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

血管内皮细胞中的一氧化氮合酶(CNOS)作用于L精氨酸合成的NO在组织灌流调节中起着重要作用。动脉粥样硬化血管内皮细胞依赖性松弛(EDR)明显降低，EDR受损被认为参与了缺血性心脏病的发病机制。本研究旨在阐明cNOS在血管内皮细胞中的调控机制。蛋白激酶C(PKC)和cAMP依赖的蛋白激酶使内皮细胞cNOS磷酸化。在培养的牛主动脉内皮细胞(BAECs)中，佛波酯(TPA和PDBu)通过PKC激活抑制A23187释放NO和ATPgS。采用核糖核酸酶保护实验和免疫印迹法研究细胞因子、机械刺激和致动脉粥样硬化脂蛋白对BAECs中cNOS基因和蛋白表达的影响。低浓度氧化低密度脂蛋白(10 mg蛋白质/毫升)或低密度脂蛋白(5 mg/毫升)可上调cNOS基因表达。此外，原位杂交和免疫组织化学显示，在WHHL兔的主动脉脂肪条纹上的内皮细胞中正常观察到cNOS基因和蛋白的表达。这些发现表明，与动脉粥样硬化相关的EDRF活性的丧失并不是由于内皮细胞cNOS表达的改变。综上所述，OX-LDL和LPC在动脉粥样硬化病变中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。

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.