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Influence of glycine on morphine-induced antinociception in mice
European Journal of Pharmacology. 179 (1990) 301-305
301
Elsevier E.IP 51250
Influence of glycine on morphine-induced antinociception in mice M. Carrara, S. Zampiron, F. Capolongo, L. Cima and P. Giusti 1 Department of Pharmacology, University of Padova, 35131 Padova, Italy and I Institute of Experimental and Chnical Medicine, Laborato~ of Pharmacology, Universit.v of Ancona, 60131 Ancona, Italy Received 2 January 1990, accepted 23 January 1990
The effects of glycine on morphine-induced antinociceptiolt were investigated in mice, using a cutaneous thermal test (hot-plate), a visceral chemical test (acetylcholine writhing test), and a locomotor activity test. When glycine (200 mg/kg p.o.) and morphine (5 mg/kg s.c.) were given together during the first 30 min, glycine first antagonized the morphine-induced antinociception then this was followed by a synergistic effect. The two-phase influence of glycine on morphine-induced antinociception may be due to the interaction of glycine with different receptors. Glycine; Morphine; Nociception (thermal); Nociception (chemical-visceral)
1. Introduction
Glycine is one of the major inhibitory neurotransmitters of the CNS and its inhibitory actions are exerted on interneurons of the spinal cord (Curtis et al., 1967; Werman et al., 1968) and brainstem (Reubi and Cuenod, 1976), and on some neurons of higher centers (Krnjevic et al., 1966). The inhibitory action of glycine on spinal neurons can be efficiently antagonized by strychnine (Curtis et al., 1968). Binding of [3H]strychnine to synaptosomal membranes from spinal cord and other CNS regions is characterized by a single class of binding sites with an affinity constant of 3-10 nM (Becker and Betz, 1987; Young and Snyder, 1974). Glycine displays affinities for this site at concentrations which are three orders of magnitude lower, i.e. K i values for displacement of [3H]strychnine binding are around 10-100 ~tM (Betz, 1987; Young and Snyder, 1974). Also, the concentration of glycine required to exert a de-
Correspondence to: P. Giusti, Department of Pharmacology. L.go Meneghetti 2, 35131 Padova. Italy.
pressant action on cultured neurons is in the micromolar range (H6sli et al., 1973). In other areas of the CNS such as cerebral cortex and basal ganglia, [3H]glycine binding displays different K D values (Probst et al., 1986; Zarbin et al., 1981). The pharmacological profile of this binding differs from that of inhibitory glycine receptors as it is most efficiently displaced by D-serine and is insensitive to strychnine (Kishimoto et al., 1981). Until recently, no physiological agonist for this binding site was known. Johnson and Ascher (1987), however, showed that glycine at concentrations as low as 10 nM potentiates the response to both glutamate and Nmethyl-D-aspartic acid ( N M D A ) in cultured mouse neurons. Although insensitive to strychnine, these effects of glycine can be antagonized by D-serine (Becker and Betz, 1987), Therefore, the glycine binding which is insensitive to strychnine may occur at a high-affinity site that modulates the interaction between glutamate and the acidic amino acid type one (AA 1) receptors. This evidence recently received further support from Bowery (1987) who pointed out that the distribution of [3H]glycine sites within the brain, de-
0014-2999/90/$03.50 ,~31990 Elsevier Science Publishers B.V. (Biomedical Division)
302 termined autoradiographically, closely matches that of [ 3H]glutamate at AA~ receptors. Besides, glycine has been reported to exert inhibitory effects on nociceptive cells, thus mimicking the actions of morphine (Curtis and Duggan, 1967; Dostrovsky and Pomeranz, 1976). Antagonistic and synergistic effects of morphine on the depressant action of glycine have been reported (Duggan et al., 1976) but an abundance of data suggests that glycine is an effective agent to increase acute morphine action and to attenuate the abstinence behaviour induced by morphine deprivation (Contrera and Tamayo, 1980). Little information is available about the CNS responses to systemic administration of glycine (Shank et al., 1973). However, it remains to be proved that a simple increase of CNS glycine concentrations enhances glycine-mediated inhibition, but certain metabolic disorders that arise from a defect in glycine cleavage are characterized clinically by weakness and lethargy (Perry et al., 1975; Von Wendt et al., 1979). On the other hand, very high doses of glycine (up to 30 g daily with food, i.e. 430 m g / k g ) have been used in hyperprolinemia and in isovaleric acidemia (Cohn et al., 1978). Moreover, an oral dose of 10 g, i.e. 140 m g / k g , has been given without side-effects in the 'glycine-tolerance' in malabsorption diseases (Nalin, 1970). The present study was carried our in order to assess the antinoception caused by the oral administration of high doses of glycine. The influence of glycine on morphine-induced antinociception was also tested.
