Role of PLCgamma 1-IP3R1 pathway in hypoxia-induced increase in itracellular calcium in pulmonary artery smooth muscle cells.

摘要

An increase in intracellular calcium concentration ([Ca2+] i) in pulmonary arterial smooth muscle cells (PASMCs) is vital for hypoxia-induced cellular responses in the lungs. However, the underlying molecular mechanisms remain incompletely understood. Here we have for the first time shown that acute hypoxia for 5 min significantly enhances the activity of total phospholipase C (PLC) in mouse resistance PAs, but not in mesenteric arteries. Western blot analysis and immunofluorescence staining reveal that PLCγ1 protein is expressed in PAs and PASMCs. The activity of PLCγ1 is augmented by acute hypoxia as well in PASMCs. Lentiviral shRNA-mediated Rieske iron-sulfur protein (RISP) knockdown in the mitochondrial complex III to inhibit reactive oxygen species (ROS) production diminishes the hypoxia-induced increase in the activity of PLCγ1 in PASMCs. Similarly, myxothiazol, an inhibitor of mitochondrial ROS production in the complex III, also reduces the hypoxia-induced response. The PLC inhibitor U73122 attenuates the hypoxia-induced vasoconstriction in PAs and hypoxia-induced increase in [Ca2+]i, in PASMCs, but its inactive analog U73433 has no effect. PLCγ1 knockdown remarkably suppresses its protein expression and the hypoxia-induced increase in [Ca2+]i, in PASMCs. Hypoxia causes an increase in the production of inositol trisphosphate (IP3). The hypoxia-induced increase in IP3 production is fully blocked by either U73122 or knock-down of PLCγ1. The IP3 receptor (IP3) antagonists 2- aminoethoxydiphenyl borate (2-APB) and xestospongin-C inhibits the hypoxia-induced increase in [Ca2+]i. Furthermore knock down IP 3R1 significantly inhibited hypoxia-induced increase in [Ca 2+]i. PLCγ1 knockdown reduces H2O 2-induced increase in [Ca2+]i. U73122, but not U73433, blocks H2O2-induced increase in [Ca 2+]i in PASMCs as well. Similarly, 2-APB and xestospongin-C also produce an inhibitory effect. Studies in chronic hypoxia exposed mice have revealed total higher PLC activity and more specifically PLCγ1 expression and activity. Furthermore, hyper contractility observed in PAs from CH mice was inhibited by PLC inhibition with U73122.;In conclusion, our findings provide the first evidence that hypoxia activates PLCγ1 by increasing RISP-dependent mitochondrial ROS production in the complex III, which causes IP3 production, IP3R 1 opening and Ca2+ release, playing an important role in hypoxia-induced Ca2+ responses in PASMCs. PLCγ1 may be involved in chronic hypoxia related pulmonary arterial hypertension and related diseases.