The release of dopamine from synaptosomes from rat striatum by the ionophores X 537A and A 23187

The antibiotics X 537A and A 23187 are negatively charged divalent cation ionophores. X 537A may, in addition, be an ionophore for amines including catecholamines. The effects of these ionophores were examined on the uptake and release of dopamine by synaptosomes prepared from rat corpus striatum. Both X 537A and A 23187, at concentrations less than 0.5 μM, release both endogenous and [³H]-dopamine from synaptosomes. They had virtually no effect on the uptake of exogenous dopamine. These compounds act by different mechanisms. X 537A causes divalent ion-independent release in which a large fraction of the effluent consists of deaminated products. X 537A, in addition, releases [³H]dopamine from rat adrenal medullary chromaffin granules. The results suggest that X 537A causes release of dopamine from intrasynaptosomal storage vesicles and perhaps is acting as a catecholamine carrier across the vesicular membrane. A 23187, on the other hand, causes a Ca²⁺-dependent release in which only a small fraction of the catechol in the effluent is deaminated. A 23187 has little effect on the release of [³H]dopamine from chromaffin granules. These results suggest that A 23187 carries Ca²⁺ into the synaptosomes and thereby initiates exocytotic release.     

Properties of [3H]taurine release from crude synaptosomal fractions of rat cerebral cortex

The release of previously accumulated [³H]taurine and [¹⁴C]GABA from crude synaptosomal (P₂) fractions isolated from rat cerebral cortex was studied using a superfusion system. The spontaneous efflux of [³H]taurine and [¹⁴C]GABA was stimulated by elevated concentrations of K⁺ (15–133 mM) in a concentration-dependent manner. This K⁺-stimulated release of [¹⁴C]GABA but not of [³H]taurine was enhanced in the presence of Ca²⁺. However, addition of 3 mM Ca²⁺ to the superfusion medium in the presence of the ionophore A 23187 resulted in a stimulation of the release of both [³H]taurine and [¹⁴C]GABA. These results are discussed in connection with the cellular localization of tourine in the central nervous system.     

Effects of ionophores A23187 and X537A on brain calcium,catecholamines and excitability

The ionophores A23187 and X537A inhibit ⁴⁵Ca uptake by rabbit brain mitochondria and synaptosomes and also stimulate the release of accumulated ⁴⁵Ca from these preparations, but have no effect on ⁴⁵Ca binding by synaptic membranes or on total brain Ca in mice. Both agents inhibit uptake and stimulate release of ³H-norepinephrine by rabbit P₂ synaptosomal preparations, while the NE and serotonin levels of mouse brain are depressed by X537A. The changes in Ca activities may be related both to the elevated thresholds for cortical after-discharge produced in cats by these ionophores, and to the ionophore induced reduction of pentylenetetrazol seizures in mice.     

Effects of X-irradiation induced loss of cerebellar granule cells on the synaptosomal levels and the high affinity uptake of amino acids.

Abstract— Crude synaptosomal (P₂) preparations were obtained from the cerebella of rats in which the granule cell population had been selectively reduced by X-irradiation treatment and from the cerebella of control animals. In the P₂ fraction from control cerebella, the level of glutamate was greater than any other of the 5 amino acids measured and was 2-fold higher than taurine, which was present at the next highest level. The content of taurine was slightly higher than that found for aspartate and was 3-fold greater than that observed for GABA. Alanine and glycine were present in the lowest amounts. The levels of glutamate and aspartate were significantly (P < 0.05) lower by 25 and 15%, respectively, in the P₂ fraction isolated from the X-irradiated cerebella in comparison to control values. The content of taurine, GABA, glycine, and alanine were not changed by the X-irradiation treatment. The uptake of 1.0 μm -l -[³H]glutamate and l -[³H]aspartate was reduced approx 20% by X-irradiation treatment, whereas the uptake of 1.0 μm -[³H]GABA and [³H]taurine was unchanged. A more detailed kinetic analysis of l -[³H]glutamate uptake revealed there was a 20% decrease in the V_max value with X-irradiation treatment and no change in the apparent K_m value. In a second study, the uptake of l -[³H]glutamate, l -[³H]aspartate and [³H]GABA was measured using P₂ fractions obtained from the cerebella of rats in which the population of granule, stellate and basket cells had been reduced by X-irradiation treatment. The uptake of 1.0μm -l -[³H]glutamate, l -[³H]aspartate and [³H]GABA was significantly (P < 0.05) reduced to 57, 68, and 59%, respectively, of control values. A more detailed kinetic analysis of [³H]GABA uptake revealed no significant change in the apparent K_m and a 35% decrease in the V_max value. The data are discussed in terms of glutamate being the excitatory neurotransmitter released from granule cells and GABA being the inhibitory neurotransmitter released from basket cells.     

