Prolonged morphine treatment increases rat brain dihydropyridine binding sites: possible involvement in development of morphine dependence

European Journal of Pharmacology, 146 (1988) 73-83

73

Elsevier

PJP 50111

Prolonged morphine treatment increases rat brain dihydropyridine binding sites: possible involvement in development of morphine dependence I Vickram Ramkumar

2 . . a n d E s a m E. E I - F a k a h a n y 3

Department of Pharmacology and Toxicology, Unwersi(v of MaD'land School of Pharmacy, Baltimore. MD 21201, U.S.A. Received 6 July 1987. revised MS received 13 October 1987, accepted 10 November 1987

Regulation of L-type Ca 2+ channels by morphine in rat brain was determined by the binding of [3H]nimodipine. Morphine, administered by subcutaneous pellet implantation, increased the density of [3 H]nimodipine binding sites in a time- and dose-dependent manner and this effect was reversible upon removal of the pellets. Increases in these dihydropyridine sites were localized to the cortex, hippocampus, hypothalamus and brainstem but not to the cerebellum and striatum. Additional experiments were performed to test the ability of different Ca 2- channel antagonists to affect naloxone-precipitated withdrawal in morphine-dependent mice and rats. These drugs effectively reduced the incidence of naloxone-induced jumping in mice and several of the withdrawal signs in rats. Taken together, our study underscores the plasticity of brain L-type Ca z + channels and suggests that their upregulation might contribute to morphine dependence. Dihydropyridine; Nimodipine; Calcium channels (L-type); Opiates; Morphine; (Dependence)

I. I n t r o d u c t i o n Several pieces of evidence support an involvement of Ca 2 ÷ in the actions of opiates. For example, acute morphine administration in rats decreases the Ca 2+ content in several brain regions (Ross and Cardenas, 1979). The loss in Ca 2+ content induced by morphine is localized to brain cellular fractions rich in nerve endings (Cardenas and Ross, 1976; Harris et al., 1977). In addition, opiates inhibit Ca 2+ uptake into synaptosomal

Supported by a grant from Miles Laboratories. 2 Recipient of the Emerson Fellowship from the University of Maryland School of Pharmacy during the course of this study. * To whom all correspondence should be addressed at present address: Department of Medicine, Duke University Medical Center, Box 3444, Durham, NC 27710, U.S.A. 3 Recipient of a Research Career Development Award from the National Institutes of Health (AG-00344).

preparations ( G u e r r e r o - M u n o z et al., 1979; K a m i k u b o et al., 1983). These effects of opiates might contribute to their analesic properties, as supported by the following findings. EGTA, a Ca 2+ chelator (Kakunaga et al., 1966) and trivalent cation Ca 2÷ channel blockers (Reddy and Yaksh, 1980), produce analgesia when administered intracerebroventricularly (i.c.v.) and intrathecally, respectively. In contrast, i.c.v, injections of Ca 2-, Mn 2- and Mg 2' potentiate the hyperalgesic response in morphine-dependent mice (Schmidt and Way, 1980), while the Ca 2+ ionophore, A23187, antagonizes fl-endorphin analgesia (Chapman and Way, 1982). The effects of chronic opiate administration differ considerably from those described above. For example, chronic morphine administration to animals increases vesicular Ca 2÷ content (Harris et al., 1977). Furthermore, K+-stimulated Ca 2~ uptake in synaptosomes is significantly increased by chronic levorphanol treatment in rats (Ross et

0014-2999/88/$03.50 '?:' 1988 Elsevier Science Publishers B.V. (Biomedical Division)

74 al., 1977) and by chronic morphine treatment in mice (Guerrero-Munoz et al., 1979). These latter changes presumably represent compensatory measures to offset the inhibitory actions by morphine on Ca 2. fluxes. Since K+-stimulated Ca 2+ uptake in synaptosomes is mediated by voltage-sensitive Ca 2+ channels (Blaustein, 1975), we decided to test the hypothesis that these channels are upregulated consequent to chronic administration of morphine in rats. It has previously been demonstrated that morphine treatment increases the density of mouse brain dihydropyridine sites (Ramkumar and El-Fakahany, 1984). In addition, the ability of Ca 2÷ channel antagonists to attenuate morphine withdrawal signs in rats and mice was determined. [3H]Nimodipine was used as a radioligand to label voltage-sensitive Ca 2+ channel (L-type) in rat brain and heart.

2. Materials and methods 2.1. Materials

Drugs were obtained from the following sources: [3H]nimodipine (130.5 Ci/mmol), New England Nuclear (Boston, MA); nifedipine, Pfizer (New York, NY); nitrendipine, nimodipine and nisoldipine were gifts from Dr. Alexander Scriabine, Miles Pharmaceuticals (West Haven, CT); verapamil, Knoll Pharmaceutical Co. (Whippany, N J); morphine, Mailinkrodt (St. Louis, MO); naloxone was a gift from Dr. Victor Nickolson, DuPont (Wilmington, DE) and clonidine, Sigma Chemical Company (St. Louis, MO). The following drugs were dissolved in sterile saline at the specified concentrations: morphine sulfate (10-50 mg/ml), naloxone hydrochloride (0.25-1.0 mg/ml) and clonidine hydrochloride (25-250 p,g/ml). The Ca 2+ channel antagonists, verapamil, nimodipine, nifedipine and nisoldipine were dissolved in polyethylene glycol to give a final concentration of 2-3 mg/ml. All drugs were prepared fresh just prior to use. 2.2. Drug treatment

