GABA FLUXES IN PRESYNAPTIC NERVE ENDINGS FROM IMMATURE RATS

Abstract— Several parameters of GABA Auxes across the synaptosomal membrane were studied using synaptosomes prepared from the brain of immature (8-day-old) rats. The following aspects of GABA carrier-mediated transport were similar in immature and mature synaptosomes: (1) magnitude of [³H]GABA accumulation; (2) GABA homoexchange in normal ionic conditions; (3) GABA homoexchange in the presence of cationic fluxes (Na⁺ and Ca²⁺ influx, K⁺ efflux) characteristic of physiological depolarization. As in adult synaptosomes (Levi & Raiteri , 1978), in these conditions the stoichiometry of GABA homoexchange was in the direction of net outward transport (efflux > influx). The essential differences between the behaviour of 8-day-old and adult synaptosomes were the following: (1) β-alanine (a glial uptake inhibitor) inhibited GABA uptake in immature synaptosomes (the inhibition being greater in crude than in purified preparations) and was without a significant effect in adult synaptosomes. DABA and ACHC (two neuronal uptake inhibitors) depressed GABA uptake more efficiently in purified than in crude immature synaptosomes, but were as effective in crude and purified nerve endings from adult animals. The data suggest a greater uptake of GABA in the‘gliosomes’contaminating the synaptosomal preparations from immature animals. (2) In immature synaptosomes prelabelled with [³H]GABA the specific radioactivity of the GABA released spontaneously or by heteroexchange (with 300 μm -OH-GABA) was the same as that present in synaptosomes, while in adult synaptosomes OH-GABA released GABA with increased specific radioactivity. The data suggest a homogeneous distribution of the [³H]GABA taken up within the endogenous GABA pool in immature, but not in mature synaptosomes. (3) In immature synaptosomes the release of GABA (radioactive and endogenous) induced by depolarization with high KC was not potentiated by Ca²⁺, unless the synaptosomes had been previously depleted of Na⁺ These data suggest that, although a Ca²⁺ sensitive pool of GABA may be present, this pool is not susceptible to being released in normal conditions, probably because the high intrasynaptosomal Na⁺ level prevents a sufficient depolarization. The possible significance of these findings in terms of functional activity of GABAergic neurotransmission in the immature brain is discussed.     

A role for calcium/calmodulin kinase(s) in the regulation of GABA exocytosis

A possible role for protein kinases in the regulation of GABA exocytosis in nerve endings was investigated. The effect on the release of the radioactive neurotransmitter ([³H]GABA) from mouse brain synaptosomes of several protein kinase inhibitors was estimated after treatment with 37 mM K⁺ in the absence of external Na⁺, a condition under which [³H]GABA release is completely Ca²⁺ dependent. Among the inhibitors one group inhibit the kinases by binding to the catalytic site (i.e. staurosporine and H7) and others (TFP, sphingosine and W7) act on the regulatory site of protein kinases. The compounds of the second group, which are reported to inhibit calmodulin dependent events and the increase in cytosolic Ca²⁺ (Ca _i ) induced by high K⁺ depolarization, were the most efficient inhibitors of [³H]GABA release. The selective inhibitor of CaMPK II, KN-62, also markedly diminished [³H]GABA release as well as the increase in Ca _i induced by high K⁺. The kinase inhibitors from the first group that are unable to diminish the increase in Ca _i induced by high K⁺ were also less efficient inhibitors of [³H]GABA release even at high concentrations. The present results indicate that at the doses tested all the drugs inhibit to some extent the release of the Ca²⁺ dependent fraction of [³H]GABA perhaps by inhibiting a CaMPK II mediated phosphorylation step triggered by depolarization and facilitated by the elevation of Ca _i . In addition, the second group of antagonists and KN-62 inhibit the elevation of Ca _i to high K⁺ thus exhibiting a higher efficiency on [³H]GABA release than the first group of antagonists.     

