Cytochrome P450 1B1 Is Essential For Angiotensin II‐ And Arachidonic Acid‐Induced Activation Of NADPH Oxidase In Vascular Smooth Muscle Cells Of Mice

Abstract

Previously, we have shown that angiotensin II (Ang II) and arachidonic acid (AA) via its metabolism by cytochrome P450 (CYP1B1) activates NADPH oxidase and generates superoxide in rat vascular smooth muscle cells (VSMCs). This study was conducted to determine the effect of Ang II and AA on NADPH oxidase activity in VSMCs from Cyp1b1-/- mice with and without transduction of adenovirus cDNA Cyp1b1. VSMCs were isolated from thoracic aorta of wild type (Cyp1b1+/+) and CYP1B1 knockout (Cyp1b1-/-) mice. And treated with Ang II (200 nM) or AA (30 mM) for 15 min. In another series of experiments, VSMCs isolated and cultured from Cyp1b1-/- mice were transduced with 120 MOI and after 24 hrs were treated with Ang II or AA for 15 min. The cells were sonicated for 10 second and ultracentrifuged at 100,000g to separate cell membranes. NADPH oxidase activity in the membranes was measured by using 5 nM lucigenin enhanced-chemiluminescence based assay. Ang II and AA increased NADPH oxidase activity in VSMC from Cyp1b1+/+ but not Cyp1b1-/- mice. However, in Cyp1b1-/- VSMCs transduced with adenovirus cDNA Cyp1b1, Ang II and AA increased NADPH oxidase activity. These data suggest that CYP1B1 is essential for Ang II-induced activation of NADPH oxidase and is most likely mediated by AA metabolites and/or lipid peroxides in VSMCs.