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Using grease-gap recording from rat neocortical slices, the GABA(B) receptor agonist baclofen elicited reversible and concentration-dependent hyperpolarizing responses (EC50=18+/-2.3 microM). The hyperpolarizations were antagonised by the GABA(B) receptor antagonist Sch 50911 [(+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid). (+)-N-1-(3-chloro-4-methoxyphenyl)ethyl-3,3-diphenylpropylamine (3-chloro,4-methoxyfendiline; 3-Cl,4-MeO-fendiline) reversibly potentiated baclofen-induced hyperpolarizing responses, which were reduced by Sch 50911, producing leftward shifts of the baclofen concentration-response curves, with a marked increase in the maximal hyperpolarization (EC50=2+/-0.5 microM). In slices preincubated with either [3H]GABA or [3H]glutamic acid, 3-Cl,4-MeO-fendiline (1 microM) potentiated the inhibitory effect of baclofen (2 microM) on the electrically evoked release of [3H]GABA and had a similar effect on the release of [3H]glutamic acid at a concentration of 0.5 microM, without affecting the basal release. These effects were blocked by Sch 50911 (10 microM). Our findings suggest that 3-Cl,4-MeO-fendiline is a potent potentiator of pre- and postsynaptic GABA(B) receptor-mediated functions.

In electrically stimulated rat neocortical brain slices preloaded with [3H]gamma-aminobutyric acid (GABA) or [3H]glutamic acid, the pharmacological actions of 1-(aminomethyl)-cyclohexaneacetic acid (gabapentin, Gp) were compared with the GABAB receptor agonists baclofen (Bac) and (3-amino-2-(S)-hydroxypropyl)-methylphosphinic acid (CGP 44532). Gabapentin, baclofen and CGP 44532 all reduced the electrically stimulated release of [3H]glutamic acid (IC50=20 microM, 0.8 microM and 2 microM, respectively). These effects were sensitive to the GABAB receptor antagonists (+)-(S)-5,5 dimethylmorpholinyl-2-acetic acid (Sch 50911) or N-3-[[1-(S)-(3,4-dichlorophenyl)ethyl]amino]-2-(S)-hydroxypropyl-P-(cyclo-hexylmethyl)-phosphinic acid (CGP 54626). By contrast, gabapentin was without effect on the release of [3H]GABA, whilst baclofen (IC50=8 microM) and CGP 44532 (IC50=1 microM) inhibited [3H]GABA release. It is concluded that gabapentin selectively activates presynaptic GABAB heteroreceptors, but not GABAB autoreceptors, and may be a useful ligand to discriminate between presynaptic GABAB receptor subtypes.

Selected neutral L-alpha-amino acids, and their dipeptides, were reversible, stereospecific, potentiators of GABA(B) receptor-mediated hyperpolarizing responses to baclofen (3-100 microM) in rat neocortical slices. These responses were sensitive to the GABA(B) receptor antagonist (+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid (Sch50911) (30 microM). Most potent were L-Leu, L-Ile and L-Phe, as were the dipeptides L-Phe-Phe and L-Phe-Leu, and less potent were L-Met, L-Val, L-Cys, L-Cystine, L-Tyr, L-Thr, L-Arg and L-Ser. Inactive were L-Trp, L-His, L-Lys and L-Pro. These potentiators gave leftward shifts of the baclofen concentration-response curves with a Hill slope of 2, and a marked increase in the maximal hyperpolarizing responses. Selected L-amino acids and dipeptides are a class of naturally occurring GABA(B) potentiators, which may be allosteric modulators.

Using grease-gap recording from rat neocortical slices, the gamma-aminobutyric acid(B) (GABA(B)) receptor agonists baclofen (3-100 microM) and SKF 97541 (3-aminopropyl-methylphosphinic acid) (1-30 microM) elicited reversible and concentration-dependent hyperpolarizing responses, with EC(50) values of 10 and 3 microM, respectively. The hyperpolarizations were antagonised by the GABA(B) receptor antagonist Sch 50911 ((+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid) (1, 5 and 10 microM). Fendiline (N-[3,3-diphenylpropyl)-alpha-methylbenzylamine) (5-50 microM) and its congeners, prenylamine (N-[3,3-diphenylpropyl)-alpha-methylphenylethylamine) (10-100 microM) and F551 (N-[3,3-diphenylpropyl)-alpha-methyl-3-methoxybenzylamine) (1-30 microM) reversibly enhanced hyperpolarizing responses to the agonists; such effects were reduced by Sch 50911. These arylalkylamines produced leftward shifts of the concentration-response curves, with a marked increase in the maximal hyperpolarization obtained, compared with the agonists alone, F551 being the most potent. These findings suggest that these arylalkylamines represent a new class of positive modulators of GABA(B) receptor-mediated function., (Copyright 2002 Elsevier Science B.V.)

