Overlapping, but not identical, discriminative stimulus effects of the neuroactive steroid pregnanolone and ethanol

Many behavioral effects of neuroactive steroids are mediated by GABA(A) receptors; however, other receptors might be involved. Ethanol has a complex mechanism of action, and many of the same receptors have been implicated in the effects of neuroactive steroids and ethanol. The goal of this study was to determine whether actions of neuroactive steroids and ethanol at multiple receptors result in similar discriminative stimulus effects. Rats discriminated 5.6 mg/kg of pregnanolone while responding under a fixed-ratio 20 schedule of food presentation. Pregnanolone, flunitrazepam and pentobarbital produced >80% pregnanolone-lever responding. In contrast, neither morphine nor the negative GABA(A) modulator beta-CCE substituted for pregnanolone up to doses that markedly decreased response rates. Ethanol substituted only in some rats; in other rats, ethanol produced <20% pregnanolone-lever responding up to rate-decreasing doses. Thus, substitution of positive GABA(A) modulators, and not morphine or beta-CCE, for pregnanolone in all rats suggests that positive modulation of GABA(A) receptors is important in the discriminative stimulus effects of pregnanolone. Although pregnanolone might have actions at other receptors, in addition to actions at GABA(A) receptors, substitution of ethanol for pregnanolone only in some rats suggests that the mechanisms of action of pregnanolone and ethanol overlap, but are not identical.     

Occupation of either site for the neurosteroid allopregnanolone potentiates the opening of the GABAA receptor induced from either transmitter binding site

Potentiating neuroactive steroids are potent and efficacious modulators of the GABA(A) receptor that act by allosterically enhancing channel activation elicited by GABA. Steroids interact with the membrane-spanning domains of the α subunits of the receptor, whereas GABA binds to pockets in the interfaces between β and α subunits. Steroid interaction with a single site is known to be sufficient to produce potentiation, but it is not clear whether effects within the same β-α pair mediate potentiation. Here, we have investigated whether the sites for GABA and steroids are functionally linked (i.e., whether the occupancy of a steroid site selectively affects activation elicited by GABA binding to the transmitter binding site within the same β-α pair). For that, we used receptors formed of mutated concatenated subunits to selectively eliminate one of the two GABA sites and one of the two steroid sites. The data demonstrate that receptors containing a single functional GABA site are potentiated by the neurosteroid allopregnanolone regardless of whether the steroid interacts with the α subunit from the same or the other β-α pair. We conclude that steroids potentiate the opening of the GABA(A) receptor induced by either agonist binding site.     

The effects of chronic haloperidol administration on GABA receptor binding

The effect of chronic administration of haloperidol for 30 days and its subsequent withdrawal for 0,4 and 8 days on the binding of [³H]-GABA and [³H]-muscimol to GABA binding sites in rat brain membranes obtained from six discrete regions of the brain and spinal cord was investigated. The chronic administration of haloperidol resulted in a marked increase in the number of GABA binding sites within the substantia nigra, but did not affect GABA binding sites in other regions of rat brain or spinal cord. The increase in GABA binding sites in the substantia nigra was evident for at least 4 days following termination of haloperidol treatment; however, 8 days following withdrawal from haloperidol, when dopaminergic supersensitivity had become maximally expressed, the number of GABA binding sites in the substantia nigra had declined and was the same as that observed in the control rats. These observations suggest that blockade of dopamine receptors in the striatum by haloperidol results in a compensatory decrease in the activity of the GABAergic strionigral system that leads to an increase in the number of GABA binding sites within the substantia nigra. This increase in the number of GABA binding sites is gradually reversed when dopamine is again allowed to interact with its receptors.     

Influence of environment on GABA receptors in muricidal rats

The influence of environment (isolation) on GABA receptor numbers ( [3H]-muscimol binding sites) and affinities was investigated in specific limbic areas known to be involved with the development of muricide. Olfactory bulbs of isolated rats were found to have identical numbers of [3H]-muscimol binding sites whether or not they were muricidal. However, in the olfactory bulbs of the aggregated animals a greater than two-fold increase was found in numbers of [3H]-muscimol binding sites in those rats that were muricidal. In the amygdala muricidal rats had a 32-34% increase in [3H]-muscimol binding sites over non-muricidal rats regardless of environment. In the septum non-muricidal rats had fewer [3H]-muscimol binding sites than muricidal rats and although the trend was evident, statistical vigor was seen only in the aggregated animals. Neither muricide nor isolation significantly influenced [3H]-muscimol binding numbers in the hypothalamus. GABA Ki values were examined in all brain regions and found to be the same in the isolated and aggregated animals whether or not they were muricidal. We concluded that environment appears to influence apparent GABA receptor numbers in the olfactory bulbs and septum whereas muricidal behavior correlates well with an increase in apparent number of GABA receptors in the amygdala. GABA receptors in the hypothalamus were not influenced by either environment or aggression.     

