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Effects of peripheral and central administration of calcium channel blocker in the naloxone-precipitated abstinence syndrome in morphine-dependent rats
European Journal of Pharmacology, 137 (1987) 9-13
9
Elsevier EJP 00740
Effects of peripheral and central administration of calcium channel blockers in the naloxone-precipitated abstinence syndrome in morphine-dependent rats J.M. Baeyens 1, E. Esposito 2, G. Ossowska 3 and R. Samanin 2,, i Department of Pharmacology, Unioersity of Granada, Medical School, 18012 Granada, Spain, 2 Istituto di Ricerche Farmacologiche 'Mario Negri' Via Eritrea 62, 20157 Milano, Italy, and 3 Department of Pharmacology, Medical Academy, 20-090 Lublin, Poland
Received 16 October 1986,revisedMS received28 January 1987, accepted17 February 1987
The effects of two calcium channel blockers (verapamil and flunarizine) were evaluated on the naloxone-precipitated syndrome in morphine-dependent rats. The withdrawal signs in saline-treated rats were mainly diarrhea, body weight loss, jumping and ptosis. On i.p. administration, verapamil and flunarizine prevented diarrhea and body weight loss but not jumping. Verapamil also reduced the incidence of ptosis at the highest dose tested (40 mg/kg). Administered i.c.v., 160 /tg verapamil reduced the body weight loss and the number of jumps without modifying diarrhea or ptosis. The results show that calcium channel blockers inhibit morphine abstinence syndrome manifestations through both peripheral and central mechanisms. Calcium channel blockers; Verapamil; Flunarizine; Morphine abstinence syndrome
I. Introduction Evidence has accumulated that at least some of the actions of morphine could be mediated through calcium antagonistic activity. Morphine has been shown to reduce the calcium content in several areas of the rat brain (Ross, 1975; Cardenas and Ross, 1976) and to inhibit synaptosomal calcium uptake in mouse and rat brain (Guerrero Munoz et al., 1979; Konno and Takayanagi, 1982; Kamikubo et al., 1983). These biochemical effects appear to be related to the behavioural actions of morphine as morphine analgesia is antagonized by calcium and calcium ionophores (Hano et al., 1964; Harris et al., 1975; Iwamoto et al., 1978; Vocci et al., 1980), whereas the calcium chelators EDTA and E G T A and the inorganic inhibitor of calcium cellular influx, lanthanum, increase the
* To whom all correspondenceshould be addressed.
analgesic effects of morphine (Hano et al., 1964; Harris et al., 1975, 1976; Vocci et al., 1980). Altered calcium disposition may also be involved in the expression of morphine physical dependence since lanthanum inhibits the abstinence jumping syndrome in morphine-dependent mice after either abrupt morphine or naloxoneprecipitated withdrawal (Harris et al., 1976). In view of the similarity between the mechanism of action of lanthanum and of calcium channel blockers we decided to study the effects of peripherally administered verapamil and flunarizine on the naloxone-precipitated morphine abstinence syndrome in rats. [3H]Verapamil binding sites have been identified in rat brain (Reynolds et al., 1983) and some behavioural actions have been reported after intracerebroventricular (i.c.v.) verapamil (Beleslin et al., 1986). We therefore decided to study the effects of i.c.v, verapamil on the morphine abstinence syndrome.
0014-2999/87/$03.50 © 1987 ElsevierSciencePublishers B.V. (BiomedicalDivision)
10
2. Materials and methods
2.4. Analysis of results
Male CD-COBS rats (Charles River, Italy) weighing about 200 g at the beginning of the experiments were used. The animals were housed four per cage with water and food available ad libitum.
The data are expressed as the proportion of animals presenting qualitatively evaluated signs (diarrhea, palpebral ptosis) and as means + S.E. for quantitatively evaluated signs (jumping, body weight loss). Differences between saline- and drug-treated animals were analysed statistically by means of Fisher's exact test (diarrhea and palpebral ptosis) and Dunnett's test (body weight loss). The non-parametric Kruskall-Wallis test was used for the data on jumping since these data are usually not normally distributed.
2.1. Induction of physical dependence On day 1 the rats received two i.p. injections (10 a.m. and 6 p.m.) of morphine HC1 10 m g / k g (calculated as free base). The dose of morphine was doubled every other day to reach daily dose of 160 m g / k g on day 7, which was continued for 3 days more. A last dose of morphine (80 mg/kg) was injected at 10 a.m. on day 11 and an abstinence syndrome was precipitated 4 h later by i.p. injection of 1 m g / k g of naloxone HC1.