The animals were assigned at random to groups and to cages. The experiments were performed blind in that during the test the operator was unaware of the drug or drugs each animal had received. All tests were performed at room temperature and took place between 10 a.m. and 3 p.m. Each animal was tested at one specific time after drug administration and was not re-used thereafter. Three independent experiments were performed to study visceral chemical antinociception (VCA), thermal antinociception (TA) and locomotor activity (LA) caused by the administration of morphine, glycine and morphine plus glycine. Each of the three experiments involved 20 groups of mice (total n = 200/experiment): five groups received morphine hydrochloride (5 m g / k g s.c.) five were treated with glycine (200 m g / k g p.o.) only and five more groups received morphine hydrochloride and glycine together. The remaining five groups received saline both orally and subcutaneously (s.c.) and were used as controls. In VCA and LA tests the responses of the groups which received the tested compounds were expressed as percentages of the response of the control group. 2.2. VCA
2. Materials and methods
VCA was evaluated by means of the acetylcholine (ACh)-induced writhing test (Collier et al., 1968). Glycine a n d / o r morphine were administered to each group of animals at least 15 min before the i.p. injection of ACh bromide (3.2 mg/kg). The number of abdominal constrictions was counted for 2 min, at 15, 30, 45, 60 and 90 min after the administration of the compounds under test.
2.1. A nimals
2.3. TA
Groups (n = 10) of female mice (Swiss strain, 20-25 g), supplied by Nossan, Italy, were housed for at least 10 days in groups of 10 (Makrolon ~Tecniplast cages) on a 12-h light-dark cycle (8 a.m.-8 p.m. light) in a temperature-controlled environment (21 + I ° C ) and allowed free access to food (GLP-Nossan pellets) and water until 1 h before the start of the experiment.
TA was determined by means of the hot-plate test (Eddy and Leinbach, 1953). Mice were placed individually on the plate maintained at 55 +_ 0.5 ° C by feedback from a surface-mounted thermocoupie. The response latency was evaluated on the basis of either hind paw lick or j u m p reaction following the contact with the plate. After three readings at 30 min intervals, response changes at
303 15, 30, 45, 60 and 90 min were assessed after administration of the compounds. The results, summarized in fig. 2, were expressed in term of average temporal differences (s) relative to the baseline. Each of the above tests employed a 'cuto f f time of 45 s at which time the animal was removed from the testing procedure to limit tissue d a m a g e and stress.
2.4. LA LA was measured with a photocell apparatus (Sodeco-Sprint, Milan, Italy). The animals were kept in individual activity cages (1 = 26, w = 26, h = 20 cm). Each cage had 10 photocells, 1 cm above the floor level, connected to an electromechanical counter. LA was measured as the n u m b e r of beam interruptions in the time periods of 0-15, 15-30, 30-45, 45-60 and 60-90 min after the administration of morphine a n d / o r glycine.
2.5. Statistical analysis The response data for the groups that received morphine and morphine plus glycine were analysed by two-way analysis of variance ( A N O V A ) . This statistical method was employed to consider treatment effect by performing single mean contrasts with the N e w m a n - K e u l s test, and the block effect due to the order of entrance of the animals into the experiment (10 blocks). However, statistical analysis showed that the latter effect was not significant. When tested for VCA, T A and LA, the control groups showed no significant changes in these parameters and are therefore not depicted in figs. 1, 2 and 3 respectively.
