s-nitro-n-acetylpenicillamine and ebselen

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

1 The role of protein kinase C (PKC) in colonic cellular injury in response to high concentrations of nitric oxide (NO) released from the donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP) was investigated. 2 Addition of SNAP (0.1-1000 microM) to the cellular suspension resulted in a dose-dependent increase in the extent of damage to isolated colonic mucosal cells as assessed by Trypan blue dye uptake and release of the lysosmal enzyme, N-acetyl-beta-glucosaminidase. SNAP treatment also resulted in an increase in cellular total PKC activity. These increases were reduced or eliminated by pretreatment of the cells with the PKC antagonists staurosporine or GF 109203X or the NO scavenger, phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). 3 PKC-alpha, PKC-delta, PKC-epsilon and PKC-zeta were detected in colonic cellular lysates by immunoblotting. However, only PKC-epsilon protein was increased in response to SNAP treatment. Furthermore, SNAP treatment resulted in activation of PKC-epsilon by causing translocation of the enzyme from the cytosolic to membrane fraction of the cell. This effect was eliminated if cells were preincubated with the NO scavenger, PTIO. 4 The extent of cellular damage in response to addition of SNAP to the incubation medium was enhanced by coincubation with the PKC activator, phorbol 12-myristate 13-acetate (PMA; 1 and 10 microM). 5 PKC activity and the extent of cell damage in response to SNAP were reduced by preincubation of the cells with the peroxyl scavenger, ebselen (0.01-10 microM). 6 These data suggest that the PKC-epsilon isoform of the enzyme mediates NO-induced damage to colonic mucosal cells. This response may occur, at least in part, due to peroxynitrite formation.

Topics: Animals; Azoles; Cell Survival; Colon; Cyclic N-Oxides; Dose-Response Relationship, Drug; Enzyme Activation; Imidazoles; Indoles; Intestinal Mucosa; Isoenzymes; Isoindoles; Male; Maleimides; Nitric Oxide; Nitric Oxide Donors; Organoselenium Compounds; Penicillamine; Protein Kinase C; Rats; Rats, Sprague-Dawley; Staurosporine; Tetradecanoylphorbol Acetate