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Cross-tolerance between D-amino acids and morphine in mice
Brain Research, 212 (198 l) 227-229 © Elsevier/North-Holland Biomedical Press
227
Cross-tolerance between D-amino acids and morphine in mice
UMBERTO FILIBECK, CLAUDIO CASTELLANO and ALBERTO OLIVERIO* Istituto di Fisiologia Generale, Universitd di Roma and Istituto di Psicobiologia e Psieofarmacologia, C N R via Reno, 1 - 00198 Roma (Italy)
(Accepted December 18th, 1980) Key words: amino acid --- analgesia - - locomotor activity --- morphine - - cross-tolerance
The effects of morphine administration on analgesia and running activity were studied in DBA/2 (DBA) and C57BL/6 (C57) mice respectively, injected with D-amino acids (D-AA) for 5 days. The results indicate a clear cross-tolerance between D-AA and morphine in DBA mice when analgesia is considered, while the stimulating effect of the opiate is not modified after D-AA pretreatment in the C57 strain.
D-amino acids (D-AAs) such as phenylalanine and leucine can cause analgesia in m a n and micO, a. Their mechanism of action seem to involve the endorphin system in that their analgesia is naloxone-reversible, and higher analgesic effects are obtained in strains of mice presenting high endorphin levels. Opiates exert stimulating effects on the l o c o m o t o r behavior of C57BL/6 (C57) mice and a lower analgesia in relation to D B A / 2 (DBA) mice irt which no running fit is evident 4. In C57 mice: (1) D-AAs do not produce an increase o f l o c o m o t o r behavior similar to the running fit induced by morphine; (2) when the analgesic effect is considered, the different sensitivity o f D B A and C57 mice was quantitatively similar to that produced by morphine in these strains; and (3) there is a synergic analgesic effect of D-AAs and morphine while no synergism is evident when the l o c o m o t o r activity is considered 1. In the present research we wanted to assess, in these two strains o f mice which represent a useful model for investigating the stimulating and analgesic effects o f opiates 4, if: (1) there is cross-tolerance between D-AAs and morphine; and (2) if cross-tolerance possibly involves both analgesia and running-fit. To study the l o c o m o t o r effects we used the toggle-floor m e t h o d previously described 4. Fifteen male mice per group (60-90 days old) were used. Each session was 30 min long. To study the antinociceptive response we used the hot-plate method to obtain a measure o f pain threshold 4. This was done by placing a mouse on a hot-plate, 55 °C ( ± 0.5 °C) and measuring the time which elapsed until the onset o f hind-pawlicking: a mouse w as removed as soon as it reacted or it failed to react after 30 sec. * To whom correspondence should be addressed: Istituto di Psicobiologia e Psicofarmacologia via Reno, 1 - 00198 Roma, Italy.
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Fig. 1. Recovery from morphine-induced or D-AA induced analgesia in DBA mice pretreated with D-AAs (morphine after D-AA) or with o-AAs (D-AA after D-AA) for 5 consecutive days. The mice were tested at 30, 60, 120 and 180 min after the injections. Trend analysis showed that the values of the group treated with morphine were significantly different from those of morphine after D-AAs (F(1.28) = 35.12, P < 0.001). Similarly the values of the group injected with D-AA only were different from those of the group pretreated with D-AA for 5 days (F (1,28) -- 58.6; P < 0.001).
In order to assess the possible cross-tolerance between o-AAs and morphine, after preliminary experiments, the following schedule was adopted: two daily injections of D-AAs (D-phenylalanine, 125 mg/kg + D-leucine, 125 mg/kg) were given for 5 consecutive days. The time of i.p. injections was 09.00 h and 19.00 h. Two different groups of mice were injected on day 5, with morphine (10 mg/kg) or D-AAs 6 h after the 09.00 h injection of D-AAs: these groups are called respectively "morphine after D-AA" and "D-AA after D-AA". Two additional groups of mice were not pretreated with o-AAs but injected with morphine 10 mg/kg or D-AAs respectively. The effects of these treatments on analgesia were assessed by testing the animals on the hot-plate test 30, 60, 120 and 180 min after the injections of morphine or D-AAs respectively. DBA mice were used in the analgesia experiments while C57 mice were tested for morphine-induced running-fit. In fact, as previously noted, the first strain represents a good model for the analgesic effect of opiates while the C57 strain may be considered as a model for the stimulating effects of morphine "%4,5. Fig. 1 shows, as previously reported, that in DBA mice both morphine and DAA induce analgesia, with a peak effect at 30 or 120 rain after the injections respectively. In the group pretreated with o-AAs for 5 days (D-AA after D-AA group) tolerance developed to the analgesic effect of D-AAs. Finally in mice pretreated with D-AAs a cross-tolerance was evident between o-AAs and morphine. Table I shows that in C57 mice morphine but not D-AAs induce a running fit. Though cross-tolerance was evident for analgesia in DBA ]nice, a pretreatment with D-AAs--which per se do not modify the activity of C57 mice - - did not alter the effects of morphine on the locomotor activity.
