European Journal of Pharmacology, 180 (1990) 129-135
129
Elsevier EJP 51280
Cortical dihydropyridine binding sites and a behavioral syndrome in morphine-abstinent rats L u c y n a A n t k i e w i c z - M i c h a l u k , J e r z y M i c h a l u k , I r e n a R o m a f l s k a , Jerzy Vetulani Department of Biochemistry, Institute of Pharmacology, Polish A eademy of Sciences, Smetna 12, PL-31-343 Krakbw, Poland
Received 19 October 1989, revisedMS received24 January 1990, accepted 13 February 1990
The density of cortical [3H]nitrendipine binding sites was elevated by over 40% in rats rendered morphine-abstinent by administration of naloxone after chronic treatment with morphine. The morphine-abstinent rats had significantly shortened response latencies in the hot-plate test. Nifedipine treatment abolished the signs of abstinence and normalized the hot-plate latencies in morphine-dependent, naloxone-treated rats. The results indicate that the symptoms of abstinence are related to a functional state of cortical dihydropyridine-sensitive calcium channels. Ca 2÷ channels; Morphine abstinence; Pain sensitivity
I. Introduction
Calcium channels in cell membranes regulate the concentration of cytosolic calcium and serve to link membrane potential changes with cellular responses (Miller, 1987; Reuter et al., 1982). As voltage-dependent calcium channels are associated with the binding sites for dihydropyridine calcium antagonists (BeUeman et al., 1981; Middlemiss, 1985; Middlemiss and Spedding, 1985), they may conveniently be studied by investigation of the specific binding of [3H]nitrendipine. Several data indicate that calcium plays an important role in nociception and in the action of opiates (Ross and Cardenos, 1979). Recent data suggest that opiate receptors are functionally coupled to voltage-sensitive ion channels and that stimulation of opiate K receptors directly, and of/~ receptors indirectly, suppresses calcium conduc-
Correspondence to: J. Vetulani, Department of Biochemistry, Institute of Pharmacology,Polish Academyof Sciences,Smetna 12, PL-31-343Krak6w, Poland.
tion (North, 1986). Morphine inhibits synaptosomal calcium uptake (Guerrero-Mu~oz et al., 1979; Kamikubo et al., 1988) and an acute injection of morphine depletes calcium in whole brain synaptosomes (Harris et al., 1977). It is possible that the inhibition of calcium uptake is related to the analgesic effect of opioids, as calcium injected into the periaqueductal gray or into the ventricles antagonizes opioid-induced analgesia and this action is facilitated by a calcium ionophore (Chapman and Way, 1982; Guerrero-Mu~oz et al., 1981; Harris et al., 1975). Conversely, the chelating agents, EGTA and EDTA, and calcium channel inhibitors enhance the analgesic effects of opioids and opioid-mediated stress analgesia (Benedek and Szikszay, 1984; Chapman and Way, 1982; Contreras et al., 1988; Harris et al., 1975; Hoffmeister and Tettenborn, 1986; Kavaliers, 1987a; Kavaliers and Ossenkopp, 1987). Calcium channel antagonists also counteract other effects of morphine, such as running fit in certain mouse strains (Kavaliers, 1987b), hypothermia in restrained rats (Benedek and Szikszay, 1984), and signs of morphine abstinence in vivo and in vitro (Baeyens et al., 1987; Bongianni et al., 1986; Caro et al.,
0014-2999/90/$03.50 © 1990 ElsevierScience Publishers B.V. (Biomedical Division)
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1988; Pellegrini-Giampietro et al., 1988; Ramkumar and El-Fakahany, 1988). The chronic effects of morphine are often opposite to the effects of a single high dose. Thus, chronic morphine treatment increases the vesicular content of calcium (Harris et al., 1977). Moreover, potassium-stimulated synaptosomal calcium uptake, which is inhibited by a single dose of morphine, is increased in mice chronically treated with morphine (Guerrero-Mu~aoz et al., 1979). Recently, Ramkumar and E1-Fakahany (1988) reported that morphine implants increased the density of [3H]nimodipine binding sites in several cerebral structures. These changes may represent compensatory responses and may be related to the development of tolerance to the actions of morphine. In this study we investigated the effect of m o r p h i n e abstinence on voltage-dependent calcium channels in the brain, using [3H]nitrendipine as a radioligand to label L-type calcium channels in the cortex and hippocampus. We also measured the effect of morphine abstinence on the responsiveness of rats to noxious stimuli in the hot-plate test, and the effect of a dihydropyridine calcium channel blocker, nifedipine, to find out if there is a relation between the changes in the density of dihydropyridine calcium channels in some brain areas and the abstinence syndrome.