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.     

Spontaneous and evoked release of [3H]taurine from a P2 subcellular fraction of the rat retina

The effects of spontaneous and evoked [³H]taurine release from a P₂ fraction prepared from rat retinas were studied. The P₂ fraction was preloaded with [³H]taurine under conditions of high-affinity uptake and then examined for [³H]taurine efflux utilizing superfusion techniques. Exposure of the P₂ fraction to high K⁺ (56 mM) evoked a Ca²⁺-independent release of [³H]taurine. Li⁺ (56 mM) and veratridine (100 M) had significantly less effect (8–15% and 15–30%, respectively) on releasing [³H]taurine compared to the K⁺-evoked release. 4-Aminopyridine (1 mM) had no effect on the release of [³H]taurine. The spontaneous release of [³H]taurine was also Ca²⁺-independent. When Na⁺ was omitted from the incubation medium K⁺-evoked [³H]taurine release was inhibited by approximately 40% at the first 5 minute depolarization period but was not affected at a second subsequent 5 minute depolarization period. The spontaneous release of [³H]taurine was inhibited by 60% in the absence of Na⁺. Substitution of Br^– for Cl^– had no effect on the release of either spontaneous or K⁺-evoked [³H]taurine release. However, substitution of the Cl^– with acetate, isethionate, or gluconate decreased K⁺-evoked [³H]taurine release. Addition of taurine to the superfusion medium (homoexchange) resulted in no significant increase in [³H]taurine efflux. The taurine-transport inhibitor guanidinoethanesulfonic acid increased the spontaneous release of [³H]taurine by approximately 40%. These results suggest that the taurine release of [³H]taurine is not simply a reversal of the carrier-mediated uptake system. It also appears that taurine is not released from vesicles within the synaptosomes but does not rule out the possibility that taurine is a neurotransmitter. The data involving chloride substitution with permeant and impermeant anions support the concept that the major portion of [³H]taurine release is due to an osmoregulatory action of taurine while depolarization accounts for only a small portion of [³H]taurine release.     

Divalent cation ionophore A23187: a potent protein synthesis inhibitor.

Divalent cation ionophore A23187 has a potent inhibitory effect on protein synthesis in the C6 rat glioma cell line. Treatment with 4 μM A23187 resulted in 93% inhibition of [1-¹⁴C]leucine label incorporation into proteins and a 61% increase in free pool labeling. Total RNA synthesis was not affected. Extracellular ionic calcium or magnesium are not required for these changes to occur. Therefore, these effects of A23187 may be a direct effect on protein synthesis or may result from release of internal stores of divalent cations. By comparison, ionophore X537A (4 μM) has only a slight inhibitory effect on protein synthesis.     

Release and exchange of neurotransmitters in synaptosomes: Effects of the ionophore A23187 and of ouabain

The effects of the ionophore A23187 and of ouabain on the release of [³H]GABA and [³H]norepinephrine were studied in superfused rat brain synaptosomes. Each of the two drugs moderately stimulated the spontaneous release of [³H]GABA, but greatly potentiated the release of [³H]GABA induced by unlabeled GABA. In contrast, the ionophore and norepinephrine showed an additive, but not a supraadditive, releasing effect on synaptosomal [³H]norepinephrine. Ouabain modestly and transiently potentiated the norepinephrine-induced [³H]norepinephrine release, which, however, was inhibited by the drug after a few minutes. It is suggested that in the new intrasynaptosomal ionic conditions determined by the two drugs, the stoichiometry of the basal homoexchange of GABA is changed in a direction favoring net outward transport.     

