GABA uptake inhibitors produce a greater antinociceptive response in the mouse tail-immersion assay than other types of GABAergic drugs

Antinociception produced by the GABA uptake inhibitors d,l- SKF-89976A and SKF-100330A was characterized and compared to that produced by other types of GABAergic drugs. Using the mouse tail-immersion assay it was found that the antinociception produced by the uptake inhibitors was antagonized by scopolamine, a cholinergic muscarinic receptor antagonist. However, neither SKF compound demonstrated any significant affinity for muscarinic receptor binding sites suggesting that they are not direct-acting cholinomimetics. In vitro uptake experiments revealed that the SKF compounds selectively inhibit GABA transport, having no effect on the accumulation of aspartic acid, glutamic acid, beta-alanine or glycine. Moreover, antinociception and GABA uptake inhibition were stereoselective for SKF-89976A, with the d-isomer being more active in both tests. When comparing antinociceptive responses at maximally effective doses it was also found that the SKF compounds were substantially more efficacious than direct-acting GABA receptor agonists or a GABA transaminase inhibitor. These data suggest that uptake inhibitors may be facilitating GABA transmission in a system that is less affected by other types of GABAergic compounds.     

Calcium-Independent γ-Aminobutyric Acid Release from Growth Cones: Role of γ-Aminobutyric Acid Transport

Neuronal growth cones isolated in bulk from neonatal rat forebrain have uptake and K(+)-stimulated release mechanisms for gamma-aminobutyric acid (GABA). Up to and including postnatal day 5, the K(+)-stimulated release of [3H]GABA and endogenous GABA is Ca2+ independent. At these ages, isolated growth cones neither contain synaptic vesicles nor stain for synaptic vesicle antigens. Here we examined the possibility that the release mechanism underlying Ca2(+)-independent GABA release from isolated growth cones is by reversal of the plasma membrane GABA transporter. The effects of two GABA transporter inhibitors, nipecotic acid and an analogue of nipecotic acid, SKF 89976-A, on K(+)-stimulated release of [3H]GABA from superfused growth cones were examined. Nipecotic acid both stimulated basal [3H]GABA release and enhanced K(+)-stimulated release of [3H]GABA, which indicates that this agent can stimulate GABA release and is, therefore, not a useful inhibitor with which to test the role of the GABA transporter in K(+)-stimulated GABA release from growth cones. In contrast, SKF 89976-A profoundly depressed both basal and K(+)-stimulated [3H]GABA release. This occurred at similar concentrations at which uptake was blocked. These observations provide evidence for a major role of the GABA transporter in GABA release from neuronal growth cones.     

On the activity of γ-aminobutyric acid and glutamate transporters in chick embryonic neurons and rat synaptosomes

The uptake of radioactive -aminobutyric acid (GABA) andd-aspartate and the effect of SKF 89976-A, a non-substrate inhibitor of the GABA transporter, on this uptake have been investigated. Neuronal cultures from eight-day-old chick embryos grown for three or six days in vitro, were used as a model. For comparison, we also used the P₂-fraction from rat. Neuronal cultures grown for three and six days expressed high-affinity uptake systems for [³H]GABA and ford-[³H]aspartate with an increasing V_max during this period. The lipophilic non-substrate GABA uptake inhibitor, SKF 89976-A, inhibited transporter mediated uptake of GABA both in cell cultures from chicken, and in P₂-fractions from rat. The results also showed that SKF 89976-A was a poor inhibitor of the uptake ofd-aspartate. We found no non-saturable uptake ofd-aspartate.     

Effect of Homo-β-Proline and Other Heterocyclic GAB A Analogues on GABA Uptake in Neurons and Astroglial Cells and on GABA Receptor Binding

