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Resistance to the Nitric Oxide/Cyclic Guanosine 5'-Monophosphate/Protein Kinase G Pathway in Vascular Smooth Muscle Cells from the Obese Zucker Rat, a Classical Animal Model of Insulin Resistance: Role of Oxidative Stress

Diabetes Unit (I.R., P.D.M., G.D., L.M., M.V., G.A., M.T.), Department of Clinical and Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, 10043 Orbassano (Turin), Italy; and Department of Genetics, Biology, and Medical Chemistry (A.B.), University of Turin, 10126 Turin, Italy

Address all correspondence and requests for reprints to: Professor Mariella Trovati, M.D., Diabetes Unit, Department of Clinical and Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, 10043 Orbassano (TO), Italy. E-mail: mariella.trovati{at}unito.it.

Some in vivo and ex vivo studies demonstrated a resistance to the vasodilating effects of nitric oxide (NO) in insulin-resistant states and, in particular, obese Zucker rats (OZR). To evaluate the biochemical basis of this phenomenon, we aimed to identify defects of the NO/cGMP/cGMP-dependent protein kinase (PKG) pathway in cultured vascular smooth muscle cells (VSMCs) from OZR and lean Zucker rats (LZR) by measuring: 1) NO donor ability to increase cGMP in the absence and presence of inhibitors of soluble guanylate cyclase (sGC) and phosphodiesterases (PDEs); 2) NO and cGMP ability to induce, via PKG, vasodilator-stimulated phosphoprotein (VASP) phosphorylation at serine 239 and PDE5 activity; 3) protein expression of sGC, PKG, total VASP, and PDE5; 4) superoxide anion concentrations and ability of antioxidants (superoxide dismutase+catalase and amifostine) to influence the NO/cGMP/PKG pathway activation; and 5) hydrogen peroxide influence on PDE5 activity and VASP phosphorylation. VSMCs from OZR vs. LZR showed: 1) baseline cGMP concentrations higher, at least in part owing to reduced catabolism by PDEs; 2) impairment of NO donor ability to increase cGMP, even in the presence of PDE inhibitors, suggesting a defect in the NO-induced sGC activation; 3) reduction of NO and cGMP ability to activate PKG, indicated by the impaired ability to phosphorylate VASP at serine 239 and to increase PDE5 activity via PKG; 4) similar baseline protein expression of sGC, PKG, total VASP, and PDE5; and 5) higher levels of superoxide anion. Antioxidants partially prevented the defects of the NO/cGMP/PKG pathway observed in VSMCs from OZR, which were reproduced by hydrogen peroxide in VSMCs from LZR, suggesting the pivotal role of oxidative stress.