In rat neocortical preparations maintained in Mg(2+)-free Krebs medium, baclofen depressed the frequency of spontaneous discharges in a concentration-dependent manner (EC(50) = 6 microM), sensitive to (3-aminopropyl)ethylphosphinic acid (CGP 36216) (100, 300 and 500 microM) (pA(2) = 3.9 +/- 0.1). By contrast, CGP 36216, up to 1 mM, was ineffective in antagonising baclofen-induced hyperpolarisations, mediated through gamma-aminobutyric acid(B) (GABA(B)) postsynaptic receptors. In electrically stimulated brain slices preloaded with [3H]GABA, CGP 36216 increased [3H]GABA release (IC(50) = 43 microM), which was reversed by baclofen (20 microM). While CGP 36216 is ineffective at GABA(B) postsynaptic receptors, it is appreciably more active at presynaptic receptors.

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.

In rat neocortical slices maintained in Mg(2+)-free Krebs medium, the gamma-aminobutyric acid (GABA(B)) receptor agonist baclofen concentration-dependently depressed the frequency of spontaneous discharges (EC(50)=12 microM). This was reversibly antagonised by (R, S)-3-amino-2-hydroxy-propyl-P-n-butyl-phosphinic acid (CGP 47332A) (25, 100, 300 microM) which produced rightwards shifts of the baclofen concentration-response curves (pA(2) value=4.8+/-0.1). In electrically stimulated slices preloaded with [3H]GABA, CGP 47332A increased its release (EC(150)=100 microM) through antagonism of GABA(B) autoreceptors. Although CGP 47332A was some six times weaker on GABA(B) auto- than on heteroreceptors, yet its congener lacking the beta-hydroxy substituent displays equal potency in both binding (IC(50)=38 microM) and GABA(B) autoreceptor functional studies (EC(150)=38 microM) as previously reported [Froestl, W., Mickel, S.J. , Von Sprecher, G., Diel, P.J., Hall, R.G., Maier, L., Strub, D., Melillo, V., Baumann, P.A., Bernasconi, R., Gentsch, C., Hauser, K., Jaekel, J., Karlsson, G., Klebs, K., Maitre, L., Marescaux, C., Pozza, M.F., Schmutz, M., Steinmann, M.W., Van Riezen, H., Vassout, A., Mondadori, C., Olpe, H.R., Waldmeier, P.C., Bittiger, H., Phosphinic acid analogues of GABA: 2. Selective, orally active GABA(B) antagonists. J. Med. Chem. 38 (1995) 3313-3331.].

In rat neocortical slices maintained in Mg2+-free Krebs medium, the gamma-aminobutyric acid (GABAB) receptor agonist baclofen concentration-dependently depressed the frequency of spontaneous discharges (EC50 = 6.1 microM). This was reversibly antagonised by 3-aminopropyl-(1,1-difluoro-n-butyl)-phosphinic acid (25, 100, 500 microM) (CGP 47654A) and 3-aminopropyl-P-(alpha-hydroxybenzyl)-phosphinic acid (CGP 46165A) (50, 100, 400 microM) which produced rightwards shifts of the baclofen concentration-response curves, with respective pA2 values of 4.9+/-0.2 and 4.6+/-0.15. Although relatively potent on GABAB heteroreceptors studied here, CGP 47654A and CGP 46165A were 5 and 50 times weaker, respectively, as GABAB autoreceptor antagonists [Froestl, W., Mickel, S.J., Von Sprecher, G., Diel, P.J., Hall, R.G., Maier, L., Strub, D., Melillo, V., Baumann, P.A., Bernasconi, R., Gentsch, C., Hauser, K., Jaekel, J., Karlsson, G., Klebs, K., Maitre, L., Marescaux, C., Pozza, M.F., Schmutz, M., Steinmann, M.W., Van Riezen, H., Vassout, A., Mondadori, C., Olpe, H.R., Waldmeier, P.C., Bittiger, H., 1995. Phosphinic acid analogues of GABA. 2. Selective, orally active GABAB antagonists. J. Med. Chem. 38: 3313-3331.], representing potentially useful ligands for differentiating GABA hetero- and autoreceptors.

