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Gamma-aminobutyric acid (GABA): a para- and/or autocrine hormone in the pituitary¹

ARTUR MAYERHOFER^*, BARBARA HÖHNE-ZELL^*, KATIA GAMEL-DIDELON^*, HEIKE JUNG, PETER REDECKER, DIETRICH GRUBE, HENRYK F. URBANSKI, BRUNO GASNIER^¶, JEAN-MARC FRITSCHY^|| and MANFRED GRATZL^*2

^* Anatomisches Institut der Universität München, Germany;
Zentrum Kinderheilkunde und Humangenetik der Medizinischen Hochschule and
Abteilung Anatomie I der Medizinischen Hochschule Hannover, Germany;
Division of Neuroscience, ORPRC-OHSU, Beaverton, Oregon 97006, USA;
^¶ CNRS UPR 1929, Institut de Biologie Physico-Chimique, Paris, France; and
^|| Institute of Pharmacology, University of Zürich, CH-8057 Zürich, Switzerland

²Correspondence: Anatomisches Institut, Biedersteinerstr. 29, 80802 München, Germany. E-mail: gratzl{at}lrz.uni-muenchen.de

SPECIFIC AIMS

The neurotransmitter gamma-aminobutyric acid (GABA) plays an important role in the regulation of pituitary gland function, but details of the underlying mechanism are poorly understood. Here we identify novel intrapituitary sources of GABA and disclose the sites of its synthesis, storage, and the distribution of its receptors in the endocrine master gland, the pituitary.

PRINCIPAL FINDINGS

1. Synthesis and storage of GABA occurs in endocrine growth hormone (GH) -producing cells of the anterior lobe and POMC-producing cells of the intermediate lobe of the pituitary
Using RT-PCR, in situ hybridization histochemistry, immunohistochemistry, and immunoelectron microscopy, the GABA-synthesizing enzyme GAD 67, the vesicular transporter VIAAT/VGAT, and GABA were detected in the rat and primate pituitary. Although GAD and VIAAT/VGAT were found in all of the endocrine cells of the intermediate lobe, they were present only in the GH-producing endocrine cells of the anterior lobe (Fig. 1 ). Immunoelectron microscopy revealed that the expression of GAD and VIAAT/VGAT in these GH cells was localized to the typical intracellular GH storage organelle. The GH-producing cell line GH3 likewise was found to express the genes for GAD 67 and VIAAT/VGAT. In addition, GAD enzyme activity was measured in rat pituitary and GH3 cells and was about 10% of the activity of brain tissue (cerebellum).

View larger version (114K): [in this window] [in a new window]   Figure 1. Colocalization of VIAAT/VGAT, GAD, and GH in the rat pituitary. Consecutive semithin sections of a rat pituitary were immunostained for GH, GAD, and VIAAT/VGAT. A blood vessel is labeled with an asterisk for orientation. Bar = 10 µm. In this example, a pituitary from a male Lewis rat was used.

2. GABA_A and/or GABA_B receptor subunits are present in all endocrine cell types of the pituitary, including GH cells
Double immunofluorescence staining was performed using antibodies directed against various GABA receptor subunits and antibodies recognizing pituitary hormones. Subsequent confocal laser scanning microscopy revealed that all of the endocrine cell types in the anterior lobe of the pituitary contain GABA_A and/or GABA_B receptors (unpublished). Besides PRL, TSH, ACTH, and LH/FSH cells, GH cells were found to contain subunits of GABA_A and/or GABA_B receptors (Fig. 2 ).

View larger version (74K): [in this window] [in a new window]   Figure 2. Example of GABAA receptor subunits in GH and other pituitary cells. Immunocytochemical detection of three immunoreactive GH cells by confocal laser microscopy (left). Two of these GH cells and another adjacent pituitary cell are immunoreactive for the 2 subunit of the GABA A receptor (middle panel). The right panel shows the computer merged images. Bar = 5 µm. In this example, a pituitary from a female Wistar rat was used.

CONCLUSIONS AND SIGNIFICANCE

Although GABA acts as a major inhibitory neurotransmitter in the brain, it also directly regulates the secretion of the pituitary hormones PRL, GH, LH/FSH, TSH, and ACTH. The sources of pituitary GABA are not well established, but it is generally assumed that pituitary GABA originates from hypothalamic neurons and there is evidence to support two possible pathways. The first involves the secretion of GABA into the portal blood and the second involves direct innervation of pituitary cells of the intermediate lobe (Fig. 3 ).

