Sildenafil inhibits chronically hypoxic upregulation of canonical transient receptor potential expression in rat pulmonary arterial smooth muscle

Abstract

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.

Sildenafil decreases rat tracheal hyperresponsiveness to carbachol and changes canonical transient receptor potential gene expression after antigen challenge

“…Sildenafil inhibited human pulmonary artery smooth muscle cell proliferation by decreasing capacitative Ca 2+ entry, with its antiproliferative effect being partially related to the effect on calcium signaling via down-regulation of TRPC1 gene expression. Additionally, chronic hypoxia elevated basal intracellular Ca 2+ levels in pulmonary artery smooth muscle cells by enhancing store-operated Ca 2+ entry through probable up-regulated TRPC1 and TRPC6 gene expression (39). According to this study, sildenafil inhibited the increases in TRPC expression in cells exposed to prolonged hypoxia, and, as expected, it decreased TRPC1 and TRPC6 expression to values found in control conditions.…”

Effects of chronic exposure to cigarette smoke on canonical transient receptor potential expression in rat pulmonary arterial smooth muscle

“…Right ventricular pressure and right ventricular hypertrophy were measured using the same method as we described previously (12). Briefly, a 23-gauge needle filled with heparinized saline was connected to a pressure transducer and inserted via the diaphragm into the right ventricle.…”

“…Chamber temperature was maintained at 37°C with an in-line heat exchanger and dual channel heater controller (SF-28 and TC-344B; Warner Instruments). After perfusion for 10 min to remove extracellular dye, we measured fura-2 fluorescence emitted at 510 nm after excitation at 340 and 380 nm (F 340, F380) in 15ϳ30 cells using a xenon arc lamp, interference filters, electronic shutter, ϫ20 fluorescence objective, and cooled charge-coupled device imaging camera, as previously described (12). Protocols were executed and data were collected on-line with InCyte software (Intracellular Imaging, Cincinnati, OH).…”

Sodium Tanshinone IIA Sulfonate Inhibits Canonical Transient Receptor Potential Expression in Pulmonary Arterial Smooth Muscle from Pulmonary Hypertensive Rats

“…Animals such as rat and mouse exposed to chronic hypoxia developed chronically hypoxia pulmonary hypertension (CHPH) in less than 3 weeks (23)(24)(25) (27,(29)(30)(31). A monocrotaline (MCT)-induced PH animal model is also commonly used to study the pathological mechanisms of PAH.…”

“…Right ventricular systolic pressure and right ventricular hypertrophy were measured using the same method as we described previously (30). Intrapulmonary vessels were visualized by H&E staining on formalin-fixed and paraffin-embedded lung cross-sections (5 mm thickness).…”