Carriers for GABA and noradrenaline uptake coexist on the same nerve terminal in rat hippocampus

European Journal of Pharmacology, 136 (1987) 303-310

303

Elsevier EJP 00728

Carriers for GABA and noradrenaline uptake coexist on the same nerve terminal in rat hippocampus Giambattista Bonanno and Maurizio Raiteri

*

lstituto di Farmacologia e Farmacognosia, UniversiM di Genova, Viale Cembrano 4, 16148 Genova, ltaly

Received 25 September 1986, revised MS received 17 December 1986, accepted 3 February 1987

y-Aminobutyric acid (GABA; 1-300 #M) increased the basal release of [3H]noradrenaline ([3H]NA) from rat hippocampal synaptosomes. The effect of GABA at low concentrations (below 10 ~M) was largely bicucullineosensitive while the sensitivity to bicuculline decreased at higher concentrations. Muscimol mimicked GABA but only below 10/~M; bicuculline antagonized the effect of muscimol. Up to 300 ~tM ( - ) b a c l o f e n did not modify [3H]NA release. The effect of GABA was potently counteracted by S K & F 89976A, S K & F 100330A and S K & F 100561, three novel inhibitors of neuronal GABA uptake. These compounds could not entirely prevent the effect of GABA, being least effective at the lowest GABA concentrations (below 10 /tM) and becoming progressively more effective when the concentrations of GABA were increased. The effect of muscimol was insensitive to S K & F 89976A. The effect of 100 /xM GABA was totally prevented when bicuculline and uptake inhibitor were added together to the superfusion medium. The results suggest that the basal release of [3H]NA can be enhanced by GABA through two mechanisms: GABA A receptor activation and penetration into NA terminals by a GABA uptake process. Thus a carrier for the uptake of NA and a carrier for the uptake of GABA appear to coexist on the same nerve terminal in rat hippocampus. GABA; Noradrenaline release; GABA uptake inhibitors; GABA receptors; Synaptosomes (superfused)

I. Introduction P r e s y n a p t i c m o d u l a t i o n of n e u r o t r a n s m i t t e r release occurs in general t h r o u g h the activation of r e c e p t o r s l o c a t e d o n the external surface of the releasing nerve t e r m i n a l s (see for recent reviews: Chesselet, 1984; Raiteri et al., 1984). However, it has been f o u n d recently that y - a m i n o b u t y r i c acid ( G A B A ) c o u l d e n h a n c e the s p o n t a n e o u s release of [3H]acetylcholine newly f o r m e d from tritiated choline in rat h i p p o c a m p u s s y n a p t o s o m e s , with no a p p a r e n t i n v o l v e m e n t of p r e s y n a p t i c G A B A rec e p t o r s ( B o n a n n o a n d Raiteri, 1986). Since the

* To whom all correspondence should be addressed: Istituto di Farmacologia e Farmaeognosia, Viale Cembrano 4, 16148 Genova, Italy.

effect of G A B A was p r e v e n t e d b y i n h i b i t o r s of the n e u r o n a l u p t a k e of the a m i n o acid, the m o s t likely i n t e r p r e t a t i o n was that, in rat h i p p o c a m p u s , an u p t a k e system selective for G A B A is l o c a t e d on cholinergic nerve terminals. This novel finding can b e i n t e r p r e t e d in various ways. F o r instance, m o d u l a t i o n of t r a n s m i t t e r release might occur in some cases b y a hitherto u n k n o w n m e c h a n i s m involving the selective transp o r t of the m o d u l a t o r into the releasing terminal. A n o t h e r p o s s i b i l i t y is that G A B A a n d acetylcholine coexist in some h i p p o c a m p a l nerve endings. It was therefore of interest to investigate w h e t h e r the presence of a G A B A t r a n s p o r t e r on a n o n - G A B A e r g i c t e r m i n a l could be a p h e n o m e n o n that applies to n e u r o t r a n s m i t t e r systems other t h a n that of acetylcholine. In the p r e s e n t work, we have e x a m i n e d the effect of G A B A o n the s p o n t a n e o u s

0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

304 release of [3H]noradrenaline ([3H]NA) from rat hippocampus synaptosomes.