Effect of baclofen on in vitro noradrenaline release from rat hippocampus and cerebellum: an action at an α2-adrenoceptor

The release of [³H]noradrenaline from rat hippocampal synaptosomes by 25 mM K⁺ and 5 μM veratridine, but not by the Ca²⁺ ionophore A23187 was depressed by baclofen. This depression was reversed by 8-Bromo-cAMP. This action of baclofen was stereospecific and mimicked both that of GABA in the presence of bicuculline and that of clonidine. The α₂-adrenoceptor antagonists yohimbine and Wy25309 antagonised the action of clonidine and baclofen but not that of GABA. Specific binding of [³H]clonidine was displaced by Wy25309 and baclofen, but not by GABA. Specific binding of [³H]GABA in the presence of Ca²⁺ was displaced by baclofen but not by Wy25309. It is concluded that baclofen is not a specific agonist at GABA_B receptors in the brain.     

Potassium-stimulated release of GABA,glycine, and taurine from the chick retina

The effect of depolarizing potassium concentration on the release of [¹⁴C]glycine, [³H]GABA, and [³⁵S]taurine was investigated in the whole chick retina and in a synaptosomal fraction prepared from the chick retina. In the whole retina, increasing potassium concentration above 40 mM resulted in an increased release of the three amino acids. The release of glycine was the most stimulated and that of taurine, the least. The potassium-evoked release of glycine and GABA was calcium dependent. In the synaptosomal fraction, 68.5 mM potassium significantly stimulated the efflux of GABA and glycine by a calcium-dependent mechanism. The release of taurine from this fraction was unaffected by high potassium.     

The ionophore X537A (Lasolocid) transiently increases acetylcholine release from rat brain invitro

The effect of X537A on acetylcholine (ACh) release was examined $\text{in vitro}$ in superfused slices of rat cerebrum and striatum. The ionophore (30 μM) induced a transient release of ACh which was not dependent on calcium in the medium. Also in contrast to K⁺-stimulated release, X537A-induced release was not sustained by 10^?5M choline in the superfusion medium and not inhibited by 5 × 10^?4M pentobarbital. The ionophore did not transport ACh or choline from an aqueous to an organic phase. Both K⁺ and X537A inhibited 1 μM (³H) choline uptake into striatal synaptosomes but this effect of X537A was more extensive and less reversible than that caused by K⁺. X537A did not inhibit choline acetyltransferase activity.     

Spontaneous and depolarization-induced efflux of hypotaurine from mouse cerebral cortex slices: Comparison with taurine and gaba

The spontaneous and potassium- or veratrine-stimulated efflux of [³⁵S]hypotaurine from superfused cerebral cortex slices of adult mice was compared with the release of [³H]taurine and [³H]GABA. Initially GABA was the fastest released. Hypotaurine was, however, eventually released fastest, since its spontaneous efflux did not slow down during superfusions as did taurine and GABA effluxes. More than 60 % of all preloaded labelled amino acids still remained in the slices after 80-min superfusions. The effluxes of all three amino acids were stimulated by potassium and veratrine depolarizations: GABA efflux most and hypotaurine efflux least. The veratrine-stimulated release of taurine was long-lasting, while all other responses started and ended abruptly. With respect to efflux properties hypotaurine resembled more GABA than taurine.     

Effects of the ionophores,X-537A and A-23187 on catecholamine release from the in vitro frog adrenal

1. The ionophore X-537A increases the rate of catecholamine release from the $\text{in vitro}$ frog adrenal.2. The ratio of epinephrine/norepinephrine measured during X-537A stimulation was the same as that during spontaneous release.3. Even when Ca⁺⁺ was removed from the Ringer, X-537A stimulated catecholamine release, but depolarization by elevated extra-cellular K⁺ was no longer effective.4. X-537A also increases the release of dopamine β-hydroxylase, suggesting that the ionophore acts, at least in part, by stimulating the exocytosis of the chrommaffin granule contents.5. Therefore, it is questionable whether the release of catecholamines by X-537A is owing to its action as a Ca⁺⁺- ionophore.6. The divalent cation ionophore, A-23187 (50μM), did not affect the rate of catecholamine release.     

