Neurochemical Research (1) 409-416 (1976)

RELEASE A N D E X C H A N G E OF N E U R O T R A N S M I T T E R S IN S Y N A P T O S O M E S : E F F E C T S OF THE I O N O P H O R E A23187 A N D OF O U A B A I N GIULIO LEVI, 1 PETER J. ROBERTS, z AND MAURIZIO RAITERI 3 aLaboratorio di Biologia Cellulare, CNR Rome 2Department of Physiology and Biochemistry University of Southhampton, England 31stituto di Farmacologia Universitd Cattolica, Rome

Accepted April 16, 1976

T h e effects of the ionophore A23187 and o f ouabain on the release o f [ ~ H ] G A B A and [~H]norepinephrine were studied in superfused rat brain synapt o s o m e s . E a c h of the two drugs moderately stimulated the s p o n t a n e o u s release of [ 3 H ] G A B A , but greatly potentiated the release o f [ 3 H ] G A B A induced by unlabeled G A B A . In contrast, the ionophore and norepinephrine s h o w e d an additive, but not a " s u p r a a d d i t i v e , " releasing effect on s y n a p t o s o m a l ['~H]norepinephrine. Ouabain modestly and transiently potentiated the norepinephrine-induced [3H]norepinephrine release, which, h o w e v e r , was inhibited by the drug after a few minutes. It is suggested that in the n e w i n t r a s y n a p t o s o m a l ionic conditions determined by the two drugs, the stoichiometry of the basal h o m o e x c h a n g e of G A B A is changed in a direction favoring net o u t w a r d transport.

INTRODUCTION The divalent cation ionophore A23187 has been shown to stimulate the release of hormones and neurotransmitters from a variety of secretory cells (1-4), including nerve endings (5,6), and is believed to act by causing an increased influx of Ca g+ into the secretory cells (5-7). The

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9 1976Plenum PublishingCorporation, 227 West 17th Street, New York, N.Y. 10011. No part of this publicationmay be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming~recording, or otherwise, without written permissionof the publisher.

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FIO. 1. Effect of ionophore A23187 and of ouabain on the spontaneous and GABAstimulated [3H] GABA release from synaptosomes. See the text for experimental details. Arrows indicate the time at which the composition of the superfusion medium was changed. (a) Each of the 4 lower curves is the average of 6 experiments (4 run in duplicate); the 2 top curves are averages of 2 duplicate experiments. (b) Each curve is the average of 2 duplicate experiments.

increased availability of "free" intraceUular calcium has been shown to trigger neurosecretory processes (8-14) by a still-unknown mechanism. Therefore, A23187 may be a potential tool for studying the mechanisms of neurotransmitter release at nerve endings. Ouabain also stimulates the release of putative transmitters in vitro, and it has been suggested that the increased release is due to an elevation of the intracellular concentration of Na + determined by the drug (15). In the present investigation, we have demonstrated that the ionophore A23187 and ouabain, which are both weak stimulators of [3H]GABA release from isolated nerve endings, greatly potentiate the release of [~H] GABA induced by unlabeled GABA. Furthermore, we have shown that the effects of the two drugs on the release of [3H]norepinephrine ([3H]NE) are qualitatively different from those observed with GABA.

E F F E C T S OF A23187 A N D O U A B A I N ON N E U R O T R A N S M I T T E R

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EXPERIMENTAL PROCEDURE Synaptosomes prepared from adult male Wistar rat cerebrum according to Gray and Whittaker (16) were resuspended in 0.32 M glucose at a protein concentration of 4 mg/ml, diluted 1:10 in a Krebs-Ringer medium (128 mM NaCI, 5 mM KCI, 2.7 mM CaCI2, 1.2 mM MgSO4, 25 mM Tris-HC1 buffer at pH 7.35), equilibrated for 10 rain at 37~ and then prelabeled for 10 min with 0.5/zM [3H]GABA (New England Nuclear Corp., spec. act. 10 Ci/mmole), or with 0.1/~M [ZH]norepinephrine (Amersham Radiochemical Centre, spec. act. 10.1 Ci/mmole). Portions (1 ml) of the suspensions were collected on DAWP 02500 Millipore filters and placed at the bottom of 6-8 parallel superfusion chambers (17) thermostated at 37~ After being washed with warm medium for a few seconds, the synaptosomes were superfused with oxygenated, glucose-containing medium for 7.5 rain, and then (see the arrows in Figs. 1 and 2) with new media containing either the drug under study or the unlabeled neurotransmitter under study, or both, as indicated in the figures. Fractions of 1 min (0.6 ml) were collected; the radioactivity released, and that remaining on the filters, was measured. In the supeffusion conditions used, reuptake of spontaneously released substrates was completely prevented (17-19). The incubation and superfusion media contained 10 /xM aminooxyacetic acid, or 12.5 /zM nialamide and 1 mM ascorbic acid, to prevent [3H]GABA and [3H]NE metabolism,

