Taurine and GABA release from mouse cerebral cortex slices: Effects of structural analogues and drugs

The effects of structural analogues, excitatory amino acids and certain drugs on spontaneous and potassium-stimulated exogenous taurine and GABA release were investigated in mouse cerebral cortex slices using a superfusion system. Spontaneous efflux of both amino acids was rather slow but could be enhanced by their uptake inhibitors. Taurine efflux was facilitated by exogenous taurine, hypotaurine, -alanine and GABA, whereas GABA, nipecotic acid and homotaurine effectively enhanced GABA release. The stimulatory potency of the analogues closely corresponded to their ability to inhibit taurine and GABA uptake, respectively, indicating that these efflux processes could be mediated by the carriers operating outwards. Glutamate induced GABA release, whereas taurine efflux was potentiated by aspartate, glutamate, cysteate, homocysteate and kainate. The centrally acting drugs, including GABA agonists and antagonists, as well as the proposed taurine antagonist TAG (6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine-1,1-dioxide), had no marked effects on spontaneous taurine and GABA release. Potassium ions stimulated dosedependently both taurine and GABA release from the slices, the responses of taurine being strikingly slow but sustained. Exogenous GABA and nipecotic acid accelerated the potassium-stimulated GABA release, whereas picrotoxin and bicuculline were ineffective. The potassium-stimulated taurine release was unaltered or suppressed by exogenous taurine and analogues, differing in this respect from GABA release. The apparent magnitude of the depolarization-induced GABA release is thus influenced by the function of membrane transport sites, but the same conclusion cannot be drawn with regard to taurine. Haloperidol and imipramine were able to affect the evoked release of both taurine and GABA.     

Seizure-susceptibility and decreased taurine transport in the genetically epileptic rat

P₂ fractions from brains of genetically seizure-susceptible (SS) rats as compared to seizure-resistant (SR) rats show decreased high affinity uptake of taurine. Uptakes of GABA and glutamate into P₂ fractions did not differ between the substrains. In neonatal SS rats that had never had a seizure, the uptake of taurine is decreased both into the whole brain in vivo and into P₂ fraction in vitro, as compared to age-matched SR rats. This indicates that decreased uptake is not a consequence of seizure activity per se. In non-seizure susceptible progeny of SS rats, the uptake of taurine into P₂ fraction did not differ significantly from that of SR rats. In kidney cortex slices from SS rats, taurine uptake is slightly greater than in slices from SR rats. We propose that the decreased taurine transport in the P₂ fraction of the brains of SS rats may reflect a defect in transport in vivo that contributes to seizure-susceptibility.     

Changes in the Uptake of GABA and Taurine During Neuronal and Glial Maturation

Abstract: The influence of the time of culture on GABA and taurine uptake was investigated in spontaneously matured cultures of glial and neuronal origins and in cultures treated with cyclic nucleotides. In the spontaneously matured cultures the capacity of the high-affinity neuronal GABA transport system increased with time in culture. Essentially opposite results were found for the uptake of GABA by glial cultures. In contrast with the neuronal uptake of GABA, the capacity of the taurine transport system was significantly decreased. Uptake of taurine into glia, however, exhibited a progressive increase with the period of culture. The values of Km, for the high-affinity systems were always found to range around 10 μM. It is suggested that, in mature cells, neuronal uptake sites are of prime importance for GABA transport, while taurine uptake may be more specifically directed towards glial cells. When cultures were treated with cyclic nucleotide derivatives, a morphological differentiation was induced, which could not be linked to a stimulation of GABA or taurine uptake systems as compared with the non-treated cultures.     

Effects of cations on taurine,hypotaurine, and GABA uptake in mouse brain slices

The effects of cations on taurine, hypotaurine and GABA uptake were studied in mouse brain slices under identical experimental conditions. The uptakes were all strictly sodium-dependent. The omission or excess of K⁺ inhibited similarly taurine, hypotaurine and GABA uptake. The effects of omission of Ca²⁺ or Mg²⁺ were less pronounced. In both normal-sodium and low-sodium media all uptakes were saturable, consisting of both low-and high-affinity transport components. TheK _m constants for both low-and high-affinity transport components of hypotaurine and GABA increased in low-sodium medium, suggesting that sodium ions are necessary for their attachment to possible carrier sites in plasma membranes. In the case of taurine, however, the translation rate rather than the affinity of carrier sites was affected in Na⁺-free media. More than two sodium ions may be involved in the transport of one hypotaurine and one GABA molecule, whereas the coupling ratio between sodium and taurine was at least three. In its cation dependence hypotaurine uptake thus resembled more GABA uptake than taurine uptake.     

