3- and 5-isoxazolol zwitterions: an ab initio molecular orbital study relating to GABA agonism and antagonism

The Hartree-Fock ab initio molecular orbital method has been applied to eight compounds: GABA (gamma-amino butyric acid) (1), its partially rigidified analog, TACA (trans-4-aminocrotonic acid) (2), six isoxazolol analogs; muscimol (5-aminomethylisoxazol-3-ol (3), THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) (4), THAZ (5,6,7,8-tetrahydro-4H-isoxazolo[4,5-d]azepin-3-ol) (5), isomuscimol (3-aminomethylisoxazol-5-ol) (6), iso-THIP (4,5,6,7-tetrahydroisoxazolo[3,4-c] pyridin-5-ol) (7), and iso-THAZ (5,6,7,8-tetrahydro-4H-isoxazolo[3,4-d]azepin-5-ol) (8). GABA is an endogenous inhibitory transmitter. The four following molecules (2), (3), (4) and (5) are agonist: they bind themselves to the GABA receptors and induce approximately the same effect as GABA. (6) is lightly agonist, presenting a lower affinity. Compounds (7) and (8) are antagonists, giving rise to convulsion. Optimized molecular conformations of GABA (1), muscimol (3) and isomuscimol (6) are discussed. Geometric and electronic parameters showing the presence of intramolecular hydrogen bonds are presented. The permutation of the heteroatoms in the isoxazole ring has no effect on the side-chain orientation explaining maybe the agonist character of isomuscimol, being able to adopt easily and exactly the active conformation. Atomic charge distributions and electronic overlap populations for all compounds have been computed in order to try to understand why their GABAergic activities can be so different. The computed values show that the 3-isoxazolol ring mimics in a good way the carboxylic function of GABA. They also illustrate the larger electronic delocalization within the 5-isoxazolol ring and therefore the resulting antagonist character, except for isomuscimol.     

Glycine Antagonists Structurally Related to 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol Inhibit Binding of [3H]Strychnine to Rat Brain Membranes

[3H]Strychnine binding to rat pons + medulla membranes was used as a measure of glycine receptors or glycine receptor-coupled chloride channels in vitro. A series of compounds structurally related to 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), which previously were shown to antagonize glycine responses in cat spinal cord, inhibited [3H]strychnine binding in micromolar concentrations. The most potent of these glycine antagonists, 5,6,7,8-tetrahydro-4H-isoxazolo[3,4-d]azepin-3-ol (iso-THAZ), was also the most potent inhibitor of [3H]strychnine binding, with a Ki of 1,400 nM. The Ki value for strychnine was 7.0 nM, whereas the Ki value for the mixed gamma-aminobutyric acid (GABA)/glycine antagonist 3 alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (RU 5135) was only 4.6 nM. Sodium chloride (1,000 mM) enhanced the affinity of strychnine, brucine, isostrychnine, and the nonselective GABA antagonist pitrazepin for [3H]strychnine binding sites, whereas the affinities of glycine, beta-alanine, and taurine were reduced. These sodium chloride shifts, however, were not predictive of antagonist or agonist properties, since the sodium chloride shift for the glycine antagonist iso-THAZ and of the other THIP-related antagonists were similar to those of the glycine-like agonists. The various sodium chloride shifts show that different groups of ligands bind to glycine receptor sites in different ways.     

STRUCTURE AND BIOLOGICAL ACTIVITY OF A SERIES OF CONFORMATIONALLY RESTRICTED ANALOGUES OF GABA

—Microelectrophoretic methods were used to study the effects on spinal neurones of a series of conformationally restricted analogues of GABA, most of which are structurally related to musci-mol (3-hydroxy-5-aminomethylisoxazole). 3-Hydroxy-5-(l-aminoethyl)isoxazole and 3-hydroxy-5-(2-aminoethyl)isoxazole were GABA-like depressants comparable in effectiveness with GABA. The inhibitors of GABA uptake 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol and nipecotic acid (piperidine-3-carboxylic acid) reversibly enhanced the depressant action of GABA. 3-Hydroxy-5-dimethylaminomethly-isoxazole, 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-d]azepm-3-ol, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol, and nipecotic acid reversibly antagonized the postsynaptic action of glycine. A structure-activity correlation was made in an indirect attempt to elucidate some comformational requirements for interaction of GABA with its postsynaptic receptor and the binding site of its uptake system. The results seem to indicate that different conformations of GABA are required for these interactions.     