2. Materials and methods
2.1. Animals The experiments were carried out with male Wistar rats, weighing 200-230 g at the beginning of experiment, kept under standard laboratory conditions (approx. 22°C, natural day-night cycle), ten to a home cage (large plastic cage with wire lid and sawdust bedding), with free access to standard laboratory food and tap water. 2.2. Drugs and drug treatment All injections were given in a volume of 4 m l / k g i.p. Morphine (hydrochloride, Polfa) was given in a daily dose of 20 m g / k g for 8 consecu-
tive days (controls received saline). The rats were tested 24 h after the last injection of morphine; some animals were injected with naloxone (hydrochloride, Endo), 2 mg/kg, 24 h after the last dose of morphine. The rats used for biochemical assay were decapitated 60 min after the naloxone injection. The control naloxone-treated group received a single injection of the drug 60 min before death. Nifedipine (Polfa), 5 mg/kg, was given 15 min before naloxone. In an acute experiment naloxone was given 30 rain, and nifedipine 45 min, before the tests. 2.3. Behavioral tests The responsiveness of rats to a painful stimulus was assessed using the hot-plate test. Twenty-four hours after the last dose of morphine, the rats were placed on a metal plate kept at 56 ° C and the latency of response (licking of hind paws or jumping) was measured. The cut-off time was 30 s. The rats challenged with naloxone received it 30 min before the test. The rats tested for 'wet-dog' shakes were observed for 60 min in wire-mesh cage and the number of shakes was recorded by two independent observers. The animals tested for diarrhea were weighed before naloxone was injected and then 90 rain later. The difference in body weight is presented as the per cent of initial body weight. The rats used for the behavioral tests were not used for the biochemical experiments to avoid the possibility that the stress of testing affected the state of the calcium channels. 2.4. Membrane preparation and receptor binding assay The brain was rapidly removed, placed on an ice-chilled porcelain plate, and the cerebral cortex and hippocampus were dissected. The tissues were homogenized at 0 ° C in 20 vol of 50 mmolfll Tris-HC1 buffer, pH (at 23 ° C) 7.6 with a Polytron disintegrator (setting 4, 10 s). The cortex from each animal was homogenized separately; the hippocampal tissue from two animals was pooled for membrane preparation.
131
The homogenate was centrifuged at 0 ° C and 1000 × g for 10 min. The supernatant was decanted and recentrifuged at 0 ° C and 25000 × g for 30 min, and the resulting pellet was resuspended in the buffer and recentrifuged under the same conditions. The pellet thus obtained (fraction P2; Whittaker and Barker, 1972) was stored at - 1 8 ° C for not longer than 48 h. For binding studies the pellet was reconstituted in the Tris-HC1 buffer to obtain a final protein concentration (measured according to Lowry et al., 1951) of approx. 1.2 mg/ml. The radioligand [3H]nitrendipine (NEN, specific activity 78.3 Ci/mmol), was prepared in six concentrations (final conc. 0.04-2 nM) in the buffer. The incubation mixture (final volume 550 /tl) consisted of 450 /zl of membrane suspension, 50/xl of a [3H]nitrendipine solution and 50/~1 of the buffer without (total binding) or with (nonspecific binding) nifedipine (final conc. 10/xM). The incubation was carried out in duplicate, in a shaking water-bath, at 25 ° C for 30 rain. Addition of the radioligand initiated the incubation which was terminated by vacuum-assisted filtration of the incubation mixture through G F / C Whatman fiberglass filters. The filters were then rinsed twice with 5 ml of ice-cold incubation buffer and placed in plastic scintillation minivials. Three
millilitres of Bray's fluid (Bray, 1960) were added and the samples were counted for radioactivity in a Beckman LS 3801 scintillation counter. Specific binding is defined as the difference between total and non-specific binding, and is expressed in f m o l / m g protein. The results were evaluated by Scatchard analysis for assessment of Bm~x and K D values.
2.5. Statistics The significance of changes between treatments was assessed with a one-way analysis of variance, followed, when appropriate (P < 0.05), with the Tukey-Kramer test. Student's t-test for paired comparisons was used to analyze body weight changes. The differences between pre- and postnaloxone weight are presented as percentages of the initial body weight.
3. Results
3.1. [~H]Nitrendipine binding 3.1.1. Binding site characteristics Over the concentration range used, all Scatchard plots were rectilinear (r coefficient ranging from
TABLE 1 The effect of chronic morphine treatment on specific [3H]nitrendipine binding to cerebral cortical and hippocampal membranes. N, number of independent Scatchard plots (six concentrations, in duplicate). F, variance ratio; df, degrees of freedom, v l / v 2. Group
N
Bmax ( f m o l / m g prot.) Mean + S.E.M.
% contr.
K d (nM) Mean ___S.E.M.
% contr.
Cerebral cortex Control Naloxone Morphine 1 Morphine 1 + naloxone F (dr 3/35)
16 8 8 7
89+ 5 93 + 8 107+ 7 127 + 13 4.81 (P < 0.01)
100 104 120 b 143 c
0.80+0.06 0.90 + 0.13 0.86 +0.06 0.90 + 0.08 0.43
100 112 108 l 12
tI ippocampus Control Naloxone Morphine 1 Morphine 1 + naloxone F (dr 3/12)
4 4 4 4
109 + 109 + 115 + 112 + 0.07
100 100 106 103
0.70 0.61 0.75 0.63 0.39
100 87 107 90
10 11 10 12
+ 0.09 + 0.09 + 0.13 + 0.10
a Morphine, 20 mg/kg, was given for 8 consecutive days; rats were killed 24 h after the last dose (morphine-dependent group) or were challenged with 2 m g / k g of naloxone and killed 60 rain later, b p < 0.1, c p < 0.01 (different from control, Tukey-Kramer test).
132 TABLE 2
TABLE 3
The effect of naloxone and nifedipine on the hot-plate latencies of morphine-naive rats. Nifedipine, 5 m g / k g , was given 45 min, and naloxone, 2 m g / k g , 30 min before the test. F, variance ratio; df, degrees of freedom, v J v 2 ; NS, not significant.
T h e effect of naloxone and nifedipine on the hot-plate latencies of rats receiving morphine chronically. Naloxone, 2 m g / k g i.p., was given on the 9th day, 24 h after the last morphine dose; the hot-plate test was carried out 30 min after naloxone administration. Nifedipine, 5 m g / k g , was given 15 min before naloxone. F, variance ratio; df, degrees of freedom, v l / v 2.