Inhibition by Quinacrine of Depolarization-Induced Acetylcholine Release and Calcium Influx in Rat Brain Cortical Synaptosomes

The effects of quinacrine on depolarization-induced [³H]acetylcholine (ACh) release and ⁴⁵Ca²⁺ influx were examined in rat brain cortical synaptosomes. Quinacrine significantly reduced the stimulated release of [³H]ACh by high K⁺ and veratridine without affecting the spontaneous efflux from the preloaded synaptosomes. Quinacrine had no effect on ionophore A23187-induced release of [³H]ACh from the synaptosomes. Quinacrine (100 μM) markedly diminished the stimulated Ca²⁺ influx by veratridine and high K⁺ but not that by “Na⁺-free.” Trifluoperazine, a potent calmodulin antagonist, inhibited both Ca²⁺ influx and ACh release induced by the depolarizing agents. Inhibitory potencies of the two drugs on ACh release and Ca²⁺ influx were compared with the antagonism of calmodulin by two drugs, suggesting that the inhibition of depolarization-induced Ca²⁺ influx and ACh release by these drugs could not be explained by the antagonism of calmodulin.     

Effects of External pH Variations on Brain Presynaptic Sodium and Calcium Channels; Repercussion on the Evoked Release of Amino Acid Neurotransmitters

The effects of external pH (pH _out) variations on the Na⁺ and on the Ca²⁺ dependent fractions of the evoked amino acid neurotransmitter release were separately investigated, using GABA as a model transmitter. In [³H]GABA loaded mouse brain synaptosomes, the external acidification (pH _out6.0) markedly decreased the Na⁺ dependent fraction of [³H]GABA release evoked by veratridine (10 M) in the absence of external Ca²⁺, as well as the Ca²⁺ dependent fraction of [³H]GABA release evoked by high (20 mM) K⁺ in the absence of external Na⁺. The depolarization-induced elevation of [Na _i ] (monitored in synaptosomes loaded with the Na⁺ indicator dye, SBFI) and the depolarization-induced elevation of [Ca _i ] (monitored in synaptosomes loaded with the Ca²⁺ indicator dye fura-2) were also markedly decreased at pH _out 6. On the contrary, the external alkalinization (pH _out 8) facilitated all the above responses. A slight increase of the baseline release of the [³H]GABA was observed when pH _out was changed from 7.4 to 8. This effect was only observed in the presence of Ca²⁺. pH _out changes from 7.4 to 6 or to 7 did not modify the baseline release of the transmitter. All the effects of pH _out variations on [³H]GABA release were independent on the presence of HCO-₃. It is concluded that external H⁺ regulate amino acid neurotransmitter release by their actions on presynaptic Na⁺ channels, as well as on presynaptic Ca²⁺ channels.     

Compartmentation and release of exogenous GABA in sheep brain synaptosomes

Exogenous tritiated -aminobutiric acid ([³H]GABA) is retained in two compartments in sheep cortex synaptosomes, corresponding to cytoplasmic and vesicular spaces, assuming that freeze-thawing the synaptosomes loaded with [³H]GABA releases the cytoplasmic [³H]GABA (81±3.9%), and that subsequent solubilization of the synaptosomes with 1% sodium cholate releases the vesicular [³H]GABA (19±3.9%). Depolarization of synaptosomes with 40 mM K⁺ in a Na⁺-medium, in the absence of Ca²⁺, releases 20.3±2.7% of the [³H]GABA retained in the synaptosomes. The [³H]GABA released under these conditions comes predominantly from the cytoplasm. The presence of 1 mM Ca²⁺ during depolarization releases and additional 13% (a total of about 33.5±9.9%) of the releasable [³H]GABA, and the [³H]GABA release which is Ca²⁺-dependent also comes mostly from the cytoplasmic compartment. When choline replaces external Na⁺, the [³H]GABA release is absolutely Ca²⁺-dependent, and the [³H]GABA released also comes mostly from the cytoplasmic pool. Therefore, it appears that [³H]GABA taken up by synaptosomes is accumulated mostly in the cytoplasmic compartment from which it is released upon depolarization. The technique described permits distinguishing the effect of different factors on the two pools of accumulated [³H]GABA.     

