Studies on the mechanism of modulation of [3H]noradrenaline release from rat hippocampal synaptosomes by GABA and benzodiazepine receptors

Release of [³H]noradrenaline from rat hippocampal synaptosomes was triggered by pulses of 25 mM K⁺, 5 μM veratridine or superfusion with the Ca²⁺ ionophore A23187. GABA with bicuculline or chlordiazepoxide depressed the release of [³H]noradrenaline evoked by depolarisation but not by the Ca²⁺ ionophore. 8 Br-cAMP with [Ca²⁺]₀ 0.3 mM had no effect on spontaneous or K⁺-evoked release of [³H]noradrenaline and completely blocked the effect of chlordiazepoxide and GABA with bicuculline. With [Ca²⁺]₀ 1 mM 8 Br-cAMP enhanced spontaneous and K⁺-evoked release of [³H]noradrenaline, and reversed the depression caused by GABA with bicuculline. GABA alone evoked Ca²⁺-dependent release of [³H]noradrenaline which was sensitive to [Cl^?]₀. The results suggest that the GABA_A-receptor mediated release of [³H]noradrenaline is due to depolarisation resulting from increased Cl^? conductance whereas the depression of depolarisation-dependent release of [³H]noradrenaline by GABA_B or benzodiazepine receptors is mediated by a cAMP-dependent decrease in the voltage-dependent Ca²⁺ conductance.     

Depolarization-evoked GABA release from myenteric plexus is partially coupled to L-, N-, and P/Q-type calcium channels

1. There are many evidences suggesting that -aminobutyrate (GABA) is an important neurotransmitter and/or neuromodulator in the gut.2. Using the myenteric plexus-longitudinal muscle preparation from the guinea pig ileum, we investigated the evoked release of [³H] GABA from enteric neurons by electrical pulses or high KCl, which occurs in a calcium-dependent and -independent way. In addition, using selective calcium channel blockers, we report the participation of distinct subtypes of calcium channels in the evoked release, showing a minor participation of L- and Q-type calcium channels, while N- and P-type have a participation of approximately 15%, each. However, regardless of the combination of Ca²⁺ channel blockers, we did not observe an inhibition greater than 50% of the calcium-dependent component of [³H] GABA release.3. Thus, while the observed Ca²⁺-independent release mostly probable occur via reversal of the membrane GABA transporter, in our conditions, a considerable portion of the Ca²⁺-dependent evoked release of [³H] GABA is not coupled to L-, N-, or P/Q-type calcium channels, suggesting the involvement of intracellular calcium stores or other ways of getting calcium across the membrane.     

γ-Aminobutyric acid modulation of acetylcholine-induced contractions of a smooth muscle from an echinoderm (Sclerodactyla briareus)

This study provides pharmacological evidence for the presence of GABAergic neurons innervating the longitudinal muscle of the body wall (LMBW) of holothurians. γ-Aminobutyric acid (GABA) A and B receptor subtypes were both present in this system and regulated spontaneous contractions as well as responses to acetylcholine (ACh) that stimulated contraction of the LMBW. GABA dose-dependently relaxed the resting tone of the LMBW. GABA (10⁻⁵ M) inhibited ACh-induced (10⁻⁴ M) contractions by 20%. The GABA B agonist, baclofen, relaxed the LMBW, an effect potentiated by GABA. Pretreatment with baclofen (10⁻⁴ M) inhibited ACh (10⁻⁴ M) contractions of the LMBW by 50%. Phaclofen, a GABA receptor B antagonist, caused a dose-dependent increase in resting tension. Phaclofen-induced (10⁻⁵ M) contractions were reversed by the addition of GABA or baclofen (10⁻⁴ M) and potentiated by the addition of another GABA B receptor antagonist, 2-hydroxy-saclofen (10⁻⁵ M). Pretreatment with phaclofen (10⁻⁵ M) caused a marked potentiation of ACh-induced (10⁻⁴ M) contractions by 101%. 2-Hydroxy-saclofen (10⁻⁵ M) had a toxic effect on the LMBW, rendering it completely unresponsive either to ACh or to a second exposure to GABA, and so exhibiting cross-desensitization. Muscimol, a GABA A receptor agonist, had no effect on the resting tension of the LMBW. Curiously, pretreatment of the muscle with muscimol (10⁻⁵ M) potentiated ACh-evoked (10⁻⁴ M) contractions by nearly 20%. Bicuculline (10⁻⁵ M), a GABA A receptor antagonist, generated large, sustained contractions and partially blocked GABA-induced (10⁻⁴ M) relaxation. Like 2-hydroxy-saclofen, bicuculline (10⁻⁵ M) had a profound cross-desensitizing effect on the LMBW to subsequent exposures to GABA and ACh. ACh was unable to potentiate the sustained contractions induced by bicuculline. Accepted: 17 September 1998     