2.2. Testing for withdrawal Before injection of naloxone the animals were placed individually for 30 min acclimatization in test chambers consisting of rectangular acryl-glass boxes (30 × 30 × 25 cm). Abstinence signs precipitated by naloxone were recorded within 20 min of naloxone injection according to a previously described procedure (Cervo et al., 1981) by observers uninformed as to the treatment.
2. 3. Drugs The drugs employed and their suppliers were as follows: verapamil HCI (Knoll Laboratories), flunarizine HC1 (Janssen Pharmaceutica), morphine HCI (Farmitalia-Carlo Erba), naloxone HC1 (Endo Laboratories). Verapamil, morphine and naloxone were dissolved in distilled water, flunarizine was suspended in 5% gum arabic. All drugs were injected i.p. in a volume of 2 ml/kg. Verapamil and flunarizine were injected respectively 30 and 60 min before naloxone, the times necessary to obtain maximum CNS concentrations in the rat (Hamann et al., 1983; Michiels et al., 1983). Verapamil was also injected i.c.v. 10 min before naloxone, in a volume of 5/~1 according to the method of Noble et al. (1967).
3. Results
3.1. Effect of i.p. verapamil and flunarizine on morphine withdrawal signs Vehicle-treated rats that had received morphine for 11 days showed mainly jumping, palpebral ptosis, diarrhea and body weight loss after naloxone injection. Other signs such as tooth chattering, wet-dog shakes, vocalization on touch, salivation and flat posture were observed less frequently and were not considered in the analysis of the data. The rats that had received verapamil (10, 20
TABLE 1 Effects of various doses of i.p. verapamil and flunarizine on j u m p i n g and body weight loss precipitated by naloxone in morphine-dependent rats. Verapamil and flunarizine were injected 30 and 60 min before naloxone, respectively. Treatment
mg/kg
Jumping a
A Body weight (g) b
Saline Verapamil Verapamil Verapamil
10 20 40
12.3+2.8 10.0 + 3.6 13.2+3.8 14.0 + 5.0
-9.2+0.9 + 2.6 + 1.7 d + 0 . 3 + 1.8 d + 7.5 + 1.5 d
Vehicle Flunarizine Flunarizine Flunarizine
20 40 80
11.15:2.6 15.05:4.2 11.65:4.4 12.7 5:2.9
-8.25:1.2 -1.0+2.1 c -2.25:3.0 c - 1.5 5:2.2 c
a Mean number of episodes 5: S.E. b Mean5:S.E. of the difference between body weight before and 2 h after naloxone. c p < 0.05 and d P < 0.01 compared with saline- or vehicletreated rats (Dunnett's test).
11 TABLE 2
TABLE 4
Effects of various doses of i.p. verapamil and flunarizine on diarrhea and ptosis precipitated by naloxone in morphine-dependent rats. Verapamil and flunarizine were injected 30 and 60 min before naloxone, respectively.
Effect of i.c.v, verapamil on diarrhea and ptosis precipitated by naloxone in morphine-dependent rats. Verapamil was injected" 10 min before naloxone, n.s. = not significantly different from saline (Fisher's exact test).
Proportion of animals with
Treatment
mg/kg
Diarrhea
Ptosis
Saline Verapamil Verapamil Verapamil
10 20 40
11/11 1/11 b 2/12 b I/II b
7/11 8/11 11/12 1/11 a
Vehicle Flunarizine Flunarizine Flunarizine
20 40 80
12/13 7/12 7/11 2/13 b
7/13 8/12 5/11 8/13
" P < 0.05 and b p < 0.01 compared with saline- or vehicletreated rats (Fisher's exact test).
and 40 mg/kg, i.p.) or flunarizine (20, 40 and 80 mg/kg, i.p.) showed the same number of jumps as did the vehicle treated rats (table 1). However, both drugs at all doses tested significantly reduced body weight loss during naloxone-precipitated withdrawal (table 1). As shown in table 2, verapamil at all doses tested also lowered the proportion of animals showing diarrhea and at highest dose it reduced the number of animals showing ptosis. In contrast, flunarizine did not affect ptosis and reduced the incidence of diarrhea (table 2).
