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Effects of yohimbine on morphine analgesia and physical dependence in the rat
European Journal of Pharmacology, 211 (1092) 35-38 © 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
35
EJP 52253
Effects of yohimbine on morphine analgesia and physical dependence in the rat V i c t o r i a Iglesias, Luis F. Alguacil, Cecilio A l a m o a n d E d u a r d o C u e n c a Department of l'hysiology and Pharmacology, Universityof'Alcald de lh'nare~, Madrid, Spare Received 24 June 1991, revised MS received 27 September 1991, accepted 5 November 1901
The effects of yohimbine on morphine analgesia and on the development of opiatc physical dependence were studied to find out more about the involvement of o~2-adrenergic mechanisms in opioid actions. Male Sprague-Dawley rats (250-300 g) were used. The acute effect of morphine (5 mg/kg i.p.) in the tail-flick test was reduced significantly by pretreatment with a single dose of yohimbine (2 mg/kg i.p.). Alone yohimbine, produced a slight hyperalgesia. Animals treated with a sustained-release preparation of morphine (300 mg/kg s.c.) showed the same sensitivity to opiate analgesia 72 h later whether they were treated concomitantly with yohimbine or not, but they exhibited fewer withdrawal symptoms upon naloxone injection after yohimbine (2 or 4 mg/kg i.p. 24, 28, 48 and 52 h after the start of systemic morphine treatment). The results obtained confirm previous data on the effects of yohimbine on morphine analgesia and reveal the importance of a2-adrenergic activation in the development of opioid physical dependence. Morphine analgesia; Morphine dependence; Yohimbine
1. Introduction There are numerous studies demonstrating the involvement of noradrenergic function in the pharmacological actions of opioids. Much work has focused on the role of a2-adrenergic mechanisms in the acute effect, tolerance and dependence elicited by opioids. There are close similarities between a 2- and opioid receptors with respect to their distribution in the central nervous system (Pazos, 1988) or the transduction systems coupled to the agonist-reccptor complexes (North et al., 1987). Some of the acute effects of opioids are blocked by az-adrenoceptor antagonists. Concerning opioid analgesia, it has been shown that the antinociceptivc actions of morphine are antagonized by pretreatment with the a2-blocker, yohimbine (Browning et al., 1982). On the other hand, a positive interaction between opioids and a2-agonists has been described and it appears to be either additive or synergistic depending on the route of administration and the nociceptive test used (Wilcox et at., 1987; Drasner and Fields, 1988; Monasky et al., 1990; Ossipov et al., 1990). o~2-Adrenoceptor density and function appear to be significantly influenced by repeated opioid administration (Smith et al., 1989). When subacute or chronic
Correspondence to: V. Iglesias, Department of Fisiologia y Farmacologia, University of Alcalfi, 28871 Alcalfi de Henares (Madrid), Spain. Tel. 34.1.889 0400. ext. 2416.
opioids are assayed in experimental pain models, cross-tolerance develops with the analgesic effects of a2-agonists (Solomon and Gebhart, 1988; Stevens et al., 1988). Moreover, withdrawal from repeated opioid administration also affects binding to a2-adrenoceptors (Garcia-Sevilla et al., 1988; Smith et al., 1989), and the action of a2-agonists in blocking signs of opiate withdrawal is well-documented (Tseng et al., 1975; Gold et al., 1978). It can be concluded from these findings that the effects of chronic opioids arc closely related with modifications of a2-adrenergic mechanisms. Nevertheless, the effect of a2-adrenoceptor blockers on opioid tolerance and dependence has been poorly studied. In vitro experiments performed in our laboratory showed an attenuation of morphine tolerance development by yohimbine (Alguacil et al., 1987). This finding encouraged us to study the possible effect of yohimbine on morphine analgesia, tolerance and dependencc in vivo, since the results could broaden the knowledge of the interactions between opioids and tx2-adrcnergic function.
