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In neocortical slices maintained in Mg(2+)-free Krebs medium, the gamma-aminobutyric acid (GABA(B)) receptor agonists baclofen, (3-amino-2(S)-hydroxypropyl)methylphosphinic acid (CGP 44532), and its (R)-enantiomer CGP 44533 depressed the frequency of spontaneous discharges in a concentration-dependent manner (EC(50)=10, 6.5, and 50 microM, respectively). These effects were reversibly antagonised by the GABA(B) receptor antagonist (+)-(S)-5,5 dimethylmorpholinyl-2-acetic acid (Sch 50911) (3, 10, and 30 microM) (average pA(2) value=6.0+/-0.2). In neocortical wedges, baclofen, CGP 44532 and CGP 44533 elicited concentration-dependent hyperpolarisations (the EC(50)s were 14, 7.5 and 16 microM, respectively) sensitive to Sch 50911 (1, 5, 10 microM) (average pA(2) value=6.0+/-0.1), whilst they also depressed ileal electrically elicited cholinergic twitch contractions (EC(50)=11, 7, and 50 microM) that were antagonised by Sch 50911 (average pA(2) value=6.0+/-0.1). In electrically stimulated brain slices preloaded with [3H]GABA, baclofen, CGP 44532 and CGP 44533 decreased [3H]GABA release (IC(50)=5, 0.45, and 10 microM); this effect was reversed by Sch 50911 (50 microM). It is concluded that CGP 44532 is a far more potent agonist at GABA(B) autoreceptors than at central or peripheral heteroreceptors.

(R,S)-4-Amino-3-(7-methylbenzo[b]furan-2-yl)-butanoic acid (7-MBFG), a new benzofuran analogue of the GABA(B) receptor agonist baclofen, has been evaluated for pharmacological activity on GABA(B) receptors in the guinea-pig isolated ileum and rat neocortical slices. 7-MBFG (300 and 500 microM) reversibly antagonized the (R,S)-baclofen induced depression of cholinergic twitch contractions in the guinea-pig ileum and shifted the concentration-response curve for baclofen to the right, in a parallel manner, giving an apparent pA2 value of 3.7+/-0.3. Likewise, 7-MBFG (300 and 500 microM) reversibly blocked the baclofen-induced suppression of spontaneous discharges, in rat neocortical slices maintained in Mg2+ -free Krebs medium, and caused a rightward, parallel shift of the baclofen concentration-response curve, giving an apparent pA2 value of 4.1+/-0.1. The compound 7-MBFG belongs to a novel, new class of antagonist at central and peripheral GABA(B) receptors, in which the antagonist properties reside in the pseudo-aromatic character of their 3-benzo[b]furan-2-yl substituents, and might provide useful leads for further development of GABA(B) receptor ligands.

The GABAB receptor antagonist saclofen (3-amino-2-(4-chlorophenyl)propylsulphonic acid) has been resolved by chiral high-performance liquid chromatography. The enantiomer (R)-saclofen, but not (S)-saclofen, reversibly antagonised the (R,S)-baclofen-induced depression of cholinergic twitch contractions in the guinea-pig ileum with an apparent pA2 of 5.3. Also, 2-hydroxy-saclofen was resolved by the same method, its (S)-enantiomer yielding an apparent pA2 of 5.0. This method provides a convenient resolution of these antagonists.

In the guinea-pig isolated ileum, both baclofen (5-100 microM) and gamma-aminobutyric acid (GABA; 5-100 microM) induced a bicuculline-insensitive depression of cholinergic twitch contractions which was reversibly and competitively antagonised by 3-amino-1-nitropropane (50-250 microM). 3-Amino-1-nitropropane (pA2 = 5.0 +/- 0.1) was twice as potent as 2-hydroxysaclofen (pA2 = 4.5 +/- 0.1), but was equipotent with 3-aminopropyl(P-diethoxymethyl)phosphinic acid (CGP 35348) (pA2 = 4.9 +/- 0.2), and did not show any partial agonist activity at these peripheral GABAA or GABAB receptor sites. In rat neocortical slices, 3-amino-1-nitropropane did not activate GABAB receptor sites or affect baclofen-induced suppression of spontaneous discharges. In the cat spinal cord, however, under in vivo conditions, the corresponding nitro analog of baclofen 3-amino-2-(4-chloro)-nitropropane was an agonist at GABAB receptor sites, although more than 60 times weaker than baclofen in depressing monosynaptic excitatory field potentials, whereas 3-amino-1-nitropropane was an extremely weak agonist at bicuculline-sensitive GABAA sites. The differing actions of 3-amino-1-nitropropane at peripheral and central GABAB receptor sites suggest heterogeneity among these receptors.

