Subunit composition and pharmacological characterization of gamma-aminobutyric acid type A receptors in frog pituitary melanotrophs

The frog pars intermedia is composed of a single population of endocrine ce lls directly innervated by gamma-aminobutyric acid (GABA)ergic nerve termin als. We have previously shown that GABA(A) acting through GABA(A) receptors , modulates both the electrical and secretory activities of frog pituitary melanotrophs. The aim of the present study was to take advantage of the fro g melanotroph model to determine the relationship between the subunit compo sition and the pharmacological properties of native GABA(A) receptors. Immu nohistochemical labeling revealed that in situ and in cell culture, frog me lanotrophs were intensely stained with alpha(2)-, alpha(3)-, gamma(2)-, and gamma(3)-subunit antisera and weakly stained with a gamma(1)-subunit antis erum. Melanotrophs were also immunolabeled with a monoclonal antibody to th e beta(2)/beta(3)-subunit. In contrast, frog melanotrophs were not immunore active for the alpha(1)-, alpha(5)-, and alpha(6)-isoforms. The effects of allosteric modulators of the GABA, receptor on GABA-activated chloride curr ent were tested using the patch-clamp technique. Among the ligands acting a t the benzodiazepine-binding site, clonazepam (EC50, 5 x 10(-9) M), diazepa m (EC50, 10(-8) M), zolpidem (EC50, 3 x 10(-8) M) and beta-carboline-3-carb oxylic acid methyl ester (EC50, 10(-6) M) were found to potentiate the whol e cell GABA-evoked current in a dose-dependent manner. Methyl-6,7-dimethoxy -4-ethyl-beta-carboline (IC50, 3 x 10(-5) M) inhibited the current, whereas Ro15-4513 had no effect. Among the ligands acting at other modulatory site s, etomidate (EC50, 2 x 10(-6) M) enhanced the GABA-evoked current, whereas 4'-chlorodiazepam (IC50, 4 x 10(-7) M), ZnCl2 (IC50, >5 x 10(-5) M), and f urosemide (IC50, >3 x 10(-4) M) depressed the response to GABA. PK 11195 di d not affect the GABA-evoked current or its inhibition by 4'-chlorodiazepam . The results indicate that the native GABA(A) receptors in frog melanotrop hs are formed by combinations of alpha(2)-, alpha(3)-, beta(2/3)-, gamma(1) -, gamma(2), and gamma(3)-subunits. The data also demonstrate that clonazep am Is the most potent, and zolpidem is the most efficient positive modulato r of the native receptors. Among the inhibitors, 4-chlorodiazepam is the mo st potent, whereas ZnCl2, is the most efficient negative modulator of the G ABA(A) receptors. The present study provides the first correlation between subunit composition and the functional properties of native GABA(A) recepto rs in nontumoral endocrine cells.