristics of ISOC are compared with CCE recorded with fura-2 under similar ionic conditions. The findings suggest that CCE is active during the sustained phase of AVP Ca signaling. We have also identified several TRPC homologs expressed in A7r5 cells and MASMC. Evidence is presented to support a role of TRPC1 as a component of the store-operated Ca entry pathway. Materials and Methods A7r5 Cell Culture. A7r5 cells were cultured as described previously (Byron and Taylor, 1993). Cells were subcultured onto rectangular (9 22-mm, number 11⁄2) glass coverslips or plastic tissue culture dishes (Corning, Acton, MA). For fura-2 fluorescence measurements, confluent cell monolayers grown on glass coverslips were used 2 to 5 days after plating. For electrophysiological recordings, cells grown on plastic tissue culture dishes were trypsinized (0.25% trypsin and 1% EDTA) and replated on 12-mm round glass coverslips. Cells were allowed to adhere for approximately 15 min and then transferred to external solution. Cells were then used for patchclamp recording within 30 min or maintained at 4°C for up to 2 h