In rat neocortical slices maintained in Mg2+-free Krebs medium, baclofen depressed the rate of spontaneous discharges in a concentration-dependent manner (EC50 = 4.5 microM). This depression was reversibly antagonised by 5-(S,R)-hydroxymethyl-5-methylmorpholinyl-2-(R,S)-acetic acid (Sch 54679) and 2-(R,S)-5-[spirocyclopentyl]-morpholinyl-acetic acid (Sch 51324) (respective pA2 values of 5.8+/-0.15 and 5.4+/-0.2). In electrically-stimulated slices preloaded with [3H]gamma-aminobutyric acid (GABA), Sch 54679 (EC50 = 3 microM) was 2.3 times more potent than Sch 51324 (EC50 = 7 microM) in increasing [3H]GABA release through antagonism of GABA(B) autoreceptors. These structurally novel analogues may be pharmacologically useful for elucidating GABA(B) receptor functions.

Cholinergic twitch responses in the guinea-pig isolated ileum, and spontaneous discharges in rat neocortical slices, were depressed by the GABA(B) receptor agonist baclofen. These actions were reversibly antagonised by the sulphonamide derivatives (R,S)-2-hydroxy-3-phthalimidopropanesulphonamide (HPIPS), 3-amino-N-benzoylpropanesulphonamide (ABPS) and 3-phthalimidopropanesulphonamide (PIPS) which produced rightwards shifts of the baclofen concentration-response curves, with pA2 values ranging between 4.1 and 4.3 in both preparations. From these results, HPIPS, ABPS and PIPS constitute a novel class of antagonists at GABA(B) hetero-receptors.

The pharmacological properties of morpholin-2-yl-phosphinic acids were evaluated on GABA(B) receptors. In rat neocortical slices maintained in Mg2+-free Krebs medium, baclofen, a GABA(B) receptor agonist, produced a concentration-dependent depression of the frequency of spontaneous discharges with an EC50 of 14 +/- 5.5 microM, which was antagonised reversibly by the morpholin-2-yl-phosphinic derivatives. The order of potency was 3-[(3S,6R)-6-[(cyclohexylmethyl)hydroxyphosphinoylmethyl- morpholin-3-yl]benzoic acid (CGP 76290A) (pA2 = 7.1 +/- 0.05) > its enantiomer 3-[(3R,6S)-6-[(cyclohexylmethyl)hydroxyphosphinoylmethyl]-++ +morpholin-3-yl]benzoic acid (CGP 76291A) (pA2 = 6.8 +/- 0.1) > cyclohexylmethyl-[(2R',5S')-5-(3-nitrophenyl)-morpholin-2-++ +ylmethyl]phosphinic acid (CGP 71978) (pA2 = 6.5 +/- 0.05) > cyclohexylmethyl-[(2R,5S)-5-phenyl-morpholin-2-ylmethyl++ +]phosphinic acid (CGP 71980) (pA2 = 6.3 +/- 0.15) > its enantiomer cyclohexylmethyl-[(2S,5R)-5-phenyl-morpholin-2-ylmethyl++ +]phosphinic acid (CGP 71979) (pA2 = 5.8 +/- 0.1). An open chain analogue of CGP 76290A, CGP 56999A (3-[1(R)-[(3-cyclohexylmethyl-hydroxyphosphinoyl)-2(S)-hydro xypropyl-amino]-ethyl]benzoic acid lithium salt) gave a pA2 of 6.6 +/- 0.2. In GABA(B) receptor binding assays, CGP 71982 (the racemic mixture of CGP 76290A and CGP 76291A), CGP 76290A, CGP 76291A, CGP 71978, CGP 71980 and CGP 71979 had IC50 values against [3H]CGP 27492 binding of 8, 1.85, 69, 124, 326 and 1460 nM, respectively. In electrically-evoked [3H]GABA release from rat cortical slices, CGP 71982, CGP 71978, CGP 71980 and its enantiomer CGP 71979, antagonised GABA(B) autoreceptors with EC150 values of 2.5, 33, 181 and 474 nM, respectively. These compounds form a novel class of potent GABA(B) receptor antagonists.

