Important role of PLC-γ1 in hypoxic increase in intracellular calcium in pulmonary arterial smooth muscle cells

Yadav VR, Song T, Joseph L, Mei L, Zheng Y-M, Wang Y-X. Important role of PLC-gamma 1 in hypoxic increase in intracellular calcium in pulmonary arterial smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 304: L143-L151, 2013. First published November 30, 2012; doi: 10.1152/ajplung.00310.2012.-An increase in intracellular calcium concentration ([Ca2+](i)) in pulmonary arterial smooth muscle cells (PASMCs) induces hypoxic cellular responses in the lungs; however, the underlying molecular mechanisms remain incompletely understood. We report, for the first time, that acute hypoxia significantly enhances phospholipase C (PLC) activity in mouse resistance pulmonary arteries (PAs), but not in mesenteric arteries. Western blot analysis and immunofluorescence staining reveal the expression of PLC-gamma 1 protein in PAs and PASMCs, respectively. The activity of PLC-gamma 1 is also augmented in PASMCs following hypoxia. Lentiviral shRNA-mediated gene knockdown of mitochondrial complex III Rieske iron-sulfur protein (RISP) to inhibit reactive oxygen species (ROS) production prevents hypoxia from increasing PLC-gamma 1 activity in PASMCs. Myxothiazol, a mitochondrial complex III inhibitor, reduces the hypoxic response as well. The PLC inhibitor U73122, but not its inactive analog U73433, attenuates the hypoxic vasoconstriction in PAs and hypoxic increase in [Ca2+] i in PASMCs. PLC-gamma 1 knockdown suppresses its protein expression and the hypoxic increase in [Ca2+](i). Hypoxia remarkably increases inositol 1,4,5-trisphosphate (IP3) production, which is blocked by U73122. The IP3 receptor (IP3R) antagonist 2-aminoethoxydiphenyl borate (2-APB) or xestospongin-C inhibits the hypoxic increase in [Ca2(+)](i). PLC-gamma 1 knockdown or U73122 reduces H2O2-induced increase in [Ca2(+)](i) in PASMCs and contraction in PAs. 2-APB and xestospongin-C produce similar inhibitory effects. In conclusion, our findings provide novel evidence that hypoxia activates PLC-gamma 1 by increasing RISP-dependent mitochondrial ROS production in the complex III, which causes IP3 production, IP3R opening, and Ca2+ release, playing an important role in hypoxic Ca2+ and contractile responses in PASMCs.