GABA and taurine sensitivity of cockroach giant interneurone synapses: Indirect evidence for the existence of a GABA uptake mechanism

The effects of exogenous GABA and taurine were studied on the cercal afferent-giant interneurone synapses (G.I. 2) located in the neuropile of the sixth abdominal ganglion of the cockroach, Periplaneta americana L. The decrease in excitatory synaptic potentials and the increase in postsynaptic membrane conductance due to GABA were enhanced by lowering the temperature of the saline, by using Na⁺ pump inhibitors, Na⁺ free salines or by agents blocking GABA uptake. The action of temperature was studied for taurine. Implications of these results for the identification of a metabolically dependent GABA uptake mechanism into glial cells are discussed.     

Effect ofl-homocysteine and derivatives on the high-affinity uptake of taurine and GABA into synaptosomes and cultured neurons and astrocytes

The effect ofl-nomocysteine and selected derivatives on the high-affinity uptake of the inhibitory neuroeffectors, GABA and taurine, was investigated in synaptosomes, and in cultured neurons and astrocytes. High-affinity uptake of taurine into synaptosomes was inhibited most effectively byl-homocysteine,Dl-homocysteine and homocystine whereas neuronal uptake was unaffected by any of the compounds tested. The high affinity uptake of taurine into astrocytes was markedly inhibited byl-homocysteine,l-homocysteic acid andl-homocystine. High-affinity GABA uptake into astrocytes was notably inhibited byl-homocystine, none of the other compounds tested causing appreciable inhibition below a concentration of 5 mM. Neuronal and synaptosomal high-affinity uptake of GABA was not significantly affected by any of the test compounds at concentrations below 5 mM. The implication of these results to the study of the mechanism of homocysteine-induced seizures and their relevance to the genetic disorder homocystinuria is discussed.     

Extracellular GABA in the Ventrolateral Thalamus of Rats Exhibiting Spontaneous Absence Epilepsy: A Microdialysis Study

Abstract: There is compelling evidence that excessive GABA-mediated inhibition may underlie the abnormal electrical activity, initiated in the thalamus, associated with epileptic absence seizures. In particular, the GABA_B receptor subtype seems to play a critical role, because its antagonists are potent inhibitors of absence seizures, whereas its agonists exacerbate seizure activity. Using a validated rat model of absence epilepsy, we have previously found no evidence of abnormal GABA_B receptor density or affinity in thalamic tissue. In the present study, we have used in vivo microdialysis to monitor changes in levels of extracellular GABA and other amino acids in this brain region. We have shown that basal extracellular levels of GABA and, to a lesser extent, taurine are increased when compared with values in nonepileptic controls. However, modifying GABAergic transmission with the GABA_B agonist (−)-baclofen (2 mg/kg i.p.), the GABA_B antagonist CGP-35348 (200 mg/kg i.p.), or the GABA uptake inhibitor tiagabine (100 µ M ) did not produce any further alteration in extracellular GABA levels, despite the ability of these compounds to increase (baclofen and tiagabine) or decrease (CGP-35348) seizure activity. These findings suggest that the increased basal GABA levels observed in this animal model are not simply a consequence of seizure activity but may contribute to the initiation of absence seizures.     

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     

UPTAKE, RELEASE AND HOMO- AND HETERO-EXCHANGE DIFFUSIONS OF INHIBITORY AMINO ACIDS IN GUINEA-PIG CEREBELLAR SLICES

Abstract— GABA, taurine and β-alanine are taken up by guinea-pig cerebellar slices by both the high-and low-affinity uptake processes, whereas glycine is taken up only by the low-affinity process. A considerable amount of labelled GABA loaded in the slice is released by unlabelled external GABA and a minute amount is released by external β-alanine, glycine and taurine. External glycine and β-alanine releases labelled glycine loaded in the slice. Labelled taurine loaded is effectively released by external taurine and β-alanine, while labelled β-alanine loaded is released only by external β-alanine.
It is suggested that hetero-exchanges which are one-directional in some cases also take place between the amino acids in addition to homo-exchanges. Therefore, high-affinity uptake processes observed with GABA and taurine could be the result of the homo-exchange diffusions, while that of β-alanine could be due to either the homo-exchange or the hetero-exchange diffusions or both.
K⁺'-evoked releases of GABA and to a lesser extent, taurine are partially dependent upon the presence of Ca⁺ in the superfusion media, whereas that of glycine and probably that of β-alanine, are not, K⁺ -evoked releases of labelled GABA and taurine are larger when loaded by their high-affinity uptake systems than by their low-affinity uptake processes. The reverse is the case with labelled glycine and β-alanine. These results do not rule out the possibility that taurine might act as a neurotransmitter in the cerebellum.     

