TRPC1 and Orai1 interact with STIM1 and mediate capacitative Ca(2+) entry caused by acute hypoxia in mouse pulmonary arterial smooth muscle cells.

Previous studies in pulmonary artery smooth muscle cells (PASMCs) showed that acute hypoxia activates capacitative Ca(2+) entry (CCE) but the molecular candidate(s) mediating CCE caused by acute hypoxia remain unclear. The present study aimed to determine if transient receptor potential canonical 1 (TRPC1) and Orai1 interact with stromal interacting molecule 1 (STIM1) and mediate CCE caused by acute hypoxia in mouse PASMCs. In primary cultured PASMCs loaded with fura-2, acute hypoxia caused a transient followed by a sustained rise in intracellular Ca(2+) concentration ([Ca(2+)](i)). The transient but not sustained rise in [Ca(2+)](i) was partially inhibited by nifedipine. Acute hypoxia also 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 rise in [Ca(2+)](i) and the increase in Mn(2+) quench rate were both inhibited in cells treated with TRPC1 antibody or TRPC1 small interfering (si)RNA, in STIM1 siRNA-transfected cells and in Orai1 siRNA-transfected cells. Moreover, overexpression of STIM1 resulted in a marked increase in [Ca(2+)](i) and Mn(2+) quench rate caused by acute hypoxia, and they were reduced in cells treated with TRPC1 antibody and in cells transfected with Orai1 siRNA. Furthermore, TRPC1 and Orai1 coimmunoprecipitated with STIM1 and the precipitation levels of TRPC1 and Orai1 were increased in cells exposed to acute hypoxia. Immunostaining showed colocalizations of TRPC1-STIM1 and Orai1-STIM1, and the colocalizations of these proteins were more apparent in acute hypoxia. These data provide direct evidence that TRPC1 and Orai1 channels mediate CCE through activation of STIM1 in acute hypoxic mouse PASMCs.

Orai1 interacts with STIM1 and mediates capacitative Ca2+ entry in mouse pulmonary arterial smooth muscle cells.

Previous studies in mouse pulmonary arterial smooth muscle cells (PASMCs) showed that cannonical transient receptor potential channel TRPC1 and stromal interaction molecule 1 (STIM1) mediate the sustained component of capacitative Ca(2+) entry (CCE), but the molecular candidate(s) that mediate the transient component of CCE remain unknown. The aim of the present study was to examine whether Orai1 mediates the transient component of CCE through activation of STIM1 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. The nifedipine-insensitive transient rise in [Ca(2+)](i) and the increase in Mn(2+) quench of fura-2 fluorescence caused by CPA were both reduced in cells treated with Orai1 siRNA. These responses to CPA were further reduced in cells treated with Orai1 and STIM1 small interfering (si)RNA. Moreover, overexpression of STIM1 enhanced the rise in [Ca(2+)](i) and the increase in Mn(2+) quench of fura-2 fluorescence caused by CPA, and these responses were reduced in cells treated with Orai1 siRNA. RT-PCR revealed Orai1 and STIM1 mRNAs, and Western blot analysis identified Orai1 and STIM1 proteins in mouse PASMCs. Furthermore, Orai1 was found to coimmunoprecipitate with STIM1, and the precipitation level of Orai1 was increased in cells subjected to store-depletion. Immunostaining revealed colocalization of Orai1 and STIM1 proteins, and the colocalization of these proteins was more apparent after store-depletion. These data provide direct evidence that the transient component of CCE is mediated by Orai1 channel as a result of STIM1 activation in mouse PASMCs.

The contribution of TRPC1 and STIM1 to capacitative Ca(2+) entry in pulmonary artery.

Capacitative calcium entry (CCE) through store-operated channels (SOCs) has been shown to contribute to the rise in intracellular calcium concentration ([Ca(2+)](i)) and mediate pulmonary artery smooth muscle contraction. CCE is activated as a result of depletion of intracellular Ca(2+) stores but there is a great deal of controversy surrounding the underlying signal that active CCE and the molecular makeup of SOCs. The discovery of canonical subgroup of transient receptor potential channels (TRPC) and recent identification of stromal-interacting molecule 1 (STIM1) protein have opened a door to the study of the identity of SOCs and the signal that activates these channels. Among all the TRPC channels, TRPC1 is widely studied in many cell types and shown to be part of SOCs components, whereas STIM1 protein is found to act as a Ca(2+) sensor in the intracellular Ca(2+) stores and activates SOCs. However, there is very little evidence for the roles of TRPC1 and STIM1 in the contribution of CCE in pulmonary artery. This chapter outlines the roles of TRPC1 and STIM1 in pulmonary artery smooth muscle cells and discusses our recent findings that TRPC1 and STIM1 are functionally interact with each other to mediate CCE in these cells. We also propose a model for the molecular makeup of SOCs formed by TRPC1 and STIM1 in pulmonary artery.

