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Modulation of nitric oxide synthase activity in brain regions: Physiological and pathophysiological implications
EVIDENCE THAT NITRIC OXIDE AND PROSTAGLANDINS ARE INVOLVED IN THE MECHANISMS OF HIV-I gpl20-INDUCED DEATH OF HUMAN NEUROBLASTOMA CELLS 1N CULTIJRE. Corasaniti M:T*.,Mclino G §., Navarra M^.. Marra R°.; Fiuazzi-Agro' A§. and Nistic6 G ^.
*t,'aculO; q/'Phmwtaqv, ~hfiversi(v o~ Reggio Calabria," ,¢;I/)I-1RC(I% Rome: ,~l)epartmen/ of l.".xperimen/al Medicine and Biochemical Science.s, ^l£rperimen/al Neurobiolog, v ('enter '5~tondino-Tor Ver,~ata", /)epm~onent o/ Biok~gv, UniveJ:s'ily of Rome "Tor l"ergata", Rome; °C,'W&IBI/I1,] ('atanzaro (ltahO. Human immunodeficiency vires type 1 (HIV-I) coat prolein gp120 has been shown to induce dealh of different ueuronal cell types in prinmry cultures. It has bccn reporled that N-methyl-D-aspartate (NMDA) receptor-gated Ca2~ entry (Dreyer cl al., 1990, Science, 248:364: Lipton ctal., 1991, Neuron, 7:11 l) is revolved in the mechanism of gpl20-induced excessive nitric oxide (NO) production which, in turn, leads cultured rat cortical neuroncs IO death (Dawson el al., I993, PNAS, 90:3256). Wc now report data from experimenls aimed to farther characterize the mechanisms through which gp120 produces cytotoxicity. Treatment of CHP 100 neuroblastoma cells with gp120 (10pM) induced an approximately 130% increase over control cell death, as determined 18 h after 50 min exposure (gp120-induccd cell dealh=16.1+l.l%, P<0.01 vs conlrol cell death=6.8+0.5%).~The lethal effects induced by gpl20 were significantly reduced in Ca 2 '-free medium and by 10 rain preincubation of CHP 100 cells with selective competitive (CGP37849, 100 ~M; LY274614, 100 4tM) and uou-compct!.five (MK801, 200 nM) anmgouisls of NMDA bnl nol non NMDA (CNQX, 100 bM), receptors. Blockade of gp120-induced cell death was also afforded by L-NAME (200 ~tM), an inhibitor of NO synthase, by Ihe D-enantiomcr (1.0 raM) of L-arginine or by haemoglobin ( 10 ~tM), a NO-trapping agent (see Moncada et al., 1991, Pharmacol. Rev., 43:109). Addition of l,-arginine (1.0 raM) reverted the prolccfive effects afforded by L-NAME The cytotoxic effects elicited by gpl20 were also prevented by indomelhacin and flufeuamic acid (10 ftM), two cyclooxygcuase iuhibitors.
