Interactions of taurine with GABAB binding sites in mouse brain.

The interactions of taurine with GABAB receptors were studied in membranes from the brain of the mouse by measuring the binding of [3H]baclofen and that of [3H]GABA in the presence of isoguvacine. Taurine displaced ligand binding to GABAB receptors concentration-dependently with an IC50 in the micromolar range. The effects of baclofen on the release of taurine and GABA from slices of cerebral cortex of the mouse were assessed using a superfusion system. Potassium-stimulated release of both [3H]taurine and [3H]GABA was unaffected by baclofen but potentiated by delta-aminovalerate. The enhancement of release of [3H]taurine by delta-aminovalerate was partially antagonized by baclofen, suggesting that baclofen-sensitive receptors could modify the release.

D-aspartate release from cerebellar astrocytes: modulation of the high K-induced release by neurotransmitter amino acids.

The properties of D-aspartate release were studied in cerebellar astrocytes (14-15 DIV) in primary cultures in the rat. The spontaneous release of D-aspartate from astrocytes was fast, being further enhanced in Na- and Ca-free (EDTA-containing) media. Kainate, quisqualate, D-aspartate and L-glutamate stimulated the release, whereas L-glutamatediethylester was inhibitory. The release was enhanced by veratridine and high K (50 mM). Substitution of chloride by acetate in the experimental medium did not change the basal release but slightly decreased the potassium-induced release, indicating that the high K-induced D-aspartate release is primarily due to depolarization of cells. The K-stimulated release was independent of extracellular Ca2+ and potentiated by kainate and quisqualate. The effect of kainate was reduced by kynurenate, and that of quisqualate by L-glutamatediethylester. Glycine, taurine and GABA were equally effective in depressing the stimulated release of D-aspartate. The inhibition of GABA could be blocked by GABA antagonists. The results suggest that inhibitory amino acids may be involved in the regulation of glutamate release from cerebellar astrocytes. A further implication is that cerebellar astrocytes possess functional glutamate receptors of kainate and quisqualate subtypes.

Displacement of excitatory amino acid receptor ligands by acidic oligopeptides.

A number of L-glutamyl and L-aspartyl dipeptides, glutathione, gamma-D-glutamylglycine and gamma-D-glutamyltaurine, were tested for their efficacy to displace ligands specific for different subtypes of excitatory amino acid receptors from rat brain synaptic membranes. In general, the L enanthiomorphs of gamma-glutamyl peptides were more potent displacers than gamma-D-glutamylglycine and -taurine but the latter were more specific for the quisqualate type of receptors. gamma-L-glutamyl-L-glutamate was the most effective dipeptide in displacing the binding of glutamate, 2-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) and 2-amino-5-phosphonoheptanoate (APH), whereas gamma-L-glutamyl-L-aspartate was the most effective in the binding of kainate. Both oxidized and reduced glutathione were inhibitory, being most potent in the binding of AMPA. gamma-L-Glutamylaminomethylsulphonate was most effective in the binding of APH. The most potent gamma-L-glutamyl peptides (glutathione, gamma-L-glutamyl-L-glutamate, -L-aspartate, and -glycine) may act as endogenous modulators of excitatory aminoacidergic neurotransmission.

Uptake and release of glycine in cerebellar granule cells and astrocytes in primary culture: potassium-stimulated release from granule cells is calcium-dependent.

The properties of [3H]glycine uptake and release were studied with cerebellar granule cells, 7-9 days in vitro, (DIV) and astrocytes, 14-15 DIV, in primary cultures. The uptake of glycine in both cell types consisted of a saturable high-affinity transport and nonsaturable diffusion. The transport constant (Km) and maximal velocity (V) were significantly higher in granule cells than in astrocytes. Uptake was strictly Na+-dependent and also markedly diminished in low-Cl medium. The specificity of the uptake was similar in both cell types. The spontaneous release of glycine from granule cells and astrocytes was fast. Homoexchange with extracellularly added glycine in granule cells suggests that the efflux is at least partly mediated via membrane transport sites in these cells. Kainate stimulated the release more effectively in neurons than in glial cells, the effect apparently being mediated by specific kainate-sensitive receptors in both cell types. The release was enhanced by veratridine and by depolarization of cell membranes by high K (50 mM) in both neurons and astrocytes. The potassium-stimulated release was partially Ca-dependent in neurons but Ca-independent in glial cells. The results suggest a functional role for glycine in both cerebellar astrocytes and glutamatergic granule cells.

Gamma-aminobutyric acid and benzodiazepine receptors in cultured cerebellar granule cells: effects of taurine and its lipophilic derivatives.

