Sodium-independent binding of [3H]cocaine in mouse striatum is serotonin related

Brain Research, 342 (1985) 145-148

145

Elsevier BRE 20994

Sodium-independent binding of pH]cocaine in mouse striatum is serotonin related MAARTEN E. A. REITH, BERNARD E. MEISLER, HENRY SERSHEN and ABEL LAJTHA Center for Neurochemistry, The Nathan S. Kline Institute for Psychiatric Research, Ward's Island, New York. N Y 10035 (U. S.A .)

(Accepted April 2nd, 1985) Key words: cocaine - - cocaine binding-- sodium - - dopamine - - serotonin - - striatum-- mice

There was a highly significant correlation between ICs0 values of various drugs in inhibiting the Na+-independent [3H]cocaine binding in the mouse striatum and their values in inhibiting the synaptosomal uptake of [3H]serotonin. In contrast, there was no correlation between the inhibition of binding in the absence of Na + and the inhibition of [3H]dopamine uptake. Lesioning of serotonergic nerve terminals with 5, 7-dihydroxytryptamine reduced the Na+-independent [3H]cocaine binding, without affecting the Na+-dependent binding. These results indicate that the bulk of the Na+-independent [3H]cocaine binding in the mouse striatum is associated with serotonergic nerve terminals. Cocaine is a moderately potent blocker of uptake of both dopamine and serotonin in the striatum 3. In the presence of 20-120 mM Na + the saturable binding of [3H]cocaine to striatal membranes appears to be associated primarily with dopaminergic nerve terminals2, 5,10. Under these conditions, the contribution of serotonergic terminals, if any, to the [3H]cocaine binding is difficult to assess, since the striatum is a predominantly dopaminergic innervated region. In the absence of Na + [3H]cocaine still binds saturably to striatal membranes 2,7 but the binding is far less than in the presence of 50 mM Na + (ref. 2); yet, in absolute amounts, the Na+-independent binding is comparable with that observed in other brain regions. Since lesioning of dopaminergic terminals by 6-hydroxydopamine does not reduce the [3H]cocaine binding in the absence of Na + while eliminating the stimulation of binding by Na + (ref. 2), it is unlikely that the Na+-independent binding merely represents the unstimulated dopamine-related site. In the present communication we present evidence that the major portion of this binding is associated with serotonergic nerve terminals. For the experiments presented in Fig. 1, adult male BALB/cBy mice (20 g, Jackson Laboratories, Bar Harbor, ME) were decapitated and the striatal tissues were rapidly removed. P2 membranes were pre-

pared as described previously 9 and suspended in icecold 25 mM Tris-HC1 (pH 7.7 at room temperature). Portions (0.5 ml, 0.5 mg of protein) were incubated at 21 °C for 20 rain with 17 nM [3H]cocaine (New England Nuclear, Boston, MA, 31.1 Ci/mmol) and amounts of unlabeled cocaine or other drugs as indicated. Non-specific binding was defined as residual binding observed in the presence of 30 ~M unlabeled cocaine, and represented on the average 21% of the total binding in the experiments presented in Fig. 1. Assays were terminated with a single manifold Millipore filtration apparatus on GF/B glass fiber filters (Whatman) pretreated with 0.05% poly-L-lysine (w/v). These filters bound only negligible amounts of [3H]cocaineS. Uptake of [3H]dopamine (New England Nuclear, 23.1 Ci/mmol) and [3H]serotonin (New England Nuclear, 27.3 Ci/mmol) into striatal synaptosomes was measured as described in previous papersS, 10. The incubations (1 ml, 0.2 mg of protein) were for 4 min with final concentrations of tritiated monoamines of 0,1 ~tM. Active uptake of [3H]dopamine and [3H]serotonin into nerve terminals was defined as total uptake minus uptake in the presence of 50 u M benztropine and 10 I~M chlorimipramine, respectively. ICs0 values (concentrations that inhibit specific binding or uptake by 50%) were estimated by linear regression analysis of log-probit plots of in-

Correspondence: M. Reith, Center for Neurochemistry, Ward's Island, New York, NY 10035, U.S.A.

