Signaling Mechanism of Cytochrome P450 1B1‐Dependent Angiotensin II‐Induced Activation of NADPH Oxidase in Vascular Smooth Muscle Cells

Abstract

Previously, we have shown that angiotensin II (Ang II) activates NADPH oxidase and generates superoxide via release of arachidonic acid (AA) and its metabolism by cytochrome P450 (CYP1B1) in rat vascular smooth muscle cells (VSMCs). This study was conducted to determine the signaling mechanism involved in CYP1B1‐dependent Ang II‐ and AA‐induced NADPH oxidase activity in VSMCs isolated from aorta of rat, and wild type (Cyp1b1+/+) and CYP1B1 knockout (Cyp1b1−/−) mice. Ang II‐and AA‐induced increase in NADPH oxidase activity, measured using lucigenin as a chemiluminescent probe, was inhibited in rat VSMCs transduced with adenovirus (Ad) shRNA CYP1B1 but not its scrambled control shRNA, and in VSMCs from Cyp1b1−/− but not Cyp1b1+/+ mice. Ang II and AA increased ERK1/2, p38 MAPK, and c‐Src activities as determined by their phosphorylation by western blot analysis, in VSMCs from rat and Cyp1b1+/+ mice; increases in the activities of these signaling molecules were inhibited by Ad shRNA CYP1B1 in rat VSMCs, and in VSMCs from Cyp1b1−/− mice. Inhibitors of ERK1/2 (U0126), p38 MAPK (SB202190), and c‐Src (PP2), blocked Ang II‐ and AA induced increase in activities of these signaling molecules and NADPH oxidase, but not CYP1B1, measured using P450‐Glo™ Assay Kit, in rat VSMCs. These data suggest that Ang II stimulates NADPH oxidase activity via AA metabolism by CYP1B1 and activation of ERK1/2, p38 MAPK, and c‐Src, in VSMCs.