GLUCOSE ELEVATIONS ALTER BRADYKININ-STIMULATED INTRACELLULAR CALCIUM ACCUMULATION IN CULTURED ENDOTHELIAL-CELLS

Objective: Diabetes selectively injures receptor-mediated endothelium- dependent relaxation. In this study, we investigated the effect of ele vated glucose concentrations on intracellular calcium (Ca-i(2+)) signa l transduction in response to stimulants of EDRF/nitric oxide release in cultured bovine aortic endothelial cells. Methods: [Ca-i(2+)] was m easured in cell suspensions using Fura-2 and fluorescence spectroscopy while nitric oxide production was evaluated using radioimmunoassay of cGMP production. Results: After 24 h exposure to 25 mM glucose in Har n's F-12 media, the increase in endothelial cell [Ca-i(2+)] in respons e to 100 nM bradykinin was attenuated by 40% while the response to ion omycin was unaltered. When RMPI medium was used, no reduction in respo nse to bradykinin was observed at 25 mM glucose, but a significant red uction in [Ca-i(2+)] signal was observed after exposure to 35 mM gluco se for a similar time period. Defective [Ca-i(2+)] signaling was also seen in cells using MEM medium. [Ca-i(2+)] signal responses to ionomyc in and NaF, a G-protein activator of extracellular calcium entry via c alcium channels, were unaltered by elevated glucose exposure. The defe ct in [Ca-i(2+)] signal was not mimicked by either mannose or sucrose, but was prevented by co-incubation with cytochalasin B to inhibit glu cose uptake. Neither superoxide dismutase nor catalase nor the extrace llular hydroxyl radical scavenger, mannitol, blocked the reduction in the bradykinin-induced increase of [Ca-i(2+)] in elevated glucose-expo sed cells; however, the reduction was completely blocked by the cell-p ermeable hydroxyl radical scavenger, dimethylthiourea. Bradykinin-stim ulated (but not ionomycin-stimulated) cGMP production within endotheli al cells or in RFL-6 detector cells was attenuated by elevated glucose exposure. Conclusions: Hyperglycemia may contribute to endothelium-de pendent relaxation in diabetes via an attenuated increase in Ca-i(2+) signal transduction for the release contribute to defective endotheliu m-dependent relaxation In diabetes via an attenuated increase in Ca, o f nitric oxide by endothelial cells. This defect possibly arises as a consequence of hydroxyl radicals formed intracellularly.