S. Luzzi

GABAA and GABAB receptor‐mediated effects in guinea‐pig ileum

1 The effects of γ‐aminobutyric acid (GABA) and related substances were examined in guinea‐pig ileum longitudinal muscle. 2 GABA at doses ranging from 10−7 m to 3 m 10−6 m elicited a relaxation while at higher doses (3 × 10−6 m — 10−4 m), as previously described, it caused a contraction followed by relaxation. 3 GABA‐induced relaxation was bicuculline‐insensitive, was mimicked by (—)‐baclofen but not by homotaurine and muscimol. The effect of baclofen was stereospecific. GABA‐ and (—)‐baclofen‐induced relaxations were dose‐dependent and their ED50 values were similar. A specific cross‐desensitization occurred between GABA and (—)‐baclofen. 4 The bicuculline‐insensitive relaxation induced by GABA and (—)‐baclofen was prevented by tetrodotoxin and hyoscine but not by phentolamine plus propranolol, naloxone or theophylline. 5 In preparations in which the muscle tone was raised by histamine or prostaglandin F2α, GABA and (—)‐baclofen induced relaxation to the same extent as before increasing the tone. If the tone was raised by DMPP, a greater bicuculline‐insensitive relaxation occurred. 6 Contraction caused by GABA was bicuculline‐sensitive and was mimicked by homotaurine and muscimol. Contraction was dose‐dependent and muscimol was about three times more potent than GABA or homotaurine. A specific cross‐desensitization occurred between the contractile effects of GABA and those of homotaurine or muscimol. 7 Bicuculline competitively antagonized the contractile effects of GABA, homotaurine and muscimol and gave closely similar pA2 values. The slope of the Schild plot for the above drugs was near 1, confirming the competitive nature of the antagonism. 8 The bicuculline‐sensitive contraction induced by GABA, homotaurine and muscimol was abolished by tetrodotoxin and was non‐competitively antagonized by hyoscine, while it was unaffected by hexamethonium, mepyramine and methysergide. 9 It is concluded that two receptors mediate the GABA effects in guinea‐pig ileum: a bicuculline‐sensitive GABAA receptor, which elicits contraction through an excitatory action on cholinergic post‐ganglionic neurones; and a bicuculline‐insensitive GABAB receptor which causes relaxation through an inhibitory presynaptic action on cholinergic post‐ganglionic neurones. We confirm that GABA, homotaurine and muscimol are GABAA agonists, while GABA and (—)‐baclofen are GABAB agonists.

Agonists, antagonists and modulators of excitatory amino acid receptors in the guinea‐pig myenteric plexus

1 The receptors for glutamic acid (L‐Glu) present in the guinea‐pig myenteric plexus‐ileal longitudinal muscle preparation have been studied by measuring the muscle contraction induced by numerous putative endogenous agonists acting at these receptors. Furthermore, the actions of different concentrations of antagonists, glycine, Mg2+ and Ca2+ on the ileal contractions induced by l‐G1u have been evaluated. 2 The EC50 values of the most common putative endogenous agonists of these receptors were: L‐Glu 1.9 × 10−5 m; L‐aspartate 8 × 10−5 m; quinolinate 5 × 10−4M; L‐homocysteate 1.4 × 10−4M; the dipeptide aspartyl‐glutamate 8 × 10−5m, while N‐acetyl‐aspartyl‐glutamate was inactive. Among the molecules used to classify excitatory amino acid receptors, N‐methyl‐D‐aspartate (NMDA) was the most potent (EC50 5 × 10−4m). Kainic and quisqualic acids were almost completely inactive. 3 The responses to L‐Glu were competitively antagonized by 2‐amino‐5‐phosphonovaleric acid. They were, also, prevented by hyoscine (10−7m) and by tetrodotoxin (3 × 10−7 m), suggesting that the L‐Glu‐induced ileal contraction was in some way dependent upon an action on the myenteric cholinergic neurones. Kynurenic acid was a non‐competitive antagonist, γ‐D‐glutamyl‐taurine (10−4m) and aminophosphonobutyric acid (10−4m) did not modify the L‐Glu‐induced contractions. 4 Glycine (10−5 m) significantly potentiated the effects of glutamate especially when the ionic composition of the superfusion medium contained concentrations of Ca2+ in the range of 0.6–1.2 mM. Strychnine 3 × 10−5 m did not modify the actions of glycine. 5 The data presented here confirm the presence of NMDA receptors in the guinea‐pig myenteric plexus, and show that these receptors, similar to those present in primary neuronal cultures may be modulated by glycine.

