Nitric oxide modulates Gi-protein expression and adenylyl cyclase signaling in vascular smooth muscle cells.
ABSTRACT 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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ABSTRACT: Pendrin is a Cl(-)/HCO(3)(-) exchanger, expressed in the apical regions of some intercalated cell subtypes, and is critical in the pressor response to angiotensin II. Since angiotensin type 1 receptor inhibitors reduce renal pendrin protein abundance in mice in vivo through a mechanism that is dependent on nitric oxide (NO), we asked if NO modulates renal pendrin expression in vitro and explored the mechanism by which it occurs. Thus we quantified pendrin protein abundance by confocal fluorescent microscopy in cultured mouse cortical collecting ducts (CCDs) and connecting tubules (CNTs). After overnight culture, CCDs maintain their tubular structure and maintain a solute gradient when perfused in vitro. Pendrin protein abundance increased 67% in CNT and 53% in CCD when NO synthase was inhibited (N(G)-nitro-l-arginine methyl ester, 100 μM), while NO donor (DETA NONOate, 200 μM) application reduced pendrin protein by ∼33% in the CCD and CNT. When CNTs were cultured in the presence of the guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (10 μM), NO donors did not alter pendrin abundance. Conversely, pendrin protein abundance rose when cAMP content was increased by the application of an adenylyl cyclase agonist (forskolin, 10 μM), a cAMP analog (8-bromo-cAMP, 1 mM), or a phosphodiesterase inhibitor (BAY60-7550, 50 μM). Since NO reduces cellular cAMP in the CNT, we asked if NO reduces pendrin abundance by reducing cAMP. With blockade of cGMP-stimulated phosphodiesterase II, NO did not alter pendrin protein abundance. We conclude that NO acts through cAMP to reduce pendrin total protein abundance by enhancing cAMP degradation.AJP Renal Physiology 07/2012; 303(6):F812-20. · 4.42 Impact Factor
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ABSTRACT: Brown adipose tissue thermogenic program includes complex molecular and structural changes. However, energetic aspects of this process are poorly depicted. We investigated time-dependent reprogramming of interscapular brown adipose tissue (IBAT) energy metabolism during cold-acclimation, as well as the effects of nitric oxide (()NO) on those changes. Rats were exposed to cold (4±1°C) for periods of 1, 3, 7, 12, 21, and 45days, and divided into three groups: control, treated with L-arginine, and treated with N(ω)-nitro-L-arginine methyl ester (L-NAME). In the early phase of cold-acclimation (up to 7days), the protein levels of all metabolic parameters and oxidative phosphorylation components were below the control. However, metabolic parameters and respiratory chain components entered a new homeostatic level in the late phase of cold-acclimation. These changes were accompanied with increased protein levels of phospho-AMP-dependent protein kinase-α (phospho-AMPKα) on the first day of cold-acclimation, and hypoxia-inducible factor-1α (HIF-1α) throughout early cold-acclimation. L-arginine positively affected protein expression of enzymes involved in glucose metabolism and β-oxidation of fatty acids in the early phase of cold-acclimation, and oxidative phosphorylation components throughout cold-acclimation. In contrast, L-NAME had the opposite effects. Results suggest that IBAT structural remodeling is followed by energy metabolism reprogramming, which control might be orchestrated by the action of AMPKα and HIF-1α. Data also indicated the involvement of L-arginine-()NO in the regulation of IBAT metabolism. Results obtained in this study might be of great importance for elucidating regulatory pathways governing energy metabolism in both physiological and pathophysiological states.Biochimica et Biophysica Acta 09/2011; 1810(12):1252-61. · 4.66 Impact Factor
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ABSTRACT: Neuronal survival has been shown to be enhanced by alpha-tocopherol and modulated by cyclic AMP (cAMP). Somatostatin (SST) receptors couple negatively to adenylyl cyclase (AC), thus leading to decreased cAMP levels. Whether alpha-tocopherol can stimulate neuronal survival via regulation of the somatostatinergic system, however, is unknown. The aim of this study was to investigate the effects of alpha-tocopherol on the SST signaling pathway in the rat dentate gyrus. To that end, 15-week-old male Sprague-Dawley rats were treated daily for 1 week with (+)-alpha-tocopherol or vehicle and sacrificed on the day following the last administration. No changes in either SST-like immunoreactivity (SST-LI) content or SST mRNA levels were detected in the dentate gyrus as a result of alpha-tocopherol treatment. A significant decrease in the density of the SST binding sites and an increase in the dissociation constant, however, were detected. The lower SST receptor density in the alpha-tocopherol-treated rats correlated with a significant decrease in the protein levels of the SST receptor subtypes SSTR1-SSTR4, whereas the corresponding mRNA levels were unaltered. G-protein-coupled-receptor kinase 2 expression was decreased by alpha-tocopherol treatment. This vitamin induced a significant increase in both basal and forskolin-stimulated AC activity, as well as a decrease in the inhibitory effect of SST on AC. Whereas the protein levels of AC type V/VI were not modified by alpha-tocopherol administration, ACVIII expression was significantly enhanced, suggesting it might account for the increase in AC activity. In addition, this treatment led to a reduction in Gialpha1-3 protein levels and in Gi functionality. alpha-Tocopherol did not affect the expression of the regulator of G-protein signaling 6/7 (RGS6/7). Finally, alpha-tocopherol induced an increase in the levels of phosphorylated cAMP response element binding protein (p-CREB) and total CREB in the dentate gyrus. Since CREB synthesis and phosphorylation promote the survival of many cells, including neurons, whereas SST inhibits the cAMP-PKA pathway, which is known to be involved in CREB phosphorylation, the alpha-tocopherol-induced reduction of SSTR observed here might possibly contribute, via increased cAMP levels and CREB activity, to the mechanism by which this vitamin promotes the survival of newborn neurons in the dentate gyrus.Neuroscience 05/2009; 162(1):106-17. · 3.12 Impact Factor