Abstract: Two groups of GABA (γ-aminobutyric acid) analogues, one comprising derivatives of β-proline and the other compounds structurally related to nipecotic acid, were investigated as potential inhibitors of high-affinity GABA transport in neurons and glial cells, as well as displacers of GABA receptor binding. In addition to cis -4-hydroxynipecotic acid, which is known as a potent inhibitor of GABA uptake, homo-β-proline was the only compound which proved to be a potent inhibitor of glial as well as neuronal GABA uptake. IC₅₀ values for GABA uptake into glial cells and brain cortex "prisms" were 20 and 75 μM, respectively, and the IC₅₀ value obtained for GABA uptake into cultured neurons was 10 μM. A kinetic analysis of the action of homo-β-proline on GABA uptake into cultured astrocytes and neurons showed that this compound acts as a competitive inhibitor of GABA uptake in both cell types. From the apparent K _m values, K _i values for homo-β-proline of 16 and 6 μM could be calculated for glial and neuronal uptake, respectively. This mechanism of action strongly suggests that homo-β-proline interacts with the GABA carriers. Furthermore, homo-β-proline also displaced GABA from its receptor with an IC₅₀ value of 0.3 μM. The cis -4-hydroxynipecotic acid analogues, cis- and trans-4-mercaptonipecotic acid, had no inhibitory effect on glial or neuronal GABA uptake. Other SH reagents, PCMB, NEM and DTNB, were shown to be relatively weak inhibitors of GABA uptake into cultured astrocytes, suggesting that SH groups are not directly involved in the interaction between GABA and its transport carrier.     

Stimulation of γ-[3H]Aminobutyric Acid Release from Cultured Mouse Cerebral Cortex Neurons by Sulphur-Containing Excitatory Amino Acid Transmitter Candidates: Receptor Activation Mediates Two Distinct Mechanisms of Release

In primary cultures of mouse cerebral cortex neurons, sulphur-containing excitatory amino acids (SAAs; namely, L-cysteine sulphinate, L-cysteate, L-homocysteine sulphinate, L-homocysteate, S-sulphocysteine) at concentrations ranging from 0.1 microM to 1 mM evoked a saturable release of gamma-[3H]aminobutyric acid ([3H]GABA) in the absence of any other depolarizing agent. All SAAs exhibited essentially similar potency (EC50, 100-150 microM) in releasing [3H]GABA although a variable profile of maximal stimulatory effect was observed when compared with basal release. The intracellular accumulation of the lipophilic cation, [3H]tetraphenylphosphonium, was significantly reduced in the presence of all SAAs, thus verifying a depolarization of the neuronal plasma membrane. SAA-stimulated release of [3H]GABA was shown to comprise two distinct components, calcium-dependent and calcium-independent, which occur after activation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Thus, all SAA-evoked responses were antagonized by the selective, competitive NMDA-receptor antagonist, 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid (IC50 range, greater than 50 microM) and the non-NMDA-receptor antagonist, 6,7-dinitroquinoxalinedione (IC50 range, 5-50 microM). Removal of magnesium ions from the superfusion medium caused a significant potentiation of SAA-evoked responses without having any effect on basal levels of [3H]GABA efflux, a result consistent with an involvement of NMDA-receptor activation. Calcium-independent release (i.e., that release remaining in the presence of 1 mM cobalt ions) was a distinct component but of smaller magnitude. Using 500 microM excitatory amino acid agonist concentrations, this component of release was (1) markedly attenuated by 15 microM SKF-89976-A, a non-transportable inhibitor of the GABA carrier, and (2) abolished when choline ions replaced sodium ions in the superfusion medium or when in the presence of excitatory amino acid receptor antagonists. These observations are clearly consistent with a receptor-mediated, depolarization-induced reversal of the GABA carrier.     

Inhibitors of the γ-aminobutyric acid transporter 1 (GAT1) do not reveal a channel mode of conduction

We expressed the γ-aminobutyric acid (GABA) transporter GAT1 (SLC6A1) in Xenopus laevis oocytes and performed GABA uptake experiments under voltage clamp at different membrane potentials as well as in the presence of the specific GAT1 inhibitors SKF-89976A and NO-711. In the absence of the inhibitors, GAT1 mediated the inward translocation of 2 net positive charges across the plasma membrane for every GABA molecule transported into the cell. This 2:1 charge flux/GABA flux ratio was the same over a wide range of membrane potentials from −110 mV to +10 mV. Moreover, when GABA-evoked (500 μM) currents were measured at −50 and −90 mV, neither SKF-89976A (5 and 25 μM) nor NO-711 (2 μM) altered the 2:1 charge flux/GABA flux ratio. The results are not consistent with previous hypotheses that (i) GABA evokes an uncoupled channel-mediated current in GAT1, and (ii) GAT1 inhibitors block the putative uncoupled current gated by GABA. Rather, the results suggest tight coupling of GAT1-mediated charge flux and GABA flux.     

