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P06 Studies on the antilipoperoxidative action ofadenosine in the liver of carbon tetrachloride treated rats
RI~SUM]~S DES COMMUNICATIONS : ADI~NOSINE
P02
ADENOSINE-INDUCED BRONCHOCONSTRICTION IN THE ALLERGIC RABBIT: EVIDENCE FOR A~-ADENOSINE RECEPTOR.
p~n~_~ ADENOSINE AND PURINE METABOLISM IN MAMMARY EPITHELIAL CELLS OF IrJIMP MICE.
Metzger. W.J., All, S., Fisher, R.H., EI-Hashim, A.Z, Mustafa, S.J.
*Barankiewicz, J., Trembacz, H. and Zwierzchowski L.
East Carolina University School of Medicine. Greenville, NC 27858.
Institute of Biochemistry and Biophysics, and Institute of Genetic and Animal Breeding, Polish Academy of Sciences, 02-532 Warsaw, Rakowiecka 36, Poland.
Recently, there has been increasinginterestin adenosineas a mediator of allergic asthma. Adenosine causes a rapid and profoundbmnchoconstriction in allergic, but not in normal rabbits (All, et al. FASEB J. 3_,1236, 1989). The rabbit model for late phase asthma has physiologic characteristics and pharmacologic sensitivity comparable to human asthma. In the present study, we estimated the contribution of the adenosine A~ vs. A~ receptorin the allergic rabbit lung using 5' N-ethylcarboxamide adenosine (NECA) and cyclopentyl adenosine (CPA) as an A2 and A~ agoniet respectively. Analogs of adenosine were given by aerosol to compare relative potency of each in causing bronchoconstriction. Neonates were immunized with ragweed to preferentially produce specific IgE and develop non-specific bronchial hyperresponsiveness (PC~o histamine). Four allergic rabbits (PC~ohistamine 4.49 +_0.31 mg/ml) were challenged with adenosine and its analogs and PCsowas determined. NECA and CPA were given three days apart followed by a histamine chaltenge to determine change in airway hyperresponsiveness. The PC~o (mg/ml) values for adenosine, NECA, and CPA in these animals were 9.85 +_ 0.86, 3.90 + 0.27 (P < 0.002), and 1.45 _+0.26 (P < 0.001), respectively (PCsofor CPA vs NECA P < 0.002). There was no significant change in the baseline airway reactivity 24 hrs. following adenosineor either of its analogs. These data suggest that adenosineinduced bronchoconstriction in this model is mediated primarily through activation of an A 1 adenosine receptor.
Purine metabolism was compared in epithelial mammary gland cells isolated from virgin, pregnant and lactating mice. Also the effect of lactogenic hormones: insulin, prolactin and hydrocortisone on adanine and adenosine metabolism were studied in mammary gland explants from pregnant mice, It was found that pudne nucleotides can be synthesized by both purine biosynthesis de nave pathway and by purina salvage pathway in isolated mammary gland cells from virgin, pregnant and lactating mice, The capacity of purina nucleotide synthesis by the salvage pathway was about 10 times higher than the rate of de novo biosynthesis in all three physiological stages of mammary gland, Adenine was salvaged mainly to ATP, although deamination of AMP was also significant. Adenosine was more efficiently deaminated than phoephorylated and large accumulat!on of hypoxanthine outside of cells was observed. Inosine was the major product of hypoxenthine metabolism, though significant amount of hypoxanthine was also incorporated into ATP and GTP. Guanine was mainly deaminated to xanthins, but a significant amount of GMP was also phosphorylated via GDP to GTP as well intereonvertad to IMP. During physiological activation of the mammary cells, which occur during pregnancy and lactation, the rate of adenine, hypoxanthine and guanine salvage, AMP, ADP, GMP, GDP phosphorylations, interconversion of IMP to AMP and to GMP and IMP dephosphorylation increased considerably in comparison with that in cells from virgin mice. Insulin, prolactin, hydrocortisone enhanced (127, 62, 25% respectively) adenine and adenosine salvage. The highest rate of enhancement of adenine and adenosine salvage was observed when insulin was used in combination with prolectin and hydrocortisone.
4~ DESENSITIZATION p~rl~ I~1.11 CYCLASE SYSTEM.
