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Dextromethorphan binding sites in rat brain: Localization and pharmacology
CHANGES MURINE
IN
OPIOID
RECEPTOR
DENSITY
ON
THE EPFECT OF MORPHINE ON THE RESPONSE TO SHEEP ERYTHROCYTES IN MICE G~F. Maximova, P.M, Clodt, A.A. Potapova G Russia State Medical University, Ostrcvitianova st.,la,117869, Institute of Pharmacology, Baltlyskaya st.,8, Moscow, Russia
SPLENOCYTES
INDUCED BY IN V I V O TREATMENT WITH MORPHINE A N D M E T H A D O N E
P. Massi, G. Patrini, G. Ricevuti*, A. Mazzone*, G. Fossati*, I. Mazzucchelli ~ , E. Gori and D. Parolaro Institute of Pharmacology, Fac. Sciences, Via Vanvitelli 32/A, 20129 Milan, Italy *Dept. Internal Medicine, IRCCS Policlinico S.Matteo, P.le Golgi, 27100, Pavia, Italy.
We studied the effect of morphine on humoral immune response in mice. Mice were immunized intraperitoneally by 5xI0 s sheep red blood cells (SRBC). Experimental groups received intraperitoneal injection of morphine (15 mg/kg) on the day of immunization or I, 2 or 3 days after immunization. Plaque forming cells (PFC) were assayed 4 days after immunization.Results were expressed as nt~bet of PFC per I0 6 splenic lymphocytes and as number of PFC per spleen. It was found that m ~ p h i n e stimulated the response from 5,5xI0 to 14,0xI0 ~ P~C per spleen when th~ drug was administrated on a day of immunization. Injections of morphine on the otheT days after immunization slightly reduced or enhanced the response but failed to achieve statistical significance. Nalox o n administration prior to morphine injection abolished the stimulating effect o f morphine. Naloxon per se had no effect on the response. It is concluded that morphine is able t o s t i m u l a t e the humoral response to SRBC in mice in vivo. The effect is probably mediated by a specific opiate receptors as it is blocked by naloxon.
We verified whether changes in opioid receptor density on murine splenocytes occur after an acute in vivo treatment with morphine (20 mg/kg s.c.) and methadone (12.5 mg/kg s.c.) at doses inducing the same analgesic effect. Flow cytofluorimetric analysis was employed to define the splenocytes subpopulation (T and B lymphocytes and macrophages) using MoAb antibody and the binding of fluoresceinyl opiate antagonist, naloxone, on opiate receptors was calculated. Both morphine and methadone reduced the density of opiate receptors on B and T tymphocytes. Specifically, 20 rain, 24 and 72 hr after the injection there was a marked reduction (about 55%) in naloxone binding; the return to baseline level was reached after 5 days for T lymphocytes and after 7 days for B lymphocytes. Concerning macrophages, besides the relative low abundance of these cells in the total splenocytes, a trend of reduction in opiate receptor density was observed after acute in vivo treatment, the effect disappearing after "/"days. The point emerging from this study is that opioids in a single dose are capable of down-regulate opiate receptors in murine splenocytes for a long period of time.
DEXTROMETHORPHAN BINDING SITES IN RAT BRAIN: LOCALIZATION AND PHARMACOLOGY P. Meoni, *F. Tortella & N.G. Bowery, Dept. Pharmacology, School of Pharmacy, London, U.K. and *W.R.A.I.R., Washington D.C., U.S.A.
P H Y S I C A L A C T I V I T Y OF H I G H D U R A T I O N INDUCES ANTIN OCICEPTION THROUGH OPIOID AND SEROTONERGIC SYSTEMS M.F. Pach¢¢0, R. Pereira, C. A. Fontes Ribeiro, T . R . A . Macedo. Inst. of Pharmacol. and Exp. Ther., Fac. of Medicine, Univ. of Coimbra, Portugal.