A novel GABA(A) receptor pharmacology: drugs interacting with the α(+) β(-) interface

GABA(A) receptors are ligand-gated chloride channels composed of five subunits that can belong to different subunit classes. The existence of 19 different subunits gives rise to a multiplicity of GABA(A) receptor subtypes with distinct subunit composition; regional, cellular and subcellular distribution; and pharmacology. Most of these receptors are composed of two α, two β and one γ2 subunits. GABA(A) receptors are the site of action of a variety of pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, neuroactive steroids, anaesthetics and convulsants. Whereas GABA acts at the two extracellular β(+) α(-) interfaces of GABA(A) receptors, the allosteric modulatory benzodiazepines interact with the extracellular α(+) γ2(-) interface. In contrast, barbiturates, neuroactive steroids and anaesthetics seem to interact with solvent accessible pockets in the transmembrane domain. Several benzodiazepine site ligands have been identified that selectively interact with GABA(A) receptor subtypes containing α2βγ2, α3βγ2 or α5βγ2 subunits. This indicates that the different α subunit types present in these receptors convey sufficient structural differences to the benzodiazepine binding site to allow specific interaction with certain benzodiazepine site ligands. Recently, a novel drug binding site was identified at the α(+) β(-) interface. This binding site is homologous to the benzodiazepine binding site at the α(+) γ2(-) interface and is thus also strongly influenced by the type of α subunit present in the receptor. Drugs interacting with this binding site cannot directly activate but only allosterically modulate GABA(A) receptors. The possible importance of such drugs addressing a spectrum of receptor subtypes completely different from that of benzodiazepines is discussed.     

Studies on the mechanism of interactions between anesthetic steroids and gamma-aminobutyric acidA receptors

Functional interactions between steroidal anesthetics and gamma-aminobutyric acidA (GABAA) receptors have been examined with 36Cl- uptake measurements in rat cerebrocortical synaptoneurosomes. The primary effect of the steroids was to enhance the affinity of GABA for its receptors without much effect on the maximal uptake rate; the ED50 for GABA decreased from 66.4 +/- 5.7 to 8.9 +/- 1.2 microM in the presence of 20 microM 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one. Stimulation of 36Cl- uptake by high concentrations of the anesthetic steroid in the absence of exogenous GABA was not due to direct stimulation of GABAA receptors, as currently proposed, but is due to enhanced action of endogenous GABA, inasmuch as the steroid markedly increases GABA affinity for the receptors. Typically, endogenous GABA was maintained at near 1 microM by a Na(+)-dependent GABA transport system in the synaptoneurosomes. Elevation of its level with nipecotic acid, a specific inhibitor of the GABA transport system, or reduction with GABase, a GABA-scavenging system, increased or decreased, respectively, the steroid-induced bicuculline-sensitive 36Cl- uptake. At low concentrations of GABA (less than 2 microM), the stimulatory effect of 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one was markedly potentiated by pentobarbital but antagonized by 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one, a partial agonist of higher affinity. These observations, along with the structure-activity relationships of steroid analogs, strongly suggest the existence of a specific binding site for the steroids in GABAA receptors and led us to propose a minimal model in which two key common functional groups of anesthetic steroids, 3 alpha-OH- and 17 beta-polar substituents, interact with GABAA receptors (probably through hydrogen bondings) while their hydrophobic backbone remains in contact with the fatty acyl chains of membrane phospholipids.     

The properties of the brain dopamine and GABA receptors in rats with chronic alcoholic intoxication with different ethanol withdrawal times]

Marked changes in dopamine and GABA receptors in the brain of rats with chronic alcohol intoxication were registered 48 h and 3 months after ethanol withdrawal. The changes in dopamine receptors characterized by the increased number of the sites of binding with ligand (Bmax) were most pronounced 48 h after ethanol withdrawal. In 3 months the properties of these receptors tended toward recovery. The changes in GABA receptors also manifested by the increase in Bmax become further pronounced 3 months after ethanol withdrawal.     

