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Effects of peroxynitrite and 3-nitro-l-tyrosine on the rat anococcygeus muscle
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EFFECTS OF PEROXYNITRITE AND 3-NITRO-L.-TYROSINE ON THE RAT ANOCOCCYGEUS MUSCLE B. Ho&r, E. Baggut, G. Soyak, B. Tunctau, i&g&l, f. Kanark Depattment of Pharmacology, Faculty of Pharmacy, University of Gazi, 06330Hipodrom, Ankara, Turkey.
PROTECTIVE EFFECT OF MELATONIN ON CELLULAR ENERGY DEPLETION IN A NON-SEPTIC SHOCK MODEL INDUCED BY ZYMOSAN JN THE RAT
Peroxynitrite (ONOO‘) generated by the reaction of nitric oxide and superoxide radical is a potent relaxant on the vascular smooth muscles and 3-nitro-L-tyrosine (NT) (a stable product of ONOO’) attenuates a-adrenoceptor agonists induced haemodynamic responses. Isolated rat anococcygeus muscle (RAM) has barn suggested as a useful praparation for studying adrenergic mechanisms and the effect of ONOO’ and NT an this musclehave not been investigated,y&. The aim of the present study was to characterize the effects of ONOO’ and NT on the isolated RAM. Cumulative administration of 0.1-1.0 mM ONOO’ synthetiaed in a quenched-flow reactor as described previously by Beckman et al. (1990) produced dosedependent relaxations on phenylephrine (2 pM)precartracted RAM. The mean values of the relaxations calculated as the percentage of precontraction were 22.Zt2.2, 47.7ti.4 and 72.3h5.0, respectively (n=6). Adminiatmtim of decomposed ONW on precontractcd tissues produced 25.1*1.7% relaxation (n=3) at 0.1 n&f, whereas at the higher concentrations (0.31.O mM) it produced a bipbasic response which was composed of an initial contraction followed by a mlaxation (19.4ti.6, 35.5i5.4 and 43.4i1.7, 56.3a2.6, respectively, n-3). lbe vehicle of ONOO’ caused dose-dependent contractions, NT (0.01-0.1 mM) incubation for 20 mitt, prior to the precontmction of tissue did not influence phenylwhtine-induced precontraction and ONOG-induced relaxations. During the precontraction plateau, administration of prazosin (a specific atblocker; 0.01-1.0 nM) inhibited phenylephrine-induced tonus, but NT did not. These results indicated that ON00 was a relaxant agent on RAM and its stable product NT is neither an a-adrenergic blocker nor an inhibitor ofrelaxations induced by ONOO’.
Instituteof Pharmacology,School ofMedicine, Universiryof Messina
326 WINE COMPLEX PHENOLS AND TANNINS (CPT) PROTECT AGAINST LIVER OXIDATIVE DNA DAMAGE INDUCED BY ZNITROPROPANE. C., M. Lodovici. G. Paganelli and P. Dolara. Department of Pharmacology, Universtty of Florence, Viale Morgagni 65, 50134, Florence Italy. The protection exerted by antioxidants against carcinogenesis mediated by oxygen radicals might be due to the prevention of oxidative DNA damage. We evaluated whether wine complex phenols and tannins (CPT) might exert a preventive effects on oxidative DNA damage induced with 2 Nitropropane (2NP), an hepatocarcinogen for which there is strong evidence of a mechanism of action mediated by oxidative DNA damage. We administered a mixture of CPT to rats 2 wk before a single challenge with 2NP (100 mg/kg i.p.). The animals were treated for 14 consecutive days by gavage with CPT (57.2 mglkgld). Controls received water alone. Hepatic levels of 6hydroxy-2-deoxyguanosine (60HdG), a reliable marker for oxidative DNA damage, were subsequently examined. The administration of CPT did not modify the liver basal levels of 60HdG, however in rats sacrificed 15 hr following 2NP injection, the levels of BOHdG, expressed as ratio 60HdG12deoxyguanosine 62dG). were significantly decreased shy CPT from 47.66 x IO- f 3.17 (S.E.) (n=5) to 33.32 x lg i 2.52 (S.E.) (n=5). Our results demonstrate that WCP have a protective effect on oxidative DNA damage induced by 2NP. This effect might be due to scavenging of active oxygen species by these polyphenolic compounds. combined to a possible elevation in the activities of phase II enzymes, as is reported in the literature for green tea polyphenols.
G.
S. Cwocrea, AP. Caputi
DNA single-strand bmakage and activation of the nucleer enxyme poly (ADP-ribose) synthetase (PARS) triggers an energy consuming, ineDicient repair cycle, which contributes to pcroxyninite-induced cellular injury. Recently, was proposed that aymosau, a non-bactetial agent, causes cellular injury by inducing the production of peroxynitrite and consequent PARS activation. Here we investigated whether in viva melatonin treatment iuhibits cellular injury induced by peroxynitrite production and PARS activation in macrophages collected from rats subjected to xymosaninduced shock. Macrophages hatvested from the peritoneal cavity exhibited a significant production of peroxynitrite, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123. Furthermore, zymosan-induced shock caused a suppression of macrophage mitochoudrial mspiration, DNA straud breakage, activation of PARS and reduction of cellular levels of NAD’. In viva treatment with melatonin (25 and 50 mgkg, intraptitoneally, 1 h a&r aymosan injection) significantly reduced in dosedependent maturer peroxynitrite formation and prevented the appearance of DNA damage, the decrease in mitoohondrial respiration, the loss of cellular levels of NAD’ and the PARS activation. Gur study support tlte view that the antioxidant and autiinflammatoy effect of melatonin is atso correlated with the inhibition of peroxynitrite production and PARS activation. Jn conclusion, melatonin may be a novel pharmacological approach to prevent cell injury in inflammation.