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FIG. 2. Effect of ionophore A23187 and of ouabain on the spontaneous and norepinephrine (NE)-stimulated [3H]NE release from synaptosomes. See the text for experimental details. Arrows indicate the time at which the composition of the superfusion medium was changed. In the experiment in (b) marked as " o u a b a i n . . . from zero time" (third entry from the top in the legend), ouabain was added to the medium at the beginning of the superfusion, whereas NE was added at min 7.5, as in the other cases. Each curve is the average of 4 duplicate experiments (a) or of 2 duplicate experiments (b).

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respectively. A23187 was dis solved in absolute ethanol and added to the superfusion media immediately before use (10 /~lJml). An equivalent amount of ethanol was added to the control media. The data on [3H]GABA release presented in Fig. 1 were obtained with purified synaptosomes. One duplicate experiment was run using a crude synaptosomal preparation (P2), with qualitatively similar results. The data on [ZH]NE release presented in Fig. 2 were obtained with crude synaptosomal preparations (P2)- One quadruplicate experiment using purified synaptosomes gave essentially identical results.

RESULTS AND DISCUSSION Figure 1 shows that the release of [~H] GABA from synaptosomes was largely stimulated by the addition of 10 /zM unlabeled GABA to the superfusion fluid. This release of radioactivity by homoexchange has already been discussed in previous studies (18,20,21). The calcium ionophore A23187 caused only a moderate enhancement of [3H] GABA release, which was not further increased by higher ionophore concentrations. Ouabain had a somewhat greater effect than A23187. When unlabeled GABA was added to the superfusion media together with either A23187 or ouabain, the release of [3H]GABA became significantly higher than the sum of the release induced by unlabeled GABA alone plus that induced by the drug alone, without any lag period. In neither case could this "supraadditive" effect be explained on the basis of an accelerated 1:1 homoexchange process of the amino acid in the presence of the drug, since both drugs inhibited the influx of [3H] GABA into synaptosomes by 50-70%. It therefore seems legitimate to conclude that the n e t outward movement of GABA from synaptosomes observed in the presence of exogenous GABA plus either A23187 or ouabain was larger than that induced by either drug alone. A consequence of this reasoning is that a large part of the inhibitory effect of A23187 and ouabain on [3H]GABA uptake might paradoxically be due to an enhancement of net release induced by the presence of extracellular amino acid. All the effects of A23187 on GABA uptake and spontaneous and stimulated release were abolished when CaCI2 was not added to the incubation and superfusion media, showing a direct involvement of the cation in the mechanism of action of the ionophore. This finding is in keeping with the concept that the ionophore acts by promoting an influx of Ca 2+ under conditions in which the concentration of extracellular Ca ~+ exceeds that of intracellular " f r e e " Ca 2+. On the other hand, none of the effects of ouabain changed significantly when Ca z+ was omitted from the media, even in the presence of 0.1 mM E GTA. Although

EFFECTS

O F A23187 A N D O U A B A I N

ON NEUROTRANSMITTER

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ouabain has been described as being able to increase the influx of 45Ca2+ into synaptosomes (23,24; however, see reference 25), it seems that changes in calcium influx are not responsible for the glycoside-induced effects on GABA transport described in the present study. Ouabain might increase the availability of intracellular " f r e e " calcium indirectly, through its effects on sodium and potassium concentrations. However, calcium is not expected to influence GABA carrier-mediated transport when the sodium concentration is relatively high (26). Martin and Smith (26) suggested that if the synaptosomal inward and outward transports of GABA have similar ionic requirements, sodium on one hand, and calcium (when the concentration of sodium is low) on the other, should stimulate the effiux of GABA from synaptosomes. It is conceivable that when the carrier-mediated transport of GABA is activated by the presence of extracellular GABA (Fig. 1), the addition of the ionophore or of ouabain, which cause an increase in the intracellular concentration of Ca 2+ and Na +, respectively, effects a change in the stoichiometry of the GABA homoexchange process. In the new ionic conditions determined by the drugs, one molecule of exogenous amino acid could drive the exodus of more than one molecule of intracellular amino acid, with a resulting net release superimposed on the release caused by the drug alone. Figure 2 shows that unlabeled norepinephrine (NE) stimulated the release of [3H]NE from supeffused synaptosomes. This effect could be due to homoexchange, as in the case of amino acids, or to a displacement from vesicle-bound stores. However, neither of these phenomena could account for the high affinity uptake of radioactive NE, as discussed in detail elsewhere (27). A23187 at the same concentration as that used in the experiments with [3H]GABA caused a substantial stimulation of [3H]NE release (Fig. 2a). The combined effect of A23187 and unlabeled NE did not cause a "supraadditive" release of the labeled amine, as was observed with GABA. Ouabain had a minimal effect on the spontaneous release of [3H]NE, and moderately potentiated the releasing effect of unlabeled NE, only during the first few minutes after the simultaneous addition of the two compounds (Fig. 2b). The releasing effect of NE was decreased, however, when the amine was added to the superfusion fluid a few minutes after ouabain (Fig. 2b), a condition in which GABA and ouabain still showed a "supraadditive" releasing effect on [SH] GABA (data not shown). Since similar mechanisms of transport at synaptosomal membranes have been described for catecholamines and for amino acids (28,29), one might expect the effects of A23187 and of ouabain on [~H]NE release to be similar to those obtained with [3H] GABA. The differences that were

