Bassil Marcel, Anand-Srivastava Madhu B
Department of Physiology, Faculty of Medicine, University of Montreal, C.P. 6128, Succ. Centre-ville, Montreal, Quebec, Canada H3C 3J7.
Free Radic Biol Med. 2006 Oct 1;41(7):1162-73. doi: 10.1016/j.freeradbiomed.2006.07.004. Epub 2006 Jul 11.
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.
我们之前已经表明，用一氧化氮（NO）合酶抑制剂N6-硝基-L-精氨酸甲酯处理大鼠4周会导致血压升高以及Gialpha蛋白水平升高。本研究旨在探讨NO是否能调节Gi蛋白的表达及相关的腺苷酸环化酶信号传导。这些研究使用了A10血管平滑肌细胞（VSMC）和来自Sprague-Dawley大鼠主动脉的原代培养细胞。用S-亚硝基-N-乙酰青霉胺（SNAP）或硝普钠（SNP）处理细胞24小时，并用免疫印迹技术测定Gialpha蛋白的表达。通过测量[α-32P]ATP生成的[32P]cAMP来测定腺苷酸环化酶活性。用SNAP（100 microM）或SNP（0.5 mM）处理细胞可使Gialpha-2和Gialpha-3的表达降低约25-40%，而不影响Gsalpha蛋白的水平。Gialpha蛋白表达的降低反映在Gi功能（受体非依赖性和依赖性）的降低上，这表现为GTPγS刺激的腺苷酸环化酶活性降低或减弱，以及血管紧张素II和C-ANP4-23（心房利钠肽（ANP）的环缺失类似物，在SNAP处理的细胞中与利钠肽受体-C（NPR-C）特异性相互作用）对腺苷酸环化酶活性的抑制。SNAP诱导的Gialpha-2和Gialpha-3蛋白表达降低未被可溶性鸟苷酸环化酶抑制剂1H[1,2,4]恶二唑[4,3-a]喹喔啉-1-酮或蛋白激酶G抑制剂KT5823阻断，但被尿酸（过氧亚硝酸盐清除剂）和Mn(III)四（苯甲酸卟啉）MnTBAP（过氧亚硝酸盐清除剂和超氧化物歧化酶模拟剂，可抑制过氧亚硝酸盐的产生）恢复至对照水平，这表明NO介导的Gialpha蛋白表达降低不依赖于cGMP，可能归因于过氧亚硝酸盐水平的升高。此外，在SNAP处理的细胞中，GTPγS、异丙肾上腺素和福斯高林介导的Gsalpha对腺苷酸环化酶的刺激作用显著增强。这些结果表明，NO通过不依赖于cGMP的机制降低了VSMC中Gialpha蛋白的表达及相关功能。从这些研究中可以推测，NO诱导的Gi蛋白水平降低以及由此导致的cAMP水平升高可能是NO调节血压的另一种机制。