Thermodynamics of Agonist and Antagonist Interaction with the Strychnine-Sensitive Glycine Receptor

The thermodynamic parameters associated with the interactions of agonists and antagonists with glycine receptors in rat spinal cord membranes were determined. The binding of the antagonist [3H]strychnine and the inhibition of strychnine binding by 11 different glycinergic ligands were examined at temperatures between 0.5 and 37 degrees C. The density of receptors was not affected by the temperature at which the incubation was performed, but the ability of glycine receptor agonists and antagonists to compete with [3H]strychnine binding varied markedly. The affinity of the receptor for the antagonists strychnine, 2-aminostrychnine, RU-5135, 5,6,7,8-tetrahydro-4H-isoxazolo[5,4-c]azepin-3-ol, and the ligands bicuculline, norharmane, and PK-8165 decreased at higher temperatures. The binding of these ligands was enthalpy-driven. In contrast, the affinity of the agonists glycine, beta-alanine, and taurine and of the antihelmintic ivermectin increased at higher temperatures, and their binding was characterized by substantial increases in entropy. In addition, temperature affected the allosteric interaction between the glycine and strychnine sites of the receptor, as indicated by changes in the Hill number of the competition curves for glycine. Our results clearly indicate that the binding of agonists and antagonists to the glycine receptor is differentially affected by temperature, probably as a consequence of the different changes induced in the receptor conformation.     

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.     

The Binding of the GAB A Agonist [3H]THIP to Rat Brain Synaptic Membranes

Abstract: THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) is a specific GABA agonist with potent analgesic properties. The binding of radioactive THIP to thoroughly washed, frozen, and thawed membranes isolated from rat brains has been studied at 2°C under sodium ion-free conditions and compared with the binding of [³H]GABA and [³H]piperidine-4-sulphonic acid ([³H]P4S). The best computer fits to the experimental data were in all cases attained with a receptor model based on three independent binding sites, of which only the high- and medium-affinity sites could be characterised satisfactorily. While the K_D values were found to be comparable for all three ligands employed, the density of the high-affinity binding site (B_M1) was, with the exception of the membranes from the cerebellum, considerably lower for [³H]THIP than for [³H]GABA and [³ H]P4S. The regional distribution of the GABA receptors, which bind [³H]THIP, was different from those recognizing [³H]GABA and [³H]P4S. A number of analogues, including asymmetric compounds with known configuration, were tested as inhibitors of the binding of [³H]GABA, [³H]muscimol, [³H]THIP, [³H]isoguvacine, and [³H]P4S. The concentrations of the asymmetric compounds required for the inhibition of [³H]P4S binding were much higher than those required for the displacement of [³H]GABA, [³H]muscimol, [³H]THIP, and [³H]isoguvacine. The comparable relative potencies of inhibitors do, however, indicate that all of the ligands bind to the GABA receptors.     

Lack of a high affinity uptake system for the GABA agonists THIP and isoguvacine in neurons and astrocytes cultured from mouse brain

The possibility that the GABA-receptor agonists isoguvacine and THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) might be taken up into brain cells via the high affinity GABA transport system was tested by incubation of cultured neurons and astrocytes in media containing either [³H]GABA, [³H]isoguvacine or [³H]THIP at different concentrations. While GABA was actively taken up into both cell types via high affinity transport mechanisms, no high affinity transport could be demonstrated for isoguvacine or THIP. Both compounds did, however, penetrate into the cells. It is concluded that isoguvacine and THIP interact with the high affinity GABA-carrier neither in neurons nor in astrocytes.     