~ITRIC OXIDR MODULATES Ca 2+ HOMEOSTASIS IN P c i 2 CELLS #Clara Sciorati, #.*Emilio Clementi, *°Teresa Granato and °Giuseppe Nistic6 #CNR C y t o p h a r m a c o l o g y Ctr., Dibit - H. Ban Raffaele, Milano; *Dept. P h a r m a c o l o g y , U n i v e r s i t y of R e g g i o Calabria, Catanzaro; °Dept. Biology, U n i v e r s i t y of Roma "Tor Vergata", Roma, Italy. N i t r i c oxide (NO) is implicated in the regulation of crucial, Ca2+-dependent events in neuronal cells (i). We h a v e i n v e s t i g a t e d the role of NO in m o d u l a t i n g b o t h Ca 2+ r e l e a s e and influx e l i c i t e d by m e m b r a n e receptors coupled to PIP 2 hydrolysis and IP 3 g e n e r a t i o n in the neurosecretory cell line, PC12 (2). Analysis of fura-2 loaded cells (2) revealed that Ca 2+ release from intracellular rapidly-exchanging Ca2÷ stores was increased in the presence of NO s y n t h a s e i n h i b i t o r s and r e d u c e d b y N O - r e l e a s i n g drugs. This effect was p a r a l l e l e d by consistent changes in !P3 production, elicited by stimulation with either v a r i o u s r e c e p t o r agonists, the G p r o t e i n a c t i v a t o r AIF4, or the Ca 2+ ionophore ionomyein. These findings suggest t h a t NO a c t i o n w a s e x e r t e d via p a r t i a l i n h i b i t i o n of p h o s p h o l i p a s e C~ activity. The storedependent compenentof the Ca 2+ influx r e s p o n s e w a s i n c r e a s e d b y active NO generation, and inhibited b y NOS blockade. In contrast, Ca 2+ influx through Second messenger-operated Ca 2+ channels was totally insensitive to the action of NO. The inhibition by NO of Ca 2+ release appears te be m e d i a t e d through cGMPdependent protein k i n a s e I activation, w h i l e the stimulation of Ca 2+ i n f l u x seems d u e to s i g n a l s different from cGMP generation. These results indicate that NO is a k e y regulator of Ca 2+ homeostasis in PC12 cells. I)T. Dawson and S. Snyder, J. Neurosci. 14:5147, 1994 2)E. Clementi et al., J. Biol. Chem. 267:2164, 1992.
THE EFFECTS OF NO SYNTHASE INHIBITORS ON MORPHINE REWARD AND ABSTINENCE SYNDROME A.Zharkovsky, T.Kivastik and T.Zharkovsky Department of Pharmacology, University of Tartu, EE2400 Tartu, Estonia
MODULATION OF NITRIC OXIDE SYNTHASE ACTIVITY IN BRAIN REGIONS: PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL IMPLICATIONS
M. Yu. Stepanichev, M.V. Onufriev, A.V. Volkov, Yu.V. Zarzhetsky, N.V. gulyaeva I n s t i t u t e of Higher Nervous A c t i v i t y and Neurophysiology RAS, B u t l e r o v S t r . , 5 A, Moscow 117865, R u s s i a Modulation of brain NO-synthase (NOS) is suggested to be of great neurophysiologioal significance. Effects of'different factors on NOS activity in rat brain regions was studied using new approaches. Two methods for quantitative NOS assay were elaborated: a) monitoring by cryogenic ESR in vitro formation of mononitrotrosyl complex in the reaction of NO with diethyrdithiooarbamate and Fe 2+, b) and fluorometric'registration of the rate of NADPH oxidation inhibited by specific NOS inhibitors. NOS activity depended on the brain structure studied, on the strain of rats, on their sex and age. Cardiac arrest during 7-i5 min (model of postresuscitation pathology) resulted in the decrease of brain NOS activity; this effect was most pronounced in cerebellum i - 2 h after resuscitation and depended on individual behavioral characteristics of rats. Treatment of rats with bacterial endotoxins did not change cerebral NOS but induced NOS activity in brain regions aften the opening o ~ blood-brain barrier by acute hyper%ensl0n.
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Nitric oxide (NO) is termed postsynaptically in response to glutamate acting upon NMDA receptor and its release is involved in many glutamate actions in the CNS. According to the results of the recent studies NO are implicated in opioid tolerance and dependence, However. it is not clear whether NO is implicated in the morphine-induced reinforcement. The present study was addressed to investigate the role of NO in morphine reward and in the development and expression of physical dependence. Therefore. we studied the effects of NO synthase inhibitors L-NG-Nitroarginine (L-NNA) and L-NG-Methylarginine (L-NMA) on morphine-induced place preference (CPP) in rats and on the signs morphine-induced abstinence in mice. NO synthase inhibitors L-NNA (5 and 20 mg/kg) and L-NMA (10 mg/kg) had no significant influence on place conditioning themselves. L-NNA (5 mg/kg) or L-NMA (10 mg/kg) had no effect on morphine-induced CPP. However. higher dose of L-NNA (20 mg/kg) significantly antagonized morphine-induced place preference. L-NMA when co-administered with morphine, significantly attenuated jumping behaviour and diarrhea in morphine-dependent mice. A trend toward an inhibition of head twitches was also observed, though, it did not achieve statistical significance. Acute administration of L-NMA or L-NNA also significantly inihibited the signs of morphine abstinence.The results of our study suggest that NO is involved in the morphine reward as well as in the development of physical dependence on opioids.
