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

Overwhelming 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.

NO production and intracellular Ca²⁺ levels ([Ca²⁺]_i) were assessed using the fluorescent probes 4,5-diaminofluorescein diacetate and fura-2 respectively.

Exposure 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⁺]_i under these conditions, since a close correlation between [Ca²⁺]_i increases 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 and d-α-tocopherol (antioxidant).

Chronic 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.