6-ketoprostaglandin-f1-alpha and linsidomine

The peroxynitrite scavenging ability of Procyanidins from Vitis vinifera L. seeds was studied in homogeneous solution and in human umbilical endothelial cells (EA.hy926 cell line) using 3-morpholinosydnonimine (SIN-1) as peroxynitrite generator. In homogeneous phase procyanidins dose-dependently inhibited 2',7'-dichloro-dihydrofluorescein (DCFH) oxidation induced by SIN-1 with an IC50 value of 0.28 microM. When endothelial cells (EC) were exposed to 5 mM SIN-1, marked morphological alterations indicating a necrotic cell death (cell viability reduced to 16 +/- 2.5%) were observed. Cell damage was suppressed by procyanidins, with a minimal effective concentration of 1 microM (cell morphology and integrity completely recovered at 20 microM). Cellular localization of procyanidins in EC was confirmed using a new staining procedure and site-specific peroxyl radical inducers: AAPH and cumene hydroperoxide (CuOOH). Endothelial cells (EC) pre-incubated with procyanidins (20 microM) and exposed to FeCl3/K3Fe(CN)6 showed a characteristic blue staining, index of a site-specific binding of procyanidins to EC. Procyanidins dose-dependently inhibit the AAPH induced lipid oxidation and reverse the consequent loss of cell viability, but were ineffective when oxidation was driven at intracellular level (CuOOH). This demonstrates that the protective effect is due to their specific binding to the outer surface of EC thus to quench exogenous harmful radicals. Procyanidins dose-dependently relaxed human internal mammary aortic (IMA) rings (with intact endothelium) pre-contracted with norepinephrine (NE), showing a maximal vasorelaxant effect (85 +/- 9%) at 50 microM (catechin: 18 +/- 2% relaxation at 50 microM). This effect was completely abolished when IMA-rings were de-endothelized and when IMA-rings with intact endothelium were pretreated with L-NMMA or with the soluble guanylate cyclase inhibitor, ODQ. Pre-incubation with indomethacin reduces (by almost 50%) the vasodilating effect of procyanidins, indicating the involvement also of a COX-dependent mechanism. This was confirmed in another set of experiments, where procyanidins dose-dependently stimulate the prostacyclin (PGI2) release, reaching a plateau between 25 and 50 microM. Finally, pre-incubation of IMA-rings with procyanidins (from 6.25 to 25 microM) resulted in a dose-dependent prevention of the endothelin-1 (ET-1) vasoconstriction. The ability of procyanidins to prevent peroxynitrite attack to vascular cells, by

Topics: 6-Ketoprostaglandin F1 alpha; Antioxidants; Biflavonoids; Catechin; Cell Survival; Cells, Cultured; Endothelin-1; Endothelium; Endothelium, Vascular; Fluoresceins; Fluorescent Dyes; Free Radicals; Humans; In Vitro Techniques; Lipid Peroxidation; Mammary Arteries; Molsidomine; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Peroxynitrous Acid; Proanthocyanidins; Seeds; Vasoconstriction; Vitis

The effects of nitric oxide (NO) and its second messenger cyclic guanosine monophosphate (cGMT) on prostacyclin (PGI2) synthesis were studied in cultured rat heart endothelial cells using three different non-enzymatic nitric oxide releasing substances as well as inhibitors of nitric oxide synthase and of soluble guanylate cyclase. Production of prostacyclin, measured as 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), was stimulated up to 1.7 fold in endothelial cells treated with the NO donors SIN-1 (3-morpholino sydnonimine), GEA 3162 (3-aryl-substituted oxatriazole imine) and GEA 3175 (3-aryl-substituted oxatriazole sulfonyl), chloride). In each case the synthesis of cGMP increase as much as 40-100 fold. An inhibitor of NO synthase, NG-nitro-L-arginine methyl ester (L-NAME), decreased the basal production of 6-keto-PGF1 alpha in non-stimulated endothelial cells, an effect that could be reversed by the NO donors SIN-1, GEA 3162 and GEA 3175. cGMP formation in the L-NAME treated endothelial cells was unaltered. The guanylate cyclase inhibitors, methylene blue (100 mumol/l) and LY83583 (100 mumol/l), caused a 1.5-10 fold increase in 6-keto-PGF1 alpha production while NO-donor-stimulated endothelial cGMP production was decreased by 10 to 90%. However, when SIN-1 was used as a stimulant, LY83583 had no significant effect on the production of cGMP. These findings support the hypothesis that NO stimulates prostacyclin production directly by activating cyclooxygenase. The results also suggest that NO could have an indirect effect on prostacyclin production via cGMP.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; Epoprostenol; Female; Guanylate Cyclase; Male; Molsidomine; Nitric Oxide; Nitric Oxide Synthase; Platelet Aggregation Inhibitors; Rats; Rats, Wistar; Triazoles; Vasodilator Agents