Radioligand binding studies in the rat (male

Sprague-Dawley, 250-275 g, Dominion Labs) were conducted in animals administered subcutaneous (s.c.) implantation of placebo or morphine pellets. Morphine pellets (75 mg base) were prepared according to the method of Gibson and Tingstad (1970). Except for an equal weight of Avicei substituted for morphine, placebo pellets contained all other ingredients present in morphine pellets. In order to determine the appropriate dose and duration of morphine treatment needed for maximal increase in [~H]nimodipine binding, rats were implanted with 1-3 morphine pellets (treated) or 3 placebo pellets (control) for 1-4 days and tissue homogenates thus obtained were used for radioligand binding studies. Results from these experiments indicated that the optimal increase in binding occurred following implantation of 3 morphine pellets for 3 days (fig. 2 of Results). Therefore, all subsequent radioligand binding experiments were performed using this treatment paradigm. The effect of morphine withdrawal on [3H]nimodipine binding in rat brain was determined by removal of pellets 1, 3 and 10 days following a 3 day treatment period. For behavioral studies, mice were each implanted with a placebo pellet (control) or a morphine pellet (treated) for 3 days. Initially, an i.p. morphine treatment schedule was attempted, as described below for rats. However, we were unable to produce an equivalent degree of dependence under this paradigm as observed via pellet implantation. Rats were injected with increasing doses of morphine over a 6 day period to produce dependence. Starting with 3 i.p. injections of 10 mg/kg on the first day, the dose of morphine was progressively increased to 20, 50, 100, 100 and 200 mg/kg on consecutive days. This latter treatment schedule was chosen over s.c. pellet implantation to avoid complicating the interpretation of the anti-withdrawal action of the Ca 2" channel antagonists, resulting from the release of morphine from pellets. In addition, naloxone-precipitated withdrawal in rats was initiated 6-8 h following the last morphine injection to limit the interference of this drug in the behavioral studies. Preliminary experiments indicated - 30% increase in brain dihydropyridine binding sites under this treatment paradigm.

75

2.3. Preparation of tissue homogenates Morphine-dependent rats were decapitated and their brains and hearts were removed rapidly on ice, rinsed and weighed. Hearts were minced with a razor blade before homogenization. Brains (without cerebellum and brainstem) were homogenized whole or dissected into the cerebral cortex, striatum, hippocampus, brainstem, hypothalamus and cerebellum according to the method of Glowinski and Iversen (1966). All tissues were homogenized at 4°C by Polytron (Brinkmann, Setting 7, 30 s) in 50 mM Tris-HCl buffer (pH 7.4). Homogenates were centrifuged at 1 000 × g for 10 min at 4°C, following which the supernatant was centrifuged at 30000 × g for 30 min. Pellets were resuspended in Tris buffer to give a working concentration of 5% (w/v) for [3H]nimodipine binding experiments.

2.4. [~H]Nimodipine binding assay Saturation studies were performed by incubating membranes (0.2 mg protein/assay tube) with 5-8 concentrations (0.05-1.0 nM) of [3H]nimodipine in a final volume of 1 ml of 50 mM Tris-HCl buffer (pH 7.4). Incubations were carried out in triplicate for 90 rain at room temperature ( 22 ° C). Due to the light sensitivity of [3H]nimodipine, experiments were performed under faint yellow lighting. Following incubation, samples were filtered under vacuum through G F / C glass fiber filters using a Cell Harvester (Brandel, Gaithersburg, MD) and then washed 3 times with 3 ml of ice-cold buffer. Each filter was extracted for at least 6 h in 4 ml of Budget Solve Scintillation fluid (Research Product International, Mount Prospect, IL) and its radioactive content was measured by liquid scintillation spectrometry in a Beckman LS6800 liquid scintillation counter at about 50% counting efficiency. Specific binding was calculated as the difference between total binding and non-specific binding determined in the presence of 1 ~M nifedipine.

2.5. Analgesic test Rats were implanted with 3 placebo pellets or 3 morphine pellets and analgesia was determined

using a hot plate at 55°C. Hind paw lick was taken as the appropriate response to the thermal nociceptive stimulus. A cut-off time of 60 s was used. Each rat was tested twice, 15 rain apart, 2, 26 and 50 h after pellet implantation

2.6. Naloxone-precipitated withdrawal in morphinedependent rats and mice Mice and rats were made dependent on morphine as described above. Morphine-dependent mice were administered vehicle (polyethylene glycol or saline), nimodipine (5 and 10 mg/kg), nisoldipine (16.6 mg/kg) or clonidine (10 #g/kg), 20 rain prior to naloxone challenge. Naloxone (1-2 mg/kg) was then administered to all groups of mice. Naloxone-precipitated withdrawal in mice, characterized by jumping, was measured over the first 5 min period following the administration of naloxone. The effects of Ca 2+ antagonists on naloxoneprecipitated withdrawal signs in the rats were also tested. Morphine-dependent rats were pretreated with either vehicle (polyethylene glycol or saline), nimodipine, verapamil, nifedipine or clonidine 20 min prior to the initiation of withdrawal with naloxone (1 mg/kg i.p.). Withdrawal signs which were assessed upon the injection of naloxone included abdominal stretching, diarrhea and fluid loss, 'wet dog' shakes, teeth chattering and ptosis. The effects of the Ca 2 ÷ antagonists and clonidine on the severity of diarrhea and fluid loss were determined by measuring the loss in body weight in the respective treatment groups 3 h following naloxone injections.

2.7. Data analysis and protein determination The maximal binding capacity (Bmax) and equilibrium dissociation constant (Kd) were obtained from Scatchard plots (Rosenthal, 1967) and leastsquares linear regression analyses of the saturation data. Statistical significance between means was determined by the Student's t-test. Significant effects of Ca 2÷ antagonists on naloxone-precipitated withdrawal signs were tested using the MannWhitney and Chi-Square tests. Protein concentrations were determined by the method of Lowry et al. (1951).

76 3. R e s u l t s =

3.1. Effect of chronic morphine treatment [~H]nimodipine binding sites in the brain

on

1

T

•

PPEL LALCEETBSO

•

0

1 MORPHINE PELLFT

A

220,

~,..~

2 MORPHINE PELLETS 3 MORPHINE PELLETS

"1 J

~_ "~2oo The b i n d i n g of [ 3 H ] n i m o d i p i n e to whole rat b r a i n h o m o g e n a t e was s a t u r a b l e a n d o f high affinity (fig. 1). M a x i m a l b i n d i n g a n d e q u i l i b r i u m dissociation c o n s t a n t s were 1 6 2 . 7 + 4 . 0 f m o l / m g p r o t e i n (N = 6 ) a n d 0.13 + 0 . 0 3 n M ( m e a n + S.E.M.), respectively. F o l l o w i n g i m p l a n t a t i o n of 3 m o r p h i n e pellets for 3 days, a significant increase in the Bm~~ to 196.0 + 7.0 f m o l / m g p r o t e i n (N = 9) was observed with no c h a n g e in K d values which averaged 0.17 + 0.01 nM.

