Activation of H2O2-induced VSOR Cl- currents in HTC cells require phospholipase Cgamma1 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 (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.
Varela
Diego
D
Centro de Estudios Moleculares de la Célula & Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
Simon
Felipe
F
Olivero
Pablo
P
Armisén
Ricardo
R
Leiva-Salcedo
Elías
E
Jørgensen
Finn
F
Sala
Francisco
F
Stutzin
Andrés
A
D000818:Animals; D001896:Boron Compounds / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects*; D045744:Cell Line, Tumor; D018118:Chloride Channels / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005799:Genes, Dominant; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D015640:Ion Channel Gating / Q000187:drug effects*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D050505:Mutant Proteins / Q000378:metabolism; D051966:Phospholipase C gamma / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011743:Pyrimidines / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
C412373:AG 1879; D001896:Boron Compounds; D018118:Chloride Channels; D004963:Estrenes; D050505:Mutant Proteins; D011743:Pyrimidines; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D004815:Epidermal Growth Factor; D006861:Hydrogen Peroxide; C109986:2-aminoethoxydiphenyl borate; D019061:src-Family Kinases; D051966:Phospholipase C gamma