A Subtype of the g-Aminobutyric AcidB Receptor Regulates Cholinergic Twitch Response in the Guinea Pig Ileum

The pharmacological profile of the g-aminobutyric acid (GABA)B receptor regulating cholinergic twitch contraction in the guinea pig ileum myenteric plexus-longitudinal muscle preparation was investigated. GABA and (2)-baclofen inhibited the contraction, exhibiting quite close potencies (pD2 for GABA 5 5.70; pD2 for (2)-baclofen 5 5.33). The compound CGP 47656 also reduced the cholinergic twitch concentration (pD2 5 5.42), but its efficacy was significantly lower than that of (2)-baclofen or GABA. Added at varying concentrations, CGP 47656 modified the concentration-response curve of (2)-baclofen as expected for a partial agonist. Phaclofen, CGP 36742, CGP 35348, and CGP 52432 behaved as competitive antagonists of (2)-baclofen, exhibiting the following pA2 values: 3.90, 4.88, 5.02, and 7.82, respectively. The compound CGP 56999 behaved as a potent noncompetitive GABAB receptor antagonist. In comparing the pharmacological profile of the ileal receptor with those of the previously characterized pharmacological subtypes of the GABAB receptor present in the central nervous system, it can be seen that the GABAB receptor inhibiting cholinergic twitch contraction in guinea pig ileum myenteric plexus-longitudinal muscle mostly resembles the receptor located on somatostatin human neocortex nerve terminals. Electrophysiological, biochemical, and functional release studies suggest that g-aminobutyric acid (GABA)B receptors are pharmacologically heterogeneous (Bowery, 1989, 1993; Kerr et al., 1990a; Bonanno and Raiteri, 1993; Mott and Lewis, 1994; Cunningham and Enna, 1996). Presynaptic release-inhibiting GABAB receptors display clear pharmacological diversity, based on their differential sensitivity to (2)baclofen and to various selective GABAB receptor antagonists (Bonanno and Raiteri, 1993). Activation of GABAB receptors was shown to mediate inhibition of electrically evoked cholinergic twitch contractions and of acetylcholine release in guinea pig ileum (Bowery et al., 1981; Kaplita et al., 1982; Giotti et al., 1983; Kleinrok and Kilbinger, 1983; Ong and Kerr, 1983; Taniyama et al., 1992, 1996). In analogy with the guinea pig, activation of GABAB receptors in human small intestine was reported to inhibit longitudinal muscle motility through an action on cholinergic neurons (Gentilini et al., 1992). The finding that baclofen could inhibit the tetrodotoxin-insensitive release of [H]acetylcholine evoked by high K (Taniyama et al., 1992, 1996), together with previous electrophysiological evidence (Cherubini and North, 1984), suggests that GABAB receptors are located on cholinergic nerve endings of the myenteric plexus. The GABAB receptors inhibiting the electrically evoked cholinergic twitch contraction in guinea pig ileum were previously reported to be phaclofenand CGP 35348-sensitive (Kerr et al., 1987; Ong et al., 1994). However, multiple phaclofenand CGP 35348-sensitive GABAB receptors were found to exist in the rat central nervous system (CNS; Gemignani et al., 1994; Bonanno et al., 1999), which justifies further pharmacological characterization of the intestinal GABAB receptors. In the present work, we determined the pharmacological profile of the GABAB receptor inhibiting the electrically evoked cholinergic twitch contraction from myenteric plexuslongitudinal muscle (MP-LM) preparations of guinea pig ileum by using a series of ligands (CGP 52432 and CGP 47656; Gemignani et al., 1994; CGP 36742; Bonanno et al., 1999) able to distinguish receptor subtypes within the phaclofenand CGP 35348-sensitive GABAB receptor group. In addition, CGP 56999, a potent antagonist supporting heterogeneity of presynaptic GABAB receptors in the rat spinal cord (Teoh et al., 1996), was used. Materials and Methods Animals and Tissue Preparation. Male guinea pigs, weighing 350 to 450 g, were sacrificed by cervical dislocation. Terminal ileum was removed after the 10 cm nearest to the ileocecal junction had Received for publication October 21, 1999. 1 This work was supported by an Italian MURST Network grant (1997). ABBREVIATIONS: GABA, g-aminobutyric acid; CNS, central nervous system; CGP 35348, 3-aminopropyl(diethoxymethyl)phosphinic acid; CGP 36742, 3-aminopropyl-n butyl phosphinic acid; CGP 47656, 3-aminopropyl-(difluoromethyl)phosphinic acid; CGP 52432, [3-[[(3,4-dichlorophenyl)methyl]amino]propyl](diethoxymethyl) phosphinic acid; CGP 56999, [3{[1-(R)-(3-carboxyphenyl)ethyl]amino}-2-(S)-hydroxy-propyl]cyclohexylmethyl-phosphinic acid; MP-LM, myenteric plexus-longitudinal muscle; SRIF, somatostatin; CCK, cholecystokinin. 