3.2. Effects of i.c.v, verapamil withdrawal signs
on morphine
Intracerebroventricular saline-treated rats that had received morphine for 11 days showed the TABLE 3 Effects of i.c.v, verapamil and body weight loss precipitated by naloxone in morphine-dependent rats. Verapamil was injected 10 min before naloxone. Treatment
#tg
Jumping a
A Body weight (g) b
Saline Verapamil Veraparnil
80 160
16.44-4.4 18.5 4- 2.8 3.1+2.9 c
-9.44-2.3 - 9.8 :!: 2.0 - 2.3 :t:2.7 d
a Mean number of episodes+S.E, b Mean4-S.E. of the difference between body weight before and 2 h after naloxone injection, c p < 0.05 compared with saline (Kruskall-Wallis test), d p < 0.05 compared with saline (Dunnett's tes 0.
Treatment
Saline Verapamil Verapamil
pg
80 160
Proportion of animals with Diarrhea
Ptosis
11/11 9 / 1 2 n.s. 7 / 1 0 n.s.
5/11 8 / 1 2 n.s. 6 / 1 0 n.s.
same naloxone-precipitated abstinence signs as intraperitoneal saline-treated rats. Verapamil i.c.v. caused no overt behavioural effects which could have interfered with the expression of the abstinence syndrome. Verapamil 160 #g i.c.v, but not 80 /xg i.c.v. significantly reduced the number of jumps and the body weight loss induced by naloxone (table 3). The incidence of ptosis and diarrhea was not affected by verapamil at any dose (table 4).
4. Discussion
Our study shows that calcium channel blockers can inhibit the expression of several naloxone-precipitated withdrawal signs in morphine-dependent rats by acting through both peripheral and central mechanisms. A recent study reported a reduction of the morphine abstinence syndrome after peripheral administration of verapamil (Bongianni et al., 1985). Our results show that flunarizine produced similar effects and that verapamil could produce some effects by a central action. Verapamil prevented diarrhea after its i.p. but not after its i.c.v, injection, which suggests that peripheral mechanisms are involved in this action. Several facts support this suggestion: (a) morphine dependence and naloxone-precipitated withdrawal can easily be obtained in isolated segments of ileum (Collier et al., 1981), (b) calcium channel binding sites have been identified in intestinal smooth muscle (Bolger et al., 1983), (c) verapamil can antagonize calcium-induced contractions in intestinal smooth muscle in vitro (Spedding, 1982). Verapamil could have prevented body weight
12 loss through its effect on diarrhea but the reduction of b o d y weight loss was p r o d u c e d by central "injections of verapamil at doses which failed to inhibit diarrhea, suggesting that this effect is at least partially mediated by a central mechanism. That verapamil acts on the C N S was even more evident in the case of jumping, which was only reduced after i.c.v, administration. This is consistent with the effect of i.c.v, l a n t h a n u m on naloxone-precipitated j u m p i n g in morphine-dependent mice (Harris et al., 1976). The lack of efficacy of verapamil after its i.p, injection could be explained by the fact that, as j u m p i n g was inhibited after 160 /zg but not after 80 /~g i.c.v., high concentrations of verapamil in the brain must be necessary to achieve inhibition. After i.p. injection of verapamil 30 m g / k g , the maximal concentration found in the brain was about 1.5 ktg/g tissue ( H a m a n n et al., 1983) and this m a y not be enough to reduce jumping. That verapamil reduced b o d y weight loss and j u m p i n g through an action on the central nervous system was not unexpected considering that there are calcium channel blocker binding sites in the rat brain (Marangos et al., 1982; M u r p h y et al., 1982; Reynolds et al., 1983; G o u l d et al., 1985) which appear to be functional (Middlemiss and Spedding, 1985; Battaini et al., 1986; Beleslin et al., 1986) and which increase in n u m b e r after chronic m o r p h i n e treatment ( R a m k u m a r and E1Fakahany, 1984). The spectrum of activity of verapamil on morphine-withdrawal signs is similar to that of clonidine that also reduces diarrhea, b o d y weight loss and prosis (Cervo et al., 1981; Meyer and Sparber, 1976). However, the effects of verapamil do not seem to be mediated through az-adrenoceptor stimulation because it has been shown that verapamil has a very low affinity for [3 H]clonidine binding sites in rat brain (Van Meel et al., 1983) and can even antagonize non-competitively the vascular effects of a2-adrenoceptor stimulants (Van Meel et al., 1983). Moreover, verapamil reduced the expression of j u m p i n g whereas clonidine did not (Cervo et al., 1981; R o m a n d i n i et al., 1984). Flunarizine also reduced the incidence of diarhea and the b o d y weight loss on i.p. injection.