Mutual Inhibition Kinetic Analysis of γ-Aminobutyric Acid, Taurine, and β-Alanine High-Affinity Transport into Neurons and Astrocytes: Evidence for Similarity Between the Taurine and β-Alanine Carriers in Both Cell Types

The transport kinetics of gamma-aminobutyric acid (GABA), taurine, and beta-alanine in addition to the mutual inhibition patterns of these compounds were investigated in cultures of neurons and astrocytes derived from mouse cerebral cortex. A high-affinity uptake system for each amino acid was demonstrated both in neurons (Km GABA = 24.9 +/- 1.7 microM; Km Tau = 20.0 +/- 3.3 microM; Km beta-Ala = 73.0 +/- 3.6 microM) and astrocytes (Km GABA = 31.4 +/- 2.9 microM, Km Tau = 24.7 +/- 1.3 microM; Km beta-Ala = 70.8 +/- 3.6 microM). The maximal uptake rates (Vmax) determined were such that, in neurons, Vmax GABA greater than Vmax beta-Ala = Vmax Tau, whereas in astrocytes, Vmax beta-Ala greater than Vmax Tau = Vmax GABA. Taurine was found to inhibit beta-alanine uptake into neurons and astrocytes in a competitive manner, with Ki values of 217 microM in neurons and 24 microM in astrocytes. beta-Alanine was shown to inhibit taurine uptake in neurons and astrocytes, also in a competitive manner, with Ki values of 72 microM in neurons and 71 microM in astrocytes. However, beta-alanine was found to be a weak noncompetitive inhibitor of neuronal and astrocytic GABA uptake, whereas in reverse experiments, GABA displayed weak noncompetitive inhibition of neuronal and astrocytic uptake of beta-alanine. Likewise, taurine was a weak noncompetitive inhibitor of GABA uptake in neurons and similarly, GABA was a weak noncompetitive inhibitor of taurine uptake into neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of 3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazol (exo-THPO) and its N-substituted analogs on GABA transport in cultured neurons and astrocytes and by the four cloned mouse GABA transporters

The system of GABA transporters in neural cells constitutes an efficient mechanism for terminating inhibitory GABAergic neurotransmission. As such these transporter are important therapeutical targets in epilepsy and potentially other neurological diseases related to the GABA system. In this study a number of analogs of 3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazol (exo-THPO), a promising lead structure for inhibitors of GABA uptake were investigated. It was found that the selectivity of N-acetyloxyethyl-exo-THPO for inhibition of the astroglial GABA uptake system was 10-fold as compared to inhibition of the neuronal GABA uptake system. Selectivity in this magnitude may provide potent anti-convulsant activity as has recently been demonstrated with the likewise glia-selective GABA uptake inhibitor, N-methyl-exo-THPO. In contrast to the competitive inhibition of GABA uptake exhibited by N-substituted analogs of 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO), nipecotic acid, and guvacine, N-4,4-diphenyl-3-butenyl(DPB)-N-methyl-exo-THPO and 4-phenylbutyl-exo-THPO exhibited non-competitive type inhibition kinetics. The lipophilic character of a number of GABA analogs was concluded by far to constitute the determining factor for the potency of these compounds as inhibitors of GAT1-mediated uptake of GABA. This finding underscores the complexity of the pharmacology of the GABA transport system, since these non-competitive inhibitors are structurally very similar to some competitive GABA uptake inhibitors. Whether these structure-activity relationships for inhibition of GABA uptake may provide sufficient information for the development of new structural leads and to what extent these compounds may be efficient as therapeutical anti-convulsant agents remain to be elucidated.     

