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Estelle Louiset, Ruth McKernan, Werner Sieghart, Hubert Vaudry, Subunit Composition and Pharmacological Characterization ofγ -Aminobutyric Acid Type A Receptors in Frog Pituitary Melanotrophs, Endocrinology, Volume 141, Issue 3, March 2000, Pages 1083–1092, https://doi.org/10.1210/endo.141.3.7397
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Abstract
The frog pars intermedia is composed of a single population of endocrine cells directly innervated by γ-aminobutyric acid (GABA)ergic nerve terminals. We have previously shown that GABA, acting through GABAA receptors, modulates both the electrical and secretory activities of frog pituitary melanotrophs. The aim of the present study was to take advantage of the frog melanotroph model to determine the relationship between the subunit composition and the pharmacological properties of native GABAA receptors. Immunohistochemical labeling revealed that in situ and in cell culture, frog melanotrophs were intensely stained withα 2-, α3-, γ2-, andγ 3-subunit antisera and weakly stained with aγ 1-subunit antiserum. Melanotrophs were also immunolabeled with a monoclonal antibody to theβ 2/β3-subunit. In contrast, frog melanotrophs were not immunoreactive for the α1-,α 5-, and α6-isoforms. The effects of allosteric modulators of the GABAA receptor on GABA-activated chloride current were tested using the patch-clamp technique. Among the ligands acting at the benzodiazepine-binding site, clonazepam (EC50, 5 × 10−9m), diazepam (EC50 , 10−8m), zolpidem (EC50, 3 × 10−8m), and β-carboline-3-carboxylic acid methyl ester (EC50, 10−6m) were found to potentiate the whole cell GABA-evoked current in a dose-dependent manner. Methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (IC50, 3 × 10−5m) inhibited the current, whereas Ro15–4513 had no effect. Among the ligands acting at other modulatory sites, etomidate (EC50, 2 × 10−6m) enhanced the GABA-evoked current, whereas 4′-chlorodiazepam (IC50, 4 × 10−7m), ZnCl2 (IC50,> 5 × 10−5m), and furosemide (IC50, >3 × 10−4m) depressed the response to GABA. PK 11195 did not affect the GABA-evoked current or its inhibition by 4′-chlorodiazepam. The results indicate that the native GABAA receptors in frog melanotrophs are formed by combinations of α2-,α 3-, β2/3-, γ1-,γ 2-, and γ3-subunits. The data also demonstrate that clonazepam is the most potent, and zolpidem is the most efficient positive modulator of the native receptors. Among the inhibitors, 4′-chlorodiazepam is the most potent, whereas ZnCl2 is the most efficient negative modulator of the GABAA receptors. The present study provides the first correlation between subunit composition and the functional properties of native GABAA receptors in nontumoral endocrine cells.