NMDA and Non-NMDA Receptor-Mediated Release of [3H]GABA from Granule Cell Dendrites of Rat Olfactory Bulb

Abstract: We have studied the effect of glutamate and the glutamatergic agonists N-methyl-d -aspartate (NMDA), kainate, and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on [³H]GABA release from the external plexiform layer of the olfactory bulb. The GABA uptake blocker nipecotic acid significantly increased the basal [³H]GABA release and the release evoked by a high K⁺ concentration, glutamate, and kainate. The glutamate uptake blocker pyrrolidine-2,4-dicarboxylate (2,4-PDC) inhibited by 50% the glutamate-induced [³H]GABA release with no change in the basal GABA release. The glutamatergic agonists NMDA, kainate, and AMPA also induced a significant [³H]GABA release. The presence of glycine and the absence of Mg²⁺ have no potentiating effect on NMDA-stimulated release; however, when the tissue was previously depolarized with a high K⁺ concentration, a significant increase in the NMDA response was observed that was potentiated by glycine and inhibited by the NMDA receptor antagonist 2-amino-5-phosphonoheptanoic acid (AP-7). The kainate and AMPA effects were antagonized by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not by AP-7. The glutamate effect was also inhibited by CNQX but not by the NMDA antagonist 2-amino-5-phosphonopentanoic acid (AP-5); nevertheless, in the presence of glycine, [³H]GABA release evoked by glutamate was potentiated, and this response was significantly antagonized by AP-5. Tetrodotoxin inhibited glutamate- and kainate-stimulated [³H]GABA release but not the NMDA-stimulated release. The present results show that in the external plexiform layer of the olfactory bulb, glutamate is stimulating GABA release through a presynaptic, receptor-mediated mechanism as a mixed agonist on NMDA and non-NMDA receptors; glutamate is apparently also able to induce GABA release through heteroexchange.     

Glutamate- and GABA-mediated neuron–satellite cell interaction in nodose ganglia as revealed by intracellular calcium imaging

In the sensory ganglia, neurons are devoid of synaptic contacts, and ganglion neurons surrounded by one of glial cells, satellite cells. Recent studies suggest that neurons and satellite cells interact through neurotransmitters. In the present study, intracellular Ca²⁺ ([Ca²⁺]_i) dynamics of neurons and satellite cells from one of viscerosensory ganglia, nodose ganglion (NG), were investigated by stimulation with glutamate and its agonist and/or the antagonist of the GABA_A receptor bicuculline. In the specimens containing neurons with satellite cells, glutamate and a metabotropic glutamate receptor (mGluR) agonist t-ACPD evoked [Ca²⁺]_i increases in both neurons and surrounding satellite cells. Moreover, bicuculline also induced [Ca²⁺]_i increases in neurons and satellite cells. However, in the isolated neurons, bicuculline did not cause an increase in [Ca²⁺]_i, suggesting that satellite cells are equipped with the ability to release GABA. In the neurons associated with satellite cells, the delay time until the onset of a response was shorter in the case of glutamate stimulation with bicuculline than that without bicuculline (107.3 ± 93.4 vs. 231.8 ± 97.0 s, p < 0.01). Furthermore, immunoreactivities for glutamate transporter, GLAST, and GABA transporter, GAT-3, were observed in both neurons and satellite cells of NG. In conclusion, the levels of [Ca²⁺]_i of NG neurons and surrounding satellite cells are increased by glutamate through at least mGluRs, and endogenous GABA modulates these responses; GABA inhibition is dependent on a close association between neurons and satellite cells. Such neuron–glia interaction in the nodose ganglion may regulate sensory information from visceral organs.     