The pharmacological properties of (+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid (Sch 50911) were evaluated on GABA(B) receptors in rat neocortical slices. The GABA(B) receptor agonist, baclofen, produced a concentration-dependent depression of the frequency of spontaneous discharges in slices maintained in Mg2+-free Krebs medium with an EC50 of 6 microM, reversibly antagonised by Sch 50911 (5, 10 and 25 microM) with an apparent pA2 of 6.0 +/- 0.1. The (-) enantiomer Sch 50910 (500 microM) and the racemic des-methyl analogue Sch 48588 (500 microM) were inactive. In slices preloaded with [3H]GABA, Sch 50911 antagonised GABA(B) autoreceptors, increasing the electrically-stimulated 3H overflow in a concentration-dependent manner, with an IC50 of 3 microM. The maximal effect (148 +/- 10.5%) was found at 10 microM, but at 50 microM the response was reduced to 67 +/- 19%. In contrast, evoked release was unaffected by Sch 50910 (100 microM) whilst Sch 48588 at 100 microM increased the overflow by 51.3 +/- 11.6%. In summary, Sch 50911 is a relatively potent antagonist of considerable potential in studies of GABA(B) receptor function.

In rat neocortical slices maintained in Mg2+-free Krebs medium, the GABAB receptor agonists baclofen and 3-aminopropylphosphinic acid dose-dependently reduced the frequency of spontaneous discharges, 3-aminopropylphosphinic acid being 10 times less potent than baclofen. These were sensitive to the antagonist CGP 52432 (3-[[3,4-dichloro-phenyl)methyl]-amino]propyl](-P-diethoxymethyl)- phosphinic acid) (1, 5 and 10 microM). The GABA uptake inhibitor NO-711 (1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-+ ++pyridinecarboxylic acid) (5 and 10 microM) produced 2.9 and 9 fold increases in the potency of 3-aminopropylphosphinic acid without affecting baclofen-induced responses. In this study, the low potency of 3-aminopropylphosphinic acid when compared to baclofen, may be attributed to its uptake by NO-711-sensitive GABA transporters.

In rat neocortical slices maintained in Mg2+-free Krebs medium, baclofen and its thienyl analogs, 4-amino-3-(5-chlorothien-2-yl)-butanoic acid (5h), 4-amino-3-(5-methylthien-2-yl)-butanoic acid (5d), 4-amino-3-(5-bromothien-2-yl)-butanoic acid (5f) and 4-amino-3-(thien-3-yl)-butanoic acid (5j) dose-dependently suppressed the spontaneous discharges, antagonised by the GABA(B) receptor antagonist 2-hydroxysaclofen (200 microM). Their relative potencies were baclofen > 5h > 5d > 5f > 5j. These heterocyclic analogs may prove useful as GABA(B) receptor agonists in functional studies.

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.

Racemic 3-amino-2-hydroxy-N-(4-nitrophenyl)-propanesulphonamide (AHPNS), a sulphonamide analog of GABA, reversibly and competitively antagonised the concentration-dependent depression of cholinergic twitch contractions by baclofen, in the electrically stimulated guinea-pig isolated ileum, with a pA2 of 4.0 +/- 0.2. In the rat neocortex, maintained in Mg(2+)-free medium, AHPNS (100-500 microM) also reversibly antagonised the baclofen (10 microM)-induced suppression of spontaneous discharges. AHPNS is a new class of GABAB receptor antagonist that has central and peripheral blocking actions.

Interactions of N-ethylmaleimide and aluminium fluoride (AlF - 4) with GABAB receptors have been examined using spontaneously discharging rat neocortical slices. The suppression of discharges by the GABAB receptor agonist baclofen (5-10 mu M) was irreversibly prevented by N-ethylmaleimide (10-50 mu M) and its analog N-phenylmaleimide (10-50 mu M), whilst superfusion of slices with NaF (10 mM) and AlCl3 (100 mu M) to form a fluoroaluminate (AlF - 4) complex markedly potentiated the action of baclofen. The lipoxygenase inhibitors, nordihydroguaiaretic acid (10-50 mu M) and eicosatetraynoic acid (10-50 mu M) or the phospholipase A2 inhibitor bromophenacylbromide (50-100 mu M) did not affect the response to baclofen. The depressant action of baclofen is evidently mediated through G-proteins, but is not dependent on arachidonic acid metabolites.