OF
CORONARY
ADENOSINE
RECEPTOR-ADENYLATE
*Current address Gensia Pharmaceuticals, Inc., San Diego, CA. USA
Hussain, T. and Mustafa, &J. Dept. of PharmacoL School of Med. East Carolina University, Greenville, NC 27858. Using a new in vitro model, we investigated the regulation of adenosine receptorsecond messenger system. Isolated coronary arteries were incubated in the absence control) and presence (treated) of 2-chloroadenosine {CAD) in culture media at 37°C. KC- and PGF2o-induced contraction and adenosine analogs, isoproterenol (ISOP)-, sodium nitroprusside (SNP)-mediated relaxation were similar in fresh and incubated arteries suggesting the viability of the tissue. CAD treatment shifted the relaxationresponses to adenosine analogs (CAD, NECA and R-PIA) to the right. The shift was both concentration- and time-dependent being maximum at 100 I~M CAD at 3 days. CAD treatment did not shift relaxation responses to ]SOP, forskolin and SNP. ISOP (10 pM, 3 days) treatment shifted the relaxation response to ISOP to the right and not to adenosine analogs. Cholera and pertussis toxins catalyzed a~P-labelling of G-proteins suggested a decreased ability of Gs and Gi activation. Western blot analysis with specific antisera did not show any changes in the quantity of c~-eubunitsof either Gs or Gi. Basal and forskolin-stimulated activity of adanylate cyclase was decreased in the treated arteries. CAD and forskolin stimulated cAMP accumulation in the coronary rings was attenuated in the CAD treated group but ISOP-induced accumulation was unchanged. The data suggest a desensitization of adenosine receptor-adenylate cyclase system via regulation of both Gs and Gi in coronary artery. (Supported by HL 27339).
p ~ n ~ l EVIDENCE AGAINST THE INVOLVEMENT OF ATP-SENSITIVE K+ CHANNELS IN I , P ' I ' ADENOSINE-MEDIATED VASODILATION OF PORCINE CORONARY ARTERY. Mustafa, S.J. and Makujina, S.R.
l~_ STUDIES ON THE ANTILIPOPEROXIDATIVEACTION OF ADENOSINE IN THE LIVER p~n~ I.IIIMI OF CARBON TETRACHLORIDE TREATED RATS. Chagoya de S&nchez, V., Y&r~ez,L., Vidrio, S., D[az-Mu#oz, It& and Hernandez-Mu#oz, R. Departamento de Bioenergetica, Institute de Fisiologfa Celular. Universidad Nacional Aut6noma de Mdxico, Apartado 70-600 04510 M~Jxico. Adenosine delayed acute hepatotoxicity induced by carbon tetrachlorids (CCI4) preventing cell necrosis possibly through its antilipoperoxidative action (1). Furthermore, it is widely accepted that the CCI4 hepatotoxicity depends on its reductive dehaloganation catalyzed by the NADPH-cytochrome P-450 reductase in the liver cell endoplasmic reticulum, forming ~CCI3,this radical react rapidly with oxygen to yield chemical reactive species of short live responsible for lipid peroxidation and covalent binding of C C I 4 cleavage products to microsomal lipids. On these bases we confirmed the effect of the nucleoside measuring its effect in lipoperoxidation in vivo by a decrease in hepatic formation of conjugated dienee as well as the irreversible binding of t'CCI,. To go further inside of the mechanism we quantified the level of NADPH in the liver, the amount of cytochrome P-450 and glucose-6-phosphatase in mierosomes as well as the in vitro metabolism of CCI,. We conclude that the nueleoside does not diminish the CCI, metabolism neither the initiation process of lipid peroxidation and its effect could be related to a decreased propagation process. 1) Bioehem. Pharmacol. 33, 2599, 1984.
P07
CHANGES ON THE LIVER REDOX STATE INDUCED BY CARBON TETRACHLORIDE ADMINISTRATION AND ITS REVERSIBILITY BY ADENOSINE. R. Hern&ndez-Mu#oz, M. Dfaz-MuFToz,F. Lopez-Barrera and V. Chagoya de Sanchez
DepL Pharmacology, East Carolina Univ., School of Medicine, Greenville, NC 27858.
Depto de Bioenerg6tica, Institute de Fisiologia Celular, UNAM M~xico City, M~xico.
