Singh A, Sventek P, Larivière R, Thibault G, Schiffrin E L
MRC Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montréal, University of Montréal, Québec, Canada.
Am J Hypertens. 1996 Sep;9(9):867-77. doi: 10.1016/s0895-7061(96)00104-5.
The inducible nitric oxide synthase (iNOS) present in vascular smooth muscle cells (VSMC) may play a role in the generation of nitric oxide (NO) in the vascular wall, regulating blood vessel tone in normotension and hypertension. In this study the effect of interleukin (IL)-1 beta, a cytokine that induces iNOS, on NO generation (measured as nitrite), cyclic guanosine monophosphate (cGMP) generation, and steady-state abundance of iNOS mRNA were examined in VSMC from 3 week old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats, during the period preceding the elevation of blood pressure. With cell density dependent variations in nitrite production eliminated, VSMC from SHR and WKY did not differ in NO generation except after prolonged incubation (30 h), when SHR cells produced less NO. However, cGMP concentrations associated with IL-1 beta stimulation were significantly smaller in SHR VSMC than in cells from WKY. IL-1 beta stimulation resulted in increased abundance of iNOS mRNA to the same extent in both WKY and SHR VSMC. Inhibitors of NOS, NG-monomethyl-L-arginine (L-NMMA) and N omega-nitro-L-arginine methyl ester (L-NAME), did not block the induction of iNOS mRNA, although nitrite production and cGMP generation were inhibited. The protein synthesis inhibitor cycloheximide and the RNA synthesis inhibitor actinomycin-D almost completely blocked the production of nitrite in cells from both strains of rats. Actinomycin-D completely blocked the induction of iNOS mRNA by IL-1 beta in cells from both strains of rats, whereas cycloheximide partially blocked its synthesis in WKY, but had no significant effect on IL-1 beta induced expression of iNOS mRNA in SHR VSMC. Thus, IL-1 beta controls iNOS gene expression at the transcriptional level, and an intermediate labile protein, whose synthesis is inhibited by cycloheximide, is required for IL-1 beta stimulated induction of iNOS mRNA transcription in WKY cells but not in SHR. We conclude that although iNOS is expressed to similar extent in VSMC of prehypertensive SHR and WKY and similar amounts of NO are initially generated, there are differences between the VSMC of SHR and WKY in the regulation of the transcription of iNOS mRNA, there is a lower sustained production of NO, and there is a reduced generation of cGMP in response to IL-1 beta stimulated NO production. These differences between VSMC from prehypertensive SHR and WKY may indicate a pathophysiological role of iNOS in early blood pressure elevation in SHR.
血管平滑肌细胞（VSMC）中存在的诱导型一氧化氮合酶（iNOS）可能在血管壁一氧化氮（NO）的生成中发挥作用，在正常血压和高血压状态下调节血管张力。在本研究中，检测了白细胞介素（IL）-1β（一种诱导iNOS的细胞因子）对3周龄自发性高血压大鼠（SHR）和Wistar-Kyoto（WKY）大鼠VSMC中NO生成（以亚硝酸盐衡量）、环磷酸鸟苷（cGMP）生成以及iNOS mRNA稳态丰度的影响，检测时间为血压升高之前。消除细胞密度依赖性亚硝酸盐产生的变化后，除了长时间孵育（30小时）后SHR细胞产生的NO较少外，SHR和WKY的VSMC在NO生成方面没有差异。然而，与IL-1β刺激相关的cGMP浓度在SHR VSMC中明显低于WKY细胞。IL-1β刺激导致WKY和SHR VSMC中iNOS mRNA丰度增加的程度相同。一氧化氮合酶抑制剂NG-单甲基-L-精氨酸（L-NMMA）和Nω-硝基-L-精氨酸甲酯（L-NAME）虽然抑制了亚硝酸盐产生和cGMP生成，但并未阻断iNOS mRNA 的诱导。蛋白质合成抑制剂环己酰亚胺和RNA合成抑制剂放线菌素-D几乎完全阻断了两种大鼠品系细胞中亚硝酸盐的产生。放线菌素-D完全阻断了IL-1β对两种大鼠品系细胞中iNOS mRNA的诱导，而环己酰亚胺部分阻断了WKY细胞中iNOS mRNA的合成，但对IL-1β诱导的SHR VSMC中iNOS mRNA的表达没有显著影响。因此，IL-1β在转录水平上控制iNOS基因表达，并且在WKY细胞中，IL-1β刺激诱导iNOS mRNA转录需要一种中间不稳定蛋白，其合成受环己酰亚胺抑制，而在SHR中则不需要。我们得出结论，尽管在高血压前期SHR和WKY的VSMC中iNOS表达程度相似，并且最初产生的NO量相似，但SHR和WKY的VSMC在iNOS mRNA转录调控方面存在差异，NO的持续产生较低，并且对IL-1β刺激的NO产生反应中cGMP生成减少。高血压前期SHR和WKY的VSMC之间的这些差异可能表明iNOS在SHR早期血压升高中的病理生理作用。
