Glucose down-regulation of cGMP-dependent protein kinase I expression in vascular smooth muscle cells involves NAD(P)H oxidase-derived reactive oxygen species.
Reduced levels of cGMP-dependent protein kinase I (PKG-I) in vasculature have been shown to contribute to diabetic vascular dysfunctions. However, the underlying mechanisms remain unknown. In this report, using primary rat aortic smooth muscle cells (VSMC), we investigated the mechanisms of glucose-mediated regulation of PKG-I expression. Our data showed that high glucose (30 mM glucose) exposure significantly reduced PKG-I production (protein and mRNA levels) as well as PKG-I activity in cultured VSMC. Glucose-mediated decreases in PKG-I levels were inhibited by a superoxide scavenger (tempol) or NAD(P)H oxidase inhibitors (diphenylene iodonium or apocynin). High glucose exposure time-dependently increased superoxide production in VSMC, which was abolished by tempol or apocynin treatment, but not by other inhibitors of superoxide-producing enzymes (L-NAME, rotenone, or oxypurinol). Total protein levels and phosphorylated levels of p47phox (an NADPH oxidase subunit) were increased in VSMC after high glucose exposure. Transfection of cells with siRNA-p47phox abolished glucose-induced superoxide production and restored PKG-I protein levels in VSMC. Treatment of cells with PKC inhibitor prevented glucose-induced p47phox expression/phosphorylation and superoxide production and restored the PKG-I levels. Decreased PKG-I protein levels were also found in femoral arteries from diabetic mice, which were associated with the decreased DEA-NONOate-induced vasorelaxation. Taken together, the present results suggest that glucose-mediated down-regulation of PKG-I expression in VSMC occurs through PKC-dependent activation of NAD(P)H oxidase-derived superoxide production, contributing to diabetes-associated vessel dysfunctions.
Liu
Shu
S
Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, KY 40536, USA.
Ma
Xueying
X
Gong
Mingcui
M
Shi
Lihua
L
Lincoln
Thomas
T
Wang
Shuxia
S
D000818:Animals; D000838:Anions; D002478:Cells, Cultured; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D015536:Down-Regulation; D004996:Ethidium / Q000031:analogs & derivatives / Q000737:chemistry; D005609:Free Radicals; D005947:Glucose / Q000378:metabolism*; D006943:Hyperglycemia / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism*; D018384:Oxidative Stress; D051381:Rats; D017382:Reactive Oxygen Species; D013481:Superoxides
D000838:Anions; D005609:Free Radicals; D017382:Reactive Oxygen Species; C067883:dihydroethidium; D013481:Superoxides; D019255:NADPH Oxidases; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; C569720:Prkg1 protein, mouse; D004996:Ethidium; D005947:Glucose