Treatment
Latency (s) Mean + S.E.M. (N)
% of control
Control Naloxone Nifedipine Nifedipine + naloxone
8.3 + 0.79 6.1 + 0.63 9.8 + 1.43 8.7 + 1.08
100 74 118 105
F (dr 3/37)
2.27 (NS)
(11) (10) (10) (10)
Treatment
Latency (s) Mean _+S.E.M. (N)
Control Morphine " Morphine ~ + naloxone Morphine ~ + naloxone + nifedipine F (df 3 / 5 6 )
0.87 to 0.95), suggesting homogeneity of [3H]nitrendipine binding sites. In control rats the density of [3H]nitrendipine binding sites in the hippocampus was 30% higher than in the cortex (P < 0.001), whereas the K d values for those sites in both brain areas were similar (table 1).
3.1.2. The effects of morphine abstinence An increase in [3H]nitrendipine binding site density of borderline significance (by - 20%, 0.1 > P > 0.05) was observed in morphine-dependent rats killed 24 h after the last dose of morphine. The density of cortical [3H]nitrendipine binding sites was significantly elevated (by over 40%, P < 0.01) in the rats with fully developed naloxoneprecipitated abstinence syndrome. No changes
% of control
8.5 + 0.68 (20) 4.9 +0.46 (10) 4.6 +_0.36 (20)
100 58 t, 54 b
9.7 +_0.86 (I0)
114
16.52 (P < 0.001)
Morphine, 20 m g / k g per day i.p., was given for 8 consecutive days. The hot-plate test was carried out 24 h after the last morphine treatment, b p < 0.01 (different from control, Tukey-K.ramer test).
were observed in the density of hippocampal [3H]nitrendipine binding sites. The Kd values remained unchanged in both brain areas (table 1).
3.2. Hot-plate latency 3.2.1. Effects of nifedipine and naloxone in naive rats Neither nifedipine nor naloxone, nor their combination significantly affected the nociceptive response (table 2).
TABLE 4 The effect of nifedipine on wet dog shakes and body weight loss induced by naloxone in morphine-dependent mice. Control rats received nine daily injections of 4 m l / k g i.p. of saline. Abstinent rats received morphine, 20 m g / k g , for 8 days and naloxone, 2 m g / k g , 24 h 'after the last morphine injection. Some of the abstinent rats received 5 m g / k g nifedipine 15 m m before naloxone. Rats tested for wet-dog shakes were observed for 60 rain, starting from the moment of the last injection. Rats tested for diarrhea were weighed immediately before the last injection and then 90 min later. All data are means + S.E.M. Oroup
Control Abstinent Abstinent, nifedipine pretreated F (df)
Body weight loss
Body weight (g) Wet dog shakes (shakes/60 rain)
Pre-naloxone
Post-naloxone
(initial body weight = 100)
2.0 + 0.9 (6) 12.0 + 1.6 (6) a
282.5 _ 4.1 (10) 249.9 + 4.5 (12) ~
281.0 ± 4.0 (10) 232.6 _+4.7 (12)
0.62 + 0.24 4.66 + 0.47 b
3.25:1.1 (6) 19.53 (2/15)
247.8+6.1 (9) ~ 15.55 (2/28)
247.8+6.3
0.00+__0.50
(9)
a p < 0.01 (different from controls, Tukey-Kramer test); h p < 0.001 (different from initial body weight, t-test for paired comparisons).
133
3.2.2. Effects in rats treated chronically with morphine The hot-plate latency of morphine-dependent rats was shortened by approx. 45%, as it was in the group treated with saline or challenged with naioxone after completion of the morphine treatment. Nifedipine treatment of morphine-abstinent rats normalized the latency of the hot-plate response (table 3). 3.2.3. The effects of nifedipine in naloxone-precipitated abstinence syndrome Chronic treatment with morphine slowed down the rate of body weight increase (table 3), but the morphine-treated rats did not display apparent signs of abstinence, such as increased wet-dog shakes or diarrhea, 24 h after the last dose. Naloxone administered to morphine-treated rats produced vigorous wet-dog shakes and diarrhea (measured as body weight loss). Nifedipine given 15 min before naloxone prevented the development of both parameters of the abstinence syndrome (table 4).