328 THE PROTECTIVE ROLE OF ENDOGENOUS GLUTATHIONE AGAINST PEROXYNITRITE-INDUCED INJURY IN CARRAGEENAN LOCAL INFLAMMATION. S. Cuzzocrea’, G. Costantino’,
B. Zingarelli’~‘,
AP. Caputi’
’Institute of Pharmacology, University qfMessina, Italy; ‘Divisron of Critical Care Medicine, Children’s Hospital Medical Center Cincinnati Ohio L!S.A. Here we have investigated the protective role of endogeoous glutathione, a known scavenger of peroxynittite, in rats subjected to catrageenau-induced pleurisy. In vivo depletion of endogenous glutathione pools with L-buthionine-(S,R)-sulfoximine (BSO, 1 g&g for 24h, intraperitoneally) enhances the cerrageenan-induced degree of pleural exudation, polymotphonuclear migration in rats subjected to carrageenan-induced pleurisy. Lung myeloperoxidase (MPO) activity and lipid peroxidation was significautly increased in BSO pretreated rats. However the inducible NO synthase in lung samples was unaffected by BSO pretrsatment. lrnmunohistochemical analysis for nitrotyrosine. a footprint of peroxyniuite, revealed a positive staining in lungs from oarrageenan-treated rats, which was massively enhanced by BSO preueatment. Furthermore, in viva BSO pretreatment signiticantly increased peroxynitrite formation as measured by the oxidation of the fluorescent dye dihydrorhodaminc 123, enhanced the appearaucc of DNA damage, the decrease in mitochondrial mspiration and partially decreased the cellular level of NADt in ex vivo macrophages harvested from the pleura) cavity of rats subjected to carrageenan-induced pleurisy. In vivo treatment with exogenous glutathionc (50 mgkg i.p.) significantly reverted the effects of BSO. Thus, endogenous glutathione plays an important protective role against carrageenan-induced local inflammation.
325
327
EFFECTS OF PEROXYNITRITE AND 3-NITRO-L.-TYROSINE ON THE RAT ANOCOCCYGEUS MUSCLE B. Ho&r, E. Baggut, G. Soyak, B. Tunctau, i&g&l, f. Kanark Depattment of Pharmacology, Faculty of Pharmacy, University of Gazi, 06330Hipodrom, Ankara, Turkey.
PROTECTIVE EFFECT OF MELATONIN ON CELLULAR ENERGY DEPLETION IN A NON-SEPTIC SHOCK MODEL INDUCED BY ZYMOSAN JN THE RAT
Peroxynitrite (ONOO‘) generated by the reaction of nitric oxide and superoxide radical is a potent relaxant on the vascular smooth muscles and 3-nitro-L-tyrosine (NT) (a stable product of ONOO’) attenuates a-adrenoceptor agonists induced haemodynamic responses. Isolated rat anococcygeus muscle (RAM) has barn suggested as a useful praparation for studying adrenergic mechanisms and the effect of ONOO’ and NT an this musclehave not been investigated,y&. The aim of the present study was to characterize the effects of ONOO’ and NT on the isolated RAM. Cumulative administration of 0.1-1.0 mM ONOO’ synthetiaed in a quenched-flow reactor as described previously by Beckman et al. (1990) produced dosedependent relaxations on phenylephrine (2 pM)precartracted RAM. The mean values of the relaxations calculated as the percentage of precontraction were 22.Zt2.2, 47.7ti.4 and 72.3h5.0, respectively (n=6). Adminiatmtim of decomposed ONW on precontractcd tissues produced 25.1*1.7% relaxation (n=3) at 0.1 n&f, whereas at the higher concentrations (0.31.O mM) it produced a bipbasic response which was composed of an initial contraction followed by a mlaxation (19.4ti.6, 35.5i5.4 and 43.4i1.7, 56.3a2.6, respectively, n-3). lbe vehicle of ONOO’ caused dose-dependent contractions, NT (0.01-0.1 mM) incubation for 20 mitt, prior to the precontmction of tissue did not influence phenylwhtine-induced precontraction and ONOG-induced relaxations. During the precontraction plateau, administration of prazosin (a specific atblocker; 0.01-1.0 nM) inhibited phenylephrine-induced tonus, but NT did not. These results indicated that ON00 was a relaxant agent on RAM and its stable product NT is neither an a-adrenergic blocker nor an inhibitor ofrelaxations induced by ONOO’.
