The GABA transporter and its inhibitors

GABA is the major inhibitory neurotransmitter in the brain and GABA re- uptake from the synaptic cleft is one important mechanism in the regulation of GABA activity. Inhibition of the re-uptake of GABA by potent and selective inhibitors of the GABA transporter enhances GABA activity. This property can be used therapeutically in for instance epilepsy or psychiatric disorders. In this paper putative structures of the GABA transporter, its mechanism of action, the progress made in the search for its amino acids involved in the binding of substrates and the SAR of inhibitors acting at the transporter will be discussed. To date only highly selective GAT-1 inhibitors are available. These compounds are lipophilic derivatives of (R)-nipecotic acid and guvacine. The most potent inhibitors of the cloned human GAT-1 are NNC-711 (IC50 = 0.04 mM) and tiagabine (IC50 = 0.07 mM). A diheteroarylvinyloxy analogue of tiagabine, 5 times more potent than tiagabine, has been reported recently. For the GAT-2, GAT-3 and BGT-1 subtypes only compounds with a small preference for one of the subtypes have been published.     

Action of bicyclic isoxazole GABA analogues on GABA transporters and its relation to anticonvulsant activity.

The inhibitory action of bicyclic isoxazole gamma-aminobutyric acid (GABA) analogues and their 4,4-diphenyl-3-butenyl (DPB) substituted derivatives has been investigated in cortical neurones and astrocytes as well as in human embryonic kidney (HEK 293) cells transiently expressing either mouse GABA transporter-1 (GAT-1), GAT-2, -3 or -4. It was found that 4,5,6,7-tetrahydroisoxazolo(4,5-c)pyridin-3-ol (THPO) and 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-c]azepin-3-ol (THAO) displayed some inhibitory activity on GAT-1 and GAT-2, where the compounds exhibited a slightly lower potency on GAT-2 compared to GAT-1. DPB substituted THPO displayed higher inhibitory potency than the parent compound regarding the ability to inhibit GABA uptake via GAT-1 and GAT-2. Concerning the inhibitory mechanism, THPO, THAO and DPB-THPO were competitive inhibitors on GAT-1 transfected HEK 293 cells and the same mechanism was observed for THPO in GAT-3 transfected cells. Regarding GABA uptake into neurones and astroglia cells THAO and DPB-THAO both displayed competitive inhibitory action. The observations that THPO, THAO as well as their DPB derivatives act as competitive inhibitors together with earlier findings such as potent anticonvulsant activity, lack of proconvulsant activity and the ability of THPO to increase extracellular GABA concentration, indicate that these bicyclic isoxazole GABA analogues and their DPB derivatives may be useful lead structures in future search for new antiepileptic drugs.     

Intramolecular cross-linking in a bacterial homolog of mammalian SLC6 neurotransmitter transporters suggests an evolutionary conserved role of transmembrane segments 7 and 8

The extracellular concentration of the neurotransmitters dopamine, serotonin, norepinephrine, GABA and glycine is tightly controlled by plasma membrane transporters belonging to the SLC6 gene family. A very large number of putative transport proteins with a remarkable homology to the SLC6 transporters has recently been identified in prokaryotes. Here we have probed structural relationships in a 'microdoman' corresponding to the extracellular ends of transmembrane segments (TM) 7 and 8 in one of these homologs, the tryptophan transporter TnaT from Symbiobacterium thermophilum. We found that simultaneous - but not individual - substitution of Ala286 at the top of TM7 and Met311 at the top of TM8 with cysteines conferred sensitivity to submicromolar concentrations of Hg(2+) as assessed in a [(3)H]tryptophan uptake assay. Because Hg(2+) can cross-link pairs of cysteines, this suggests close proximity between TM 7 and 8 in the tertiary structure of TnaT as previously suggested for the mammalian counterparts. Furthermore, the inhibition of uptake upon cross-linking the two cysteines provides indirect support for a conserved conformational role of these transmembrane domains in the transport process. It was not possible, however, to transfer to TnaT binding sites for another metal ion, Zn(2+), that we previously engineered in the dopamine (DAT) and GABA (GAT-1) transporters between TM 7 and 8. This suggests that the structure of the TM7/8 microdomain is not identical with that of DAT and GAT-1. Hence, our data also emphasize possible structural differences that should be taken into account when interpreting future data on bacterial homologs of the SLC6 transporters.     