Analogues of Azepinomycin as Inhibitors of Guanase

Abstract

Synthesis and biochemical screening against guanase of analogues of the naturally occurring guanase inhibitor azepinomycin (2) are reported. Compound e-amino-5,6,7,8,-tetrahydro-4H-imidazo[4,5-e][1,4]diazepine-5,8-dione (3) was synthesized in six steps commencing with 1-benzyl-5-nitroimidazole-4-carboxylic acid (5). Compound 3 and its synthetic precursor 3-benzyl-6-(N-benzyloxycarbonyl)amino-5,6,7,8-tetrahydro-4H-imidazo[4,5-e][1,4]diazepine-5,8-dione (12) were screened against rabbit liver guanase. Both were found to be moderate inhibitors of the enzyme with K₁′s in the range of 10^?4 M.     

Modulation of [3H]Diazepam Binding in Rat Cortical Membranes by GABAA Agonists

GABAA receptor agonists modulate [3H]diazepam binding in rat cortical membranes with different efficacies. At 23 degrees C, the relative potencies for enhancement of [3H]diazepam binding by agonists parallel their potencies in inhibiting [3H]gamma-aminobutyric acid [( 3H]GABA) binding. The agonist concentrations needed for enhancement of [3H]diazepam binding are up to 35 times higher than for [3H]GABA binding and correspond closely to the concentrations required for displacement of [3H]bicuculline methochloride (BMC) binding. The maximum enhancement of [3H]diazepam varied among agonists: muscimol = GABA greater than isoguvacine greater than 3-aminopropane sulphonic acid (3APS) = imidazoleacetic acid (IAA) greater than 4,5,6,7-tetrahydroisoxazolo (4,5,6)-pyridin-3-ol (THIP) = taurine greater than piperidine 4-sulphonic acid (P4S). At 37 degrees C, the potencies of agonists remained unchanged, but isoguvacine, 3 APS, and THIP acquired efficacies similar to GABA, whereas IAA, taurine, and P4S maintained their partial agonist profiles. At both temperatures the agonist-induced enhancement of [3H]diazepam binding was reversible by bicuculline methobromide and by the steroid GABA antagonist RU 5135. These results stress the importance of studying receptor-receptor interaction under near-physiological conditions and offer an in vitro assay that may predict the agonist status of putative GABA receptor ligands.     

Diazotization and Thiocyanate Differentiate Agonists from Antagonists for the High- and Low-Affinity Receptors of γ-Aminobutyric Acid

The differentiation of high- and low-affinity postsynaptic gamma-aminobutyric acid (GABA) receptors was examined in a washed cortical membrane preparation of the rat. The selective elimination of the high- and low-affinity GABA sites by the chaotropic anion thiocyanate and diazotization by p-diazobenzenesulfonic acid (DSA), respectively, offered two model systems for the separate sites. The [3H]GABA displacing potencies of some GABA agonists [GABA, 4,5,6,7-tetrahydro- isoxazole [4,5c]pyridine-3-ol (THIP), and muscimol] and antagonists [bicuculline methiodide (BCM), 3-alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (R-5135), and d-tubocurarine] and their slope factors were examined in these model systems and in control membranes. The displacing potency of the agonists was increased in the DSA-pretreated membranes and decreased in the presence of thiocyanate. The displacing potency of the antagonists was shifted in an opposite manner. The chaotropic effect of thiocyanate was reversible and not additive with the inhibitory effect of diazotization on the specific binding of GABA. Inhibition of specific GABA binding by pyridoxal-5-phosphate (PLP) could not be protected by GABA antagonists (BCM and R-5135) but only by agonists. The results can be interpreted in the framework of a dual (agonist-antagonist) receptor model, postulating a hydrophobic accessory site at the low-affinity GABA receptor. The effect of thiocyanate on the GABA receptor may result in the exposure of the hydrophobic accessory sites.     