4. Discussion
The main finding of the present study is that the state of naloxone-precipitated morphine abstinence in the rat is accompanied by a significant increase in the density of [3H]nitrendipine binding sites in cortical membranes, concomitant with an increase in pain responsiveness and expression of characteristic behavioral changes and diarrhea. If [3H]nitrendipine binding reflects the functional state of voltage-dependent calcium channels (as postulated by Middlemiss and Spedding (1985), our results provide strong support for the notion that cortical voltage-dependent calcium channels play an important role in the mechanisms of abstinence. The involvement of voltage-dependent calcium channels in the opiate abstinence syndrome is also suggested by the observation that the main behavioral characteristics of the state of abstinence, i.e. increased sensitivity to pain in the hot-plate test, body weight loss (due presumably to diarrhea), and characteristic behavior (wet-dog
shakes), were completely prevented by a dihydropyridine calcium channel blocker given shortly before the opiate antagonist. This finding, which corroborates the results of others (Baeyens et al., 1987; Bongianni et al., 1986; Caro et al., 1988; Pellegrino-Giampietro et al,, 1988; Ramkumar and El-Fakahany, 1988), suggests that calcium channel blockers should be tested in the treatment of opiate addicts. The present study confirms an earlier report of Ramkumar and El-Fakahany (1988) that chronic administration of morphine leads to an increase in the density of rat brain dihydropyridine binding sites. The difference between their results and ours is small. We observed less marked changes in morphine-dependent animals in which full behavioral abstinence syndrome had not been precipitated with naloxone: no increase in hippocampal [3H]nitrendipine binding, and a non-significant (0.05 < P < 0.1) increase in the cortex (although quantitatively comparable to that observed by Ramkumar and EI-Fakahany (1988)). A close scrutiny of the data of the latter authors suggests that the increase in the density of calcium channels during morphine tolerance is not a robust phenomenon, and that it is very dependent on the dose and time of treatment. Thus, Ramkumar and E1-Fakahany (1988) observed significant increases in dihydropyridine binding site density only in rats implanted with three 75 mg morphine pellets, while no changes were observed in animals treated with one or two pellets. Moreover, the effect ceased to be significant if the treatment lasted more than 3 days. In our experiment morphine was administered in a manner mo(,e resembling its clinical and illicit use, i.e. the dose was relatively low and the period of treatment was much longer. As the increase in the density of [3H]nitrendipine binding sites took place 60 min after naloxone was injected into the morphine-dependent rat, it seems that the change may be attributed to the degree of the state of abstinence rather than dependence. Although the animals tested 24 h after the last injection of morphine without naloxone challenge did not display vigorous wet-dog shakes, the possibility that they already were in a state of mild abstinence cannot be excluded. The increase in their responsiveness to pain stimuli measured with
134
the hot-plate test strongly suggests such a possibility. As mentioned in the Introduction, several data indicate that calcium antagonizes the antinociceptire action of opiates and suggest the involvement of calcium in pain perception (see, e.g., Chapman and Way, 1982; Crowder et al., 1986; GuerreroMu~aoz et al., 1981; Harris et al., 1975; Kamikubo et al., 1988; Miller, 1987; Ross and Cardenos, 1979). These reports show that opioid-induced analgesia is accompanied by a decrease in synaptosomal calcium concentrations. Intraventricular administration of calcium, however, does not affect hot-plate or tail-flick responses (GuerreroMufioz et al., 1981). However, Ueda et al. (1987), who found that naloxone enhanced the potassium-evoked entry of calcium into synaptosomes and the release of substance P in spinal cord of mice with implanted morphine pellets but not from control mice, concluded that the jumping that occurs after naloxone administration to morphine-dependent mice is caused by elevated substance P release due to increased calcium entry into nerve terminals. The present results demonstrate that the increase in the [3H]nitrendipine binding site density in the cortical membranes of abstinent rats is accompanied by a marked reduction in the hot-plate latency, and that a similar, though less marked, relationship exis'ts in 'dependent' rats. These results suggest that the increase in pain perception may result from facilitation of calcium access into the neurons.
Acknowledgements This study was supported by Grant No. CPBP 06.02.I.2. The technical assistance of Mrs. M. Kafel and Mr. K. Michalski is gratefully acknowledged.
References Baeyens, J.M., E. F~posito, G. Ossowska and R. Samanin, 1987, Effects of peripheral and central administration of calcium channel blockers in the naloxone-precipitated abstinence syndrome in morphine-dependent rats, European J. Pharmacol. 137, 9.
Belleman, P., D. Ferrz, F. Lubbecke and H. Glossmarm, 1981, 3H-Nitrendipine, a potent calcium antagonist binds with high affinity to cardiac membranes, Arzneim. Forsch. 31, 2064. Benedek, G. and M. Szikszay, 1984, Potentiation of thermoregulatory and analgesic effects of morphine by calcium antagonists, Pharmacol. Res. Commun. 16, 1009. Bongianni, F., V. Carla, F. Moroni and D.E. PellegriniGiampietro, 1986, Calcium channel inhibitors suppress the morphine-withdrawal syndrome in rats, Br. J. Pharrnacol. 88, 561. Bray, G.A., 1960, A simple efficient tiquid scintillator for counting solutions in a liquid scintillation counter, Anal. Biochem. ], 279. Caro, G., M. Barrios and J.M. Bayens, 1988, Dose-dependent and stereoselective antagonism by diltiazem of naloxoneprecipitated morphine abstinence after acute morphine dependence in vivo and in vitro, Life Sci. 43, 1523. Chapman, D. and E. Way, 1982, Modification of endorphin/ enkephalin analgesia and stress-induced analgesia by divalent cations, a cation chelator and a ionophore, Br. J. Pharmacol. 75, 389. Contreras, E., L. Tamao and M. Amigo, 1988, Calcium channeI antagonists increase morphine-induced analgesia and antagonize morphine tolerance, European J. Pharmacol. 148, 463. Crowder, J., D. Norris and H. Bradford, 1986, Morphine inhibition of calcium fluxes, neurotransmitter release and protein and lipid phosphorytation in brain slices and synaptosomes, Neuropharmaco~ogy 35, 2501. Guerrero-Mufioz, F., C. Adames, Z. Fearon and E.L. Way, 1981, Calcium-opiate antagonism in the periaqueductal grey (PGA) region, European J. Pharmacol. 76, 417. Guerrero-Mu~oz, F., K. Cerreta, M. Guerrero and E. Way, 1979, Effect of morphine on synaptosomal Ca ++ uptake, J. Pharmacol. Exp. Ther. 209, 132. Harris, R.A., H.H. Loh and E.L. Way, 1975, lZffect of divalent cations, cation chelators and an ionophore on morphine analgesia and tolerance, J. Pharmacol. Exp. Ther. 195, 488. Harris, R.A., H. Yamamoto, H. Loh and E.L. Way, 1977, Discrete changes in brain calcium with morphine analgesia, tolerance-dependence and abstinence, Life Sci. 20, 501. Hoffmeister, F. and D. Tettenborn, 1986, Calcium agonists and antagonists of dihydropyridine type: antinociceptive effects, interference with opiate-/~-receptor agonists and nearopharmacological action in rodents, Psychopharmacology 90, 299. Kamikubo, K., M. Niwa, H. Fugimnra and K. Miura, ]983, Morphine inhibits depolarization-dependent calcium uptake by synaptosomes, European J. Pharmacol. 95, 149. Kavaliers, M., 1987a, Aggression and defeat-induced opioid analgesia in mice are modified by calcium channel antagonists and agonists, Neurosci. Lett. 74, 107. KavaU.ers, M., 1987b, Stimulatory influences of calcium channel antagonists on stress-induced opioid analgesia and locomotor activity, Brain Res. 408, 403.
