Molecular and Physiological Evidence for Functional γ-Aminobutyric Acid (GABA)-C Receptors in Growth Hormone-secreting Cells

Abstract

The neurotransmitter γ-aminobutyric acid (GABA), released by hypothalamic neurons as well as by growth hormone- (GH) and adrenocorticotropin-producing cells, is a regulator of pituitary endocrine functions. Different classes of GABA receptors may be involved. In this study, we report that GH cells, isolated by laser microdissection from rat pituitary slices, possess the GABA-C receptor subunit ρ2. We also demonstrate that in the GH adenoma cell line, GH3, GABA-C receptor subunits are not only expressed but also form functional channels. GABA-induced Cl- currents were recorded using the whole cell patch clamp technique; these currents were insensitive to bicuculline (a GABA-A antagonist) but could be induced by the GABA-C agonist cis-4-aminocrotonic acid. In contrast to typical GABA-C mediated currents in neurons, they quickly desensitized. Ca2+i recordings were also performed on GH3 cells. The application of either GABA or cis-4-aminocrotonic acid led to Ca2+ transients of similar amplitude, indicating that the activation of GABA-C receptors in GH3 cells may cause membrane depolarization, opening of voltage-gated Ca2+ channels, and a subsequent Ca2+ influx. Our results point at a role for GABA in pituitary GH cells and disclose an additional pathway to the one known via GABA-B receptors. The neurotransmitter γ-aminobutyric acid (GABA), released by hypothalamic neurons as well as by growth hormone- (GH) and adrenocorticotropin-producing cells, is a regulator of pituitary endocrine functions. Different classes of GABA receptors may be involved. In this study, we report that GH cells, isolated by laser microdissection from rat pituitary slices, possess the GABA-C receptor subunit ρ2. We also demonstrate that in the GH adenoma cell line, GH3, GABA-C receptor subunits are not only expressed but also form functional channels. GABA-induced Cl- currents were recorded using the whole cell patch clamp technique; these currents were insensitive to bicuculline (a GABA-A antagonist) but could be induced by the GABA-C agonist cis-4-aminocrotonic acid. In contrast to typical GABA-C mediated currents in neurons, they quickly desensitized. Ca2+i recordings were also performed on GH3 cells. The application of either GABA or cis-4-aminocrotonic acid led to Ca2+ transients of similar amplitude, indicating that the activation of GABA-C receptors in GH3 cells may cause membrane depolarization, opening of voltage-gated Ca2+ channels, and a subsequent Ca2+ influx. Our results point at a role for GABA in pituitary GH cells and disclose an additional pathway to the one known via GABA-B receptors. γ-Aminobutyric acid (GABA) 1The abbreviations used are: GABA, γ-aminobutyric acid; CACA, cis-4-aminocrotonic acid; GH, growth hormone; TSH, thyroid-stimulating hormore; RT, reverse transcription.1The abbreviations used are: GABA, γ-aminobutyric acid; CACA, cis-4-aminocrotonic acid; GH, growth hormone; TSH, thyroid-stimulating hormore; RT, reverse transcription. is widely distributed in the central nervous system (1Sivilotti L. Nistri A. Prog. Neurobiol. 1991; 36: 35-92Crossref PubMed Scopus (457) Google Scholar). There, generally, it inhibits neuronal firing and contributes to stabilization of the membrane resting potential by acting on GABA-A, -B, and -C receptors. The term “GABA-C” receptor, which refers to bicuculline- and baclofen-insensitive ionotropic GABA receptors formed by ρ subunits, is controversial, and GABA-C receptors may simply be a subset of GABA-A channels (2Barnard E.A. Skolnick P. Olsen R.W. Mohler H. Sieghart W. Biggio G. Braestrup C. Bateson A.N. Langer S.Z. Pharmacol. Rev. 1998; 50: 291-313PubMed Google Scholar). GABA-C receptors are located in certain areas of the central nervous system and in the retinas of various species. They form Cl- channels, assumed to organize in either homo- or heteromers of the different ρ subunits (3Enz R. Biol. Chem. 2001; 382: 1111-1122Crossref PubMed Scopus (59) Google Scholar). Outside of the central nervo