Effect of hyperosmotic conditions on the expression of the betaine-GABA-transporter (BGT-1) in cultured mouse astrocytes.

The adaptation of cells to hyperosmotic conditions involves accumulation of organic osmolytes to achieve osmotic equilibrium and maintenance of cell volume. The Na+ and Cl(-)-coupled betaine/GABA transporter, designated BGT-1, is responsible for the cellular accumulation of betaine and has been proposed to play a role in osmoregulation in the brain. BGT-1 is also called GAT2 (GABA transporter 2) when referring to the mouse transporter homologue. Using Western Blotting the expression of the mouse GAT2 protein was investigated in astrocyte primary cultures exposed to a growth medium made hyperosmotic (353+/-2.5 mosmol/kg) by adding sodium chloride. A polyclonal anti-BGT-1 antibody revealed the presence of two characteristic bands at 69 and 138 kDa. When astrocytes were grown for 24 h under hyperosmotic conditions GAT2 protein was up-regulated 2-4-fold compared to the level of the isotonic control. Furthermore, the expected dimer of GAT2 was also up-regulated after 24 h under the hyperosmotic conditions. The [3H]GABA uptake was examined in the hyperosmotic treated astrocytes, and characterized using different selective GABA transport inhibitors. The up-regulation of GAT2 protein was not affecting total GABA uptake but the hyperosmotic condition did change total GABA uptake possibly involving GAT1. Immunocytochemical studies revealed cell membrane localization of GAT2 throughout astroglial processes. Taken together, these results indicate that astroglial GAT2 expression and function may be regulated by hyperosmolarity in cultured mouse astrocytes, suggesting a role of GAT2 in osmoregulation in neural cells.

Selective inhibitors of GABA uptake: synthesis and molecular pharmacology of 4-N-methylamino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol analogues.

A series of lipophilic diaromatic derivatives of the glia-selective GABA uptake inhibitor (R)-4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol [(R)-exo-THPO, 4] were synthesized via reductive amination of 3-ethoxy-4,5,6,7-tetrahydrobenzo[d]isoxazol-4-one (9) or via N-alkylation of O-alkylatedracemic 4. The effects of the target compounds on GABA uptake mechanisms in vitro were measured using a rat brain synaptosomal preparation or primary cultures of mouse cortical neurons and glia cells (astrocytes), as well as HEK cells transfected with cloned mouse GABA transporter subtypes (GAT1-4). The activity against isoniazid-induced convulsions in mice after subcutaneous administration of the compounds was determined. All of the compounds were potent inhibitors of synaptosomal uptake the most potent compound being (RS)-4-[N-(1,1-diphenylbut-1-en-4-yl)amino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (17a, IC50 = 0.14 microM). The majority of the compounds showed a weak preference for glial, as compared to neuronal, GABA uptake. The highest degree of selectivity was 10-fold corresponding to the glia selectivity of (R)-N-methyl-exo-THPO (5). All derivatives showed a preference for the GAT1 transporter, as compared with GAT2-4, with the exception of (RS)-4-[N-[1,1-bis(3-methyl-2-thienyl)but-1-en-4-yl]-N-methylamino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (28d), which quite surprisingly turned out to be more potent than GABA at both GAT1 and GAT2 subtypes. The GAT1 activity was shown to reside in (R)-28d whereas (R)-28d and (S)-28d contributed equally to GAT2 activity. This makes (S)-28d a GAT2 selective compound, and (R)-28d equally effective in inhibition of GAT1 and GAT2 mediated GABA transport. All compounds tested were effective as anticonvulsant reflecting that these compounds have blood-brain barrier permeating ability.

First demonstration of a functional role for central nervous system betaine/{gamma}-aminobutyric acid transporter (mGAT2) based on synergistic anticonvulsant action among inhibitors of mGAT1 and mGAT2.