Craviso et al [Mol. Pharmacol. 23 619 (1993)] first described the presence of high and low affinity binding sites for dextromethorphan (DM), the non-opioid antitussive agent, in guinea-pig and rat brain. We are currently attempting to determine the location and pharmacology of these binding sites in rodent and human brain using receptor autoradiography. In the present study we report' data obtained in rat brain. Brain sections (10~m) were cut at q8°C and thaw-mounted on to gelatin-coated glass slides. 3H-DM (10nM, 81 Ci/mmol) binding was performed by incubation (60 min, 4°C) in Tris HC1 buffer (50raM, pH 7.4) with or without NaC1 (50raM). Sections were subsequently washed for 2.5 min in buffer containing 0.1M choline chloride and 0.1% triton X-100. Regional binding densities determined from autoradiograms (Hyperfilm, 3 weeks exposure) were classified as low (0-71), medium (71-142) and high (142-320 fmoles/mg protein). High levels of binding were observed in the medial mammillary nucleus, dorsal raph6 and superior collicus with medium levels in central grey area, interpeduncular nucleus, dorsal tegmental area, inferior colliculus, anterior hypothalamus and pons. Binding in these areas was dependent on Na÷ since in its absence, binding was decreased by as much as 75%. DM binding to the hippocampal formation (CA1, CA3, dentate gyrus) and occipital cortex was unaffected by Na+. 3-(3-hydroxyphenyl)-N-(1-propyl) piperidine (PPP, 100nM), (KD at Na÷ -independent 3H-DM binding sites = 22nM) failed to alter the Na+-dependent binding, whereas phenytoin (1001aM) reduced the binding at this site by approximately 50%. These results reveal the existence of a Na÷ -dependent subpopulation of DM binding sites which is unaffected by PPP.
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The aim of the present study was to verify in rats if prolonged physical exercise can induce antinociception through the opioid and/or serotonergic systems. Male Wistar rats were trained to swim for 40 min.The tail-flick latency or the pressure needed to cause pain (Randall and Sellito Apparatus) was measured, before and after the forced swimming test; before this test l m g / k g naloxone, l m g / k g ketanserin, l m g / k g ondansetron, 20mg/kg L-tryptophan (Ltrypt), 20mg/kg L - 5 - h y d r o x y t r y p t o p h a n (5-HTP), 100 mg/kg lisine acetylsalicilate or 5 mg/kg morphine were administered subcutaneously. A similar number of rats was subjected to the same p r o t o c o l except the swimming test. The following results were obtained: a) The swimming test induced a significant antinociception, which was inhibited by naloxone, ketanserin or ondansetron; b) L - T r y p t or 5-HTP did not significantly changed the pain threshold at rest; c)Lisine acetylsalicilate and principally morphine caused a significant antinociceptive effect; d) After the r e s t period (instead of exercise) naloxone, ketanserin or ondansetron did n o t show a significant effect. In conclusion, prolonged physical stress in rats induces antinociception through the production of endogenous opioids and 5-HT. The antinociceptive effect of this amine was performed through 5-HT2 and 5-HT3 receptors. L - T r y p t and 5-HTP display opposite effects at the periphery or in the CNS.
119--
IN
OPIOID
RECEPTOR
DENSITY
ON
THE EPFECT OF MORPHINE ON THE RESPONSE TO SHEEP ERYTHROCYTES IN MICE G~F. Maximova, P.M, Clodt, A.A. Potapova G Russia State Medical University, Ostrcvitianova st.,la,117869, Institute of Pharmacology, Baltlyskaya st.,8, Moscow, Russia
SPLENOCYTES
INDUCED BY IN V I V O TREATMENT WITH MORPHINE A N D M E T H A D O N E
P. Massi, G. Patrini, G. Ricevuti*, A. Mazzone*, G. Fossati*, I. Mazzucchelli ~ , E. Gori and D. Parolaro Institute of Pharmacology, Fac. Sciences, Via Vanvitelli 32/A, 20129 Milan, Italy *Dept. Internal Medicine, IRCCS Policlinico S.Matteo, P.le Golgi, 27100, Pavia, Italy.
We studied the effect of morphine on humoral immune response in mice. Mice were immunized intraperitoneally by 5xI0 s sheep red blood cells (SRBC). Experimental groups received intraperitoneal injection of morphine (15 mg/kg) on the day of immunization or I, 2 or 3 days after immunization. Plaque forming cells (PFC) were assayed 4 days after immunization.Results were expressed as nt~bet of PFC per I0 6 splenic lymphocytes and as number of PFC per spleen. It was found that m ~ p h i n e stimulated the response from 5,5xI0 to 14,0xI0 ~ P~C per spleen when th~ drug was administrated on a day of immunization. Injections of morphine on the otheT days after immunization slightly reduced or enhanced the response but failed to achieve statistical significance. Nalox o n administration prior to morphine injection abolished the stimulating effect o f morphine. Naloxon per se had no effect on the response. It is concluded that morphine is able t o s t i m u l a t e the humoral response to SRBC in mice in vivo. The effect is probably mediated by a specific opiate receptors as it is blocked by naloxon.
