Original Contribution FUNCTIONAL ASSOCIATION OF NOX1 WITH P22PHOX IN VASCULAR SMOOTH MUSCLE CELLS IBRAHIM R. HANNA,* LULA L. HILENSKI,* ANNA DIKALOVA,* YOSHIHIRO TANIYAMA,* SERGEY DIKALOV ,* ALICIA L YLE,* MARK T. QUINN, y BERNARD LASSE ` GUE,* and KATHY K. GRIENDLING* * Division of Cardiology, Department of Medicine, Emory University, Atlanta, GA 30322; and y Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717, USA (Received 19 May 2004; Revised 27 July 2004; Accepted 12 August 2004) Available online 27 August 2004 Abstract — The vascular NAD(P)H oxidases constitute important sources of ROS in the vessel wall and have been implicated in vascular disease. Vascular smooth muscle cells (VSMCs) from conduit arteries express two gp91phox homologs, Nox1 and Nox4, of which Nox1 is agonist-sensitive. Because p22phox has been shown to be functionally important in vascular cells stimulated with vasoactive hormones, the relationship of Nox1 and p22phox was investigated in VSMCs from rat and human aortas. Coimmunoprecipitation studies demonstrated that p22phox and hemagglutinin- tagged Nox1 associate in unstimulated VSMCs. These findings were confirmed by confocal microscopy, showing colocalization of the two proteins in their native states in the plasma membrane and submembrane areas of the cell. NADPH-driven superoxide production, as measured by electron spin resonance using 1-hydroxy-3-carboxypyrrolidine as a spin probe, is dependent on the coexpression of both subunits, suggesting the importance of the association for the functional integrity of the enzyme. These results indicate that in contrast to the neutrophil enzyme, VSMCs can use Nox1 rather than gp91phox as a catalytic center in the p22phox-based oxidase and that these two proteins are preassembled at or near the plasma membrane and submembrane vesicular structures in unstimulated cells. D 2004 Elsevier Inc. All rights reserved. Keywords — NAD(P)H oxidase, Nox, Vascular smooth muscle, p22phox, Superoxide, Reactive oxygen species, Free radicals INTRODUCTION Reactive oxygen species (ROS) play a central role in vascular pathobiology. They are produced by and affect all cell types in the vessel wall [1–5]. A major source of these highly reactive molecules is the NAD(P)H oxidase family of enzymes distributed throughout the artery. The composite structures of these oxidases have been modeled after the well-studied neutrophil enzyme, with some important differences [6]. The phagocytic oxidase is composed of at least five subunits, with two trans- membrane proteins, p22phox and gp91phox, that asso- ciate in a one-to-one stoichiometry to form cytochrome b 558 [7–9]. Upon neutrophil stimulation, the three cytosolic components p47phox, p67phox, and the small G-protein Rac translocate to the membrane and assemble with cytochrome b 558, resulting in enzyme activation and burst production of ROS. The h-chain of the enzyme, gp91phox, comprises the electron transfer activity, whereas p22phox acts as a stabilizing and regulatory subunit [10]. The NAD(P)H oxidase subunits expressed in vascular cells vary depending on cell type and vessel size. Vascular smooth muscle cells (VSMCs) derived from large conduit vessels possess a functional p22phox-containing flavin oxidase despite the absence of the catalytic center gp91phox [10]. In recent years, novel structural homologs of gp91phox have been identified in VSMCs, namely Nox1 and Nox4 [11,12]. Nox1 mediates NAD(P)H- dependent production of ROS in rat aortic smooth muscle cells (RASMs) stimulated with angiotensin II and PDGF [12], whereas the function of Nox4 in vascular cells remains unclear. Current research is centered on identify- ing the binding partners of these catalytic subunits, based 1542 Address correspondence to: Kathy K. Griendling, Division of Cardiology, Emory University, 319 WMB, 1639 Pierce Drive, Atlanta, GA 30322, USA. Fax: (404) 727 3585; E-mail: kgriend@emory.edu. Free Radical Biology & Medicine, Vol. 37, No. 10, pp. 1542–1549, 2004 Copyright D 2004 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter doi:10.1016/j.freeradbiomed.2004.08.011