0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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Fig. 1. IC50values of drugs in inhibiting [3H]dopamine uptake, [3H]serotonin uptake, and Na+-independent pH]cocaine binding in mouse striatum. See text for numbering of the compounds.

hibition by 4 concentrations of each drug assayed in triplicate in two separate tissue preparations. The following drugs, n u m b e r e d as in Fig. 1, with their sources in parentheses, were used: 1, cocaine hydrochloride (Mallinckrodt); 2, ( + ) - p s e u d o c o c a i n e hydrogen tartrate (Merck); 3, ( + ) - n e o p s e u d o c o c a i n e hydrogen tartrate (Merck); 4, norcocaine (National Institute on Drug A b u s e ) ; 5, N-allyinorcocaine (Dr. K. A. Nieforth, Storrs, CT); 6, benzoyttropine (Dr. S. B. Ross, Astra); 7, b e n z o y l - p s e u d o t r o p i n e (Dr. S. B. Ross, Astra); 8, W I N 35,428 ( W i n t h r o p ) ; 9. W I N 35,065-2 ( W i n t h r o p ) : 10, W I N 35,140 (Winthrop); 11, W I N 35,004 (Winthrop); 12. W I N 35~065-3 ( W i n t h r o p ) : 13, benztropine mesylate (Merck Sharp and D o h m e ) ; 14, D - a m p h e t a m i n e (Smith Kline and French); 15, m e t h y l p h e n i d a t e (National Institute on D r u g A b u s e ) ; and 16, imipramine hydrochloride (Geigy). In a g r e e m e n t with a competitive mechanism of inhibition of cocaine binding, pseudo Hill coefficients between 0.8 and 1.2 were observed for c o m p o u n d s 1,2, 5 and 11-16. The remaining drugs displayed coefficients lower than 0.8 in this series of experiments. F o r most drugs, the

slopes of the serotonin uptake inhibition curves were similar to the slope found for cocaine: s t e e p e r curves were observed for c o m p o u n d s 3 - 5 and 12, and a more shallow curve was found for 16. The slopes of the d o p a m i n e uptake inhibition curves of most drugs were similar to the slope for cocaine; steeper curves were displayed by 2, 3 and 9, and more shallow curves by 6, 10 and 14. F o r the experiment presented in Table I, mice were treated with 5,7-dihydroxytryptamine creatinine sulfate (Sigma). A n amount of 75 u g of the neurotoxin was administered intracisternally in a volume of I(IM of sterile 0.9% NaCl containing ascorbic acid (1 mg/ml) to mice under p e n t o b a r b i t a l anesthesia (30 mg/kg i.p.) 1 h after an i.p. injection of desipramine (25 mg/kg); control animals were p r e t r e a t e d with desipramine but received 0.9% NaCI instead of 5,7-dihydroxytryptamine. Animals received another injection of neurotoxin or placebo 9 days later; they were killed 12 days after the second injection. The entire experiment involved 25 lesioned and 25 control mice. Striatal tissues of 5 animals were pooled and homogenized in 16 vols. of ice-cold 0,25 M sucrose, the p H of which was made 7.4 by addition of sufficient Tris-HCP. The h o m o g e n a t e was centrifuged at 1000 g for 10 min, The supernatant was the starting material for assaying both m o n o a m i n e uptake and [3H]cocaine binding. F o r the uptake, a porTABLE 1 EfJeets of treatment with 5.7-dihydroxytrvptamme on striatal dopamine uptake, serotonin uptake, und cocaine binding

Mice were injected intraeisternally with saline or 5.7-dihydroxytryptamine (5.7-DHT) after desipramine treatment tsee text). Data are means z S.E.M. of 5 separate samples assayed in triplicate: each sample consisted of a pool of striatal tissues from 5 animals.

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147 tion of the supernatant was diluted with the above ice-cold 0.25 M sucrose buffer, and E D T A was added to give a final concentration of 0.1 mM. For the binding, the remaining supernatant was centrifuged at 17,000 g for 20 min. The resulting P2 pellet was resuspended in ice-cold 25 mM Tris-HC1, pH 7.7 at room temperature. Protein in all final preparations was estimated by the method of Lowry as described previouslyS. There was no significant correlation (Fig. 1) (r = 0,37: n = 16; 0.10 < P < 0.20) between the IC50 values of the drugs in inhibiting the uptake of [3H]dopamine and their values in inhibiting the Na+-inde pendent binding of [-~H]cocaine in the striatum. This result, together with the inability of 6-hydroxydopamine to reduce the Na+-independent [3H]cocaine binding 2, indicates that the binding in the absence of Na + is not related to dopamine uptake. In contrast, there was a highly significant correlation (r = 0.81; n = 16; P < 0.001) between the inhibition of [3H]serotonin uptake and the inhibition of pH]cocaine binding measured in the absence of Na +, suggesting an association between this binding and serotonergic nerve terminals. More direct evidence for such an association was obtained in experiments with mice whose serotonergic nerve terminals were lesioned by 5,7-dihydroxytryptamine. The neurotoxin had no effect on dopaminergic nerve terminals in the striatum as measured by the synaptosomal uptake of [3H]dopamine (Table I). In contrast, there was a statistically significant reduction of 50% in the synaptosomal uptake of [3H]serotonin. Mice seem to be less sensitive to the neurotoxic effect of intracisternal 5,7-dihydroxytryptamine than rats l,~.l~, but for comparison with our previous work 7 m we used mice in the present experiments. The 50% reduction in serotonin uptake was accompanied by a decrease of 35% in the binding of [3H]cocaine measured in the absence of Na+ (0.02 < P < 0.05; Table I). The effect on binding was due to a decrease in the Bmax;in this experiment and two subsequent repeats the K,l in control and lesioned tissues was the same (mean 453 nM, range 342-576 nM, calculated with the L I G A N D program a from