GABA transporter type 1 (GAT-1) uptake inhibition reduces stimulated aspartate and glutamate release in the dorsal spinal cord in vivo via different GABAergic mechanisms

Mechanisms through which the reported antinociceptive activity of GABA re-uptake inhibitors is mediated (and where on the sensory neuraxis) have not been defined. Here, microdialysis in the anaesthetised rat was used to examine the effect of selective GABA transporter type 1 (GAT-1) inhibition on basal and evoked amino acid release in the dorsal spinal cord. Reverse dialysis of the selective GAT-1 inhibitor NO-711 (10–300 μM) induced a concentration-related increase in extracellular GABA (maximal threefold of basal levels) without affecting other amino acids. Employing an S2/S1 paradigm, release evoked by brief high (45 mM) K⁺-induced depolarisation of aspartate and glutamate, but not GABA or glycine, was found to be significantly reduced by reverse dialysis of NO-711 (300 μM). Co-administration of selective antagonists for GABA_A or GABA_B receptors ((+)-bicuculline (100 μM) or SCH 50911 (100 μM), respectively) prevented the GAT-1 inhibition-induced reduction of evoked aspartate. In contrast, while (+)-bicuculline also antagonised the reduction of evoked glutamate, SCH 50911 (up to 1 mM) was without effect. Inhibition of GAT-1 re-uptake was further found to play a permissive role in autoinhibitory effects on GABA release mediated through GABA_A and GABA_B receptors. These data demonstrate that augmentation of GABAergic neurotransmission by re-uptake inhibition activates pharmacologically distinguishable inhibitory influences on aspartate and glutamate release in the dorsal spinal cord. Thus, inhibition of spinal pro-nociceptive neurotransmitter release may contribute to the analgesic action of this drug class.     

The effects of amino acids and antagonists on the isolated hemisected spinal cord of the immature rat.

1 Records of ventral and dorsal root polarity of the isolated hemisected spinal cord of the 3-9 day old rat showed that respective dose-dependent depolarizations of motoneurones (VR responses) and primary afferent terminals (DR responses) were produced by both acidic and neutral amino acids in the presence of procaine (1 mM) or tetrodotoxin (0.1 micron). 2 Of the four neutral amino acids, gamma-aminobutyrate (GABA), glycine, taurine, and beta-alanine, GABA was the most effective in producing DR responses and glycine the most effective in producing VR responses. Only taurine depressed the electrical activity recorded from ventral roots. 3 The DR responses produced by GABA, beta-alanine and taurine were all antagonized by bicuculline (5 micron) and picrotoxin (5 micron). Bicuculline was more selective than picrotoxin in antagonizing VR responses produced by GABA. 4 Strychnine (1 micron) antagonized VR responses produced by glycine beta-alanine and taurine without affecting responses produced by GABA. DR responses to the neutral amino acids were unaffected by strychnine.     

A novel antagonist, phenylbenzene omega-phosphono-alpha-amino acid, for strychnine-sensitive glycine receptors in the rat spinal cord.

1. 3-[2'-Phosphonomethyl[1,1'-biphenyl]-3-yl]alanine (PMBA) is a novel glycine antagonist at strychnine-sensitive receptors. The chemical structure of PMBA, possessing both a glycine moiety and a phosphono group, is quite different from that of strychnine. 2. In the spinal motoneurone of newborn rats, glycine (100 microM-1 mM) induced depolarizing responses in a concentration-dependent manner. PMBA effectively inhibited depolarizing responses to glycine and other agonists, such as taurine and beta-alanine. The dose-response curves for glycine were shifted to the right in an almost parallel manner (pA2 value: 5.30 +/- 0.23, n = 5) by PMBA which was about 60 times less potent than strychnine (pA2 value: 7.08 +/- 0.21, n = 5) as a glycine antagonist. 3. PMBA (1-100 microM) did not interact with modulatory glycine sites on N-methyl-D-aspartate (NMDA) receptors, which suggests a high selectivity of PMBA for strychnine-sensitive glycine receptors. At considerably high concentrations (0.1 mM-1 mM), PMBA depressed responses to GABA (pA2 value: 3.57 +/- 0.24, n = 3). 4. PMBA inhibited the binding of [3H]-strychnine to synaptosomes from adult rat spinal cords; the IC50 values of PMBA, glycine and strychnine were 8 +/- 2, 9 +/- 3 and 0.08 +/- 0.04 microM, respectively (n = 5) for [3H]-strychnine (4.8 nM). 5. PMBA is a central excitant drug with relatively high potency and selectivity and should be useful as a pharmacological probe for analysing the mechanisms underlying physiological functions of glycine receptors.     

