Intermittent Hypoxia Elicits a Rapid Up‐Regulation of Cav3.2 T‐type Ca2+ Channels Mediated by Reactive Oxygen Species

Abstract

We previously reported that intermittent hypoxia (IH) augmented Cav3.1 and Cav3.2 T‐type calcium channel subunit expression in adrenal chromaffin cells from neonatal rats. In the present study, we examined the mechanism of T‐type Ca2+ channel up‐regulation by IH in HEK cells stably expressing the Cav3.2 subunit. HEK cells were exposed to between 10–120 cycles of IH (one cycle is 30 sec at 1.5% O2 followed by 5 min at 20% O2). Whole cell patch‐clamp recordings showed that IH treatment increased peak CaV3.2 current density in a stimulation dependent manner and was not reversed for at least 16 hours following re‐oxygenation. IH also augmented plasma membrane expression of CaV3.2 protein levels, as determined by immunoblot. IH elevated ROS levels and ROS scavengers (MnTmPyP; 50μM or PEG‐catalase 500 U/ml) inhibited IH augmented Cav3.2 currents. Xanthine (250 μM)/xanthine oxidase (50 mU/ml) which generate ROS exogenously, mimicked the effects of IH. NADPH oxidase (NOX) are the major source of ROS, mediating cellular responses to IH. NOX activity and protein levels were up‐regulated by IH in HEK cells. The NOX inhibitors apocyanin (500 μM) and AEBSF (100μM), as well as silencing of NOX2, prevented IH induced up‐regulation of CaV3.2 current. These results demonstrate that in HEK cells expressing Cav3.2, ROS generated by NOX mediates IH‐increased CaV3.2 T‐type Ca2+ channel expression. (Supported by NIH‐HL‐90554, HL‐86493).