2. Materials and methods
2.1. Effect of yohimbine on the analgesic effect of morphine after acute opioid exposure S p r a g u c - D a w l e y male rats (250-300 g) were used. The animals were restrained in plastic holders and their basal nociccptive thresholds were established with
36 the 'tail-flick' method (D'Amour and Smith, 1971) using a heat-radiating lamp. Animals showing latencies greater than 10 s were not used for this study. After baseline thresholds were determined, the rats were assigned at random to an experimental group and they were pretreated i.p. either with saline or yohimbine (2 mg/kg). Morphine (5 m g / k g i.p.) or saline was injected 5 rain later and tail-flick [atencies were redetermined 30, 60 and 120 min after this treatment. Drugs and saline were injected in a volume of 10 ml/kg.
15.0 13.5 12.0 10.5
+I
9.0 7.5
I
6.0 4.5
1
3.0 1.5
2.2. Effect of yohimbine on the analgesic effect of morphine after prolonged opioid exposure Basal nociceptive thresholds were established as explained above. The animals were given s.c. a sustained-release suspension containing 300 m g / k g of morphine (Alguacil et al., 1989) or the corresponding vehicle. Saline or yohimbine (2 or 4 mg/kg) was injected i.p. 24, 28, 48 and 52 h after morphine suspension administratkm. Latencies in the tail-flick test were redctermined 72 h after morphine treatment, and the analgesic effect of an acute dose of morphine (5 m g / k g i.p.) was tested at this time as previously described.
2.3. Lffect of yohimbine on morphine withdrawal After tail-flick testing, the chronically treated animals received I m g / k g of naloxone and they were placed in plastic chambers (Bliisig et al., 1973) for study of opioid withdrawal signs during two consecutive periods of 15 min. The number of escape attempts and wet-dog shakes werc recorded. The presence of other withdrawal signs was scored with one point and the sum of these points was defined as a global withdrawal index. The signs included in this parameter were diarrhea, chromodacryorrhea, syalorrhea, rhinorrhea, ptosis, ejaculation, teeth-chattering, vocalization on touch, irritability, paw tremor and tachypnea (maximum score = Ill.
i 0
i 30
i 60
i 120
Fig. 1. Latencies in the tail-flick test in animals pretreated i.p. with saline or yohimbine (2 m g / k g ) and treated 5 rain later i.p. with saline or morphine (5 m g / k g ) . (©) Saline+saline, n = 10; (11) saline+ morphine, n = lO; ( • ) yohimbine + saline, n = 9 and (el yohimbine + morphine, n = 10. * P < 0.05 vs. saline + saline. Ordinate: latency (s); abscissa: time (mink
bine (2 mg/kg). By itself yohimbine produced hyperalgcsia at the dose tested. The baseline threshold in the morphine suspension groups was still significantly elevated compared to that in the control group after 3 days. Repeated treatment with yohimbine failed to affect tail-flick latencies in rats exposed to the opioid for 72 h (fig. 2). Rats treated with the morphine suspension displayed marked withdrawal signs upon naloxone injection. This behavior was reduced by repeated administration of yohimbine in a dose-dependent fashion and
LATENCY [sec) 15.o
13.5
t
12.0 1 10.5 1
7.5 6.0 9.0 t
2.4. Statistics Statistical comparison between different treatments and dosages was performed by analysis of variance (ANOVA) procedures followed by a multiple range test (least significant differences). The results were considered significant when the probability level was less than 0.05.
3. Results
As shown in fig. 1, the acute effect of morphine (5 mg/kg) in the tail-flick test was antagonized by yohim-
4.5
o
3'o
5o
11o
TIME [min) Fig. 2. Effect of an acute dose of morphine (5 m g / k g ) in the tail-flick test with animals pretreated 72 h before with a suspension of morphine (300 m g / k g ) s.c.) or the corresponding vehicle. The animals received yohimbine (2 or 4 m g / k g i.p.) or saline 24, 28, 48 and 52 h after the administration of the morphine suspension or vehicle. (©) Vehicle+saline, n = 13; (el morphine + saline, n ~ 12; ( I ) morphine+yohimbine 2 mg/kg, n = 10; ( D ) morphine+yohimbine 4 mg/kg, n = 10. Asterisks indicate significant differences between the morphine suspension groups and the control (vehicle+saline: P < 0.05).