RELEASE OF [3H]GABA FROM IN VITRO PREPARATIONS: COMPARISON OF THE EFFECT OF DABA AND β-ALANINE ON THE K+ AND PROTOVERATRINE STIMULATED RELEASE OF [3H]GABA FROM BRAIN SLICES AND SYNAPTOSOMES1

It has been proposed that the major portion of [³H]GABA released from rat cortical slices upon exposure to high K⁺ comes from a neuronal pool. Using carrier mediated exchange diffusion of DABA or β-alanine in the superfusion medium for GABA in the slice as a technique for manipulating neuronal and glial pools of GABA, it was found that DABA but not β-alanine substantially reduced the K⁺ stimulated release of [³H]GABA. The present study using synaptosomes as an in vitro model of the nerve ending was undertaken to ascertain whether this neuronal pool of releasable [³H]GABA was associated with a specific transmitter pool in nerve endings. A continuous superfusion system employing a Ca²⁺ pulse to produce a calcium coupled release (Levy et al, 1973) was used to study the effect of two concentrations (20 μm , 1 mm ) of DABA and β-alanine on the release of [³H]GABA from synaptosomes. In contrast to the results in slices, DABA at both concentrations had no effect on the release of [³H]GABA from synaptosomes in spite of evidence that exchange diffusion was occurring. With protoveratrine as the releasing agent there was no effect of DABA on the release of [³H]GABA from either slices or synaptosomes. The results suggest that the major portion of [³H]GABA released from cortical slices by high K⁺ comes from a non-transmitter pool in the neuron. Use of K⁺ stimulated release of amino acids from cortical slices as a criterion for neurotransmitter function must be viewed with caution.     

Voltage-Sensitive Ca2+ Channels Involved in Nicotinic Receptor-Mediated [3H]Dopamine Release from Rat Striatal Synaptosomes

Abstract: The potent nicotinic agonist anatoxin-a elicits mecamylamine-sensitive [³H]dopamine release from striatal synaptosomes, and this action is both Na⁺ and Ca²⁺ dependent and is blocked by Cd²⁺. This suggests that stimulation of presynaptic nicotinic receptors results in Na⁺ influx and local depolarisation that activates voltage-sensitive Ca²⁺ channels, which in turn provide the Ca²⁺ for exocytosis. Here we have investigated the subtypes of Ca²⁺ channels implicated in this mechanism. [³H]Dopamine release evoked by anatoxin-a (1 µM) was partially blocked by 20 µM nifedipine, whereas KCl-evoked release was insensitive to the dihydropyridine. However, a ⁸⁶Rb⁺ efflux assay of nicotinic receptor function suggested that nifedipine has a direct effect on the receptor, discrediting the involvement of L-type channels. The N-type Ca²⁺ channel blocker ω-conotoxin GVIA (1 µM) blocked anatoxin-a-evoked [³H]dopamine release by 60% but had no significant effect on ⁸⁶Rb⁺ efflux; release evoked by both 15 and 25 mM KCl was inhibited by only 30%. The P-type channel blocker ω-agatoxin IVA (90 nM) also inhibited KCl-evoked release by ～30%, whereas anatoxin-a-evoked release was insensitive. The Q-type channel blocker ω-conotoxin MVIIC (1 µM) had no effect on either stimulus. These results suggest that presynaptic nicotinic receptors on striatal nerve terminals promote [³H]dopamine release by activation of N-type Ca²⁺ channels. In contrast, KCl-evoked [³H]dopamine release appears to involve both N-type and P-type channels.     