2. Materials and methods

Adult male Sprague-Dawley rats (200-250 g) were used. Crude synaptosomes were prepared from hippocampus as previously described (Marchi and Raiteri, 1985). Briefly, the tissue was homogenized in 40 volumes of 0.32 M sucrose buffered at pH 7.4 with phosphate. The homogenate was centrifuged (5 min, 1000 × g) to remove nuclei and debris and the synaptosomes were isolated from the supernatant by centrifugation at 12000 × g for 20 min. The synaptosomal pellet was then resuspended in a physiological medium of the following composition (mM): NaC1 125, KCI 3, CaCI 2 1.2, MgSO 4 1.2, N a H 2 P O 4 1, N a H C O 3 22, glucose 10 (aeration with 95% 02 and 5% CO 2 at 37°C) pH 7.2-7.4. Protein was measured by a modification of the method of Lowry (Petersen, 1977). Synaptosomes were incubated with [3H]NA (final concentration 0.08 /~M) for 15 rain at 3 7 ° C and aliquots of the suspension were layered on 0.65 /~m Millipore filters (0.20-0.35 m g / p r o t e i n per filter) at the bottom of 20 parallel superfusion chambers (Raiteri et al., 1974; Raiteri and Levi, 1978, for technical details). Superfusion was started with standard medium at a rate of 0.6 m l / m i n and, after 34 min had been allowed for the system to equilibrate, 5 separate 2 rain fractions were collected. The synaptosomes were exposed to GABA, muscimol, ( - ) b a c l o f e n , leucine, valine or a-aminoisobutyric acid at the end of the second fraction collected; bicuculline, S K & F 89976A, S K & F 100561 and S K & F 100330A were added 8 min before GABA. In some experiments on [3H]NA release, the fractions were collected into vials containing 100 /~1 of a protective solution (1.5% EDTA, 1% ascorbic acid and 0.001% unlabeled NA); the [3H]NA present in each fraction and that remaining in the filters at the end of the superfusion was measured following separation from 3Hdeaminated metabolites on Bio-Rex 70 columns, according to the method of Smith et al. (1975).

The release of tritium into the superfusate samples was calculated as percent of the radioactivity content of synaptosomes at the start of the respective collection period. The effects of the drugs on amine release were evaluated by calculating the ratio between the percent efflux in the fraction corresponding to the maximal effect of GABA (in general the fourth fraction collected) and the percent efflux in the second fraction (see fig. 1, inset). This ratio was compared to the corresponding ratio obtained under control conditions. The two-tailed Student's t-test was used for comparison of mean values. [3H]NA (44 C i / m m o l ) was obtained from Amersham Radiochemical Centre (U.K.); GABA, L-leucine, L-valine and a-aminoisobutyric acid were from Serva (Heidelberg, FRG); (+)bicuculline from Sigma (St. Louis, USA). The following drugs were generous gifts from the companies indicated: ( - ) b a c l o f e n (Ciba-Geigy, Basel, Switzerland); muscimol (Zambon Farmaceutici, Milan, Italy); S K & F 89976A (N-(4,4-diphenyl-3butenyl)-nipecotic acid), S K & F 100330A (N(4,4-diphenyl-3-butenyl)-guvacine) and SK & F 100561 (N-(4,4-diphenyl-3-butenyl)-homo-/~-proline) (Smith Kline & French, Welwyn, England).

3. Results

3.1. Effects of GABA on the spontaneous release of [-~H]NA from rat hippocampus synaptosomes When synaptosomes prepared from rat hippocampus and prelabeled with [3H]NA were exposed in superfusion to exogenous GABA there was an increase in the spontaneous outflow of radioactivity. The effect of GABA was concentration-dependent within the range of concentrations (1-300/~M) examined (fig. 1). Neutral amino acids like leucine, valine and a-aminoisobutyric acid tested at 100-300/~M, did not augment significantly the release of [3H]NA (data not shown). In order to ascertain whether the GABA-induced increase of tritium outflow reflected an enhancement of [3H]NA release, the 3H-metabolites and [3H]NA were separated both in the frac-

