s-nitro-n-acetylpenicillamine and 3-(5--hydroxymethyl-2--furyl)-1-benzylindazole

Carbon monoxide (CO) modulates several physiological functions through activation of a cGMP-dependent pathway similar to that of nitric oxide (NO). Here we investigated the possible involvement of soluble guanylate cyclase in the anti-aggregatory effect of micromolar concentrations of CO released by a novel, water-soluble, CO releasing molecule (CORM) in human platelets.. Human platelet aggregation was induced by collagen or thrombin, and the effects of CO releasing molecule (CORM-3) and an NO donor on platelet aggregation were compared.. CORM-3 liberated CO in a time- and concentration-dependent manner as evidenced by the formation of carbon monoxy myoglobin (MbCO) using a spectrophotometric assay. When added to washed platelets, CORM-3 (10-300 microM) inhibited collagen- and thrombin-induced aggregation in a concentration-dependent manner. The anti-aggregatory effect of CORM-3 was reversed by deoxy-Mb (50 microM). Interestingly, in the presence of an inhibitor of guanylate cyclase (ODQ, 5 microM), inhibition of collagen-induced aggregation by CORM-3 was not blocked but potentiated. Under the same experimental conditions, inhibition of platelet aggregation by an NO donor (SNAP, 1 microM) was prevented by ODQ. In collagen-induced or thrombin-induced platelet aggregation, a stimulator of guanylate cyclase (YC-1, 0.3 microM) did not alter the effect of CORM-3, whereas it markedly potentiated the inhibition of platelet aggregation mediated by SNAP. Notably, CORM-3-induced inhibition of platelet aggregation was of similar degree when platelets were activated by a low (20 mU/ml) or by high concentration of thrombin (100-200 mU/ml), whereas NO donors (SNP and SNAP)- or carbaprostacylin (cPGI(2))-induced effects were considerably attenuated when platelets were activated by high concentrations of thrombin.. Inhibition of platelet aggregation by CO released by a novel, water-soluble CORM is not mediated by activation of soluble guanylate cyclase. In contrast to NO and PGI(2), CO effectively inhibits platelets even when cells are activated excessively. We suggest that despite the fact that CO is not a potent inhibitor of platelet activation, it may gain importance when NO and PGI(2) alone are insufficient to overcome excessive platelet activation.

Topics: Analysis of Variance; Blood Platelets; Carbon Monoxide; Collagen; Dose-Response Relationship, Drug; Guanylate Cyclase; Humans; Indazoles; Nitric Oxide Donors; Organometallic Compounds; Oxadiazoles; Penicillamine; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Quinoxalines; Spectrophotometry; Statistics, Nonparametric; Thrombin; Time Factors

The effect of 2-phenyl-1,2-benzisoselenazol-3(2H)-one (ebselen) on nitric oxide (NO) mediated responses and NO generation from NO donors were studied in vitro. In precontracted rat isolated anococcygeus muscles, relaxations induced by NO donors, electrical field stimulation and 5-[1-(phenylmethyl)-1H-indazole-3-yl]-2-furanmethanol (YC-1) were significantly inhibited by ebselen (100 microM), whereas responses elicited by papaverine and theophylline were not affected; those by 8-bromo-cyclic-guanosine-monophosphate (8-Br-cGMP) were slightly enhanced. NO generation from NO gas aqueous solution or acidified nitrite was not affected, but that from S-nitroso-N-acetyl-penicillamine (SNAP) was attenuated by ebselen, and the attenuation was reserved by glutathione. Both glutathione and cupric sulphate altered the ultraviolet spectrum of ebselen. These findings suggest that ebselen at high concentrations nonselectively inhibited NO-mediated responses, possibly through inhibiting soluble guanylate cyclase. Ebselen does not appear to directly interact with NO, but it may inhibit NO release from nitrosothiols by a thiol- and/or copper-dependent mechanism.

Topics: Animals; Aorta; Azoles; Copper; Cyclic GMP; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Electric Stimulation; Endothelium, Vascular; Enzyme Activators; Enzyme Inhibitors; Glutathione; In Vitro Techniques; Indazoles; Isoindoles; Male; Muscle Relaxation; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Organoselenium Compounds; Papaverine; Penicillamine; Phosphodiesterase Inhibitors; Rats; Rats, Sprague-Dawley; Spectrophotometry, Ultraviolet; Vasodilation

Nitric oxide (NO) performs a central role in biological systems, binding to the heme site of soluble guanylyl cyclase (sGC), leading to enzyme activation and elevation of intracellular levels of cGMP. Organic nitrates, in particular, nitroglycerin (GTN), are clinically important nitrovasodilators that function as NO-mimetics in biological systems. Comparison of sGC activation data with electrochemically measured rates of NO release for genuine NO donors, NONOates and nitrosothiols, yields an excellent correlation between the EC(50) for sGC activation and the rate constant for NO release, k(NO). However, activation of sGC by GTN and the nitrates has very different characteristics, including the requirement for specific added thiols, for example, cysteine. The reaction of GTN with cysteine in anaerobic solution yields NO slowly, and NO release, measured by chemiluminescence detection, is quenched by added metal ion chelator. The generation of NO under aerobic conditions is 100-fold slower than the anaerobic reaction. Furthermore, NO release from the reaction of GTN with cysteine in phosphate buffer is too slow to account for sGC activation by GTN/cysteine. The slow rate of the chemical reaction to release NO suggests that nitrates can activate sGC by an NO-independent mechanism. In contrast to the genuine NO donors, GTN behaves as a partial agonist with respect to sGC activation, but in the presence of the allosteric sGC activator, YC-1, GTN exhibits full agonist activity.

Topics: Animals; Aorta; Cysteine; Enzyme Activation; Enzyme Activators; Enzyme Inhibitors; Glutathione; Guanylate Cyclase; Hydrazines; Indazoles; Molecular Mimicry; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Nitroso Compounds; Penicillamine; Rats; S-Nitrosoglutathione; Solubility

This study aimed to examine effects of varied organic phosphates on activities of soluble guanylate cyclase (sGC). The enzyme was purified from bovine lung. Physiologically relevant concentrations of ATP, 2,3-bisphosphoglyceric acid and inositol hexakisphosphate inhibited its enzyme activities under steady-state conditions as well as those determined under stimulation with S-nitroso-N-acetylpenicillamine, a nitric oxide donor, carbon monoxide or YC-1. Lineweaver-Burk plot analyses revealed that these three organic phosphates act as competitive inhibitors. Other organic phosphates such as cardiolipin and sphingomyelin but not inorganic phosphates exhibited such inhibitory actions. These results suggest that organic phosphates serve as inhibitors for sGC-dependent signaling events.

Topics: 2,3-Diphosphoglycerate; Adenosine Triphosphate; Animals; Binding, Competitive; Carbon Monoxide; Cattle; Enzyme Activators; Enzyme Inhibitors; Guanylate Cyclase; In Vitro Techniques; Indazoles; Kinetics; Nitric Oxide; Nitric Oxide Donors; Organophosphorus Compounds; Penicillamine; Phytic Acid; Signal Transduction; Solubility