Turnover and release of GABA in rat cortical slices: Effect of a GABA-T inhibitor,gabaculine

The turnover and release of endogenous and labeled GABA were followed in rat cortical slices after incubation with [³H]GABA. High performance liquid chromatography was used to measure endogenous GABA and to separate [³H]GABA from its metabolites. During superfusion with 3 mM K⁺ the slices rapidly lost their [³H]GABA content while maintaining constant GABA levels. Exposure to 50 mM K⁺ for 25 min caused an initial rapid rise in the release of both endogenous and [³H]GABA followed by a more rapid decline in the release of the latter. The specific activity of released GABA was two to four times higher than that in the slices. Depolarization lead to a net synthesis of GABA. The GABA-T inhibitor, gabaculine, (5 M) in vitro arrested the metabolism of [³H]GABA and rapidly doubled the GABA content but did not significantly increase the high K⁺ evoked release of endogenous GABA. In vivo pretreatment with 0.5 mM/kg gabaculine quadrupled GABA content and increased both the spontaneous and evoked release of endogenous GABA but while its Ca²⁺-dependent release increased by 50%, the Ca²⁺-independent release was enhanced sevenfold. This large Ca²⁺-independent release of GABA is likely to have different functional significance from the normal Ca²⁺-dependent release.     

GABA Induces Functionally Active Low-Affinity GABA Receptors on Cultured Cerebellar Granule Cells

The effect of γ-aminobutyric acid (GABA) and its agonists muscimol and 4,5,6,7-tetrahydroisoxazolo[5-4-c]pyridin-3-ol (THIP) on the development of GABA receptors on cerebellar granule cells was studied by cultivation of the cells in media containing these substances. It was found that the presence of 50 μM GABA in the culture media led to the induction of low-affinity GABA receptors (K_D 546 ± 117 nM) in addition to the high-affinity receptors (K_D 7 ± 0.5 nM) which were present regardless of the presence of GABA in the culture media. The functional activity of the GABA receptors was tested by investigating the ability of GABA to modulate evoked glutamate release from the cells. It was found that GABA could inhibit evoked glutamate release (ED₅₀ 10 ± 3 (μM) only when the cells had been cultured in the presence of 50 νM GABA, 50 μM muscimol, or 150 μM THIP, i.e., under conditions where low-affinity GABA receptors were present on the cells. This inhibitory effect of GABA could be blocked by 120 μM bicuculline and mimicked by 50 μM muscimol or 150 μM THIP whereas 150 μM (-)-baclofen had no effect. It is concluded that GABA acting extracellularly induces formation of low-affinity receptors on cerebellar granule cells and that these receptors are necessary for mediating an inhibitory effect of GABA on evoked glutamate release. The pharmacological properties of these GABA receptors indicate that they belong to the so-called GABA_A receptors.     

THE EFFECT OF BICUCULLINE, METRAZOL, PICROTOXIN AND STRYCHNINE ON THE RELEASE OF [3H]GABA FROM RAT BRAIN SLICES

Each of the four convulsants used significantly influenced the release of [³H]-GABA from brain slices, without affecting [³H]GABA uptake. Bicuculline (10^?5M, but not 10-fold higher or lower concentrations) potentiated the electrically evoked release of [³H]GABA but not the resting release, whereas metrazol (10^?4 to 10^?6 M) was found to inhibit resting but not electrically evoked release. Strychnine (10^?4 and 10^?5 M) and picro-toxin (10^?4 M) inhibited electrically evoked release.     