This study was conducted to determine whether ATP-sensitive K+ (K+A~p)channets are involved in adenosine-mediated vasodilation. Ring segments of porcine coronary artery were suspended in muscle chambers containing Krebs-Henseleit buffer (pH 7.4: 95% Oz+5%COz). After an initial equilibration period of 60 min during which the rings were placed under 2-5g of resting tension, the tissues were repeatedly challenged with 35-50 mM KCI until constant and reproducible contractions were achieved. Endothelial integrity was assessed by the ability of 100 nM bradykinin to cause relaxation. Pinscidil and glibenclamide were the respective K÷A~channel agenist and antagonist employed. Adenosine, 2-chloroadenosine (CAD), 5'-N-ethylcarboxamidoadenoeine(NECA), R(-)-N6(2-phenylisopropyl)adenosine (R-PIA),N6-cyclopentyladenoeine (CPA) (10nM-100 ftM), sodium nitropruseide SNP) (lnM-10 ,uM) and pinacidil (10nM-10~.tM) produced concentration-dependent relaxat ons n denuded r rigs contracted with KCI, endothelin-1 or PGFzo A~ agonists (R-PIA and CPA) failed to elicit vasoconstriction. Incubation of tissues with 0.3-5~tM glibenclamide for 30 rain did not cause a significant change in the force generated by the contracting agents, Glibenclamide caused e parallel rightward shift of the concentration-response curve to pinacidil but did not alter the responses elicited by SNP or adenosine and its analogs. Such was also the case with rings possessing functional endothelium. The data suggests that K*A~channels are net involved in the mechanism by which adenosine and its analogs elicit their vasedilatory response in isolated porcine coronary artery. (Supported by HL 27339).
The role of changes on the redox state in both, eytoeolic and mitochondrial compartments, has been widely established in the physio-pathology of ethanol-induced liver damage. However, ethanol-induced shiff of redox state seems to be more involved in the acute ethanol ingestion, rather than in the chronic effects mediated by this hepatotoxin. In this regard, it is interesting that changes on the redox state could modulate, at least in part, the collagen synthesis, which is accumulated in the chronic liver disease. We have studied the effects of carbon tetrachloride (CCI4) on the liver redox state, during the generation of liver fibrosis, and the protective action of adenosine administration on these effects. The CCI4 produced a marked shift of both, cytosolic and mitochondrial redex state, toward a more reduced potential, which was more drastic in the mitochondrial compartment. In the latter, the redox state was also calculated through the equilibrium Of the reaction mediated by the glutamic dehydrogenaee, giving very similar results. Another redox-palr metabolites, namely reduced and oxidized glutathione, were also markedly affected by the CCI4, suggesting that during liver fibrosis generation, the liver metabolism is altered by a more reduced cellular redex state. Simultaneously adenosine administration to CC[4-treated rats, largely avoided these effects on the redox state. These data strongly correlates with the adenosine actions of preserving the energy charge, mitochondrial function and amelioration of liver fibrosis, which have been found in livers of animals chronically treated with CCI4 plus adenosine.
247
P02
ADENOSINE-INDUCED BRONCHOCONSTRICTION IN THE ALLERGIC RABBIT: EVIDENCE FOR A~-ADENOSINE RECEPTOR.
p~n~_~ ADENOSINE AND PURINE METABOLISM IN MAMMARY EPITHELIAL CELLS OF IrJIMP MICE.
Metzger. W.J., All, S., Fisher, R.H., EI-Hashim, A.Z, Mustafa, S.J.
*Barankiewicz, J., Trembacz, H. and Zwierzchowski L.
East Carolina University School of Medicine. Greenville, NC 27858.
Institute of Biochemistry and Biophysics, and Institute of Genetic and Animal Breeding, Polish Academy of Sciences, 02-532 Warsaw, Rakowiecka 36, Poland.
Recently, there has been increasinginterestin adenosineas a mediator of allergic asthma. Adenosine causes a rapid and profoundbmnchoconstriction in allergic, but not in normal rabbits (All, et al. FASEB J. 3_,1236, 1989). The rabbit model for late phase asthma has physiologic characteristics and pharmacologic sensitivity comparable to human asthma. In the present study, we estimated the contribution of the adenosine A~ vs. A~ receptorin the allergic rabbit lung using 5' N-ethylcarboxamide adenosine (NECA) and cyclopentyl adenosine (CPA) as an A2 and A~ agoniet respectively. Analogs of adenosine were given by aerosol to compare relative potency of each in causing bronchoconstriction. Neonates were immunized with ragweed to preferentially produce specific IgE and develop non-specific bronchial hyperresponsiveness (PC~o histamine). Four allergic rabbits (PC~ohistamine 4.49 +_0.31 mg/ml) were challenged with adenosine and its analogs and PCsowas determined. NECA and CPA were given three days apart followed by a histamine chaltenge to determine change in airway hyperresponsiveness. The PC~o (mg/ml) values for adenosine, NECA, and CPA in these animals were 9.85 +_ 0.86, 3.90 + 0.27 (P < 0.002), and 1.45 _+0.26 (P < 0.001), respectively (PCsofor CPA vs NECA P < 0.002). There was no significant change in the baseline airway reactivity 24 hrs. following adenosineor either of its analogs. These data suggest that adenosineinduced bronchoconstriction in this model is mediated primarily through activation of an A 1 adenosine receptor.