Study of the Mechanism of Release of [3H]GABA from a Teleost Retina In Vitro

Abstract: γ-Aminobutyric acid (GABA) is thought to be a neurotransmitter in the vetebrate retina. We studied the voltage and Ca²⁺ dependency of the process of release of [³H]GABA from the retina of the teleost Eugenes plumieri, using a microsuperfusion technique. Two depolarizing agents, veratridine and high potassium, produced a concentration-dependent release of [³H]GABA. The veratridine effect was inhibited in Na⁺-free solution, but was not affected by 1 μM tetrodotoxin. A substantial inhibition (about 75%) of the veratridine-and potassium-stimulated release of [³H] GABA occurred in Ca²⁺-free medium. Inhibitors of the Ca²⁺ channel, such as Mg²⁺(20 mM), La³⁺ (0.1 mM), and methoxy-verapamil (4 μM-0.4 mM), inhibited the veratridine-and K⁺-stimulated release. However, Co²⁺ and Cd²⁺ caused a potentiation and no change of the K⁺-and veratridine-stimulated release, respectively. This release process is apparently specific, since both depolarizing agents were unable to release [³H]methionine, a nontransmitter amino acid, under the same experimental conditions. Autoradio-graphic studies with [³H]GABA, using the same incubation conditions as for the release experiments, showed a high density of silver grains over the horizontal cells with almost no accumulation by amacrine cells and Muller cells. β-Alanine and nipecotic acid were used as two relative specific inhibitors of the glial and neuronal GABA uptake mechanisms, respectively. Only a small heteroexchange with [³H]GABA was found with β-alanine, and no inhibition of the subsequent veratridine-stimulated release. On the other hand, nipecotic acid produced a strong heteroexchange with [³H]GABA and lacked the capacity to induce the veratridine-stimulated release of [³H]GABA. These results suggest a voltage-and Ca²⁺-dependent neuronal release of [³H]GABA from retina.     

Relationship between synaptosomal uptake and release of [14C]gaba, [14C]diaminobutyric acid and [14C]beta-alanine.

[¹⁴C]GABA is taken up by rat brain synaptosomes via a high affinity, Na⁺-dependent process. Subsequent addition of depolarizing levels of potassium (56.2 MM) or veratridine (100 μM) stimulates the release of synaptosomal [¹⁴C]GABA by a process which is sensitive to the external concentration of divalent cations such as Ca²⁺, Mg²⁺, and Mn²⁺. However, the relatively smaller amount of [¹⁴C]GABA taken up by synaptosomes in the absence of Na⁺ is not released from synaptosomes by Ca²⁺ -dependent, K ⁺-stimulation. [¹⁴C]DABA, a competitive inhibitor of synaptosomal uptake of GABA (Iversen & Johnson , 1971) is also taken up by synaptosomal fractions via a Na ⁺ -dependent process; and is subsequently released by Ca²⁺ -dependent, K⁺-stimulation. On the other hand, [¹⁴C]β-alanine, a purported blocker of glial uptake systems for GABA (Schon & Kelly , 1974) is a poor competitor of GABA uptake into synaptosomes. Comparatively small amounts of [¹⁴C] β-alanine are taken up by synaptosomes and no significant amount is released by Ca²⁺ -dependent, K⁺-stimulation. These data suggest that entry of [¹⁴C]GABA into a releasable pool requires external Na⁺ ions and maximal evoked release of [¹⁴C]GABA from the synaptosomal pool requires external Ca²⁺ ions. The GABA analogue, DABA, is apparently successful in entering the same or similar synaptosomal pool. The GABA analogue, β-alanine, is not. None of the compounds or conditions studied were found to simultaneously affect both uptake and release processes. Compounds which stimulated release (veratridine) or inhibited release (magnesium) were found to have minimal effect on synaptosomal uptake. Likewise compounds (DABA) or conditions (Na⁺-free medium) which inhibited uptake, had little effect on release.     

Vesicular and carrier-mediatied depolarization-induced release of [3H]GABA: Inhibition by amiloride and verapamil

The Ca²⁺-dependent, presumably exocytotic fraction of the [³H]GABA released by depolarization is dissected from the depolarization-induced Na⁺-dependent, carrier-mediated fraction of [³H]GABA release in mouse brain synaptosomes. GABA homoexchange is prevented by the [³H]GABA carrier blocker, DABA. The absence of external Na⁺ completely abolishes the release of the carrier-mediated, presumably cytoplasmic release of [³H]GABA induced by homoexchange and heteroexchange with GABA and DABA, respectively. The carrier-mediated, Na⁺-dependent fraction of the depolarization-induced release of [³H]GABA is resistant to tetrodotoxin (TTX) but is sensitive to amiloride and verapamil. The Ca²⁺-dependent fraction of the [³H]GABA released by high K⁺ depolarization is also completely abolished by amiloride (from 300 M) and sensitive to verapamil (30 M), but in contrast is insensitive to the absence of external Na⁺ and to DABA. On the basis of these results we conclude that amiloride and verapamil inhibit high K⁺-induced release of [³H]GABA by antagonizing the entrance of Ca²⁺ (and possibly Na⁺ when external Ca²⁺ is absent) through a population of voltage sensitive presynaptic Ca²⁺ channels activated by depolarization.Depto. de Biología Molecular Instituto de Investigaciones Biomédicas UNAM.     