saturation data obtained with pooled preparations of each group). The results of the lesion experiment indicate that the bulk of this binding is associated with serotonergic nerve terminals. The lack of effect of 5,7-dihydroxytryptamine on either [3H]dopamine uptake or [3H]cocaine binding in the presence of 50 mM Na ÷ is in agreement with earlier findings that indicate that the Na+-dependent binding is associated with dopaminergic nerve terminals in the striatum 2,5.m. Although the binding in the presence of 50 mM Na + includes Na+-independent binding, the latter is only a small portion of the former: it may be even smaller than indicated in Table I. since there is some evidence that 50 mM Na ÷ inhibits the Na+-in dependent binding in the striatum of 6-hydroxydopamine-lesioned rats 2. It is therefore to be expected that the decrease in Na+-independent binding by 5,7dihydroxytryptamine, as compared with the binding measured in the presence of 50 mM Na +, is too small to be detected within experimental error. The present results show that binding of [3H]cocaine in the striatum is complex. [3H]Cocaine labels both dopamine uptake-related sites and serotonin uptake-related sites, and it is conceivable that inhibition of serotonin uptake by cocaine underlies some of cocaine's effects. There was a time when inhibition of norepinephrine uptake was considered the main action of cocaine, and cocaine is still routinely used for this purpose in experiments on electrically evoked release of norepinephrine in superfusion designs. More recent studies emphasize the inhibition of dopamine uptake by cocaine 2,5.m. The present results point out the possibility that the ability of cocaine to inhibit the neuronal uptake of serotonin has a role in the effect of cocaine on the nervous system. In this context it is of interest that both the increase in locomotor activity and the reduction in the serotonin content of the pons-medulla as a result of cocaine are attenuated bv concomitant administration of 5-hydroxytryptophan plus decarboxylase inhibitor6. This work was supported by Grant DA 03025 from the National Institute on Drug Abuse.

4~ 1 Breese, G. R. and Mueller, R. A., Alterations in the neur~toxicity of 5,7-dihydroxytryptamine by pharmacologic agents in adult and developing rats, Ann. N.Y. Acad. Sci.. 305 (1978) 160, 2 Kennedy, L. T. and Hanbauer, I., Sodium-sensitive cocaine binding to rat striatal membrane: possible relationship to dopamine uptake sites, J. Neurochem., 41 (1983) 172. 3 Koe, B. K., Molecular geometry of inhibitors of the uptake of catecholamines and serotonin in synaptosomal preparations of rat brain, J. Pharrnacol. exp. Ther., 199 (1976) 649. 4 Munson, P. J. and Rodbard, D., LIGAND: a versatile computerized approach for characterization of ligand-binding systems, Analyt. Biochem., 107 (1980) 220. 5 Pimoule, C., Schoemaker, H., Javoy-Agid, F,, Scatton, B., Agid, Y. and Langer, S. Z., Decrease in [-~H]cocaine binding to the dopamine transporter in Parkinson's disease, Eutop. J. Pharmacol., 95 (1983) 145. 6 Pradhan, S. N., Battacharyya, A. K. and Pradhan, S., Serotonergic manipulation of the behavioral effects of cocaine

in rats, Cornmun. Psychopharmacol., 2 (!978)4.'~1 7 Reith, M. E. A., Sershen, H. and Eajtha, A,, Saturat~lc pH]cocaine binding in central nervous system of mouse, Life Sci., 27 (1980) 1055. 8 Reith, M, E. A., Sershen, H., Allen, D. 1,. ~md l_ajtha, A , A portion of [3H]cocaine binding in brain is associated with serotonergic neurons, Molec. Pharmacol., 23 (1983) 600. 9 Reith, M. E. A., Meisler, B. E., Sershen, H. and Lajtha, A., [3H]Cocaine binding in brain is inhibited by Fris (hydroxymethyl) aminomethane, J. Neurosci. Meth., 12 (1984) 151. 10 Sershen, H., Reith, M. E. A., Hashim, A. and Lajtha, A., Reduction of dopamine uptake and cocaine binding in mouse striatum by N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine, Europ. J. Pharmacol., 102 (1984) 175. 11 Stewart, R. M., Gerson, S. C., Sperk, G_ Campbell, A. and Baldessarini, R. J., Biochemical, behavioral, and pharmacologic studies of the effects of dihydroxytryptamines in the rodent brain, Ann. N. Y. Acad, Sci., 305 (1978) 198.