37 SCORE 25.0
•
22-5 20.0 17-5 15.o 12.5 10.0
*
*
*
•
7.5 5.0 "
+
2-5-
o
6 ESCAPE ATTEMPTS WET-DOG SNAKES GLOBAL INDEX WITHDRAWAL SIGNS
Fig. 3. Withdrawal signs elicited by naloxone administration (l m g / k g i.p.) to the animals previously used in the tail-flick test. ([]) Vehicle +saline, n = 1 3 : ( m ) m o r p h i n e + s a l i n e , n = 1 2 ; ( D ) m o r p h i n e + yohimbine 2 m g / k g , n = 10; (El) m o r p h i n e + y o h i m b i n e 4 m g / k g , n = 10. * P < 0.05 vs. vehicle + saline; ÷ P < 0.05 vs. morphine + saline.
it was abolished by the higher dosage of the az-adrenoceptor antagonist (fig. 3).
4. Discussion
Our results are very similar to those obtained by Browning et ai. (1982) showing that systemic yohimbine significantly blocks the analgesic effect of systemic morphine in the tail-flick test. Previous studies performed with this test have shown that the intrathecai (i.t.) administration of yohimbine reduces the analgesic effect of morphine when the opiate is injected in supraspinal areas of the central nervous system (Camarata and Yaksh, 1985); on the contrary, i.t. yohimbine does not modify the antinociceptive action of i.t. morphine (Solomon and Gebhart, 1988). These findings suggest that yohimbine decreases opiate analgesia by blocking at the spinal level the function of descending inhibitory pathways which are activated by the opiate at supraspinal sites (Wigdor and Wilcox, 1987). In addition to confirming the results of Browning et al. we also obtained a hyperalgesic effect of yohimbine, which suggests the existence of tonic inhibitory activity of descending noradrenergic pathways under basal conditions. It is consistent with this that Sagen and Proudfit (1984) reported that nociceptive thresholds can be reduced by i.t. administration of a2-adrenoceptor antagonists. Prolonged exposure to opiates has been shown to produce cross-tolerance with ae-agonists in several tests (Yamazaki and Kaneto, 1985; Stevens et al., 1988; Solomon and Gebhart, 1988). These findings are in agreement with the reduction of a2-adrenoceptor density in several areas of the central nervous system found after chronic administration of morphine (Smith et al., 1989). Down-regulation of a2-adrenoceptors ap-
pears to be one of the adaptative mechanisms that respond to the complex effects of opiates on the function of noradrenergic pathways (Copeland and Pradhan, 1988). In this situation, it was interesting to study the effect of a2-adrenoceptor blockade on opiate tolerance and dependence, since this treatment could prevent some of the biochemical changes associated with adaptation to chronic morphine. Indeed, previous reports from our laboratory showed that yohimbine could effectively antagonize morphine tolerance in the guinea-pig ileum (Alguacil et al., 1987). Also, the group of Kaneto found that yohimbine delays the development of morphine tolerance in the tail-pinch test (Kihara and Kaneto, 1986). In the present experiments, prolonged exposure to morphine did not give rise to a marked degree of tolerance, since the baseline threshold was higher than the value observed in the control group. The nociceptive action of a test dose of morphine was slightly reduced after the prolonged exposure to the opiate, but the magnitude of this reduction did not suggest the development of robust opioid tolerance. Consequently, the effect of yohimbine on morphine tolerance could not be clearly established in our experiments and other methodology is needed. However, it is interesting that the sensitivity to opioid analgesia after 72 h of morphine exposure did not seem to be influenced by yohimbine treatment. Despite the apparent absence of opioid tolerance, the systemic morphine