Noxal is a central component of the smooth muscle NADPH oxidase in mice

NADPH oxidase is the most important source of oxygen-derived radicals (ROS) in the vascular wall. In vascular smooth muscle cells (VSMC), NADPH oxidase is characterized by the expression of the membrane subunit Nox1, which is activated by cytoplasmic proteins binding to its activation domain. We set out to identify the cytoplasmic protein involved in NADPH oxidase activation in mouse VSMC. Western blot analysis revealed that human endothelial cells and leukocytes but not VSMC from the aorta of the rat and the mouse express the classic NADPH oxidase activator p67phox. In mouse VSMC, however, the p67phox homologue Noxal was detected. Using antibodies generated against mouse Noxa I, the protein was observed in the cytosolic fraction of mouse VSMC with a molecular weight of about 51 kDa. Immunohistochemistry revealed that Noxa I is expressed in the smooth muscle layer but not in endothelium or the adventitia of the mouse carotid artery. Fluorescent fusion proteins of Noxal were observed to be expressed in the cytoplasm of VSMC and coexpression of the NADPH oxidase organizer Noxol targeted the complex to membrane. An antisense plasmid of Noxal attenuated the endogenous Noxal protein expression in VSMC. This plasmid attenuated the ROS formation in mouse VSMC as detected using L012 chemiluminescence and prevented the agonist-induced ROS production in response to basic fibroblast growth factor and epidermal growth factor. In conclusion, these data indicate that Noxal replaces p67phox in VSMC and plays a central role in the activation of the NADPH oxidase in the vascular wall. (c) 2006 Elsevier Inc. All rights reserved.