Instituteof Pharmacology,School ofMedicine, Universiryof Messina
326 WINE COMPLEX PHENOLS AND TANNINS (CPT) PROTECT AGAINST LIVER OXIDATIVE DNA DAMAGE INDUCED BY ZNITROPROPANE. C., M. Lodovici. G. Paganelli and P. Dolara. Department of Pharmacology, Universtty of Florence, Viale Morgagni 65, 50134, Florence Italy. The protection exerted by antioxidants against carcinogenesis mediated by oxygen radicals might be due to the prevention of oxidative DNA damage. We evaluated whether wine complex phenols and tannins (CPT) might exert a preventive effects on oxidative DNA damage induced with 2 Nitropropane (2NP), an hepatocarcinogen for which there is strong evidence of a mechanism of action mediated by oxidative DNA damage. We administered a mixture of CPT to rats 2 wk before a single challenge with 2NP (100 mg/kg i.p.). The animals were treated for 14 consecutive days by gavage with CPT (57.2 mglkgld). Controls received water alone. Hepatic levels of 6hydroxy-2-deoxyguanosine (60HdG), a reliable marker for oxidative DNA damage, were subsequently examined. The administration of CPT did not modify the liver basal levels of 60HdG, however in rats sacrificed 15 hr following 2NP injection, the levels of BOHdG, expressed as ratio 60HdG12deoxyguanosine 62dG). were significantly decreased shy CPT from 47.66 x IO- f 3.17 (S.E.) (n=5) to 33.32 x lg i 2.52 (S.E.) (n=5). Our results demonstrate that WCP have a protective effect on oxidative DNA damage induced by 2NP. This effect might be due to scavenging of active oxygen species by these polyphenolic compounds. combined to a possible elevation in the activities of phase II enzymes, as is reported in the literature for green tea polyphenols.
G.
S. Cwocrea, AP. Caputi
DNA single-strand bmakage and activation of the nucleer enxyme poly (ADP-ribose) synthetase (PARS) triggers an energy consuming, ineDicient repair cycle, which contributes to pcroxyninite-induced cellular injury. Recently, was proposed that aymosau, a non-bactetial agent, causes cellular injury by inducing the production of peroxynitrite and consequent PARS activation. Here we investigated whether in viva melatonin treatment iuhibits cellular injury induced by peroxynitrite production and PARS activation in macrophages collected from rats subjected to xymosaninduced shock. Macrophages hatvested from the peritoneal cavity exhibited a significant production of peroxynitrite, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123. Furthermore, zymosan-induced shock caused a suppression of macrophage mitochoudrial mspiration, DNA straud breakage, activation of PARS and reduction of cellular levels of NAD’. In viva treatment with melatonin (25 and 50 mgkg, intraptitoneally, 1 h a&r aymosan injection) significantly reduced in dosedependent maturer peroxynitrite formation and prevented the appearance of DNA damage, the decrease in mitoohondrial respiration, the loss of cellular levels of NAD’ and the PARS activation. Gur study support tlte view that the antioxidant and autiinflammatoy effect of melatonin is atso correlated with the inhibition of peroxynitrite production and PARS activation. Jn conclusion, melatonin may be a novel pharmacological approach to prevent cell injury in inflammation.
328 THE PROTECTIVE ROLE OF ENDOGENOUS GLUTATHIONE AGAINST PEROXYNITRITE-INDUCED INJURY IN CARRAGEENAN LOCAL INFLAMMATION. S. Cuzzocrea’, G. Costantino’,
B. Zingarelli’~‘,
AP. Caputi’
’Institute of Pharmacology, University qfMessina, Italy; ‘Divisron of Critical Care Medicine, Children’s Hospital Medical Center Cincinnati Ohio L!S.A. Here we have investigated the protective role of endogeoous glutathione, a known scavenger of peroxynittite, in rats subjected to catrageenau-induced pleurisy. In vivo depletion of endogenous glutathione pools with L-buthionine-(S,R)-sulfoximine (BSO, 1 g&g for 24h, intraperitoneally) enhances the cerrageenan-induced degree of pleural exudation, polymotphonuclear migration in rats subjected to carrageenan-induced pleurisy. Lung myeloperoxidase (MPO) activity and lipid peroxidation was significautly increased in BSO pretreated rats. However the inducible NO synthase in lung samples was unaffected by BSO pretrsatment. lrnmunohistochemical analysis for nitrotyrosine. a footprint of peroxyniuite, revealed a positive staining in lungs from oarrageenan-treated rats, which was massively enhanced by BSO preueatment. Furthermore, in viva BSO pretreatment signiticantly increased peroxynitrite formation as measured by the oxidation of the fluorescent dye dihydrorhodaminc 123, enhanced the appearaucc of DNA damage, the decrease in mitochondrial mspiration and partially decreased the cellular level of NADt in ex vivo macrophages harvested from the pleura) cavity of rats subjected to carrageenan-induced pleurisy. In vivo treatment with exogenous glutathionc (50 mgkg i.p.) significantly reverted the effects of BSO. Thus, endogenous glutathione plays an important protective role against carrageenan-induced local inflammation.