treatment was able to produce a marked degree of physical dependence. Under these conditions, a dose-dependent inhibition of opioid withdrawal responses was obtained when yohimbine was repeatedly administered to the animals. This blockade affected withdrawal signs originating either in the spinal cord or at higher levels in the central nervous system (Buccafusco, 1990). Accordingly, the development of physical dependence to morphine seems to be closely related with a2-adrenoceptor alterations which can be reversed by blocking these sites. These and other results on the effects of a2-agonists on opioid withdrawal lead to the conclusion that both the establishment and the expression of opioid physical dependence are profoundly altered by a2-adrenoceptor ligands. An acute dose of the a2-agonist, clonidine, reduces the magnitude of morphine withdrawal (Tseng et al., 1975) while the simultaneous administration of clonidine and morphine fails to modify the development of dependence (Cervo et al., 1981). Conversely, an acute dose of yohimbine potentiates the effect of morphine withdrawal (Dwoskin et al., 1983) while repeated administration of the antagonist prevents the development of dependence. The mechanism of the antiwithdrawal actions of acute clonidine has been extensively studied both in experimental models and in man (Garcia-Sevilla et al., 1988). A similar study on the mechanisms underlying the action of yohimbine could
38 contribute to the understanding of the interactions of az-adrenoceptor function with the adaptative res p o n s e s e l i c i t e d by c h r o n i c o p i o i d s .
References Alguacil, L.F., C. Alamo, C. Santos and E. Cuenca, 1987, Yohimbine reduces morphine tolerance in guinca-pig ileum, Life Sci. 41), 155. Alguacil, L.F., C. Alamo, V. lglesias and E. Cuenca, 1989, An automated method for the evaluation of jumping activity in mice. Effects of clonidine on morphine withdrawal, Moth. Find. Exp. Clin. Pharmacol. I1,677. Bl~isig, J., A. Herz, K. Reinhold and S. Zieglg~ingsberger, 1973, Development of physical dependence on morphine in respect to time and dosage and quantification of the precipitated withdrawal syndrome in rats, Psychopharmacologia 33, 19. Browning, D.L., A. Livingston and B. Morris, 1982, Interactions of drugs active at opiate receptors and drugs active at ~re-receptors on various test systems, Br. J. Pharmacol. 77. 487. Buccafusco, J.J., 1990, Participation of different brain regions in the anti-narcotic withdrawal action of clonidine in the dependent rat, Brain Res. 513, 8. Camarata, P.J. and T.L. Yaksh, 1985, Characterization of the spinal adrenergic receptors mediating the spinal effects produced by the midroinjection of morphine into the pcriaqueductal gray, Brain Res. 336, 133. Cervo, L., C. Rochat, S. Romandini and R. Samanin. 1981, Evidence of a preferential role of brain serotonin in thc mechanisms leading to naloxone-precipitated compulsive jumping in morphine-dependent rats, Psychopharmacology 74, 271. Copeland, R.L. and S.N. Pradhan, 1988, Effect of morphine on central neurotransmitters in the rat, Biogenic Amines 5, 205. D'Amour, F.E. and D.L. Smith, 1941, A method for determining loss of pain sensation. J. Pharmacol. Exp. Thor. 72, 74. Drasner, K. and H.L. Fields. 1988, Synergy between the antinociceplive effects of intrathecal clonidine and systemic morphine in the rat, Pain 32, 309. Dwoskin, L.P., B.S. Neal and S.B. Sparber, 1983, Yohimbine exacerbates and clonidinc attenuates acute morphine withdrawal in rats, Eur. J. Pharmacol. 90, 269. Garcia-Sevilla, J.A., i. Ulibarri, L. Ugedo and M. Guti6rrez, 1988, Alpha-2-adrenoceptor modulation in opiate addiction, Rev. Farmacol. Clin. Exp. 5 (Suppl. 1), 31.