We verified whether changes in opioid receptor density on murine splenocytes occur after an acute in vivo treatment with morphine (20 mg/kg s.c.) and methadone (12.5 mg/kg s.c.) at doses inducing the same analgesic effect. Flow cytofluorimetric analysis was employed to define the splenocytes subpopulation (T and B lymphocytes and macrophages) using MoAb antibody and the binding of fluoresceinyl opiate antagonist, naloxone, on opiate receptors was calculated. Both morphine and methadone reduced the density of opiate receptors on B and T tymphocytes. Specifically, 20 rain, 24 and 72 hr after the injection there was a marked reduction (about 55%) in naloxone binding; the return to baseline level was reached after 5 days for T lymphocytes and after 7 days for B lymphocytes. Concerning macrophages, besides the relative low abundance of these cells in the total splenocytes, a trend of reduction in opiate receptor density was observed after acute in vivo treatment, the effect disappearing after "/"days. The point emerging from this study is that opioids in a single dose are capable of down-regulate opiate receptors in murine splenocytes for a long period of time.
DEXTROMETHORPHAN BINDING SITES IN RAT BRAIN: LOCALIZATION AND PHARMACOLOGY P. Meoni, *F. Tortella & N.G. Bowery, Dept. Pharmacology, School of Pharmacy, London, U.K. and *W.R.A.I.R., Washington D.C., U.S.A.
P H Y S I C A L A C T I V I T Y OF H I G H D U R A T I O N INDUCES ANTIN OCICEPTION THROUGH OPIOID AND SEROTONERGIC SYSTEMS M.F. Pach¢¢0, R. Pereira, C. A. Fontes Ribeiro, T . R . A . Macedo. Inst. of Pharmacol. and Exp. Ther., Fac. of Medicine, Univ. of Coimbra, Portugal.
Craviso et al [Mol. Pharmacol. 23 619 (1993)] first described the presence of high and low affinity binding sites for dextromethorphan (DM), the non-opioid antitussive agent, in guinea-pig and rat brain. We are currently attempting to determine the location and pharmacology of these binding sites in rodent and human brain using receptor autoradiography. In the present study we report' data obtained in rat brain. Brain sections (10~m) were cut at q8°C and thaw-mounted on to gelatin-coated glass slides. 3H-DM (10nM, 81 Ci/mmol) binding was performed by incubation (60 min, 4°C) in Tris HC1 buffer (50raM, pH 7.4) with or without NaC1 (50raM). Sections were subsequently washed for 2.5 min in buffer containing 0.1M choline chloride and 0.1% triton X-100. Regional binding densities determined from autoradiograms (Hyperfilm, 3 weeks exposure) were classified as low (0-71), medium (71-142) and high (142-320 fmoles/mg protein). High levels of binding were observed in the medial mammillary nucleus, dorsal raph6 and superior collicus with medium levels in central grey area, interpeduncular nucleus, dorsal tegmental area, inferior colliculus, anterior hypothalamus and pons. Binding in these areas was dependent on Na÷ since in its absence, binding was decreased by as much as 75%. DM binding to the hippocampal formation (CA1, CA3, dentate gyrus) and occipital cortex was unaffected by Na+. 3-(3-hydroxyphenyl)-N-(1-propyl) piperidine (PPP, 100nM), (KD at Na÷ -independent 3H-DM binding sites = 22nM) failed to alter the Na+-dependent binding, whereas phenytoin (1001aM) reduced the binding at this site by approximately 50%. These results reveal the existence of a Na÷ -dependent subpopulation of DM binding sites which is unaffected by PPP.
--
The aim of the present study was to verify in rats if prolonged physical exercise can induce antinociception through the opioid and/or serotonergic systems. Male Wistar rats were trained to swim for 40 min.The tail-flick latency or the pressure needed to cause pain (Randall and Sellito Apparatus) was measured, before and after the forced swimming test; before this test l m g / k g naloxone, l m g / k g ketanserin, l m g / k g ondansetron, 20mg/kg L-tryptophan (Ltrypt), 20mg/kg L - 5 - h y d r o x y t r y p t o p h a n (5-HTP), 100 mg/kg lisine acetylsalicilate or 5 mg/kg morphine were administered subcutaneously. A similar number of rats was subjected to the same p r o t o c o l except the swimming test. The following results were obtained: a) The swimming test induced a significant antinociception, which was inhibited by naloxone, ketanserin or ondansetron; b) L - T r y p t or 5-HTP did not significantly changed the pain threshold at rest; c)Lisine acetylsalicilate and principally morphine caused a significant antinociceptive effect; d) After the r e s t period (instead of exercise) naloxone, ketanserin or ondansetron did n o t show a significant effect. In conclusion, prolonged physical stress in rats induces antinociception through the production of endogenous opioids and 5-HT. The antinociceptive effect of this amine was performed through 5-HT2 and 5-HT3 receptors. L - T r y p t and 5-HTP display opposite effects at the periphery or in the CNS.
119--