Search

Metabotropic gamma-aminobutyric acid(B) (GABAB) receptors for the major inhibitory transmitter GABA, together with metabotropic glutamate (mGLuRs) receptors, the extracellular calcium-sensing receptors (CaSRs), some V2R pheromone receptors and T1R taste receptors, belong to the family of 3 G-protein-coupled receptors (GPCRs). GABAB receptors are known to control neuronal excitability and modulate synaptic neurotransmission, playing a very important role in many physiological activities. These receptors are widely expressed and distributed in the nervous system and have been implicated in a variety of neurodegenerative and pathophysiological disorders including epilepsy, spasticity, chronic pain, depression, schizophrenia and drug addiction. To form a functional receptor entity, GABAB receptors must exist as a heterodimer consisting of GABAB1 and GABAB2 receptor subtypes with two 7-transmembrane proteins, and these subunits arise from distinct genes. The GABAB1 subunit binds the endogenous ligand within its extracellular N-terminus, whilst the GABAB2 subunit is not only essential for the correct trafficking of the GABAB1 subunit to the cell surface, but is also responsible for the interaction of the receptor with its cognate G-protein. Allosteric modulation has recently been recognized as an alternative pharmacological approach to gain selectivity in drug action. It is now generally accepted that modulators acting at the allosteric sites provide a novel perspective for the development of subtype-selective agents acting at GPCRs. These agents interact with allosteric binding sites quite separate from the highly conserved agonist binding region. In this review, we present a new class of phenylalkylamines, based on the lead compound fendiline, that are potent positive potentiators of GABAB receptor-mediated function and discuss their putative clinical applications. It is proposed that these new modulators may have therapeutic value in GABAB receptor pharmacology and are capable of selectively modifying GABAB receptor function. The allosteric modulators are offering an attractive and novel means to identify new leads, that are devoid of side effects associated with GABAB receptor agonists, and may, therefore, represent a major advance in the drug discovery process.

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 Mg2+-free Kreb's medium, the effects of Ca2+ concentration on repetitive spontaneous discharges and on GABA(B) receptor-mediated responses were investigated. Over a concentration range of 0.3-2.4 mM Ca2+, there was a reduction of discharge amplitude, with a 50 +/- 6.5% reduction in 0.3 mM Ca2+, whilst the burst frequency remained unaffected. Baclofen, the GABA(B) receptor agonist, produced a concentration-dependent depression of discharge frequency, reversibly antagonised by the antagonist (+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid (Sch 50911). The EC50 value for baclofen in 2.4 mM Ca2+ was 11 microM, while the EC50 values in 0.3, 0.6, 1.2, and 1.8 mM Ca2+ were 1.3, 2.5, 3.6, and 10 microM, respectively, resulting in 8.5, 4.4, 3.1, and 1.1-fold leftward shifts. This enhanced action of baclofen in low extracellular Ca2+ concentrations in the neocortex may be the result of a lower concentration gradient which reinforces the action of baclofen.

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, 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.

Release of excitatory transmitter from boutons on crayfish nerve terminals was inhibited by (R,S)-baclofen, an agonist at GABAB receptors. Baclofen had no postsynaptic actions as it reduced quantal content without affecting quantal amplitude. The effect of baclofen increased with concentration producing 18% inhibition at 10 microM; EC50, 50% inhibition at 30 microM; maximal inhibition, 85% at 100 microM and higher. There was no desensitization, even with 200 or 320 microM baclofen. Phaclofen, an antagonist at GABAB receptors, competitively antagonized the inhibitory action of baclofen (KD = 50 microM, equivalent to a pA2 = 4.3 +/- 0.1). Phaclofen on its own at concentrations below 200 microM had no effect on release, whereas at 200 microM phaclofen itself increased the control level of release by 60%, as did 2-hydroxy-saclofen (200 microM), another antagonist at GABAB receptors. This increase was evidently due to antagonism of a persistent level of GABA in the synaptic cleft, since the effect was abolished by destruction of the presynaptic inhibitory fiber, using intra-axonal pronase. We conclude that presynaptic GABAB receptors, with a pharmacological profile similar to that of mammalian GABAB receptors, are involved in the control of transmitter release at the crayfish neuromuscular junction.