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Fig. 1. Effect of G A B A on the basal release of [3H]NA ( O ) or total tritium (O) in rat hippocampus synaptosomes. Synaptosomes were labeled with [3H]NA and exposed to G A B A in superfusion as described under Methods, Fractions were collected and counted for radioactivity. In some experiments separation between [3H]NA and 3H-metabolites was carried out according to Smith et al. (1975). The data presented are m e a n s + S.E.M. of 4-10 experiments run in duplicate. Inset: effect of 100 /tM G A B A on the basal efflux of [3H]NA (El) or 3H-deaminated metabolites (11). The synaptosomes were labeled with [3H]NA then were superfused for 38 rain with standard medium which was then replaced with a new medium containing 100 ~ M of G A B A (see arrow). Fractions were collected every 2 rain and aliquots were used to measure the total radioactivity and to evaluate the [3H]NA content. The superfused tissue was extracted with 1 N hydrochloric acid: formic acid 85:15 v / v and aliquots were analyzed for total tritium and [3H]NA. The release of 3H-deaminated metabolites was calculated by subtracting the radioactivity accounted for by [3H]NA from the total radioactivity released. The curve presented is the average of three experiments in triplicate.

tions collected during superfusion and in the synaptosomes at the end of superfusion. The inset of fig. 1 illustrates the results obtained when synaptosomes were exposed to 100 # M GABA. Before the addition of G A B A the fractional release of [3H]NA and that of 3H-metabolites were almost identical. However, when G A B A was added to the superfusion medium (see arrow), the en-

h a n c e m e n t of tritium outflow was totally accounted for by unmetabolized [3 H]NA. The data obtained with other concentrations of GABA (3, 10 and 30 #M) and expressed as percent increase of [3H]NA release over control, are reported in fig. 1. In conclusion, G A B A at the concentrations used in this study caused the release of intact [3H]NA; therefore we refer in the remainder of

306

the text to the GABA-induced tritium efflux in excess of the baseline release as GABA-induced [3H]NA release.

/~M but the effect of muscimol reached a plateau at 10 t~M. No enhancing effect on the release of [3H]NA could be observed with ( - ) b a c l o f e n , tested at 100 or 300/~M.

3.2. Experiments with drugs selective for GABA A or GABA 8 receptors

3.3. Effects of GABA uptake inhibitors on the GABA-induced release of [ 3H]NA

The antagonism by bicuculline of the GABAinduced [3H]NA release is shown in fig. 2. Although the GABA A receptor antagonist could not be used at concentrations higher than 100 # M because it itself increased the release of tritium, the results in fig. 2, particularly those obtained with 10/~M GABA, indicate that only a portion of the GABA-induced [3H]NA release was bicuculline-sensitive. The bicuculline-insensitive portion of the GABA effect appears to be higher at 100 /~M than at 10 /~M GABA. When 3 /~M GABA was used, the effect of the amino acid was entirely bicuculline-sensitive (fig. 4). Muscimol enhanced the spontaneous release of [3H]NA (fig. 2). The effect of muscimol was quantitatively similar to that of GABA at the concentrations lower than 10

The enhancement of [3H]NA release evoked by GABA was counteracted by three drugs, S K & F 89976A, S K & F 100561 and S K & F 100330A (fig. 3), which are novel inhibitors of GABA uptake (Yunger et al., 1984). These compounds have been reported to be more potent inhibitors of [3H]GABA uptake than the parent amino acids guvacine and nipecotic acid (Yunger et al., 1984). Moreover, they appear to be quite selective inhibitors of GABA uptake in rat brain synaptosomes: the IC50 of S K & F 89976A against 1 /~M [3H]GABA uptake was about 0.2 /~M whereas it was higher than 100 ~M against the uptake of [3H]NA (0.1 /~M), [3H]dopamine (0.1 #M) or [3H]serotonin (0.03 /.LM) (data not shown). The

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Fig. 4. Effects of (+)bicuculline and SK&F 89976A on the enhancement of [3H]NA release induced by GABA or muscimol. Experimental details as in the legend to fig. 1. (+)Bicuculline and SK&F 89976A, separately or together, were added to the superfusion medium 8 min before GABA or muscimol. The data presented are m e a n s + S.E.M. of 3-6 experiments in triplicate. * P < 0.01; ** P < 0.001 compared to the control obtained using GABA alone. * P < 0.01; ** P < 0.001 compared to the respective control obtained using GABA in presence of 100 ~M bicuculline; * P < 0.001 compared to the respective control obtained using G A B A in presence of 10 # M SK&F 89976A.