Gold, M.S., D.E. Redmond, Jr. and H.D. Klebcr. 1978, Clonidine blc~cks acute opiate-withdrawal symptoms, Lancet 2, 599. Kihara, "I. and H. Kaneto. 1986, Important role of adrenergic function in the development of analgesic tolerance to morphine in mice, Jap. J. Pharmacol. 42, 419. Monasky, M.S., A.R. Zinsmcister, C.W. Stevens and T.L. Yaksh, 1990, Interaction of intrathecal morphine and ST-91 on antinociception in the rat: dose-response analysis, antagonism and clearance, J. Pharmacol. Exp. Ther. 254, 383. North, R.A., J.T. Williams, A. Surprenant and M.J. Christie, 1987, Mu and delta receptors belong to a family of receptors that are coupled to potassium channels, Proc. Natl. Acad. Sci. U.S.A. 84, 5487. Ossipov, M.tI., S. Harris, P. Lloyd and E. Messinco, 1990, An isobolographic analysis of the antinociceptive effect of systemically and intrathecally administered combinations of clonidine and opiates. J. Pharmacol. Exp. Ther. 255. 1107. Pazos, A., 1988, Anatomical distribution of opioid receptors and adrenoceptors in the brain of mammals, Rev. Farmacol. Clin. Exp. 5 (Suppl. 1). 11. Sagen, J. and H.K. Proudfit, 1984, Effect of intrathecally administered noradrcnergic antagonists on nociception in the rat, Brain Res. 310, 295. Smith, C.B., H.C. Moises, R.N. Spengler and P.J. Hollingsworth, 1989, Changes in o:2-adrenoceptor numbcr and function in brains of morphine-dependent rats, Eur. J. Pharmacol. 161, 111. Solomon, R.E. and G.F. Gebhart, 1988, Intrathecal morphine and clonidine: antinociceptivc tolerance and cross-tolerance and effects on blood pressure, J. Pharmacol. Exp. Ther. 245, 444. Stevens, C.W., M.S. Monasky and T.L. Yaksh, 1988, Spinal infusion of opiate and a 2 agonists in rats: tolerance and cross-tolerance studies, J. Pharmacol. Exp. Ther. 244, 63. Tseng, L.F., H.H. Ia)h and E.T. Wei, 1975, Effects of clonidine on morphine withdrawal signs in the rat, Eur. J. Pharmacol. 30, 93. Wigdor, S. and G.L. Wilcox, 1987, Central and systemic morphineinduced antinociception in mice: contribution of descending serotonergic and noradrenergic pathways, J. Pharmacol. Exp. Ther. 242, 90. Wilcox, G.L., K.lt. Carlsson, A. Jochim and 1. Jurna, 1987, Mutual p~tentiation of antinociceptive effects of morphine and clonidine on motor and sensory responses in rat spinal cord, Brain Res. 405, 84. Yamazaki, A. and 1t. Kaneto, 1985, Single dose tolerance to the analgesic effect of clonidine and cross-tolerance between morphine and clonidine, Jap. J. Pharmacol. 39, 461.