135 Kavaiiers, M. and K.-P. Ossenkopp, 1987, Calcium channel involvement in magnetic field inhibition of morphine-induced analgesia, Naunyn-Schmiedeb. Arch. Pharmacol. 336, 308. Lowry, O.H., N.J. Rosebrough, A.L. Farr, ILL Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Middlemiss, D., 1985, The calcium channel activator, Bay K8644, enhances K ÷ evoked efflux of acetylcholine and noradrenaline from brain slices, Naunyn-Schmiedeb. Arch. Pharmacol. 331, 114. Middlemiss, D. and M. Spedding, 1985, A functional correlate for the dihydropyridine binding site in rat brain, Nature 314, 94. Miller, R.J., 1987, Multiple calcium channels and neuronal functions, Science 325, 46. North, R.A., 1986, Opioid receptor types and membrane ion channels, Trends Neurosci. 9, 114. Pellegrini-Giampietro, D.E., L. Bacciotini, V. Carlh and F. MoroN, 1988, Morphine withdrawn in cortical slices: suppression by Ca 2 %channel inhibitors of abstinence-induced [3Hl-noradrenaline release, Br. J. Pharmacol. 93, 535.
Ramkumar, V. and E. E1-Fakahany, 1988, Prolonged morphine treatment increases rat brain dihydropyridine binding sites: possible involvement in development of morphine dependence, European J. Pharmacol. 146, 73. Reuter, H., C.F. Stevens, R.W. Tsiern and G. Yellen, 1982, Properties of single calcium channels in cardiac cell culture, Nature 297, 501. Ross, D.H. and H.L. Cardenos, 1979, Nerve cell calcium as a mes~nger for opiate and endorphin actions, Adv. Biochem. PsychopharmacoL 20, 301. Ueda, H., S. Tamura, M. Satoh and H. Takagi, 1987, Excess release of substance P from the spinal cord of mice during morphine withdrawal and involvement of the enhancement of presynaptic Ca 2+ entry, Brain Res. 425, 101. Whittaker, V.P. and L.A. Barker, 1972, The subcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles, in: Methods of Neurochemistry, Vol. 2, ed. R. Fried (Marcel Dekker, New York) p. 1.
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Elsevier EJP 51280
Cortical dihydropyridine binding sites and a behavioral syndrome in morphine-abstinent rats L u c y n a A n t k i e w i c z - M i c h a l u k , J e r z y M i c h a l u k , I r e n a R o m a f l s k a , Jerzy Vetulani Department of Biochemistry, Institute of Pharmacology, Polish A eademy of Sciences, Smetna 12, PL-31-343 Krakbw, Poland
Received 19 October 1989, revisedMS received24 January 1990, accepted 13 February 1990
The density of cortical [3H]nitrendipine binding sites was elevated by over 40% in rats rendered morphine-abstinent by administration of naloxone after chronic treatment with morphine. The morphine-abstinent rats had significantly shortened response latencies in the hot-plate test. Nifedipine treatment abolished the signs of abstinence and normalized the hot-plate latencies in morphine-dependent, naloxone-treated rats. The results indicate that the symptoms of abstinence are related to a functional state of cortical dihydropyridine-sensitive calcium channels. Ca 2÷ channels; Morphine abstinence; Pain sensitivity
I. Introduction
Calcium channels in cell membranes regulate the concentration of cytosolic calcium and serve to link membrane potential changes with cellular responses (Miller, 1987; Reuter et al., 1982). As voltage-dependent calcium channels are associated with the binding sites for dihydropyridine calcium antagonists (BeUeman et al., 1981; Middlemiss, 1985; Middlemiss and Spedding, 1985), they may conveniently be studied by investigation of the specific binding of [3H]nitrendipine. Several data indicate that calcium plays an important role in nociception and in the action of opiates (Ross and Cardenos, 1979). Recent data suggest that opiate receptors are functionally coupled to voltage-sensitive ion channels and that stimulation of opiate K receptors directly, and of/~ receptors indirectly, suppresses calcium conduc-
Correspondence to: J. Vetulani, Department of Biochemistry, Institute of Pharmacology,Polish Academyof Sciences,Smetna 12, PL-31-343Krak6w, Poland.