Carrier-mediated and carrier-independent release of serotonin from isolated central nerve endings

The release of serotonin elicited by Ca²⁺-dependent stimuli (depolarization, ionophore A23187) from rat brain synaptosomes previously labelled with the radioactive indoleamine was not affected by the presence of the serotonin carrier blocker chlorimipramine. In contrast, other releasing stimuli, such as superfusion with a Na⁺-free medium or exposure to various releasing drugs (fenfluramine, p-chloroamphetamine, tryptamine and mianserin, both in normal Krebs-Ringer medium and in low-Na⁺ medium), evoked efflux of serotonin from nerve endings which was prevented by chlorimipramine. The results indicate that serotonin can be released from central nerve endings by two mechanisms, differentially affected by the blockade of the membrane carrier system: the characteristics of the Ca²⁺-dependent release are compatible with an exocytotic mechanism, whereas the release induced by lack of Na⁺ or by phenylethylamines and tryptamine appears to occur by outward transport mediated by the membrane carrier.     

ω-Aga IVA selectively inhibits the calcium-dependent fraction of the evoked release of [3H]GABA from synaptosomes

The effect of -Aga IVA, a P-type Ca²⁺ channel blocker, on the release of the inhibitory neurotransmitter GABA and on the elevation of Ca_i induced by depolarization was investigated in [³H]GABA and fura-2 preloaded mouse brain synaptosomes, respectively. Two strategies (i.e. 20 mM external K⁺ and veratridine) that depolarize by different mechanisms the preparation were used. High K⁺ elevates Ca_i and induces [³H]GABA release in the absence of external Na⁺ and in the presence of TTX, conditions that abolish veratridine induced responses. The effect of -Aga IVA on the Ca²⁺ and Na⁺ dependent fractions of the depolarization evoked release of [³H]GABA were separately investigated in synaptosomes depolarized with high K⁺ in the absence of extermal Na⁺ and with veratridine in the absence of external Ca²⁺, respectively. The Ca²⁺ dependent fraction of the evoked release of [³H]GABA and the elevation of Ca²⁺ induced by high K⁺ are markedly inhibited (about 50%) in synaptosomes exposed to -Aga IVA (300 nM) for 3 min before depolarization, whereas the Na⁺ dependent, Ca²⁺ independent carrier mediated release of [³H]GABA induced by veratridine, which is sensitive to verapamil and amiloride, is not modified by -Aga IVA. Our results indicate that an -Aga IVA sensitive type of Ca²⁺ channel is highly involved in GABA exocytosis.     

Effect of α-Latrotoxin on Acetylcholine Release and Intracellular Ca2+ Concentration in Synaptosomes: Na+-Dependent and Na+-Independent Components

Abstract: We studied the effect of α-latrotoxin (αLTX) on [¹⁴C]acetylcholine ([¹⁴C]ACh) release, intracellular Ca²⁺ concentration ([Ca²⁺]_i), plasma membrane potential, and high-affinity choline uptake of synaptosomes isolated from guinea pig cortex. αLTX (10^?10-10^?8M) caused an elevation of the [Ca²⁺]_i as detected by Fura 2 fluorescence and evoked [¹⁴C]ACh efflux. Two components in the action of the toxin were distinguished: one that required the presence of Na⁺ in the external medium and another that did not. Displacement of Na⁺ by sucrose or N-methylglucamine in the medium considerably decreased the elevation of [Ca²⁺]_i and [¹⁴C]ACh release by αLTX. The Na⁺-dependent component of the αLTX action was obvious in the inhibition of the high-affinity choline uptake of synaptosomes. Some of the toxin action on both [Ca²⁺]_i and [¹⁴C]ACh release remained in the absence of Na⁺. Both the Na⁺-dependent and the Na⁺-independent components of the αLTX-evoked [¹⁴C]ACh release partly required the presence of either Mg²⁺ or Ca²⁺. The nonneurotransmitter [¹⁴C]choline was released along with [¹⁴C]ACh, but this release did not depend on the presence of either Na⁺ or Ca²⁺, indicating nonspecific leakage through the plasma membrane. We conclude that there are two factors in the release of ACh from synaptosomes caused by the toxin: (1) cation-dependent ACh release, which is related to (a) Na⁺-dependent divalent cation entry and (b) Na⁺-independent divalent cation entry, and (2) nonspecific Na⁺- and divalent cation-independent leakage.     