35
EJP 52253
Effects of yohimbine on morphine analgesia and physical dependence in the rat V i c t o r i a Iglesias, Luis F. Alguacil, Cecilio A l a m o a n d E d u a r d o C u e n c a Department of l'hysiology and Pharmacology, Universityof'Alcald de lh'nare~, Madrid, Spare Received 24 June 1991, revised MS received 27 September 1991, accepted 5 November 1901
The effects of yohimbine on morphine analgesia and on the development of opiatc physical dependence were studied to find out more about the involvement of o~2-adrenergic mechanisms in opioid actions. Male Sprague-Dawley rats (250-300 g) were used. The acute effect of morphine (5 mg/kg i.p.) in the tail-flick test was reduced significantly by pretreatment with a single dose of yohimbine (2 mg/kg i.p.). Alone yohimbine, produced a slight hyperalgesia. Animals treated with a sustained-release preparation of morphine (300 mg/kg s.c.) showed the same sensitivity to opiate analgesia 72 h later whether they were treated concomitantly with yohimbine or not, but they exhibited fewer withdrawal symptoms upon naloxone injection after yohimbine (2 or 4 mg/kg i.p. 24, 28, 48 and 52 h after the start of systemic morphine treatment). The results obtained confirm previous data on the effects of yohimbine on morphine analgesia and reveal the importance of a2-adrenergic activation in the development of opioid physical dependence. Morphine analgesia; Morphine dependence; Yohimbine
1. Introduction There are numerous studies demonstrating the involvement of noradrenergic function in the pharmacological actions of opioids. Much work has focused on the role of a2-adrenergic mechanisms in the acute effect, tolerance and dependence elicited by opioids. There are close similarities between a 2- and opioid receptors with respect to their distribution in the central nervous system (Pazos, 1988) or the transduction systems coupled to the agonist-reccptor complexes (North et al., 1987). Some of the acute effects of opioids are blocked by az-adrenoceptor antagonists. Concerning opioid analgesia, it has been shown that the antinociceptivc actions of morphine are antagonized by pretreatment with the a2-blocker, yohimbine (Browning et al., 1982). On the other hand, a positive interaction between opioids and a2-agonists has been described and it appears to be either additive or synergistic depending on the route of administration and the nociceptive test used (Wilcox et at., 1987; Drasner and Fields, 1988; Monasky et al., 1990; Ossipov et al., 1990). o~2-Adrenoceptor density and function appear to be significantly influenced by repeated opioid administration (Smith et al., 1989). When subacute or chronic
Correspondence to: V. Iglesias, Department of Fisiologia y Farmacologia, University of Alcalfi, 28871 Alcalfi de Henares (Madrid), Spain. Tel. 34.1.889 0400. ext. 2416.
opioids are assayed in experimental pain models, cross-tolerance develops with the analgesic effects of a2-agonists (Solomon and Gebhart, 1988; Stevens et al., 1988). Moreover, withdrawal from repeated opioid administration also affects binding to a2-adrenoceptors (Garcia-Sevilla et al., 1988; Smith et al., 1989), and the action of a2-agonists in blocking signs of opiate withdrawal is well-documented (Tseng et al., 1975; Gold et al., 1978). It can be concluded from these findings that the effects of chronic opioids arc closely related with modifications of a2-adrenergic mechanisms. Nevertheless, the effect of a2-adrenoceptor blockers on opioid tolerance and dependence has been poorly studied. In vitro experiments performed in our laboratory showed an attenuation of morphine tolerance development by yohimbine (Alguacil et al., 1987). This finding encouraged us to study the possible effect of yohimbine on morphine analgesia, tolerance and dependencc in vivo, since the results could broaden the knowledge of the interactions between opioids and tx2-adrcnergic function.
2. Materials and methods
2.1. Effect of yohimbine on the analgesic effect of morphine after acute opioid exposure S p r a g u c - D a w l e y male rats (250-300 g) were used. The animals were restrained in plastic holders and their basal nociccptive thresholds were established with
36 the 'tail-flick' method (D'Amour and Smith, 1971) using a heat-radiating lamp. Animals showing latencies greater than 10 s were not used for this study. After baseline thresholds were determined, the rats were assigned at random to an experimental group and they were pretreated i.p. either with saline or yohimbine (2 mg/kg). Morphine (5 m g / k g i.p.) or saline was injected 5 rain later and tail-flick [atencies were redetermined 30, 60 and 120 min after this treatment. Drugs and saline were injected in a volume of 10 ml/kg.
15.0 13.5 12.0 10.5
+I
9.0 7.5
I
6.0 4.5
1
3.0 1.5
2.2. Effect of yohimbine on the analgesic effect of morphine after prolonged opioid exposure Basal nociceptive thresholds were established as explained above. The animals were given s.c. a sustained-release suspension containing 300 m g / k g of morphine (Alguacil et al., 1989) or the corresponding vehicle. Saline or yohimbine (2 or 4 mg/kg) was injected i.p. 24, 28, 48 and 52 h after morphine suspension administratkm. Latencies in the tail-flick test were redctermined 72 h after morphine treatment, and the analgesic effect of an acute dose of morphine (5 m g / k g i.p.) was tested at this time as previously described.