308 S K & F compounds were much more potent than bicuculline in preventing the effect of GABA. For instance, the effect of 100 ~ M GABA was inhibited equally by 1 /~M S K & F 89976A and by 100 # M bicuculline (see fig. 3 vs. fig. 2). Similarly to bicuculline, however, the S K & F compounds were unable to counteract completely the GABA-induced [3H]NA release. A plateau effect was reached at about 10/~M of the uptake blocker, an indication that a portion of the G A B A effect was insensitive to the G A B A uptake inhibitors. Opposite to what we observed in the case of bicuculline, the portion of the G A B A effect insensitive to the S K & F compounds appeared to be greater at 10 /~M than at 100 ~ M GABA. Moreover, the effect of 3 btM G A B A (which was totally bicucullinesensitive) could not be significantly affected by S K & F 89976A (fig. 4).

3.4. Effects of a combination of bicuculline and GABA uptake inhibitor on the GABA-induced [3H]NA release Figure 4 shows that the GABA-induced [3 H]NA release was almost totally abolished when bicuculline and S K & F 89976A were present concomitantly in the superfusion medium. In contrast, the enhancement of [3H]NA release caused by muscimol was insensitive to S K & F 89976A but was almost totally prevented by bicuculline.

4. Discussion

Most of the experiments described here were carried out with synaptosomes prepared from rat hippocampus and prelabeled with 0.08 ttM [3H]NA. At this low concentration of label and considering that the hippocampus contains very few dopamine terminals, a selective uptake of [ 3 H ] N A b y the N A c a r r i e r p r e s e n t on noradrenergic nerve endings could be reasonably assumed. The involvement of serotonergic nerve terminals possibly labeled by [3H]NA should be excluded Since G A B A did not evoke any release from hippocampal synaptosomes prelabeled with [3H]serotonin (Bonanno and Raiter i, in press). The release from prelabeled synaptosomes was

studied with a technique of superfusion which has been shown to minimize indirect effects between transmitters (see for details: Raiteri and Levi, 1978). Thus the first conclusion to be drawn from the present work could be that, in rat hippocampus, GABA enhances the spontaneous release of newly taken up N A by acting directly on NA terminals. There is increasing evidence that GABA acts on two distinct receptors: a G A B A A receptor, which can be blocked by bicuculline and activated by muscimol and a G A B A B receptor, which is bicuculline-insensitive but can be activated by baclofen (Enna, 1983; Bowery et al., 1984). The finding that ( - ) b a c l o f e n had no effect on the spontaneous release of [3H]NA (fig. 2) indicates that an involvement of G A B A a receptors in the GABA-induced [3H]NA release is unlikely. On the other hand, the GABA A agonist muscimol enhanced the release of [3H]NA, and bicucuHine antagonized both muscimol and GABA, which indicates involvement of G A B A A receptors. However, the following observations allow the conclusion that activation of G A B A A receptors located on N A terminals is not the only mechanism involved in the effect of GABA: (a) bicuculline antagonized only in part the effect of the amino acid except when GABA was used at relatively low concentrations (fig. 4); (b) a comparison between the concentration-response relation of G A B A and muscimol (see figs. 1 and 2) shows that the effect of the GABA A agonist which was at least as potent as G A B A in the low concentration range reached a plateau at about 10 /~M, while the effect of G A B A continued to increase; (c) the effect of muscimol was almost totally antagonized by bicuculline. Thus, the effect of G A B A on [3H]NA release includes a component which does not seem to be mediated by GABA A receptors. That G A B A could enhance the spontaneous release of [3H]NA from rat hippocampus synaptosomes through activation of G A B A A receptors had been reported previously by Fung and Fillenz (1983). However, these authors did not pay attention to the fact that bicuculline antagonized only part (about 50%) of the G A B A effect. In the absence of bicuculline, the GABA-induced [3H]NA release was counteracted by drugs