Tetanus toxin treatment in vitro inhibits the release of GABA from rat cerebral cortex slices evoked by high K+ and Na+-free media

The influence of tetanus toxin in vitro on the release of exogenous [³H]GABA was studied with rat cerebral cortex slices. The influx, long-term accumulation and spontaneous efflux of GABA were not modified by the toxin. The release induced by high K⁺ (50 mM) medium from the superfused slices pretreated with the toxin was significantly inhibited in a time- and dose-dependent fashion. This release was attenuated during superfusion with Ca²⁺-free medium and the toxin no longer affected the remaining Ca²⁺-independent release. The release induced by Na⁺-free media did not require extracellular Ca²⁺ ions, and the toxin inhibited the release both with and without Ca²⁺. The toxin treatment had no marked influence on the ouabain (20 μM) or veratrine (25–50 μM)-induced release of GABA. The toxin treatment in vitro appears to modify some step(s) in the stimulated release of GABA without affecting its unstimulated membrane transport. Tetanus toxin may thus prove a valuable tool in studying the mechanisms of the release of GABA and possibly other inhibitory transmitters in synapses of the central nervous system.     

Role of External and Internal Calcium on Heterocarrier-Mediated Transmitter Release

Abstract: Release-regulating heterocarriers exist on brain nerve endings. We have investigated in this study the mechanisms involved in the neurotransmitter release evoked by GABA heterocarrier activation. GABA increased the basal release of [³H]acetylcholine and [³H]noradrenaline from rat hippocampal synaptosomes and of [³H]dopamine from striatal synaptosomes. These GABA effects, insensitive to GABA receptor antagonists, were prevented by inhibiting GABA uptake but not by blocking noradrenaline, choline, or dopamine transport. Lack of extracellular Ca²⁺ or addition of tetrodotoxin selectively abolished the GABA-evoked release of [³H]noradrenaline, leaving unaffected that of [³H]acetylcholine or [³H]dopamine. 1,2-Bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) or vesamicol attenuated the release of [³H]acetylcholine elicited by GABA. Reserpine, but not BAPTA-AM, prevented the effect of GABA on [³H]dopamine release. Autoreceptor activation inhibited the GABA-evoked release of [³H]noradrenaline but not that of [³H]acetylcholine or [³H]dopamine. It is concluded that (a) the release of [³H]noradrenaline consequent to activation of GABA heterocarriers sited on noradrenergic terminals meets the criteria of a conventional exocytotic process, (b) the extracellular [Ca²⁺]-independent releases of [³H]acetylcholine and [³H]dopamine appear to occur from vesicles possibly through involvement of intraterminal Ca²⁺, and (c) autoreceptor activation only affects heterocarrier-mediated vesicular release linked to entry of extracellular Ca²⁺.     

RELATIONSHIP BETWEEN Ca2+-DEPENDENT AND INDEPENDENT RELEASE OF [3H]GABA EVOKED BY HIGH K+, VERATRIDINE OR ELECTRICAL STIMULATION FROM RAT CORTICAL SLICES