2.3. Lffect of yohimbine on morphine withdrawal After tail-flick testing, the chronically treated animals received I m g / k g of naloxone and they were placed in plastic chambers (Bliisig et al., 1973) for study of opioid withdrawal signs during two consecutive periods of 15 min. The number of escape attempts and wet-dog shakes werc recorded. The presence of other withdrawal signs was scored with one point and the sum of these points was defined as a global withdrawal index. The signs included in this parameter were diarrhea, chromodacryorrhea, syalorrhea, rhinorrhea, ptosis, ejaculation, teeth-chattering, vocalization on touch, irritability, paw tremor and tachypnea (maximum score = Ill.
i 0
i 30
i 60
i 120
Fig. 1. Latencies in the tail-flick test in animals pretreated i.p. with saline or yohimbine (2 m g / k g ) and treated 5 rain later i.p. with saline or morphine (5 m g / k g ) . (©) Saline+saline, n = 10; (11) saline+ morphine, n = lO; ( • ) yohimbine + saline, n = 9 and (el yohimbine + morphine, n = 10. * P < 0.05 vs. saline + saline. Ordinate: latency (s); abscissa: time (mink
bine (2 mg/kg). By itself yohimbine produced hyperalgcsia at the dose tested. The baseline threshold in the morphine suspension groups was still significantly elevated compared to that in the control group after 3 days. Repeated treatment with yohimbine failed to affect tail-flick latencies in rats exposed to the opioid for 72 h (fig. 2). Rats treated with the morphine suspension displayed marked withdrawal signs upon naloxone injection. This behavior was reduced by repeated administration of yohimbine in a dose-dependent fashion and
LATENCY [sec) 15.o
13.5
t
12.0 1 10.5 1
7.5 6.0 9.0 t
2.4. Statistics Statistical comparison between different treatments and dosages was performed by analysis of variance (ANOVA) procedures followed by a multiple range test (least significant differences). The results were considered significant when the probability level was less than 0.05.
3. Results
As shown in fig. 1, the acute effect of morphine (5 mg/kg) in the tail-flick test was antagonized by yohim-
4.5
o
3'o
5o
11o
TIME [min) Fig. 2. Effect of an acute dose of morphine (5 m g / k g ) in the tail-flick test with animals pretreated 72 h before with a suspension of morphine (300 m g / k g ) s.c.) or the corresponding vehicle. The animals received yohimbine (2 or 4 m g / k g i.p.) or saline 24, 28, 48 and 52 h after the administration of the morphine suspension or vehicle. (©) Vehicle+saline, n = 13; (el morphine + saline, n ~ 12; ( I ) morphine+yohimbine 2 mg/kg, n = 10; ( D ) morphine+yohimbine 4 mg/kg, n = 10. Asterisks indicate significant differences between the morphine suspension groups and the control (vehicle+saline: P < 0.05).
37 SCORE 25.0
•
22-5 20.0 17-5 15.o 12.5 10.0
*
*
*
•
7.5 5.0 "
+
2-5-
o
6 ESCAPE ATTEMPTS WET-DOG SNAKES GLOBAL INDEX WITHDRAWAL SIGNS
Fig. 3. Withdrawal signs elicited by naloxone administration (l m g / k g i.p.) to the animals previously used in the tail-flick test. ([]) Vehicle +saline, n = 1 3 : ( m ) m o r p h i n e + s a l i n e , n = 1 2 ; ( D ) m o r p h i n e + yohimbine 2 m g / k g , n = 10; (El) m o r p h i n e + y o h i m b i n e 4 m g / k g , n = 10. * P < 0.05 vs. vehicle + saline; ÷ P < 0.05 vs. morphine + saline.
it was abolished by the higher dosage of the az-adrenoceptor antagonist (fig. 3).
