Molecular and Physiological Evidence for Functional {gamma}-Aminobutyric Acid (GABA)-C Receptors in Growth Hormone-secreting Cells*

Katia Gamel-Didelon {ddagger}, Lars Kunz {ddagger}, Karl Josef Föhr §, Manfred Gratzl {ddagger} and Artur Mayerhofer {ddagger} 

From the {ddagger} Anatomisches Institut der Universität München, 80802 München, Germany and the §Klinik für Anästhesiologie, Universität Ulm, 89070 Ulm, Germany

Received for publication, February 19, 2003 , and in revised form, March 25, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {rho}2. 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
{gamma}-Aminobutyric acid (GABA)1 is widely distributed in the central nervous system (1). 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 {rho} subunits, is controversial, and GABA-C receptors may simply be a subset of GABA-A channels (2). 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 {rho} subunits (3). Outside of the central nervous system and retina, the expression of GABA-C receptor subunits was reported based on RT-PCR analysis performed in rat peripheral tissues, namely in gonadal endocrine tissues, adrenal gland, placenta, and small intestine (4), and was also found by immunohistochemistry in human neuroendocrine midgut tumor cells (5). In these cells, GABA-C receptors were shown to be functional by studying Ca2+i levels and hormone release. In addition, functional GABA-C receptors were also observed in pituitary thyroid-stimulating hormone (TSH) cells using electrophysiological techniques (6). GABA, produced by growth hormone- (GH) secreting cells (7), acts as an autocrine regulator of GH levels via GABA-B receptors (8). In the present study, we report that GH cells also express GABA-C receptor subunits, which form functional receptors in a rat GH-producing cell line, GH3.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals—Pituitary glands and brains were obtained from Sprague-Dawley rats bred at the Technische Universität München. They were painlessly killed under ether anesthesia, according to institutional animal care guidelines. The tissues were removed and processed as described previously (7, 8).

Culture Procedures of Rat GH3 Cells—The culture procedures applied for GH3 cells were described (8). Briefly, the cells were grown in F12-Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal calf serum (PAA Laboratories, Linz, Austria). Since GH3 cells produce GABA, the patch clamp and Ca2+ measurements required regular renewal of the medium. Positive recordings were obtained only up to day 6 of culture, when the cells did not yet form a confluent monolayer.

Immunohistochemistry and Laser Microdissection of Rat Pituitary Cells—Immunohistochemical methods were performed as described previously (8). For staining GH and TSH cells, the following antisera were used: monkey anti-GH (diluted 1:500; courtesy Dr. Parlow, National Hormone and Pituitary Program, Torrance, CA) and rabbit anti-TSH (1:20,000; Chemicon International, Temecula, CA), respectively, and as secondary antibodies, biotin-labeled goat anti-human and goat anti-rabbit (diluted 1:500; Dianova, Hamburg, Germany). 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, 9, 10).

RNA Isolation and RT-PCR—RNA isolation and RT-PCRs from GH3 cells, laser-dissected GH and TSH cells, or rat tissues were performed as reported (8). Oligonucleotide primers (Table I) were chosen to encompass exon-intron boundaries to detect possible genomic DNA contamination; for amplifying {rho}2 cDNA from the laser-dissected samples, nested primers were required. For all experiments, the nature of the amplified cDNAs was confirmed by direct sequencing using one of the oligonucleotide primers (AGOWA, Berlin, Germany).


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TABLE I
Oligonucleotide primers used for PCR

 

Immunocytochemistry—GH3 cells were cultivated on glass coverslips (2 x 104 cells/coverslip) for 1 day. They were then 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 für Biochemie, Universität Erlangen-Nürnberg, Germany). This antiserum was raised against {rho}1 but was shown to recognize {rho}2 as well (12). Immunoreactivity was visualized using a fluorescein isothiocyanate-labeled secondary goat antirabbit antiserum (diluted 1:200; Dianova, Hamburg, Germany). For control purposes, either the specific antiserum was omitted or incubations with rabbit normal serum (1:10,000; 1:20,000; 1:40,000) were carried out instead. Sections were examined with a Zeiss Axiovert microscope (Zeiss, Jena, Germany), equipped with a fluorescein isothiocyanate filter set.