3. Results
3.1. VCA (fig. 1) Glycine given orally up to 200 m g / k g , caused no statistically significant effects on the abdominal writhes (not shown). The m a x i m u m inhibition ( > 80%) of abdominal constrictions with morphine, 5 m g / k g s.c., was obtained 15 min after drug treatment, while the m a x i m u m inhibition ( > 85%) was obtained after 90 min in presence of glycine (200 m g / k g p.o.). The s.c. and p.o. administration of the same volume of saline to the control groups produced no significant changes of the nociceptive response. The same happened with the s.c. administration of m o r p h i n e in the presence of p.o. administered saline.
3.2. TA (fig. 2) The administration of glycine also did not produce an increase in antinociceptive response in TA, evaluated by the hot-plate test. As in VCA, the development and the intensity of the TA effect showed a different pattern when morphine was given alone and when it was given simultaneously with glycine. Morphine injected alone produced a non-significant degree of antinociception within 10 to 30 min after its administration, in contrast to morphine and glycine given together. The combination of morphine and glycine produced a sig-
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4~
IO
2.6. Drug administration Morphine hydrochloride and glycine, dissolved in distilled water to avoid any interference of co-administered sodium ions, were administered s.c. and p.o., respectively, in a volume of 10 m l / k g . The animals in the control groups received the same volume of saline.
Fig. 1. Visceral chemical antinociception (writhing test) caused by the administration of morphine hydrochloride (11 5 mg/kg s.c.) alone or associated with glycine ([] 200 mg/kg p.o.). The results are expressed as inhibition (%) of abdominal constrictions compared with those in the control groups. Each column represents the mean score + S.E. of a group of 10 mice. Asterisks indicate that at the time of the test there was a significant difference between the groups of treated-animals ( * P < 0.01).
304
0
.
Fig. 2. Thermal antinociception (hot-plate test) caused by the administration of morphine hydrochloride ( I 5 mg/kg s.c.) alone or associated with glycine (~ 200 mg/kg p.o.). Each column represents the mean score+ S.E. of 10 mice. Asterisks indicate that at the time of the test there was a significant difference between the two groups of animals (* P < 0.01). The initial reaction time was subtracted in all tests.
nificantly higher degree of a n t i n o c i c e p t i o n (P < 0.01) within 45, 60 and 90 min after its administration than did m o r p h i n e given alone. The s.c. a n d p.o. a d m i n i s t r a t i o n of the same v o l u m e of saline to the control groups p r o d u c e d no signific a n t changes of the nociceptive response• T h e s a m e h a p p e n e d with the s.c. a d m i n i s t r a t i o n of m o r p h i n e in the presence of p.o. a d m i n i s t e r e d saline. 3.3. LA T h e b e h a v i o u r p a t t e r n of the mice treated with glycine alone was not different from that of the controls. A n i m a l s treated with m o r p h i n e alone showed the typical sedated pattern, which is presented q u a n t i t a t i v e l y in fig. 3. W h e n m o r p h i n e
Fig. 3. Modifications in locomotor activity induced by morphine hydrochloride (ll 5 mg/kg) alone or associated with glycine ([] 200 mg/kg). The results are expressed as variations (%) from control groups. Each column represents the mean score+S.E, of l0 mice. Asterisks indicate that at the time of the test there was a significant difference between the two groups of treated animals ( * P < 0.01 ).
a n d glycine were a d m i n i s t e r e d together a significant increase (P < 0.01) in m o t o r activity was observed after 0-15 a n d 15-30 min in the a n i m a l s that received a c o m b i n a t i o n of the two c o m p o u n d s in c o m p a r i s o n to those that received only morphine. The s.c. and p.o. a d m i n i s t r a t i o n of the same volume of saline to the c o n t r o l g r o u p s d i d not p r o d u c e any significant changes of the nociceptive response. T h e same h a p p e n e d with the s.c. a d m i n i s t r a t i o n of m o r p h i n e in the presence of p.o. a d m i n i s t e r e d saline.