Abstract— It has been reported that the release of GABA by high K⁺ is Ca²⁺-dependent while release induced by veratridine or electrical stimulation has been frequently found to be independent of Ca²⁺. To see the source of Ca²⁺-dependent and independent release of GABA, cortical slices which had accumulated [³H]GABA were exposed to 50 mm -K⁺ or 50 μm -veratridine for 48min. In the presence of Ca²⁺ the 2 agents released approx the same amount of [³H]GABA but tetrodotoxin (TTX) abolished release induced only by veratridine, while omission of Ca²⁺ reduced release induced only by 50mm -K⁺. Pre-exposure of the slices for 48min to 50mm -K⁺ in the presence of Ca²⁺ reduced the second release by 50mm -K⁺ by 77% and that by veratridine by 74%, suggesting that in the presence of Ca²⁺ the 2 depolarizing agents release [³H]GABA from the same pool. Pre-exposure to 50mm -K⁺ in the absence of Ca²⁺ reduced the second release by 50mm -K⁺ or by veratridine only by 37 and 27% respectively, indicating that most of the reduction in release was the result of a depletion of releasable [³H]GABA stores. The second exposure to 50mm -K⁺ in the absence of Ca²⁺ reduced the evoked release further, while exposure to veratridine in the absence of Ca²⁺, after depletion of the stores, enhanced release 2.7 times. Electrical stimulation (64 Hz, 2 ms, 40 mA, alternating polarity) during 24min in the presence of Ca” caused an initial 5-fold increase in efflux, which declined subsequently. In the absence of Ca²⁺, instead of a rapid increase, a slow but smaller increase in the efflux of [³H]GABA was found. TTX almost completely abolished the electrically evoked increase in release. Pre-treatment with 50mm -K⁺ reduced the electrically evoked release by 94% but electrical stimulation in the absence of Ca²⁺ after depletion of releasable stores doubled this release. Results suggest that in the presence of Ca²⁺, high K⁺, veratridine and electrical stimulation release [³H]GABA from the same Ca²⁺-dependent store, but in the absence of Ca²⁺ veratridine and electrical stimulation enhance the release from a Ca²⁺-independent store, probably as a result of an increased influx of Na⁺.     

Nigericin-induced Na+/H+ and K+/H+ exchange in synaptosomes: Effect on [3H]GABA release

The effect of the putative K⁺/H⁺ ionophore, nigericin on the internal Na⁺ concentration ([Na _i ]), the internal pH (pH _i ), the internal Ca²⁺ concentration ([Ca _i ]) and the baseline release of the neurotransmitter, GABA was investigated in Na⁺-binding benzofuran isophtalate acetoxymethyl ester (SBFIAM), 2′,7′-bis(carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM), fura-2 and [³H]GABA loaded synaptosomes, respectively. In the presence of Na⁺ at a physiological concentration (147 mM), nigericin (0.5 μM) elevates [Na _i ] from 20 to 50 mM, increases thepH _i , 0.16 pH units, elevates four fold the [Ca _i ] at expense of external Ca²⁺ and markedly increases (more than five fold) the release of [³H]GABA. In the absence of a Na⁺ concentration gradient (i.e. when the external Na⁺ concentration equals the [Na _i ]), the same concentration (0.5 μM) of nigericin causes the opposite effect on thepH _i (acidifies the synaptosomal interior), does not modify the [Na _i ] and is practically unable to elevate the [Ca _i ] or to increase [³H]GABA release. Only with higher concentrations of nigericin than 0.5 μM the ionophore is able to elevate the [Ca _i ] and to increase the release of [³H]GABA under the conditions in which the net Na⁺ movements are eliminated. These results clearly show that under physiological conditions (147 mM external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore, and all its effects are triggered by the entrance of Na⁺ in exchange for H⁺ through the ionophore itself. Nigericin behaves as a K⁺/H⁺ ionophore in synaptosomes just when the net Na⁺ movements are eliminated (i.e. under conditions in which the external and the internal Na⁺ concentrations are equal). In summary care must be taken when using the putative K⁺/H⁺ ionophore nigericin as an experimental tool in synaptosomes, as under standard conditions (i.e. in the presence of high external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore.     

Is the concentration of γ-aminobutyric acid in the nerve terminal regulated via product inhibition of glutamic acid decarboxylase?