In the guinea-pig isolated ileum, (RS)-(+/-)-baclofen induced a depression of cholinergic twitch contractions, reversibly and competitively antagonised by (S)-2-hydroxysaclofen (pA2 = 5.2 +/- 0.2), but not by (R)-2-hyroxysaclofen. The depression of excitatory field potentials by baclofen ( 5 mu M) in rat CA1 hippocampal slices was antagonised by (S)-2-hydroxysaclofen (100 mu m) (pA2 = 4.3), whilst in rat neocortex, (S)-2-hyroxysaclofen (50-500 mu M) antagonised the baclofen (10 mu M)-induced suppression of spontaneous discharges, the (R)-enantiomer being inactive. These results show that (S)-2-hydroxysaclofen is the active antagonist at central and peripheral GABAB receptors.

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.

In the guinea-pig isolated ileum, both baclofen and gamma-guanidinobaclofen elicited dose-dependent depression of cholinergic twitch contractions, sensitive to the GABAB receptor antagonists phaclofen and 2-hydroxysaclofen. gamma-Guanidinobaclofen was 5 times less potent than R,S-(+/-)-baclofen in depressing the contractions. The corresponding GABA analogs, guanidinoacetic acid, beta-guanidinopropionic acid and gamma-guanidinobutanoic acid were inactive. In rat neocortical slices maintained in Mg(2+)-free medium, baclofen (1-50 microM) reduced the amplitude and rate, whilst gamma-guanidinobaclofen (1 mM) has a very weak GABAB receptor agonist action, 100 times weaker than baclofen. gamma-Guanidinobaclofen is therefore a GABAB receptor agonist, more potent at peripheral than central GABAB receptors.

Interactions of amiloride with GABAB receptors have been examined using spontaneously discharging rat neocortical slices. These discharges were suppressed by the GABAB receptor agonist baclofen (10 microM), and were prevented by amiloride and its analogs 5-(N,N-dimethyl)-amiloride, 5-(N-methyl-N-isobutyl)-amiloride and benzamil, but not by triamterene (100-500 microM). Each of these also increased the spontaneous discharge rate and reduced the discharge amplitude. The action of amiloride and its analogs in preventing the action of baclofen, may involve allosteric modification of the receptor binding sites via guanine nucleotide-binding proteins, or an indirect effect through antagonism of co-activated adenosine A1 receptors.

The activity of the nitropropane analog of baclofen, 3-amino-2-(4-chlorophenyl)-nitropropane (N-BAC), has been examined at central and peripheral GABAB receptors. N-BAC was less potent than baclofen as a GABAB receptor agonist in depressing repetitive twitch contractions in the guinea-pig isolated ileum (IC50s for baclofen = 4.1 +/- 1.3 microM; N-BAC = 9.2 +/- 0.3 microM) and vas deferens (IC50s for baclofen = 30 microM; N-BAC = 100 microM), competitively antagonised by phaclofen, 2-hydroxysaclofen and CGP 35348 (3-aminopropyl-P-di-ethoxymethylphosphinic acid). In the ileum, the pA2 values for CGP 35348 with baclofen (4.7 +/- 0.2) and N-BAC (4.6 +/- 0.3) were not significantly different (P > 0.05), indicating that both agonists activate the same receptor type. By contrast, in rat neocortical slices, N-BAC was 20 times weaker than baclofen in attenuating spontaneous discharges, sensitive to CGP 35348, whilst it was 100 times less potent than baclofen in depressing evoked CA1 population spikes in the hippocampus. This new GABAB receptor agonist, N-BAC, is thus more active at peripheral than central GABAB receptors.

R-(-)-beta-phenyl-GABA has been compared at GABAB receptors using cortical and ileal preparations. R-(-)-beta-phenyl-GABA (EC50 = 25 microM) was a less potent full agonist than R,S-(+/-)-baclofen (EC50 = 2.5 microM), in depressing CA1 population spikes of rat hippocampal slices, and 5 times less potent in attenuating the spontaneous discharges of rat neocortex. However, R-(-)-beta-phenyl-GABA (100-400 microM) was only a weak partial agonist in the ileum. All these actions were sensitive to CGP 35348 (3-aminopropyl-(P-diethoxymethyl)-phosphinic acid) and therefore mediated by GABAB receptors.