Fhinarizine was a less potent inhibitor of diarrhea than verapamil, in contrast to their similar potency as antagonists of calcium-induced contractions in isolated intestinal smooth muscle (Spedding, 1982). However, it should be considered that, although flunarizine and verapamil share the same mechanism of action, they show differences in their capacity to displace [3H]nitrendipine from calcium channel blocker binding sites (Bolger et al., 1983) and in their functional actions (Spedding, 1982; Spedding and Berg, 1984) on intestinal smooth muscle. These differences may underlie their different potency to block diahrrea in morphine-abstinent rats.
Acknowledgements This work was supported by CNR Contract No. 85.00754.56 and project No. 635 RegJone Lombardia (Medicina Preventiva).
References Battaini, F., S. Govoni, S. Di Giovine and M. Trabucchi, 1986, Neurotensin effect on dopamine release and calcium transport in rat striatum: interactions with diphenylalkylamine calcium antagonists, Naunyn-Schmiedeb. Arch. Pharmacol. 332, 267. Beleslin, D.B., R. Samardzic, D. Jovanovic-micic and B. Terzic, 1986, Verapamil-induced behavioral autonomic and motor effects in cats, Pharmacol. Biochem. Behav. 24, 329. Bolger, G.T., P. Gengo, R. Klockowski, E. Luchowski, H. Siegel, R.A. Janis, A.M. Triggle and D.J. Triggle, 1983, Characterization of binding of the C a 2 + channel antagonist, [3H]nitrendipine, to guinea pig ileal smooth muscle, J. Pharmacol. Exp. Ther. 225, 291. Bongianni, F., V. Carla, F. Moroni and D. Pellegrini, 1985, Ca2+-channel inhibitors prevent morphine withdrawal syndrome in rats, Br. J. Pharmacol. 86, 529 P. Cardenas, H.L. and D.H. Ross, 1976, Calcium depletion of synaptosomes after morphine treatment, Br. J. Pharmacol. 57, 521. Cervo, L., C. Rochat, S. Romandini and R. Samanin, 1981, Evidence of a preferential role of brain serotonin in the mechanisms leading to naloxone-precipitated compulsive jumping in morphine dependent rats, Psychopharmacology 74, 271. Collier, H.O.J., N.J. Cuthbert and D.L. Francis, 1981, Model of opiate dependence in the guinea-pig isolated ileum, Br. J. Pharmacol. 73, 921. Gould, R.J., K.M.M. Murphy and S.H. Snyder, 1985, Autoradiographic localization of calcium channel antagonist re-
13 ceptors in rat brain with [3H]nitrendipine, Brain Res. 330, 217. Guerrero Munoz, F., K.V. Cerreta, M.L. Guerrero and E.L. Way, 1979, Effect of morphine on synaptosomal Ca 2+ uptake, J. Pharmacol. Exp. Ther. 209, 132. Hamann, S.R., G.D. Todd and R.G. McAllister Jr., 1983, The pharmacology of verapamil. V. Tissue distribution of verapamil and norverapamil in rat and dog, Pharmacology 27, 1. Hano, K., H. Kaneto and T. Kakunaga, 1964, Significance of calcium ion in the morphine analgesia, Jap. J. Pharmacol. 14, 227. Harris, R.A., H.H. Loh and E.L. Way, 1975, Effects of divalent cations, cation chelators and an ionophore on morphine analgesia and tolerance, J. Pharmacol. Exp. Ther. 195, 488. Harris, R.A., H.H. Loh and E.L. Way, 1976, Antinociceptive effects of lanthanum and cerium in nontolerant and morphine tolerant-dependent animals, J. Pharmacol. Exp. Ther. 196, 288. Iwamoto, E.T., R.A. Harris, H.H. Loh and E.L. Way, 1978, Antinociceptive responses after microinjection of morphine or lanthanum in discrete rat brain sites, J. Pharmacol. Exp. Ther. 206, 46. Kamikubo, K., M. Niwa, H. Fujimura and K. Miura, 1983, Morphine inhibits depolarization-dependent calcium uptake by synaptosomes, European J. Pharmacol. 95, 149. Konno, F. and I. Takayanagi, 1982, Effect of morphine on the stimuli-induced calcium uptake into synaptosomes isolated from morphine-tolerant rats, Jap. J. Pharmacol. 32, 625. Marangos, P.J., J. Patel, C. Miller and A.M. Martino, 1982, Specific calcium antagonist binding sites in brain, Life Sci. 31, 1575. Meyer, D.R. and S.B. Sparber, 1976, Clonidine antagonizes body weight loss and other symptoms used to measure withdrawal in morphine pelleted rats given naloxone, Pharmacologist 18, 236. Michiels, M., R. Hendriks, F. Knaeps, R. Woestenborghs and J. Heykants, 1983, Absorption and tissue distribution of flunarizine in rats, pigs and dogs, Arzneim. Forsch. 33, 1135. Middlemiss, D.N. and M. Spedding, 1985, A functional correlate for the dihydropyridine binding site in rat brain, Nature 314, 94.