Fractions of synaptosomes were used to study the regulation of -aminobutyric acid (GABA) synthesis. The isolated synaptosomes were superfused in media of various compositions. [³H]GABA and GABA released into the medium or remaining in the synaptosomes were analyzed by liquid scintillation and HPLC techniques. Different conditions, designed to increase the GABA efflux rate were used: the rate of superfusion was varied and the concentrations of K⁺ and Ca²⁺ were altered. Stimulation of GABA efflux was paralleled with an increased synthesis of GABA, since, in spite of the increased GABA efflux, a relatively constant intraterminal level was found. The findings suggest that the intraterminal concentration of GABA and thus also its synthesis is regulated via product inhibition. In addition, [³H]GABA, exogenous, and GABA, endogenous, responded to external stimulae (Ca²⁺, veretradine, various GABA concentrations and the glutaminase inhibitor diazo-nor-leucine) in a way which was compatible with them being localized in and/or released from different compartments.     

GABA efflux from synaptosomes: Effects of membrane potential,and external GABA and cations

Summary Presynaptic GABAergic nerve terminals accumulate -aminobutyric acid (GABA) by a sodium-dependent carrier mechanism in which two Na⁺ are co-transported with one GABA. We have examined the influence of external GABA and cations on GABA efflux from³H-GABA loaded rat brain synaptosomes, to determine whether or not the carriers can also mediate GABA efflux. We observed that, in Ca-free media (to minimize Ca-dependent evoked release), external GABA promotes GABA efflux when the medium contains Na⁺, butinhibits GABA efflux in the absence of Na⁺. The efflux of GABA into Ca-free media is stimulated by depolarization (either with veratridine or increased external K⁺). These data, and published data on the internal Na⁺ dependence of GABA efflux into Ca-free media, indicate that exiting GABA is cotransported with Na⁺. The stimulatory effect of depolarization is consistent with efflux of Na⁺ along with the uncharged GABA. The (carrier-mediated) efflux is also stimulated when the carriers cycle inward with Na⁺+GABA. The inhibitory effect of GABA in Na⁺-free media implies that GABA can bind to unloaded carriers and that the carriers loaded only with GABA cycle very slowly, if at all. Our data, and data from the literature, can be fitted to a simple model with sequential binding of solutes: external GABA binds to the carrier first, and only the free or fully-loaded (with 2Na⁺+1GABA) carriers can cycle. Other binding sequences and random binding, do not fit the experimental observations.     

A reevaluation of veratridine as a tool for studying the depolarization-induced release of neurotransmitters from nerve endings

The effect of veratridine on neurotransmitter release was studied using rat brain synaptosomes superfused at 37°C. Veratridine (5–75 M) caused a concentration-dependent release of [³H]GABA from prelabeled synaptosomes in the presence of 2.7 mM Ca²⁺. In the whole range of veratridine concentrations, the release of [³H]GABA elicited by the drug was substantially increased rather than decreased in the absence of Ca²⁺ or with Ca²⁺ concentrations of 0.45 and 0.9 mM. The release of the amino acid was inhibited more by 5.4 mM than by 2.7 mM Ca²⁺. The effect on endogenous (chemically measured) GABA was similar to that on [³H]GABA. The inhibitory effect of Ca²⁺ on the veratridine-induced release of [³H]GABA was consistently seen in a variety of experimental conditions except one, namely when the experiment was run at room temperature (22–23°C) rather than at physiological temperature (37°C). In fact, at 22–23°C the release of GABA evoked by the alkaloid was somewhat potentiated by Ca²⁺. At 37°C, glutamate appeared to behave similarly to GABA, whereas the veratridine-induced release of [³H]noradrenaline and [³H]dopamaine was largely Ca²⁺-dependent. The mechanism of the release of transmitters elicited by veratridine is discussed. It is concluded that the evoked release of GABA and glutamate is due more to the veratridine-induced depolarization (Na⁺ influx) than to the accompanying influx of Ca²⁺, and it is suggested that the inhibitory effect of Ca²⁺ on the overall release of amino acids is due to the antagonism exerted by the divalent cation on the veratridine action at the Na⁺ channel. In contrast, in the case of catecholamines, the influx of Ca²⁺ would have a prominent role in triggering exocytotic release, whereas the depolarization itself would have slight or no importance.