View larger version (31K): [in this window] [in a new window]   Figure 3. Neuronal, neurohaemal, and novel para/autocrine control of pituitary function by GABA.

Tools available for the study of GABAergic systems have included immunodetection of GABA, measurement of glutamic acid decarboxylase (GAD) activity, and analysis of the cellular localization of the GABA-synthesizing enzymes GAD 65 and GAD 67. Recently, a novel approach to studying the GABAergic system has become available, and is based on the finding that a vesicular transporter named VIAAT (vesicular inhibitory amino acid transporter) or VGAT (vesicular GABA transporter) specifically transports GABA and glycine. In particular, antibodies recognizing VIAAT/VGAT have proved to be valuable in revealing the localization of the GABA storage sites in synaptic vesicles of nerve terminals of the brain. In the present study, we used both the established and the novel methodologies to study GABA synthesis and storage and GABA receptor expression in the endocrine master gland, the pituitary. The results obtained show that two so far unrecognized types of endocrine cells in the pituitary gland have the capacity to synthesize and store GABA. These are the POMC cells of the intermediate lobe and the GH cells of the anterior lobe (Figs. 1 and 3) .

Studies using GABA agonists and antagonists provided evidence for a specific function of GABA to affect hormone secretion induced by the hypothalamic-releasing hormones. These and additional studies showed that GABA_A, GABA_B, and GABA_C receptor genes are expressed in the pituitary. In contrast, only a few of these GABA receptors have been directly localized at the cellular level in the pituitary. These are GABA_C receptors, which have been detected on TSH-secreting cells in the rat anterior lobe, and GABA_A receptors on melanotrophs of the frog intermediate lobe. Our results clearly indicate that GABA_A and/or GABA_B receptors exist in all of the pituitary cell types in which hormone secretion is known to be influenced by GABA. A more detailed investigation of the distribution of various GABA receptor subtypes and the composition of their subunits in the rat pituitary is currently in progress.

The finding that pituitary GABA is derived both from novel intrapituitary, as well as extrapituitary sources, and that GABA receptors are expressed in essentially all pituitary endocrine cell types, including GH cells, implies that GABA plays a fundamental role in regulating pituitary function. Since GH cells and POMC cells have the capacity to produce GABA, they most likely represent the key cell types in the intrapituitary GABAergic system. The immunoelectron microscopical colocalization of GAD and VIAAT/VGAT in the typical intracellular storage organelle of GH indicates that GABA and GH are likely to be synthesized and stored together. In the present study, we focused only on GH cells, and have no comparable detailed information about the POMC cells. As is generally accepted, intracellular calcium triggers exocytosis in GH and other endocrine cells. GH cells are regarded as pacemaker cells since intracellular calcium oscillations in GH cells occur spontaneously and can be modulated by hypothalamic factors, including GHRH and somatostatin. The integration of these signals in the pacemaker cells causes rhythmic GH secretion patterns, which are well established in rodents and primates. The presence of GH and GABA in the same subcellular compartment and the rhythmic secretion of GH imply that GABA may also be released with parallel rhythmicity. That functional GABA receptors exist in pituitary endocrine cells suggests that GABA, derived from GH cells, may control pulsatile release of hormones of neighboring endocrine cells in a paracrine manner. In addition, the expression of GABA receptors on GH cells may be indicative of an autocrine feedback loop. The specific details of these proposed novel mechanisms, as well as the regulatory function of GABA derived from POMC cells, remain to be elucidated.

In summary, the control of pituitary function by GABA is exerted at different hierarchic levels (Fig. 3) . First, in the hypothalamus the function of neurons that secrete releasing factors (e.g. GnRH, GHRH, TRH) is regulated by GABAergic neurons. Second, in addition to this indirect control, GABAergic hypothalamic neurons extend their axons to the median eminence to form pericapillary terminals from where GABA can access the portal circulation and reach the pituitary. Third, hypothalamic GABAergic axons terminate on endocrine cells of the intermediate lobe. Together with GABA released into the portal circulation, direct innervation has been regarded as the exclusive source of regulatory GABA in the pituitary. Our study now shows the unexpected presence of additional sources of GABA in the rat and monkey pituitary, namely, GH cells of the anterior lobe and POMC cells of the intermediate lobe. These cells are likely to be the centerpiece of a novel basic mechanism in the regulation of pituitary endocrine function.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0546fje ; to cite this article, use FASEB J. (February 20, 2001) 10.1096/fj.00-0546fje

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