In pulmonary arterial smooth muscle cells (PASMCs), Ca2+ influx through store-operated Ca2+ channels thought to be composed of canonical transient receptor potential (TRPC) proteins is an important determinant of intracellular free calcium concentration ([Ca2+](i)) and pulmonary vascular tone. Sildenafil, a type V phosphodiesterase inhibitor that increases cellular cGMP, is recently identified as a promising agent for treatment of pulmonary hypertension. We previously demonstrated that chronic hypoxia elevated basal [Ca2+](i) in PASMCs due in large part to enhanced store-operated Ca2+ entry (SOCE); moreover, ex vivo exposure to prolonged hypoxia (4% O2 for 60 h) upregulated TRPC1 and TRPC6 expression in PASMCs. We examined the effect of sildenafil on basal [Ca2+](i), SOCE, and the expression of TRPC in PASMCs under prolonged hypoxia exposure. We also examined the effect of sildenafil on TRPC1 and TRPC6 expression in pulmonary arterial smooth muscle (PA) from rats that developed chronically hypoxic pulmonary hypertension (CHPH). Compared with vehicle control, treatment with sildenafil (300 nM) inhibited prolonged hypoxia induced increases of 1) basal [Ca2+](i), 2) SOCE, and 3) mRNA and protein expression of TRPC in PASMCs. Moreover, sildenafil (50 mg . kg(-1) . day(-1)) inhibited mRNA and protein expression of TRPC1 and TRPC6 in PA from chronically hypoxic (10% O2 for 21 days) rats, which was associated with decreased right ventricular pressure and right ventricular hypertrophy. Furthermore, we found, in PASMCs exposed to prolonged hypoxia, that knockdown of TRPC1 or TRPC6 by their specific small interference RNA attenuated the hypoxic increases of SOCE and basal [Ca2+]i, suggesting a cause and effect link between increases of TRPC1 and TRPC6 expression and the hypoxic increases of SOCE and basal [Ca2+]i. These results suggest that sildenafil may alter basal [Ca2+](i) in PASMCs by decreasing SOCE through downregulation of TRPC1 and TRPC6 expression, thereby contributing to decreased vascular tone of pulmonary arteries during the development of CHPH.

from an EF-hand motif in the NH 2 -terminal region of STIM1 located within the SR/ER lumen, translocation of STIM1 proteins to form oligomeric collections termed "puncta" in portions of the SR/ER membrane close to SOC in plasma membrane, and interactions of STIM1 with SOC and/or associated regulatory proteins that lead to channel activation and SOCE (13,59,62). Indeed, it has been proposed that fulfillment of these functions by STIM1 should define SOC (58). Closely related to STIM1 is STIM2, a 105-kDa protein detected in SR/ER but not plasma membrane, which has 61% structural homology with STIM1, including an EF-hand domain, a protein-protein interaction site known as the sterile ␣-motif domain, and a coiled-coils membrane spanning region within an ezrin/radixin/moesin (ERM) domain (9). The function of STIM2 is not as well understood as that of STIM1. Some studies indicate that STIM2 has little or no effect on SOCE (22,30,36,51), whereas others suggest that STIM2 may inhibit STIM1 (50), act to maintain basal cytoplasmic and SR/ER luminal Ca 2ϩ (4), or regulate store-dependent and -independent activation of SOC (35). Although STIM2 was detected in airway and coronary arterial smooth muscle (36, 51), expression in PASMC has not been reported.In a previous study of distal and proximal pulmonary arteries (24), we found that STIM1 expression, SOCE, and [Ca 2ϩ ] i responses to hypoxia but not KCl were greater in myocytes from distal arteries, which are thought to be the major locus of HPV (45,47). In this study, we examined STIM2 expression and used RNA interference to evaluate the roles of STIM1 and STIM2 in SOCE and [Ca 2ϩ ] i responses to acute hypoxia in distal PASMC. METHODSIsolation and culture of rat distal PASMC. Animal protocols were approved by the Animal Care and Use Committee of the Johns Hopkins Medical Institutions. PASMC were harvested from distal (Ͼ4th generation) intrapulmonary arteries of anesthetized (pentobarbital, 65 mg/kg ip) male Wistar rats (300 -500 g body wt) and cultured