异丙肾上腺素导致的心肌损伤大鼠心肌牛磺酸转运障碍和牛磺酸防治作用

目的:观察异丙肾上腺素(ISO)导致心肌损伤大鼠的心肌组织牛磺酸跨膜转运功能及牛磺酸转运体(TAUT)和半胱氨酸亚磺酸脱羧酶(CSD)的改变.方法:用[~3H]标记的牛磺酸测定心肌牛磺酸转运及其与心肌浆膜结合功能;竞争性RT-PCR测定TAUT和CSD mRNA的含量.结果:ISO组与对照组比,心肌[~3H]-牛磺酸摄入减少,释放量增加,心肌浆膜最大结合速率(B_(max))减少,TAUT和CSD mRNA水平减少.牛磺酸治疗组与ISO组比,心肌牛磺酸摄入量明显增加,释放量减少,B_(max)值增加,TAUT和CSDmRNA含量增加,各组间K_d值无明显差异.结论:ISO诱导的心肌损伤存在牛磺酸转运障碍及TAUT和CSD基因的下调,外源性给予牛磺酸对心肌损伤有明显防治作用.     

Depolarizing actions of γ-aminobutyric acid and related compounds on rat superior cervical ganglia in vitro

1 Potential changes in rat superior cervical ganglia were recorded in vitro with surface electrodes.2 gamma-aminobutyric acid (GABA) produced a transient, low-amplitude ganglion depolarization at rest, and a transient hyperpolarization in ganglia depolarized by carbachol. Depolarization was not prevented by preganglionic denervation. The log dose-response curve for depolarization was sigmoid with a mean ED(50) of 12.5 muM.3 The ganglion was depolarized in similar manner by the following compounds (mean molar potencies relative to GABA (=1) in brackets): 3-aminopropane sulphonic acid (3.4), gamma-amino-beta-hydroxybutyric acid (0.27), beta-guanidino-propionic acid (0.12), guanidinoacetic acid (0.057), delta-aminovaleric acid (0.048), beta-alanine (0.01), 2,4-diaminobutyric acid, gamma-guanidinobutyric acid, taurine and N-methyl-GABA (all <0.01). The following compounds did not depolarize the ganglion at 10 mM concentrations: alpha- and beta-amino-n-butyric acids, alpha-amino-iso-butyric acid, glycine and glutamic acid.4 Depolarization declined in the continued presence of GABA. Ganglia thus ;desensitized' to GABA showed a diminished response to other amino acids but not to carbachol.5 The effect of GABA was not antagonized by hyoscine and hexamethonium in combination, in concentrations sufficient to block responses to carbachol.6 Responses to GABA were blocked more readily than those to carbachol by bicuculline (IC(50), 14 muM) and picrotoxin (IC(50), 37 muM). Strychnine (IC(50), 73 muM) was a relatively weak and less selective GABA-antagonist.7 It is concluded that sympathetic ganglion cells possess receptors for GABA and related amino acids which are (a) different from the acetylcholine receptors and (b) similar to GABA receptors in the central nervous system.     

The role of glycine residues in the function of human organic anion transporter 4

Human organic anion transporter 4 (hOAT4) belongs to a superfamily of organic ion transporters that play critical roles in the body disposition of clinically important drugs, including anti-HIV therapeutics, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. In this study, we investigated the role of conserved glycine residues in hOAT4 function. We mutagenized each of the six glycine residues (at positions 11, 241, 383, 388, 400, and 466) to serine, and their functional properties were analyzed in COS-7 cells by measuring the uptake of [(3)H]estrone sulfate. Our results showed that mutants G11S, G383S, G388S, and G466S exhibited transport activities comparable with those of wild-type hOAT4. In contrast, mutants G241S and G400S almost completely lost transport function. We then further characterized Gly-241 and Gly-400 by mutagenizing these residues to amino acids with varying sizes of side chains, including alanine, valine, and leucine. We demonstrated that increasingly larger side chains at positions 241 and 400 increasingly impaired hOAT4 function. Cell-surface biotinylation using an impermeant biotinylating reagent showed that mutations of Gly-241 and Gly-400 interfered with the trafficking of the transporter onto cell surface. Immunofluorescence analysis of mutant-transfected cells confirmed these results. Substitutions of amino acids with large side chains at positions 241 and 400 resulted in decreased V(max) and increased K(m.) These results suggest that Gly-241 and Gly-400 are important both in targeting the transporter to the plasma membrane and in substrate binding. This is the first identification and characterization of critical amino acid residues in hOAT4 and may provide important insights into the structure-function relationships of the organic ion transporter family.     

