Activation of H2O2-induced VSOR CI(-)currents in HTC cells require phospholipase C gamma 1 phosphorylation and Ca2+ mobilisation

Volume-sensitive outwardly rectifying (VSOR) Cl- channels participate in several physiological processes such as regulatory volume decrease, cell cycle regulation, proliferation and apoptosis. Recent evidence points to a significant role of hydrogen peroxide (H2O2) in VSOR Cl- channel activation. The aim of this study was to determine the signalling pathways responsible for H2O2-induced VSOR Cl- channel activation. In rat hepatoma (HTC) cells, H2O2 elicited a transient increase in tyrosine phosphorylation of phospholipase C gamma 1 (PLC gamma 1) that was blocked by PP2, a Src-family protein kinases inhibitor. Also, H2O2 triggered an increase in cytosolic [Ca2+] that paralleled the time course of PLC gamma 1 phosphorylation. The H(2)O(2)induced [Ca2+](i) rise was prevented by the generic phospholipase C (PLC) inhibitor U73122 and the inositol 1,4,5-trisphosphate-receptor (IP3R) blocker 2-APB. In line with these results, manoeuvres that prevented PLC gamma 1 activation and/or [Ca2+](i) rise, abolished H2O2-induced VSOR Cl- currents. Furthermore, in cells that overexpress a phosphorylation-defective dominant mutant of PLC gamma 1, H2O2 did not induce activation of VSOR Cl- currents. All these H2O2-induced effects were independent of extracellular Ca2+. Our findings suggest that activation of PLC gamma 1 and subsequent Ca-i(2+) mobilisation mediate H2O2-induced VSOR Cl- currents, indicating that H2O2 operates via redox-sensitive signalling pathways akin to those activated by osmotic challenges. Copyright (c) 2007 S. Karger AG, Basel.