Segments of the isolated proximal ileum of the guinea-pig were set up in the organ bath to record longitudinal muscle contractions. Both gamma-aminobutyric acid (GABA) and 5-hydroxytryptamine induced neurally mediated cholinergic contractile responses; desensitization to 5-HT did not consistently depress contractile responses to GABA, whilst quipazine antagonized the neuronal responses induced by 5-HT without affecting responses to GABA. It is thus concluded that 5-HT does not mediate GABA-induced responses in the ileum.

Contractions of the guinea-pig ileum elicited by GABA were reversibly antagonised by various concentrations of picrotoxinin and bicuculline. This antagonism was specific for GABA-induced responses, responses to ACh, BK, and HA being unaffected. The chloride ion channel blockers, furosemide, 2 x 10-4 mol/l, and piretanide, 5.5 x 10-5 mol/l, substantially reduced GABA-induced contractions of the ileum but not those elicited by ACh or electrical stimulation of the cholinergic nerves. The action of GABA in stimulating the intrinsic cholinergic nerves appears to be Cl- dependent.

In isolated segments of the guinea-pig ileum, there was: (a) an early, short-lived (less than 20 s) depression by gamma-aminobutyric acid (GABA) of contractile responses to 5-hydroxytryptamine (5-HT), acetylcholine(ACh), or nicotine, also seen with 3-amino-1-propanesulphonic acid (3APS) or muscimol in place of GABA, and sensitive to bicuculline, picrotoxinin or piretanide, and (b) a delayed, longer-lasting (30 s-1 min) depression of responses to 5-HT and nicotine, but not exogenously applied ACh, also seen with baclofen and only antagonised by delta-aminovaleric acid (DAVA). At 25 degrees C, all these effects were still observed but slowed, whilst at 37 degrees C after cold storage (6 degrees C) overnight, the early, short-lived depression was reduced or eliminated, yet the delayed depression was enhanced. It is concluded that the early, short-lived depression is mediated through GABAA-receptor sites, and the delayed, longer-lasting depression through GABAB-receptor sites on neurones of the myenteric plexus; effects consistent with GABA being a neurotransmitter in the enteric nervous system.

Localization of [3H]GABA in the guinea-pig myenteric plexus has been studied using light microscopic autoradiography. In the presence of beta-alanine, 10(-3) M, an inhibitor of glial cell high affinity GABA transport, [3H]GABA was transported by a high affinity uptake system into neuronal elements of the plexus. Scattered neurones accumulating [3H]GABA showed localization of silver grains over the soma and axonal processes. In addition a large population of uptake sites for [3H]GABA was found within the secondary and tertiary meshworks of the plexus so that dense accumulations of silver grains were observed localized over distinct 'tracts' within all three meshworks of the plexus. These results are considered to provide strong evidence for GABAergic neurons in the enteric nervous system.

Cortisol had a biphasic action on GABAA-receptor-mediated contractile responses, enhancing at picomolar concentrations (1-10 pM) and inhibiting at higher concentrations (10-1000 nM). There was a sinistral shift of the GABA dose-response curve in the presence of 10 pM cortisol, with a significant potentiation of the GABA-induced contractions over the lower dose range of GABA (3-30 microM), whereas 100 nM cortisol caused a non-parallel dextral shift of the GABA dose-response curve, with a depression of the maximum GABA response indicative of non-competitive antagonism. Cortisol at various concentrations did not affect GABAB-receptor-mediated ileal relaxations, or the baclofen-induced depression of twitch contractions to transmural stimulation. Such concentrations of cortisol also did not affect ileal responses to exogenously applied acetylcholine or cholinergic twitch contractions themselves. These results suggest that cortisol is a specific, and very potent modulator at GABAA-receptor complexes in the guinea pig ileum.

In the guinea-pig isolated ileum, both gamma-aminobutyric acid (GABA) and baclofen induced a dose-dependent depression of cholinergic twitch contractions to transmural stimulation, sensitive to delta-aminovaleric acid (DAVA) and phosphonobaclofen (phaclofen). beta-Phenyl-GABA (BPG) antagonised this depressant action of baclofen and GABA, whilst itself weakly depressing ileal twitch contractions, an effect insensitive to DAVA or phaclofen, and thus unrelated to any GABAB-receptor-mediated effects. These results suggest that the baclofen receptors in the ileum that are antagonised by BPG differ from either of baclofen receptors in the spinal cord, where the presynaptic receptors are blocked by phaclofen and the postsynaptic receptors are insensitive to phaclofen, with BPG having baclofen-like actions at both sites. Interaction of BPG and baclofen with different receptor populations may explain the differing therapeutic actions of these compounds.

