The involvement of ryanodine‐sensitive Ca2+ release (RsCR) in bradykinin (Bk)‐induced Ca2+ release, capacitative Ca2+ entry (CCE) and nitric oxide synthase (NOS) activation was assessed in freshly isolated bovine coronary artery endothelial cells. Using deconvolution microscopy fura‐2 was found throughout the whole cytosol, while the cell membrane impermeable dye FFP‐18 was exclusively in the cell membrane. Thus, perinuclear ([Ca2+]pn) and subplasmalemmal Ca2+ concentration ([Ca2+]sp) were monitored using fura‐2 and FFP‐18. Inhibition of Na+−Ca2+ exchange by lowering extracellular Na+ concentration augmented the Bk‐induced [Ca2+]pn signal in Ca2+‐free solution. This effect was abolished when RsCR was prevented with 25 μmmu;mol l−1 ryanodine, while inhibition of RsCR had no effect on Bk‐induced increase in [Ca2+]pn without inhibition of Na+−Ca2+ exchange. Initiating RsCR by 200 nmol l−1 ryanodine increased [Ca2+]sp, while [Ca2+]pn remained constant. However, when Na+−Ca2+ exchange was prevented, ryanodine was also able to elevate [Ca2+]pn. Blockage of RsCR diminished Ca2+ extrusion in response to stimulation with Bk in normal Na+‐containing solution. Inhibition of RsCR blunted Bk‐activated CCE, while inhibition of Na+−Ca2+ exchange during stimulation enhanced CCE. Although direct activation of RsCR failed to activate NOS, inhibition of RsCR diminished the effect of ATP and Bk on NOS, while the effect of thapsigargin remained unchanged. These data suggest that during stimulation subplasmalemmal RsCR occurs, which contributes to the activities of CCE and NOS. Thus, the function of the subplasmalemmal Ca2+ control unit must be extended as a regulator for CCE and NOS.

No abstract

No abstract

Little is known about the mechanism(s) of endothelial dysfunction in diabetes. In this study, the effect of nonenzymatic glycated LDL, a phenomenon induced by elevated D-glucose levels associated with diabetes, on porcine aortic endothelial cells was investigated. Two fractions of LDL from diabetic patients were separated by affinity column chromatography and are referred to herein as fraction alpha (nonglycated LDL) and fraction beta (glycated LDL). Incubation of endothelial cells for 24 h with total LDL isolated from diabetic subjects (dLDL) increased the release of superoxide anions (*O2-) by fivefold, while no effect of LDL isolated from healthy individuals (nLDL) was found. Fraction beta, but not fraction alpha, evoked the *O2- release. In vitro-glycated LDL mimicked the effect of dLDL/fraction beta on *O2- release that correlated with its degree of glycation (R2 = 0.96). Moreover, nitric oxide (NO) stability (measured with a porphyrinic-based electrode) and NO bioactivity (measured by its ability to elevate cellular cGMP levels) were reduced in cells treated with dLDL by 46 and 41%, respectively. dLDL (but not nLDL or fraction alpha) abolished shear stress-induced L-arginine uptake. The inhibitory effect of dLDL on shear stress-induced L-arginine uptake was mimicked by in vitro-glycated LDL. The efficiency of in vitro-glycated LDL to diminish shear stress-evoked L-arginine uptake correlated with the extent of glycation (R2 = 0.88). Moreover, dLDL, but not nLDL or fraction alpha, reduced shear stress-mediated cGMP formation and NOx production by 47 and 88%, respectively. This effect was also mimicked by in vitro-glycated LDL, correlating with its degree of glycation (R2 = 0.86). Under these experimental conditions, glycated LDL reduced shear stress-induced increase in NO synthesis by inhibition of shear stress-stimulated L-arginine uptake and NO bioactivity due to increased endothelial cell *O2- release. These properties may contribute to the reduced vasodilatory response and the vascular complications in diabetes.

The effects of acute hyperglycemia on endothelial Ca2+ signaling, formation of endothelium-derived relaxing factor (EDRF) and bioactivity of EDRF were investigated. Hyperglycemia increased 2,5-tert-butyl-1,4-hydrochinone (BHQ)-initiated Ca2+ signaling and EDRF formation in a concentration-dependent manner. The effect of elevated D-glucose on Ca2+/EDRF response could be diminished by co-incubation with the antioxidants vitamin E, probucol, GSH, vitamin C and superoxide dismutase. Convincingly, hyperglycemic conditions yielded an increase in superoxide anion release from endothelial cells and the superoxide anion-generating mixture xanthine oxidase/hypoxanthine mimicked the effect of hyperglycemia on Ca2+/EDRF signaling. Besides an enhanced formation of the vasodilatatory NO compound EDRF, hyperglycemia enhanced NO degradation by endothelial cells and, thus, reduced bioactivity of EDRF. We suggest that vasoactivity during acute hyperglycemia depends on the superoxide anion scavenging properties of the vascular wall. In acute hyperglycemia and early stages of diabetes, radical scavenging capacity may be suitable to protect NO degradation, resulting in an enhanced vasodilation. In contrast, decreased free radical scavenging properties of the vasculature in prolonged hyperglycemia and in later stages of diabetes might promote NO degradation by an overshoot of superoxide anions, resulting in an attenuation of endothelium-dependent vasodilation.