Abstract 2333: Localization of NADPH Oxidase-4 is Crucial in Determining the Oxygen Sensitivity of TASK-1 Channel in Primary Human Pulmonary Artery Smooth Muscle Cells
Hypoxic pulmonary vasoconstriction (HPV) is a physiological response of small pulmonary arteries that diverts mixed venous blood away from hypoxic alveoli, thus optimizing the matching of perfusion and ventilation and preventing arterial hypoxemia. Acute hypoxic inhibition of potassium (K+) channels is a critical step in regulatory processes designed to link lowering of oxygen levels to cellular responses in pulmonary artery smooth muscle cells (PASMCs) and to pulmonary vasoconstriction. The resting membrane potential and consequently the intracellular calcium concentration are determined by TASK-1 channels in human PASMC. However, the involvement of TASK-1 in HPV is still unknown. In the present study, we investigated the role of NADPH oxidase-4 and its functional subunit in determining the oxygen sensitivity of TASK-1 channel in primary human PASMCs. We employed the whole-cell patch-clamp technique combined with small interfering (si)RNA, quantitative RT-PCR, immunostaining and confocal microscopy in human PASMCs. Our data show that moderate hypoxia (pO2 app. 40mmHg) significantly depolarized primary human PASMCs and reversibly inhibited TASK-1 current to 51±3% (n=22, p<0.001) calculated at 0 mV. Silencing NADPH oxidase-4 with siRNA abolished the effect of hypoxia (n=25) on TASK-1. Silencing of the functional subunit of p22phox (n=22) revealed the same effect, whereas the scrambled siRNA (n=15) failed to attenuate the hypoxia-induced inhibition. The localization of the reactive oxygen species (ROS) producing NADPH oxidase-4 is crucial for the hypoxic inhibition of TASK-1. Using immunostaining followed by confocal microscopy we found the NADPH oxidase-4 is suited in the endoplasmic reticulum and nucleus of the human PASMCs. This is the first report indicating the functional requirement of ROS producing NADPH oxidase-4 and its subunit p22phox in determining the acute hypoxic-sensitivity of TASK-1 channels in native human PASMCs. These results may lead to better understanding of the redox mediated regulation behind oxygen sensitive channels.