INHIBITION OF GABA UPTAKE IN RAT BRAIN SLICES BY NIPECOTIC ACID, VARIOUS ISOXAZOLES AND RELATED COMPOUNDS

—A variety of isoxazoles structurally related to muscimol (3-hydroxy-5-aminomethylisoxazole) were tested as inhibitors of the uptake of GABA and some other amino acids in rat brain slices, and of the activity of the GABA-metabolizing enzymes l -glutamate 1-carboxylyase and GABA:2-oxo-glutarate aminotransferase. A bicyclic derivative, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol, proved to be a more potent inhibitor of GABA uptake than muscimol. Structure-activity studies on this derivative, which appeared to be a competitive inhibitor of GABA uptake, led to the findings that nipecotic acid (piperidine-3-carboxylic acid) is a powerful non-competitive inhibitor of GABA uptake, and that perhydro-1,2-oxazine-6-carboxylic acid is a relatively weak competitive inhibitor of GABA uptake.     

GABA agonists

Summary This review describes the development of GABA receptor agonists with no detectable affinity for other recognition sites in GABA-mediated synapses. The key compounds are THIP, isoguvacine, and piperidine-4-sulphonic acid (P4S), developed via extensive structural modifications of the potent but not strictly specific GABA agonist muscimol. The structural parameters, which have to be considered in the design of GABA agonists are discussed on the basis of the structures and biological activities of these GABA agonists and a number of related compounds.A model, which summarizes our present knowledge of the structure of the postsynaptic GABA receptor complex, is presented, and the interaction of GABA agonists with various sites in this complex is discussed. Of particular interest are the effects of GABA agonists on the binding of diazepam to the benzodiazepine binding site, assumed to be a structural unit of the GABA receptor complex. While rigid molecules like THIP are capable of activating the GABA receptors, a certain degree of conformational mobility of GABA agonists apparently is a prerequisite for stimulation of diazepam binding in vitro at 0 °C. These findings suggest that GABA receptor functions involve conformational changes of certain elements of the receptor complex.Some aspects of the pharmacology of GABA agonists are discussed, including the attempts to develop GABA agonists with desirable pharmacokinetic and toxicological characteristics. While muscimol is a toxic compound, THIP is well tolerated by animals, and in contrast to isoguvacine, THIP penetrates into the brain after systemic administration to animals, a difference which can be explained on the basis of their protolytic properties. The attempts to develop pro-drugs of isoguvacine capable of penetrating the blood-brain barrier with subsequent decomposition in the brain tissue to isoguvacine are described.     

Neurochemical correlates of gamma-aminobutyrate (GABA) inhibition in cat visual cortex

High affinity binding of [3H]gamma-aminobutyric acid (GABA) to neuronal membranes from different parts of cat visual cortex was tested for sensitivity to GABA(A) agonists isoguvacine and THIP, GABA(A) antagonist SR95531 and GABA(B) agonist baclofen. Some of the GABA(A)-binding sites were found to have a very low affinity for THIP, suggesting the presence and, possibly, uneven distribution of "non-synaptic" GABA(A) receptors in cat visual cortex. There were no differences in Km and Vmax values of high affinity uptake of GABA and in the potency of K(+)-stimulated release of GABA, between primary and association cortices. Consequently, the present results indicate that despite the anatomical and physiological differences between the primary and association feline visual cortices the neurochemical characteristics of GABAergic inhibition are very similar in the two regions.     