Alterations in benzodiazepine and GABA receptor binding in rat brain following systemic injection of kainic acid

The binding of gamma-aminobutyric acid (GABA) and benzodiazepine to receptors was examined in regions of rat brain at various times after subcutaneous injection of kainic acid (KA, 15 mg/kg). The animals exhibited pronounced convulsions 90 min-4 hr after this treatment. During this period (2 hr after the injection of kainic acid) no alterations in the binding of [3H]-GABA or [3H]flunitrazepam to receptors were detected in the frontal cortex, the hippocampus or the amygdala-pyriform cortex. After recovery from the acute convulsive phase, the rats appeared to be hyperexcitable, hyperactive, and displayed marked aggression and occasional clonic convulsions one to 80 days later. During this period a marked increase (80-200%) in the number of binding sites for GABA in the amygdala-pyriform cortex occurred but this was associated with a slow decrease in the number of binding sites for [3H]flunitrazepam to 70% control value at 3 weeks. Binding of the "peripheral"-type of benzodiazepine ligand, [3H]-Ro5-4864, was increased to 450% of control 3 weeks after injection. In addition, the ability of GABA to stimulate the binding of [3H]flunitrazepam was reduced when measured 3 days after the injection of kainic acid. It is suggested that the long-term behavioural syndrome observed in kainic acid-treated rats, as well as the reduced effectiveness of diazepam in preventing seizures in animals treated with kainic acid, (Czuczwar, Turski, Turski and Kleinrock, 1981) may be explained in part by a reduction in the number of neuronal benzodiazepine receptors and a "desensitization" of the GABA receptors which are coupled to benzodiazepine receptors.     

2-Phenyl-imidazo[1,2-a]pyridine derivatives as ligands for peripheral benzodiazepine receptors: stimulation of neurosteroid synthesis and anticonflict action in rats.

Selective activation of peripheral benzodiazepine receptors (PBRs) in adrenal cells and brain oligodendrocytes promotes steroidogenesis. Three 2-phenyl-imidazo[1,2-a]pyridine derivatives (CB 34, CB 50 and CB 54) have now been investigated with regard to their selectivity for PBRs and their ability to stimulate central and peripheral steroidogenesis in rats. The three CB compounds (10(-10)-10(-4) M) potently inhibited the binding of the PBR ligand [3H]-PK 11195 to brain and ovary membranes in vitro, without substantially affecting [3H]-flunitrazepam binding to central benzodiazepine receptors. These compounds (10(-7)-10(-4) M) also had little or no marked effects on GABA-evoked Cl- currents in voltage-clamped Xenopus oocytes expressing human alpha1beta2gamma2S GABA(A) receptors. In addition, they failed to affect ligands binding to GABA(B), D1/D2 dopamine, muscarinic acetylcholine, N-methyl-D-aspartic acid and opiate receptors. Intraperitoneal administration of CB compounds (3-50 mg kg(-1)) induced a dose-dependent increase in the concentrations of neuroactive steroids in plasma and brain. The brain concentrations of pregnenolone, progesterone, allopregnanolone and allotetrahydrodeoxycorticosterone (THDOC) showed maximal increases in 96+/-3, 126+/-14, 110+/-12 and 70+/-13% above control, respectively, 30 to 60 min after injection of CB 34 (25 mg kg(-1)). CB 34 also increased the brain concentrations of neuroactive steroids in adrenalectomized-orchiectomized rats, although to a lesser extent than in sham-operated animals, suggesting that CB compounds stimulate brain steroidogenesis independently of their effects on peripheral tissues. The increase in brain and plasma neurosteroid content induced by CB 34 was associated with a marked anticonflict effect in the Vogel test. Our results indicate that the three CB compounds tested are specific and potent agonists at peripheral benzodiazepine receptors, and that they stimulate steroidogenesis in both the brain and periphery.     