In a recent study, EF1502 [N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo [d]isoxazol-3-ol], which is an N-substituted analog of the GAT1-selective GABA uptake inhibitor exo-THPO (4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol), was found to inhibit GABA transport mediated by both GAT1 and GAT2 in human embryonic kidney (HEK) cells expressing the mouse GABA transporters GAT1 to 4 (mGAT1-4). In the present study, EF1502 was found to possess a broad-spectrum anticonvulsant profile in animal models of generalized and partial epilepsy. When EF1502 was tested in combination with the clinically effective GAT1-selective inhibitor tiagabine [(R)-N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid] or LU-32-176B [N-[4,4-bis(4-fluorophenyl)-butyl]-3-hydroxy-4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol], another GAT1-selective N-substituted analog of exo-THPO, a synergistic rather than additive anticonvulsant interaction was observed in the Frings audiogenic seizure-susceptible mouse and the pentylenetetrazol seizure threshold test. In contrast, combination of the two mGAT1-selective inhibitors, tiagabine and LU-32-176B, resulted in only an additive anticonvulsant effect. Importantly, the combination of EF1502 and tiagabine did not result in a greater than additive effect in the rotarod behavioral impairment test. In subsequent in vitro studies conducted in HEK-293 cells expressing the cloned mouse GAT transporters mGAT1 and mGAT2, EF1502 was found to noncompetitively inhibit both mGAT1 and the betaine/GABA transporter mGAT2 (K(i) of 4 and 5 muM, respectively). Furthermore, in a GABA release study conducted in neocortical neurons, EF1502 did not act as a substrate for the GABA carrier. Collectively, these findings support a functional role for mGAT2 in the control of neuronal excitability and suggest a possible utility for mGAT2-selective inhibitors in the treatment of epilepsy.

Role of the betaine/GABA transporter (BGT-1/GAT2) for the control of epilepsy.

Inactivation of gamma-aminobutric acid (GABA) as a neurotransmitter is mediated by diffusion in the synaptic cleft followed by binding to transporter sites and translocation into the intracellular compartment. The GABA transporters of which four subtypes have been cloned (GAT1-4) are distributed at presynaptic nerve endings as well as extrasynaptically on astrocytic and neuronal elements. This anatomical arrangement of the transporters appears to be of critical functional importance for the maintenance of GABAergic neurotransmission. Pharmacological characterization of the GABA transporters using a large number of GABA analogs having restricted conformation and lipophilic character has been of instrumental importance for elucidation of the functional importance of the different transporters. One such analog EF1502 (N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-methylamino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol) has been shown to selectively inhibit GAT1 (GABA transporter 1) and GAT2/BGT-1 (betaine/GABA transporter). Moreover, this GABA analog exhibits an unusually high efficiency as an anticonvulsant suggesting a novel role of the betaine/GABA transporter in epileptic seizure control. It is hypothesized that extrasynaptic actions of GABA may be involved in this phenomenon.

GABA transporters as drug targets for modulation of GABAergic activity.

The identification and subsequent development of the GABA transport inhibitor tiagabine has confirmed the important role that GABA transporters play in the control of CNS excitability. Tiagabine was later demonstrated to be a selective inhibitor of the GABA transporter GAT1. Although selective for GAT1, tiagabine lacks cell type selectivity and is an equipotent inhibitor of neuronal and glial GAT1. To date, four GABA transporters have been cloned, i.e., GAT1-4. The finding that some of these display differential cellular and regional expression patterns suggests that drugs targeting GABA transporters other than GAT1 might offer some therapeutic advantage over GAT1 selective inhibitors. Furthermore, it is particularly interesting that several recently defined GABA transport inhibitors have been demonstrated to display a preferential selectivity for the astrocytic GAT1 transporter. That cellular heterogeneity of GAT1 plays a role in the control of CNS function is confirmed by the demonstration that inhibition of astrocytic GABA uptake is highly correlated to anticonvulsant activity. At the present time, a functional role for the other GABA transporters is less well defined. However, recent findings have suggested a role for the mouse GAT2 (homologous to the human betaine transporter) in the control of seizure activity. In these studies, the non-selective GAT1 and mouse GAT2 transport inhibitor EF1502 (N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol) was found to exert a synergistic anticonvulsant action when tested in combination with the GAT1 selective inhibitors tiagabine and LU-32-176B (N-[4,4-bis(4-fluorophenyl)-butyl]-3-hydroxy-4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol). Additional studies will be required to define a role for the other GABA transporters and to further identify the functional importance of their demonstrated cellular and regional heterogeneity. A summary of these and other issues are discussed in this brief review.

