Role of Hypoxia Inducible Factor (HIF) and NADPH oxidases in regulation of Kv channels in pulmonary artery smooth muscle cells

Rationale: Hypoxia sensitive voltage gated K++ channels have been widely hypothesised for their role in regulating the contractility and growth of pulmonary artery smooth muscle cells (PASMC). Acute hypoxia leads to the inhibition of the hypoxia sensitive Kv channels whereas chronic hypoxia down-regulates their expression at both, mRNA and protein level in isolated rat PASMCs. Regarding the regulation of various Kv channels in hypoxia the role of reactive oxygen species (ROS) has been discussed but the mechanism of its action is still unclear.

Objectives: We investigated the role of NADPH oxidases and ROS in regulating the expression of Kv channels through hypoxia inducible transcription factors (HIF).

Methods and Results: Studies were performed on isolated rat PASMC and murine lung sections. Isolated rat PASMC showed an increased protein level of NOX4 and a simultaneous decrease in expression of Kv2.1 channel upon exposure to hypoxia (72h, 1%O2). RNA interference against NOX4 resulted in an increase in the protein level of Kv2.1 under normoxic conditions. This was accompanied by a decrease in the protein expression of a splice variant of HIF-2 alpha. The upstream region of Kv2.1 mRNA was found to contain three hypoxia responsive element (HRE) sites which were screened for their binding to HIF transcription factors using an electro mobility shift assay (EMSA). EMSA results showed that one of the HRE binding sites showed hypoxia specific shift which disappeared on incubation with HIF-2 alpha antibody and treatment with NADPH oxidase inhibitors such as DPI (Diphenyleneiodonium).

Conclusion: Our results show that NADPH oxidases and ROS can be an important regulator of expression of Kv channels via hypoxia inducible factors.