Store-Operated Channels Mediate Ca ²⁺ Influx and Contraction in Rat Pulmonary Artery

Abstract

Abstract-Cation channels activated by Ca 2ϩ store depletion have been proposed to mediate Ca 2ϩ influx in vascular smooth muscle cells. The aim of this study was to determine if store-operated channels have a functional role in pulmonary artery smooth muscle cells (PASMCs). In intact rat pulmonary artery rings, cyclopiazonic acid (CPA) produced a sustained contraction that was resistant to inhibition by nifedipine, but abolished in Ca 2ϩ -free solution and 50% blocked in the presence of 6 mol/L Cd 2ϩ , 10 mol/L Ni 2ϩ , 600 mol/L La 3ϩ , and 7 mol/L SKF96365. In freshly isolated PASMCs loaded with fura-2, CPA increased the intracellular Ca 2ϩ concentration by stimulating dihydropyridine-resistant Ca 2ϩ influx, which was Ϸ50% blocked by 10 mol/L Ni 2ϩ and 7 mol/L SKF96365. In perforated-patch recordings, CPA activated a sustained inward current at negative membrane potentials, which persisted in cells dialyzed with BAPTA, showed a near linear dependence on membrane potential when Cs ϩ was the main intracellular cation, and was blocked by Ni 2ϩ , Cd 2ϩ , and SKF96365 at concentrations preventing contraction. The current showed a bimodal dependence on extracellular Ca 2ϩ , being enhanced 2-fold in the absence of Ca 2ϩ and around 10-fold on reducing Ca from 1.8 to 0.2 mmol/L. RT-PCR revealed the expression of Trp1, Trp3, Trp4, Trp5, and Trp6 mRNA, whereas immunostaining identified Trp1, Trp3, Trp4, and Trp6 channel proteins in isolated PASMCs. At least one of these subunits may contribute to cation channels in PASMCs, which are activated by store depletion to bring about Ca 2ϩ influx and contraction.

Properties of a store-operated nonselective cation channel in airway smooth muscle

“…To determine if a similar mechanism operates in ASM, on-cell measurements of single channel currents were conducted both prior to and during passive store depletion with either CPA or BAPTA-AM. In all cell-attached patches examined, there was evidence of spontaneous single channel activity (,44% exhibiting robust activity), which was inhibit CPA-activated membrane currents and/or CCE in several tissues [16,21,27]. In the present study, application of 10 and 100 mM La 3+ reversibly inhibited the BAPTA-AMevoked current in a dose-dependent manner.…”

“…CPA enhances Ca 2+ entry in several other ASM preparations presumably through activation of Ca 2+ -permeable NSCCs [10,15,16,24]. Basal NSCC currents have also been described in isolated smooth muscle cells from rabbit portal vein [25], ear artery [26] and coronary artery [22] as well as rat pulmonary artery [27].…”

Enhanced expression of transient receptor potential channels in idiopathic pulmonary arterial hypertension

“…Functional TRPC channels exist in native cells as homo-or heterotetramers composed of two or more TRPC subunits (29,42,43). Studies have shown that overexpression of TRPC genes in mammalian cells leads to the formation of Ca 2ϩ permeable cation channels activated by both ligand-receptor interaction and store depletion (29,31,33,(42)(43)(44)(45)(46)(47)(51)(52)(53)(54)(55).…”

“…A central aspect of pulmonary vascular remodeling is medial hypertrophy caused by sustained pulmonary vasoconstriction (2-4), excessive pulmonary artery smooth muscle cell (PASMC) proliferation (5), and inhibited PASMC apoptosis (6, 7), resulting in a narrowed vascular lumen and increased PVR. Although its etiology remains unclear, elevated levels of circulating mitogens, dysfunction or down-regulation of receptors and ion channels, upregulation of transporters, and heightened activity of elastases and glycoproteins have been implicated in IPAH (5,6,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) Transient receptor potential (TRP) channel genes may encode subunits that form receptor-(ROC) and store-(SOC) operated Ca 2ϩ channels in many cell types, including PASMC and pulmonary artery endothelial cells (PAEC) (28,(30)(31)(32)(33)(34). Ca 2ϩ entry through ROC and SOC increases [Ca 2ϩ ] cyt , allowing for phosphorylation of signal transduction proteins and transcription factors (23,24,(35)(36)(37)(38), that are essential for the progression of the cell cycle (21).…”

“…Transient receptor potential (TRP) channel genes may encode subunits that form receptor-(ROC) and store-(SOC) operated Ca 2ϩ channels in many cell types, including PASMC and pulmonary artery endothelial cells (PAEC) (28,(30)(31)(32)(33)(34). Ca 2ϩ entry through ROC and SOC increases [Ca 2ϩ ] cyt , allowing for phosphorylation of signal transduction proteins and transcription factors (23,24,(35)(36)(37)(38), that are essential for the progression of the cell cycle (21).…”