Purine metabolism was compared in epithelial mammary gland cells isolated from virgin, pregnant and lactating mice. Also the effect of lactogenic hormones: insulin, prolactin and hydrocortisone on adanine and adenosine metabolism were studied in mammary gland explants from pregnant mice, It was found that pudne nucleotides can be synthesized by both purine biosynthesis de nave pathway and by purina salvage pathway in isolated mammary gland cells from virgin, pregnant and lactating mice, The capacity of purina nucleotide synthesis by the salvage pathway was about 10 times higher than the rate of de novo biosynthesis in all three physiological stages of mammary gland, Adenine was salvaged mainly to ATP, although deamination of AMP was also significant. Adenosine was more efficiently deaminated than phoephorylated and large accumulat!on of hypoxanthine outside of cells was observed. Inosine was the major product of hypoxenthine metabolism, though significant amount of hypoxanthine was also incorporated into ATP and GTP. Guanine was mainly deaminated to xanthins, but a significant amount of GMP was also phosphorylated via GDP to GTP as well intereonvertad to IMP. During physiological activation of the mammary cells, which occur during pregnancy and lactation, the rate of adenine, hypoxanthine and guanine salvage, AMP, ADP, GMP, GDP phosphorylations, interconversion of IMP to AMP and to GMP and IMP dephosphorylation increased considerably in comparison with that in cells from virgin mice. Insulin, prolactin, hydrocortisone enhanced (127, 62, 25% respectively) adenine and adenosine salvage. The highest rate of enhancement of adenine and adenosine salvage was observed when insulin was used in combination with prolectin and hydrocortisone.
4~ DESENSITIZATION p~rl~ I~1.11 CYCLASE SYSTEM.
OF
CORONARY
ADENOSINE
RECEPTOR-ADENYLATE
*Current address Gensia Pharmaceuticals, Inc., San Diego, CA. USA
Hussain, T. and Mustafa, &J. Dept. of PharmacoL School of Med. East Carolina University, Greenville, NC 27858. Using a new in vitro model, we investigated the regulation of adenosine receptorsecond messenger system. Isolated coronary arteries were incubated in the absence control) and presence (treated) of 2-chloroadenosine {CAD) in culture media at 37°C. KC- and PGF2o-induced contraction and adenosine analogs, isoproterenol (ISOP)-, sodium nitroprusside (SNP)-mediated relaxation were similar in fresh and incubated arteries suggesting the viability of the tissue. CAD treatment shifted the relaxationresponses to adenosine analogs (CAD, NECA and R-PIA) to the right. The shift was both concentration- and time-dependent being maximum at 100 I~M CAD at 3 days. CAD treatment did not shift relaxation responses to ]SOP, forskolin and SNP. ISOP (10 pM, 3 days) treatment shifted the relaxation response to ISOP to the right and not to adenosine analogs. Cholera and pertussis toxins catalyzed a~P-labelling of G-proteins suggested a decreased ability of Gs and Gi activation. Western blot analysis with specific antisera did not show any changes in the quantity of c~-eubunitsof either Gs or Gi. Basal and forskolin-stimulated activity of adanylate cyclase was decreased in the treated arteries. CAD and forskolin stimulated cAMP accumulation in the coronary rings was attenuated in the CAD treated group but ISOP-induced accumulation was unchanged. The data suggest a desensitization of adenosine receptor-adenylate cyclase system via regulation of both Gs and Gi in coronary artery. (Supported by HL 27339).
p ~ n ~ l EVIDENCE AGAINST THE INVOLVEMENT OF ATP-SENSITIVE K+ CHANNELS IN I , P ' I ' ADENOSINE-MEDIATED VASODILATION OF PORCINE CORONARY ARTERY. Mustafa, S.J. and Makujina, S.R.
