Upregulation of Intermediate-Conductance Ca(2+)-Activated K(+) Channels (KCNN4) in Porcine Coronary Smooth Muscle Requires NADPH Oxidase 5 (NOX5)

AIMS: NADPH oxidase (NOX) is the primary source of reactive oxygen species (ROS) in vascular smooth muscle cells (SMC) and is proposed to play a key role in redox signaling involved in the pathogenesis of cardiovascular disease. Growth factors and cytokines stimulate coronary SMC (CSMC) phenotypic modulation, proliferation, and migration during atherosclerotic plaque development and restenosis. We previously demonstrated that increased expression and activity of intermediate-conductance Ca(2+)-activated K(+) channels (KCNN4) is necessary for CSMC phenotypic modulation and progression of stenotic lesions. Therefore, the purpose of this study was to determine whether NOX is required for KCNN4 upregulation induced by mitogenic growth factors. METHODS AND RESULTS: Dihydroethidium micro-fluorography in porcine CSMCs demonstrated that basic fibroblast growth factor (bFGF) increased superoxide production, which was blocked by the NOX inhibitor apocynin (Apo). Apo also blocked bFGF-induced increases in KCNN4 mRNA levels in both right coronary artery sections and CSMCs. Similarly, immunohistochemistry and whole cell voltage clamp showed bFGF-induced increases in CSMC KCNN4 protein expression and channel activity were abolished by Apo. Treatment with Apo also inhibited bFGF-induced increases in activator protein-1 promoter activity, as measured by luciferase activity assay. qRT-PCR demonstrated porcine coronary smooth muscle expression of NOX1, NOX2, NOX4, and NOX5 isoforms. Knockdown of NOX5 alone prevented both bFGF-induced upregulation of KCNN4 mRNA and CSMC migration. CONCLUSIONS: Our findings provide novel evidence that NOX5-derived ROS increase functional expression of KCNN4 through activator protein-1, providing another potential link between NOX, CSMC phenotypic modulation, and atherosclerosis.