Neurosteroid analogues. 10. The effect of methyl group substitution at the C-6 and C-7 positions on the GABA modulatory and anesthetic actions of (3alpha,5alpha)- and (3alpha,5beta)-3-hydroxypregnan-20-one

The planar 5alpha-reduced steroid (3alpha,5alpha)-3-hydroxypregnan-20-one and the nonplanar 5beta-reduced steroid (3alpha,5beta)-3-hydroxypregnan-20-one act at GABA(A) receptors to induce general anesthesia. The structural features of the binding sites for these anesthetic steroids on GABA(A) receptors have not been determined. To determine how structural modifications at the steroid C-6 and C-7 positions effect the actions of these anesthetic steroids, an axial or equatorial methyl group was introduced at these positions. The analogues were evaluated (1) in [(35)S]-tert-butylbicyclophosphorothionate binding experiments, (2) in electrophysiological experiments using rat alpha(1)beta(2)gamma(2L) GABA(A) receptors expressed in Xenopus laevis oocytes, and (3) as tadpole anesthetics. The effects of methyl group substitution in the 5alpha- and 5beta-reduced series of compounds were strikingly similar. In both series, a 6beta-Me group gave compounds with actions similar to or greater than those of the parent steroids. A 6alpha-, 7beta- or 7alpha-Me substituent resulted in reduced potency for inhibition of radioligand binding, GABA(A) receptor modulation and tadpole anesthesia. Because of the similar effects of methyl group substitution in the two series of compounds and previous results from other studies showing that structural modifications in the steroid D ring/side chain region produce similar effects regardless of the stereochemistry of the A,B-ring fusion, we propose that either the 3alpha-hydroxyl groups of planar and nonplanar anesthetic steroids hydrogen bond to different amino acids on GABA(A) receptors or that this critical hydrogen bonding group interacts with membrane lipids instead of the receptor.     

Regulation of inhibitory synaptic transmission by a conserved atypical interaction of GABA(A) receptor beta- and gamma-subunits with the clathrin AP2 adaptor

The number of surface and synaptic GABA(A) receptors is an important determinant of inhibitory synapse strength. Surface receptor number is in part controlled by removal of receptors from the membrane by interaction with the clathrin adaptor AP2. Here we demonstrate that there are two binding sites for AP2 in the gamma2-subunit: a Yxxvarphi type motif specific to gamma2-subunits and a basic patch AP2 binding motif, that is also found in GABA(A) receptor beta-subunits. Blocking GABA(A) receptor-AP2 interactions using a peptide that inhibits AP2 binding to GABA(A) receptors via the conserved basic patch mechanism increases synaptic responses within minutes, whereas simultaneously blocking both binding mechanisms has an additive effect. These data suggest that multiple AP2 internalization signals control the levels of surface and synaptic GABA(A) receptors to regulate synaptic inhibition.     

Stress-protection action of beta-phenyl(GABA): involvement of central and peripheral type benzodiazepine binding sites

Forced swimming stress caused a significant increase in the density of central type benzodiazepine binding sites in rat cerebral cortex and hippocampus. The number of peripheral type benzodiazepine binding sites was also enhanced on blood platelets. The affinity of neither central nor peripheral type benzodiazepine binding sites was changed considerably after swimming stress. Pretreatment of rats with beta-(phenyl)GABA (100 mg/kg), a GABAB agonist, almost completely eliminated the described changes of the both types of benzodiazepine binding sites caused by swimming stress. In an elevated plus-maze model of anxiety beta-(phenyl)GABA itself was inactive but like diazepam effectively counteracted the behavioural effects of DMCM, a beta-carboline derivative with anxiogenic properties. The possible involvement of benzodiazepine receptors in the mechanism of action of beta-(phenyl)GABA is discussed.     

Progesterone and estrogens in rat brain: modulation of GABA (gamma-aminobutyric acid) receptor activity

Our data indicate that estrogens and progesterone can regulate the number of GABA receptors (as detected by [3H]muscimol binding assay) in rat brain. Both hormones act in selected areas. The extent of the effect (up to 160% increase) and the number of areas responsive suggest that sex hormones may play a very important role in the regulation of the functions of GABAergic transmission in the central nervous system.     

