Vaziri N D, Wang X Q
Division of Nephrology, Department of Medicine, University of California, Irvine, Calif 92697, USA.
Hypertension. 1999 Dec;34(6):1237-41. doi: 10.1161/01.hyp.34.6.1237.
Earlier studies have demonstrated that nitric oxide (NO) exerts a fast-acting inhibitory influence on endothelial NO synthase (eNOS) enzymatic activity in isolated vascular tissue preparations. The present study was designed to examine the possible effect of NO on eNOS protein expression in cultured endothelial cells and intact animals. Human coronary endothelial cells were incubated with S-nitroso-N-acetyl-penicillamine (SNAP, an NO donor), oxyhemoglobin (HGB, an NO trapping agent), SNAP plus HGB, or inactive vehicle (control). In other experiments, cells were treated with 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor), 1H-[1,2, 4]oxadiazolo-[4,3-2]quinoxalin-1-one (ODQ, a guanylate cyclase inhibitor), SNAP plus ODQ, 8-bromo-cGMP (8-Br-cGMP, a cell-permeable cGMP compound), 8-Br-cGMP plus HGB, or inactive vehicle in order to discern the effect of cGMP. The incubations were conducted for 24 hours, and total nitrate plus nitrite production and eNOS protein abundance (Western analysis) were measured. To determine the effect of NO on eNOS expression in vivo, rats were treated with either the NO donor isosorbide dinitrate or placebo by gastric gavage for 48 hours, and aortic eNOS protein expression was examined. The NO donor SNAP markedly depressed, whereas the NO scavenger HGB significantly raised, eNOS protein expression. The downregulatory action of SNAP was completely abrogated by HGB. Phosphodiesterase inhibitor and 8-Br-cGMP downregulated, whereas the guanylate cyclase inhibitor ODQ upregulated eNOS protein expression. The downregulatory action of SNAP was completely overcome by the guanylate cyclase inhibitor ODQ, and the upregulatory action of the NO scavenger HGB was abrogated by 8-Br-cGMP. Administration of NO donor resulted in a marked downregulation of aortic eNOS protein expression in intact animals, thus confirming the in vitro findings. NO serves as a negative-feedback regulator of eNOS expression via a cGMP-mediated process.
早期研究表明，一氧化氮（NO）对离体血管组织制剂中的内皮型一氧化氮合酶（eNOS）酶活性具有快速起效的抑制作用。本研究旨在探讨NO对培养的内皮细胞和完整动物中eNOS蛋白表达的可能影响。将人冠状动脉内皮细胞与S-亚硝基-N-乙酰青霉胺（SNAP，一种NO供体）、氧合血红蛋白（HGB，一种NO捕获剂）、SNAP加HGB或无活性载体（对照）一起孵育。在其他实验中，用3-异丁基-1-甲基黄嘌呤（一种磷酸二酯酶抑制剂）、1H-[1,2,4]恶二唑并-[4,3-b]喹喔啉-1-酮（ODQ，一种鸟苷酸环化酶抑制剂）、SNAP加ODQ、8-溴-cGMP（8-Br-cGMP，一种可透过细胞的cGMP化合物）、8-溴-cGMP加HGB或无活性载体处理细胞，以辨别cGMP的作用。孵育24小时后，测量总硝酸盐加亚硝酸盐产量和eNOS蛋白丰度（蛋白质印迹分析）。为了确定NO对体内eNOS表达的影响，通过胃管给大鼠灌胃NO供体硝酸异山梨酯或安慰剂48小时，并检测主动脉eNOS蛋白表达。NO供体SNAP显著降低，而NO清除剂HGB显著升高eNOS蛋白表达。HGB完全消除了SNAP的下调作用。磷酸二酯酶抑制剂和8-溴-cGMP下调，而鸟苷酸环化酶抑制剂ODQ上调eNOS蛋白表达。鸟苷酸环化酶抑制剂ODQ完全克服了SNAP的下调作用，而8-溴-cGMP消除了NO清除剂HGB的上调作用。给完整动物施用NO供体导致主动脉eNOS蛋白表达显著下调，从而证实了体外研究结果。NO通过cGMP介导的过程作为eNOS表达的负反馈调节因子。