4. D i s c u s s i o n
A c c o r d i n g to Yaksh (1984) a n d Y a k s h a n d N o u e i h e d (1985), different s u b t y p e s of o p i a t e receptors, /t, ~ a n d ~, K are involved in the T A responses e v a l u a t e d in the hot plate test and in the V C A responses evaluated in the writhing test respectively. In mice, glycine (200 m g / k g p.o.) affected the time course of V C A (fig. 1) a n d T A (fig. 2) a n t i n o c i c e p t i o n i n d u c e d by m o r p h i n e (5 m g / k g s.c.) administration. The morphine-induced changes in L A were also influenced by glycine (fig. 3). In the T A test (fig. 2) glycine r e d u c e d the a n t i n o c i c e p t i v e action of m o r p h i n e after 15 a n d 30 min when they were a d m i n i s t e r e d together but the effect was not significant, while glycine signific a n t l y (P < 0.01) r e d u c e d the activity of m o r p h i n e in the V C A (fig. 1) a n d L A (fig. 3) tests, within the same time period. A f t e r 45, 60 a n d 90 min following the a d m i n i s t r a t i o n of m o r p h i n e , glycine significantly (P < 0.01) increased the effect of m o r p h i n e in the T A and V C A tests. But the LA was significantly r e d u c e d (P < 0.01) in mice that received the two c o m p o u n d s . The results o b t a i n e d indicate that glycine produces an a n t a g o n i s t i c a n d synergistic effect on some p h a r m a c o l o g i c a l actions of m o r p h i n e . In particular, when m o r p h i n e and glycine were given together, an a n t a g o n i s t i c effect of glycine was observed d u r i n g the first 30 min. T h e synergistic effect b e c a m e evident later. The first e x c i t a t o r y response to glycine m a y have been d u e to its interaction with the strychnine-insensitive b i n d i n g site, an effect which is m o d u l a t e d b y n a n o m o l a r c o n c e n t r a t i o n s of glycine.
305
The late inhibitory response of glycine may have been due to its interaction with the strychnine-sensitive receptor, which requires micromolar concentrations of glycine. The dual influence of glycine on morphine-induced antinociception, already stressed by Duggan et al. (1976), may thus be related to the kinetics and binding characteristics of glycine.
References Becker, C.-M. and H. Betz, 1987, Strychine: useful probe or not?, Trends Pharmacol. Sci. 8, 204. Betz, H., 1987, Biology and structure of the mammalian glycine receptor, Trends Neurosci. 10, 113. Bowery, N.G., 1987, Glycine-binding sites and N M D A receptors, Nature 326, 338. Cohn, R.M., M. Judkoff, R. Rothman and S. Segal, 1978, Isovaleric acidemia: use of glycine therapy in neonates, New Engl. J. Med. 299, 996. Collier, H.O.J., L.C. Dineen, C.A. Johnson and C. Schneider, 1968, The abdominal constriction response and its suppression by analgesic drugs in the mouse, Br. J. Pharmacol. Chemother. 32, 295. Contrera, E. and L. Tamayo, 1980, Effects of glycine, /5'-alanine and diazepam upon morphine-tolerant-dependent mice, J. Pharm. Pharmacol. 32, 336. Curtis, D.R. and A.W. Duggan, 1967. The depression of spinal inhibition by morphine, Agents Actions 1, 14. Curtis, D.R., L. H6sli and G.A.R. Johnson, 1967, Inhibition of spinal neurons by glycine, Nature 215, 1502. Curtis, D.R., L. Ht~sli and G.A.R. Johnson, 1968, A pharmacological study of the depression of spinal neurones by glycine and related aminoacids, Exp. Brain Res. 6, 1. Dostrovsky, J.O. and B. Pomeranz, 1976, Interaction of iontophoretically applied morphine with responses of interneurons in cat spinal cord, Exp. Neurol. 52, 325. Duggan, A.W., J. Davies and J.C. Hall, 1976, Effects of opiate agonist and antagonist on central neurons of the cat, J. Pharmacol. Exp. Ther. 196, 107. Eddy, N.B. and D. Leinbach, 1953, Synthetic analgesic. Ii. Dithienylbutenyl and dithienylbutylamine, J. Pharmacol. Exp. Ther. 107, 385. Fl~sli, L., E. H~sli and P.S. Andres, 1973, Nervous tissue culture. A mc~lel to study action and uptake of putative neurotransmitter such as aminoacids, Brain Res. 62, 597.
