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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.

Bicuculline-insensitive receptors for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), GABAB receptors, are a distinct subclass of receptors that mediate depression of synaptic transmission and contribute to neuronal inhibition. When activated, these receptors reduce transmission at excitatory and inhibitory synapses, as a result of an increase in K+ conductance, or a decrease in voltage-dependent Ca2+ currents. They are also linked to G-proteins, or intracellular effector systems in a very complex manner. The recent development of highly specific and potent agonists and antagonists for these receptors has led to a much better understanding of their physiology and pharmacology, including their heterogeneity, as well as their molecular biology. Over the past year, expression and cloning studies have contributed to major advances in characterizing GABAB receptor structure, with the discovery of the amino acid sequences of GABABR1a/R1b splice variants and GABABR2 receptors. These isoforms are widely distributed throughout the nervous system, and can be functionally expressed. Importantly, GABABR2 receptors can form a heteromeric assembly with GABABR1 proteins to operate as a heterodimer that displays robust coupling to inward-rectifying K+ channels, as well as inhibition of forskolin-stimulated adenylate cyclase activity. Further insights underlying the mechanisms of GABAB receptor functions can now be gained, leading ultimately to the therapeutic potential of drugs acting at these sites. It is increasingly clear that new information on GABAB receptor molecular structure will provide a plethora of targets for pharmaceutical intervention in areas such as drug addiction, nociception and absence seizures. This review summarizes the renewed efforts, and highlights the recent advances emerging in this field.

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, 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.

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.

(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.

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.

The effects of five phosphonic derivatives of GABA on the release of [3H]-GABA from rat neocortical slices, preloaded with [3H]-GABA, were investigated. Phaclofen and 4-aminobutylphosphonic acid (4-ABPA) increased the overflow of [3H] evoked by electrical stimulation (2 Hz) in a concentration-dependent manner, with similar potencies (phaclofen EC50=0.3 mmol/l, 4-ABPA EC50=0.4 mmol/l). At 3 mmol/l, phaclofen increased the release of [3H]-GABA by 82.6+/-8.6%, and 4-ABPA increased the release by 81.3+/-9.0%. 2-Amino-ethylphosphonic acid (2-AEPA) increased the overflow of [3H] by 46.8+/-10.9% at the highest concentration tested (3 mmol/l). In contrast, the lower phosphonic homologue 3-aminopropylphosphonic acid (3-APPA), and 2-amino-2-(p-chlorophenyl)-ethylphosphonic acid (2-CPEPA), a baclofen analogue, did not modify the stimulated overflow. These results suggest that phaclofen, 4-ABPA and 2-AEPA are antagonists at GABA(B) autoreceptors, the latter being the weakest antagonist, whilst neither 3-APPA nor 2-CPEPA are active at these receptors. Since phaclofen, 4-ABPA and 2-CPEPA are antagonists and 3-APPA a partial agonist/antagonist on GABA(B) heteroreceptors, the lack of effect of 3-APPA and 2-CPEPA on [3H]-GABA release in this study suggests that GABA(B) autoreceptors may be pharmacologically distinct from the heteroreceptors.

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.

1. While GABAB antagonists have been examined in vitro, very few have been tested in vivo. A range of GABAB antagonists were tested against baclofen-induced muscle relaxation and hypothermia. 2. The GABAB antagonists exhibited a range of in vivo activity profiles. 3. CGP 35348 showed clear antagonist effects, while BPBA and 4-ABPA appeared to have agonist properties. 4. Phaclofen, 2-hydroxysaclofen, 3-APPA and 9G seemed to have little effect in this system at the doses tested. 5. Differences between in vivo and in vitro activity could be explained by differences in blood-brain barrier permeability, or possible differences in affinities for the sub-classes of GABAB receptors.

GABAB receptors are a distinct subclass of receptors for the major inhibitory transmitter 4-aminobutanoic acid (GABA) that mediate depression of synaptic transmission and contribute to the inhibition controlling neuronal excitability. The development of specific agonists and antagonists for these receptors has led to a better understanding of their physiology and pharmacology, highlighting their diverse coupling to different intracellular effectors through Gi/G(o) proteins. This review emphasises our current knowledge of the neurophysiology and neurochemistry of GABAB receptors, including their heterogeneity, as well as the therapeutic potential of drugs acting at these sites.

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.

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.

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.