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An approach to understand the pathophysiological role of endothelial constitutive nitric oxide synthase

了解内皮组成型一氧化氮合酶病理生理作用的方法

基本信息

批准号：
06454292
负责人：
YOKOYAMA Mitsuhiro
金额：
$ 4.61万
依托单位：
Kobe University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

NO synthesized from L-arginine by the action of constitutive NO synthase (cNOS) in vascular endothelial cell plays an important role in the regulation of tissue perfusion. Endothelium-dependent relaxation (EDR) is markedly reduced in atherosclerotic arteries and the impairment of EDR is thought to be involved in the pathogenesis of ischemic heart disease. The purpose of the present study is to clarify the regulatory mechanisms of cNOS in vascular endothelial cells. Endothelial cNOS was phosphorylated by protein kinase C (PKC) and cAMP-dependent protein kinase. In cultured bovine aortic endothelial cells (BAECs) , phorbol esters such as TPA and PDBu inhibited NO release by A23187 as well as ATPgS through PKC activation. We employed RNase protection assay and immunoblotting to elucidate the effect of cytokine, mechanical stimuli and atherogenic lipoprotein on the expression of cNOS mRNA and protein levels in BAECs. Low concentration of ox-LDL (10mg protein/mL) or LPC (5mg/mL) upregulated cNOS mRNA levels. Furthermore, in situ hybridization and immunohistochemistry revealed that the cNOS mRNA and protein expression was normally observed in the endothelial cells overlying aortic fatty streaks in WHHL rabbits. These findings suggest that loss of EDRF activity associated with atherosclerosis is not due to an alteration of endothelial cNOS expression. In summary, ox-LDL and LPC in atherosclerotic lesion may cause endothelial dysfunction by the inhibition of receptor-mediated intracellular signaling pathway.

血管内皮细胞中组成型一氧化氮合酶（constitutive NO synthase，cNOS）作用下由L-精氨酸合成的NO在组织灌注调节中起重要作用。内皮依赖性舒张功能（EDR）在动脉粥样硬化中显著降低，EDR受损被认为与缺血性心脏病的发病机制有关。本研究旨在阐明cNOS在血管内皮细胞中的调控机制。内皮细胞cNOS被蛋白激酶C（PKC）和cAMP依赖性蛋白激酶磷酸化。在培养的牛主动脉内皮细胞（BAECs），佛波醇酯，如TPA和PDBu抑制NO释放A23187以及ATPgS通过PKC激活。我们采用RNase保护试验和免疫印迹法研究了细胞因子、机械刺激和致动脉粥样硬化脂蛋白对BAECs cNOS mRNA和蛋白表达的影响。低浓度ox-LDL（10 mg蛋白/mL）或LPC（5 mg/mL）可上调cNOSmRNA水平。原位杂交和免疫组化结果显示，WHHL兔主动脉脂肪纹上的内皮细胞cNOSmRNA和蛋白表达正常。这些发现表明，与动脉粥样硬化相关的EDRF活性的丧失不是由于内皮cNOS表达的改变。综上所述，动脉粥样硬化病变中ox-LDL和LPC可能通过抑制受体介导的细胞内信号通路而导致内皮功能障碍。