3.2. Effect of increasing the dose and duration of morphine treatment on [ 3H]nimodipine binding sites in the brain M o r p h i n e increased [ 3 H ] n i m o d i p i n e b i n d i n g sites in whole b r a i n h o m o g e n a t e s in a dose- a n d

z ; 2007- O ~ E s ~ ~

'.

i! .° 0

~-

~--

I

I

I

~

.

I

I

11

[3HINIMODIPINE'[nMI /

,q

=.~

•

A.

\o\ o ~ *oc

\

,

40 BOUND

,

80

~ 180

~m ~

1SO

Zm ,1'

x

I 1-2 DURATIONOF O:

l I 3 4. PELLET IMPLANTATION.

(DAYS)

Fig. 2. Dose and time dependence of morphine-induced increase in [3H]nimodipine binding sites in rat brain. Rats were implanted with 3 placebo pellets or 1-3 morphine pellets (75 mg morphine each) for 1-4 days. Following treatment, rats were killed and their brains prepared for [3H]nimodipine binding experiments. These experiments were performed in triplicate by incubating tissues with 6-8 concentrations of the radioligand, as described in Materials and methods. Each point represents the mean + S.E.M. of 2-9 rats. * Statistically different from rats treated with placebo pellets for 1-2 days (P< 0.05). ** Statistically different from rats treated with placebo pellets for 3 days (P < 0.05).

A

,,o.2 0.4 o.s o.e 1.o 1.~

i'°°°

ol

'

• MORPHINE

L

o\

~

t i m e - d e p e n d e n t m a n n e r (fig. 2). M a x i m a l increases in Bma× were o b s e r v e d following i m p l a n t a tion of 3 pellets for 3 days. Therefore, all subsequent r a d i o l i g a n d b i n d i n g e x p e r i m e n t s were perf o r m e d u n d e r these t r e a t m e n t conditions. I m p l a n tation of p l a c e b o pellets over a similar time p e r i o d h a d no effect on the B. . . . but increased the K d slightly ( d a t a not shown).

3.3. Time course of analgesia in morphine-treated rats

,%\,

120

( Imol/me

160 200 240 pxotein )

Fig. 1. Increase in [3H]nimodipine binding sites in rat brain following morphine pellet implantation. Rats were implanted with 3 morphine pellets for 3 days. Brain homogenates were prepared and incubated in triplicate with 6 different concentrations of [3H]nimodipine (0.05-1.2 nM) as described in Materials and methods. This figure is a representative plot of 11 and 4 experiments with similar results, obtained from placebo- and morphine-treated rats. respectively.

R a t s i m p l a n t e d with 3 m o r p h i n e pellets d e m o n s t r a t e d analgesia to the thermal nociceptive stimuli p r o d u c e d b y a hot plate (table 1). A n a l gesia persisted for up to at least 26 h after m o r p h i n e pellet i m p l a n t a t i o n , b u t tolerance to this effect was o b s e r v e d b y 50 h (table 1). R a t s imp l a n t e d with p l a c e b o pellets d e m o n s t r a t e d no analgesic responses c o m p a r e d to naive c o n t r o l s

77 TABLE 1 Duration of morphine analgesia in the rat following implantation of 3 morphine pellets. Rats were implanted with 3 placebo pellets or 3 morphine pellets (75 mg morphine base each) and analgesia was determined using a hot plate at 55 o C. Hind paw lick was taken as the appropriate response to the thermal nociceptive stimulus. Each rat was tested twice, 15 min apart, 2, 26 and 50 h after pellet implantation. Latency time, in seconds, is presented as the mean+S.E.M, from duplicate determinations at each time point. Latency for naive controls was 12.7-+0.6 ( N = 5 ) . Number of rats tested between parentheses. Treatment

Placebo Morphine

r~ r~

"6 t..,

E o

Latency time (s) 2h ~

26h a

50h a

12.0+ 1.7 (5) 42.8+4.4 (6) "

12.7+ 1.7 (5) 42.4-t-3.3 (6) "

10.9+0.8 (5) 16.7+2.9 (6)

Lr.,

a Treatment duration, b Statistically different from control (P < 0.05).

©

(table 1). Values for naive controls averaged 12.7 ± 0.6 s (mean ± S.E.M.).

3.4. Effect of morphine treatment on the binding sites of [ 3H]nimodipine in various brain regions [3H]Nimodipine binding in homogenates from different brain regions was saturable and of high affinity (fig. 3, table 2). Scatchard plots were linear in all the regions studied, suggesting the interaction of this ligand with a homogenous population of dihydropyridine receptors. Morphine treatment increased the density of [3 H]nimodipine binding sites in the cerebral cortex,

60

120

BOUND

180

O

40

(fmol/mq

80

19.O

protein)

Fig. 3. Brain regional distribution of morphine-induced increase in [3H]nimodipine sites. Rats were implanted with 3 placebo pellets or 3 morphine pellets (75 mg each) for 3 days, followin 8 which brains were dissected into different regions and tissue homogenates from each region prepared for binding experiments. [3H]Nimodipine binding studies were performed in triplicate by incubating the tissue with 6-8 concentrations of the radioligand (0.05-1.0 nM). The Scatchard plots shown are representatives from each brain region.

hippocampus, brainstem and hypothalamus (fig. 3, table 2). No significant increases in binding sites were observed in the striatum or cerebellum.