229 TABLE I Effects of morphine, D-AAs and morphine after D-AA on the running activity of C57 mice tested in a toggle-floor box
Morphine induced an increase in locomotor activity in relation with controls (F (1,56) = 468.8; P < 0.00l) while D-AAs were ineffective (F(1,56) = 3.9; P > 0.05). The performance of mice pretreated with D-AAs and injected with morphine was not different from that of mice injected with morphine alone (F(1.56) = 2.3; P > 0.05). Treatment
Mean number of crossh~gs
Saline Morphine 10 mg/kg D-AAs 125 mg/kg Morphine 10 mg/kg after D-AAs 125 mg/kg
61.06 :k 2.87 212.13 ± 7.14 74,86 ~k 2.3 201.33 :k 5.70
I n general, the present findings indicate that a clear cross-tolerance between DA A s a n d m o r p h i n e is evident i n mice when analgesia is considered. O n the contrary, the stimulating effects of opiates are n o t modified after a similar p r e t r e a t m e n t with DA A s . These data further support previous findings 1 indicating that distinct mecha-
nisms a n d receptors are implicated i n the stimulating a n d analgesic effects of morphine, This fact may be ascribed to a n effect of o - A A s o n the e n d o r p h i n mechanisms involved i n analgesia b u t n o t o n those related to central stimulation. The existence of cross tolerance between n o n - a d d i c t i n g D-AAs a n d m o r p h i n e may have interesting clinical implications. This research was supported by a grant of the Italian Ministry of Health.
1 Alleva, E., Castellano, C. and Oliverio, A., Effects of L- and D-amino acids on analgesia and locomotor activity of mice: their interaction with morphine, Brain Research, 198 (1980) 249-252. 2 Bigler, E. D. and Eidelberg, E., Nigrostriatal effects of morphine in two mouse strains, Life Sci., 19 (1976) 1399-1406. 3 Cheng, R. S. S. and Pomeranz, B., Correlation of genetic differences in endorphin systems and analgesic effects of D-amino acids in mice, Brabz Research, 177 (1979) 583-587. 4 Oliverio, A. and Castellano, C., Genotype-dependent sensitivity and tolerance to morphine and heroin: dissociation between opiate induced running and analgesia in the mouse, Psychopharmacologia (Bed.), 39 (1974) 13-22. 5 Oliverio, A., Castellano, C., Racagni, F., Spano, P. F., Trabucchi, M. and Cattabeni, F., Genetic aspects in narcotic action. In M. L. Adler, L. Manara and R. Samanin (Eds.), Factors. Affecting the/tction of Narcotics, Raven Press, New York, 1978, pp. 7-18.
227
Cross-tolerance between D-amino acids and morphine in mice
UMBERTO FILIBECK, CLAUDIO CASTELLANO and ALBERTO OLIVERIO* Istituto di Fisiologia Generale, Universitd di Roma and Istituto di Psicobiologia e Psieofarmacologia, C N R via Reno, 1 - 00198 Roma (Italy)
(Accepted December 18th, 1980) Key words: amino acid --- analgesia - - locomotor activity --- morphine - - cross-tolerance
The effects of morphine administration on analgesia and running activity were studied in DBA/2 (DBA) and C57BL/6 (C57) mice respectively, injected with D-amino acids (D-AA) for 5 days. The results indicate a clear cross-tolerance between D-AA and morphine in DBA mice when analgesia is considered, while the stimulating effect of the opiate is not modified after D-AA pretreatment in the C57 strain.
D-amino acids (D-AAs) such as phenylalanine and leucine can cause analgesia in m a n and micO, a. Their mechanism of action seem to involve the endorphin system in that their analgesia is naloxone-reversible, and higher analgesic effects are obtained in strains of mice presenting high endorphin levels. Opiates exert stimulating effects on the l o c o m o t o r behavior of C57BL/6 (C57) mice and a lower analgesia in relation to D B A / 2 (DBA) mice irt which no running fit is evident 4. In C57 mice: (1) D-AAs do not produce an increase o f l o c o m o t o r behavior similar to the running fit induced by morphine; (2) when the analgesic effect is considered, the different sensitivity o f D B A and C57 mice was quantitatively similar to that produced by morphine in these strains; and (3) there is a synergic analgesic effect of D-AAs and morphine while no synergism is evident when the l o c o m o t o r activity is considered 1. In the present research we wanted to assess, in these two strains o f mice which represent a useful model for investigating the stimulating and analgesic effects o f opiates 4, if: (1) there is cross-tolerance between D-AAs and morphine; and (2) if cross-tolerance possibly involves both analgesia and running-fit. To study the l o c o m o t o r effects we used the toggle-floor m e t h o d previously described 4. Fifteen male mice per group (60-90 days old) were used. Each session was 30 min long. To study the antinociceptive response we used the hot-plate method to obtain a measure o f pain threshold 4. This was done by placing a mouse on a hot-plate, 55 °C ( ± 0.5 °C) and measuring the time which elapsed until the onset o f hind-pawlicking: a mouse w as removed as soon as it reacted or it failed to react after 30 sec. * To whom correspondence should be addressed: Istituto di Psicobiologia e Psicofarmacologia via Reno, 1 - 00198 Roma, Italy.
