H2O2 regulates recombinant Ca2+ channel alpha1C subunits but does not mediate their sensitivity to acute hypoxia.
Acute hypoxic inhibition of the pore-forming alpha(1C) subunit of the L-type Ca(2+) channel mediates hypoxic arterial vasodilatation, a physiological response which matches tissue O(2) demand and supply in the systemic vasculature. In numerous O(2)-sensing cell types, reactive O(2) species (ROS) have been proposed as mediators linking lowered O(2) levels with the appropriate cellular response. In this study, we examined the roles of H(2)O(2) and NADPH oxidase as mediators of hypoxic inhibition of recombinant alpha(1C) subunits. Human cardiac L-type Ca(2+) channel alpha(1C) subunits were stably expressed in HEK 293 cells. Ca(2+) currents were recorded using the whole-cell configuration of the patch-clamp technique. Bath application of 100microM H(2)O(2) significantly enhanced depolarisation-evoked Ca(2+) currents in a voltage-dependent manner, while dialysis with 1000Uml(-1) catalase reduced these currents. In the presence of catalase, hypoxic inhibition of Ca(2+) currents was not significantly different compared to non-dialysed controls. The NADPH oxidase inhibitors diphenylene iodonium (10microM) and phenylarsine oxide (5microM) were without effect on either basal Ca(2+) currents or responses to hypoxia. Thus, endogenous production of H(2)O(2) regulates the alpha(1C) subunit. However, neither suppression of H(2)O(2) levels nor inhibition of NADPH oxidase is involved in O(2)-dependent regulation of the Ca(2+) channel.
Hudasek
Kristin
K
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ont., Canada L8S 4K1.
Brown
Stephen T
ST
Fearon
Ian M
IM
D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002374:Catalase / Q000494:pharmacology; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D004594:Electrophysiology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology*; D009206:Myocardium / Q000737:chemistry; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D018408:Patch-Clamp Techniques; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D020746:Calcium Channels, L-Type; D021122:Protein Subunits; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D002374:Catalase; D019255:NADPH Oxidases