tion (North, 1986). Morphine inhibits synaptosomal calcium uptake (Guerrero-Mu~oz et al., 1979; Kamikubo et al., 1988) and an acute injection of morphine depletes calcium in whole brain synaptosomes (Harris et al., 1977). It is possible that the inhibition of calcium uptake is related to the analgesic effect of opioids, as calcium injected into the periaqueductal gray or into the ventricles antagonizes opioid-induced analgesia and this action is facilitated by a calcium ionophore (Chapman and Way, 1982; Guerrero-Mu~oz et al., 1981; Harris et al., 1975). Conversely, the chelating agents, EGTA and EDTA, and calcium channel inhibitors enhance the analgesic effects of opioids and opioid-mediated stress analgesia (Benedek and Szikszay, 1984; Chapman and Way, 1982; Contreras et al., 1988; Harris et al., 1975; Hoffmeister and Tettenborn, 1986; Kavaliers, 1987a; Kavaliers and Ossenkopp, 1987). Calcium channel antagonists also counteract other effects of morphine, such as running fit in certain mouse strains (Kavaliers, 1987b), hypothermia in restrained rats (Benedek and Szikszay, 1984), and signs of morphine abstinence in vivo and in vitro (Baeyens et al., 1987; Bongianni et al., 1986; Caro et al.,
0014-2999/90/$03.50 © 1990 ElsevierScience Publishers B.V. (Biomedical Division)
130
1988; Pellegrini-Giampietro et al., 1988; Ramkumar and El-Fakahany, 1988). The chronic effects of morphine are often opposite to the effects of a single high dose. Thus, chronic morphine treatment increases the vesicular content of calcium (Harris et al., 1977). Moreover, potassium-stimulated synaptosomal calcium uptake, which is inhibited by a single dose of morphine, is increased in mice chronically treated with morphine (Guerrero-Mu~aoz et al., 1979). Recently, Ramkumar and E1-Fakahany (1988) reported that morphine implants increased the density of [3H]nimodipine binding sites in several cerebral structures. These changes may represent compensatory responses and may be related to the development of tolerance to the actions of morphine. In this study we investigated the effect of m o r p h i n e abstinence on voltage-dependent calcium channels in the brain, using [3H]nitrendipine as a radioligand to label L-type calcium channels in the cortex and hippocampus. We also measured the effect of morphine abstinence on the responsiveness of rats to noxious stimuli in the hot-plate test, and the effect of a dihydropyridine calcium channel blocker, nifedipine, to find out if there is a relation between the changes in the density of dihydropyridine calcium channels in some brain areas and the abstinence syndrome.
2. Materials and methods
2.1. Animals The experiments were carried out with male Wistar rats, weighing 200-230 g at the beginning of experiment, kept under standard laboratory conditions (approx. 22°C, natural day-night cycle), ten to a home cage (large plastic cage with wire lid and sawdust bedding), with free access to standard laboratory food and tap water. 2.2. Drugs and drug treatment All injections were given in a volume of 4 m l / k g i.p. Morphine (hydrochloride, Polfa) was given in a daily dose of 20 m g / k g for 8 consecu-
tive days (controls received saline). The rats were tested 24 h after the last injection of morphine; some animals were injected with naloxone (hydrochloride, Endo), 2 mg/kg, 24 h after the last dose of morphine. The rats used for biochemical assay were decapitated 60 min after the naloxone injection. The control naloxone-treated group received a single injection of the drug 60 min before death. Nifedipine (Polfa), 5 mg/kg, was given 15 min before naloxone. In an acute experiment naloxone was given 30 rain, and nifedipine 45 min, before the tests. 2.3. Behavioral tests The responsiveness of rats to a painful stimulus was assessed using the hot-plate test. Twenty-four hours after the last dose of morphine, the rats were placed on a metal plate kept at 56 ° C and the latency of response (licking of hind paws or jumping) was measured. The cut-off time was 30 s. The rats challenged with naloxone received it 30 min before the test. The rats tested for 'wet-dog' shakes were observed for 60 min in wire-mesh cage and the number of shakes was recorded by two independent observers. The animals tested for diarrhea were weighed before naloxone was injected and then 90 rain later. The difference in body weight is presented as the per cent of initial body weight. The rats used for the behavioral tests were not used for the biochemical experiments to avoid the possibility that the stress of testing affected the state of the calcium channels. 2.4. Membrane preparation and receptor binding assay The brain was rapidly removed, placed on an ice-chilled porcelain plate, and the cerebral cortex and hippocampus were dissected. The tissues were homogenized at 0 ° C in 20 vol of 50 mmolfll Tris-HC1 buffer, pH (at 23 ° C) 7.6 with a Polytron disintegrator (setting 4, 10 s). The cortex from each animal was homogenized separately; the hippocampal tissue from two animals was pooled for membrane preparation.
131
The homogenate was centrifuged at 0 ° C and 1000 × g for 10 min. The supernatant was decanted and recentrifuged at 0 ° C and 25000 × g for 30 min, and the resulting pellet was resuspended in the buffer and recentrifuged under the same conditions. The pellet thus obtained (fraction P2; Whittaker and Barker, 1972) was stored at - 1 8 ° C for not longer than 48 h. For binding studies the pellet was reconstituted in the Tris-HC1 buffer to obtain a final protein concentration (measured according to Lowry et al., 1951) of approx. 1.2 mg/ml. The radioligand [3H]nitrendipine (NEN, specific activity 78.3 Ci/mmol), was prepared in six concentrations (final conc. 0.04-2 nM) in the buffer. The incubation mixture (final volume 550 /tl) consisted of 450 /zl of membrane suspension, 50/xl of a [3H]nitrendipine solution and 50/~1 of the buffer without (total binding) or with (nonspecific binding) nifedipine (final conc. 10/xM). The incubation was carried out in duplicate, in a shaking water-bath, at 25 ° C for 30 rain. Addition of the radioligand initiated the incubation which was terminated by vacuum-assisted filtration of the incubation mixture through G F / C Whatman fiberglass filters. The filters were then rinsed twice with 5 ml of ice-cold incubation buffer and placed in plastic scintillation minivials. Three
millilitres of Bray's fluid (Bray, 1960) were added and the samples were counted for radioactivity in a Beckman LS 3801 scintillation counter. Specific binding is defined as the difference between total and non-specific binding, and is expressed in f m o l / m g protein. The results were evaluated by Scatchard analysis for assessment of Bm~x and K D values.