We have evaluated the contributions of nitric oxide (NO) and prostacyclin (PGI2) in the in vivo antiplatelet effects of clinically useful nitrovasodilators. In rats, intravenous infusion of three NO donors, glyceryl trinitrate, sodium nitroprusside, or 3'-morpholinosydnonimine, the stable metabolite of molsidomine, released 6-keto PGF1alpha (the stable metabolite of PGI2) and inhibited ex vivo human platelet aggregation to adenosine diphosphate by at least 80%. In in vitro studies, glyceryl trinitrate, sodium nitroprusside, and 3'-morpholinosydnonimine, at clinically attainable concentrations, increased cyclooxygenase activity in endothelial cells (EC), which resulted in a four- to sixfold release of 6-keto PGF1alpha. Pretreatment of the EC with hemoglobin which binds to and inactivates the biological actions of NO, but not by methylene blue (MelB), attenuated the NO-mediated PGI2 from the EC by at least 70%. Release of 6-keto PGF1alpha by the NO donors increased the ability of these compounds to inhibit thrombin-induced human platelet aggregation by at least 10 times; this potentiation was inhibited by hemoglobin but not by MeB. MeB blocked the direct anti-platelet effect of the NO donors in the absence of EC. In summary, we have demonstrated that NO, directly as well as together with an NO-driven cyclooxygenase activation (and hence PGI2), release contributes to the marked anti-platelet effects observed after the in vivo administration of clinically used nitrovasodilators.

Topics: 6-Ketoprostaglandin F1 alpha; Adenosine Diphosphate; Animals; Aorta; Cattle; Cells, Cultured; Endothelium, Vascular; Enzyme Activation; Epoprostenol; Hemoglobins; Humans; Kinetics; Male; Methylene Blue; Models, Cardiovascular; Molsidomine; Nitric Oxide; Nitroglycerin; Nitroprusside; Penicillamine; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Sprague-Dawley; S-Nitroso-N-Acetylpenicillamine; Vasodilation; Vasodilator Agents; Verapamil

1. Modulation of prostaglandin biosynthesis in vivo by either exogenous or endogenous nitric oxide (NO) has been studied in the rat using arachidonic acid (AA)-induced paw oedema and measuring both the foot volume and the amount of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), the stable metabolite of prostacyclin (PGI2), in the oedematous fluid recovered from inflamed paws. 2. Paw injections of 150 or 300 nmol of AA were virtually inactive whereas 600 nmol produced a moderate oedema which was greatly reduced by the NO synthase inhibitor L-NG-nitro arginine methyl ester (L-NAME, 100 nmol/paw) and the NO scavenger haemoglobin (Hb, 30 mumol/paw), but unaffected by the inhibitor of the soluble guanylate cyclase, methylene blue (Mb, 3 mumol/paw) and L-arginine (15 mumol/paw). 3. The NO-donors (10 mumol/paw) 3-morpholino-sydnonimine-hydrochloride (SIN-1), S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and sodium nitroprusside (SNP) significantly potentiated the paw oedema induced by AA (300 nmol/paw). 4. SIN-1 (2.5, 5 and 10 mumol/paw) produced a significant dose-dependent increase of the oedema induced by AA which was correlated with increased amounts of 6-keto-PGF1 alpha in the fluid recovered from inflamed paws. 5. Both oedema and prostaglandin biosynthesis induced by the combination AA+SIN-1 were greatly suppressed by either Hb (30 mumol/paw) or indomethacin (3 mumol/paw or 5 mg kg-1 s.c.) but unaffected by Mb (3 mumol/paw). 6. In LPS-treated rats (6 mg kg-1, i.p.) doses of AA inactive in normal animals produced a remarkable oedema which was reduced by L-NAME or Hb, unaffected by Mb and increased by L-arginine.7. These results demonstrate that NO increases prostaglandin biosynthesis in vivo through a guanosine 3': 5'-cyclic monophosphate (cyclic GMP)-independent mechanism and suggest that the interaction between NO synthase and cyclo-oxygenase (COX) pathways may represent an important mechanism for the modulation of the inflammatory response.

