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

Katia Gamel-Didelon,Lars Kunz,Karl Josef Föhr,Manfred Gratzl,Artur Mayerhofer

doi:10.1074/jbc.m301729200

Katia Gamel-Didelon, Lars Kunz + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m301729200

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Abstract

The neurotransmitter gamma-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 rho2. 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.

Highlights

␥-Aminobutyric acid (GABA)1 is widely distributed in the central nervous system [1]
We report that growth hormone- (GH) cells express GABA-C receptor subunits, which form functional receptors in a rat GH-producing cell line, GH3
Expression of GABA-C Receptors in Rat Pituitary GH Cells and in GH3 Cells—GABA-C receptors are present outside the central nervous system and retina, for example in the pituitary gland

Summary

MATERIALS AND METHODS

Animals—Pituitary glands and brains were obtained from SpragueDawley rats bred at the Technische Universitat Munchen. Immunohistochemistry and Laser Microdissection of Rat Pituitary Cells—Immunohistochemical methods were performed as described previously [8]. The subsequent microdissection of immunostained cells from rat pituitaries was performed using the PALM௡ Robot-MicroBeam technology (P.A.L.M. GmbH, Bernried, Germany) and following methods described (8 –10). Immunocytochemistry—GH3 cells were cultivated on glass coverslips (2 ϫ 104 cells/coverslip) for 1 day They were fixed and handled as described previously [11]. For immunolocalization of GABA-C receptors, a polyclonal antibody produced in rabbit was used (diluted 1:50; courtesy Dr Enz, Institut fur Biochemie, Universitat ErlangenNurnberg, Germany). This antiserum was raised against ␳1 but was shown to recognize ␳2 as well [12].

TABLE I Oligonucleotide primers used for PCR

RESULTS

DISCUSSION