Effect of Nipecotic Acid, a γ-Aminobutyric Acid Transport Inhibitor, on the Turnover and Release of γ-Aminobutyric Acid in Rat Cortical Slices

Abstract: To see the effect of a γ-aminobutyric acid GABA uptake inhibitor on the efflux and content of endogenous and labeled GABA, rat cortical slices were first labeled with [³H]GABA and then superfused in the absence or presence of 1 mM nipecotic acid. Endogenous GABA released or remaining in the slices was measured with high performance liquid chromatography, which was also used to separate [³H]GABA from its metabolites. In the presence of 3 mM K⁺, nipecotic acid released both endogenous and [³H]GABA, with a specific activity four to five times as high as that present in the slices. The release of labeled metabolite(s) of [³H]GABA was also increased by nipecotic acid. The release of endogenous GABA evoked by 50 mM K⁺ was enhanced fourfold by nipecotic acid but that of [³H]GABA was only doubled when expressed as fractional release. In a medium containing no Ca²⁺ and 10 mM Mg²⁺, the release evoked by 50 mMK⁺ was nearly suppressed in either the absence or the presence of nipecotic acid. In the absence of nipecotic acid electrical stimulation (bursts of 64 Hz) was ineffective in evoking release of either endogenous or [³H]GABA, but in the presence of nipecotic acid it increased the efflux of endogenous GABA threefold, while having much less effect on that of [³H]GABA. Tetrodotoxin (TTX) abolished the effect of electrical stimulation. Both high K⁺ and electrical stimulation increased the amount of endogenous GABA remaining in the slices, and this increase was reduced by omission of Ca²⁺ or by TTX. The results suggest that uptake of GABA released through depolarization is of major importance in removing GABA from extracellular spaces, but the enhancement of spontaneous release by nipecotic acid may involve intracellular heteroexchange. Depolarization in the presence of Ca²⁺ leads to an increased synthesis of GABA, in excess of its release, but the role of this excess GABA remains to be established.     

Interacting Presynaptic k-Opioid and GABAA Receptors Modulate Dopamine Release from Rat Striatal Synaptosomes

Abstract— The presynaptic regulation of stimulated dopa-mine release from superfused rat striatal synaptosomes by opioids and γ-aminobutyric acid (GABA) was studied. It was found that in addition to dopamine D₂ autoreceptors, calcium-dependent K⁺-stimulated [³H]dopamine release was inhibited through activation of a homogeneous population of k -opioid receptors in view of the potent inhibitory effect of the k -selective agonist U69.593 (EC₅₀ 0.2 nM) and its antagonism by norbinaltorphimine. Neither μ-nor δ-selective receptor agonists affected release of [³H]-dopamine. In addition, GABA potently inhibited the evoked [³H]dopamine release (EC₅₀ 0.4 nM) through activation of GABA_A receptors in view of the GABA-mimicking effect of muscimol, the sensitivity of its inhibitory effect to picro-toxin and bicuculline, and the absence of an effect of the GABA_B receptor agonist baclofen. In the presence of a maximally effective concentration of GABA, U69,593 did not induce an additional release-inhibitory effect, indicating that these receptors and the presynaptic D₂ receptor are colocalized on the striatal dopaminergic nerve terminals. The excitatory amino acid agonists N-methyl-d -aspartate and kainate, as well as the cholinergic agonist carbachol, stimulated [³H]dopamine release, which was subject to k -opioid receptor-mediated inhibition. In conclusion, striatal dopamine release is under regulatory control of multiple excitatory and inhibitory neurotransmitter by activation of colocalized presynaptic receptors for excitatory amino acids, acetylcholine, dopamine, dynorphins, and GABA within the dopaminergic nerve terminals. Together, these receptors locally control ongoing dopamine neurotransmission.     