In the guinea-pig isolated ileum, beta-(2-thienyl)-gamma-aminobutyric acid (BTG; 100-500 microM) reversibly and competitively (pA2 = 4.3 +/- 0.1) antagonised the baclofen-induced (5-100 microM) depression of cholinergic twitch contractions, but not that to adenosine or morphine. By contrast, in rat neocortical slice preparations, BTG (100-500 microM) acted as an agonist, abolishing the frequency and amplitude of spontaneous discharges, sensitive to 2-hydroxysaclofen (100-500 microM). BTG exhibits differential actions at GABAB receptors in brain and periphery.

In guinea-pig isolated ileal preparations, the 5-methylthien-2-yl (5d), 5-bromothien-2-yl (5f) and 5-chlorothien-2-yl (5h) analogs of baclofen depressed twitch responses to field stimulation in a dose-dependent manner. These actions were reversibly and competitively antagonised by 2-hydroxysaclofen but not by naloxone, phentolamine, propranolol or theophylline. The relative potencies (EC50 values) were baclofen (10 microM) greater than 5h (40 microM) greater than 5d (80 microM) greater than 5f (120 microM). These analogs represent a novel class of specific GABAB receptor agonists which, like baclofen, should readily enter the brain.

A new series of GABAB receptor antagonists, based on short-chain baclofen analogues has been investigated. In guinea-pig isolated ileal preparations, the GABAB receptor-mediated, baclofen-induced depression of cholinergic twitch responses was reversibly and competitively antagonised by the short-chain baclofen analogues 3-amino-3-(p-chlorophenyl)propionic acid (apparent pA2 = 3.5), 2-amino-2-(p-chlorophenyl)ethanephosphonic acid (apparent pA2 = 3.8), and 2-amino-2-(p-chlorophenyl)ethanesulphonic acid apparent pA2 = 4.0). The corresponding des-chloro analogues were all less active. These compounds represent another class of GABAB receptor antagonists which may cross the blood-brain barrier.

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.

In the isolated ileum of the guinea-pig, gamma-aminobutyric acid (GABA) elicited both GABAA- and GABAB-receptor-mediated responses. Barbiturates significantly potentiated the GABAA receptor-induced contractions over the lower dose range of applied GABA (less than 50 microM), without affecting the GABAB-receptor-induced 'after-relaxation', their relative potencies being thiopentone (ThP) greater than pentobarbitone (PB) greater than barbitone (Bb) greater than phenobarbitone (PhB). Also, higher doses of ThP, PB and Bb elicited bicuculline- and picrotoxinin-sensitive GABA-like contractions. PB shifted the dose-response curve for GABA in the presence of bicuculline methochloride to the left without altering the slope or the dose ratio, and not only displaced the dose-response curve for GABA in the presence of picrotoxinin but also restored the slope and the maximum response towards that of the control GABA-induced response. These results further characterise the GABAA-receptor sites of the guinea-pig enteric nervous system, where GABA is evidently a neurotransmitter.

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.

The actions of gamma-aminobutyric acid (GABA) and its analogues, 3-amino-1-propanesulphonic acid (3APS) and baclofen, have been investigated using isolated segments of the guinea-pig ileum and distal colon. GABA and 3APS, but not baclofen, induced GABAA-receptor mediated effects; prompt, dose-dependent contractions of the ileum which were antagonised by bicuculline, picrotoxinin, piretanide, tetramethylenedisulphotetramine, atropine and tetrodotoxin. Baclofen and GABA, but not 3APS, induced a dose-dependent GABAB-receptor mediated depression of electrically elicited twitch contractions of the ileum, unaffected by the GABAA-receptor antagonists or by antagonism of adenosine, adrenergic, opiate or nicotinic receptors. In the distal colon, baclofen and GABA caused a bicuculline- and picrotoxinin-insensitive depression of spontaneous cholinergic contractions. Desensitization to GABA and baclofen, and cross-desensitization to both agonists was observed. Combined antagonism of GABAA-receptors and desensitization to baclofen slowed pellet expulsion to the same extent as GABA desensitization alone, indicating that both GABAA- and GABAB-receptor sites are involved in this modification of peristalsis by GABA.