Neurosteroid analogues. 14. Alternative ring system scaffolds: GABA modulatory and anesthetic actions of cyclopenta[b]phenanthrenes and cyclopenta[b]anthracenes

Although the structural features of binding sites for neuroactive steroids on gamma-aminobutryic acid type A (GABA A) receptors are still largely unknown, structure-activity studies have established a pharmacophore for potent enhancement of GABA A receptor function by neuroactive steroids. This pharmacophore emphasizes the importance of the position and stereochemistry of hydrogen-bonding groups on the steroid. However, the importance of the steroid ring system in mediating hydrophobic interactions with the GABA A receptor is unclear. We have taken the cyclopenta[ b]phenanthrene (tetracyclic compounds with a nonlinear ring system different from that of steroids) and cyclopenta[ b]anthracene (tetracyclic molecules with a linear 6-6-6-5 carbocyclic ring system) ring systems and properly substituted them to satisfy the pharmacophore requirements of the critical hydrogen-bond donor and acceptor groups found in neuroactive steroids. We have found these cyclopenta[ b]phenanthrene and cyclopenta[ b]anthracene analogues to have potent activity at the GABA A receptor, rivaling that of the most potent steroid modulators. Single-channel analysis of electrophysiological data indicates that similarly substituted analogues in the different ring systems affect the kinetic components of macroscopic currents in different ways. Mutations to the hydrogen bonding amino acids at the putative steroid binding site (alpha1Q241L mutation and alpha1N407A/Y410F double mutation) produce similar effects on macroscopic current amplitude by the different ring system analogues suggesting that the different kinetic effects are explained by the precise interactions of each analogue with the same binding site(s).     

Lack of allosteric modulation of striatal GABA(A) receptor binding and function after cocaine sensitization

GABA(A) receptor binding after repeated cocaine has been shown to be either increased as indicated by benzodiazepine binding or decreased as indicated by convulsant-site binding. We measured the GABA binding site with [3H]-muscimol binding to GABA(A) receptors and found no differences between saline- and cocaine-sensitized rats. Allosteric modulation of [3H]-muscimol binding with flunitrazepam was also unchanged after cocaine sensitization. In addition, [3H]-flunitrazepam binding and allosteric modulation of [3H]-flunitrazepam binding with GABA was unchanged after 1 day withdrawal from repeated cocaine. GABA(A) receptor function and allosteric modulation of GABA(A) receptor function measured by GABA-stimulated Cl(-) uptake was also unchanged after withdrawal from repeated cocaine. Finally, in vitro cocaine reduced GABA(A) receptor function in striatal microsacs of saline- and cocaine-treated rats. In conclusion, repeated cocaine did not change the coupling of the GABA(A) receptor between the GABA and benzodiazepine (BZD) binding site after 1 day withdrawal.     

Cyclodextrins sequester neuroactive steroids and differentiate mechanisms that rate limit steroid actions

BACKGROUND AND PURPOSE: Neuroactive steroids are potent modulators of GABA(A) receptors and are thus of interest for their sedative, anxiolytic, anticonvulsant and anaesthetic properties. Cyclodextrins may be useful tools to manipulate neuroactive effects of steroids on GABA(A) receptors because cyclodextrins form inclusion complexes with at least some steroids that are active at the GABA(A) receptor, such as (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha5alphaP, allopregnanolone). EXPERIMENTAL APPROACH: To assess the versatility of cyclodextrins as steroid modulators, we investigated interactions between gamma-cyclodextrin and neuroactive steroids of different structural classes. KEY RESULTS: Both a bioassay based on electrophysiological assessment of GABA(A) receptor function and optical measurements of cellular accumulation of a fluorescent steroid analogue suggest that gamma-cyclodextrin sequesters steroids rather than directly influencing GABA(A) receptor function. Neither a 5beta-reduced A/B ring fusion nor a sulphate group at carbon 3 affected the presumed inclusion complex formation between steroid and gamma-cyclodextrin. Apparent dissociation constants for interactions between natural steroids and gamma-cyclodexrin ranged from 10-60 microM. Although gamma-cyclodextrin accommodates a range of natural and synthetic steroids, C(11) substitutions reduced inclusion complex formation. Using gamma-cyclodextrin to remove steroid not directly bound to GABA(A) receptors, we found that cellular retention of receptor-unbound steroid rate limits potentiation by 3alpha- hydroxysteroids but not inhibition by sulphated steroids. CONCLUSIONS AND IMPLICATIONS: We conclude that gamma-cyclodextrins can be useful, albeit non-specific, tools for terminating the actions of multiple classes of naturally occurring neuroactive steroids.     