TABLE 2 Effect of morphine treatment on [3H]nimodipine binding sites in different brain regions and heart of the rat =. Number of animals tested between parentheses. Brain region

Cortex Striatum Hippocampus Brainstem Hypothalamus Cerebellum Heart

B,,~ (fmol/mg protein)

K d (nM)

Placebo

Morphine

Placebo

Morphine

140.7 + 11.6 (7) 167.1+ 9.8 (7) 174.0 + 12.9 (6) 47.1 -+ 5.4(5) 83.9+ 9.4(5) 47.1 + 7.9 (3) 240.2+ 4.1 (4)

169.1 + 7.3 (8) b 177.4+ 8.8 (7) 214.7 + 8.7 (7) " 62.7+ 5.1 (5) b 105.2+ 5 . 7 ( 5 ) " 51.2 + 2.7 (3) 214.7+13.5 (4)

0.18+0.02 0.18+0.01 0.17 _+0.01 0.15-+0.05 0.14+0.02 0.17 + 0.03 0.12_+0.01

0.13 +0.01 " 0.13_+0.01 " 0.12 + 0.01 b 0.13-+0.13 0.14+0.02 0.14 + 0.0g 0.12_+0.01

" Values are expressed as the means + S.E.M. b Statistically different from control (P < 0.05).

78

In the cortex, striatum and hippocampus, a decrease in the K d was also observed consequent to morphine treatment (table 2). [3H]Nimodipine binding in heart homogenates was also saturable and of high affinity. In this tissue, however, morphine treatment had no effect on receptor density of affinity (table 2).

3.5. Reversibility of the effects of morphine on [¢H]nimodipine binding in the brain upon removal of morphine pellets Rats implanted with morphine pellets showed a significant increase in the density of dihydropyridine sites in whole brain homogenates, compared to the values obtained in placebo-treated rats (table 3 and as demonstrated in fig. 1). Removal of morphine pellets on day 3 and allowing the animals to remain drug-free for various periods of time, resulted in a slow return of the dihydropyridine sites towards control levels. No significant reversal was obtained 1 and 2 days after pellet removal. However, by day 10, almost complete recovery to control level was observed (table 3). In this set of experiments, morphine treatment also increased the K d of [3H]nimodipine binding in whole brains, with a further increase observed 1 day after withdrawal of the morphine pellets. This parameter returned to control levels by day 3 (table 3).

TABLE 3 Time course of reversal of morphine-induced increase in [3H]nimodipine binding in rat brain % Status

B.... (fmol/rfig protein)

K,j (nM)

N

Control Morphinedependent

167.1 + 5.6

0.13+0.03

11

201.7+ 11.1 b

Withdrawal duration (days) 1 181.5-t- 7.7 3 190.8 ± 7.7 10 170.8 ± 1.8 ~

0.22+0.04

4

0.29 ± 0.02 b 0.14 ±0.01 0.08±0.01

4 3 3

'~ Values are presented as the mean ±S.E.M. t, Statistically significant difference from control (P < 0.05). ~ Statistically significant difference from morphine-dependent rats (P < 0.05).

3.6. Effect of dihydropyridine Ca 2 + antagonists on naloxone-precipitated withdrawal in morphine-dependent mice Injections of naloxone (2.0 mg/kg) induced a characteristic jumping behavior in morphine-dependent mice. This withdrawal sign was observed soon after naloxone injections and persisted for at least 15 min. In order to quantitate the severity of naloxone-precipitated withdrawal, the number of jumps elicited in the first 5 min of naloxone treatment was counted. As observed in table 4, nimodipine decreased the severity of this withdrawal sign initiated by naloxone. Following pretreatment with 5 and 10 mg/kg nimodipine, the number of jumps observed per 5 min (expressed as a percentage of vehicle) were 56 + 43 and 35 + 40 (mean + S.D.), respectively. Nisoldipine, another dihydropyridine Ca 2+ antagonist, was also effective in reducing the incidence of jumping at the single dose tested (16.6 mg/kg). 3. 7. Effects of Ca 2 ~ antagonists on naloxone-precipitated withdrawal in morphine-dependent rats Rats made dependent on morphine and then injected with naloxone (1 mg/kg i.p.) demonstrated a number of withdrawal signs (table 5).

TABLE 4 Inhibition of naloxone-precipitatcd withdrawal j u m p i n g in the mouse by pretrcatment with Ca -,+ antagonists and clonidine. Mice were made dependent on morphine by pellet implantation for 3 days. Dependent mice were pretreated with i.p. injections of vehicle or Ca-" ÷ antagonists at the indicated doses 20 min prior to naloxone challenge. Jumping was determined over the first 5 min following naloxone injections. Values are expressed as a percentage (means + S.D.) of vehicle-pretreated mice. N u m b e r of mice tested between parentheses. Treatment

Percent of vehicle

Vehicle Nisoldipine (16.6 m g / k g ) Nimodipine (5 m g / k g ) Nimodipine (10 m g / k g )

100 35 ± 35 56 +43 35 + 4 0

(16) (9) ~ (7) " (13) ~.b

~' Statistically different from vehicle-pretreated mice administered naloxone (P < 0.05), using the Mann-Whitney U-test. Clonidine (10 p.g/kg) completely abolished j u m p i n g in this experimental paradigm, h Statistically different from mice protreated with 5 m g / k g nimodipine (P < 0.05).

79 TABLE 5

TABLE 6

Inhibition of naloxone-precipitated withdrawal signs in the rat followingpretreatment with Ca 2~ antagonists and clonidine a.b. Rats were made dependent on morphine by i.p. injections over a 6 day period, as described in Materials and methods. Dependent rats were pretreated with vehicle, Ca 2' antagonists or clonidine, at the indicated concentrations, 20 min prior to naloxone challenge. The incidence or severity of withdrawal signs was determined for 30 min following naloxone injections.

Inhibition of naloxone-precipitated weight loss in morphinedependent rats by Ca 2- channel antagonists ". Number of rats tested between parentheses.

Withdrawal signs

Vehicle

Nimodipine (10 mg/ kg i.p.)

Clonidine (1(30 p.g/ kg i.p.)