Murphy, K.M.M., R.J. Gould and S.H. Snyder, 1982, Autoradiographic visualization of [3 H]nitrendipine binding sites in rat brain: localization to synaptic zones, European J. Pharmacol. 81,517. Noble, E.P., R.J. Wurtman and J. Axelrod, 1967, A simple and rapid method for injecting 3H-norepinephrine into the lateral ventricle of the rat brain, Life Sci. 6, 281. Ramkumar, V. and E.E. El-Fakahany, 1984, Increase in [3H]nitrendipine binding sites in the brain in morphinetolerant mice, European J. Pharmacol. 102, 371. Reynolds, I.J., R.J. Gould and S.H. Snyder, 1983, [3H]Verapamil binding sites in brain and skeletal muscle: regulation by calcium, European J. Pharmacol. 95, 319. Romandini, S., L. Cervo and R. Samanin, 1984, Evidence that drugs increasing 5-hydroxytryptamine transmission block jumping but not wet dog shakes in morphine-abstinent rats: a comparison with clonidine, J. Pharm. Pharmacol. 36, 68. Ross, D.H., 1975, Tolerance to morphine-induced calcium depletion in regional brain areas: Characterization with reserpine and protein synthesis inhibitors, Br. J. Pharmacol. 55, 431. Spedding, M., 1982, Assessment o f ' Ca 2 +-antagonist' effects of drugs in K+-depolarized smooth muscle. Differentiation of antagonist subgroups, Naunyn-Schmiedeb. Arch. Pharmacol. 318, 234. Spedding, M. and C. Berg, 1984, Interactions betweeen a 'calcium channel agonist', Bay K 8644, and calcium antagonists differentiate calcium antagonist subgroups in K+-depolarized smooth muscle, Naunyn-Schmiedeb. Arch. Pharmacol. 328, 69. Van Meel, J.C.A., J. Qian, P.B.M.W.M. Timmermans and P.A. Van Zwieten, 1983, Differential inhibition of a2-adrenoceptor mediated pressor responses by (+)- and ( - ) verapamil in pithed rats, J. Pharm. Pharmacol. 35, 500. Vocci, F.J., S.P. Welch and W.L. Dewey, 1980, Differential effects of divalent cations, cation chelators and an ionophore (A23187) on morphine and dibutyryl guanosine 3':5'-cyclic monophosphate antinociception, J. Pharmacol. Exp. Ther. 214, 463.