*t,'aculO; q/'Phmwtaqv, ~hfiversi(v o~ Reggio Calabria," ,¢;I/)I-1RC(I% Rome: ,~l)epartmen/ of l.".xperimen/al Medicine and Biochemical Science.s, ^l£rperimen/al Neurobiolog, v ('enter '5~tondino-Tor Ver,~ata", /)epm~onent o/ Biok~gv, UniveJ:s'ily of Rome "Tor l"ergata", Rome; °C,'W&IBI/I1,] ('atanzaro (ltahO. Human immunodeficiency vires type 1 (HIV-I) coat prolein gp120 has been shown to induce dealh of different ueuronal cell types in prinmry cultures. It has bccn reporled that N-methyl-D-aspartate (NMDA) receptor-gated Ca2~ entry (Dreyer cl al., 1990, Science, 248:364: Lipton ctal., 1991, Neuron, 7:11 l) is revolved in the mechanism of gpl20-induced excessive nitric oxide (NO) production which, in turn, leads cultured rat cortical neuroncs IO death (Dawson el al., I993, PNAS, 90:3256). Wc now report data from experimenls aimed to farther characterize the mechanisms through which gp120 produces cytotoxicity. Treatment of CHP 100 neuroblastoma cells with gp120 (10pM) induced an approximately 130% increase over control cell death, as determined 18 h after 50 min exposure (gp120-induccd cell dealh=16.1+l.l%, P<0.01 vs conlrol cell death=6.8+0.5%).~The lethal effects induced by gpl20 were significantly reduced in Ca 2 '-free medium and by 10 rain preincubation of CHP 100 cells with selective competitive (CGP37849, 100 ~M; LY274614, 100 4tM) and uou-compct!.five (MK801, 200 nM) anmgouisls of NMDA bnl nol non NMDA (CNQX, 100 bM), receptors. Blockade of gp120-induced cell death was also afforded by L-NAME (200 ~tM), an inhibitor of NO synthase, by Ihe D-enantiomcr (1.0 raM) of L-arginine or by haemoglobin ( 10 ~tM), a NO-trapping agent (see Moncada et al., 1991, Pharmacol. Rev., 43:109). Addition of l,-arginine (1.0 raM) reverted the prolccfive effects afforded by L-NAME The cytotoxic effects elicited by gpl20 were also prevented by indomelhacin and flufeuamic acid (10 ftM), two cyclooxygcuase iuhibitors.
~ITRIC OXIDR MODULATES Ca 2+ HOMEOSTASIS IN P c i 2 CELLS #Clara Sciorati, #.*Emilio Clementi, *°Teresa Granato and °Giuseppe Nistic6 #CNR C y t o p h a r m a c o l o g y Ctr., Dibit - H. Ban Raffaele, Milano; *Dept. P h a r m a c o l o g y , U n i v e r s i t y of R e g g i o Calabria, Catanzaro; °Dept. Biology, U n i v e r s i t y of Roma "Tor Vergata", Roma, Italy. N i t r i c oxide (NO) is implicated in the regulation of crucial, Ca2+-dependent events in neuronal cells (i). We h a v e i n v e s t i g a t e d the role of NO in m o d u l a t i n g b o t h Ca 2+ r e l e a s e and influx e l i c i t e d by m e m b r a n e receptors coupled to PIP 2 hydrolysis and IP 3 g e n e r a t i o n in the neurosecretory cell line, PC12 (2). Analysis of fura-2 loaded cells (2) revealed that Ca 2+ release from intracellular rapidly-exchanging Ca2÷ stores was increased in the presence of NO s y n t h a s e i n h i b i t o r s and r e d u c e d b y N O - r e l e a s i n g drugs. This effect was p a r a l l e l e d by consistent changes in !P3 production, elicited by stimulation with either v a r i o u s r e c e p t o r agonists, the G p r o t e i n a c t i v a t o r AIF4, or the Ca 2+ ionophore ionomyein. These findings suggest t h a t NO a c t i o n w a s e x e r t e d via p a r t i a l i n h i b i t i o n of p h o s p h o l i p a s e C~ activity. The storedependent compenentof the Ca 2+ influx r e s p o n s e w a s i n c r e a s e d b y active NO generation, and inhibited b y NOS blockade. In contrast, Ca 2+ influx through Second messenger-operated Ca 2+ channels was totally insensitive to the action of NO. The inhibition by NO of Ca 2+ release appears te be m e d i a t e d through cGMPdependent protein k i n a s e I activation, w h i l e the stimulation of Ca 2+ i n f l u x seems d u e to s i g n a l s different from cGMP generation. These results indicate that NO is a k e y regulator of Ca 2+ homeostasis in PC12 cells. I)T. Dawson and S. Snyder, J. Neurosci. 14:5147, 1994 2)E. Clementi et al., J. Biol. Chem. 267:2164, 1992.