Isoguvacine Binding, Uptake, and Release: Relation to the GABA System

Isoguvacine (1,2,3,6-tetrahydropyridine-4-car-boxylic acid) is a GABA (γ-aminobutyric acid) agonist with limited conformational flexibility. In these studies we investigated the binding, uptake, and release of [³H] isoguvacine by use of tissue preparations of rat CNS, comparing the results with similar studies of [³H]GABA. The results from these investigations indicate that isoguvacine binds to membrane preparations of rat forebrain with pharmacological characteristics similar to the post-synaptic GABA recognition site; that it is transported into synaptosomal preparations by an uptake system similar to the high-affinity GABA uptake system; and that recently accumulated isoguvacine is released in a Ca²⁺-dependent manner and by heteroexchange with external GABA. The ability of isoguvacine and γ-hydroxybutyric acid to decrease the K⁺-stimulated Ca²⁺-dependent release process was also investigated. The results indicate that isoguvacine interactions have many of the biochemical features of GABA synaptic function, isoguvacine being, however, less potent than GABA.     

Biological Efficiency of AMP Deaminase Inhibitor: 3-[2-(3-CARBOXY-4-BROMO-5,6,7,8-Tetrahydronaphthyl)Ethyl]-3,6,7,8-Tetrahydroimidazo[4,5]-[1,3]Diazepin-8-OL

AMP deaminase could be a potential target for treatment of heart disease but experimental evaluation of this concept is difficult due to limited availability of inhibitors with proven efficiency in biological systems. This study evaluated the effect of 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol, an AMP deaminase inhibitor (AMPDI) on the pathways of nucleotide metabolism in perfused rat heart. We show that AMPDI at 0.3 mM concentration effectively inhibits AMP deaminase in this experimental model.     

Effects of GABA, THIP, and potassium on excitability of myelinated axons of isolated amphibian spinal roots

The effects of direct applications of GABA (gamma-aminobutyric acid) and the GABAA agonist, THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) on the excitability of myelinated axons of individual dorsal and ventral spinal roots (lumbar VI and (or) VII) of the isolated bullfrog peripheral nerve are reported. Increases evoked by the GABA agonists (0.01-10 mM) in the amplitude of half-maximal A-fiber compound action potentials indicate the presence of depolarizing responses with apparently greater localization to the dorsal roots, and a sensitivity to GABA twofold greater than that for THIP. The changes evoked by GABA and THIP, as well as potassium have components that closely resemble those of sensory and motor fibers in the more distal, desheathed nerve bundle but are smaller and delayed, differences attributable to a closely attached root sheath that acts as a diffusion barrier. These results confirm the likely existence of GABAA receptors on both dorsal and ventral spinal roots.     

Synthesis of thiophenecarboxamides, thieno[3,4-c]pyridin-4(5H)-ones and thieno[3,4-d]pyrimidin-4(3H)-ones and preliminary evaluation as inhibitors of poly(ADP-ribose)polymerase (PARP)

Inhibitors of poly(ADP-ribose)polymerase (PARP) inhibit repair of damaged DNA and thus potentiate radiotherapy and chemotherapy of cancer. Treatment of 3-cyanothiophene with potassium nitrate and concentrated sulphuric acid gave 5-nitrothiophene-3-carboxamide. 4-Nitrothiophene-2-carboxamide and 5-nitrothiophene-2-carboxamide were formed similarly from 2-cyanothiophene. Reduction with tin(II) chloride gave the corresponding aminothiophenecarboxamide salts which were isolated via their N-Cbz derivatives. Lithiation of 3,4-dibromothiophene at -116 degrees C and quenching with alkyl chloroformates gave 4-bromothiophene-3-carboxylates, which were hydrolysed to 4-bromothiophene-3-carboxylic acid. Hurtley reactions with the enolates of pentane-2,4-dione and of 1-phenylbutane-1,3-dione, followed by acyl cleavage, led to 4-(2-oxopropyl)thiophene-3-carboxylic acid and 4-phenacylthiophene-3-carboxylic acid, respectively. Condensation with ammonia in acetic acid gave 6-methyl- and 6-phenylthieno[3,4-c]pyridin-4-ones, which were selectively nitrated at the 1- and 7-positions or were dinitrated. Ethyl 4-acetamido- and 4-benzamido-thiophene-3-carboxylates were cyclised to 2-methyl- and 2-phenyl-thieno[3,4-d][1,3]oxazin-4-ones, respectively. Ring-opening with ammonia and recyclisation led to 2-substituted thieno[3,4-d]pyrimidin-4-ones. The aminothiophenecarboxamides are analogues of 3-aminobenzamide, a selective inhibitor of poly(ADP-ribose)polymerase (PARP); the thienopyridinones and the thienopyrimidinones are analogues of isoquinolin-1-ones and quinazolin-4-ones, respectively, which inhibit this enzyme. In preliminary assays, several thienopyridinones and thienopyrimidinones showed potent inhibitory activity against PARP.     

