Activation of H<sub>2</sub>O<sub>2</sub>-Induced VSOR Cl<sup>-</sup> Currents in HTC Cells Require Phospholipase Cγ1 Phosphorylation and Ca<sup>2+</sup> Mobilisation

Abstract

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 (H(2)O(2)) in VSOR Cl(-) channel activation. The aim of this study was to determine the signalling pathways responsible for H(2)O(2)-induced VSOR Cl(-) channel activation. In rat hepatoma (HTC) cells, H(2)O(2) elicited a transient increase in tyrosine phosphorylation of phospholipase Cgamma1 (PLCgamma1) that was blocked by PP2, a Src-family protein kinases inhibitor. Also, H(2)O(2) triggered an increase in cytosolic [Ca(2+)] that paralleled the time course of PLCgamma1 phosphorylation. The H(2)O(2)-induced [Ca(2+)](i) rise was prevented by the generic phospholipase C (PLC) inhibitor U73122 and the inositol 1,4,5-trisphosphate-receptor (IP(3)R) blocker 2-APB. In line with these results, manoeuvres that prevented PLCgamma1 activation and/or [Ca(2+)](i) rise, abolished H(2)O(2)-induced VSOR Cl(-) currents. Furthermore, in cells that overexpress a phosphorylation-defective dominant mutant of PLCgamma1, H(2)O(2) did not induce activation of VSOR Cl(-) currents. All these H(2)O(2)-induced effects were independent of extracellular Ca(2+). Our findings suggest that activation of PLCgamma1 and subsequent Ca(2+)(i) mobilisation mediate H(2)O(2)-induced VSOR Cl(-) currents, indicating that H(2)O(2) operates via redox-sensitive signalling pathways akin to those activated by osmotic challenges.