228 30
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20
Z >. 0 Z t--
10-
_1
~o
8b
~o "rIME
~13o
IN M I N
Fig. 1. Recovery from morphine-induced or D-AA induced analgesia in DBA mice pretreated with D-AAs (morphine after D-AA) or with o-AAs (D-AA after D-AA) for 5 consecutive days. The mice were tested at 30, 60, 120 and 180 min after the injections. Trend analysis showed that the values of the group treated with morphine were significantly different from those of morphine after D-AAs (F(1.28) = 35.12, P < 0.001). Similarly the values of the group injected with D-AA only were different from those of the group pretreated with D-AA for 5 days (F (1,28) -- 58.6; P < 0.001).
In order to assess the possible cross-tolerance between o-AAs and morphine, after preliminary experiments, the following schedule was adopted: two daily injections of D-AAs (D-phenylalanine, 125 mg/kg + D-leucine, 125 mg/kg) were given for 5 consecutive days. The time of i.p. injections was 09.00 h and 19.00 h. Two different groups of mice were injected on day 5, with morphine (10 mg/kg) or D-AAs 6 h after the 09.00 h injection of D-AAs: these groups are called respectively "morphine after D-AA" and "D-AA after D-AA". Two additional groups of mice were not pretreated with o-AAs but injected with morphine 10 mg/kg or D-AAs respectively. The effects of these treatments on analgesia were assessed by testing the animals on the hot-plate test 30, 60, 120 and 180 min after the injections of morphine or D-AAs respectively. DBA mice were used in the analgesia experiments while C57 mice were tested for morphine-induced running-fit. In fact, as previously noted, the first strain represents a good model for the analgesic effect of opiates while the C57 strain may be considered as a model for the stimulating effects of morphine "%4,5. Fig. 1 shows, as previously reported, that in DBA mice both morphine and DAA induce analgesia, with a peak effect at 30 or 120 rain after the injections respectively. In the group pretreated with o-AAs for 5 days (D-AA after D-AA group) tolerance developed to the analgesic effect of D-AAs. Finally in mice pretreated with D-AAs a cross-tolerance was evident between o-AAs and morphine. Table I shows that in C57 mice morphine but not D-AAs induce a running fit. Though cross-tolerance was evident for analgesia in DBA ]nice, a pretreatment with D-AAs--which per se do not modify the activity of C57 mice - - did not alter the effects of morphine on the locomotor activity.
229 TABLE I Effects of morphine, D-AAs and morphine after D-AA on the running activity of C57 mice tested in a toggle-floor box
Morphine induced an increase in locomotor activity in relation with controls (F (1,56) = 468.8; P < 0.00l) while D-AAs were ineffective (F(1,56) = 3.9; P > 0.05). The performance of mice pretreated with D-AAs and injected with morphine was not different from that of mice injected with morphine alone (F(1.56) = 2.3; P > 0.05). Treatment
Mean number of crossh~gs
Saline Morphine 10 mg/kg D-AAs 125 mg/kg Morphine 10 mg/kg after D-AAs 125 mg/kg
61.06 :k 2.87 212.13 ± 7.14 74,86 ~k 2.3 201.33 :k 5.70
I n general, the present findings indicate that a clear cross-tolerance between DA A s a n d m o r p h i n e is evident i n mice when analgesia is considered. O n the contrary, the stimulating effects of opiates are n o t modified after a similar p r e t r e a t m e n t with DA A s . These data further support previous findings 1 indicating that distinct mecha-
nisms a n d receptors are implicated i n the stimulating a n d analgesic effects of morphine, This fact may be ascribed to a n effect of o - A A s o n the e n d o r p h i n mechanisms involved i n analgesia b u t n o t o n those related to central stimulation. The existence of cross tolerance between n o n - a d d i c t i n g D-AAs a n d m o r p h i n e may have interesting clinical implications. This research was supported by a grant of the Italian Ministry of Health.
1 Alleva, E., Castellano, C. and Oliverio, A., Effects of L- and D-amino acids on analgesia and locomotor activity of mice: their interaction with morphine, Brain Research, 198 (1980) 249-252. 2 Bigler, E. D. and Eidelberg, E., Nigrostriatal effects of morphine in two mouse strains, Life Sci., 19 (1976) 1399-1406. 3 Cheng, R. S. S. and Pomeranz, B., Correlation of genetic differences in endorphin systems and analgesic effects of D-amino acids in mice, Brabz Research, 177 (1979) 583-587. 4 Oliverio, A. and Castellano, C., Genotype-dependent sensitivity and tolerance to morphine and heroin: dissociation between opiate induced running and analgesia in the mouse, Psychopharmacologia (Bed.), 39 (1974) 13-22. 5 Oliverio, A., Castellano, C., Racagni, F., Spano, P. F., Trabucchi, M. and Cattabeni, F., Genetic aspects in narcotic action. In M. L. Adler, L. Manara and R. Samanin (Eds.), Factors. Affecting the/tction of Narcotics, Raven Press, New York, 1978, pp. 7-18.