9
Elsevier EJP 00740
Effects of peripheral and central administration of calcium channel blockers in the naloxone-precipitated abstinence syndrome in morphine-dependent rats J.M. Baeyens 1, E. Esposito 2, G. Ossowska 3 and R. Samanin 2,, i Department of Pharmacology, Unioersity of Granada, Medical School, 18012 Granada, Spain, 2 Istituto di Ricerche Farmacologiche 'Mario Negri' Via Eritrea 62, 20157 Milano, Italy, and 3 Department of Pharmacology, Medical Academy, 20-090 Lublin, Poland
Received 16 October 1986,revisedMS received28 January 1987, accepted17 February 1987
The effects of two calcium channel blockers (verapamil and flunarizine) were evaluated on the naloxone-precipitated syndrome in morphine-dependent rats. The withdrawal signs in saline-treated rats were mainly diarrhea, body weight loss, jumping and ptosis. On i.p. administration, verapamil and flunarizine prevented diarrhea and body weight loss but not jumping. Verapamil also reduced the incidence of ptosis at the highest dose tested (40 mg/kg). Administered i.c.v., 160 /tg verapamil reduced the body weight loss and the number of jumps without modifying diarrhea or ptosis. The results show that calcium channel blockers inhibit morphine abstinence syndrome manifestations through both peripheral and central mechanisms. Calcium channel blockers; Verapamil; Flunarizine; Morphine abstinence syndrome
I. Introduction Evidence has accumulated that at least some of the actions of morphine could be mediated through calcium antagonistic activity. Morphine has been shown to reduce the calcium content in several areas of the rat brain (Ross, 1975; Cardenas and Ross, 1976) and to inhibit synaptosomal calcium uptake in mouse and rat brain (Guerrero Munoz et al., 1979; Konno and Takayanagi, 1982; Kamikubo et al., 1983). These biochemical effects appear to be related to the behavioural actions of morphine as morphine analgesia is antagonized by calcium and calcium ionophores (Hano et al., 1964; Harris et al., 1975; Iwamoto et al., 1978; Vocci et al., 1980), whereas the calcium chelators EDTA and E G T A and the inorganic inhibitor of calcium cellular influx, lanthanum, increase the
* To whom all correspondenceshould be addressed.
analgesic effects of morphine (Hano et al., 1964; Harris et al., 1975, 1976; Vocci et al., 1980). Altered calcium disposition may also be involved in the expression of morphine physical dependence since lanthanum inhibits the abstinence jumping syndrome in morphine-dependent mice after either abrupt morphine or naloxoneprecipitated withdrawal (Harris et al., 1976). In view of the similarity between the mechanism of action of lanthanum and of calcium channel blockers we decided to study the effects of peripherally administered verapamil and flunarizine on the naloxone-precipitated morphine abstinence syndrome in rats. [3H]Verapamil binding sites have been identified in rat brain (Reynolds et al., 1983) and some behavioural actions have been reported after intracerebroventricular (i.c.v.) verapamil (Beleslin et al., 1986). We therefore decided to study the effects of i.c.v, verapamil on the morphine abstinence syndrome.
0014-2999/87/$03.50 © 1987 ElsevierSciencePublishers B.V. (BiomedicalDivision)
10
2. Materials and methods
2.4. Analysis of results
Male CD-COBS rats (Charles River, Italy) weighing about 200 g at the beginning of the experiments were used. The animals were housed four per cage with water and food available ad libitum.
The data are expressed as the proportion of animals presenting qualitatively evaluated signs (diarrhea, palpebral ptosis) and as means + S.E. for quantitatively evaluated signs (jumping, body weight loss). Differences between saline- and drug-treated animals were analysed statistically by means of Fisher's exact test (diarrhea and palpebral ptosis) and Dunnett's test (body weight loss). The non-parametric Kruskall-Wallis test was used for the data on jumping since these data are usually not normally distributed.
2.1. Induction of physical dependence On day 1 the rats received two i.p. injections (10 a.m. and 6 p.m.) of morphine HC1 10 m g / k g (calculated as free base). The dose of morphine was doubled every other day to reach daily dose of 160 m g / k g on day 7, which was continued for 3 days more. A last dose of morphine (80 mg/kg) was injected at 10 a.m. on day 11 and an abstinence syndrome was precipitated 4 h later by i.p. injection of 1 m g / k g of naloxone HC1.
2.2. Testing for withdrawal Before injection of naloxone the animals were placed individually for 30 min acclimatization in test chambers consisting of rectangular acryl-glass boxes (30 × 30 × 25 cm). Abstinence signs precipitated by naloxone were recorded within 20 min of naloxone injection according to a previously described procedure (Cervo et al., 1981) by observers uninformed as to the treatment.
2. 3. Drugs The drugs employed and their suppliers were as follows: verapamil HCI (Knoll Laboratories), flunarizine HC1 (Janssen Pharmaceutica), morphine HCI (Farmitalia-Carlo Erba), naloxone HC1 (Endo Laboratories). Verapamil, morphine and naloxone were dissolved in distilled water, flunarizine was suspended in 5% gum arabic. All drugs were injected i.p. in a volume of 2 ml/kg. Verapamil and flunarizine were injected respectively 30 and 60 min before naloxone, the times necessary to obtain maximum CNS concentrations in the rat (Hamann et al., 1983; Michiels et al., 1983). Verapamil was also injected i.c.v. 10 min before naloxone, in a volume of 5/~1 according to the method of Noble et al. (1967).