Effect of Taurine on Neurotransmitter Release from Insect Synaptosomes

The effect of taurine on the release of [3H]acetylcholine ([3H]ACH) and [3H]gamma-aminobutyric acid ([3H]GABA) from preloaded locust synaptosomes has been studied. Veratridine (100 microM) and K+ (100 mM) both evoked [3H]ACh release and this was reduced in a concentration-dependent manner by taurine (5, 10, and 20 mM). In contrast to this, veratridine induced no observable release of [3H]GABA, and the response to K+ was slight. In the presence of taurine, however, a concentration-dependent enhancement of [3H]GABA release was observed. Since nipecotic acid (1 mM), an inhibitor of neuronal GABA uptake, also revealed [3H]GABA release induced by veratridine, it is suggested that both this effect and that of taurine are due to prevention of GABA reuptake. These results suggest that taurine may act as a neuromodulator in insects.     

Deletion of the γ-Aminobutyric Acid Transporter 2 (GAT2 and SLC6A13) Gene in Mice Leads to Changes in Liver and Brain Taurine Contents

The GABA transporters (GAT1, GAT2, GAT3, and BGT1) have mostly been discussed in relation to their potential roles in controlling the action of transmitter GABA in the nervous system. We have generated the first mice lacking the GAT2 (slc6a13) gene. Deletion of GAT2 (both mRNA and protein) neither affected growth, fertility, nor life span under nonchallenging rearing conditions. Immunocytochemistry showed that the GAT2 protein was predominantly expressed in the plasma membranes of periportal hepatocytes and in the basolateral membranes of proximal tubules in the renal cortex. This was validated by processing tissue from wild-type and knockout mice in parallel. Deletion of GAT2 reduced liver taurine levels by 50%, without affecting the expression of the taurine transporter TAUT. These results suggest an important role for GAT2 in taurine uptake from portal blood into liver. In support of this notion, GAT2-transfected HEK293 cells transported [³H]taurine. Furthermore, most of the uptake of [³H]GABA by cultured rat hepatocytes was due to GAT2, and this uptake was inhibited by taurine. GAT2 was not detected in brain parenchyma proper, excluding a role in GABA inactivation. It was, however, expressed in the leptomeninges and in a subpopulation of brain blood vessels. Deletion of GAT2 increased brain taurine levels by 20%, suggesting a taurine-exporting role for GAT2 in the brain.     

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     

Alterations in the Content of Amino Acid Neurotransmitters Before the Onset and During the Course of Methoxypyridoxine-Induced Seizures in Individual Rabbit Brain Regions

In rabbits, generalized seizures were induced by methoxypyridoxine, and changes in amino acid concentrations of 15 brain regions were investigated before seizure onset and during the course of sustained epileptiform activity. As previously reported, gamma-aminobutyric acid (GABA) concentration decreased preictally in most regions. At the same time, taurine level was elevated in the hypothalamus, thalamus, hippocampus, caudatum, and frontal cortex. After 90 min of seizures, it was significantly decreased in the hypothalamus, periaqueductal grey, substantia nigra, frontal cortex, and cerebellum. Glycine content was reduced preictally only in the substantia nigra; after seizure onset its concentration rose in all brain areas. Glutamate content in the frontal cortex decreased before seizure onset; after 1.5 h of seizures, its concentration in cerebellum, caudatum, and hippocampus was reduced. Aspartate level was decreased in most areas after sustained seizures; in putamen, however, it was elevated. In contrast, glutamine content increased preictally in the superior colliculus and in all brain areas by approximately 200% after 90 min of seizures. Alanine and valine content also rose markedly in most brain areas after prolonged seizures, and threonine showed the same tendency. The single brain regions were observed to respond to methoxypyridoxine in highly individualistic ways. For example, the glycine content of the substantia nigra, which is believed to utilize this amino acid as a neurotransmitter, decreased preictally. The potential importance of the superior colliculus in seizure induction is considered in view of the early rise in glutamine level. The antagonistic preictal behavior of taurine and GABA is discussed with respect to synthesis, uptake from the blood, and antiepileptic properties.     