CLC-3 chloride channels in the pulmonary vasculature.

Volume-sensitive outwardly rectifying anion channels (VSOACs) are expressed in pulmonary artery smooth muscle cells (PASMCs) and have been implicated in cell proliferation, growth, apoptosis and protection against oxidative stress. In this chapter, we review the properties of native VSOACs in PASMCs, and consider the evidence that ClC-3, a member of the ClC superfamily of voltage dependent Cl- channels, may be responsible for native VSOACs in PASMCs. Finally, we examine whether or not native VSOACs and heterologously expressed ClC-3 channels function as bona fide chloride channels or as chloride/proton antiporters.

TRPC1 and STIM1 mediate capacitative Ca2+ entry in mouse pulmonary arterial smooth muscle cells.

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.

Cell culture alters Ca2+ entry pathways activated by store-depletion or hypoxia in canine pulmonary arterial smooth muscle cells.

Previous studies have shown that, in acutely dispersed canine pulmonary artery smooth muscle cells (PASMCs), depletion of both functionally independent inositol 1,4,5-trisphosphate (IP(3))- and ryanodine-sensitive Ca(2+) stores activates capacitative Ca(2+) entry (CCE). The present study aimed to determine if cell culture modifies intracellular Ca(2+) stores and alters Ca(2+) entry pathways caused by store depletion and hypoxia in canine PASMCs. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured in fura 2-loaded cells. Mn(2+) quench of fura 2 signal was performed to study divalent cation entry, and the effects of hypoxia were examined under oxygen tension of 15-18 mmHg. In acutely isolated PASMCs, depletion of IP(3)-sensitive Ca(2+) stores with cyclopiazonic acid (CPA) did not affect initial caffeine-induced intracellular Ca(2+) transients but abolished 5-HT-induced Ca(2+) transients. In contrast, CPA significantly reduced caffeine- and 5-HT-induced Ca(2+) transients in cultured PASMCs. In cultured PASMCs, store depletion or hypoxia caused a transient followed by a sustained rise in [Ca(2+)](i). The transient rise in [Ca(2+)](i) was partially inhibited by nifedipine, whereas the nifedipine-insensitive transient rise in [Ca(2+)](i) was inhibited by KB-R7943, a selective inhibitor of reverse mode Na(+)/Ca(2+) exchanger (NCX). The nifedipine-insensitive sustained rise in [Ca(2+)](i) was inhibited by SKF-96365, Ni(2+), La(3+), and Gd(3+). In addition, store depletion or hypoxia increased the rate of Mn(2+) quench of fura 2 fluorescence that was also inhibited by these blockers, exhibiting pharmacological properties characteristic of CCE. We conclude that cell culture of canine PASMCs reorganizes IP(3) and ryanodine receptors into a common intracellular Ca(2+) compartment, and depletion of this store or hypoxia activates voltage-operated Ca(2+) entry, reverse mode NCX, and CCE.

Mobilization of sarcoplasmic reticulum stores by hypoxia leads to consequent activation of capacitative Ca2+ entry in isolated canine pulmonary arterial smooth muscle cells.

Capacitative Ca2+ entry (CCE) has been speculated to contribute to Ca2+ influx during hypoxic pulmonary vasoconstriction (HPV). The aim of the present study was to directly test if acute hypoxia causes intracellular Ca2+ concentration ([Ca2+]i) rises through CCE in canine pulmonary artery smooth muscle cells (PASMCs). In PASMCs loaded with fura-2, hypoxia produced a transient rise in [Ca2+]i in Ca2+-free solution, indicating Ca2+ release from the intracellular Ca2+ stores. Subsequent addition of 2 mm Ca2+ in hypoxia elicited a sustained rise in [Ca2+]i, which was partially inhibited by 10 microm nisoldipine. The dihydropyridine-insensitive rise in [Ca2+]i was due to increased Ca2+ influx, because it was abolished in Ca2+-free solution and hypoxia was shown to significantly enhance the rate of Mn2+ quench of fura-2 fluorescence. The dihyropyridine-insensitive rise in [Ca2+]i and the increased rate of Mn2+ quench of fura-2 fluorescence were inhibited by 50 microm SKF 96365 and 500 microm Ni2+, but not by 100 microm La3+ or 100 microm Gd3+, exhibiting pharmacological properties characteristic of CCE. In addition, predepletion of the intracellular Ca2+ stores inhibited the rise in [Ca2+]i induced by hypoxia. These results provide the first direct evidence that acute hypoxia, by causing Ca2+ release from the intracellular stores, activates CCE in isolated canine PASMCs, which may contribute to HPV.