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observed instead might have several explanations. One likely possibility is that very little, if any, " f r e e " amine is present in the synaptosomal cytoplasm (30-32). The release of [3H]NE induced by exogenous, unlabeled NE may thus be due to a displacement of the radioactive amine from endogenous binding sites, rather than to a strict homoexchange process located at the plasma membrane. A23187 would not be expected to potentiate the exogenous amine-induced release of [3H]amine, unless it increased the cytoplasmic content of " f r e e " labeled amine. In this respect, it is pertinent to recall that depolarization of synaptosomes causes a calcium-dependent release of unmetabolized [3H]NE (33), a finding that would be difficult to explain if the amine were released from the storage vesicles into the cytoplasm, where it would be rapidly deaminated by monoaminoxidases. Moreover, the ionophore A23187 has been shown to release unmetabolized dopamine from striatal synaptosomes (5). Except during the first few minutes, ouabain decreased, rather than potentiated, the stimulatory effect of unlabeled NE on the release of [3H]NE (Fig. 2b). This effect of ouabain may be due to the fact that the high intracellular Na + concentration caused by the drug prevents the intracellular binding of the newly taken up unlabeled amine, and consequently slows down the displacement of the previously accumulated [3H]amine. The early stimulatory effect of ouabain on the NEinduced [3H]NE release (Fig. 2b) might be due to a temporary increase of free labeled amine in the cytoplasm, consequent to an Na+-induced detachment from intracellular binding sites. This increased free amine could rapidly exit from the particles through an accelerated exchange diffusion process. The effects of the ionophore and of ouabain on the GABA-stimulated [~H]GABA release described in the present study might be relevant to the understanding of biochemical mechanisms underlying synaptic function. If phenomena similar to those observed with isolated synaptosomes were also present in nerve terminals of the living brain, one could suggest that the low concentrations of GABA maintained in the synaptic cleft by the homoexchange process (18,20,22) operating in normal, resting conditions might subserve the function of potentiating the release of GABA when the physiological stimulus reaches the synapse and causes an inward current of sodium and calcium. ACKNOWLEDGMENTS We thank Dr. Graziella Rusca and Mr. Alberto Coletti for assistance. Peter J. Roberts participated in this work during a leave of absence from

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the D e p a r t m e n t o f P h y s i o l o g y & B i o c h e m i s t r y of the U n i v e r s i t y o f S o u t h a m p t o n , U . K . , a n d w a s s u p p o r t e d b y the R o y a l S o c i e t y a n d E M B O . T h e i o n o p h o r e A23187 w a s a k i n d gift o f Eli L i l l y a n d Co. T h i s s t u d y was p a r t l y s u p p o r t e d b y R e s e a r c h G r a n t N o . 922 o f the N o r t h A t l a n t i c T r e a t y O r g a n i z a t i o n , a n d G r a n t N o . CT.74.0024904 o f the Italian National Research Council.