Effects of Arachidonic Acid on Glutamate and γ-Aminobutyric Acid Uptake in Primary Cultures of Rat Cerebral Cortical Astrocytes and Neurons

The effects of arachidonic acid on glutamate and gamma-aminobutyric acid (GABA) uptake were studied in primary cultures of astrocytes and neurons prepared from rat cerebral cortex. The uptake rates of glutamate and GABA in astrocytic cultures were 10.4 nmol/mg protein/min and 0.125 nmol/mg protein/min, respectively. The uptake rates of glutamate and GABA in neuronal cultures were 3.37 nmol/mg protein/min and 1.53 nmol/mg protein/min. Arachidonic acid inhibited glutamate uptake in both astrocytes and neurons. The inhibitory effect was observed within 10 min of incubation with arachidonic acid and reached approximately 80% within 120 min in both types of culture. The arachidonic acid effect was not only time-dependent, but also dose-related. Arachidonic acid, at concentrations of 0.015 and 0.03 mumol/mg protein, significantly inhibited glutamate uptake in neurons, whereas 20 times higher concentrations were required for astrocytes. The effects of arachidonic acid were not as deleterious on GABA uptake as on glutamate uptake in both astrocytes and neurons. In astrocytes, GABA uptake was not affected by any of the doses of arachidonic acid studied (0.015-0.6 mumol/mg protein). In neuronal cultures, GABA uptake was inhibited, but not to the same degree observed with glutamate uptake. Lower doses of arachidonic acid (0.03 and 0.015 mumol/mg protein) did not affect neuronal GABA uptake. Other polyunsaturated fatty acids, such as docosahexaenoic acid, affected amino acid uptake in a manner similar to arachidonic acid in both astrocytes and neurons. However, saturated fatty acids, such as palmitic acid, exerted no such effect. The significance of the arachidonic acid-induced inhibition of neurotransmitter uptake in cultured brain cells in various pathological states is discussed.     

On the presence of 3H-GABA uptake mechanism in bovine spermatozoa

In the present study, existence of (3)H-GABA uptake mechanism in bovine spermatozoa and the modulation of (3)H-GABA transport by GABA itself were evaluated. The hypothesis was tyrosine phosphorylation affects transporter (GAT) function. (3)H-GABA uptake assays were performed on bovine spermatozoa and it resulted to be temperature- and time-dependent and K(m) was 1.48muM. Uptake was inhibited by the metabolic inhibitor ouabain and different blockers of GAT-1 (beta-alanine, l-DABA, nipecotic acid, tiagabine). Extracellular GABA up-regulated GABA transport, while the addition of SKF89976A, a high affinity inhibitor of the rat brain GABA transporter, reduced GABA uptake. Tyrosine phosphorylation affects transporter function since genistein, a broad-spectrum tyrosine kinase inhibitor, decreased (3)H-GABA uptake. Reduction in uptake did not occur in the presence of daidzein, an inactive genistein analogue. Furthermore, the genistein-mediated reduction in transport could be prevented by the tyrosine phosphatase inhibitor pervanadate. The action of these drugs on GABA transport is likely mediated through the GABA transporter GAT-1 since SKF89976A blocked a majority of GABA uptake. Wash-out experiments indicated that the genistein effect was reversible. When the experiments were conducted using "in vitro" capacitated spermatozoa there was no detectable uptake. Present results demonstrate that the carrier-mediated GABA uptake system in bovine spermatozoa modulates its function in response to extracellular GABA, that changes in lipid distribution and membrane composition which occur during capacitation eliminates GABA uptake and suggest the involvement of tyrosine phosphorylation in GABA transport.     

Inhibition of transporter mediated γ-aminobutyric acid (GABA) release by SKF 89976-A,a GABA uptake inhibitor,studied in a primary neuronal culture from chicken

The effect of SKF 89976-A, a lipophilic non-substrate inhibitor of the -aminobutyric acid (GABA) transporter, on the release of radioactive GABA andd-aspartate has been studied. Neuronal cultures from 8 day old chick embryos, grown for six days, served as a model. The cultures were incubated with [³H]d-aspartate and [¹⁴C] GABA with the subsequent addition of high or low concentrations of SKF 89976-A. Finally the cultures were exposed to differently composed media for either 30 or 300 seconds. The release was quantified, using liquid scintillation counting. The efflux of [³H]d-aspartate and [¹⁴C] GABA was increased by [K⁺] and time, and a minimum value was obtained at [Ca²⁺] 1.05 mM. The release of both [³H]d-aspartate and [¹⁴C] GABA was inhibited by SKF 89976-A. The obtained results indicate that transporter mediated processes are the major mechanisms of transmitter release in the investigated model.     