Glutamic Acid and γ-Aminobutyric Acid Modulate Each Other's Release Through Heterocarriers Sited on the Axon Terminals of Rat Brain

Abstract: The effects of γ-aminobutyric acid (GABA) on the spontaneous release of endogenous glutamic acid (Glu) or aspartic acid (Asp) and the effects of Glu on the release of endogenous GABA or [³H]GABA were studied in superfused rat cerebral cortex synaptosomes. GABA increased the outflow of Glu (EC₅₀17.2 μM) and Asp (EC₅₀ 18.4 μM). GABA was not antagonized by bicuculline or picrotoxin. Neither muscimol nor (-)-baclofen mimicked GABA. The effects of GABA were prevented by GABA uptake inhibitors and were Na⁺ dependent. Glu enhanced the release of [³H]GABA (EC₅₀ 11.5 μM) from cortical synaptosomes. Glu was not mimicked by the glutamate receptor agonists N-methyl-d -aspartic, kainic, or quisqualic acid. The Glu effect was decreased by the Glu uptake inhibitor D-threo-hydroxyaspartic acid (THA) and it was Na⁺ sensitive. Similarly to Glu, D-Asp increased [³H]GABA release (EC₅₀ 9.9 μM), an effect blocked by THA. Glu also increased the release of endogenous GABA from cortex synaptosomes. In this case the effect was in part blocked by the (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 6-cyano-7-nitroquinoxaiine-2, 3-dione, whereas the 6-cyano-7-nitroquinoxaline- 2, 3-dione-insensitive portion of the effect was prevented by THA. GABA increased the [³H]D-Asp outflow (EC₅₀ 13.7 μM) from hippocampal synaptosomes in a muscimol-, (-)- baclofen-, bicuculline-, and picrotoxin-insensitive manner. The GABA effect was abolished by blocking GABA uptake and was Na⁺ dependent. Glu increased the release of [³H]- GABA from hippocampal synaptosomes (EC₅₀ 7.1 μM) in an N-methyl-d -aspartic acid-, kainic acid-, or quisqualic acid-insensitive way. The effect of Glu was prevented by THA and was Na⁺ dependent. As in the cortex, the effect of Glu was mimicked by D-Asp in a THA-sensitive manner. It is proposed that high-affinity GABA or Glu heterocarriers are sited respectively on glutamatergic or GA- BAergic nerve terminals in rat cerebral cortex and hippocampus. The uptake of GABA may modulate Glu and Asp release, whereas the uptake of Glu may modulate the release of GABA. The existence of these heterocarriers is in keeping with the reported colocalization of GABA and Glu in some cortical and hippocampal neurons. Preliminary data suggest that these mechanisms may also be present in rat cerebellum and spinal cord.     

Increased GABA Release in the Human Brain Cortex as a Potential Pathogenetic Basis of Hyperosmolar Diabetic Coma

Human cerebral cortical slices preincubated with [³H]GABA, [³H]noradrenaline, or 5-[³H]hydroxytryptamine and superfused with Krebs solution or Mg²⁺-free Krebs solution were used to investigate the influence of increased D-glucose concentrations on the release of these [³H]-neurotransmitters evoked by high K⁺ content or NMDA receptor activation, respectively. An increase in level of D-glucose (normal content, 11.1 mM) by 32, 60, and/or 100 mM (a range characteristic for hyperosmolar diabetic coma) increased the [³H]GABA release and inhibited the [³H]noradrenaline release evoked by both methods of stimulation. The K⁺-induced 5-[³H]hydroxytryptamine release was also inhibited by high D-glucose content. Blockade of GABA_B receptors by p-(3-aminopropyl)-p-diethoxymethylphosphinic acid (CGP 35348) attenuated the inhibitory effect of high D-glucose content on the K⁺-evoked release of [³H]noradrenaline and 5-[³H]hydroxy-tryptamine, suggesting that the effect on monoamine release is, at least to a major part, the result of the increased GABA release and, as a consequence, of an increased GABA concentration at inhibitory GABA_B receptors. The membrane-impermeable sorbitol mimicked the increasing effect of D-glucose on [³H]GABA release and its inhibitory effect on 5-[³H]hydroxytryptamine release. However, dimethyl sulfoxide, which is known to permeate rapidly through biological membranes, had no effect at concentrations equiosmolar to D-glucose. It is concluded that a reduction in brain cell volume caused by increased extracellular, compared with cytoplasmic, osmolarity is crucial for the changes in neuronal function observed at high D- glucose and sorbitol content, In view of the fact that GABA is the main inhibitory neurotransmitter in the brain, the increased GABA release may be assumed to contribute to the pathogenesis of hyperosmolar diabetic coma.     