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.

(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.

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.

Participation of adenosine receptors in the depression of synaptic transmission during hypoxia, and the production of multiple populations spikes in the pyramidal neurons following hypoxia, has been investigated in the CA1 area of the rat hippocampal slice. A method is presented for analysing such hyperexcitability, using input/output curves of the second population spike. This method provides evidence that rebound hyperexcitability following hypoxia or prolonged adenosine-mediated inhibition results from an increase in excitability of the CA1 pyramidal neurons rather than from an increase in excitatory neurotransmitter release. Hypoxia-induced depression of the synaptic components of evoked field potentials was blocked in a concentration dependent manner by the selective A1 receptor antagonist 8-cyclopentyltheophylline (8-CPT), demonstrating extracellular accumulation of adenosine during hypoxia. Upon reoxygenation of slices following 30 min hypoxia, multiple population spikes were evoked by a single orthodromic stimulus in slices that exhibited only a single population spike prior to hypoxia. Such post-hypoxic hyperexcitability was not prevented by superfusion of slices with 8-CPT during hypoxia. Depression of synaptic transmission by 30 min superfusion of slices with 50 microM adenosine was also followed, upon washout, by the appearance of multiple population spikes. However, such hyperexcitability could not be produced by superfusion with adenosine analogues selective for A1 receptors, cyclopentyladenosine, selective for A2a receptors, 2-p-(2-carboxyethyl)phenetheylamino-5'-ethylcarboxamidoadenosine (CGS 21680), or active at A2a and A2b receptors, N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine, suggesting that adenosine receptors other than the A1, A2a or A2b subtypes are involved in its generation.

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, 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 comparison has been made of the effects of carbon monoxide (CO) or nitrogen (N2) exposure on synaptic transmission in the hippocampal slice. CA1 field potentials, evoked by Schaffer collateral stimulation, were unaffected by superfusion of slices with artificial cerebral spinal fluid (ACSF) equilibrated with either 15% CO or 15% N2 for 120 min. However, superfusion with hypoxic ACSF equilibrated with either 85% CO or 85% N2 caused a rapid depression of synaptic transmission. Reperfusion with control ACSF following 30 min hypoxia led to recovery of evoked responses and a slight hyperexcitability. In the hippocampal slice, synaptic transmission, as assessed by input/output curves, was not different during or following hypoxia induced by exposure to CO or N2. In the short term, CO is not toxic.

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.

Mink lung epithelial cells were transfected with c-myc and activated H-ras genes. The transfected sublines formed colonies in soft agar and were tumorigenic when injected subcutaneously into athymic nude mice. DNA synthesis was measured in each of the cell lines by 3H-thymidine incorporation and in the parent line there was dose related stimulation of DNA synthesis by epidermal growth factor (EGF) and inhibition by transforming growth factor-beta (TGF-beta). The c-myc transfected line had a reduced inhibitory response to TGF-beta and an exaggerated stimulatory response to EGF whereas the activated H-ras1 transfected line did not respond to TGF-beta or EGF. The activated H-ras1 transfected line was significantly more resistant to doxorubicin (ID50, 4.4 nM) and vincristine (ID50, 4.9 nM) than the parent mink lung epithelial cell line (ID50, 2.7 nM and 2.4 nM respectively). It would appear that oncogene transfection can alter the sensitivity of mink lung epithelial cells to both exogenous growth factors and cytotoxic drugs.

