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 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.


METHODS
[Ca2+i] was measured 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 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.


CONCLUSIONS
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.
Pieper
G M
GM
Department of Transplant Surgery, Medical College of Wisconsin, Milwaukee 53226 USA.
Dondlinger
L
L
D000818:Animals; D001011:Aorta; D001920:Bradykinin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D003571:Cytochalasin B / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology*; D007424:Intracellular Fluid / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D013268:Stimulation, Chemical
D007476:Ionophores; D019384:Nucleic Acid Synthesis Inhibitors; D009569:Nitric Oxide; D003571:Cytochalasin B; D015759:Ionomycin; D006152:Cyclic GMP; D005947:Glucose; D001920:Bradykinin; D002118:Calcium