Brain Research, 521 (1990) 347-351 Elsevier

347

BRES 24147

Inhibition of serotonin uptake into mouse brain synaptosomes by ionophores and ion-channel agents Maarten E.A. Reith and Cathy A. O'Reilly Center for Neurochemistry, The Nathan S. Kline Institute for Psychiatric Research, Ward's Island, New York, NY 10035 (U.S.A.)

(Accepted 13 March 1990) Key words: Synaptosome; Serotonin; Ion channel; Ionophore

[sH]Serotonin uptake into mouse cerebrocortical synaptosomes was decreased by the K÷ ionophore valinomycin, the K ÷ and Na ÷ ionophore gramicidin, and the proton ionophore carbonylcyanide m-chlorophenylhydrazone. The Na+/H ÷ exchanger monensin reduced uptake at non-depolarizing concentrations. Uptake was also decreased by inhibition of the Na+,K+-ATPase with ouabain and by tetrodotoxin-sensitive activation of voltage-dependent sodium channels with veratridine, batrachotoxin and scorpion venom. In contrast, the Ca2÷ channel agents BAY K8644 and nimodipine were ineffective. The effect of reducing the Na ÷ gradient depended upon whether the internal Na ÷ concentration was raised (i.e. by scorpion venom, monensin) or the external Na ÷ concentration was lowered (37 mM NaC1 in the medium). The action of 5-hydroxytryptamine (serotonin; 5-HT) is terminated by carrier-mediated uptake into serotonergic nerve terminals 22. It is generally accepted that serotonin uptake requires external Na + and is stimulated by external CI- and internal K ÷ 2,10,16,19,20. The uptake in brain appears to be electroneutral as is the case in bovine blood platelets, a tissue often used as a model system for the 5-HT carrier in the brain 21'z3. Preparations of plasma membrane vesicles, devoid of storage granules, represent a less complex system as compared to synaptosomes allowing for the manipulation of internal and external ion concentrations 8"16'19. Conclusions derived from such preparations should, however, also be evaluated in crude synaptosomes as the purification process may inadvertently alter some of the properties of the carrier. In the present study, such crude synaptosomes are treated with ionophores, ion-channel agents, and the Na+,K+-ATPase inhibitor ouabain in order to induce changes in ion gradients across the synaptosomal membrane. Although this approach has been taken in the characterization of the synaptosomal uptake of norepinephrine 25, dopamine 7, and glutamate 24, there is no such information for serotonin uptake with the exception of the effects of Ca 2÷ channel drugs (dihydropyridine compounds, or verapamil and diltiazem) 5"18. Batrachotoxin (BTX) was donated by Dr. J.W. Daly (NIH, Bethesda, MD). The calcium-channel activator BAY K8644 (1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylic acid methyl

ester) and calcium-channel blocker nimodipine (1,4dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-dicarboxylic acid 2-methoxyethyl-l-methylethyl ester) w e r e donated by Miles Institute for Preclinical Pharmacology, West Haven, CT; [3H]5-HT (30.4 Ci/mmol) was from New England Nuclear (Boston, MA). All other drugs, including scorpion (Leiurus quinquestriatus) venom (ScVenom), tetrodotoxin (TI'X), monensin, valinomycin, gramicidin, and veratridine were from Sigma (St. Louis, MO). Male BALB/cBy mice, 8-12 weeks of age, weighing 21-24 g, from the breeding colony of our institute were used. Fresh cerebral cortex was homogenized in 8 vols of ice-cold 0.32 M sucrose containing 0.1 mM NazEDTA and sufficient Tris-HC1 to achieve pH 7.4 (ref. 9) with a motor-driven Teflon pestle and glass homogenizer (0.5 mm clearance). The homogenate was centrifuged at 1000 g for 10 min at 0-4 °C, and the cloudy supernatant was used as the crude synaptosomal preparation. Samples (50 pl, 0.2-0.4 mg of protein) of the crude synaptosomal suspension were incubated for 1 min at 37 °C with 25/xl of water with or without inhibitor (5-HT, drug) and 425 /xl of gassed (100% oxygen) buffer, containing (in mM): NaCI 122, KC1 5, MgSO4 1.2, glucose 10, CaCI 2 1, ascorbic acid 1, nialamide 0.01, Tris-HCl 20, and [3H]5HT (final concentration consisting of approximately 4 nM [3H]5-HT from New England Nuclear and 20 nM unlabeled 5-HT), pH 7.4, at room temperature. Stocks of drug (in ethanol or water, see below), 5-HT (in the above

Correspondence: M.E.A. Reith, Center for Neurochemistry, the Nathan S. Kline Institute for Psychiatric Research, Ward's Island, New York, NY 10035, U.S.A.