Topics: 6-Ketoprostaglandin F1 alpha; Amino Acid Oxidoreductases; Animals; Arachidonic Acid; Arginine; Capillary Permeability; Edema; Epoprostenol; Lipopolysaccharides; Male; Methylene Blue; Molsidomine; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Rats; Rats, Wistar; Vasodilator Agents

The role of nitric oxide (NO) synthesis was investigated in endotoxin (LPS) tolerance induced in rats by intraperitoneal injection of a sublethal dose of Salmonella enteritidis LPS (100 micrograms/kg intraperitoneally). Peritoneal macrophages were harvested 6 and 24 h after LPS injection and stimulated in vitro with LPS. LPS significantly stimulated arachidonic acid metabolism, as assessed by 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) levels, and NO production, as assessed by nitrite, in macrophages collected from control rats. In macrophages from tolerant rats LPS-stimulated 6-keto-PGF1 alpha production was significantly reduced, while nitrite production was increased compared to control macrophages (p < .001). In in vivo mortality studies, rats that were pretreated 24 h earlier with sublethal LPS were resistant to the lethal effect of a subsequent dose of LPS (15 mg/kg intravenously) in comparison to control rats (p < .001). NG-Nitro-L-arginine-methyl ester, an inhibitor of NO synthase, decreased mean survival time in control rats and abrogated the resistance to the lethal effect of LPS in tolerant rats. In contrast, molsidomine, a NO donor, improved survival in control rats but did not modify the resistance to the lethal dose of LPS in tolerant rats. The results suggest that sustained NO synthesis may be a beneficial mechanism for the induction of LPS tolerance.

Topics: 6-Ketoprostaglandin F1 alpha; Analysis of Variance; Animals; Arachidonic Acids; Arginine; Cells, Cultured; Drug Tolerance; Endotoxins; Lethal Dose 50; Lipopolysaccharides; Macrophages, Peritoneal; Male; Molsidomine; NG-Nitroarginine Methyl Ester; Nitric Oxide; omega-N-Methylarginine; Rats; Reference Values; Salmonella enteritidis

The modulating effects of exogenous and endogenous nitric oxide (NO) on the cardiac anaphylactic reaction and eicosanoid release were investigated in isolated perfused sensitized guinea-pig hearts using 3-morpholinosydnonimine (SIN-1), the active metabolite of molsidomine, as NO-donor and NG-nitro-L-arginine (NNA) as an inhibitor of NO biosynthesis. Infusion of SIN-1 (final concentrations in the perfusates 0.3 or 1.0 mmol/l) elevated coronary flow under basal conditions as well as during cardiac anaphylaxis, while NNA (0.1 mmol/l) decreased basal coronary flow and aggravated the anaphylactic coronary constriction. Both drugs did not modify the characteristic biphasic profile of the coronary constriction after antigen challenge with an initial more severe phase followed by a less pronounced long-lasting flow reduction. Neither SIN-1 nor NNA affected spontaneous heart rate. However, while NNA tended to prolong the duration of antigen-induced arrhythmias, SIN-1 (1 mmol/l) had an inhibitory effect. This protection might be related to the increased coronary flow in the presence of SIN-1. SIN-1 inhibited anaphylactic release of cysteinyl-leukotrienes (LT) and 6-keto-prostaglandin (PG) F1 alpha, but did not influence thromboxane (TX) B2 release. On the other hand, NNA (0.1 mmol/l) inhibited anaphylactic release of TXB2, but had only marginal effects on the release of cysteinyl-LT and 6-keto-PGF1 alpha. The results suggest that exogenous and endogenous NO functionally antagonize the effects of vasoconstrictor mediators released after antigen challenge. Additional effects of high concentrations of SIN-1 and NNA on antigen-induced eicosanoid release could modulate the vascular actions of these drugs during cardiac anaphylaxis.

Topics: 6-Ketoprostaglandin F1 alpha; Anaphylaxis; Animals; Antihypertensive Agents; Arginine; Coronary Circulation; Guinea Pigs; Heart; Leukotriene B4; Male; Molsidomine; Nitroarginine; Perfusion; Thromboxane B2; Vasodilator Agents