4. Discussion
Our results are very similar to those obtained by Browning et ai. (1982) showing that systemic yohimbine significantly blocks the analgesic effect of systemic morphine in the tail-flick test. Previous studies performed with this test have shown that the intrathecai (i.t.) administration of yohimbine reduces the analgesic effect of morphine when the opiate is injected in supraspinal areas of the central nervous system (Camarata and Yaksh, 1985); on the contrary, i.t. yohimbine does not modify the antinociceptive action of i.t. morphine (Solomon and Gebhart, 1988). These findings suggest that yohimbine decreases opiate analgesia by blocking at the spinal level the function of descending inhibitory pathways which are activated by the opiate at supraspinal sites (Wigdor and Wilcox, 1987). In addition to confirming the results of Browning et al. we also obtained a hyperalgesic effect of yohimbine, which suggests the existence of tonic inhibitory activity of descending noradrenergic pathways under basal conditions. It is consistent with this that Sagen and Proudfit (1984) reported that nociceptive thresholds can be reduced by i.t. administration of a2-adrenoceptor antagonists. Prolonged exposure to opiates has been shown to produce cross-tolerance with ae-agonists in several tests (Yamazaki and Kaneto, 1985; Stevens et al., 1988; Solomon and Gebhart, 1988). These findings are in agreement with the reduction of a2-adrenoceptor density in several areas of the central nervous system found after chronic administration of morphine (Smith et al., 1989). Down-regulation of a2-adrenoceptors ap-
pears to be one of the adaptative mechanisms that respond to the complex effects of opiates on the function of noradrenergic pathways (Copeland and Pradhan, 1988). In this situation, it was interesting to study the effect of a2-adrenoceptor blockade on opiate tolerance and dependence, since this treatment could prevent some of the biochemical changes associated with adaptation to chronic morphine. Indeed, previous reports from our laboratory showed that yohimbine could effectively antagonize morphine tolerance in the guinea-pig ileum (Alguacil et al., 1987). Also, the group of Kaneto found that yohimbine delays the development of morphine tolerance in the tail-pinch test (Kihara and Kaneto, 1986). In the present experiments, prolonged exposure to morphine did not give rise to a marked degree of tolerance, since the baseline threshold was higher than the value observed in the control group. The nociceptive action of a test dose of morphine was slightly reduced after the prolonged exposure to the opiate, but the magnitude of this reduction did not suggest the development of robust opioid tolerance. Consequently, the effect of yohimbine on morphine tolerance could not be clearly established in our experiments and other methodology is needed. However, it is interesting that the sensitivity to opioid analgesia after 72 h of morphine exposure did not seem to be influenced by yohimbine treatment. Despite the apparent absence of opioid tolerance, the systemic morphine treatment was able to produce a marked degree of physical dependence. Under these conditions, a dose-dependent inhibition of opioid withdrawal responses was obtained when yohimbine was repeatedly administered to the animals. This blockade affected withdrawal signs originating either in the spinal cord or at higher levels in the central nervous system (Buccafusco, 1990). Accordingly, the development of physical dependence to morphine seems to be closely related with a2-adrenoceptor alterations which can be reversed by blocking these sites. These and other results on the effects of a2-agonists on opioid withdrawal lead to the conclusion that both the establishment and the expression of opioid physical dependence are profoundly altered by a2-adrenoceptor ligands. An acute dose of the a2-agonist, clonidine, reduces the magnitude of morphine withdrawal (Tseng et al., 1975) while the simultaneous administration of clonidine and morphine fails to modify the development of dependence (Cervo et al., 1981). Conversely, an acute dose of yohimbine potentiates the effect of morphine withdrawal (Dwoskin et al., 1983) while repeated administration of the antagonist prevents the development of dependence. The mechanism of the antiwithdrawal actions of acute clonidine has been extensively studied both in experimental models and in man (Garcia-Sevilla et al., 1988). A similar study on the mechanisms underlying the action of yohimbine could
38 contribute to the understanding of the interactions of az-adrenoceptor function with the adaptative res p o n s e s e l i c i t e d by c h r o n i c o p i o i d s .
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