A Possible Role of the Non-GAT1 GABA Transporters in Transfer of GABA From GABAergic to Glutamatergic Neurons in Mouse Cerebellar Neuronal Cultures

Cultures of dissociated cerebellum from 7-day-old mice were used to investigate the mechanism involved in synthesis and cellular redistribution of GABA in these cultures consisting primarily of glutamatergic granule neurons and a smaller population of GABAergic Golgi and stellate neurons. The distribution of GAD, GABA and the vesicular glutamate transporter VGlut-1 was assessed using specific antibodies combined with immunofluorescence microscopy. Additionally, tiagabine, SKF 89976-A, betaine, β-alanine, nipecotic acid and guvacine were used to inhibit the GAT1, betaine/GABA (BGT1), GAT2 and GAT3 transporters. Only a small population of cells were immuno-stained for GAD while many cells exhibited VGlut-1 like immuno-reactivity which, however, never co-localized with GAD positive neurons. This likely reflects the small number of GABAergic neurons compared to the glutamatergic granule neurons constituting the majority of the cells. GABA uptake exhibited the kinetics of high affinity transport and could be partly (20%) inhibited by betaine (IC₅₀ 142 μM), β-alanine (30%) and almost fully (90%) inhibited by SKF 89976-A (IC₅₀ 0.8 μM) or nipecotic acid and guvacine at 1 mM concentrations (95%). Essentially all neurons showed GABA like immunostaining albeit with differences in intensity. The results indicate that GABA which is synthesized in a small population of GAD-positive neurons is redistributed to essentially all neurons including the glutamatergic granule cells. GAT1 is not likely involved in this redistribution since addition of 15 μM tiagabine (GAT1 inhibitor) to the culture medium had no effect on the overall GABA content of the cells. Likewise the BGT1 transporter cannot alone account for the redistribution since inclusion of 3 mM betaine in the culture medium had no effect on the overall GABA content. The inhibitory action of β-alanine and high concentrations of nipecotic acid and guvacine on GABA transport strongly suggests that also GAT2 or GAT3 (HUGO nomenclature) could play a role.     

Inhibitors of the GABA uptake systems

Summary This review describes a novel class of heterocyclic GABA uptake inhibitor with no affinity for the GABA receptors. The parent compound nipecotic acid is a potent inhibitor of neuronal and glial GABA uptake, and nipecotic acid is a substrate for the transport carriers concerned. The structurally related cyclic amino acids guvacine and cis-4-hydroxynipecotic acid are also potent inhibitors of both GABA transport systems. Even minor structural alterations of these compounds result in considerable or complete loss of activity. Whereas homonipecotic acid is a weak but selective inhibitor of glial GABA uptake, homoguvacine is virtually inactive. Similarly the lower homologues of nipecotic acid and guvacine, -proline and 3-pyrroline-3-carboxylic acid, respectively, show some selectivity with respect to inhibition of glial GABA uptake, but these compounds are much weaker than the parent compounds. The bicyclic compounds THPO and THAO, in which the carboxyl groups of nipecotic acid and homonipecotic acid have been replaced by 3-isoxazolol units are moderately potent and practically specific inhibitors of glial GABA uptake. cis-4-Mercaptonipecotic acid is considerably weaker than the closely related analogue cis-4-hydroxynipecotic acid, but the former compound may interact irreversibly with the GABA transport carriers.The results demonstrate a pronounced substrate specificity of the glial and in particular the neuronal GABA transport system. It is evident that the GABA molecule is transported in a conformation different from that, in which it activates its receptors. These findings are of importance for the development of drugs for selective pharmacological regulation of the functions of central GABA-mediated synapses in certain neurological diseases.     

High-Affinity Uptake of (RS)-Nipecotic Acid in Astrocytes Cultured from Mouse Brain. Comparison with GABA Transport

Abstract: (RS)-Nipecotic acid is taken up into cultured astrocytes by a saturable high-affinity transport system with a K_m, of 28.8 ± 2.8 μM and a V_max of 0.294 ± 0.022 nmol × min⁻¹× [mg cell protein]⁻¹. The uptake which represents a net inward transport was sodium-dependent, requiring translocation of one sodium ion for each molecule of nipecotic acid taken up. The most potent inhibitors of GABA uptake into astrocytes (GABA, (R)-nipecotic acid, (3RS,4SR)-4-hydroxynipecotic acid, and guvacine) were shown to be potent inhibitors of nipecotic acid uptake (IC₅₀) 20, 25, 25, and 50 μm respectively), GABA being a competitive inhibitor. (S)-2,4-Diaminobutyric acid was a more efficient inhibitor than β-alanine of glial uptake of (RS)-nipecotic acid. It is concluded that astroglial uptake of (RS)-nipecotic acid and GABA is mediated by the same transport system.     