Johnson, J.W. and P. Ascher, 1987, Glycine potentiates the N M D A responses in cultured mouse brain neurons, Nature 325. 529. Kishimoto, H.. J.R. Simon and M.H. Aprison, 1981, Determination of the equilibrium dissociation constants of number of glycine binding sites in several areas of the rat central nervous system, using a sodium-interdependent system, J. Neurochem. 37, 1015. Krnjevic. K., M. Randic and D.W. Straughan, 1966, Pharmacology of cortical inhibition, J. Physiol. London 184, 78. Nalin, D.R., 1970, Effect of glycine and glucose on sodium and water adsorption in patients with cholera, Gut 11,768. Perry, T.L., N. Urquhart, J. MacLean, M.E. Evans, S. Hansen, A.G. Davidson, D.A. Applegarth, P.J. MacLoed and J.E. Lack, 1975, Nonketonic hyperglycinemia: glycine accumulation due to the absence of glycine cleavage , N. Engl. J. Med. 292, 1269. Probst, A., R. Cortes and J.M. Placios, 1986, The distribution of glycine receptors in the h u m a n brain. Unlight microscopic autoradiographic study using [3 H]strychnine, Neuroscience 17, 11. Reubi, J.C. and M. Cuenod, 1976, Release of exogenous glycine in the pigeon optic tectum during stimulation of a midbrain nucleus, Brain Res. 112, 347. Shank, R.P., M.H. Aprison and C.F. Baxter, 1973, Precursor of glycine in the nervous system: Comparison of specific activities in glycine and other amino acids after administration of (U-laC)glucose, ( 3 ,4- 14C)glucose, (l-uaC)glucose, (U-14C)serine or (l,5-14C)citrate to the rat, Brain Res. 52 301. Von Wendt, L., A. Hirvasniemi and S. Similar, 1979, Nonketonic hyperglycinemia: a genetic study of 13 Finnish families, Clin. Gene 15, 411. Werman, R., R.A. Davidoff and M.H. Aprison, 1968, Inhibitory actions of glycine on spinal neurons in the cat. J. Neurophysiol. 31, 81. Yaksh, T.L., 1984, Multiple opioid receptor systems in brain and spinal cord: Part 1, European J. Anaesthesiol. 1, 171. Yaksh, T.L. and R. Noueihed, 1985, The physiology and pharmacology of spinal opiates, Ann. Rev. Pharmacol. Toxicol. 25, 433. Young, A.B. and S.H. Snyder, 1974, Strychnine binding in rat spinal cord membranes ass~x:iated with the synaptic glycine receptor: cooperativity of glycine interactions, Mol. Pharmacol. 10, 790. Zarbin, M.A., J.K. Wamsley and M.J. Kuhar, 1981, Glycine receptor: light microscopic autoradiographic localization with [3H]strychnine, J. Neurosci. 1, 532.
301
Elsevier E.IP 51250
Influence of glycine on morphine-induced antinociception in mice M. Carrara, S. Zampiron, F. Capolongo, L. Cima and P. Giusti 1 Department of Pharmacology, University of Padova, 35131 Padova, Italy and I Institute of Experimental and Chnical Medicine, Laborato~ of Pharmacology, Universit.v of Ancona, 60131 Ancona, Italy Received 2 January 1990, accepted 23 January 1990
The effects of glycine on morphine-induced antinociceptiolt were investigated in mice, using a cutaneous thermal test (hot-plate), a visceral chemical test (acetylcholine writhing test), and a locomotor activity test. When glycine (200 mg/kg p.o.) and morphine (5 mg/kg s.c.) were given together during the first 30 min, glycine first antagonized the morphine-induced antinociception then this was followed by a synergistic effect. The two-phase influence of glycine on morphine-induced antinociception may be due to the interaction of glycine with different receptors. Glycine; Morphine; Nociception (thermal); Nociception (chemical-visceral)
1. Introduction
Glycine is one of the major inhibitory neurotransmitters of the CNS and its inhibitory actions are exerted on interneurons of the spinal cord (Curtis et al., 1967; Werman et al., 1968) and brainstem (Reubi and Cuenod, 1976), and on some neurons of higher centers (Krnjevic et al., 1966). The inhibitory action of glycine on spinal neurons can be efficiently antagonized by strychnine (Curtis et al., 1968). Binding of [3H]strychnine to synaptosomal membranes from spinal cord and other CNS regions is characterized by a single class of binding sites with an affinity constant of 3-10 nM (Becker and Betz, 1987; Young and Snyder, 1974). Glycine displays affinities for this site at concentrations which are three orders of magnitude lower, i.e. K i values for displacement of [3H]strychnine binding are around 10-100 ~tM (Betz, 1987; Young and Snyder, 1974). Also, the concentration of glycine required to exert a de-
Correspondence to: P. Giusti, Department of Pharmacology. L.go Meneghetti 2, 35131 Padova. Italy.