0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

348 buffer), and [3H]5-HT (in the above buffer) were kept on ice and added to the incubation mixtures just before the synaptosomes. The reaction was terminated by filtration through Whatman GF/F filters presoaked in 0.05% (w/v) poly-L-lysine (M r 15 000-30 000). After washing twice with 4 ml of ice-cold buffer (same as described above minus the ascorbic acid and nialamide), the filters were transferred to counting vials. Determination of radioactivity and protein (by the method of Lowry) was done as described previously 16. Specific uptake of [3H]5-HT was defined as total uptake minus non-specific uptake in the presence of 50 ktM chlorimipramine. This non-specific uptake was approximately 14% of the total uptake without preincubation, and 23% with a 10-min preincubation. Filter binding was equal for samples with and without chlorimipramine and amounted to 6% of the total uptake without preincubation and 12% with preincubation. Preincubation of synaptosomes for 10 rain at 37 °C by itself reduced total uptake of [3H]5-HT by about 40% but had no effect on non-specific uptake. Uptake was linear with time up to 1 rain, and with protein up to 0.9 mg per assay. In saturation analysis, the [3H]5-HT concentration ranged from 20 to 250 nM. Affinity (Kin)

and maximal velocity (V ..... ) were estimated with the non-linear least-squares curve-fitting computer program L I G A N D 15. Valinomycin, gramicidin, monensin, veratridine, BTX, BAY K8644, and nimopidine were dissolved in ethanol. Samples of these stocks were added to the incubation mixtures resulting in final ethanol concentrations not exceeding 5% (v/v). Control experiments showed that these ethanol concentrations did not by themselves affect the uptake of [3H]5-HT. When crude synaptosomal preparations were added to incubation mixtures containing [3H]5-HT and either 2.5 or 2 0 / t M of the K + ionophore valinomycin, a small but significant reduction in uptake was observed (approximately 86-89% of control values) (open bars, Fig. 1). Preincubation of the synaptosomes for 10 min (hatched bars) with 20/~M valinomycin resulted in an appreciably greater reduction to 67% of control. The H + ionophore CCCP decreased uptake to a 37% level without preincubation. Both valinomycin and CCCP, at the concentrations used, are expected to collapse the membrane potential as well as ion gradients 6"12"17. The present results do not indicate which effect underlies the reduc-

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349 concentrations as low as 0.03 /zM, monensin reduced uptake to 58% of control, while at concentrations of 1 and 10/zM it lowered uptake to approximately 15% of control. Without preincubation, higher monensin concentrations were required to achieve the same effects. Addition of synaptosomes to incubation mixtures containing the sodium-channel activator veratridine led to decreases in uptake in a concentration-dependent manner (open bars, Fig. 2). The reduction in uptake observed in the presence of 50/zM veratridine (40% of control value) was counteracted by the copresence of 1 /zM TTX (78% of control). BTX, a slowly acting sodium-channel activator, had no effect without preincuhation (open bar), but severely impaired uptake after preincubation in a "VFX-sensitive manner (hatched bars, Fig. 2). Abolishing the inactivation of sodium channels by ScVenom resulted in appreciable reductions in uptake that were counteracted by TTX. The latter effect was less pronounced at higher ScVenom concentrations, suggesting an interaction between the action of ScVenom and that of TTX as has been suggested for BTX and TTX 4. The effect of TTX indicates that the decrease in 5-HT uptake observed with veratridine, BTX, or ScVenom is

tion in 5-HT uptake, but the postulated electroneutrality of 5-HT uptake 16'19'21 favors the latter. Gramicidin, an ionophore for both K + and Na t (ref. 11), had only a slight effect when added together with the synaptosomes at 5/~M, but inhibited uptake completely at 50/~M. With a 10-min preincubation period, 15 and 50 /~M of gramicidin decreased uptake to 14% and 4% of control, respectively. The effect of gramicidin (50/zM) was unaltered by coaddition of the sodium-channel blocker TTX at 1 ~M, a concentration that by itself had no effect with or without preincubation (Fig. 1), indicating that voltage-dependent sodium channels were not involved. Inhibition of Na+,K+-ATPase by ouabain had little or no effect without preincubation (Fig. 1). In contrast, uptake was reduced to 57% of control after preincubation with 100/~M ouabain and to 16% with 200 /~M ouabain. Gramicidin and ouabain collapse Na t gradients as well as K ÷ gradients, resulting in depolarization 1'3. However, the observed reduction in 5-HT uptake may be due to the effect on Na ÷ gradients because preincubation with non-depolarizing 3,13 concentrations of monensin, a N a t / H + exchanger, resulted in a concentration-dependent reduction of uptake (Fig. 1). At