3. Results
3.1. Effect of i.p. verapamil and flunarizine on morphine withdrawal signs Vehicle-treated rats that had received morphine for 11 days showed mainly jumping, palpebral ptosis, diarrhea and body weight loss after naloxone injection. Other signs such as tooth chattering, wet-dog shakes, vocalization on touch, salivation and flat posture were observed less frequently and were not considered in the analysis of the data. The rats that had received verapamil (10, 20
TABLE 1 Effects of various doses of i.p. verapamil and flunarizine on j u m p i n g and body weight loss precipitated by naloxone in morphine-dependent rats. Verapamil and flunarizine were injected 30 and 60 min before naloxone, respectively. Treatment
mg/kg
Jumping a
A Body weight (g) b
Saline Verapamil Verapamil Verapamil
10 20 40
12.3+2.8 10.0 + 3.6 13.2+3.8 14.0 + 5.0
-9.2+0.9 + 2.6 + 1.7 d + 0 . 3 + 1.8 d + 7.5 + 1.5 d
Vehicle Flunarizine Flunarizine Flunarizine
20 40 80
11.15:2.6 15.05:4.2 11.65:4.4 12.7 5:2.9
-8.25:1.2 -1.0+2.1 c -2.25:3.0 c - 1.5 5:2.2 c
a Mean number of episodes 5: S.E. b Mean5:S.E. of the difference between body weight before and 2 h after naloxone. c p < 0.05 and d P < 0.01 compared with saline- or vehicletreated rats (Dunnett's test).
11 TABLE 2
TABLE 4
Effects of various doses of i.p. verapamil and flunarizine on diarrhea and ptosis precipitated by naloxone in morphine-dependent rats. Verapamil and flunarizine were injected 30 and 60 min before naloxone, respectively.
Effect of i.c.v, verapamil on diarrhea and ptosis precipitated by naloxone in morphine-dependent rats. Verapamil was injected" 10 min before naloxone, n.s. = not significantly different from saline (Fisher's exact test).
Proportion of animals with
Treatment
mg/kg
Diarrhea
Ptosis
Saline Verapamil Verapamil Verapamil
10 20 40
11/11 1/11 b 2/12 b I/II b
7/11 8/11 11/12 1/11 a
Vehicle Flunarizine Flunarizine Flunarizine
20 40 80
12/13 7/12 7/11 2/13 b
7/13 8/12 5/11 8/13
" P < 0.05 and b p < 0.01 compared with saline- or vehicletreated rats (Fisher's exact test).
and 40 mg/kg, i.p.) or flunarizine (20, 40 and 80 mg/kg, i.p.) showed the same number of jumps as did the vehicle treated rats (table 1). However, both drugs at all doses tested significantly reduced body weight loss during naloxone-precipitated withdrawal (table 1). As shown in table 2, verapamil at all doses tested also lowered the proportion of animals showing diarrhea and at highest dose it reduced the number of animals showing ptosis. In contrast, flunarizine did not affect ptosis and reduced the incidence of diarrhea (table 2).
3.2. Effects of i.c.v, verapamil withdrawal signs
on morphine
Intracerebroventricular saline-treated rats that had received morphine for 11 days showed the TABLE 3 Effects of i.c.v, verapamil and body weight loss precipitated by naloxone in morphine-dependent rats. Verapamil was injected 10 min before naloxone. Treatment
#tg
Jumping a
A Body weight (g) b
Saline Verapamil Veraparnil
80 160
16.44-4.4 18.5 4- 2.8 3.1+2.9 c
-9.44-2.3 - 9.8 :!: 2.0 - 2.3 :t:2.7 d
a Mean number of episodes+S.E, b Mean4-S.E. of the difference between body weight before and 2 h after naloxone injection, c p < 0.05 compared with saline (Kruskall-Wallis test), d p < 0.05 compared with saline (Dunnett's tes 0.
Treatment
Saline Verapamil Verapamil
pg
80 160
Proportion of animals with Diarrhea
Ptosis
11/11 9 / 1 2 n.s. 7 / 1 0 n.s.