The actions of 2-hydroxy-saclofen (2-OH-S), a recently developed analog of baclofen, were studied at presynaptic GABAB receptors in the rat hippocampal slice. Baclofen (0.5-20 microM) reduces the amplitude of excitatory postsynaptic potentials (EPSPs) recorded from hippocampal CA1 pyramidal neurons. In the presence of 200-500 microM 2-OH-S, the synaptic depressant action of baclofen is significantly reduced. These data show that 2-OH-S is an effective antagonist at presynaptic GABAB receptors on excitatory terminals in the hippocampus.

In the guinea-pig isolated ileum, cortisone at 0.001-10 nM induced a non-competitive, dose-dependent antagonism of GABAA-receptor-mediated contractile responses to applied GABA, depressing the maximum contractile response to GABA (100 microM), without affecting contractile responses to acetylcholine or cholinergic twitch contractions. At higher concentrations (greater than 10 nM), cortisone depressed contractile responses to acetylcholine (10-100 nM) and cholinergic twitch responses to transmural stimulation. Cortisone is thus the most potent non-competitive antagonist at GABAA-receptor complexes in the guinea-pig ileum. From molecular modelling, sterically there appeared little difference between cortisone and cortisol, the latter being an enhancer of GABAA-receptor-mediated action in the ileum. However, there were significant differences in electrostatic potentials between the two steroids, due to the different levels of oxidation at C11 which may contribute to such opposing actions.

The actions of the GABA analog 3-aminopropanephosphinic acid (3-APA) were studied in the guinea-pig isolated ileal preparation and at synapses between cultured rat hippocampal neurons. Like the GABAB receptor agonist, baclofen, 3-APA inhibited the electrically evoked ileal twitch. The EC50 for 3-APA was 0.8 microM; the EC50 for baclofen was 9 microM. In addition, the depressant responses to 3-APA and baclofen were blocked by the GABAB receptor antagonists phaclofen, saclofen, 2-hydroxy-saclofen and delta-aminovaleric acid. 3-APA also mimicked the presynaptic action of baclofen at GABAergic synapses between embryonic rat hippocampal neurons in culture. 3-APA reduced the amplitude of inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) by greater than 50% at a concentration of 1 microM, while baclofen reduced synaptic transmission to a similar degree at 10 microM. 3-APA did not alter membrane conductance, nor did the drug alter postsynaptic responses to GABA. These data show that 3-APA is a potent agonist at presynaptic GABAB receptors in the periphery and on GABAergic neurons from the central nervous system. The activity of 3-APA at central postsynaptic GABAB receptors remains to be studied.

The inhibition of the binding of the GABAB agonist [3H](-)-baclofen to rat cerebellar membranes by some sulfonic and phosphonic acid analogues of GABA has been studied. These analogues have been shown to act as GABAB antagonists in the rat cortical wedge and the guinea-pig isolated ileum preparations. The order of potency of phaclofen (IC50 118 microM), 2-hydroxysaclofen (IC50 5.1 microM) and saclofen (IC50 7.8 microM) as inhibitors of [3H](-)-baclofen binding was similar to the order of potency of these compounds as GABAB antagonists, whereas 3-aminopropylphosphonic acid (IC50 1.5 microM) and 4-aminobutyl-phosphonic acid (IC50 3.9 microM) were much more potent than anticipated from their relatively weak GABAB antagonist actions. These results indicate that inhibition of [3H](-)-baclofen binding to rat cerebellar membranes does not reflect antagonist activity at GABAB receptors seen in the rat cortical wedge preparation or the guinea-pig isolated ileum preparation. This may indicate a heterogeneity of GABAB binding and receptor sites.

Baclofen reduced the frequency and aborted the bursts of spontaneous paroxysmal discharges in rat neocortical slices maintained in magnesium-free medium. This action was prevented by pretreatment with barium or caesium, which each increased the ictaform burst frequency, amplitude and duration. 4-Amino-pyridine also increased the burst frequency but reduced the amplitude and did not completely prevent the action of baclofen. Evidently baclofen suppresses such discharges by opening potassium channels normally involved in limiting the burst activity.