pressant action on cultured neurons is in the micromolar range (H6sli et al., 1973). In other areas of the CNS such as cerebral cortex and basal ganglia, [3H]glycine binding displays different K D values (Probst et al., 1986; Zarbin et al., 1981). The pharmacological profile of this binding differs from that of inhibitory glycine receptors as it is most efficiently displaced by D-serine and is insensitive to strychnine (Kishimoto et al., 1981). Until recently, no physiological agonist for this binding site was known. Johnson and Ascher (1987), however, showed that glycine at concentrations as low as 10 nM potentiates the response to both glutamate and Nmethyl-D-aspartic acid ( N M D A ) in cultured mouse neurons. Although insensitive to strychnine, these effects of glycine can be antagonized by D-serine (Becker and Betz, 1987), Therefore, the glycine binding which is insensitive to strychnine may occur at a high-affinity site that modulates the interaction between glutamate and the acidic amino acid type one (AA 1) receptors. This evidence recently received further support from Bowery (1987) who pointed out that the distribution of [3H]glycine sites within the brain, de-
0014-2999/90/$03.50 ,~31990 Elsevier Science Publishers B.V. (Biomedical Division)
302 termined autoradiographically, closely matches that of [ 3H]glutamate at AA~ receptors. Besides, glycine has been reported to exert inhibitory effects on nociceptive cells, thus mimicking the actions of morphine (Curtis and Duggan, 1967; Dostrovsky and Pomeranz, 1976). Antagonistic and synergistic effects of morphine on the depressant action of glycine have been reported (Duggan et al., 1976) but an abundance of data suggests that glycine is an effective agent to increase acute morphine action and to attenuate the abstinence behaviour induced by morphine deprivation (Contrera and Tamayo, 1980). Little information is available about the CNS responses to systemic administration of glycine (Shank et al., 1973). However, it remains to be proved that a simple increase of CNS glycine concentrations enhances glycine-mediated inhibition, but certain metabolic disorders that arise from a defect in glycine cleavage are characterized clinically by weakness and lethargy (Perry et al., 1975; Von Wendt et al., 1979). On the other hand, very high doses of glycine (up to 30 g daily with food, i.e. 430 m g / k g ) have been used in hyperprolinemia and in isovaleric acidemia (Cohn et al., 1978). Moreover, an oral dose of 10 g, i.e. 140 m g / k g , has been given without side-effects in the 'glycine-tolerance' in malabsorption diseases (Nalin, 1970). The present study was carried our in order to assess the antinoception caused by the oral administration of high doses of glycine. The influence of glycine on morphine-induced antinociception was also tested.
The animals were assigned at random to groups and to cages. The experiments were performed blind in that during the test the operator was unaware of the drug or drugs each animal had received. All tests were performed at room temperature and took place between 10 a.m. and 3 p.m. Each animal was tested at one specific time after drug administration and was not re-used thereafter. Three independent experiments were performed to study visceral chemical antinociception (VCA), thermal antinociception (TA) and locomotor activity (LA) caused by the administration of morphine, glycine and morphine plus glycine. Each of the three experiments involved 20 groups of mice (total n = 200/experiment): five groups received morphine hydrochloride (5 m g / k g s.c.) five were treated with glycine (200 m g / k g p.o.) only and five more groups received morphine hydrochloride and glycine together. The remaining five groups received saline both orally and subcutaneously (s.c.) and were used as controls. In VCA and LA tests the responses of the groups which received the tested compounds were expressed as percentages of the response of the control group. 2.2. VCA
2. Materials and methods
VCA was evaluated by means of the acetylcholine (ACh)-induced writhing test (Collier et al., 1968). Glycine a n d / o r morphine were administered to each group of animals at least 15 min before the i.p. injection of ACh bromide (3.2 mg/kg). The number of abdominal constrictions was counted for 2 min, at 15, 30, 45, 60 and 90 min after the administration of the compounds under test.