THE EFFECTS OF NO SYNTHASE INHIBITORS ON MORPHINE REWARD AND ABSTINENCE SYNDROME A.Zharkovsky, T.Kivastik and T.Zharkovsky Department of Pharmacology, University of Tartu, EE2400 Tartu, Estonia
MODULATION OF NITRIC OXIDE SYNTHASE ACTIVITY IN BRAIN REGIONS: PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL IMPLICATIONS
M. Yu. Stepanichev, M.V. Onufriev, A.V. Volkov, Yu.V. Zarzhetsky, N.V. gulyaeva I n s t i t u t e of Higher Nervous A c t i v i t y and Neurophysiology RAS, B u t l e r o v S t r . , 5 A, Moscow 117865, R u s s i a Modulation of brain NO-synthase (NOS) is suggested to be of great neurophysiologioal significance. Effects of'different factors on NOS activity in rat brain regions was studied using new approaches. Two methods for quantitative NOS assay were elaborated: a) monitoring by cryogenic ESR in vitro formation of mononitrotrosyl complex in the reaction of NO with diethyrdithiooarbamate and Fe 2+, b) and fluorometric'registration of the rate of NADPH oxidation inhibited by specific NOS inhibitors. NOS activity depended on the brain structure studied, on the strain of rats, on their sex and age. Cardiac arrest during 7-i5 min (model of postresuscitation pathology) resulted in the decrease of brain NOS activity; this effect was most pronounced in cerebellum i - 2 h after resuscitation and depended on individual behavioral characteristics of rats. Treatment of rats with bacterial endotoxins did not change cerebral NOS but induced NOS activity in brain regions aften the opening o ~ blood-brain barrier by acute hyper%ensl0n.
--59--
Nitric oxide (NO) is termed postsynaptically in response to glutamate acting upon NMDA receptor and its release is involved in many glutamate actions in the CNS. According to the results of the recent studies NO are implicated in opioid tolerance and dependence, However. it is not clear whether NO is implicated in the morphine-induced reinforcement. The present study was addressed to investigate the role of NO in morphine reward and in the development and expression of physical dependence. Therefore. we studied the effects of NO synthase inhibitors L-NG-Nitroarginine (L-NNA) and L-NG-Methylarginine (L-NMA) on morphine-induced place preference (CPP) in rats and on the signs morphine-induced abstinence in mice. NO synthase inhibitors L-NNA (5 and 20 mg/kg) and L-NMA (10 mg/kg) had no significant influence on place conditioning themselves. L-NNA (5 mg/kg) or L-NMA (10 mg/kg) had no effect on morphine-induced CPP. However. higher dose of L-NNA (20 mg/kg) significantly antagonized morphine-induced place preference. L-NMA when co-administered with morphine, significantly attenuated jumping behaviour and diarrhea in morphine-dependent mice. A trend toward an inhibition of head twitches was also observed, though, it did not achieve statistical significance. Acute administration of L-NMA or L-NNA also significantly inihibited the signs of morphine abstinence.The results of our study suggest that NO is involved in the morphine reward as well as in the development of physical dependence on opioids.