Anticonvulsant effects of some inhibitory neurotransmitter amino acids

The anticonvulsive effects of GABA, taurine, and glycine were investigated on several chemically-induced and genetic seizure models. Intravenous injections of either GABA, taurine, or glycine provided protection against 3-mercaptopropionic acid (MPA)-induced convulsions in adult Swiss mice. GABA was partially effective against isonicotinic acid hydrazide and was without effect against bicuculline-induced convulsions bProlonged administration of glycine prevented MPA-induced convulsions but not electrically induced seizures or seizures induced by strychnine or metrazol.Intragastric glycine protected young audiogenic seizure-susceptible DBA/2 mice against all three phases of sound-induced convulsions (wild running, clonic and tonic seizure), but GABA and taurine provided little or no protection. With increase of glycine, the cerebral levels of glutamine and serine also increased, but that of glutamic acid decreased. The endogenous glutamic and glycine levels were slightly higher in the brains of the audiogenic seizure-susceptible DBA/2 mice than in that of the resistant BALB/Cy strain.     

Modulatory Action of Taurine on the Release of GABA in Cerebellar Slices of the Guinea Pig

Abstract: For the purpose of demonstrating the action of taurine as a neuromodulator in addition to its suggested neurotransmitter function, the effects of taurine and muscimol on the depolarization-induced Ca-dependent release of [³H]γ-aminobutyric acid (pH]GABA) and l -[³H]glutamate in cerebellar slices from guinea pigs were investigated. The release of [³H]GABA was found to be greatly decreased by a GABA agonist, muscimol, and by taurine, but not by glycine. The release of l -[³H]glutamate was little affected by taurine. The release of [³H]GABA was enhanced by bicuculline and strychnine, but not by picrotoxin, and the suppressive action of muscimol on the GABA release was antagonized by bicuculline, picrotoxin, and strychnine, suggesting the possible existence of presynaptic autoreceptors for GABA in the cerebellum. The suppressive action of taurine on the release of [³H]GABA, on the other hand, was blocked only by bicuculline. These results suggest that taurine reduced the release of [³H]GABA from cerebellar slices by acting on the GABA autoreceptors or, more likely, on other types of receptors that are sensitive to bicuculline. As a possible mechanism for this modulatory action of taurine, the blockade by this amino acid of the influx of Ca²⁺ into cerebellar tissues was tentatively suggested.     

Mutual interactions in the transport of taurine,hypotaurine, and GABA in brain slices

The mutual interactions and the effects of GABA on the saturable transport components of taurine and hypotaurine were investigated with mouse brain slices. The low-affinity taurine transport was competitively inhibited by both hypotaurine and GABA. Hypotaurine did not alter the kinetic parameters of high-affinity taurine uptake, whereas there occurred some stimulation with GABA, possibly by heteroexchange. Taurine had no significant effects on high-affinity hypotaurine uptake, whereas the low-affinity component was reduced by both taurine and GABA, GABA strongly interfered with the high-affinity hypotaurine uptake, being the preferred substrate in simultaneous uptake experiments. The results confirm that taurine, hypotaurine, and GABA are transported into brain slices by only one two-component system with affinities highest for GABA and lowest for taurine.     

Hypotaurine transport in brain slices

Hypotaurine uptake was compared to taurine and GABA uptakes in brain slices under identical experimental conditions. The slices effectively concentrated both hypotaurine and GABA from the medium, whereas taurine was taken up more slowly. The uptakes of these three structurally related amino acids were all saturable, consisting of one low-and one high-affinity transport component. The kinetic parameters of hypotaurine uptake were of the same order of magnitude as those of GABA uptake. All uptake systems were sensitive to temperature, metabolic poisons, and sodium omission. Hypotaurine uptake was inhibited by GABA,l-2,4-diaminobutyric acid (l-DABA), cysteic acid, and -alanine, but not by taurine. Taurine uptake was strongly reduced by hypotaurine, -alanine, andl-DABA, as well as by GABA, whereas GABA uptake was affected only by cystamine,l-DABA, and nipecotic acid.The uptake processes of hypotaurine, taurine, and GABA were thus fairly similar and showed properties characteristic for neurotransmitter uptake. Hypotaurine uptake resembled more GABA than taurine uptake. The present inhibition studies suggest that there may exist only one common two-component transport system for these three amino acids.     