Wet dog shakes Ptosis Teeth chattering Abdominal stretching Diarrhea

3.4 (9) 100 7.0

1.3 (9) ~ 55 " 3.0 ~

1.0 (6) ¢ 0¢ 3.8 ~

0.9 2.7

0.5 ~ 0.1 "

0~ 0

a All withdrawal signs were measured over a 30 min period. Wet dog shakes, teeth chattering and abdominal stretching are presented as the average number of occurrences. The percentage of animals showing ptosis and the mean severity score for diarrhea (0 = no diarrhea, 3 = severe diarrhea) are also presented, b Statistical comparisons between vehicle- and drug-treated groups were made using the Mann-Whitney U-test for all signs except ptosis, which was analysed by the ChiSquare test. ¢ Statistically different from vehicle-pretreated rats administered naloxone (P < 0.05). Number in parentheses represents the number of animals tested.

P r e t r e a t m e n t with saline or polyethylene glycol 20 m i n prior to n a l o x o n e injection had n o effect on the incidence of withdrawal symptoms. However, p r e t r e a t m e n t of m o r p h i n e - d e p e n d e n t rats with n i m o d i p i n e (10 m g / k g ) reduced the incidence of n a l o x o n e - p r e c i p i t a t e d withdrawal signs. F o r example, the n u m b e r of wet dog shakes, teeth chattering, a b d o m i n a l stretching a n d the severity of diarrhea were significantly reduced. Furthermore, following n i m o d i p i n e p r e t r e a t m e n t , the n u m b e r of rats showing ptosis was significantly less as c o m p a r e d to vehicle-pretreated rats a d m i n istered naloxone. Clonidine, a drug k n o w n to be effective in blocking m o r p h i n e withdrawal ( G o l d et al., 1978) was included as a s t a n d a r d to compare the effects of n i m o d i p i n e . This drug completely abolished the occurrence of ptosis, abd o m i n a l stretching a n d diarrhea. It was also partially effective against wet dog shakes a n d teeth chattering.

Drug pretreatment

% of control body weight

Vehicle Nifedipine (20 mg/kg) Nimodipine (10 mg/kg) Verapamil (10 mg/kg Ckmidine (10 #g/kg) Clonidine (100 ~tg/kg)

93.0+0.9 (8) 90.1 +_0.6 (3) b 98.3 + 0.9 (9) ~ 96.7+0.9 (3) h 98.0+_0.4 (7) h 99.5 +0.9 (6) h

Morphine-dependent rats were pretreated by i.p. injections of the various drugs 20 min prior to naloxone (1 mg/kg i.p.) challenge. Weight loss was determined 3 h after naloxone injection, b Statistically significant difference from vehicle (P < 0.05).

The effects of different Ca 2+ c h a n n e l antagonists and c l o n i d i n e against n a l o x o n e - p r e c i p i t a t e d weight loss in m o r p h i n e - d e p e n d e n t rats is shown in table 6. M o r p h i n e - d e p e n d e n t rats pretreated with either saline or p o l y e t h y l e n e glycol 20 min prior to n a l o x o n e injection showed a n average loss in weight of - 7% of starting body weight ( - 20-25 g) after 3 h. Nifedipine, n i m o d i p i n e a n d verapamil were effective in blocking this loss in body weight, c o m p a r e d to vehicle. In this respect, the effect of these drugs was c o m p a r a b l e to clonidine. In addition to weight loss, verapamil was tested for its effects o n other withdrawal parameters but was found to be less effective than the other Ca 2~ c h a n n e l a n t a g o n i s t s tested (data not shown).

4. Discussion O u r results d e m o n s t r a t e an u p r e g u l a t i o n of dih y d r o p y r i d i n e b i n d i n g sites in rat b r a i n conseq u e n t to p r o l o n g e d m o r p h i n e treatment. This increase was both dose- a n d t i m e - d e p e n d e n t , reversible a n d localized to specific b r a i n regions. F u r t h e r m o r e , we have shown that several Ca 2~ c h a n n e l antagonist, a d m i n i s t e r e d i.p., can att e n u a t e n a l o x o n e - p r e c i p i t a t e d withdrawal signs in mice a n d rats. The time d e p e n d e n c e of the observed increase in Ca 2~- c h a n n e l s is suggestive of a slow a d a p t a tion of the b r a i n to c h r o n i c m o r p h i n e a d m i n i s t r a -

80 tion and might reflect the requirement of de novo protein synthesis for the expression of additional channels. Interestingly, concomitant daily administration of cycloheximide (an inhibitor of protein synthesis) and morphine abolishes the development of tolerance and physical dependence in mice (Loh et al., 1969). Optimal increases in [3H]nimodipine binding observed by day 3 of morphine treatment (table 1) correlate well with the development of tolerance (Way et al., 1969) and dependence (Yoburn et al., 1985; Bongianni et al., 1986) on morphine. Similarly, the optimal dose (3 × 75 mg pellets) used in our study is comparable to that used by Yoburn and coworkers (1985) to produce the highest blood level of morphine, in addition to a high degree of physical dependence. The distribution of dihydropyridine receptors in brain has been previously characterized (Marangos et al., 1982; Gould et al., 1985) and bears good similarities to that observed in table 2. Morphine treatment produced increases in these sites in the cerebral cortex, hippocampus, hypothalamus and brainstem, regions which are rich in precursors of endogenous opioid peptides (Khachaturian et al., 1985) and opioid receptors (Goodman and Snyder, 1982; Itzhak et al., 1982). No effect was observed in the cerebellum, a region which contains a low density of opioid receptors (Pert et al., 1976). Nevertheless, we were unable to demonstrate significant changes in the striatum, which contains high levels of both/~ and ~ opioid receptors (McLean et al., 1986). The reason for this lack of effect is not clear. However, Thayer et al. (1987) recently reported that K--simulated entry of Ca 2 ÷ into striatal neurons is less sensitive to inhibition by nitrendipine as compared to hippocampal neurons. Jncreases in dihydropyridine binding sites observed in the present study were smaller than those previously observed by us in mouse brain (Ramkumar and EI-Fakahany, 1984). The reason for this is not clear. However, it is possible that these differences might be species related. In addition to effects on Bmax, morphine treatment produced significant reductions in K d values obtained in the cerebral cortex, striatum and hippocampus (table 2). However, these changes are