Glycinergic inhibition in thalamus revealed by synaptic receptor blockade

Using juvenile rat brain slices, we examined the possibility that strychnine-sensitive receptors for glycine-like amino acids contributed to synaptic inhibition in ventrobasal thalamus, where gamma-aminobutyrate (GABA) is the prevalent inhibitory transmitter. Ventrobasal nuclei showed staining for antibodies against alpha1 and alpha2 subunits of the glycine receptor. Exogenously applied glycine, taurine and beta-alanine increased membrane conductance, effects antagonized by strychnine, indicative of functional glycine receptors. Using glutamate receptor antagonists, we isolated inhibitory postsynaptic potentials and currents (IPSPs and IPSCs) evoked by high-threshold stimulation of medial lemniscus. Like the responses to glycine agonists, these synaptic responses reversed near E(Cl). In comparative tests with GABA receptor antagonists, strychnine attenuated inhibition in a majority of neurons, but did not alter slow, GABA(B) inhibition. For complete blockade, the majority of fast IPSPs required co-application of strychnine with bicuculline or gabazine, GABA(A) receptor antagonists. Strychnine acting with an IC50 approximately = 33 nM, eliminated residual fast inhibition during selective GABA(A) receptor blockade with gabazine. The latency of onset for IPSPs was compatible with polysynaptic pathways or prolonged axonal propagation time. Strychnine lacked effects on monosynaptic, GABAergic IPSPs from zona incerta. The specific actions of strychnine implicated a glycine receptor contribution to fast inhibition in somatosensory thalamus.     

Glycine 154 of the equilibrative nucleoside transporter, hENT1, is important for nucleoside transport and for conferring sensitivity to the inhibitors nitrobenzylthioinosine, dipyridamole, and dilazep

hENT1 and hENT2 are members of the human equilibrative nucleoside transporter family. hENT1 is ubiquitously expressed and plays an important role in the disposition and pharmacological activity of nucleoside drugs and nucleosides, such as adenosine. hENT2 is expressed in only a few tissues (e.g. muscle). hENT1 and hENT2 differ in their affinity for nucleoside substrates and in their sensitivity to inhibitors, such as nitrobenzylthioinosine (NBMPR). hENT1 has higher (or equal) affinity to hENT2 for all natural nucleosides except inosine. hENT1 is also more sensitive to NBMPR inhibition (IC50 approximately 0.4-8 nM) when compared with hENT2 (IC50 approximately 2.8 microM). This difference in inhibition potency is substantially dependent on the difference in amino acid at position 154 in hENT1 (glycine) and hENT2 (serine). Since NBMPR competitively inhibits nucleoside transporter activity, we hypothesized that G154 may also play a role in the transport of natural nucleosides and in the inhibition by other hENT1 inhibitors, dipyridamole (DP), and dilazep (DZ). Our results, using a yeast expression system, demonstrate that substituting glycine 154 of hENT1 with serine of hENT2 converts hENT1 to a transporter that exhibits partial characteristics of hENT2. For example, this conversion reduces sensitivity of hENT1 to the inhibitors NBMPR, DP, and DZ and reduces its transport affinity for the natural nucleosides cytidine and adenosine. However, this conversion renders hENT1 less sensitive to inhibition by anti-HIV drugs azidothymidine, dideoxyinosine, and the nucleobase, hypoxanthine. Collectively, these results suggest that glycine 154 plays an important role in the transport of nucleosides and in sensitivity to the inhibitors NBMPR, DP, and DZ.     