REFERENCES 1. GARCIA,A.G., KIRPEKAR, S.M., and PRAT,J.C. (1975) A calcium ionophore stimulating the secretion of catecholamines from the cat adrenal. J. Physiol. 244, 253-262. 2. HELLMAN, B. (1975) Modifying actions of calcium ionophores on insulin release. Biochim. Biophys. Acta 399, 157-169. 3. WOLLHEIM, C.B., BLONDEL, B., TRUEHEART, P.A., RENOLD, A.E., and SHARP, W.G. (1975) Calcium induced insulin release in monolayer culture of endocrine pancreas. J. Biol. Chem. 250, 1354--1360. 4. L1CHTENSTEIN,L.M. (1975) The mechanism of basophile histamine release induced by antigen and by the calcium ionophore A23187. J. Immunol. 144, 1692-1699. 5. HOEZ, R.W. (1975) The release of dopamine from synaptosomes from rat striatum by the ionophores X537A and A23187. Biochim. Biophys. Acta 375, 138-152. 6. THOA, N.B., COSTA,J.L., MOSS, J., and KOPIN, I,J. (1974) Mechanism of release of norepinephrine from peripheral adrenergic neurons by the calcium ionophores X537A and A23187. Life Sci. 14, 1705-1719. 7. FOREMAN,J.C., MONGAR,J.L., and GOMPERTS, B.D. (1973) Calcium ionophores and movement of calcium ions following the physiological stimulus to a secretory process. Nature 245, 249-251. 8. KATZ, B., and MILEDI, R. (1970) Further study of the role of calcium in synaptic transmission. J. Physiol. 207,789--801. 9. BLAUSTEIN, M.P., JORNSON, E.M., and NEEDEEMAN, P. (1972) Calcium-dependent norepinephrine release from presynaptic nerve endings in vitro. Proc. Nat. Acad. Sci. U.S.A. 69, 2237-2240. 10. BLAUSTEIN, M.P. (1975). Effects of potassium, veratridine and scorpion venom on calcium accumulation and transmitter release by nerve terminals in vitro. J. Physiol. 247, 617-655. 11. LEVY, W.B., HAYCOCK,J.W., and COTMAN,C.W. (1974) Effects of polyvalent cations on stimulus-coupled secretion of 14C-gamma-amino-butyric acid from isolated brain synaptosomes. Mol. Pharmacol. 10, 438-449. 12. REDBURN, D.A., and COTMAN, C.W. (1974) Calcium-dependent release of [t4C]GABA from vinblastine and colchicine treated synaptosomes. Brain Res. 73, 550-557. 13. NICKLAS,W.J., PUSh,N, S., and BERL, S. (1973) Effect of vinblastine and colchicine on uptake and release of putative transmitters by synaptosomes and on brain actomyosin-like protein. J. Neurochem. 20, 109-121. 14. OSBORNE,R.H., and BRADFORD,H.F. (1975) The influence of sodium, potassium and lanthanum on aminoacid release from spinal medullary synaptosomes. J. Neurochem. 25, 35-41.

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15. I-~AMMERSTAD,J.P., and CUTLER, R.W.P. (1972) Sodium ion movements and the spontaneous and electrically stimulated release of 3H-GABA and 14C-glutamic acid from rat cortical slices. Brain Res. 47,401--413. 16. GRAY, E.G., and WHITTAKER,V.P. (1962) The isolation of nerve endings from brain: An electron microscopic study of cell fragments derived by homogenization and centrifugation. J. Anat. 96, 79-88. 17. RAITERI,M., AN6ELINI, F., and LEVI, G. (1974) A simple apparatus for studying the release of neurotransmitters from synaptosomes. Eur. J. Pharmacol. 25,411-414. 18. RAITERI,M., FEDERICO,R., COLETTI, A., and LEVI, G. (1975) Release and exchange studies relating to synaptosomal uptake of GABA. J. Neurochem. 24, 1243-1250. 19. RAITEm, M., BERTOLLINI,A., AN6ELINI, F., and LEVl, G, (1975) D-amphetamine as a releaser or reuptake inhibitor of biogenic amines in synaptosomes. Eur. J. Pharmacol. 34, 189-195. 20. LEVI, G., and RAITERI, M. (1974) Exchange of neurotransmitter amino acid at nerve endings can simulate high affinity uptake. Nature 250, 735-737. 21. SIMON, J.R., MARTIN, D.L., and KROLL, M. (1974) Sodium-dependent efflux and exchange of GABA in synaptosomes. J. Neurochem. 23, 981-991. 22. LEvi, G., PACE, U., and RAITERI, M. (1976) Uptake and exchange of GABA and glutamate in isolated nerve endings. In: Levi, G., Battistin, L., and Lajtha, A. (eds.), Transport Phenomena in the Nervous System: Physiological and Pathological Aspects Vol. 69 of Advancement in Experimental Medicine and Biology, Plenum Press, New York, pp. 273-289. 23. SWANSON,P.D., ANDERSON,L., and STAHL, W.L. (1974) Uptake of calcium ions by synaptosomes from rat brain. Biochim. Biophys. Acta 356, 174-183. 24. GODDARD, G.A., and ROBINSON, J.D. (1975) Calcium fluxes in rat brain synaptosomes. Fed. Proc. Fed. Am. Soc. Exp. Biol. 34, 715. 25. KAMINO,K., UYESAI