Nitric oxide modulates Gi-protein expression and adenylyl cyclase signaling in vascular smooth muscle cells

Department of Physiology, Faculty of Medicine, University of Montreal, C.P. 6128, Succ. Centre-ville, Montreal, Quebec, Canada H3C 3J7.
Free Radical Biology and Medicine (Impact Factor: 5.74). 11/2006; 41(7):1162-73. DOI: 10.1016/j.freeradbiomed.2006.07.004
Source: PubMed


We have previously shown that treatment of rats with the nitric oxide (NO) synthase inhibitor N6-nitro-L-arginine methyl ester for 4 weeks resulted in the augmentation of blood pressure and enhanced levels of Gialpha proteins. The present studies were undertaken to investigate if NO can modulate the expression of Gi proteins and associated adenylyl cyclase signaling. A10 vascular smooth muscle cells (VSMC) and primary cultured cells from aorta of Sprague-Dawley rats were used for these studies. The cells were treated with S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) for 24 h and the expression of Gialpha proteins was determined by immunobloting techniques. Adenylyl cyclase activity was determined by measuring [32P]cAMP formation for [alpha-32P]ATP. Treatment of cells with SNAP (100 microM) or SNP (0.5 mM) decreased the expression of Gialpha-2 and Gialpha-3 by about 25-40% without affecting the levels of Gsalpha proteins. The decreased expression of Gialpha proteins was reflected in decreased Gi functions (receptor-independent and -dependent) as demonstrated by decreased or attenuated forskolin-stimulated adenylyl cyclase activity by GTPgammaS and inhibition of adenylyl cyclase activity by angiotensin II and C-ANP4-23, a ring-deleted analog of atrial natriuretic peptide (ANP) that specifically interacts with natriuretic peptide receptor-C (NPR-C) in SNAP-treated cells. The SNAP-induced decreased expression of Gialpha-2 and Gialpha-3 proteins was not blocked by 1H[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase, or KT5823, an inhibitor of protein kinase G, but was restored toward control levels by uric acid, a scavenger of peroxynitrite and Mn(111)tetralis (benzoic acid porphyrin) MnTBAP, a peroxynitrite scavenger and a superoxide dismutase mimetic agent that inhibits the production of peroxynitrite, suggesting that NO-mediated decreased expression of Gialpha protein was cGMP-independent and may be attributed to increased levels of peroxynitrite. In addition, Gsalpha-mediated stimulation of adenylyl cyclase by GTPgammaS, isoproterenol, and forskolin was significantly augmented in SNAP-treated cells. These results indicate that NO decreased the expression of Gialpha protein and associated functions in VSMC by cGMP-independent mechanisms. From these studies, it can be suggested that NO-induced decreased levels of Gi proteins and resultant increased levels of cAMP may be an additional mechanism through which NO regulates blood pressure.

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    • "A similar effect of SNAP (s-nitroso-N-acetylpencillamine), another NO donor, as well as L-NAME, on ET-1-induced ERK1/2 phosphorylation in A10 VSMC has also been reported in studies from our laboratory (Fig. 2A and 2B) [76]. Since SNAP raises intracellular cGMP levels via sGC in A10 VSMC [65,66], additional studies demonstrated that 8-Br-cGMP (8-bromoguanosine 3’, 5’-cyclic monophosphate), a non-hydro-lyzable analogue of cGMP, mimicked the effect of SNAP and SNP and inhibited ET-1 stimulated ERK1/2 phosphorylation (Fig. 2C) [76]. Moreover, the ability of ODQ (1H-[1,2,4] oxadiazolo[4,3,-a]quino-xalin-1-one), a selective sGC inhibitor, to reverse SNAP-induced attenuation of ERK1/2 phosphorylation (Fig. 2D) [76] established a role of activated sGC and cGMP in mediating the effects of these NO donors on ET-1-induced ERK signaling. "
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