0022-3565/00/2931-0042$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 293, No. 1 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 293:42–47, 2000 42 at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from been discarded and strips of myenteric plexus with the longitudinal muscle attached (MP-LM) were prepared according to Paton and Vizi (1969). Single Twitch Stimulation. Four MP-LM strips (3–4 cm in length) from each animal were mounted in separate 3-ml organ baths perfused with Tyrode’s solution of the following composition: 136.9 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl2, 1.04 mM MgCl2, 11. 9 mM NaHCO3, 0.4 mM NaH2PO4, and 10 mM glucose, at 37°C, and continuously oxygenated (95% O2, 5% CO2, pH 7.2–7.4). An initial tension of 0.5 g was applied, and the longitudinal muscle activity was recorded through an isometric force transducer. After 120-min perfusion at 1 ml/min, longitudinal muscle repetitive twitch contractions (developing tension of 1.2–2.3 g) were evoked by field stimulation (alternate rectangular pulses, 0.1 Hz, 1-ms duration, 550–600 mA) delivered from two platinum electrodes. The use of submaximal voltage was a precaution against the effects of drugs being masked by supramaximal pulses (see Fosbraey and Johnson, 1980). The effects of drugs were measured as percent variation of the cholinergic twitch contraction height. To avoid desensitization (Bowery et al., 1981; Ong and Kerr, 1983), agonists were applied at 30-min intervals; when the response to any agonist concentration had reached a maximum, tissues were washed and perfused at 1 ml/min for 20 min. Two complete concentration-response curves were conducted in the same tissue. When the effects of antagonists on the agonist inhibition of twitch contraction were evaluated, after a first concentration-response curve for the agonist was completed in standard medium, a second concentration-response curve was made in standard medium (control) or in the presence of the antagonists (one antagonist concentration only per tissue). Antagonists were added to the perfusion medium 20 min before their effects were evaluated. When the effects of adding CGP 47656 on (2)-baclofen was evaluated, after a first concentration-response curve for (2)baclofen was completed, a second concentration-response curve was made for (2)-baclofen alone (control) or by adding (2)-baclofen together with CGP 47656 at various concentrations. In each experiment, a preparation was run as control; in the experimental conditions used in control preparations, the agonist EC50 value in the second curve did not significantly differ from the EC50 value in the first curve. Data obtained in first (agonists alone) and second (in the presence of antagonists) curves are given in the figures. In preliminary experiments, the effects of compounds on longitudinal muscle contractile responses to acetylcholine were evaluated in MP-LM preparations. Data Analysis. Concentration-response curves for agonist inhibition of the twitch contraction were analyzed by a four-parameter logistic function analysis (SigmaPlot software). pD2 values of agonists were measured as 2log EC50 values (EC50 is the concentration producing 50% of the drug maximum effect). The pA2 value of CGP 52432 was measured according to Arunlakshana and Schild (1959); when the concentration range (equal to or less than 10-fold) of the antagonists precluded construction of a Schild plot to provide true pA2 values, apparent pA2 values were determined using the relationship pA2 5 log (EC50 ratio 2 1) 2 log (B) in the presence of at least two different antagonist concentrations (B). The pKB value for CGP 47656 antagonism of (2)-baclofen was estimated from agonist dose ratios producing half-maximum responses in accordance with the model for partial agonism: pKB 5 log (EC50 ratio 2 1) 2 log (B) in the presence of two different CGP 47656 concentrations (B). Mean 6 S.E. values of determination in n separate experiments are indicated throughout. The statistical significance of the differences between mean values was assessed by the Student’s t test. A probability level of P , .05 was taken as statistically significant. Drugs. Acetylcholine hydrochloride, GABA, and (2)-bicuculline methobromide were purchased from Sigma Chemical Co. (St. Louis, MO). Phaclofen was purchased from Tocris Cookson (Bristol, UK). (2)Baclofen, 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP 35348), 3-aminopropyl-n butyl phosphinic acid (CGP 36742), 3-aminopropyl(difluoromethyl)phosphinic acid (CGP 47656), [3-[[(3,4-dichlorophenyl) methyl]amino]propyl](diethoxymethyl) phosphinic acid (CGP 52432), and [3-{[1-(R)-(3-carboxyphenyl)ethyl]amino}-2-(S)-hydroxy-propyl]cyclohexyl-methyl-phosphinic acid (CGP 56999) were gifts from Novartis (Basel, Switzerland). All compounds were dissolved in distilled water or