High D-Glucose–Induced Changes in Endothelial Ca²⁺/EDRF Signaling are Due to Generation of Superoxide Anions Free

Pretreatment of porcine aortic endothelial cells with high D-glucose results in enhanced endothelium-derived relaxing factor (EDRF) formation (39%) due to increased endothelial Ca2+ release (57%) and Ca²⁺ entry (97%) to bradykinin. This study was designed to investigate the intracellular mechanisms by which high D-glucose affects endothelial Ca²⁺/EDRF response. The aldose-reductase inhibitors, sorbinil and zopolrestat, failed to diminish high D-glucose-mediated alterations in Ca²⁺/EDRF response, suggesting that aldose-reductase does not contribute to high D-glucose-initiated changes in Ca²⁺/EDRF signaling. Pretreatment of cells with the nonmetabolizing D-glucose analog, 3-O-methylglucopyranose (3-OMG), mimicked the effect of high D-glucose on Ca²⁺ release (41%) and Ca²⁺ entry (114%) to bradykinin, associated with elevated EDRF formation (26%). High D-glucose and 3-OMG increased superoxide anion (O₂⁻) formation (133 and 293%, respectively), which was insensitive to inhibitors of cyclooxygenase (5,8,11,14-eicosatetraynoic acid [ETYA], indomethacin), lipoxygenase (ETYA, gossypol, nordihydroguaiaretic acid [NDGA]), cytochrome P450 (NDGA, econazole, miconazole), and nitric oxide (NO) synthase (L-omega N-nitroarginine), while it was diminished by desferal, a metal chelator. The gamma-glutamyl-cysteine-synthase inhibitor, buthioninesulfoximine (BSO), also increased formation of O₂⁻ by 365% and mimicked the effect of high D-glucose on Ca²⁺/EDRF signaling. The effects of high D-glucose, 3-OMG, and BSO were abolished by co-incubation with superoxide dismutase. Like high D-glucose, pretreatment with the O₂⁻-generating system, xanthine oxidase/hypoxanthine, elevated bradykinin-stimulated Ca2+ release (+10%), Ca²⁺ entry (+75%), and EDRF (+73%). We suggest that prolonged exposure to pathologically high D-glucose concentration results in enhanced formation of O₂⁻, possibly due to metal-mediated oxidation of D-glucose within the cells. This overshoot of O₂⁻ enhances agonist-stimulated Ca²⁺/EDRF signaling via a yet unknown mechanism.