GABA transporters and GABA-transaminase as drug targets.

The fine-tuning and homeostatic balance of the GABAergic inhibitory tone in the central nervous system (CNS) is a prerequisite for controlling the excitatory neurotransmission. This principal mechanism for controlling excitation is inhibition which has been the topic of intensive research covering all known functional entities of the GABAergic synapse. The therapeutical scope for targeting the GABA system covers a large number of neurological and psychiatric disorders. This review focuses on the major inactivation systems for GABAergic neurotransmission, the GABA transporters (GATs) and the GABA catabolic enzyme GABA -transaminase (GABA-T) as drug targets. Tiagabin and Vigabatrin, two anti-epileptic drugs on the market today, specifically inhibit GABA transport and metabolism, respectively. However, previous and recent evidence has clearly demonstrated the importance and differential functional roles of glial and neuronal GABA uptake and the metabolic fate of the sequestered neurotransmitter GABA in these cells. Moreover, the diverse expression patterns of the GABA transporters, in combination with development of GAT inhibitors with novel pharmacological profiles may initiate a renaissance for these inactivation systems as drugs targets. In particular, further research to elucidate the specialized physiological function of the GATs combined with their differential spatial expression could be of fundamental importance for the understanding of concerted action with regard to the fine-tuning of the GABAergic inhibitory tone. As such, selective targeting and modulation of GABA transporter subtypes and cell-specific GABA uptake and metabolism is of therapeutical interest in GABA-related CNS disorders, including epilepsy.

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.

Correlation between anticonvulsant activity and inhibitory action on glial gamma-aminobutyric acid uptake of the highly selective mouse gamma-aminobutyric acid transporter 1 inhibitor 3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazole and its N-alkylated analogs.

The inhibitory effect of 3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazole (exo-THPO) and its N-methylated (N-methyl-exo-THPO) and N-ethylated (N-ethyl-exo-THPO) analogs, derived from gamma-aminobutyric acid (GABA) and 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) on GABA transport was investigated using cultured neocortical neurons (GABA-ergic) and astrocytes and cloned mouse GABA transporters GAT1-4 expressed in human embryonic kidney (HEK) 293 cells. Anticonvulsant activity was assessed after i.c.v. administration to Frings audiogenic seizure-susceptible mice. Anticonvulsant activity of the O-pivaloyloxymethyl prodrug of N-methyl-exo-THPO was assessed after i.p. administration. Results from these studies were compared with those obtained from similar studies with the novel anticonvulsant drug tiagabine, which acts via inhibition of GABA transport. exo-THPO and its N-alkyl analogs inhibited neuronal, astrocytic, and GAT1-mediated GABA transport but not GABA uptake mediated by GAT2-4. N-Methyl-exo-THPO was 8-fold more potent as an inhibitor of astrocytic versus neuronal GABA uptake. The IC(50) value for inhibition of GABA uptake by GAT1 closely reflected its IC(50) value for inhibition of neuronal uptake. Tiagabine was approximately 1000-fold more potent than exo-THPO and its alkyl derivatives as an inhibitor of GABA uptake in cultured neural cells and GAT1-expressing HEK 293 cells. exo-THPO, its alkylated analogs, and tiagabine displayed a time- and dose-dependent inhibition of audiogenic seizures after i.c.v. administration. N-Methyl-exo-THPO was the most potent anticonvulsant among the exo-THPO compounds tested and only slightly less potent than tiagabine. The findings suggest a correlation between anticonvulsant efficacy and selective inhibition of astroglial GABA uptake. Furthermore, results obtained with the N-methyl-exo-THPO prodrug demonstrate the feasibility of developing a glial-selective GABA uptake inhibitor with systemic bioavailability.