Effects of depressant amino acids and antagonists on an in vitro spinal cord preparation from the adult rat

A mature sacrococcygeal in vitro spinal preparation from the rat has been used to demonstrate effects of neutral amino acids and their antagonists. gamma-Aminobutanoate (GABA), glycine and taurine (0.5-5 mM) produced dose-dependent depression of spontaneous paroxysmal activity generated in Mg2+ -free medium. The depressant effect of GABA was antagonised selectively by picrotoxin (25-50 microM) and the depressant effects of glycine and taurine were antagonised selectively by strychnine (0.2 microM). Glycine (0.5-5 mM) had a dose-dependent depolarizing action which was present at the central ends of isolated ventral roots. gamma-Aminobutanoate and taurine, had only weak depolarizing actions on ventral root fibres. Depolarizing responses to glycine showed a marked fading. Reduction in the fading appeared to be responsible for a paradoxical potentiation of glycine-induced depolarizations, which occurred in the presence of strychnine (0.2-2 microM). Strychnine (2-10 microM), picrotoxin (10-50 microM) or bicuculline (10 microM) had little or no effect on the amplitude, duration or latency of the monosynaptic component of ventral root reflexes evoked by supramaximal stimulation of dorsal roots (DR-VRP). However all three antagonists introduced slow, NMDA receptor mediated, components to these ventral root potentials. Picrotoxin and bicuculline, but not strychnine, reversibly depressed the dorsal root potential evoked from an adjacent dorsal root (DR-DRP). The depressant actions of 2-amino-5-phosphonopentanoate (AP5), kynurenate and 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) revealed both NMDA and non-NMDA receptor mediated components in the dorsal root potential.     

Hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells

We have reported previously that taurine transporter (TauT) mediates γ-aminobutyric acid (GABA) as a substrate in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells). This study investigates how TauT-mediated GABA transport is regulated in TR-iBRB2 cells under hypertonic conditions. [3H]GABA uptake by TR-iBRB2 cells exposed to 12 h- to 24 h-hypertonic culture medium was significantly greater than that of isotonic culture medium. [3H]GABA uptake by TR-iBRB2 cells was Na(+)-, Cl(-)-, and concentration-dependent with a Michaelis-Menten (K(m)) constant of 3.5 mM under isotonic conditions and K(m) of 0.324 and 5.48 mM under hypertonic conditions. Under hypertonic conditions, [3H]GABA uptake by TR-iBRB2 cells was more potently inhibited by substrates of TauT, such as taurine and β-alanine, than those of GABA transporters such as GABA, nipecotic acid, and betaine. These results suggest that an unknown high-affinity GABA transport process and TauT-mediated GABA transport are enhanced under hypertonic conditions. In conclusion, hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells.     

Novel secoergoline derivatives inhibit both GABA and glutamate uptake in rat brain homogenates: synthesis, in vitro pharmacology, and modeling

Three of twelve secoergoline derivatives (Z ethyl 4-[(ethoxycarbonylmethyl)methylamino]-2-methyl-3-phenylpent-2-enoate, 8; ethyl 1,6-dimethyl-3-oxo-5-phenyl-1,2,3,6-tetrahydropyridine-2-carboxylate, 9; Z methyl 4-[(methoxycarbonylmethyl)methylamino)-2-methyl-3-phenylpent-2-enoate, 11), containing bioisosteric sequences of GABA and Glu, inhibited both GABA and Glu transport through cerebrocortical membranes specifically. Compounds 8, 9, and 11 appeared to be equipotent inhibitors of GABA and Glu transport with IC50 values between 270 and 1100 microM, whereas derivatives 1-7, 10, and 12 were without effects. In the presence of GABA and Glu transport-specific nontransportable inhibitors, inhibition of GABA and Glu transport by 8, 9, and 11 proceeded in two phases. The two phases of inhibition were characterized by IC50 values between 4 and 180 nM and 360-1020 microM and different selectivity sequences. These findings may indicate the existence of some mechanism possibly mediated by a previously unrecognized GABA-Glu transporter. Derivatives with the cis, but not the trans configuration of bulky ester groups (8 vs 7 and 11 vs 12) showed significant inhibitory effect (IC50 values of 270 microM vs >1000 microM and 1100 microM vs >1000 microM on GABA transport, respectively). The cis-trans selectivity can be explained by docking these secoergolines in a three-dimensional model of the second and third transmembrane helices of GABA transporter type 1.     

