Chronic exposure to high glucose impairs bradykinin-stimulated nitric oxide production by interfering with the phospholipase-C-implicated signalling pathway in endothelial cells: evidence for the involvement of protein kinase C
Chronic exposure to high glucose impairs bradykinin-stimulated nitric oxide production by interfering with the phospholipase-C-implicated signalling pathway in endothelial cells: evidence for the involvement of protein kinase C
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DOI：
10.1007/s00125-004-1589-y
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发表时间：
2004-12
期刊：
影响因子：
8.2
通讯作者：
Y. Tang;G. Li
中科院分区：
文献类型：
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作者：
Y. Tang;G. Li
目的/假说令人信服的证据表明，糖尿病患者的内皮细胞功能障碍以内皮依赖性松弛减弱为特征，但其潜在的分子机制尚不清楚。由于内皮细胞产生一氧化氮(NO)是内皮细胞介导的血管松弛的主要环节，我们研究了高糖对NO产生的影响，以及与此相关的信号通路的可能改变。方法用荧光探针4，5-二氨基荧光素二乙酸酯和Fura-2分别检测NO的产生和细胞内钙离子水平([Ca+]i)。结果培养的牛主动脉内皮细胞暴露于高糖5天或10天后，可显著降低缓激肽(但不是钙离子载体)诱导的NO产生，且呈时间和剂量依赖性。这可能是由于在这些条件下缓激肽引起的[Ca~(2+)]_i升高有所减弱，因为在完整的牛主动脉内皮细胞中观察到[Ca~(2+)]_i升高与NO生成密切相关。缓激肽促进的细胞内钙动员和细胞外钙内流均受到影响。此外，在高糖培养条件下，缓激肽诱导的磷脂酶C产物Ins(1，4，5)P3的形成也受到抑制。这种异常不是由于肌醇磷脂的减少，而可能是由于缓激肽受体数量的减少。高糖引起的NO生成、[Ca~(2+)]i升高和缓激肽受体数目的变化可被蛋白激酶C抑制剂和D-生育酚(antioxidant).Conclusions/interpretationChronic暴露于高糖环境中而在很大程度上被逆转，这可能是由于蛋白激酶C过度激活而削弱了磷脂酶-C介导的钙信号转导。NO释放的这种缺陷可能导致内皮依赖性松弛减弱，从而导致糖尿病心血管疾病的发生。
Aims/hypothesisOverwhelming evidence indicates that endothelial cell dysfunction in diabetes is characterised by diminished endothelium-dependent relaxation, but the matter of the underlying molecular mechanism remains unclear. As nitric oxide (NO) production from the endothelium is the major player in endothelium-mediated vascular relaxation, we investigated the effects of high glucose on NO production, and the possible alterations of signalling pathways implicated in this scenario.MethodsNO production and intracellular Ca2+levels ([Ca2+]i) were assessed using the fluorescent probes 4,5-diaminofluorescein diacetate and fura-2 respectively.ResultsExposure of cultured bovine aortic endothelial cells to high glucose for 5 or 10 days significantly reduced NO production induced by bradykinin (but not by Ca2+ionophore) in a time- and dose-dependent manner. This was probably due to an attenuation in bradykinin-induced elevations of [Ca2+]iunder these conditions, since a close correlation between [Ca2+]iincreases and NO generation was observed in intact bovine aortic endothelial cells. Both bradykinin-promoted intracellular Ca2+mobilisation and extracellular Ca2+entry were affected. Moreover, bradykinin-induced formation of Ins(1,4,5)P3, a phospholipase C product leading to increases in [Ca2+]i, was also inhibited following high glucose culture. This abnormality was not attributable to a decrease in inositol phospholipids, but possibly to a reduction in the number of bradykinin receptors. The alterations in NO production, the increases in [Ca2+]i, and the bradykinin receptor number due to high glucose could be largely reversed by protein kinase C inhibitors andd-α-tocopherol (antioxidant).Conclusions/interpretationChronic exposure to high glucose reduces NO generation in endothelial cells, probably by impairing phospholipase-C-mediated Ca2+signalling due to excess protein kinase C activation. This defect in NO release may contribute to the diminished endothelium-dependent relaxation and thus to the development of cardiovascular diseases in diabetes.