Homotaurine: a GABAB antagonist in guinea‐pig ileum

1 Homotaurine (3‐aminopropane sulphonic acid) did not inhibit the twitch response in guinea‐pig ileum longitudinal muscle whilst γ‐aminobutyric acid (GABA) and (−)‐baclofen evoked dose‐dependent inhibitions. 2 The inhibitory effects of GABA and (−)‐baclofen were prevented in the presence of homotaurine 2 × 10−4 and 10−3 M. 3 The log dose‐effect curves of GABA and (−)‐baclofen were shifted in a parallel manner compatible with competitive antagonism. The pA2 of homotaurine with GABA (4.22 ± 0.05) and (−)‐baclofen (4.26 ± 0.1) were the same. 4 Homotaurine did not antagonize the inhibitory effects of morphine (ED50 4 × 10−7 M), noradrenaline (ED50 10−6 M) or ATP (ED50 1.5 × 10−5 M). 5 The inferior homologue of homotaurine, taurine 10−3 M, did not modify the inhibitory effects of GABA and (−)‐baclofen. 6 Picrotoxin 5 × 10−5 M antagonized GABAA receptor‐mediated contraction but did not affect GABAB receptor‐mediated inhibition. At the same concentration the drug did not influence the antagonistic action of homotaurine, thus showing no GABAA receptor‐mediated interference. 7 It may be concluded that homotaurine is a competitive antagonist of GABAB mediated effects in the guinea‐pig ileum.

Modulatory activity of GABAB receptors on cholinergic tone in guinea-pig distal colon.

1 The effect of γ‐aminobutyric acid (GABA) administration was studied in both in vitro and in vivo preparations of the guinea‐pig distal colon. 2 In in vitro preparations GABA (10−7 − 10−3m) elicited a dose‐dependent relaxation; a decrease in the spontaneous contractions was sometimes observed. 3 The effect of GABA was mimicked by (—)‐baclofen, which gave a dose‐response curve overlapping that of GABA, while (+)‐baclofen was about one hundred times less potent. 4 The relaxation responses induced by the above drugs were antagonized by 5‐aminovaleric acid (5 × 10−4m), which did not affect adenosine‐induced relaxation, but they were insensitive to bicuculline (10−5m) and picrotoxin (10−5m). Moreover, they were prevented by tetrodotoxin (6 × 10−7m). In hyoscine (10−7m)‐pretreated preparations, GABA still evoked a small relaxation response (approx. 10% of the maximum) that was bicuculline‐sensitive. 5 Desensitization to GABA (10−5m) was observed. A specific cross‐desensitization occurred between GABA (10−5m) and (—)‐baclofen (10−5m). 6 In in vivo preparations, GABA (10 μmol kg−1) and (—)‐baclofen (5 μmol kg−1) produced a dose‐related inhibition of basal tone, while (+)‐baclofen (5 μmol kg−1) had much less effect (about 25%). A decrease in the spontaneous contractions was sometimes observed. 7 The relaxant effect of GABA and (—)‐baclofen persisted in guinea‐pigs pretreated (1–2 min) with picrotoxin (1.6 μmol kg−1), whereas it was significantly reduced in animals injected 1 min beforehand with 5‐aminovaleric acid (0.2 mmol). 8 The maximal relaxant effect induced by GABA and (—)‐baclofen did not differ from that of atropine (0.9 μmol kg−1) and after atropine administration GABA had no further inhibitory effect. 9 Relaxation responses induced by GABA and (—)‐baclofen still occurred after blockade of nicotinic receptors by hexamethonium (0.17 mmol kg−1), which itself caused an increase in the basal tone. 10 When the tone was increased by topical application of physostigmine (40 μg), GABA and (—)‐baclofen induced a greater relaxation than that obtained in basal conditions. 11 It is concluded that GABA, both in vitro and in vivo administration, inhibits cholinergic tone in guinea‐pig distal colon and that this effect is mediated mainly by activation of GABAB receptors. Further experiments are required to ascertain the possible physiological role of a GABA‐releasing neuronal system in the colon in vivo.