The depolarizing responses to GABA in rat sensory ganglia in vivo and in vitro. A study of the role of glial uptake.

Intracellular recordings of neurones in dorsal root ganglia (DRG) have demonstrated that GABA transport into DRG satellite cell does not affect neuronal responses to this amino acid. The experiments were performed with transport-resistant GABA analogues and with inhibition of GABA uptake. GABA response characteristics can thus be considered exclusively neuronal in origin.     

Regional Selectivity of a γ-Aminobutyric Acid-Induced [3H]Acetylcholine Release Sensitive to Inhibitors of γ-Aminobutyric Acid Uptake

The effects of gamma-aminobutyric acid (GABA) on the release of [3H]acetylcholine ([3H]ACh) were studied in synaptosomes prepared from rat hippocampus, cerebral cortex, hypothalamus, and striatum and prelabelled with [3H]choline. When synaptosomes were exposed in superfusion to exogenous GABA (0.01-0.3 mM) the basal release of newly synthesized [3H]ACh was increased in a concentration-dependent way in hippocampus, cortex, and hypothalamus nerve endings. In contrast, the release of [3H]ACh was not significantly affected by GABA in striatal synaptosomes. The effect of GABA was not antagonized significantly by bicuculline or picrotoxin. Muscimol caused only a slight not significant increase of [3H]ACh release when tested at 0.3 mM whereas, at this concentration, (-)-baclofen was totally inactive. The GABA-induced release of [3H]ACh was counteracted by SKF 89976A, SKF 100561, and SKF 100330A, three strong and selective GABA uptake inhibitors. The data suggest that, in selective areas of the rat brain, GABA causes release of [3H]ACh following penetration into cholinergic nerve terminals through a GABA transport system.     

STRUCTURE-ACTIVITY STUDIES ON THE INHIBITION OF GABA BINDING TO RAT BRAIN MEMBRANES BY MUSCIMOL AND RELATED COMPOUNDS

Abstract— A series of compounds structurally related to muscimol (5-aminomethyl-3-isoxazolol) was tested as inhibitors of the sodium-independent binding of GABA to membranes from rat brain. Muscimol, 5-(l-aminoethyl)-3-isoxazolol, 5-(2-aminoethyl)-3-isoxazolol (homomuscimol), and the bicyclic derivative 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) were relatively potent inhibitors of GABA binding. THIP is an analogue of muscimol locked in a folded conformation. The structurally related compound 1,2,3,6-tetrahydropyridine-4-carboxylic acid (isoguvacine), a semirigid analogue of trans-4-aminocrotonic acid, was also a potent inhibitor of GABA binding. Apart from muscimol, these inhibitors of GABA binding did not influence the sodium-dependent,'high-affinity' uptake of GABA in rat brain slices, whereas the potent GABA uptake inhibitors guvacine and nipecotic acid did not influence GABA binding. The present results support previous findings that different conformational modes of GABA interact with GABA postsynaptic receptors and the neuronal GABA transport system in rat brain, and indicate that the 'active conformation' of GABA with respect to the receptors is partially folded and almost planar. Based on a comparison of the present results with previous in vivo studies the structural requirements for GABA-like activity in rat cerebral cortex and cat spinal cord seem to be somewhat different.     

