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.
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.
Russo
I
I
Diabetes Unit, Department of Clinical and Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, 10043 Orbassano (TO), Italy.
Del Mese
P
P
Doronzo
G
G
Mattiello
L
L
Viretto
M
M
Bosia
A
A
Anfossi
G
G
Trovati
M
M
D000818:Animals; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000502:physiology*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology*; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000378:metabolism; D004195:Disease Models, Animal; D007333:Insulin Resistance; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D009569:Nitric Oxide / Q000502:physiology*; D018384:Oxidative Stress; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D011924:Rats, Zucker; D015398:Signal Transduction / Q000502:physiology*; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; D010726:Phosphodiesterase Inhibitors; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D009569:Nitric Oxide; D017869:Cyclic GMP-Dependent Protein Kinases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D006152:Cyclic GMP