Patch Clamp Whole Cell Recordings—Patch clamp measurements were performed on GH3 cells grown on glass coverslips for 3–6 days. The cells were voltage-clamped at -80 mV, and whole cell currents were recorded (sampling rate, 100 Hz; low pass filter, 30 Hz) at room temperature (~22 °C) utilizing an EPC-9 amplifier (HEKA Elektronik, Lambrecht, Germany). Borosilicate patch pipettes (DMZ-Universal Puller; Zeitz, Augsburg, Germany) showed a tip resistance of 3–5 megaohms. The "extracellular" bath solution contained 140 mM NaCl, 3 mM KCl, 1 mM CaCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4). The pipette solution contained 130 mM KCl, 5 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 2 mM EGTA, and 10 mM HEPES (pH 7.4).

Calcium Measurements—Ca2+ measurements were performed on GH3 cells, up to day 6 of culture and before reaching confluency, as described (13). Briefly, the cells were loaded with Fura-2/AM (2,5 µM, dissolved in Me2SO) for 30 min at 37 °C in a standard external solution consisting of 140 mM NaCl, 2.7 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 6 mM glucose, and 12 mM HEPES (pH 7.3). Fluorescence measurements were performed with the Zeiss Fast Fluorescence Photometry System (MPM-FFP, Zeiss, Oberkochen, Germany). The excitation wavelength was switched, at 400 Hz, between 340 and 380 nm using appropriate interference filters (bandwidth, 10 nm). The emitted light (505–530 nm) was monitored after averaging with a final time resolution of 80 ms. Ca2+ levels are given in the figures as fluorescence ratios obtained from alternating excitation at 340 and 380 nm.

Drug Application—For both patch clamp and Ca2+ measurements, a combination of global and local bath perfusion was installed that generated a continuous fluid stream containing the agent but confining it to a small volume. Fast pressurized perfusion systems equipped with magnetic valves were used for drug application as described previously (13, 14). At each experiment, bath solution was applied first to test for mechanical interference by the mere approaching flow of solutions. GABA, cis-4-aminocrotonic acid (CACA), bicuculline, and baclofen (Tocris, Ellisville, MO) were utilized at a concentration of 100 µM as reported previously (6).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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. There, TSH cells were shown to possess functional GABA-C channels (6). Therefore, we used TSH cells as a reference for our experiments. Rat pituitary sections were immunostained either for GH or for TSH and subsequently submitted to laser microdissection followed by RT-PCR experiments (Fig. 1). GH and TSH immunoreactive cells were harvested (Fig. 1A), RNA was extracted, and nested RT-PCRs for {rho}2 were performed (Fig. 1B). Since additional tissue surrounds the cells of interest after microdissection, control samples in which GH/TSH immunoreactive cells were destroyed by laser shots before excision were also analyzed. The {rho}2 subunit was detected in GH cells and, as expected, in TSH cells, but not in the controls.



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FIG. 1.
Pituitary GH cells express the GABA-C subunit {rho}2. A, section of a rat pituitary used for laser microdissection of either GH- or TSH-secreting cells identified beforehand by immunohistochemistry. Groups of positive cells (here immunostained TSH cells; corresponding to TSH+ in panel B) were isolated from the surrounding tissue by a laser beam (1a) and subsequently catapulted (1b) into the cap of a microfuge tube. Each sample contained the equivalent of 30–50 immunoreactive cells. As a control, immunostained cells were destroyed (corresponding to TSH- in panel B; 2, arrows) by the laser before catapulting. The scale bar is equivalent to 40 µm. B, RNA extracted from the laser-dissected cells was subsequently submitted to nested RT-PCR amplifications. GABA-C receptor {rho}2 was detected in GH (lane GH+) and in TSH (lane TSH+) cells but not in their respective negative controls (lanes GH- and TSH-). Sequencing confirmed {rho}2 identity.

 

GH3 cells are derived from a rat GH pituitary adenoma (15) and are widely used as a model for the study of pituitary somatotrophs. We examined whether GH3 cells also possess GABA-C receptors. RT-PCR experiments identified {rho}1 and {rho}2 subunit mRNAs in GH3 cells (Fig. 2A). GABA-C receptor protein at GH3 cell membranes was shown by immunocytochemistry (Fig. 2B). The antiserum, which recognizes both {rho}1 and {rho}2, showed a staining, which was confined to the plasma membrane of the cells (Fig. 2B, left panel). Omitting the antiserum resulted in a weak homogeneous staining pattern (Fig. 2B, right panel). Further controls were performed by incubating the cells with rabbit normal serum (dilutions ranging from 1:10,000 to 1:40,000). These experiments led to a homogeneous nonspecific staining within the cells (data not shown) and argue for the specificity of the membrane-associated immunoreactivity obtained using the anti-GABA-C receptor antiserum.