l~_ STUDIES ON THE ANTILIPOPEROXIDATIVEACTION OF ADENOSINE IN THE LIVER p~n~ I.IIIMI OF CARBON TETRACHLORIDE TREATED RATS. Chagoya de S&nchez, V., Y&r~ez,L., Vidrio, S., D[az-Mu#oz, It& and Hernandez-Mu#oz, R. Departamento de Bioenergetica, Institute de Fisiologfa Celular. Universidad Nacional Aut6noma de Mdxico, Apartado 70-600 04510 M~Jxico. Adenosine delayed acute hepatotoxicity induced by carbon tetrachlorids (CCI4) preventing cell necrosis possibly through its antilipoperoxidative action (1). Furthermore, it is widely accepted that the CCI4 hepatotoxicity depends on its reductive dehaloganation catalyzed by the NADPH-cytochrome P-450 reductase in the liver cell endoplasmic reticulum, forming ~CCI3,this radical react rapidly with oxygen to yield chemical reactive species of short live responsible for lipid peroxidation and covalent binding of C C I 4 cleavage products to microsomal lipids. On these bases we confirmed the effect of the nucleoside measuring its effect in lipoperoxidation in vivo by a decrease in hepatic formation of conjugated dienee as well as the irreversible binding of t'CCI,. To go further inside of the mechanism we quantified the level of NADPH in the liver, the amount of cytochrome P-450 and glucose-6-phosphatase in mierosomes as well as the in vitro metabolism of CCI,. We conclude that the nueleoside does not diminish the CCI, metabolism neither the initiation process of lipid peroxidation and its effect could be related to a decreased propagation process. 1) Bioehem. Pharmacol. 33, 2599, 1984.
P07
CHANGES ON THE LIVER REDOX STATE INDUCED BY CARBON TETRACHLORIDE ADMINISTRATION AND ITS REVERSIBILITY BY ADENOSINE. R. Hern&ndez-Mu#oz, M. Dfaz-MuFToz,F. Lopez-Barrera and V. Chagoya de Sanchez
DepL Pharmacology, East Carolina Univ., School of Medicine, Greenville, NC 27858.
Depto de Bioenerg6tica, Institute de Fisiologia Celular, UNAM M~xico City, M~xico.
This study was conducted to determine whether ATP-sensitive K+ (K+A~p)channets are involved in adenosine-mediated vasodilation. Ring segments of porcine coronary artery were suspended in muscle chambers containing Krebs-Henseleit buffer (pH 7.4: 95% Oz+5%COz). After an initial equilibration period of 60 min during which the rings were placed under 2-5g of resting tension, the tissues were repeatedly challenged with 35-50 mM KCI until constant and reproducible contractions were achieved. Endothelial integrity was assessed by the ability of 100 nM bradykinin to cause relaxation. Pinscidil and glibenclamide were the respective K÷A~channel agenist and antagonist employed. Adenosine, 2-chloroadenosine (CAD), 5'-N-ethylcarboxamidoadenoeine(NECA), R(-)-N6(2-phenylisopropyl)adenosine (R-PIA),N6-cyclopentyladenoeine (CPA) (10nM-100 ftM), sodium nitropruseide SNP) (lnM-10 ,uM) and pinacidil (10nM-10~.tM) produced concentration-dependent relaxat ons n denuded r rigs contracted with KCI, endothelin-1 or PGFzo A~ agonists (R-PIA and CPA) failed to elicit vasoconstriction. Incubation of tissues with 0.3-5~tM glibenclamide for 30 rain did not cause a significant change in the force generated by the contracting agents, Glibenclamide caused e parallel rightward shift of the concentration-response curve to pinacidil but did not alter the responses elicited by SNP or adenosine and its analogs. Such was also the case with rings possessing functional endothelium. The data suggests that K*A~channels are net involved in the mechanism by which adenosine and its analogs elicit their vasedilatory response in isolated porcine coronary artery. (Supported by HL 27339).
The role of changes on the redox state in both, eytoeolic and mitochondrial compartments, has been widely established in the physio-pathology of ethanol-induced liver damage. However, ethanol-induced shiff of redox state seems to be more involved in the acute ethanol ingestion, rather than in the chronic effects mediated by this hepatotoxin. In this regard, it is interesting that changes on the redox state could modulate, at least in part, the collagen synthesis, which is accumulated in the chronic liver disease. We have studied the effects of carbon tetrachloride (CCI4) on the liver redox state, during the generation of liver fibrosis, and the protective action of adenosine administration on these effects. The CCI4 produced a marked shift of both, cytosolic and mitochondrial redex state, toward a more reduced potential, which was more drastic in the mitochondrial compartment. In the latter, the redox state was also calculated through the equilibrium Of the reaction mediated by the glutamic dehydrogenaee, giving very similar results. Another redox-palr metabolites, namely reduced and oxidized glutathione, were also markedly affected by the CCI4, suggesting that during liver fibrosis generation, the liver metabolism is altered by a more reduced cellular redex state. Simultaneously adenosine administration to CC[4-treated rats, largely avoided these effects on the redox state. These data strongly correlates with the adenosine actions of preserving the energy charge, mitochondrial function and amelioration of liver fibrosis, which have been found in livers of animals chronically treated with CCI4 plus adenosine.
247