In vivo release patterns and cardiovascular properties of inhibitory and excitatory amino acids in the hypothalamus

Summary The posterior hypothalamus of conscious, freely moving rats was superfused with artificial cerebrospinal fluid through a push-pull cannula and the release of amino acids was determined in the superfusate. Under basal conditions, the release rates of taurine, GABA and glutamate fluctuated according to ultradian rhythms with different frequencies. Hypothalamic superfusion with veratridine or high concentrations of potassium choride enhanced the release rates of taurine, GABA and glutamate in a concentration-dependent way. Tetrodotoxin decreased the basal release rates of the three amino acids. The release of arginine was not influenced significantly by these compounds. A fall of blood pressure elicited by intravenous infusion of nitroprusside decreased the release rates of GABA and taurine and enhanced the release of glutamate. Infusion of noradrenaline increased blood pressure and release rates of GABA and taurine, while the release of glutamate was not influenced. Neither the pressor, nor the depressor responses to drugs influenced the release of arginine in the hypothalamus. It is concluded that the inhibitory amino acids taurine and GABA released from hypothalamic neurons possess a tonic hypotensive function. The excitatory amino acid glutamate, released from glutamatergic neurons of the hypothalamus, seems to possess a hypertensive function in counteracting a fall of blood pressure.This work was supported by the Fonds zur Förderung der wissenschaftlichen Forschung. These results were presented at the Third International Congress on Amino Acids, Vienna, August 1993     

Effects of the ionophores X537A and A23187 on GABA,glycine, and taurine release from chick retinal subcellular fractions

The ionophore X537A at concentrations of 5–20 M stimulated the release of [³H]GABA and [³⁵S]taurine, from retinal subcellular crude nuclear (P₁) and crude synaptosomal (P₂) fractions. The release of [³H]GABA increased 114% and 136% over control values in P₁ and P₂ fractions, respectively. The efflux of [³⁵S]taurine from P₁ was increased by 45% and that from P₂ by 21%. X537A increased⁴⁵Ca²⁺ uptake in the P₂ fraction but not in the P₁ fraction. The effect of X537A on the amino acid release was not dependent on the presence of exogenous calcium. X537A did not affect [³H]GABA or [³⁵S]taurine uptake by the retinal fractions. A23187 enhanced [³H]GABA release from P₁ and P₂ by 52% and 105%, respectively. The ionophore also increased [¹⁴C]glycine liberation in both P₁ (35%) and P₂ (50%) but failed to stimulate [³⁵S]taurine release. A23187 produced a transient increase of⁴⁵Ca²⁺ uptake of 38% in P₁ and 30% in P₂. The effects of A23187 on the release of amino acids were calcium dependent. The amino acid uptake was not affected by the ionophore. These results are consisent with the suggested neurotransmitter role for GABA at the outer synaptic layer and for GABA and glycine at the inner synaptic layer of the retina. A neurotransmitter role for taurine is not supported by the present results.     

The Effect of Colchicine and Cytochalasin B on the Release of Taurine from the Chick Retina

Abstract: The effect of colchicine (0.5 mM) and of cytochalasin B (10⁻⁴ M) on the release of [³⁵S]taurine from the isolated chick retina, upon stimulation by 68.5 mM-KCl, 10⁻⁵ M-veratridine and 10 mM-glutamate, was studied. Cytochalasin and colchicine effects on taurine release were compared with those on K⁺-stimulated release of [³H]dopamine and [³H]GABA. Colchicine caused a marked decrease of the [³⁵S]taurine release evoked by the three stimulatory agents; it also decreased [³H]dopamine release without affecting that of [³H]GABA. Cytochalasin B significantly decreased the efflux of [³⁵S]taurine stimulated by glutamate or veratridine without altering that evoked by 68.5 mM-KCl. Cytochalasin practically suppressed the [³H]dopamine-stimulated release and slightly decreased that of [³H]GABA. This drug produced a high increase in the spontaneous release of labeled GABA and taurine. These results suggest that the release of taurine and GABA from the chick retina probably occurs through different mechanisms. It is suggested that taurine release may be related to a process involving contractile proteins.