Glucose elevations alter bradykinin-stimulated intracellular calcium accumulation in cultured endothelial cells

Diabetes selectively injures receptor-mediated endothelium-dependent relaxation. In this study, we investigated the effect of elevated glucose concentrations on intracellular calcium (Ca2+i) signal transduction in response to stimulants of EDRF/nitric oxide release in cultured bovine aortic endothelial cells.

[Ca2+i] was measured in cell suspensions using Fura-2 and fluorescence spectroscopy while nitric oxide production was evaluated using radioimmunoassay of cGMP production.

After 24 h exposure to 25 mM glucose in Ham's F-12 media, the increase in endothelial cell [Ca2+i] in response to 100 nM bradykinin was attenuated by 40% while the response to ionomycin was unaltered. When RMPI medium was used, no reduction in response to bradykinin was observed at 25 mM glucose, but a significant reduction in [Ca2+i] signal was observed after exposure to 35 mM glucose for a similar time period. Defective [Ca2+i] signaling was also seen in cells using MEM medium. [Ca2+i] signal responses to ionomycin and NaF, a G-protein activator of extracellular calcium entry via calcium channels, were unaltered by elevated glucose exposure. The defect in [Ca2+i] signal was not mimicked by either mannose or sucrose, but was prevented by co-incubation with cytochalasin B to inhibit glucose uptake. Neither superoxide dismutase nor catalase nor the extracellular hydroxyl radical scavenger, mannitol, blocked the reduction in the bradykinin-induced increase of [Ca2+i] in elevated glucose-exposed cells; however, the reduction was completely blocked by the cell-permeable hydroxyl radical scavenger, dimethylthiourea. Bradykinin-stimulated (but not ionomycin-stimulated) cGMP production within endothelial cells or in RFL-6 detector cells was attenuated by elevated glucose exposure.

Hyperglycemia may contribute to defective endothelium-dependent relaxation in diabetes via an attenuated increase in Ca2+i signal transduction for the release of nitric oxide by endothelial cells. This defect possibly arises as a consequence of hydroxyl radicals formed intracellularly.