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FIG. 2.
GABA-C receptors in GH3 cells. A, RNA samples extracted from GH3 cells, rat whole pituitary (Pit.), and brain were reverse-transcribed and used for PCR using specific primers for GABA-C {rho}1- and {rho}2-subunits. Brain samples served as positive controls; PCRs performed without template were negative (Co.). Sequencing of the PCR products confirmed their identity. B, the use of an antiserum recognizing both {rho}1- and {rho}2-subunits allowed localization of GABA-C receptors at GH3 cell membranes as seen by immunofluorescence microscopy. Immunoreactivity was detected in most cells; the arrow points to membrane-associated staining (left panel). In the control shown, the primary antiserum was omitted (right panel). The scale bar is equivalent to 10 µm.

 

Electrophysiology of GABA-C Receptors in GH3 Cells—The whole cell patch clamp technique was applied to single GH3 cells to test the functionality of the putative GABA-C receptors. Application of 100 µM GABA induced an inward whole cell current at a holding potential of -80 mV in about 70% of the cells (n = 13; Fig. 3A). The GABA-induced current was rapidly and almost completely desensitizing during GABA application. GABA was also capable of eliciting the current in the presence of the GABA-A antagonist bicuculline (n = 4; 100 µM; Fig. 3C), even when bicuculline was applied about 1 min prior to GABA. There was no significant difference between the maximum amplitudes (paired t test) for 100 µM GABA (0.8 ± 0.3 pA/picofarads; mean ± S.D.) and for 100 µM GABA + 100 µM bicuculline (0.9 ± 0.8 pA/picofarads). The channel giving rise to the GABA-induced Cl- current was identified as GABA-C receptor because of its activation by the specific GABA-C receptor agonist CACA (100 µM) in all GABA-sensitive cells tested (n = 6; Fig. 3B). The specific peak current activated by GABA or CACA was in the range of 0.5–5.0 pA/picofarads and thereby comparable with the GABA-C current observed in TSH cells (6). Activation of the current by GABA or CACA was repeatable several times in the same cell after washing with agonist-free bath solution for about 2 min (Fig. 3, A and B). The peak amplitude did not decrease in the course of the experiment in contrast to the rundown of the GABA-C receptor currents reported in TSH cells (6).



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FIG. 3.
Induction of GABA-C whole cell Cl- currents in GH3 cells. As shown in A, application of 100 µM GABA induced a quickly desensitizing inward current. As shown in B, in the same cell, 100 µM CACA elicited a current of similar amplitude and kinetics. As shown in C, GABA induced the current also in presence of the specific GABA-A antagonist bicuculline (Bic; 100 µM). The cells were clamped at -80 mV under symmetrical Cl- concentrations (ECl {approx} 0). The horizontal bar and thereby the duration of drug application represent a time period of 10 s.

 

The Activation of GABA-C Receptors Provokes Intracellular Ca2+ Transients—We performed fluorimetric measurements of cytosolic Ca2+ concentrations in GH3 cells (Fig. 4). Both GABA and the GABA-C-specific agonist CACA (100 µM of each) induced Ca2+ transients of similar amplitude (n = 9 cells). However, the use of baclofen (GABA-B receptors agonist) did not lead to Ca2+ transients (data not shown). The Ca2+ transients recorded in GH3 cells using either GABA or CACA are most probably due to Ca2+ influx through voltage-gated Ca2+ channels (16). Indeed, both KCl- and GABA-induced Ca2+ transients are almost completely blocked by Gd3+ (500 µM), a blocker of voltage-dependent Ca2+ channels (preliminary studies, data not shown).



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FIG. 4.
Intracellular Ca2+ transients of GH3 cells in response to CACA and GABA. Single GH3 cells (n = 9) were subjected to the consecutive application of CACA and GABA (100 µM each) for 5 s. Both drugs induced a rapid increase of intracellular free Ca2+. Upon drug removal, Ca2+ levels returned to basal values. Note that the GABA-application is preceded by a spontaneous Ca2+ signal, commonly observed in GH3 cells. Changes in intracellular free Ca2+ concentrations are given as the recorded 340/380-nm ratios.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, the expression of GABA-C receptors is reported in endocrine cells, namely in the pituitary GH cells and in their corresponding cell line, GH3. These receptors are functional in GH3 cells: the current induced by GABA or by the typical GABA-C agonist, CACA, is characterized by its insensitivity to the GABA-A antagonist bicuculline and by its quick desensitization. The opening of GABA-C channels may induce a membrane depolarization as suggested by an increase in Ca2+i levels measured by fluorometry on GH3 cells.