Novel 17beta-substituted conformationally constrained neurosteroids that modulate GABA A receptors

The goal of this study was to develop a series of allopregnanolone analogues substituted by conformationally constrained 17beta side chains to obtain additional information about the structure-activity relationship of 5alpha-reduced steroids to modulate GABA(A) receptors. Specifically, we introduced alkynyl-substituted 17beta side chains in which the triple bond is either directly attached to the 17beta-position or to the 21-position of the steroid skeleton. Furthermore, we investigated the effects of C22 and C20 modification. The in vitro binding affinity for the GABA(A) receptor of the new analogues was measured by allosteric displacement of the specific binding of [(3)H]4'-ethynyl-4-n-propyl-bicycloorthobenzoate (EBOB) to GABA(A) receptors on synaptosomal membranes of rat cerebellum. An allosteric binding model that has been successfully applied to ionotropic glycine receptors was employed. The most active derivative is (20R)-17beta-(1-hydroxy-2,3-butadienyl)-5alpha-androstane-3-ol (20), which possesses low nanomolar potency to modulate cerebellar GABA(A) receptors and is 71 times more active than the control compound allopregnanolone. Theoretical conformational analysis was employed in an attempt to correlate the in vitro results with the active conformations of the most potent of the new analogues.     

Stress-protective Action of β-Phenyl(GABA): Involvement of Central and Peripheral Type Benzodiazepine Binding Sites

Forced swimming stress caused a significant increase in the density of central type benzodiazepine binding sites in rat cerebral cortex and hippocampus. The number of peripheral type benzodiazepine binding sites was also enhanced on blood platelets. The affinity of neither central nor peripheral type benzodiazepine binding sites was changed considerably after swimming stress. Pretreatment of rats with β-(phenyl)GABA (100 mg/kg), a GABA_B agonist, almost completely eliminated the described changes of the both types of benzodiazepine binding sites caused by swimming stress. In an elevated plus-maze model of anxiety β-(phenyl)GABA itself was inactive but like diazepam effectively counteracted the behavioural effects of DMCM, a β-carboline derivative with anxiogenic properties. The possible involvement of benzodiazepine receptors in the mechanism of action of β-(phenyl)GABA is discussed.     

GABAA receptor sites modulating catecholamine secretion in the rat adrenal gland: evidence from 3H-muscimol autoradiography and in vivo functional studies

The occurrence and distribution of specific 3H-muscimol binding sites, most probably identical with A type gamma-aminobutyric acid (GABA) receptors, were studied in sections of the rat adrenal gland by light microscope autoradiography. Specific binding was found primarily in the adrenal medulla, in association with chromaffin cells. A limited number of binding sites was also observed within the adrenal cortex. In urethane-anaesthetized hexamethonium-pretreated rats, intravenous GABA produced a set of 'excitatory' cardiovascular effects (increase in heart rate, force of contraction and blood pressure) which were mimicked by intravenous muscimol but not by intravenous baclofen, and were antagonized by pretreatment with bicuculline. The cardiovascular excitatory effects of intravenous GABA were unaffected by reserpine pretreatment, markedly reduced by administration of phentolamine plus propranolol, and almost completely abolished by adrenalectomy. Our findings indicate the presence of GABA receptor sites on adrenal chromaffin cells, whose excitation can produce changes in cardiovascular function.     

GABAA receptors in the rat stomach may mediate mucoprotective effects

The occurrence and characteristics of binding sites specific for gamma-aminobutyric acid (GABA) and muscimol in the rat stomach were examined by biochemical and autoradiographic techniques, and the effects of GABAergic model compounds on gastric ulceration induced by chemical irritation was studied in intact and unilaterally vagotomized rats. Specific binding sites for [3H]GABA and [3H]muscimol, which showed the characteristics of GABAA receptors, were demonstrated on gastric membranes. Specific muscimol binding sites were found in all regions of the stomach and were present in both the mucosal layer and the remaining tissue of the stomach. Oral pretreatment of the rats with GABA, selective GABAA receptor agonists, or inhibitors of GABA degradation protected the gastric mucosa against the ulcers induced by acidified ethanol (chemical irritant), in both intact and vagotomized rats. These findings are consistent with the view that a subpopulation of GABAA receptors in the rat stomach may mediate the anti-ulcer effect.