2.1. A nimals
2.3. TA
Groups (n = 10) of female mice (Swiss strain, 20-25 g), supplied by Nossan, Italy, were housed for at least 10 days in groups of 10 (Makrolon ~Tecniplast cages) on a 12-h light-dark cycle (8 a.m.-8 p.m. light) in a temperature-controlled environment (21 + I ° C ) and allowed free access to food (GLP-Nossan pellets) and water until 1 h before the start of the experiment.
TA was determined by means of the hot-plate test (Eddy and Leinbach, 1953). Mice were placed individually on the plate maintained at 55 +_ 0.5 ° C by feedback from a surface-mounted thermocoupie. The response latency was evaluated on the basis of either hind paw lick or j u m p reaction following the contact with the plate. After three readings at 30 min intervals, response changes at
303 15, 30, 45, 60 and 90 min were assessed after administration of the compounds. The results, summarized in fig. 2, were expressed in term of average temporal differences (s) relative to the baseline. Each of the above tests employed a 'cuto f f time of 45 s at which time the animal was removed from the testing procedure to limit tissue d a m a g e and stress.
2.4. LA LA was measured with a photocell apparatus (Sodeco-Sprint, Milan, Italy). The animals were kept in individual activity cages (1 = 26, w = 26, h = 20 cm). Each cage had 10 photocells, 1 cm above the floor level, connected to an electromechanical counter. LA was measured as the n u m b e r of beam interruptions in the time periods of 0-15, 15-30, 30-45, 45-60 and 60-90 min after the administration of morphine a n d / o r glycine.
2.5. Statistical analysis The response data for the groups that received morphine and morphine plus glycine were analysed by two-way analysis of variance ( A N O V A ) . This statistical method was employed to consider treatment effect by performing single mean contrasts with the N e w m a n - K e u l s test, and the block effect due to the order of entrance of the animals into the experiment (10 blocks). However, statistical analysis showed that the latter effect was not significant. When tested for VCA, T A and LA, the control groups showed no significant changes in these parameters and are therefore not depicted in figs. 1, 2 and 3 respectively.
3. Results
3.1. VCA (fig. 1) Glycine given orally up to 200 m g / k g , caused no statistically significant effects on the abdominal writhes (not shown). The m a x i m u m inhibition ( > 80%) of abdominal constrictions with morphine, 5 m g / k g s.c., was obtained 15 min after drug treatment, while the m a x i m u m inhibition ( > 85%) was obtained after 90 min in presence of glycine (200 m g / k g p.o.). The s.c. and p.o. administration of the same volume of saline to the control groups produced no significant changes of the nociceptive response. The same happened with the s.c. administration of m o r p h i n e in the presence of p.o. administered saline.
3.2. TA (fig. 2) The administration of glycine also did not produce an increase in antinociceptive response in TA, evaluated by the hot-plate test. As in VCA, the development and the intensity of the TA effect showed a different pattern when morphine was given alone and when it was given simultaneously with glycine. Morphine injected alone produced a non-significant degree of antinociception within 10 to 30 min after its administration, in contrast to morphine and glycine given together. The combination of morphine and glycine produced a sig-
6~
4~
IO
2.6. Drug administration Morphine hydrochloride and glycine, dissolved in distilled water to avoid any interference of co-administered sodium ions, were administered s.c. and p.o., respectively, in a volume of 10 m l / k g . The animals in the control groups received the same volume of saline.
Fig. 1. Visceral chemical antinociception (writhing test) caused by the administration of morphine hydrochloride (11 5 mg/kg s.c.) alone or associated with glycine ([] 200 mg/kg p.o.). The results are expressed as inhibition (%) of abdominal constrictions compared with those in the control groups. Each column represents the mean score + S.E. of a group of 10 mice. Asterisks indicate that at the time of the test there was a significant difference between the groups of treated-animals ( * P < 0.01).
304
0
.