Effect of baclofen on different models of bronchial hyperreactivity in the guinea-pig

In this paper we report an inhibitory effect of (−)-baclofen on many models of bronchial hyperreactivity bothin vivo andin vitro. (−)-Baclofen protects guinea-pigs from the anaphylactic bronchospasm induced in sensitized animals by an ovalbumin aerosol and from that induced by aerosols of histamine and PGF2α. Moreover (−)-baclofen reduces the TXA2 and TXB2 output induced by ovalbumin from isolated sensitized guinea-pig lungs. On the other hand (−)-baclofen does not show antihistaminic, anticholinergic or antiprostaglandinic action on isolated tracheal preparations. It is concluded that baclofen can provide protection from bronchial hyperreactivity possibly through a modulation of autonomic nervous system activity.

Effects of GABA agonists on Herxheimer microshock in guinea pigs

In this paper we describe the first observation of GABA inhibition in an experimental model of asthmain vivo. Guinea-pigs were actively sensitized with ovalbumin i.p. and 20 days later the Herxheimer microshock was performed. GABA and (−)-baclofen injected 20 min previously significantly prevented the development of microshock. Therefore GABAergic drugs appear to modulatein vivo anaphylactic reaction. The value of this observation with regard to the physiopathology and therapy of asthma remains to be elucidated.

Effect of various GABA-receptor agonists and antagonists on anaphylactic histamine release in the guinea-pig ileum

In this paper we confirm the previously reported inhibition by GABA of anaphylactic histamine release from isolated guinea-pig ileum longitudinal muscle. Moreover we report that: A) GABA-inhibition of anaphylactic histamine release is mimicked both by GABA-A and GABA-B agonists; both GABA-A and GABA-B antagonists are effective in reversing GABAs inhibitory effect; B) the effect is exerted specifically by GABA-ergic drugs: taurine and β-alanine are ineffective; C) the GABA-ergic effect seems not to involve cholinergic and adrenergic transmission. It is concluded that it might be interesting to assess the clinical value of GABA-ergic drugs in allergic gut disorders.

Influence of GABA on anaphylactic histamine releasein vitro

In this preliminary report an unreported inhibitory action of GABA on anaphylactic reaction has been described. In a functional model (Schultz-Dale reaction) GABA has been demonstrated to inhibit the antigen-evoked contraction. This effect depends on a modulation of anaphylactic histamine release. The phenomenon is dose-dependent and requires a period of time to develop. Since GABAergic neurons are present in the preparation, it is possible to speculate that GABA receptors are involved in this inhibitory action. However, pharmacological analysis of the phenomenon has to be carried out, especially in view of the latency of GABA to develop its effect.

Inhibition of anaphylactic histamine releasein vitro by GABA

Inhibitory effect of GABA on anaphylactic histamine releasein vitro is not mimicked by 2-aminoethansulphonic acid (taurine), an aminoacid unrelated to GABA neurotransmission.Tetrodotoxin (TTX) 6×10−7M, a concentration known to block neuronal mechanism but not to modify muscle membrane and anaphylactic histamine release, strongly prevented the inhibition caused by GABA in the Schultz-Dale reaction and in anaphylactic histamine release.The inhibitory effect of GABA on anaphylactic reactionin vitro thus appears to be specific for this aminoacid and is neurogenic in nature, in that it requires integrity of neuronal mechanisms.

Glycine-related amino acids stereoselectively affect N-methyl-d-aspartate receptor-mediated contractions of guinea pig ileum: Comparison with the inhibition of strychnine-insensitive [3H]glycine binding to rat cortical membranes

Ten microM glycine, D-serine and D-alanine potentiated L-glutamate (30 microM)-induced contractions of the guinea pig ileum by an average of 35, 53 and 24%, respectively. On the contrary, D-cysteine, at the same concentration, caused a 21% inhibition of the contractile response to L-glutamate. This inhibitory effect of D-cysteine was abolished by 10 microM glycine. The corresponding L-isomers of these amino acids, namely L-serine, L-alanine and L-cysteine and the other amino acids tested, possessed negligible activity or were inactive in this test. The IC50 values of the same compounds for strychnine-insensitive binding of [3H]glycine (20 nM) to cortical membranes from the brain of the rat were: 0.26 microM, glycine; 1.2 microM, D-serine; 2.1 microM, D-alanine; 8.6 microM, D-cysteine; 51 microM, L-serine; 90 microM, L-alanine; greater than 1000 microM, L-cysteine. On the whole, these results point out a strict requirement for stereoselectivity for both of the effects examined. In addition, the results obtained in the ileum preparation suggest that D-cysteine may act as an antagonist, rather than as an agonist at the glycine site which regulates the responses of N-methyl-D-aspartate receptors.