Nitration and Functional Loss of Voltage-Gated K  Channels in Rat Coronary Microvessels Exposed to High Glucose Hongwei Li, 1 David D. Gutterman, 2,3 Nancy J. Rusch, 3,4 Aaron Bubolz, 2,3 and Yanping Liu 2,3 Coronary microvessels generate reactive oxygen spe- cies in response to high glucose (HG), resulting in vasodilator defects involving an impaired function of vascular K  channels. Inhibition of voltage-gated K  (K v ) channels by peroxynitrite (ONOO  ), formed by the interaction of superoxide and nitric oxide, may contrib- ute to impaired dilation. The present study investigated whether HG induces ONOO  formation to mediate ni- tration and impairment of K v channels in rat small coronary arteries (RSCAs). Exposure to ONOO  re- duced the dilator influence of K v channels in RSCAs. Patch-clamp studies revealed that ONOO  diminished whole-cell and unitary K v currents attributable to the K v 1 gene family in smooth muscle cells. Subsequently, immunohistochemically detected enhancement of nitro- tyrosine residues in RSCAs that were cultured in HG (23 mmol/l) compared with normal glucose (5.5 mmol/l) for 24 h correlated with the nitration of K v 1.2 channel -subunits. HG-induced nitrotyrosine formation was par- tially reversed by scavenging ONOO  . Finally, RSCAs that were exposed to HG for 24 h showed a loss of K v channel dilator influence that also was partially re- stored by the ONOO  scavengers urate and ebselen. We conclude that ONOO  generated by HG impairs K v channel function in coronary microvessels, possibly by nitrating tyrosine residues in the pore-forming region of the K v channel protein. Diabetes 53:2436 –2442, 2004 I n type 2 diabetes, endothelium-dependent vasodila- tor responses are impaired in both the macro- and the microvasculature, possibly related to the forma- tion of reactive oxygen species (ROS). The presence of hyperglycemia per se fosters the local formation of superoxide (O 2 ˙ - ) (1,2), which may interact with nitric oxide (NO) to form the highly reactive oxidant peroxyni- trite (ONOO - ). Indeed, recent evidence has emphasized that NO derived from inducible NO synthase under condi- tions of high glucose (HG) may contribute to vasodilator dysfunction by driving the generation of ONOO - (3). In turn, ONOO - may promote oxidative and nitrosative tis- sue damage (4), in part by nitration of tyrosine residues to impair the function of multiple proteins, including K + channels that mediate vasodilation (5,6). In this regard, most studies have focused on ONOO - - induced inhibition of high-conductance Ca 2+ -activated K + (K Ca ) channels as a mechanism of vasodilator impairment (5,6). However, a targeted inhibition of K Ca channels by ONOO - cannot account for some of the profound vasodi- lator defects that are induced by HG and seem to relate more closely to an impaired function of voltage-gated K + (K v ) channels. For example, vasodilator responses to forskolin and isoproterenol rely on cAMP-induced activa- tion of K v channels to mediate relaxation, but these responses are markedly blunted in rat small coronary arteries (RSCAs) that are exposed to HG for 24 h (7). In addition, the activity and expression levels of K v channels are sensitive to oxidant stress (8), and although K v chan- nels emanate from at least 11 gene families (K v 1–K v 11), the K v 1 “Shaker-type” family channels that are densely ex- pressed in vascular smooth muscle cells (VSMCs) may be particularly susceptible to open-channel block by redox agents (9,10). Collectively, these findings provide a com- pelling reason to investigate whether ROS associated with HG alter K v channel function. The present study tested the hypothesis that HG induces endogenous ONOO - forma- tion in RSCAs, resulting in nitration and impairment of K v channels and vasodilator function. RESEARCH DESIGN AND METHODS Preparation of RSCAs. Seven-week-old male Sprague-Dawley rats (Harlan, Madison, WI) were anesthetized with sodium pentobarbital (60 mg/kg i.p.). RSCAs (internal diameter 150 –200 m) were dissected from the left ventricle. Some RSCAs were incubated in culture media supplemented with either 5.5 mmol/l D-glucose (normal glucose [NG]) or 23 mmol/l D-glucose (high glucose [HG]), or 5.5 mmol/l D-glucose plus 17.5 mmol/l L-glucose (LG) for osmotic control as described previously (11). All rats were housed in the Association for Assessment and Accreditation of Laboratory Animal Care–approved Biomedical Resource Center at the Medical College of Wisconsin, and all protocols were approved by the Animal Care Committee. Formation of ONOO  . Authentic ONOO - was synthesized according to published methods (5,6). The amount of ONOO - in the stock solution was determined spectrophotometrically using the reported extinction coefficient for ONOO - (1,670 mol  l -1  cm -1 ). Before each application, an aliquot of the stock solution was diluted in 1 mmol/l NaOH and rapidly added to the vessel chamber to achieve a final concentration of 5 mol/l. Decomposed ONOO - From the 1 Heart and Vessel Diseases Center, Beijing Friendship Hospital, Affiliate of Capital University of Medical Sciences, Beijing, People’s Republic of China; 2 Department of Medicine, The Medical College of Wisconsin and The Veterans Administration Medical Center, Milwaukee, Wisconsin; the 3 Cardio- vascular Center, The Medical College of Wisconsin and The Veterans Admin- istration Medical Center, Milwaukee, Wisconsin; and the 4 Department of Pharmacology & Toxicology, The Medical College of Wisconsin and The Veterans Administration Medical Center, Milwaukee, Wisconsin. Address correspondence and reprint requests to Yanping Liu, MD, PhD, Cardiovascular Center, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. E-mail: ypliu@mcw.edu. Received for publication 19 March 2004 and accepted in revised form 8 June 2004. 4-AP, 4-aminopyridine; COR, correolide; HG, high glucose; K Ca , Ca 2+ -acti- vated K + ;K v , voltage-gated K + ; LG, L-glucose; L-NAME, N  -nitro-L-arginine methyl ester; MnTBAP, manganese [III] tetrakis 4-benzoic acid porphyrin; NG, normal glucose; ROS, reactive oxygen species; RSCA, rat small coronary artery; VSMC, vascular smooth muscle cell. © 2004 by the American Diabetes Association. 2436 DIABETES, VOL. 53, SEPTEMBER 2004