309 ( S K & F 89976A, S K & F 100561 and S K & F 100330A) which are potent and selective inhibitors of the uptake of G A B A into rat brain synaptosomes (Yunger et al., 1984; present results). As observed with bicuculline, part of the effect of G A B A was insensitive to the G A B A uptake inhibitors. Interestingly, however, when used together at their respective maximally effective concentration, bicuculline and S K & F 89976A functioned in an additive way towards GABA, indicating that the two compounds were acting through different mechanisms. Although the possibility that G A B A acted through a novel non-GABA A nonG A B A B receptor subtype (at which the above uptake inhibitors would be potent antagonists) cannot be ruled out, the finding that the three compounds had strong in vivo anticonvulsant activity and did not displace [3H]muscimol (Yunger et al., 1984) or [3H]GABA from their binding sites (Pittaluga and Raiteri, unpublished data) makes it unlikely that they also have GABA receptor antagonist activity. Thus, on the basis of the present results, G A B A can apparently enhance the spontaneous release of NA, not only by activating G A B A A receptors, but also by a mechanism involving uptake of GABA. Where is this G A B A uptake system located? If we assume that [3H]NA labels N A terminals selectively and that indirect effects are unlikely in superfused synaptosomes, the answer is that the G A B A carrier is located on N A terminals. In other words, there seems to exist in rat hippocampus a special type of nerve terminal possessing not only a transport system for N A but also a carrier for G A B A uptake. The possibility that a non-selective low affinity amino acid transport site is involved is unlikely since a number of neutral amino acids such as leucine, valine or a-aminoisobutyric acid did not evoke [3H]NA release. It is impossible to say whether the GABA A receptors are located on the same nerve terminals that possess the G A B A transport site. In fact, N A terminals carrying a G A B A A presynaptic receptor and N A terminals endowed with a G A B A uptake system may exist separately. The G A B A A receptor-mediated mechanism however appears to operate essentially in the lower range of the G A B A

concentrations examined in this work. The effect of GABA at 3 # M was totally bicuculline-sensitive but was unaffected by the G A B A uptake inhibitors (fig. 4). One can only speculate about the reason why a noradrenergic terminal should possess a G A B A carrier. It is well known that modulators of transmitter release generally act through activation of presynaptic receptors located on the external membrane of nerve endings. However, the enhancement of N A release observed when the G A B A carrier is activated raises the possibility that transmitter release is also regulated by a mechanism involving the entry of the modulator into the releasing terminal. As a second hypothesis, considering that transmitter substances can coexist in the same neuron (Chan-Palay, 1977; H~Skfelt et al., 1980; Cuello, 1982), the simultaneous presence of uptake systems for N A and for G A B A on the same nerve ending would be compatible with coexistence of the two transmitter. However, it has to be kept in mind that the N A terminals in the hippocampus b e l o n g to an extrinsic p a t h w a y , whereas GABAergic neurons are largely intrinsic. Therefore, the nerve endings co-storing GABA and NA, if any, could only exist in limited numbers. In conclusion, the basal release of N A from rat hippocampus nerve endings can be enhanced by G A B A through a mechanism which apparently involves the uptake of the amino acid by a G A B A carrier located on N A terminals. The enhancement of the basal release of acetylcholine induced by GABA in the same brain region was interpreted similarly (Bonanno and Raiteri, 1986). The coexistence on the same nerve terminal of two uptake carriers each of which was thought to be present separately on, and to represent a typical property of a particular family of nerve endings is a new finding which is open to various interpretations and therefore deserves further investigation.

Acknowledgements This work was supported by grants from the Italian Ministry of Education and from the Italian National Research Council. The authors wish to thank Miss Daniela Asaro and Miss Graziella Pellegrini for their skillful technical help and Mrs.

310 Maura Agate for her expert assistance in preparing the manuscript.

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