The effects of taurine and some lipophilic derivatives of taurine on binding to GABA and benzodiazepine receptors were studied in intact cerebellar granule cells. The phenylsuccinylimido derivatives of taurine appeared to increase the binding of muscimol in micromolar concentrations, while taurine decreased it slightly. Only minor changes were seen in the basal binding of flunitrazepam, whereas stimulation of the binding by GABA was strongly reduced by piperidino, benzamido and phenyl-succinylimidotaurine with taurine itself again showing only a weak effect. Diphenylhydantoin, which bears structural resemblance to the phenylsuccinylimido group, had a strong effect on the stimulated binding of flunitrazepam and it also slightly reduced the basal level of binding. Thus, it seems possible that the effects of the phenylsuccinylimido derivatives of taurine on the binding of flunitrazepam were due to this chemical structure and not to the taurine-like core of the molecules. The phthalimido derivative of taurine, taltrimide, which has been tested in clinical trials with epileptic patients, did not show any activity in the binding studies.

Release of endogenous taurine and gamma-aminobutyric acid from brain slices from the adult and developing mouse.

The spontaneous and potassium-stimulated release of endogenous taurine and gamma-aminobutyric acid (GABA) from cerebral cortex and cerebellum slices from adult and developing mice was studied in a superfusion system. The spontaneous release of GABA was of the same magnitude in slices from adult and developing mice, but the spontaneous release of taurine was considerably greater in the adults. The potassium-stimulated release of GABA from cerebral cortex slices was about five times greater in adult than in 3-day-old mice, but the potassium-stimulated release of taurine was more than six times greater in 3-day-old than in adult mice. In cerebellar slices from 7-day-old mice, potassium stimulation also evoked a massive release of taurine, whereas the evoked release from slices from adult mice was rather negligible. Also in cerebellar slices the potassium-stimulated release of GABA exhibited the opposite quantitative pattern. The stimulated release of both GABA and taurine was partially calcium dependent. The results suggest that taurine may be an important regulator of excitability in the developing brain.

Release of taurine from cultured cerebellar granule cells and astrocytes: co-release with glutamate.

The properties of the release of preloaded [3H]taurine and endogenous taurine were studied with cultured cerebellar granule cells (7-8 days in vitro) and astrocytes (14-15 days in vitro) from the rat. The spontaneous release of taurine from both cell types was slow. The release from both neurons and astrocytes was significantly enhanced by 0.1 mM veratridine, the stimulatory effect being more pronounced in granule cells than in astrocytes. No homo or heteroexchange with extracellularly added taurine or its structural analogues could be detected, suggesting that the efflux is probably not mediated via the membrane transport sites. Kainate stimulated the release more from granule cells than from astrocytes, the effect apparently being mediated by kainate-sensitive receptors. Depolarization of cell membranes by 50 mM K+ induced co-release of endogenous taurine and glutamate from both cell types. Preloaded [3H]taurine was readily released from astrocytes by potassium stimulation. Stimulated release occurred from granule cells if they had been cultured for 4 days with the label but not from the cells preloaded for only 15 min.

Release of taurine and GABA from cerebellar slices from developing and adult mice.

The properties of the release of exogenous radioactive taurine and GABA from cerebellar slices from developing and adult mice were investigated using a superfusion system. Potassium stimulation (50 mM K+) caused, approximately, a 1.4-fold enhancement in the release of preloaded taurine from slices from adult mice, while the response to potassium in 7-day-old mice was about 6-fold. The potassium stimulation caused, approximately, a 3-fold increase in the release of preloaded GABA from cerebellar slices from 7-day-old mice, whereas the enhancement was about 10-fold in the adult. The actual molar amount of taurine released from the immature cerebellum was strikingly large, about 16 times larger than the amount of GABA released upon the same stimulus. Spontaneous taurine efflux was potentiated by taurine and GABA, the responses being more pronounced in the 7-day-old cerebellum, suggesting that the immature cerebellum is more prone to stimulation by homo- and heteroexchange than the mature cerebellum. Potassium-stimulated taurine release was inhibited by GABAergic substances in the adult but not in the developing cerebellum. Potassium-stimulated GABA release from the adult cerebellum was greatly increased by GABA and also moderately by muscimol and bicuculline, the effect of the latter being antagonized by taurine and hypotaurine. Taurine was thus able to modulate GABA release through bicuculline-sensitive receptors, but this modulation was not evident in cerebellar slices from 7-day-old mice. An exposure of the slices to sodium-free media greatly enhanced taurine and GABA release in both age groups. The stimulated release of GABA generally exhibited a similar calcium dependency in the adult and 7-day-old cerebella but in 7-day-old mice the stimulated release of taurine was not strictly calcium-dependent.