UPTAKE AND RELEASE OF TAURINE FROM CEREBRAL CORTEX SLICES AND THEIR SUBCELLULAR COMPARTMENTS

—Cortex slices, synaptosomes and C-6 glioma cells were used to study [³⁵S]taurine uptake and its electrically-stimulated release. After exposure to taurine at two concentrations, the synaptosome preparation subsequently derived from the slices contained 41% of the particle-bound taurine and 16% of the total in the tissue. The uptake of [¹⁴C]GABA by C-6 glioma cells was inhibited 3-fold more by β-alanine than by l -DABA, whilst synaptosome preparations showed the opposite pattern, l -DABA being 2 or 3 times more effective than β-alanine. [³⁵S]Taurine uptake inhibition by l -DABA was low for synaptosomes and C-6 glioma, whereas β-alanine showed considerable effect on C-6 glioma (41%) and slices of white matter (ependyma; 50%). Synaptosome preparations showed little effect with β-alanine. When 30 min rather than 5 min incubations were employed, β-alanine depressed [³⁵S]taurine uptake by cortex slices by 30%. Taurine was taken up by a calcium-dependent mechanism and subcellular fractionation indicated that the synaptosome fraction showed losses commensurate with the net taurine release when low stimulation currents were used.     

Diaminobutyric acid: A tool for discriminating between carrier-mediated and non-carrier-mediated release of GABA from synaptosomes?

The carrier-mediated transport of GABA in rat brain synaptosomes was strongly and permanently inhibited byl-2,4-diaminobutyric acid (DAB). In order to discriminate between carrier-mediated and non-carrier-mediated release of [³H]GABA, synaptosomes prelabeled with 0.5 M [³H]GABA in the presence of 100 M DAB, or with 0.2 M [³H]GABA without DAB, were superfused in conditions stimulating the release of [³H]GABA. Only the release elicited by unlabeled GABA or DAB (by homo- and heteroexchange, respectively) was strongly inhibited in DAB-pretreated synaptosomes. The spontaneous release and the release induced by 56 mM KCl in the presence of CaCl₂, by the ionophore A23187, by ouabain, by lack of K⁺, or by purified black widow spider toxin were unaffected or only barely decreased in DAB-treated synaptosomes, and therefore do not seem to be mediated by the DAB-blocked GABA carrier.     

Function of taurine transporter (Slc6a6/TauT) as a GABA transporting protein and its relevance to GABA transport in rat retinal capillary endothelial cells

The purpose of this study was to identify the uptake mechanism of γ-aminobutyric acid (GABA) via taurine transporter (Slc6a6/TauT) and its relationship with GABA transport at the inner BRB. Rat Slc6a6/TauT-transfected HeLa cells exhibited Na⁺-, Cl⁻-, and concentration-dependent [³H]GABA uptake with a K_m of 1.5 mM. Taurine, β-alanine, and GABA markedly inhibited Slc6a6/TauT-mediated uptake of [³H]GABA. The uptake of [³H]GABA by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) was Na⁺-, Cl⁻-, and concentration-dependent with a K_m of 2.0 mM. This process was more potently inhibited by substrates of Slc6a6/TauT, taurine and β-alanine, than those of GABA transporters, GABA and betaine. In the presence of taurine, there was competitive inhibition with a K_i of 74 μM. [³H]Taurine also exhibited competitive inhibition with a K_i of 1.8 mM in the presence of GABA. In conclusion, rat Slc6a6/TauT has the ability to use GABA as a substrate and Slc6a6/TauT-mediated GABA transport appears to be present at the inner BRB.     