In Vivo Changes in γ-Aminobutyric Acid Output from the Cerebral Cortex Induced by Inhibitors of γ-Aminobutyric Acid Uptake and Metabolism

Abstract: The effects of inhibitors of γ-aminobutyric acid (GABA) metabolism or uptake on GABA output from the cerebral cortex was studied by means of a collecting cup placed on the exposed cortex of rats anaesthetized with urethane. GABA was identified and quantified by a mass-fragmentographic method. Ethanolamine-O-sulphate (10⁻² M ) applied directly on the cerebral cortex caused a long-lasting twofold increase in GABA output, whereas dl -2, 4-diaminobutyric acid (5 × 10⁻³ M ) caused a sevenfold increase and β -alanine was inactive. The results indicate that glial uptake has little effect on GABA inactivation in the cerebral cortex. The inhibition of neuronal uptake seems a more effective tool to increase GABA concentration in the synaptic cleft, and consequently also in GABA output, than the inhibition of GABA metabolism.     

Presynaptic Mechanisms Underlying the γ-Aminobutyric Acid-Evoked Receptor-Independent Release of [3H]Norepinephrine in Rat Hippocampus

The effects of gamma-aminobutyric acid (GABA) on the spontaneous efflux of [3H]norepinephrine ([3H]NE) were studied in synaptosomes prepared from rat hippocampus and prelabelled with [3H]NE. It had been observed previously that, when synaptosomes were exposed in superfusion to GABA, the basal release of the tritiated catecholamine was enhanced, apparently with no involvement of the known GABA receptors. The mechanisms underlying this effect have now been investigated. The potency of GABA as a releaser of [3H]NE was decreased by lowering the Na+ content of the superfusion medium, and its effect disappeared at 23 mM Na+. The GABA-induced [3H]NE release was counteracted by the GABA uptake inhibitor N-(4,4-diphenyl-3-butenyl)nipecotic acid (SKF 89976A), but it was unaffected by the NE uptake blockers desmethylimipramine and nisoxetine. The GABA-induced release of [3H]NE was Ca2+-dependent and tetrodotoxin-sensitive. The data support the hypothesis that GABA provoked [3H]NE release by a novel mechanism which involves penetration into the noradrenergic nerve terminals through a GABA carrier located on the NE terminals themselves. This uptake process might be electrogenic and provoke depolarization of the nerve terminals, causing an exocytotic release of [3H]NE.     

Uptake of Benzoic Acid and Chloro-Substituted Benzoic Acids by Alcaligenes denitrificans BRI 3010 and BRI 6011

The mechanism of uptake of benzoic and 2,4-dichlorobenzoic acid (2,4-DCBA) by Alcaligenes denitrificans BRI 3010 and BRI 6011 and Pseudomonas sp. strain B13, three organisms capable of degrading various isomers of chlorinated benzoic acids, was investigated. In all three organisms, uptake of benzoic acid was inducible. For benzoic acid uptake into BRI 3010, monophasic saturation kinetics with apparent K(infm) and V(infmax) values of 1.4 (mu)M and 3.2 nmol/min/mg of cell dry weight, respectively, were obtained. For BRI 6011, biphasic saturation kinetics were observed, suggesting the presence of two uptake systems for benzoic acid with distinct K(infm) (0.72 and 5.3 (mu)M) and V(infmax) (3.3 and 4.6 nmol/min/mg of cell dry weight) values. BRI 3010 and BRI 6011 accumulated benzoic acid against a concentration gradient by a factor of 8 and 10, respectively. A wide range of structural analogs, at 50-fold excess concentrations, inhibited benzoic acid uptake by BRI 3010 and BRI 6011, whereas with B13, only 3-chlorobenzoic acid was an effective inhibitor. For BRI 3010 and BRI 6011, the inhibition by the structural analogs was not of a competitive nature. Uptake of benzoic acid by BRI 3010 and BRI 6011 was inhibited by KCN, by the protonophore 3,5,3(prm1), 4(prm1)-tetrachlorosalicylanilide (TCS), and, for BRI 6011, by anaerobiosis unless nitrate was present, thus indicating that energy was required for the uptake process. Uptake of 2,4-DCBA by BRI 6011 was constitutive and saturation uptake kinetics were not observed. Uptake of 2,4-DCBA by BRI 6011 was inhibited by KCN, TCS, and anaerobiosis even if nitrate was present, but the compound was not accumulated intracellularly against a concentration gradient. Uptake of 2,4-DCBA by BRI 6011 appears to occur by passive diffusion into the cell down its concentration gradient, which is maintained by the intracellular metabolism of the compound. This process could play an important role in the degradation of xenobiotic compounds by microorganisms.