Rat neocortical slices maintained in Mg2(+)-free Krebs medium developed spontaneous paroxysmal discharges which were attenuated or suppressed by the gamma-aminobutyric acid-B (GABAB) receptor agonist baclofen, occasionally accompanied by a slight hyperpolarisation, and antagonised by the specific GABAB-receptor antagonist, 2-OH-saclofen. Over the same dose range, the GABA-analogue 3-amino-propylphosphinic acid (3-APA) caused a marked, prompt hyperpolarisation with little or no effect on the frequency of the discharges, although their amplitude was attenuated. In the presence of 2-OH-saclofen, 3-APA still induced a hyperpolarisation but the amplitude of the discharges was no longer affected. This marked difference in action between baclofen and 3-APA in the rat neocortical slices suggests there may be a heterogeneity of GABAB-receptors.

1. The phosphono-analogues of gamma-aminobutyric acid (GABA), 4-amino-butylphosphonic acid (4-ABPA), 3-amino-2-(4-chlorophenyl)-propylphosphonic acid (phaclofen) and 3-amino-2-cyclohexylpropyl-phosphonic acid, each antagonized the GABA- and baclofen-induced GABAB-receptor-mediated depression of twitch responses to transmural stimulation in the guinea-pig isolated ileum, in a concentration-dependent, reversible and surmountable manner (apparent pA2 = 4.0 +/- 0.1, 4 +/- 0.2 and 3.7 +/- 0.2 respectively, compared with 3.9 +/- 0.1 for delta-aminovaleric acid). No such activity was found in a variety of related analogues. 2. By contrast, 3-amino-propylphosphonic acid (3-APPA) behaved as a partial agonist, itself partly depressing ileal twitch contractions in a manner sensitive to 4-ABPA and phaclofen, as well as antagonizing the depression of the ileal twitch by GABA and baclofen (apparent pA2 = 4.0 +/- 0.2). 3. Both 4-ABPA and phaclofen also antagonized the baclofen-induced depression of the twitch in the guinea-pig isolated vas deferens (apparent pA2 = 4.0 +/- 0.1 for each), whilst 3-APPA behaved as a partial agonist, slightly depressing the vas twitch, and antagonised the baclofen-induced depression of the twitch (apparent pA2 = 3.9 +/- 0.1). 4. None of these phosphono-analogues exhibited any action at ileal GABAA-receptors, nor influenced the ileal twitch depression with morphine, adenosine or noradrenaline, suggesting their selectivity as antagonists at GABAB-receptors.

Gamma-aminobutyric acid (GABA) and its receptors are found in a wide range of peripheral tissues, including parts of the peripheral nervous system, endocrines, and non-neural tissues such as smooth muscle and the female reproductive system. In all these, both GABAA- and GABAB-receptor types are found, with good evidence for a physiological role in the gut, pancreatic islets and the urinary bladder. In some tissues, the pharmacology of GABA-induced actions is quite atypical and should be further explored with the newer ligands and modulators for GABAA- and GABAB-receptors.

1--Following preloading with [3H]-gamma-aminobutyric acid ([3H]-GABA), in the presence of beta-alanine to inhibit glial uptake of the label, electrical stimulation caused a frequency-dependent release of tritium as [3H]-GABA from isolated longitudinal-muscle myenteric-plexus preparations of the guinea-pig ileum and colon. 2--The electrically evoked efflux of [3H]-GABA was Ca2+-dependent, virtually abolished by preventing neuronal conduction with tetrodotoxin, and markedly reduced by preloading with [3H]-GABA in the presence of nipecotic acid which is an inhibitor of high affinity GABA-uptake. Veratridine and KCl were less effective than electrical stimulation in evoking [3H]-GABA release. 3--It is concluded that the electrically stimulated efflux of [3H]-GABA originated from GABAergic neurones of the myenteric plexus which had taken up the label. 4--These results provide further evidence to support the suggestion that GABA is a transmitter in the mammalian enteric nervous system.

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.