5/11 8 / 1 2 n.s. 6 / 1 0 n.s.
same naloxone-precipitated abstinence signs as intraperitoneal saline-treated rats. Verapamil i.c.v. caused no overt behavioural effects which could have interfered with the expression of the abstinence syndrome. Verapamil 160 #g i.c.v, but not 80 /xg i.c.v. significantly reduced the number of jumps and the body weight loss induced by naloxone (table 3). The incidence of ptosis and diarrhea was not affected by verapamil at any dose (table 4).
4. Discussion
Our study shows that calcium channel blockers can inhibit the expression of several naloxone-precipitated withdrawal signs in morphine-dependent rats by acting through both peripheral and central mechanisms. A recent study reported a reduction of the morphine abstinence syndrome after peripheral administration of verapamil (Bongianni et al., 1985). Our results show that flunarizine produced similar effects and that verapamil could produce some effects by a central action. Verapamil prevented diarrhea after its i.p. but not after its i.c.v, injection, which suggests that peripheral mechanisms are involved in this action. Several facts support this suggestion: (a) morphine dependence and naloxone-precipitated withdrawal can easily be obtained in isolated segments of ileum (Collier et al., 1981), (b) calcium channel binding sites have been identified in intestinal smooth muscle (Bolger et al., 1983), (c) verapamil can antagonize calcium-induced contractions in intestinal smooth muscle in vitro (Spedding, 1982). Verapamil could have prevented body weight
12 loss through its effect on diarrhea but the reduction of b o d y weight loss was p r o d u c e d by central "injections of verapamil at doses which failed to inhibit diarrhea, suggesting that this effect is at least partially mediated by a central mechanism. That verapamil acts on the C N S was even more evident in the case of jumping, which was only reduced after i.c.v, administration. This is consistent with the effect of i.c.v, l a n t h a n u m on naloxone-precipitated j u m p i n g in morphine-dependent mice (Harris et al., 1976). The lack of efficacy of verapamil after its i.p, injection could be explained by the fact that, as j u m p i n g was inhibited after 160 /zg but not after 80 /~g i.c.v., high concentrations of verapamil in the brain must be necessary to achieve inhibition. After i.p. injection of verapamil 30 m g / k g , the maximal concentration found in the brain was about 1.5 ktg/g tissue ( H a m a n n et al., 1983) and this m a y not be enough to reduce jumping. That verapamil reduced b o d y weight loss and j u m p i n g through an action on the central nervous system was not unexpected considering that there are calcium channel blocker binding sites in the rat brain (Marangos et al., 1982; M u r p h y et al., 1982; Reynolds et al., 1983; G o u l d et al., 1985) which appear to be functional (Middlemiss and Spedding, 1985; Battaini et al., 1986; Beleslin et al., 1986) and which increase in n u m b e r after chronic m o r p h i n e treatment ( R a m k u m a r and E1Fakahany, 1984). The spectrum of activity of verapamil on morphine-withdrawal signs is similar to that of clonidine that also reduces diarrhea, b o d y weight loss and prosis (Cervo et al., 1981; Meyer and Sparber, 1976). However, the effects of verapamil do not seem to be mediated through az-adrenoceptor stimulation because it has been shown that verapamil has a very low affinity for [3 H]clonidine binding sites in rat brain (Van Meel et al., 1983) and can even antagonize non-competitively the vascular effects of a2-adrenoceptor stimulants (Van Meel et al., 1983). Moreover, verapamil reduced the expression of j u m p i n g whereas clonidine did not (Cervo et al., 1981; R o m a n d i n i et al., 1984). Flunarizine also reduced the incidence of diarhea and the b o d y weight loss on i.p. injection.
Fhinarizine was a less potent inhibitor of diarrhea than verapamil, in contrast to their similar potency as antagonists of calcium-induced contractions in isolated intestinal smooth muscle (Spedding, 1982). However, it should be considered that, although flunarizine and verapamil share the same mechanism of action, they show differences in their capacity to displace [3H]nitrendipine from calcium channel blocker binding sites (Bolger et al., 1983) and in their functional actions (Spedding, 1982; Spedding and Berg, 1984) on intestinal smooth muscle. These differences may underlie their different potency to block diahrrea in morphine-abstinent rats.
Acknowledgements This work was supported by CNR Contract No. 85.00754.56 and project No. 635 RegJone Lombardia (Medicina Preventiva).
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