Presynaptic Inhibition in Cuneate blocked by GABA Antagonists

BICUCULLINE has been shown to have an action essentially similar to Picrotoxin in antagonizing both synaptically evoked postsynaptic inhibition and the depressant action of γ-amino-butyric acid (GABA) on cuneate neurones¹. This supports the hypothesis that GABA is the postsynaptic inhibitory transmitter in the cuneate². However, evidence³ indicates that GABA has a dual action in the cuneate, not only depressing the excitability of postsynaptic neurones, but also increasing the excitability of primary afferent terminals in a manner which might be expected of a presynaptic inhibitory transmitter. The experiments reported here show that the alkaloids bicuculline and picrotoxin block presynaptic inhibition and that this action is consistent with them exerting a GABA-antagonist action at primary afferent terminals.     

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.     

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.     

Effects of Anoxia on the Stimulated Release of Amino Acid Neurotransmitters in the Cerebellum In Vitro

Abstract: The effect of anoxia and ischemia on the release of amino acid transmitters from cerebellar slices induced by veratridine or high [K⁺] was studied. Synaptic specificity was tested by examining the tetradotoxin (TTX)-sensitive and the Ca²⁺-dependent components of stimulated release. Evoked release of endogenous amino acids was investigated in addition to more detailed studies on the stimulated efflux of preloaded [¹⁴C]GABA and d -[³H]aspartate (a metabolically more stable anologue of acidic amino acids).[¹⁴C]GABA release evoked by either method of stimulation was unaffected by periods of up to 35 min of anoxia and declined moderately by 45 min. In contrast, induced release of d -[³H]Asp increased markedly during anoxia to a peak at about 25 min, followed by a decline when anoxia was prolonged to 45 min. Evidence was obtained that the increased evoked efflux of d -[³H]Asp from anoxic slices was not due to impaired reuptake of the released amino acid and that it was completely reversible by reoxygenation of the slices. Results of experiments examining the evoked release of endogenous amino acids in anoxia were consistent with those obtained with the exogenous amino acids. Only 4 of the 10 endogenous amino acids studied exhibited TTX-sensitive veratridine-induced release under aerobic conditions (glutamate, aspartate, GABA, and glycine). Anoxia for 25 min did not affect the stimulated efflux of these amino acids with the exception of glutamate, which showed a significant increase. Compared with anoxia, effects of ischemia on synaptic function appeared to be more severe. Veratridine-evoked release of [¹⁴C]GABA was already depressed by 10 min and that of d -[³H]Asp showed a modest elevation only at 5 min. Stimulated release of d -Asp and labelled GABA declined progressively after 5 min. These findings were compared with changes in tissue ATP concentrations and histology. The latter studies indicated that in anoxia the earliest alterations are detectable in glia and that nerve terminals were the structures by far the most resistant to anoxic damage. The results thus indicated that evoked release of amino acid transmitters in the cerebellum is compromised only by prolonged anoxia in vitro. In addition, it would appear that the stimulated release of glutamate is selectively accentuated during anoxia. This effect may have a bearing on some hypoxic behavioral changes and, perhaps, also on the well-known selective vulnerability of certain neurons during hypoxia.     