Fig. 2. Thermal antinociception (hot-plate test) caused by the administration of morphine hydrochloride ( I 5 mg/kg s.c.) alone or associated with glycine (~ 200 mg/kg p.o.). Each column represents the mean score+ S.E. of 10 mice. Asterisks indicate that at the time of the test there was a significant difference between the two groups of animals (* P < 0.01). The initial reaction time was subtracted in all tests.
nificantly higher degree of a n t i n o c i c e p t i o n (P < 0.01) within 45, 60 and 90 min after its administration than did m o r p h i n e given alone. The s.c. a n d p.o. a d m i n i s t r a t i o n of the same v o l u m e of saline to the control groups p r o d u c e d no signific a n t changes of the nociceptive response• T h e s a m e h a p p e n e d with the s.c. a d m i n i s t r a t i o n of m o r p h i n e in the presence of p.o. a d m i n i s t e r e d saline. 3.3. LA T h e b e h a v i o u r p a t t e r n of the mice treated with glycine alone was not different from that of the controls. A n i m a l s treated with m o r p h i n e alone showed the typical sedated pattern, which is presented q u a n t i t a t i v e l y in fig. 3. W h e n m o r p h i n e
Fig. 3. Modifications in locomotor activity induced by morphine hydrochloride (ll 5 mg/kg) alone or associated with glycine ([] 200 mg/kg). The results are expressed as variations (%) from control groups. Each column represents the mean score+S.E, of l0 mice. Asterisks indicate that at the time of the test there was a significant difference between the two groups of treated animals ( * P < 0.01 ).
a n d glycine were a d m i n i s t e r e d together a significant increase (P < 0.01) in m o t o r activity was observed after 0-15 a n d 15-30 min in the a n i m a l s that received a c o m b i n a t i o n of the two c o m p o u n d s in c o m p a r i s o n to those that received only morphine. The s.c. and p.o. a d m i n i s t r a t i o n of the same volume of saline to the c o n t r o l g r o u p s d i d not p r o d u c e any significant changes of the nociceptive response. T h e same h a p p e n e d with the s.c. a d m i n i s t r a t i o n of m o r p h i n e in the presence of p.o. a d m i n i s t e r e d saline.
4. D i s c u s s i o n
A c c o r d i n g to Yaksh (1984) a n d Y a k s h a n d N o u e i h e d (1985), different s u b t y p e s of o p i a t e receptors, /t, ~ a n d ~, K are involved in the T A responses e v a l u a t e d in the hot plate test and in the V C A responses evaluated in the writhing test respectively. In mice, glycine (200 m g / k g p.o.) affected the time course of V C A (fig. 1) a n d T A (fig. 2) a n t i n o c i c e p t i o n i n d u c e d by m o r p h i n e (5 m g / k g s.c.) administration. The morphine-induced changes in L A were also influenced by glycine (fig. 3). In the T A test (fig. 2) glycine r e d u c e d the a n t i n o c i c e p t i v e action of m o r p h i n e after 15 a n d 30 min when they were a d m i n i s t e r e d together but the effect was not significant, while glycine signific a n t l y (P < 0.01) r e d u c e d the activity of m o r p h i n e in the V C A (fig. 1) a n d L A (fig. 3) tests, within the same time period. A f t e r 45, 60 a n d 90 min following the a d m i n i s t r a t i o n of m o r p h i n e , glycine significantly (P < 0.01) increased the effect of m o r p h i n e in the T A and V C A tests. But the LA was significantly r e d u c e d (P < 0.01) in mice that received the two c o m p o u n d s . The results o b t a i n e d indicate that glycine produces an a n t a g o n i s t i c a n d synergistic effect on some p h a r m a c o l o g i c a l actions of m o r p h i n e . In particular, when m o r p h i n e and glycine were given together, an a n t a g o n i s t i c effect of glycine was observed d u r i n g the first 30 min. T h e synergistic effect b e c a m e evident later. The first e x c i t a t o r y response to glycine m a y have been d u e to its interaction with the strychnine-insensitive b i n d i n g site, an effect which is m o d u l a t e d b y n a n o m o l a r c o n c e n t r a t i o n s of glycine.
305
The late inhibitory response of glycine may have been due to its interaction with the strychnine-sensitive receptor, which requires micromolar concentrations of glycine. The dual influence of glycine on morphine-induced antinociception, already stressed by Duggan et al. (1976), may thus be related to the kinetics and binding characteristics of glycine.
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