Anticonvulsants but not general anesthetics have differential blocking effects on different T-type current variants

The sensitivity to anticonvulsants and anesthetics of Ca(2+) currents arising from alpha1G and alpha1H subunits was examined in stably transfected HEK293 cells. For comparison, in some cases blocking effects on dorsal root ganglion (DRG) T currents were also examined under identical ionic conditions. The anticonvulsant, phenytoin, which partially blocks DRG T current, blocked alpha1G current completely but with weaker affinity ( approximately 140 microM). Among different cells, alpha1H current exhibited either of two responses to phenytoin. In one subpopulation of cells, phenytoin produced a partial, higher affinity block (IC(50) approximately 7.2 microM, maximum block approximately 43%) similar to that in DRG neurons. In other cells, phenytoin produced complete, but lower affinity, blockade (IC(50) approximately 138 microM, maximum block approximately 89%). Another anticonvulsant, alpha-methyl-alpha-phenylsuccinimide (MPS), blocked DRG current partially, but blocked both alpha1G and alpha1H currents completely with weaker affinity ( approximately 1.7 mM). These data suggest that higher affinity blockade of T-type currents by phenytoin and MPS may require additional regulatory factors that can contribute to native T-type channels. In contrast, anesthetics blocked all T current variants similarly and completely. Block of alpha1G current by anesthetics had the following order of potency: propofol (IC(50) approximately 20.5 microM) > etomidate ( approximately 161 microM) = octanol ( approximately 160 microM) > isoflurane ( approximately 277 microM) > ketamine ( approximately 1.2 mM), comparable with results on DRG T currents. Barbiturates completly blocked alpha1G currents with potency [thiopental ( approximately 280 microM), pentobarbital ( approximately 310 microM), phenobarbital ( approximately 1.54 mM)] similar to that in DRG cells. The effects of propofol, octanol, and pentobarbital on alpha1H currents were indistinguishable from effects on alpha1G currents.     

Synthesis of novel GABA uptake inhibitors. 3. Diaryloxime and diarylvinyl ether derivatives of nipecotic acid and guvacine as anticonvulsant agents.

(3R)-1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-piperidinecarboxylic acid 1 (tiagabine, Gabitril) is a potent and selective gamma-aminobutyric acid (GABA) uptake inhibitor with proven anticonvulsant efficacy in humans. This drug, which has a unique mechanism of action among marketed anticonvulsant agents, has been launched for add-on treatment of partial seizures with or without secondary generalization in patients >12 years of age. Using this new agent as a benchmark, we have designed two series of novel GABA uptake inhibitors of remarkable potency, using a putative new model of ligand interaction at the GABA transporter type 1 (GAT-1) uptake site. This model involves the postulated interaction of an electronegative region in the GABA uptake inhibitor with a positively charged domain in the protein structure of the GAT-1 site. These two novel series of anticonvulsant agents contain diaryloxime or diarylvinyl ether functionalities linked to cyclic amino acid moieties and were derived utilizing the new model, via a series of design steps from the known 4,4-diarylbutenyl GABA uptake inhibitors. The new compounds are potent inhibitors of [(3)H]-GABA uptake in rat brain synaptosomes in vitro, and their antiepileptic potential was demonstrated in vivo by their ability to protect against seizures induced by the benzodiazepine receptor inverse agonist methyl 4-ethyl-6,7-dimethoxy-beta-carboline-3-carboxylate (DMCM) in mice. From structure-activity studies of these new GABA uptake inhibitors, we have shown that insertion of an ether oxygen in conjugation with the double bond in tiagabine (K(i) = 67 nM) improves in vitro potency by 5-fold to 14 nM.     

Development of tolerance to the effects of vigabatrin (gamma-vinyl-GABA) on GABA release from rat cerebral cortex, spinal cord and retina.