Spinal mechanism of micturition reflex inhibition by naftopidil in rats

Aim

We investigated the spinal mechanism through which naftopidil inhibits the micturition reflex by comparing the effects of noradrenaline and naftopidil in rats.

Methods

The following were investigated: the influence of oral naftopidil on plasma monoamine and amino acid levels, the distribution of oral 14C-naftopidil, the effects of intravenous (IV) or intrathecal (IT) injection of noradrenaline or naftopidil on isovolumetric bladder contractions, amino acid levels in the lumbosacral spinal cord after IT noradrenaline or naftopidil, and the effects of IT naftopidil and strychnine and/or bicuculline on isovolumetric bladder contractions.

Key findings

Oral naftopidil decreased the plasma adrenaline level, while it increased the serotonin and glycine levels. After oral administration, 14C-naftopidil was detected in the spinal cord and cerebrum, as well as in plasma and the prostate gland. When the bladder volume was below the threshold for isovolumetric reflex contractions, IV (0.1 mg) or IT (0.1 μg) noradrenaline evoked bladder contractions, but IV (1 mg) or IT (0.01–1 μg) naftopidil did not. When the bladder volume was above the threshold for isovolumetric reflex contractions, IV or IT noradrenaline transiently abolished bladder contractions. IT noradrenaline decreased the levels of glycine and gamma-aminobutyric acid (GABA) in the lumbosacral cord, while IT naftopidil increased the GABA level. IT strychnine and/or bicuculline blocked the inhibitory effect of IT naftopidil on bladder contractions.

Significance

Naftopidil inhibits the micturition reflex by blocking α1 receptors, as well as by the activation of serotonergic, glycinergic, and GABAergic neurons in the central nervous system.     

Effects of taurine on cell morphology and expression of low-affinity GABA receptors in cultured cerebellar granule cells

Effects of taurine and THIP were studied on the development of cultured cerebellar granule cells with regard to GABA receptor expression and morphological development. Culturing in the presence of taurine or THIP led to the formation of low affinity GABA receptors as revealed from Scatchard analysis of [³H]GABA binding. This formation of receptors was susceptible to inhibition upon culturing in the simultaneous presence of taurine and bicuculline demonstrating the involvement of the high affinity GABA receptors which are present on the cells regardless of the culture condition. Superfusion experiments on cells cultured under the different conditions demonstrated that the low affinity GABA receptors expressed after culturing in the presence of THIP or taurine mediated an inhibition by GABA of evoked transmitter release from the granule cells. Cells cultured in either plain culture media or in the presence of taurine were indistinguishable with respect to the number of neurite extending cells observed after 4 days in culture. In contrast, culturing in the presence of THIP increased the number of neurite extending cells by 8% relative to the controls.Special issue dedicated to Dr. Paola S. Timiras     

Taurine release modified by GABAergic agents in hippocampal slices from adult and developing mice

Summary. In order to characterize the possible regulation of taurine release by GABAergic terminals, the effects of several agonists and antagonists of GABA receptors on the basal and K⁺-stimulated release of [³H]taurine were investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice using a superfusion system. Taurine release was concentration-dependently potentiated by GABA, which effect was reduced by phaclofen, saclofen and (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) at both ages, suggesting regulation by both GABA_B and GABA_C receptors. The involvement of GABA_A receptors could not be excluded since the antagonist bicuculline was able to affect both basal and K⁺-evoked taurine release. Furthermore, several GABA_B receptor effectors were able to inhibit K⁺-stimulated taurine release in the adults, while the GABA_C receptor agonists trans-4-aminocrotonic acid (TACA) and cis-4-aminocrotonic acid (CACA) potentiated this release. The potentiation of taurine release by agents acting on the three types of GABA receptors in both adult and developing hippocampus further indicates the involvement of transporters operating in an outward direction. This inference is corroborated by the moderate but significant inhibition of taurine uptake by the same compounds. Received June 28, 1999, Accepted August 31, 1999     

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⁺.