2.5. Statistics The significance of changes between treatments was assessed with a one-way analysis of variance, followed, when appropriate (P < 0.05), with the Tukey-Kramer test. Student's t-test for paired comparisons was used to analyze body weight changes. The differences between pre- and postnaloxone weight are presented as percentages of the initial body weight.
3. Results
3.1. [~H]Nitrendipine binding 3.1.1. Binding site characteristics Over the concentration range used, all Scatchard plots were rectilinear (r coefficient ranging from
TABLE 1 The effect of chronic morphine treatment on specific [3H]nitrendipine binding to cerebral cortical and hippocampal membranes. N, number of independent Scatchard plots (six concentrations, in duplicate). F, variance ratio; df, degrees of freedom, v l / v 2. Group
N
Bmax ( f m o l / m g prot.) Mean + S.E.M.
% contr.
K d (nM) Mean ___S.E.M.
% contr.
Cerebral cortex Control Naloxone Morphine 1 Morphine 1 + naloxone F (dr 3/35)
16 8 8 7
89+ 5 93 + 8 107+ 7 127 + 13 4.81 (P < 0.01)
100 104 120 b 143 c
0.80+0.06 0.90 + 0.13 0.86 +0.06 0.90 + 0.08 0.43
100 112 108 l 12
tI ippocampus Control Naloxone Morphine 1 Morphine 1 + naloxone F (dr 3/12)
4 4 4 4
109 + 109 + 115 + 112 + 0.07
100 100 106 103
0.70 0.61 0.75 0.63 0.39
100 87 107 90
10 11 10 12
+ 0.09 + 0.09 + 0.13 + 0.10
a Morphine, 20 mg/kg, was given for 8 consecutive days; rats were killed 24 h after the last dose (morphine-dependent group) or were challenged with 2 m g / k g of naloxone and killed 60 rain later, b p < 0.1, c p < 0.01 (different from control, Tukey-Kramer test).
132 TABLE 2
TABLE 3
The effect of naloxone and nifedipine on the hot-plate latencies of morphine-naive rats. Nifedipine, 5 m g / k g , was given 45 min, and naloxone, 2 m g / k g , 30 min before the test. F, variance ratio; df, degrees of freedom, v J v 2 ; NS, not significant.
T h e effect of naloxone and nifedipine on the hot-plate latencies of rats receiving morphine chronically. Naloxone, 2 m g / k g i.p., was given on the 9th day, 24 h after the last morphine dose; the hot-plate test was carried out 30 min after naloxone administration. Nifedipine, 5 m g / k g , was given 15 min before naloxone. F, variance ratio; df, degrees of freedom, v l / v 2.
Treatment
Latency (s) Mean + S.E.M. (N)
% of control
Control Naloxone Nifedipine Nifedipine + naloxone
8.3 + 0.79 6.1 + 0.63 9.8 + 1.43 8.7 + 1.08
100 74 118 105
F (dr 3/37)
2.27 (NS)
(11) (10) (10) (10)
Treatment
Latency (s) Mean _+S.E.M. (N)
Control Morphine " Morphine ~ + naloxone Morphine ~ + naloxone + nifedipine F (df 3 / 5 6 )
0.87 to 0.95), suggesting homogeneity of [3H]nitrendipine binding sites. In control rats the density of [3H]nitrendipine binding sites in the hippocampus was 30% higher than in the cortex (P < 0.001), whereas the K d values for those sites in both brain areas were similar (table 1).
3.1.2. The effects of morphine abstinence An increase in [3H]nitrendipine binding site density of borderline significance (by - 20%, 0.1 > P > 0.05) was observed in morphine-dependent rats killed 24 h after the last dose of morphine. The density of cortical [3H]nitrendipine binding sites was significantly elevated (by over 40%, P < 0.01) in the rats with fully developed naloxoneprecipitated abstinence syndrome. No changes
% of control
8.5 + 0.68 (20) 4.9 +0.46 (10) 4.6 +_0.36 (20)
100 58 t, 54 b
9.7 +_0.86 (I0)
114
16.52 (P < 0.001)
Morphine, 20 m g / k g per day i.p., was given for 8 consecutive days. The hot-plate test was carried out 24 h after the last morphine treatment, b p < 0.01 (different from control, Tukey-K.ramer test).
were observed in the density of hippocampal [3H]nitrendipine binding sites. The Kd values remained unchanged in both brain areas (table 1).
3.2. Hot-plate latency 3.2.1. Effects of nifedipine and naloxone in naive rats Neither nifedipine nor naloxone, nor their combination significantly affected the nociceptive response (table 2).
TABLE 4 The effect of nifedipine on wet dog shakes and body weight loss induced by naloxone in morphine-dependent mice. Control rats received nine daily injections of 4 m l / k g i.p. of saline. Abstinent rats received morphine, 20 m g / k g , for 8 days and naloxone, 2 m g / k g , 24 h 'after the last morphine injection. Some of the abstinent rats received 5 m g / k g nifedipine 15 m m before naloxone. Rats tested for wet-dog shakes were observed for 60 rain, starting from the moment of the last injection. Rats tested for diarrhea were weighed immediately before the last injection and then 90 min later. All data are means + S.E.M. Oroup
Control Abstinent Abstinent, nifedipine pretreated F (df)
Body weight loss
Body weight (g) Wet dog shakes (shakes/60 rain)
Pre-naloxone
Post-naloxone
(initial body weight = 100)
2.0 + 0.9 (6) 12.0 + 1.6 (6) a
282.5 _ 4.1 (10) 249.9 + 4.5 (12) ~
281.0 ± 4.0 (10) 232.6 _+4.7 (12)
0.62 + 0.24 4.66 + 0.47 b
3.25:1.1 (6) 19.53 (2/15)
247.8+6.1 (9) ~ 15.55 (2/28)
247.8+6.3
0.00+__0.50
(9)
a p < 0.01 (different from controls, Tukey-Kramer test); h p < 0.001 (different from initial body weight, t-test for paired comparisons).