Two novel beta-(benzo[b]furan) analogues of baclofen (4-amino-3-benzo[b]furan-2-ylbutanoic acid, 9G, and 4-amino-3-(5-methoxybenzo[b]furan-2-yl)butanoic acid, 9H) antagonised the baclofen-induced depression of twitch contractions in the guinea-pig isolated ileum (estimated apparent pA2 3.9 and 4.1 respectively); both 9G and 9H also antagonised in a dose-dependent manner the baclofen-induced reduction of repetitive paroxysmal discharges in rat neo-cortical slice preparations maintained in Mg2+-free Krebs solution. These benzofurans evidently represents a new class of GABAB-receptor antagonist.

In preloaded, isolated segments of duodenum, jejunum, ileum and colon, ethylenediamine (EDA) caused a 3-mercaptopropionic acid (3-MPA)-sensitive release of [3H]GABA. Both EDA and GABA caused a contraction and a delayed 'after-relaxation' in the ileum, but only a delta-aminovaleric acid (DAVA)-sensitive relaxation in other intestinal segments; these actions of EDA were reduced by 3-MPA and therefore due to release of endogenous GABA. Baclofen induced a DAVA-sensitive relaxation in all tissues. Evidently, GABA modulates cholinergic excitation of the smooth muscle at all levels of the intestine.

Possible involvement of gamma-aminobutyric acid (GABA) in intestinal motility of the guinea-pig has been investigated using the speed of propulsion of faecal pellets along isolated segments of guinea-pig distal colon, and the rate of pellet expulsion from freshly excised colon. GABA antagonism, by bicuculline or by tachyphylaxis to GABA, substantially reduced intestinal motility and generally reduced the amplitude of the reflex contraction associated with pellet propulsion. These results support the view that GABAergic mechanisms may be involved in the propulsive activity of the guinea-pig distal colon.

Axons of the rat isolated cervical vagus nerve responded to gamma-aminobutyric acid (GABA) and 5-hydroxytryptamine (5-HT) with a readily reversible dose-dependent depolarization. Prior application of 5-HT at 2 or 30 microM equally depressed the depolarizing responses to GABA, shifting the GABA dose-response curve to the right and depressing the maximum; by contrast, responses to 5-HT were affected only by large doses of GABA (greater than 300 microM), and to a lesser degree. In addition, the fade in responses to high doses of GABA was also reduced in the presence of 5-HT. It is suggested that such all-or-none depressive actions may explain the blockade of GABA-induced responses by 5-HT observed in other tissues such as the guinea-pig ileum.

Both baclofen and gamma-aminobutyric acid (GABA) induced a dose-dependent depression of cholinergic twitch contractions in the transmurally stimulated guinea-pig isolated ileum. Over a range of 0.6-2.4 mM Ca2+, the degree of depression was inversely related to the Ca2+ concentration, with an increased sensitivity and sinistral shift of the dose-response curve at lower Ca2+ concentrations. Partial occupation of Ca2+ channels by Ruthenium Red (0.1 microM) also potentiated the depressive responses to baclofen and GABA. It is concluded that these agonists, acting through GABAB receptors, limit the availability of Ca2+ required for neurotransmitter release in myenteric motor nerves.

4,6,6-Trimethylcaprolactam antagonised GABAA receptor-mediated contractile responses in guinea-pig isolated ileum, displacing the GABA dose-response curve to the right in a non-parallel manner, and causing a depression of the maximum response. Pentobarbitone not only potentiated the GABAA receptor-mediated contractions but also reversed this non-competitive antagonism by 4,6,6-trimethylcaprolactam, shifting the dose-response curve for GABA to the left and restoring the maximum response. It is conclude that this caprolactam acts at the picrotoxin-barbiturate site on the Cl(-)-ionophore complex.

Pregnenolone and its sulphate had concentration-dependent dual actions on gamma-aminobutyric acid (GABAA)-receptor-mediated ileal contractile responses, enhancing at 0.001-1 microM but inhibiting at 10 microM. There was a sinistral shift of the GABA dose-response curve in the presence of 0.01 microM pregnenolone, with a significant potentiation over the lower concentration range of GABA (3-30 microM), whilst 10 microM pregnenolone depressed the curve by 50% without a significant change in the EC50, in a manner resembling non-competitive inhibition with picrotoxin. Pregnenolone and its sulphate evidently have modulatory actions at ileal GABAA-receptor complexes in keeping with those seen using neurochemical studies in the central nervous system.