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Fig. 2. Effect of ion channel agents on synaptosomal [3H]serotonin uptake. The abbreviations used are VERA (veratridine), BTX (batrachotoxin), ScV (scorpion venom), BAYK (BAY K8644), NIMO (nimodipine), and TFX (tetrodotoxin). The TTX data are repeated from Fig. 1 for comparison. Other details are as in Fig. 1. *P < 0.05 compared with no drug addition (as Fig. 1). ~P < 0.05 compared with no preincubation (as Fig. 1).

3511 due to opening of voltage-dependent sodium channels. No effects were observed when synaptosomes were preincubated with the Ca 2+ channel activator BAY K8644 (12.5/~M) and the Ca 2+ channel blocker nimodi-

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for concentrations of dihydropyridine compounds as high as 50 ~tM. In contrast, other Ca 2+ channel agents such as bepridil and verapamil have been reported to inhibit 5-HT uptake into brain synaptosomes with IC50 values of approximately 5 /~M (refs. 5 and 18). In comparison, diltiazem is a relatively weak inhibitor of 5-HT uptake 5~ ~. In this context it is of interest to note that bepridil and verapamil interact with the voltage-dependent sodium channel as measured by inhibition of batrachotoxinin A 20-a-benzoate binding (IC50 values of 1 and 3 ~M, respectively); diltiazem is considerably weaker in this respect t4. if these compounds inhibit 5-HT uptake by interacting with the sodium channel, they do so by a mechanism that does not necessarily involve blockade of Na ÷ influx, because T T X has no effect on uptake (Figs. 1 and 2). A saturation analysis of 5-HT uptake was performed to assess the effect of diminishing the normal Na + gradient ([Na+]out > [Na+]i~) by various means. Raising [Na+]in

1 Blaustein, M.P. and Goldring, J.M., Membrane potentials in pinched-off presynaptic nerve terminals monitored with a fluorescent probe: evidence that synaptosomes have potassium diffusion potentials, J. Physiol., 247 (1975) 589-615. 2 Bogdanski, D.E, Tissari, A.H. and Brodie, B.B., Mechanism of transport and storage of biogenic amines. Ill. Effects of sodium and potassium on kinetics of 5-hydroxytryptamine and norepinephrine transport by rabbit synaptosomes, Biochim. Biophys. Acta, 219 (1979) 189-199. 3 Brosemer, R.W., Effects of inhibitors of Na ~ ,K+-ATPase on the membrane potentials and neurotransmitter effiux in rat brain slices, Brain Research, 334 (1985) 125-137. 4 Brown, G.B., [3H]batrachotoxinin-A benzoate binding to voltage-sensitive sodium channels: inhibition by the channel blockers tetrodotoxin and saxitonin, J. Neurosci., 6 (1986) 2064-20711. 5 Brown, N.L., Sirugue, O. and Worcel, M., The effects of some slow channel blocking drugs on high affinity serotonin uptake by rat brain synaptosomes, Eur. J. Pharmacol., 123 (1986) 161-165. 6 Greveling, C.R., McNeal, E.T., McCulloh, D.H. and Daly, J.W., Membrane potentials in cell-free preparations from guinea pig cerebral cortex: effect of depolarizing agents and cyclic nucleotides, J. Neurochem., 35 (1980) 922-932. 7 Holz, R.W. and Coyle, J.T., The effects of various salts, temperature, and the alkaloids veratridine and batrachotoxin on the uptake of [3H]dopamine into synaptosomes from rat striatum, Mol. Pharmacol., 10 (1974) 746-758. 8 Kanner, B.I., Active transport of ~-aminobutyric acid by membrane vesicles isolated from rat brain, Biochemistry, 17 119781 1207-121 I. 9 Koe, B.K., Molecular geometry of inhibitors of the uptake of catecholamines and serotonin in synaptosomal preparations of rat brain, J. Pharmacol. Exp. Ther., 199 11976) 649-661. 10 Kuhar, M.J. and Zarbin, M.A., Synaptosomal transport: a chloride dependence for choline, GABA, glycine and several other compounds. J. Neurochem., 31 (1978) 251 256.

by ScVenom (5/~g/ml) reduced the V..... (from 3.6 to 1.8 pmol.(mg protein) t.min t), but did not change the K m (52 nM) (data not shown). Likewise, raising [Na+]in by

without changing the Vmax. This may indicate that there are mechanistically distinct inhibitory effects on uptake produced by increasing internal Na + or by decreasing external Na +. In conclusion, the present results underscore the importance of ion gradients in the synaptosomal uptake of 5-HT, as is the case for synaptosomal uptake of norepinephrine25 and dopamine 7. This is consonant with the model developed for 5-HT uptake into porcine blood platelet vesicles2~ in which sodium and chloride ions are translocated inwardly along with 5-HT, and potassium ions outwardly upon return of the carrier. It also fits in with the postulated existence of different conformational states of the carrier d e p e n d e n t upon the ionic environment2,11, 2o.