Modulation of GABAergic neurotransmission in the brain by dipeptides.

The effects of endogenous and synthetic peptides containing GABA or its analogues on the GABA/benzodiazepine/chloride ionophore complex. GABAB receptor, Cl fluxes, GABA release and GABA uptake were studied using synaptic membranes, crude synaptoneurosomal preparations and slices prepared from the rat and mouse brain. The sodium-independent binding of GABA was strongly inhibited by GABA-histidine, followed by gamma-glutamyl-homotaurine, GABA-glycine and gamma-glutamyl-GABA. The binding of diazepam was slightly enhanced by the same peptides. The peptides alone had no effect on the chloride fluxes, but GABA-histidine, gamma-glutamyl-GABA and GABA-glycine enhanced while gamma-glutamyl-homotaurine and GABA-taurine inhibited GABA-stimulated chloride uptake. GABA-histidine was the most effective displacer of baclofen binding, but gamma-glutamylhomotaurine was entirely ineffective. The uptake of GABA was markedly inhibited in synaptosomal preparations by GABA-histidine, while all other peptides were less effective. gamma-Glutamyl-taurine attenuated but gamma-glutamyl-homotaurine and GABA-glycine enhanced the potassium-stimulated release of GABA. The present actions of GABA-histidine in vitro may be of significance for GABAergic neurotransmission in vivo.

Effect of aminooxyacetic acid on the release of preloaded [3H]GABA and radioactive metabolites from slices of developing mouse brain.

The release of [3H]gamma-aminobutyric acid (GABA) and its radioactive metabolites from slices of the cerebral cortex, cerebellum, striatum and brain stem of developing and adult mice was studied. The slices were incubated and superfused in the absence and presence of the GABA aminotransferase (GABA-T) inhibitor aminooxyacetic acid (AOAA). Exposure to 100 microM AOAA totally inhibited GABA-T and all radioactivity released from slices was in authentic GABA. In studies on developing brain the 10-microM concentration was also effective enough, except in cerebellar slices. In the absence of AOAA the major part of radioactivity spontaneously released from slices of adult cerebral cortex and cerebellum was tritiated water and still about one third part in the presence of 10 microM AOAA. Potassium stimulation induced only the release of radioactive GABA but not labeled metabolites in both presence and absence of AOAA. AOAA reduced the stimulation-induced release of GABA. It is recommended that the use of GABA-T inhibitors should be discontinued in release experiments. Then labeled GABA must be separated in the effluents from its radioactive breakdown products.

L-carnitine uptake by mouse brain synaptosomal preparations: competitive inhibition by GABA.

The uptake of L-carnitine was characterized in mouse brain synaptosomal preparations, with an emphasis on mutual interactions with GABA uptake systems. The uptake consisted of nonsaturable diffusion and one saturable energy- and sodium-dependent component. GABA, L-DABA and nipecotate were strong and hypotaurine and homotaurine moderate inhibitors of the uptake. The inhibition by GABA was shown to be competitive. GABA uptake contained two saturable transport components, high- and low-affinity. It was most strongly inhibited by nipecotate and L-DABA, but also by carnitine and hypotaurine. The high-affinity uptake of GABA was competitively inhibited by carnitine, but the inhibition of the low-affinity uptake of GABA was of the mixed type. The results suggest that GABA and carnitine share the same carrier system at synaptosomal membranes. However, GABA is the preferred substrate and the carnitine concentrations which significantly inhibited GABA uptake exceed the physiological carnitine levels in vivo.

Effects of taurine on the influx and efflux of calcium in brain slices of adult and developing mice.

45Ca2+ influx was reduced by 10-mM taurine during a 5-min incubation in slices from the cerebral cortex, cerebellum and brainstem of adult mice. In both adult and developing cerebral cortex and cerebellar slices a 30-min exposure to 1.0-mM taurine was likewise effective on resting Ca2+ influx whereas in slices depolarized by 50 mM K+ the influx was not affected. Ca2+ efflux from adult mouse cerebral cortex slices was affected by extracellular Na+ (0-180 mM) but not by K+ (50 mM) or taurine (1 mM). In cerebral cortex slices of 3-day-old mice K+ depolarization stimulated Ca2+ efflux, which effect was antagonized by 1.0 mM taurine. The results suggest that taurine is able to modify Ca2+ influx and efflux in both adult and developing brain but only at relatively high concentrations.