Effects of membrane peroxidation on [3H]acetylcholine release in rat cerebral cortical synaptosomes

[³H]Acetylcholine (ACh) release, malonaldehyde formation and⁴⁵calcium-uptake were measured in rat cerebral cortical nerve terminal that were exposed to various concentrations of ferrous and ascorbate ions. At a constant molar ratio of 25:1, ferrous:ascorbate, these ions increased malonaldehyde (MA) synthesis in a concentration-dependent manner. Treatment with these ions in the same ratio also induced a dose-related inhibition of the K⁺-depolarization-induced release of newly synthesized [³H]ACh. Combined exposure to Fe²⁺/ascorbate also reduced calcium ionophore A23187-induced [³H]ACh release. Neither ferrous nor ascorbate ions alone altered depolarization-or ionophore-induced [³H]ACh release over this concentration range. Depolarization- and A23187-induced⁴⁵calcium uptake were not affected by peroxidation, suggesting that membrane peroxidation influenced some process in the release-process subsequent to calcium influx in a manner similar to what is observed during aging.     

Effects of amino acids on calcium uptake by glial and neuroblastoma cells

The uptake of [⁴⁵Ca] has been studied in clonal glial and neuronal cells. It was somewhat more efficient in the neuroblastoma clone M₁ compared to glial clones. In all cases [⁴⁵Ca] uptake was shown to depend on the phosphate concentration in the incubation medium. It was decreased by the ionophore A 23187 at 200 μM concentration in both neuronal and glial clones. The influence of amino acids some of which are putative neurotransmitters was investigated; the interactions between [⁴⁵Ca] uptake and these amino acids were related to their concentration and the type of cells used (neuronal or glial). L-aspartate and taurine for example had two opposite effects on [⁴⁵Ca] uptake by the glial clone NN at two different concentrations; they could therefore play a role in the control of calcium level in the synaptic cleft.     

Comparison of synaptosomal and glial uptake of pipecolic acid and GABA in rat brain

The active uptake of [³H]pipecolic acid increased with incubation time and its uptake at 3 min was half of that at 20 min. [¹⁴C]GABA uptake rose earlier, and its uptake at 3 min was almost 80% of that at 20 min. On the other hand, a ratio (pellet/medium) of [³H]pipecolic acid uptake into glial cell-enriched fractions, was much less (0.4–0.6) than that of [¹⁴C]GABA (25.8–74.1). GABA, 10^–4 M, and pipecolic acid, 10^–4 M, produced a significant inhibition of [³H]pipecolic acid uptake into P₂ fractions. Pipecolic acid, 10^–4 M, significantly reduced the synaptosomal and glial uptake of [¹⁴C]GABA. GABA, 10^–4 M, affected neither spontaneous nor high K⁺-induced release of [³H]pipecolic acid from brain slices. It is suggested that pipecolic acid is involved in either synaptic transmission or in its modulation at GABA synapses in the central nervous system.     

Net taurine transport and its inhibition by a taurine antagonist

P₂-fractions were isolated from rat brain, and used to study net taurine transport. The fractions were incubated in increasing concentrations of [³H]taurine and the intraterminal concentration measured by liquid scintillation and amino acid analysis. The membrane potential of the isolated fractions was estimated using⁸⁶Rb⁺ as a marker for intracellular K⁺. Taurine was synthesized in the P₂-fraction when incubated in taurine free medium. At external taurine concentrations below 370 M a significant amount of the endogenous taurine was released to the incubation medium. Net taurine uptake into the P₂-fraction was achieved at external taurine concentrations exceeding 370 M. The taurine antagonist 6-aminomethyl-3-methyl-4H, 1, 2, 4-benzothiadiazine-1, 1-dioxide (TAG) competitively inhibited taurine and [³H]taurine transport into the P₂-fraction. As the external concentration of taurine was increased, the accumulation of⁸⁶Rb⁺ into the P₂-fraction was facilitated. This indicated an increasing hyperpolarization of the neuronal membrane as taurine transport shifted from release towards uptake. TAG reduced the hyperpolarization that paralleled taurine accumulation, in a dose dependent manner. Our results indicate that relatively low transmembranal gradients of taurine may be maintained by an electrogenic taurine transporter having a large transport capacity. Such a transporter may well serve the needs of osmotic regulation, i.e. to transport large amounts of taurine in any direction across the neuronal membrane.