133
3.2.2. Effects in rats treated chronically with morphine The hot-plate latency of morphine-dependent rats was shortened by approx. 45%, as it was in the group treated with saline or challenged with naioxone after completion of the morphine treatment. Nifedipine treatment of morphine-abstinent rats normalized the latency of the hot-plate response (table 3). 3.2.3. The effects of nifedipine in naloxone-precipitated abstinence syndrome Chronic treatment with morphine slowed down the rate of body weight increase (table 3), but the morphine-treated rats did not display apparent signs of abstinence, such as increased wet-dog shakes or diarrhea, 24 h after the last dose. Naloxone administered to morphine-treated rats produced vigorous wet-dog shakes and diarrhea (measured as body weight loss). Nifedipine given 15 min before naloxone prevented the development of both parameters of the abstinence syndrome (table 4).
4. Discussion
The main finding of the present study is that the state of naloxone-precipitated morphine abstinence in the rat is accompanied by a significant increase in the density of [3H]nitrendipine binding sites in cortical membranes, concomitant with an increase in pain responsiveness and expression of characteristic behavioral changes and diarrhea. If [3H]nitrendipine binding reflects the functional state of voltage-dependent calcium channels (as postulated by Middlemiss and Spedding (1985), our results provide strong support for the notion that cortical voltage-dependent calcium channels play an important role in the mechanisms of abstinence. The involvement of voltage-dependent calcium channels in the opiate abstinence syndrome is also suggested by the observation that the main behavioral characteristics of the state of abstinence, i.e. increased sensitivity to pain in the hot-plate test, body weight loss (due presumably to diarrhea), and characteristic behavior (wet-dog
shakes), were completely prevented by a dihydropyridine calcium channel blocker given shortly before the opiate antagonist. This finding, which corroborates the results of others (Baeyens et al., 1987; Bongianni et al., 1986; Caro et al., 1988; Pellegrino-Giampietro et al,, 1988; Ramkumar and El-Fakahany, 1988), suggests that calcium channel blockers should be tested in the treatment of opiate addicts. The present study confirms an earlier report of Ramkumar and El-Fakahany (1988) that chronic administration of morphine leads to an increase in the density of rat brain dihydropyridine binding sites. The difference between their results and ours is small. We observed less marked changes in morphine-dependent animals in which full behavioral abstinence syndrome had not been precipitated with naloxone: no increase in hippocampal [3H]nitrendipine binding, and a non-significant (0.05 < P < 0.1) increase in the cortex (although quantitatively comparable to that observed by Ramkumar and EI-Fakahany (1988)). A close scrutiny of the data of the latter authors suggests that the increase in the density of calcium channels during morphine tolerance is not a robust phenomenon, and that it is very dependent on the dose and time of treatment. Thus, Ramkumar and E1-Fakahany (1988) observed significant increases in dihydropyridine binding site density only in rats implanted with three 75 mg morphine pellets, while no changes were observed in animals treated with one or two pellets. Moreover, the effect ceased to be significant if the treatment lasted more than 3 days. In our experiment morphine was administered in a manner mo(,e resembling its clinical and illicit use, i.e. the dose was relatively low and the period of treatment was much longer. As the increase in the density of [3H]nitrendipine binding sites took place 60 min after naloxone was injected into the morphine-dependent rat, it seems that the change may be attributed to the degree of the state of abstinence rather than dependence. Although the animals tested 24 h after the last injection of morphine without naloxone challenge did not display vigorous wet-dog shakes, the possibility that they already were in a state of mild abstinence cannot be excluded. The increase in their responsiveness to pain stimuli measured with
134
the hot-plate test strongly suggests such a possibility. As mentioned in the Introduction, several data indicate that calcium antagonizes the antinociceptire action of opiates and suggest the involvement of calcium in pain perception (see, e.g., Chapman and Way, 1982; Crowder et al., 1986; GuerreroMu~aoz et al., 1981; Harris et al., 1975; Kamikubo et al., 1988; Miller, 1987; Ross and Cardenos, 1979). These reports show that opioid-induced analgesia is accompanied by a decrease in synaptosomal calcium concentrations. Intraventricular administration of calcium, however, does not affect hot-plate or tail-flick responses (GuerreroMufioz et al., 1981). However, Ueda et al. (1987), who found that naloxone enhanced the potassium-evoked entry of calcium into synaptosomes and the release of substance P in spinal cord of mice with implanted morphine pellets but not from control mice, concluded that the jumping that occurs after naloxone administration to morphine-dependent mice is caused by elevated substance P release due to increased calcium entry into nerve terminals. The present results demonstrate that the increase in the [3H]nitrendipine binding site density in the cortical membranes of abstinent rats is accompanied by a marked reduction in the hot-plate latency, and that a similar, though less marked, relationship exis'ts in 'dependent' rats. These results suggest that the increase in pain perception may result from facilitation of calcium access into the neurons.
Acknowledgements This study was supported by Grant No. CPBP 06.02.I.2. The technical assistance of Mrs. M. Kafel and Mr. K. Michalski is gratefully acknowledged.
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