1. The effects of acute and chronic vigabatrin (gamma-vinyl-GABA) (GVG) administration on gamma-aminobutyric acid (GABA) levels and release in rat cortical slices, spinal cord slices and retinas were studied. 2. GVG (250 mgkg-1 i.p.) administered to rats 18 h before death (acute administration) produced an almost 3 fold increase in GABA levels of the cortex and spinal cord and a 6 fold increase in retinal GABA. The levels of glutamate, aspartate, glycine and taurine were unaffected. 3. When GVG (250 mgkg-1 i.p.) was administered daily for 17 days (chronic administration) a similar (almost 3 fold) increase in cortical GABA occurred but the increases in spinal and retinal GABA were reduced by approximately 40%. 4. Acute administration of GVG strikingly increased the potassium-evoked release (KCl 50 mM) of GABA from all three tissues. This enhanced evoked release was reduced by about 50% in tissues taken from rats that had been chronically treated with GVG. 5. Acute administration of GVG reduced GABA-transaminase (GABA-T) activity by approximately 80% in cortex and cord and by 98% in the retina. Following the chronic administration of GVG, there was a trend for GABA-T activities to recover (significant only in cortex). Acute administration of GVG had no effect on glutamic acid decarboxylase (GAD) activity in cortex or spinal cord. However, chronic treatment resulted in significant decreases in GAD activity in both the cortex and cord (35% and 50% reduction respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of two taurine receptor subtypes on the primary afferent terminal of frog spinal cord.

1. Effects of taurine on primary afferent terminals in the frog spinal cord were examined by a sucrose-gap method applied to a dorsal root (9th or 10th segment). 2. In a normal Ringer solution, taurine (1 mM, applied for 5s at a rate of 0.04 ml s-1, 0.2 mumol) caused a hyperpolarization, but a higher concentration (10 mM, applied at the same rate, 2.0 mumol) caused a biphasic response consisting of a hyperpolarization followed by a slow onset depolarization. A similar biphasic response could also be observed in tetrodotoxin-treated preparations. 3. When the concentration of extracellular Mg2+ was increased up to 9.0 mM, the depolarizing response to taurine was augmented. The rate of the augmentation was dependent upon the extracellular Mg2+ concentration. 4. The depolarizing effect was selectively antagonized by bicuculline in concentrations (10-30 microM) that had no significant antagonizing action on gamma-aminobutyric acid (GABA)-induced depolarization. On the other hand the hyperpolarizing effect of taurine was selectively reduced by strychnine (0.1 microM) which had no antagonizing effect on responses to glycine. 5. These results suggest that in the frog spinal cord there are at least two subtypes of taurine receptor whose pharmacological profiles resemble GABA and glycine receptors in the mammalian central nervous system, and whose sensitivity may be modulated by extracellular Mg2+.     

N-酰基-1-苄基四氢异喹啉衍生物的合成和GABA转运蛋白抑制作用

目的设计合成N-酰基-1．（3，4-二甲氧基）苄基-6，7-二甲氧基-1，2，3，4-四氢异喹啉类化合物。评价其抑制GABA转运蛋白对GABA的摄取作用。方法经核磁共振氢谱、质谱、高分辨质谱和红外光谱确证目标物的结构，并经体外GAT-1竞争抑制性结合试验进行活性筛选。结果与结论合成了4个目标化合物，初步活性试验表明，化合物1c和1d对GAT-1的抑制作用要大于阳性对照药（R）-3-哌啶甲酸，显示较强的GAT-1抑制作用。     

Guanidinoethyl sulphonate is a glycine receptor antagonist in striatum

1. Guanidinoethyl sulphonate (GES) is an analogue of taurine and an inhibitor of taurine transport. Interactions of GES with GABA(A) and glycine receptors are studied by whole cell recording and fast drug application in isolated striatal neurons of the mouse. 2. We confirm that GES is a weak agonist at GABA(A) receptors, and is able to antagonize GABA-evoked responses. GES did not gate GlyR. 3. GES antagonized glycine responses in a concentration-dependent and surmountable manner. Glycine dose-response curves were shifted to the right by GES (0.5 mM), yielding EC(50)s and Hill coefficients of 62 micro M and 2.5 in control, 154 micro M and 1.3 in the presence of GES. 4. GlyR-mediated taurine responses were competitively antagonized by GES. Taurine dose-response curves, in contrast to the glycine dose-response curves were shifted by GES to the right in a parallel manner. 5. The GlyR-block by GES was not voltage-dependent. 6. In contrast to our findings in the mouse, in rat striatal neurons which lack expression of the alpha3 GlyR subunit, GES shifted the glycine dose-response curve to the right in a parallel way without affecting the maximal response. Subtype-specificity of the GES action at GlyR must await further investigation in artificial expression systems. 7. We conclude that GES is a competitive antagonist at GlyR. The antagonistic action of GES at inhibitory ionotropic receptors can explain its epileptogenic action. Care must be taken with the interpretation of data on GES evoked taurine release.