The work was supported by Grant DA 03025 from the National Institute on Drug Abuse.

11 Lee, J.D. and Shih, J.C., Evidence for two conformational states of 5-hydroxytryptamine carrier in rat cortical synaptosomes, Neuropharmacology, 26 (19871 1667-1671. 12 Li, P.P. and White, T.D., Rapid effects of veratridine, tetrodotoxin, gramicidin D, valinomycin and NaCN on the Na ÷ , K ÷ and ATP contents of synaptosomes, J. Neurochem., 28 (19771 967-975. 13 Lichtshtein, D., Kaback, H.R. and Blume, A.J., Use of a lipophilic cation for determination of membrane potential in neuroblastoma-glioma hybrid cell suspensions, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 650-654. 14 McNeal, E.T., Lewandowski, G.A., Daly, J.W. and Creveling, C.R., [3H]batrachotoxinin A 20 a-benzoate binding to voltagesensitive sodium channels: a rapid and quantitative assay for local anesthetic activity in a variety of drugs, J. Med. Chem., 28 11985) 381-388. 15 Munson, P.J. and Rodbard, D., LIGAND: a versatile computerized approach for characterization of ligand-binding systems, Anal. Biochem., 107 11980) 2211-239. 16 O'Reilly, C.A. and Reith, M.E.A., Uptake of [3H]serotonin into plasma membrane vesicles from mouse cortex, J. Biol. Chem., 263 (1988) 6115-6121. 17 Ramos, S., Grollman, E.E, Lazo, P.S., Dyer, S.A., Habig, W.H., Hardegree, M.C., Kaback, H.R. and Kohn, L.D., Effects of tetanus toxin on the accumulation of the permeant lipophilic cation tetraphenylphosphonium by guinea pig brain synaptosomes, Proc. Natl. Acad. Sci. U.S.A., 76 11979) 4783-4787. 18 Rehavi, M., Carmi, R. and Weizman, A., Tricyclic antidepressants and calcium channel blockers: interactions at the (-)desmethoxyverapamil binding site and the serotonin transporter. Eur. J. Pharrnacol., 155 (1988) 1-9. 19 Reith, M.E.A., Zimany, I. and O'Reilly, C.A., Role of ions and membrane potential in uptake of serotonin into plasma membrane vesicles from mouse brain, Biochem. Pharrnacol.. 38 (1989) 2091-21197.

351 20 Ross, S.B., The characteristics of serotonin uptake systems. In N.N. Osborne (Ed.), Biology of Serotonergic Transmission, Wiley, New York, 1982, pp. 169-195. 21 Rudnick, G. and Nelson, P.J., Platelet 5-hydroxytryptamine transport, an electroneutral mechanism coupled to potassium, Biochemistry, 17 (1978) 4739-4742. 22 Shaskan, E.G. and Snyder, S.H., Kinetics of serotonin accumulation into slices from rat brain: relationship to catecholamine uptake, J. Pharmacol. Exp. Ther., 175 (1970) 404-418. 23 Stahl, S.M. and Meltzer, H.Y., A kinetic and pharmacologic

analysis of 5-hydroxytryptamine transport by human platelets and platelet storage granules: comparison with central serotonergic neurons, J. Pharmacol. Exp. Ther., 205 (1978) 118-132. 24 Taylor, C.A., Tsai, C. and Lehmann, J., Sodium fluxes modulating neuronal glutamate uptake: differential effects of local anesthetic and anticonvulsant drugs, J. Pharmacol. Exp. Ther., 244 (1988) 666-672. 25 White, T.D., Inhibition of synaptosomal noradrenaline uptake by veratridine, gramicidin D and valinomycin, J. Neurochem., 29 (1977) 193-198.

Inhibition of serotonin uptake into mouse brain synaptosomes by ionophores and ion-channel agents.

[3H]Serotonin uptake into mouse cerebrocortical synaptosomes was decreased by the K+ ionophore valinomycin, the K+ and Na+ ionophore gramicidin, and t...
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