small, being less than 2-fold. Generally, a less than 2-fold alteration in K d is considered within the limits of experimental variation (Burt, 1980). Withdrawal of morphine also led to changes in K o, albeit in a direction opposite to that observed above (table 3). These discrepant changes in K d are difficult to reconcile. Removal of morphine pellets following the development of physical dependence in rats initiates withdrawal signs (e.g. teeth chattering, wet dog shakes, etc.). These signs are known to persist for about 10 days after morphine pellet removal (Bl~isig et al., 1973), longer than the persistence of the upregulation of dihydropyridine sites observed in this work (table 3). The temporal similarities between the increase in L-type Ca 2+ channels and the emergence of withdrawal signs suggest a common link. This prompted us to test whether blockade of Ca 2. channels by Ca 2+ antagonists will attenuate morphine withdrawal signs. Pretreatment of morphine-dependent rats and mice with Ca 2+ channel antagonists reduced the incidence of withdrawal signs elicited by naloxone. For example, the dihydropyridines nimodipine and nisoldipine attenuated naloxone-induced jumping in mice (table 4). In this respect, these drugs were not as effective as clonidine, an a2-adrenoceptor agonist used in the treatment of opioid withdrawal (Gold et al., 1978). The effect of Ca 2÷ antagonists in morphine-dependent rats is comparable to those of clonidine (tables 5, 6). Similar findings for the Ca 2 ~ antagonists nimodipine and verapamil have recently been published (Bongianni et ai., 1986). Together, these lines of evidence provide indirect support for the involvement of dihydropyridinesensitive Ca 2+ channels in the brain in mediating, in part, morphine withdrawal syndrome. Relevant comparisons between these doses of Ca 2+ channel antagonists used in the present study with those needed to produce cardiovascular effects are difficult, since in a majority of the latter studies the dihydropyridines were administered via the oral, intramuacular or intravenous routes. The amounts of drug reaching the systemic circulation following administration via these different routes are likely not equivalent. It has recently been shown that the Ca 2+ chan-

81 nel antagonist nimodipine possess significant cerebral vasodilatory action (Kanda and Flaim, 1986). Even though there is no evidence to implicate this action in the anti-withdrawal effect of nimodipine, such a possibility cannot be ruled out. As evident from our results, the Ca 2÷ channel antagonists did not completely abolish opiate withdrawal signs. Thus, we cannot preclude the contribution of other mechanisms in mediating opioid withdrawal. A number of changes in the brain produced by chronic morphine treatment has recently been reviewed (Redmond and Krystal, 1984). It is not clear whether blockade of naloxone-induced diarrhea and weight loss by Ca 2+ channel antagonists is a manifestation of the intrinsic property of these drugs on gastrointestinal (GI) smooth muscle that is totally unrelated to the withdrawal phenomenon. These antagonists are potent inhibitors of GI smooth muscle Ca 2. channels and this property mediates their antidiarrheal action. Nevertheless, it is possible that morphine treatment also upregulates Ca 2+ channels in the GI tract, resulting in enhanced GI activity following naloxone challenge. This implies that the antidiarrheal effect of the Ca 2÷ channel antagonists is relevant to morphine withdrawal. Recent evidence suggests a functional role of the dihydropyridine sites in the brain (Ramkumar and EI-Fakahany, 1986). These sites are localized on neuronal cell bodies (Miller, 1987; Thayer et al., 1987), where they appear to modulate the activity of L-type Ca 2+ channels (Nowycky et al., 1985). This subtype of Ca 2+ channels in nervous tissue is sensitive to blockade by dihydropyridine Ca 2+ channel antagonists and activation by the Ca 2+ channel agonist, Bay K8644 (Nowycky et al., 1985; Thayer et al., 1987). Their postsynaptic localization suggests that these channels do not contribute to the enhancement of depolarization-induced Ca 2+ uptake in synaptosomes prepared from opioid-dependent animals (Ross et al., 1977; Guerrero-Munoz et al., 1979). However, it is possible that morphine treatment increases the density of synaptosomal (N-type, dihydropyridine insensitive) Ca 2÷ channels as well. Morphine inhibits synaptosomal Ca 2÷ uptake and neurotransmitter release (Crowder et al., 1986), processes

mediated by N-type Ca 2+ channels (Miller, 1987). Thus, it is conceivable that prolonged blockade of these channels might lead to their upregulation. The mechanisms underlying the increase in dihydropyridine Ca 2+ antagonist binding sites in the brain are not clear. One speculation is that such an increase might reflect a compensation to prolonged inhibition of Ca 2+ channel by morphine. While morphine-induced inhibition of Ca 2. uptake into synaptosomes presumably reflect blockade of synaptosomal N-type channels, morphine might also inhibit the activity of L-type Ca 2+ channels. For example, morphine activates a hyperpolarizing K + current in neurons (Cherubini and North, 1985), thereby reducing the responsiveness of the neurons to voltage activation. This might lead to decreased activation of voltage-sensitive Ca 2+ channels (both L and N types). Prolonged blockade of voltage-sensitive Ca 2+ channel appears insufficient to account for their upregulation. For example, prolonged administration of Ca 2+ antagonists decreases, instead of increases, the number of brain dihydropyridine sites (Panza et al., 1985). Similarly, long-term treatment with neuroleptic drugs known to interact with dihydropyridine sites in brain (Gould et ai., 1983) leads to a decrease in the density of these sites (Ramkumar and El-Fakahany, unpublished observations). The level of cyclic AMP appears essential for the expression of Ca 2. channel activity in neuronal tissue (Fedulova et al., 1981; Nirenberg et al., 1983; Armstrong and Eckert, 1987). Acute opioid administration decreases adenylate cyclase activity in neuronal clonal (NG108-15) cells (Sharma et al., 1975; Griffin et al., 1985). On the other hand, chronic exposure of these cells to opioids leads to increases in both basal and stimulated cyclic AMP formation (Griffin et al., 1985), probably resulting from a loss of tonic inhibition of adenylate cyclase mediated by the guanine nucleotide regulatory protein (G i). Chronic elevation of cyclic AMP (by addition of dibutrylyl cyclic AMP or the stimulatory agonist prostaglandin El) in NG108-15 cell cultures leads to the expression of dihydropyridine sites together with increases in 45Ca2+ uptake into these cells (Nirenberg et al., 1983). Similarly, Schmid et al. (1985) have observed modulation of