Release of taurine, GABA and dopamine from rat striatal slices: mutual interactions and developmental aspects.

The spontaneous and potassium-stimulated release of preloaded taurine and GABA from striatal slices of adult and 7-day-old rats were studied using a superfusion system. Particular attention was paid to mutual interactions of taurine and GABA with dopamine in the release processes. Potassium stimulation (50 mM) enhanced taurine release more in the immature than in the adult striatum, whereas the response was the opposite with GABA release. Spontaneous taurine efflux was increased by dopamine and apomorphine, whereas stimulated release was suppressed by these agents in both age groups. This dopamine effect was partially antagonized by haloperidol, suggesting that dopaminergic systems were able to modify taurine release, possibly via dopaminergic receptors. Dopamine and apomorphine had similar but more inconsistent effects on striatal GABA release, which were not, however, mediated through conventional dopamine receptors. Stimulation with 25 mM K+ caused an 11-fold increase in striatal dopamine release: this effect was potentiated by taurine, while the actions of GABA on dopamine release were variable.

Glutamate release from cerebellar granule cells differentiating in culture: Modulation of the K(+)-stimulated release by inhibitory amino acids.

The properties of l-[(3)H]glutamate release with an emphasis on the modulation by inhibitory amino acids of the potassium-induced release were studied with cerebellar granule cells from 7-day-old rats cultured for 7 or 14 days. Spontaneous glutamate release from cells grown for 7 days was fast, being slightly enchanced in Na(+)-free medium. l-Glutamate, kainate and quisqualate stimulated the release whereas N-methyl-d-aspartate and taurine were without any effect. The potassium-evoked glutamate release was Ca(2+)-dependent and potentiated by l-glutamate and quisqualate. Stimulated release was strongly depressed by glutamatediethylester. This inhibition was antagonized by GABA but not by taurine. GABA and its structural analogues taurine, hypotaurine, ?-alanine and glycine were all equally effective in depressing stimulated glutamate release. The inhibition by GABA could be blocked by GABA antagonist. Both K(+)-evoked release and the kainate-induced release of glutamate were significantly greater in 14-day-old than in 7-day-old cultures, but the other properties of release were similar. The demonstration of calcium-dependent and potassium-stimulated glutamate release from cerebellar granule cells is consonant with the proposed neurotransmitter role of glutamate in these cells. The release could be modulated by both glutamatergic substances and inhibitory amino acids, the effect of GABA probably being mediated by GABAergic receptors.

Taurine and GABA release from mouse cerebral cortex slices: potassium stimulation releases more taurine than GABA from developing brain.

The release of exogenous taurine and gamma-aminobutyric acid (GABA) was studied with slices from the developing mouse cerebral cortex. The spontaneous efflux of GABA increased with the cerebral GABA content during postnatal development, while the spontaneous efflux of taurine was approximately the same in both neonate and adult mice, in spite of a several-fold higher cerebral taurine content in the former. GABA, taurine and their structural analogues caused marked homo- and hetero-trans-stimulation of the release in both adult and developing mice, probably via membrane transport sites. The release was greatly enhanced by both 0.01 mM veratridine and exposure to sodium-free medium, the effects being more pronounced with GABA in the adults and with taurine in the neonates. The excitatory amino acids homocysteate, aspartate and kainate enhanced taurine release particularly from the developing cerebral cortex but were not effective on GABA release in the adults. The potassium stimulation of taurine release had a strikingly slow time course in both adult and developing mice. The responses in GABA release were also fairly slow in the neonates. Potassium stimulation evoked a large release of GABA in adult but not in developing mice. The evoked taurine release was in developing mice several-fold greater than the evoked GABA release, decreasing in magnitude with age. The potassium-stimulated release was only partially calcium dependent, more so with GABA in the adults and with taurine in the neonates, but a high magnesium ion concentration inhibited the release of both amino acids more strongly in the latter age group. Verapamil (0.1 mM) almost abolished the potassium stimulation of GABA release in both adult and neonate mice and was more effective on taurine release in neonate mice. The results suggest that taurine, not GABA, is the major inhibitor of excitability in developing mouse brain.

Co-operativity in sodium-independent taurine binding to brain membranes in the mouse.

Sodium-independent taurine binding to mouse brain membranes treated twice with Triton X-100 exhibited properties of positive co-operativity, suggesting that two or more taurine molecules interact at the binding site. The proposed taurine antagonist 6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine-1,1-dioxide and the new anticonvulsant taurine derivative taltrimide as well as glycine and GABA with their antagonists displaced taurine binding, strychnine being the most effective.