David I.B. Kerr

Phaclofen: a peripheral and central baclofen antagonist

Phaclofen, the phosphonic acid derivative of baclofen, reversibly antagonized the depression of the cholinergic twitch response of the guinea pig ileum and distal colon by either baclofen or GABA. When administered microelectrophoretically, phaclofen reversibly blocked the presumed presynaptic reduction by baclofen of the monosynaptic excitation of spinal interneurones by impulses in primary afferent fibres of the cat but did not block the postsynaptic depressant action of baclofen on these neurones. Phaclofen may thus be useful in determining the physiological significance of central and peripheral bicuculline-insensitive receptors with which GABA and (-)-baclofen interact.

GABAA- and GABAB-receptor-mediated modification of intestinal motility

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.

GABA-receptors in peripheral tissues

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.

Uptake and stimulus-evoked release of [3H]-γ-aminobutyric acid by myenteric nerves of guinea-pig intestine

1 Following preloading with [3H]‐γ‐aminobutyric acid ([3H]‐GABA), in the presence of β‐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.

The actions of 2-hydroxy-saclofen at presynaptic GABAB receptors in the rat hippocampus

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.

The effect of GABA antagonism on propulsive activity of the guinea-pig large 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.

Blockade of the late IPSP in rat CA1 hippocampal neurons by 2-hydroxy-saclofen

The effects of the GABAB receptor antagonist 2-hydroxy-saclofen were studied using intracellular recording of synaptic potential from CA1 hippocampal neurons. 2-Hydroxy-saclofen (50-200 microM) reversibly blocked the late, GABAB receptor-mediated inhibitory postsynaptic potential (IPSP) but not the early, GABAA receptor-mediated IPSP. In addition, the hyperpolarizing response to baclofen was reduced by similar concentrations of 2-hydroxy-saclofen. This suggests that 2-hydroxy-saclofen is a potent antagonist at postsynaptic GABAB receptors on hippocampal neurons.

Evidence that ethylenediamine acts in the isolated ileum of the guinea-pig by releasing endogenous GABA.

1 Ethylenediamine (EDA) released [3H]‐γ‐aminobutyric acid ([3H]‐GABA) in a dose‐dependent manner from the isolated preloaded ileum of the guinea‐pig maintained in Krebs‐bicarbonate solution (pH 7.4, 37°C), in the presence of β‐alanine and amino‐oxyacetic acid (AOAA) to prevent GABA uptake into glial cells and catabolism. This release was reversibly prevented by 3‐mercaptopropionic acid (3‐MPA), also in a dose‐dependent manner. 2 In the isolated ileal preparations of the guinea‐pig maintained in Krebs‐bicarbonate solution, EDA induced a dose‐dependent transient, cholinergic contractile response (GABAA‐receptor‐mediated effect), followed by an ‘after‐relaxation’ (GABAB‐receptor‐mediated effect). EDA also induced a transient contraction superimposed on repetitive twitch responses to electrical transmural stimulation of the cholinergic neurones, followed by a depression of the twitch contractions. 3 This GABAA‐receptor‐mediated contraction was antagonized by bicuculline methochloride and picrotoxinin, whilst the GABAB‐receptor‐mediated ‘after‐relaxation’, and depression of cholinergic twitch contractions, was susceptible to antagonism by δ‐aminovaleric acid. The pA2 value for bicuculline methochloride antagonism of EDA was estimated to be 5.8, identical with that for GABA. 4 3‐Mercaptopropionic acid also prevented these pharmacological actions induced by EDA without affecting responses to GABA, 3‐aminopropranesulphonic acid, muscimol, baclofen or the twitch responses to transmural stimulation. 5 It is concluded that EDA releases both [3H]‐GABA and endogenous GABA in the guinea‐pig ileum, thus providing further evidence that GABA is a transmitter in the enteric nervous system.

Arylalkylamines are a novel class of positive allosteric modulators at GABAB receptors in rat neocortex

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.

Autoradiographic study of the distribution of [3H]γ-aminobutyrate-accumulating neural elements in guinea-pig intestine: Evidence for a transmitter function of γ-aminobutyrate

Abstract High affinity uptake, and the distribution of 3H-radiolabelled γ-aminobutyrate (GABA), cis-3-aminocyclohexanecarboxylic acid, β-alanine, proline, and leucine have been examined autoradiographically in laminar preparations of the myenteric plexus from the guinea-pig intestine. Following labelling with [3H]proline and [3H]leucine, which are incorporated into neurons, silver grains were concentrated over recognisable perikarya in the ganglia and meshworks of the plexus, whilst [3H]GABA labelled a smaller proportion of neurons and their processes. Specificity of labelling in the sites of [3H]GABA-uptake was established using combinations of labelled and unlabelled GABA, β-alanine, and cis-3-aminocyclohexanecarboxylic acid, substrates for gial or neuronal high affinity GABA uptake systems. Only myenteric neurons and their processes were labelled significantly by [3H]GABA and its analogue cis-3-[3H]aminocyclohexanecarboxylic acid. Using autoradiographs of laminar preparations and paraffin sections, [3H]GABA labelling was found over nerve fibre bundles that could be traced from their ganglionic origins through the interconnecting meshworks of the myenteric plexus into the innervation of the deep muscular plexus of the circular muscle layer where GABA is evidently concerned with prejunctional modulation of transmitter release. The extensive but selective distribution of [3H]GABA high affinity uptake sites in neural elements of the guinea-pig myenteric plexus is consistent with GABA being an enteric neurotransmitter.