82

the density and activity of skeletal muscle voltage-sensitive Ca 2-~ channels depending on the level of cyclic AMP. The basis for the increase in channel density appears to be an increase in activity at the level of messenger RNA (Nirenberg et al., 1983), leading to de novo synthesis of Ca 2÷ channels. It might be speculated that elevation of cyclic AMP levels in vivo in morphine-dependent rats might ultimately lead to the observed enhancement of dihydropyridine sites. Our finding that the brain dihydropyridine sites can be regulated by morphine is not unique to this drug. Evidences of regulation of these sites by various pharmacological manipulations have been reviewed by us (Ramkumar and E1-Fakahany, 1986). More recently, increases in dihydropyridine-sensitive Ca 2. channels have been observed consequent to alcohol treatment (Messing et al., 1986; Dolin et al., 1987). Thus, it is becoming increasingly clear that dihydropyridine sites in the CNS are an important target of drug action. In summary, we have demonstrated that chronic morphine treatment upregulates rat brain dihydropyridine binding sites. Furthermore, Ca 2 + channel antagonists were effective in reducing the incidence and severity of morphine withdrawal. This suggests a role of dihydropyridine-sensitive Ca 2+ channels in the mediation of morphine withdrawal signs.

Acknowledgements The authors would like to thank Linda Scherich for her excellent secretarial assistance. In addition, we are grateful to Drs. N. Khazan, G.A. Young and G. Hollenbeck and Mr. Michael Gentry and Mr. Walter Thompson for their invaluable assistance.

References Armstrong, D. and R. Eckert, 1987, Voltage-activated calcium channels that must be phosphorylated to respond to membrane depolarization, Proc. Natl. Acad. Sci. U.S.A. 84, 2518. Bl~sig, J., A. He~, K. Reinhold and S. Zieglg~insberger, 1973, Development of physical dependence on morphine in

respect to the time and dosage and quantification of the precipitated withdrawal syndrome in rats. Psychopharmacologia 33, 19. Blaustein, M.P., 1975, Effect of potassium, veratridine and scorpion venom on calcium accumulation and transmitter release by nerve terminals in vitro, J. Physiol. 247, 617. Bongianni, F., V. Carla, F. MoronJ and D.E. PellegriniGiampietro, 1986. Calcium channel inhibitors suppress the morphine-withdrawal syndrome in rats, Br. J. Pharmacol. 88, 561. Burr, D.R., 1980, Basic receptor methods ii. Problems of interpretation in binding studies, in: Receptor Binding Techniques (Society of Neuroscience, Washington, DC) p. 53. Cardenas, H.L. and D.H. Ross, 1976, Calcium depletion of synaptosomes after morphine treatment, Br. J. Pharmacol. 57. 521. Chapman, D.B. and E.L. Way, 1982, Modification of endorphin/enkephalin analgesia and stress-induced analgesia by divalent cations, a cation chelator and an ionophore, Br. J. Pharmacol. 75. 389. Cherubini, E. and R.A. North, 1985, ~ and ~ opioids inhibit transmitter release by different mechanisms, Prec. Natl. Acad. Sci. U.S.A. 82, 1860. Crowder, J.M., D.K. Norris and H.F. Bradford, 1986, Morphine inhibition of calcium fluxes, neurotransmitter release and protein and lipid phosphorylation in brain slices and synaptosomes. Neuropharmacology 35, 2501. Dolin, S., H. Little, H. Hudspith, C. Pagonis and J. Littleton, 1987, Increased dihydropyridine-sensitive calcium channels in rat brain may underlie ethanol physical dependence, Neuropharmacology 26, 275. Fedulova, S.A., P.J. Kostyok and N.S. Veselovsky, 1981, Calcium channels in somatic membrane of the rat dorsal root ganglion neurons, effect of cAMP, Brain Res. 214, 210. Gibson, R.D. and J.E. Tingstad, 1970, Formulation of a morphine implantation pellet suitable for tolerance-physical dependence studies in mice, J. Pharmacol. Sci. 59, 426. Glowinski, J. and L.L. Iversen, 1966, Regional studies of catecholamines in the rat brain 1. The disposition of [ 3H]norepinephrine, [ 3H]dopamine and [ 3H]DOPA in various regions of the brain. J. Neurochem. 13, 655. Gold, M.S., D.E. Redmond, Jr. and H.D. Kleber, 1978, Clonidine blocks acute opiate withdrawal symptoms, Lancet 2, 599. Goodman, R.R. and S.H. Snyder, 1982, Kappa opiate receptor localized by autoradiography to deep layers of cerebral cortex: relation to sedative effects. Proc. Natl. Acad. Sci. U.S.A. 79, 5703. Gould, R.J., K.M.M. Murphy, l.J. Reynolds and S.H. Snyder, 1983, Antischizophrenic drugs of the diphenylbutylpiperidine type act as calcium channel antagonists, Proc. Natl. Acad. Sci. U.S.A. 80, 5122. Gould, R.J., K.M.M. Murphy and S.H. Snyder, 1985, Autoradiographic localization of calcium channel antagonist receptors in rat brain with [3H]nitrendipine, Brain Res. 330, 217.