Microiontophoretically applied THIP effects upon nociceptive responses of neurons in medial thalamus

Sprague-Dawley rats anesthetized with urethane were used to study the single cell responses of medial thalamic neurons following noxious input and their interactions with gamma-aminobutyric acid (GABA) agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) and morphine sulfate applied microintophoretically . The majority of the medial thalamic neurons responded to noxious stimulation by an increase in their firing rate. Local application of both THIP and morphine attenuated the spontaneous and the noxious evoked responses of these neurons. The possibility of a role for GABA in mediating nonopiate pain suppression is discussed.     

Amplification by glycine of the effect of the GABA transport inhibitor THPO on synaptosomal GABA level

Mice were injected intramuscularly (2 mmol/kg) with the glia-selective GABA uptake inhibitor 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) 60 min prior to sacrifice, or with glycine (10 mmol/kg) 45 min before death, or with a combination of both. After decapitation of the animals, the brains were removed and synaptosomes prepared and analyzed for content of GABA, taurine, glutamine, serine, glutamate and aspartate. While no differences as compared with control animals were found for aspartate, serine and glutamine, synaptosomal GABA levels were increased significantly after injections with either THPO or glycine. The individual effects of THPO and glycine were found to be additive. Taurine levels were decreased to a similar extent in animals which had received either THPO alone or THPO in conjunction with glycine. Treatment with THPO and glycine in combination led to a decrease in the synaptosomal glutamate content. The findings are consistent with the previously observed synergistic anticonvulsant actions of THPO and glycine being mediated via the GABA neurotransmitter system.     

Effects of Inhibitors of Protein Synthesis and Intracellular Transport on the γ-Aminobutyric Acid Agonist-Induced Functional Differentiation of Cultured Cerebellar Granule Cells

The effect of inhibitors of protein synthesis (actinomycin D, cycloheximide), proteases (leupeptin), and intracellular transport (colchicine, monensin) on the gamma-aminobutyric acid (GABA) agonist [4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP)]-induced changes in morphological differentiation and GABA receptor expression was investigated in cultured cerebellar granule cells. After 4 days in culture the neurons were exposed to the inhibitors for 6 h in the simultaneous presence of THIP. Subsequently, cultures were either fixed for electron microscopic examination or used for preparation of membranes for [3H]GABA binding assays. In some experiments the functional activity of the newly induced low-affinity GABA receptors was assessed by investigation of the ability of GABA to inhibit neurotransmitter release from the neurons. These experiments were performed to differentiate between an intracellular and a plasma membrane localization of the receptors. In all experiments cultures treated with THIP alone served as controls. The inhibitors of protein synthesis totally abolished the ability of THIP to induce low-affinity GABA receptors. In contrast, the inhibitors of intracellular transport as well as the protease inhibitor did not affect this parameter. However, studies of effects of GABA on transmitter release from monensin-treated cultures showed that transmitter release could not be inhibited by GABA in these cells in spite of the presence of low-affinity GABA receptors in the membrane preparations. This indicates that the low-affinity receptors were not located in the plasma membrane. This is in good agreement with the corresponding morphological findings, that monensin treatment led to an intense vacuolization of the Golgi apparatus, thereby preventing intracellular transport of the newly synthesized GABA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)