Effect of protein kinase C and phospholipase A(2) inhibitors on the impaired ability of human diabetic platelets to cause vasodilation

1. The aim of this study was to examine the mechanism of impaired platelet-mediated endothelium-dependent vasodilation in diabetes. Exposure of human platelets to high glucose in vivo or in vitro impairs their ability to cause endothelium-dependent vasodilation. While previous data suggest that the mechanism for this involves increased activity of the cyclo-oxygenase pathway, the signal transduction pathway mediating this effect is unknown. 2. Platelets from diabetic patients as well as normal platelets and normal platelets exposed to high glucose concentrations were used to determine the role of the polyol pathway, diacylglycerol (DAG) production, protein kinase C (PKC) activity and phospholipase A(2) (PLA(2)) activity on vasodilation in rabbit carotid arteries. 3. We found that two aldose-reductase inhibitors, tolrestat and sorbinil, caused only a modest improvement in the impairment of vasodilation by glucose exposed platelets. However, sorbitol and fructose could not be detected in the platelets, at either normal or hyperglycaemic conditions. We found that incubation in 17 mM glucose caused a significant increase in DAG levels in platelets. Furthermore, the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused significant impairment of platelet-mediated vasodilation. The PKC inhibitors calphostin C and H7 as well as inhibitors of PLA(2) activity normalized the ability of platelets from diabetic patients to cause vasodilation and prevented glucose-induced impairment of platelet-mediated vasodilation in vitro. 4. These results suggest that the impairment of platelet-mediated vasodilation caused by high glucose concentrations is mediated by increased DAG levels and stimulation of PKC and PLA(2) activity

Characterization of NS 2028 as a specific inhibitor of soluble guanylyl cyclase

1. The haeme-containing soluble guanylyl cyclase (α(1)β(1)-heterodimer) is a major intracellular receptor and effector for nitric oxide (NO) and carbon monoxide (CO) and mediates many of their biological actions by increasing cyclic GMP. We have synthesized new oxadiazolo-benz-oxazins and have assessed their inhibitory actions on guanylyl cyclase activity in vitro, on the formation of cyclic GMP in cultured cells and on the NO-dependent relaxation of vascular and non-vascular smooth muscle. 2. Soluble guanylyl cyclase, purified to homogeneity from bovine lung, was inhibited by 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS 2028) in a concentration-dependent and irreversible manner (IC(50) 30 nM for basal and 200 nM for NO-stimulated enzyme activity). Evaluation of the inhibition kinetics according to Kitz & Wilson yielded a value of 8 nM for K(i), the equilibrium constant describing the initial reversible reaction between inhibitor and enzyme, and 0.2 min(−1) for the rate constant k3 of the subsequent irreversible inhibition. Inhibition was accompanied by a shift in the soret absorption maximum of the enzyme's haem cofactor from 430 to 390 nm. 3. S-nitroso-glutathione-enhanced soluble guanylyl cyclase activity in homogenates of mouse cerebellum was inhibited by NS 2028 (IC(50) 17 nM) and by 17 structural analogues in a similar manner, albeit with different potency, depending on the type of substitution at positions 1, 7 and 8 of the benzoxazin structure. Small electronegative ligands such as Br and Cl at position 7 or 8 increased and substitution of the oxygen at position 1 by -S-,- NH- or -CH(2)- decreased the inhibition. 4. In tissue slices prepared from mouse cerebellum, neuronal NO synthase-dependent activation of soluble guanylyl cyclase by the glutamate receptor agonist N-methyl-D-aspartate was inhibited by NS 2028 (IC(50) 20 nM) and by two of its analogues. Similarly, 3-morpholino-sydnonimine (SIN-1)-elicited formation of cyclic GMP in human cultured umbilical vein endothelial cells was inhibited by NS 2028 (IC(50) 30 nM). 5. In prostaglandin F(2α)-constricted, endothelium-intact porcine coronary arteries NS 2028 elicited a concentration-dependent increase (65%) in contractile tone (EC(50) 170 nM), which was abolished by removal of the endothelium. NS 2028 (1 μM) suppressed the relaxant response to nitroglycerin from 88.3±2.1 to 26.8±6.4% and induced a 9 fold rightward shift (EC(50) 15 μM) of the concentration-relaxation response curve to nitroglycerin. It abolished the relaxation to sodium nitroprusside (1 μM), but did not affect the vasorelaxation to the K(ATP) channel opener cromakalim. Approximately 50% of the relaxant response to sodium nitroprusside was recovered after 2 h washout of NS 2028. 6. In phenylephrine-preconstricted, endothelium-denuded aorta of the rabbit NS 2028 (1 μM) did not affect relaxant responses to atrial natriuretic factor, an activator of particulate guanylyl cyclase, or forskolin, an activator of adenylyl cyclase. 7. NO-dependent relaxant responses in non-vascular smooth muscle were also inhibited by NS 2028. The nitroglycerin-induced relaxation of guinea-pig trachea preconstricted by histamine was fully inhibited by NS 2028 (1 μM), whereas the relaxations to terbutaline, theophylline and vasoactive intestinal polypeptide (VIP) were not affected. The relaxant responses to electrical field stimulation of non-adrenergic, non-cholinergic nerves in the same tissue were attenuated by 50% in the presence of NS 2028 (1 μM). 8. NS 2028 and its analogues, one of which is the previously characterized 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), appear to be potent and specific inhibitors of soluble guanylyl cyclase present in various cell types. Oxidation and/or a change in the coordination of the haeme-iron of guanylyl cyclase is a likely inhibitory mechanism