83 Griffin, M.T., P.Y. Law and H.H. Loh, 1985, Involvement of both inhibitory and stimulatory guanine nucleotide binding proteins in the expression of chronic opiate regulation of adenylate cyclase activity in NGI08-15 cells, J. Neurochem. 45, 1585. Guerrero-Munoz, F., K.V. Cerreta, M.L. Guerrero and E.L. Way, 1979, Effect of morphine on synaptosomal C a ' + uptake, J. Pharmacol. Exp. Ther. 209, 132. Harris, R.A., H. Yamamoto, H.H. Loh and E.L. Way, 1977. Discrete changes in brain calcium with morphine analgesia, tolerance-dependence, and abstinence, Life Sci. 20, 501. Itzhak, Y.. K.A. Bonnet, J. Groth, J.M. Hiller and E.J. Simon, 1982, Multiple opiate binding sites in human brain regions: evidence for kappa and sigma sites, Life Sci. 31, 1363. Kakunaga, T., H. Kaneto and K. Hano, 1966, Pharmacologic studies on analgesia, VII. Significance of the calcium ion in morphine analgesia, J. Pharmacol. Exp. Ther. 153, 134. Kamikubo, K., M. Niwa, H. Fugimura and K. Miura, 1983, Morphine inhibits depolarization-dependent calcium uptake by synaptosomes, European J. Pharmacol. 95, 149. Kanda, K. and F.F. Flaim, 1986, Effects of nimodipine on cerebral blood in conscious rat, J. Pharmacol. Exp. Ther. 236, 41. Khachaturian, H., M.E. Lewis, M.K.H. Schafer and S.J. Watson, 1985, Anatomy of the CNS opioid systems, Trends Neurosci. 8, I l l . Loh, H.H., F.H. Shen and E.L. Way, 1969, Inhibition of morphine tolerance and physical dependence development and brain serotonin synthesis by cyclohexamide, Biochem. Pharmacol. 18, 2711. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randal, 1951, Protein measurement with Folin phenol reagent, J. Biol. Chem. 193, 265. Marangos, P.J., J. Patel, C. Miller and A.M. Martino, 1982, Specific calcium antagonist binding sites in the brain, Life Sci. 31, 1575. McLean, S., R.B. Rothman and M. Harkenham, 1986, Autoradiographic localization of mu and delta opiate receptors in the forebrain of the rat, Br~tin Res. 378, 49. Messing, R.O., C.L. Carpenter, I. Diamond and D.A. Greenberg, 1986, Ethanol regulates Ca 2- channels in clonal neuronal cells, Proc. Natl. Acad. Sci. U.S.A. 83, 6213. Miller, R.J., 1987, Multiple calcium channels and neuronal function, Science 235, 46. Nirenberg, N., S. Wilson, H. Higashida, A. Rotter, K. Krueger, N. Busis, R. Ray, J.G. Kenimer and M. Adler, 1983, Modulation of synapse formation by cyclic adenosine monophosphate, Science 222, 794. Nowycky, M.C., A.P. Fox and R.W. Tsien, 1985, Three types of neuronal calcium channel with different calcium agonist sensitivity, Nature 316, 440.

Panza, G., J.A. Grebb, E. Sanna, A.C. Wright and I. Hanbauer, 1985, Evidence for downregulation of [3H]nitrendipine recognition sites in mouse brain after long-term treatment with nifedipine or verapamil, Neuropharmacology 24, 1113. Pert, C.B., M. Kuhar and S.H. Snyder, 1976, Opiate receptor: autoradiographic localization in rat brain, Proc. Natl. Acad. Sci. U.S.A. 73, 3729. Ramkumar, V. and E.E. E1-Fakahany, 1984, Increase in [3H]nitrendipine binding sites in the brain in morphinetolerant mice, European, J. Pharmacol. 102, 371. Ramkumar, V. and E.E. EI-Fakahany, 1986, The current status of the dihydropyridine calcium channel antagonist binding sites in the brain, Trends Pharmacol. Sci. 7, 171. Reddy, S.V.R. and T.L. Yaksh, 1980, Antinoceptive effects of lanthanum, neodymium and europium following intracranial administration, Neuropharmacology 19, 181. Redmond, D.E., Jr. and J.H. Krystal, 1984. Multiple mechanisms of withdrawal from opioid drugs, Ann. Rev. Neurosci. 7, 443. Rosenthal, H.E., 1967, A graphic method for the determination and presentation of binding parameters in complex systems, Anal. Biochem 20, 525. Ross, D.H. and H.L. Cardenas, 1979, Nerve cell calcium as a messenger for opiate and endorphin actions, Adv. Biochem. Psychopharmacol. 20, 301. Ross, D.H., S.C. Lynn and H.L. Cardenas, 1977, Ions, opiate and cellular adaptation, in: Alcohol and Opiates: Neurochemical and Behavioral Mechanisms, ed. K. Blum (Academic Press. New York) p. 265. Schmid, A., J.-F. Renaud and M. Lazdunski, 1985, Short term and long term effects of/3-adrenergic effectors and cyclic AMP on nitrendipine-sensitive voltage-dependent Ca 2+ channels of skeletal muscle, J. Biol. Chem. 260, 13041. Schmidt, W.K. and E.L. Way, 1980, Hyperalgesic effects of divalent cations and antinociceptive effects of a calcium chelator in naive and morphine-dependent mice, J. Pharmacol. Exp. Ther. 212, 22. Sharma, S.K., M. Nirenberg and W.A. Klee, 1975, Morphine receptors as regulators of adenylate cyclase activity, Prec. Natl. Acad. Sci. U.S.A. 72, 590. Thayer, S.A., S.N. Murphy and R.J. Miller, 1987, Widespread distribution of dihydropyridine-sensitive calcium channels in the central nervous system, Mol. Pharmacol. 30, 505. Way, E.L., H.H. Loh and F.H. Shen, 1969, Simultaneous quantitative assessment of morphine tolerance and physical dependence, J. Pharmacol. Exp. Ther. 167, 1. Yoburn, B.C., J. Chen, T. Huang and C.E. lnturrisi, 1985, Pharmacokinetics and pharmacodynanucs of subcutaneous morphine pellets in the rat, J. Pharmacol. Exp. Ther. 235, 282.