Brain Research, 539 (1991) 191-195 Elsevier
191
BRES 16251
Cholinergic nerve terminals of human cerebral cortex possess a G A B A transporter whose activation induces release of acetylcholine Giambattista Bonanno 1, Antonio Ruelle 2, Gian Carlo Andrioli 2 and Maurizio Raiteri 1 Ilstituto di Farmacologia e Farmacognosia, Universitd degli Studi di Genova, Genova (Italy) and 2Divisione di Neurochirurgia, Ospedali Galliera, Genova (Italy) (Accepted 14 August 1990) Key words: ~-Aminobutyric acid uptake; Acetylcholine release; Human neocortex; Release regulation; Transmitter coexistence
The effect of ~,-aminobutyric acid (GABA) on the release of [3H]acetylcholine ([3H]ACh) from human cerebral cortex nerve terminals was investigated using synaptosomes prepared from neurosurgical specimens (which had to be removed to reach deeply located tumors) prelabeled with [3H]choline and exposed in superfusion to varying concentrations of GABA. The amino acid (3-100 /~M) increased in a concentration-dependent manner (maximal effect: 40%; ECso = 14.7 gM) the release of [3H]ACh but not that of [3H]choline. The GABA A receptor agonist muscimol (up to 100 gM) did not increase significantly the release of [3H]ACh. Accordingly, the effect of GABA was insensitive to the GABA A receptor antagonist bicuculline. The release of [3H]ACh was not affected by the GABA B receptor agonist (-)-baclofen (100-300 gM). The GABA-induced [3H]ACh release was counteracted by two inhibitors of GABA uptake, N-(4, 4-diphenyl-3-butenyl)nipecotic acid (SKF 89976A) and nipecotic acid. Moreover, the enhancing effect of GABA on [3H]ACh release was clearly Na+-dependent and was reduced by almost 90% in presence of 23 mM NaCI. The data indicate that, similarly to what had been observed in the rat, cholinergic nerve terminals in the human cerebral cortex possess a GABA transporter. Activation of this carrier brings about release of newly synthesized ACh. GABA and ACh might co-exist in some cerebrocortical nerve endings in the vertebrate brain, including man. INTRODUCTION During a recent study of the effects of y-aminobutyric acid ( G A B A ) on the release of acetylcholine ( A C h ) in the rat brain an unexpected observation was made. The amino acid a u g m e n t e d the release of [3H]ACh from rat h i p p o c a m p u s s y n a p t o s o m e s p r e l a b e l e d with [3H]choline; however, this effect was not m e d i a t e d by a G A B A e r g i c presynaptic h e t e r o r e c e p t o r . The G A B A - i n d u c e d [3H]A C h release was instead selectively blocked by inhibitors of G A B A uptake. This finding led to the conclusion that, in the rat brain, nerve terminals exist which not only possess a choline t r a n s p o r t e r linked to A C h synthesis, but also a G A B A carrier, activation of which brings about release of A C h 2. Reciprocally, the release of e n d o g e n o u s G A B A from rat h i p p o c a m p u s synaptosomes was found to be enhanced by choline, an effect which was p r e v e n t e d by hemicholinium-3, a blocker of the high affinity choline uptake 13. These novel findings m a y be variously interpreted. O n e possibility is that some neurons express a heterocarrier instead of a h e t e r o r e c e p t o r to permit release modulation. These neurons might even co-store G A B A and A C h and therefore possess co-carriers having not
only the well-known r e u p t a k e function but also that of permitting reciprocal regulation of release between the two co-transmitters. A l t h o u g h the mechanisms underlying this p h e n o m e non are now being actively investigated in our l a b o r a t o r y using experimental animals, any further e x p e r i m e n t a t i o n would obviously be most justified if the process also occurs in the human brain. W e have therefore investigated the effects of G A B A on the release of A C h in fresh specimens of human cerebral cortex.
MATERIALS AND METHODS Characteristic of human specimens Samples of human cerebral cortex were obtained from patients undergoing neurosurgery. The tissues used were removed by the surgeon to reach deeply located tumors. The samples represented parts of frontal (2), temporal (2), parietal (1) and occipital (2) lobes and were obtained from 4 male and 3 female patients (aged 49-61 years). The tissues were obtained and processed separately on different days. After premedication with atropine and meperidine, anesthesia was induced with Penthotal and maintained with 70% nitrous oxide in 30% oxygen and 0.5-1% isoflurane. Pancuronium was employed to obtain muscular relaxation. Preparation of synaptosomes Immediately after removal, the tissue was placed in a physiolog-
Correspondence: M. Raiteri, Istituto di Farmacologia e Farmacognosia, Universit~t degli Studi di Genova, Viale Cembrano 4, 16148 Genova, Italy. 0006-8993/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Di~,ision)
192 ical salt solution kept at 0-4 °C and a synaptosomal fraction was obtained within 60 min. Crude synaptosomes were prepared essentially as previously described J6. The synaptosomal pellet was then resuspended in a physiological medium having the following composition (mM): NaCI 125, KCI 3, CaCI 2 1.2, MgSO 4 1.2, NaH2PO 4 1, NaHCO 3 22, glucose 10 (aerated with 95% 02 and 5% CO 2 at 37 °C), pH 7.2-7.4. Protein was measured according to Bradford 4.
Experiments of release Synaptosomes were incubated at 37 °C for 15 min in the presence of 0.3 /~M ([3H]choline. Identical aliquots of the synaptosomal suspension (ranging between 0.4 and 0.8 mg protein in the different experiments) were then layered at the bottom of parallel superfusion chambers maintained at 37 °C TM. Superfusion was started with standard medium aerated with 95% O 2 and 5% CO 2 at a rate of 0.6 ml/min and continued for 48 min. After 33 min to obtain a constant outflow of tritium, five 3-min fractions were collected. GABA, muscimol or (-)-baclofen was added to the medium at the end of the second fraction. Bicuculline, N-(4,4-diphenyl-3-butenyl)-nipecotic acid (SKF 89976A), nipecotic acid or hemicholinium-3 was added 8 min before GABA. The Na+-deprived media were introduced 18 min before G A B A (NaCI replaced by an iso-osmotic concentration of sucrose). Radioactivity was measured in each fraction and in the superfused filters.
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