gamma-Aminobutyric-acid (GABA)-mimetic responses were induced by ethylenediamine (EDA) in the isolated ileum of the guinea-pig maintained in bicarbonate buffered Krebs-Henseleit (KBC) solution, pH 7.4, 37 degrees C, the responses consisting of a contraction followed by a relaxation. There were no such responses to EDA in bicarbonate-free phosphate buffered (KPO) or HEPES buffered (KHO) Krebs solution, gassed with 100% O2, pH 7.4, 37 degrees C, yet the ileum responded to GABA in bicarbonate-free Krebs solution. Similar GABA-mimetic responses were induced by EDA in the isolated ileum maintained in bicarbonate-free KPO or KHO modified Krebs solution, gassed with O2, if HCO3- (5mM) was first added immediately before the test dose of EDA (0.1-1 mM), the threshold [HCO3-]being 2 mM for EDA-induced responses in these preparations. However, ileal GABA-mimetic responses were induced in bicarbonate-free KPO or KHO solutions by EDA that had been pretreated with carbon dioxide, where the final [HCO3-]in the bath did not exceed 25 microM. Ethylenediamine monocarbamate (synthetic EDAC) released [3H]-GABA from preloaded segments of ileum maintained in bicarbonate-free KPO or KHO solution containing amino-oxyacetic acid and beta-alanine, the release being sensitive to 3-mercaptopropionic acid which prevents GABA release. EDA itself did not evoke any such release in the absence of bicarbonate, but released [3H]-GABA from segments maintained in KBC solution. 4 GABA-mimetic responses were induced by EDAC in the isolated ileum maintained in bicarbonate-free KPO solution, as was a delta-aminovalerate-sensitive depression of ileal twitch responses elicited by transmural stimulation, all of which were also sensitive to 3-mercaptopropionic acid. 5 It is concluded that GABA-mimetic responses to EDA in the isolated ileum of the guinea-pig, maintained in normal Krebs bicarbonate medium, result from the release of endogenous GABA by ethylenediamine monocarbamate formed through the rapid reaction of EDA with the carbon dioxide of bicarbonate buffered Krebs solution. Furthermore, in the ileum, HCO3 ions per se are not necessary for this GABA-releasing property of EDA if the latter is first converted to the monocarbamate, since syntheticethylenediamine monocarbamate elicits ileal GABA-mimetic responses in the total absence of bicarbonate.

Saclofen (the direct sulphonic analogue of baclofen) is a competitive antagonist of baclofen at GABAB receptors in guinea pig ileum and rat cortical slices (estimated pA2 = 5.3), at least twice as potent as 2-hydroxy-saclofen (pA2 = 5). A series of related sulphonic analogues also antagonised baclofen in the guinea pig ileum, including 2-hydroxy-saclofen amide (pA2 = 3.3), 3-amino-2-hydroxy-2-phenyl-propylsulphonic acid (pA2 = 3.5), 3-amino-2-(benzo-(b)-furan-2-yl)-propylsulphonic acid (pA2 = 4.3), and 3-amino-2-(4-chlorophenyl)-prop-1-enesulphonic acid (pA2 = 2.5-3), but none were more active than saclofen which is the most potent specific GABAB antagonist yet found.

2-hydroxy-saclofen (2-OH-S), a sulphonic analogue of baclofen, slightly increased the twitch height and reversibly antagonised the GABA- and baclofen-induced depression of twitch contractions in the guinea pig vas deferens and isolated ileum, causing a parallel dextral shift in the baclofen dose-response curve in a competitive manner (pA2 = 5.0) in the latter tissue. 2-OH-S (10-50 microM) reversibly elevated the spike height and antagonised the baclofen (8-20 microM)-induced suppression of ictal discharges in rat cortical slices superfused in Mg2+-free Krebs solution, the spike height declining to control level within 15 min of washout. The antagonism by 2-OH-S on GABAB receptor-mediated actions is selective, as 2-OH-S did not affect depressive responses to adenosine or morphine, or contractile responses to GABA (GABAA receptor-mediated), acetylcholine and carbachol in the ileum. Compared to phaclofen, 2-OH-S is a more potent competitive antagonist of GABAB receptor-mediated actions in the central and peripheral nervous system.

Rhythmic neurally mediated spontaneous contractions of the longitudinal muscle in the isolated ileum of the guinea pig, sensitive to tetrodotoxin and atropine, were depressed and most often abolished by the GABAA receptor antagonists, bicuculline methiodide, RU 5135, and picrotoxin, a Cl- -ionophore blocker, as well as by GABA desensitization. 3-Mercaptopropionic acid, known to prevent GABA release, also reduced these naturally occurring spontaneous contractions. All these strongly indicate a physiological involvement of endogenous GABA in the control of spontaneous rhythmic activity in the intestine.