In the pituitary gland, all types of GABA receptors are expressed (7, 8, 17), and GABA, produced either by the hypothalamus or by the pituitary itself (7, 18, 19), is known to be involved in the regulation of hormone levels (8, 20, 21). Interestingly GABA-C receptors were demonstrated previously in pituitary TSH cells (6). Therefore, these cells served as positive controls in our study. We detected the {rho}2 subunit in GH and, as expected, in TSH cells. Boue-Grabot et al. (6) showed that TSH cells possess functional GABA-C channels, reported the absence of {rho}1 in follicle-stimulating hormone, adrenocorticotropin, and prolactin cells, but did not investigate GH cells. By identifying {rho}2 in TSH and GH cells, we confirm and extend their results.

We verified by RT-PCR and immunocytochemistry that GH3 cells also express GABA-C receptors. Then, performing patch clamp recordings on single GH3 cells, we could prove functional GABA-C receptors. The application of either GABA or CACA (100 µM of each) induced a Cl- current insensitive to bicuculline, exhibiting an untypical fast desensitization but lacking rundown. Similar inactivating GABA-C currents were observed in rat TSH cells (6) but also in bipolar cells of the carp retina (22). Usually, GABA-C receptor activation is regarded to entail sustained Cl- currents (23, 24, 25) in contrast to the typically desensitizing GABA-A receptor (26, 27, 28). Most likely, variations in the subunit composition (29), species-dependent protein sequence differences, or/and tissue-specific splicing variants (6) account for these different properties. In addition, we reported previously the presence of the GABA-A receptor subunit {gamma}2 in rat GH cells (7). The possibility that heterooligomerization might occur among {rho} and GABA-A subunits is under debate (30, 31) and may also explain various electrogenic profiles.

In this report, we show that pituitary GH cells express GABA-C receptors and that they are functional in a corresponding tumor cell line, GH3. What could be the physiological role of GABA-C receptors in GH-secreting cells? A gut neuroendocrine tumor cell line STC-1 was also shown to possess functional GABA-C receptors (5, 6). In STC-1 cells, GABA increases Ca2+i levels by acting on GABA-C receptors. It is also known that the activation of GABA-B receptors influences Ca2+i concentrations in neurons (32). Since GH3 cells bear functional GABA-B receptors as well (8), one can suppose that GABA may control Ca2+i levels via both metabotropic and ionotropic mechanisms. To test this hypothesis, we performed fluorimetric measurements of cytosolic Ca2+ concentrations in GH3 cells. We showed that both GABA and CACA (100 µM of each) induced Ca2+ transients of similar amplitude. However, preliminary experiments using baclofen (a GABA-B receptor agonist) did not result in Ca2+ transients. These observations argue for a control of Ca2+i levels by GABA via ionotropic routes.

A possible mechanistic explanation for the Ca2+ transients can be deduced from results obtained in developing neurons. There, GABA acts as a trophic substance. Via GABA-A channels, GABA can depolarize the cell membrane when the Cl- reversal potential is positive to the resting membrane potential (33, 34, 35). Thus, it can provoke Ca2+ transients via the activation of voltage-operated Ca2+ channels (5, 36). The Ca2+ transients recorded in GH3 cells, using either GABA or CACA, are most probably due to Ca2+ influx through voltage-gated Ca2+ channels (16). Indeed, preliminary results showed that both KCl- and GABA-induced Ca2+ transients are almost completely blocked by Gd3+ (500 µM), a blocker of voltage-dependent Ca2+ channels. We propose that GABA action on GABA-C receptors leads to membrane depolarization and Ca2+ influx in GH3 cells, suggesting an excitatory function of GABA in endocrine cells.

In summary, the presence of multiple GABA receptor subtypes in GH cells, with different properties and GABA sensitivities, suggests that GABA may have several functions in these cells (e.g. regulation of chloride conductance, membrane potential, control of Ca2+i concentrations, modulation of hormone secretion). Our present results are in accordance with recent reports indicating new roles of neurotransmitters, including GABA, in various nonneuronal tissues. This is best illustrated in the pancreas, where the neurotransmitter glutamate is secreted by {alpha}-cells and triggers the Ca2+-dependent exocytosis of GABA from {beta}-cells. Once secreted, GABA in turn binds to GABA-A receptors on {alpha}-cells, where it acts as a paracrine inhibitor for glucagon secretion (37). Thus, signaling molecules originally thought to be restricted to the central nervous system appear to be produced and active in important endocrine systems, namely the anterior pituitary and the pancreas.


    FOOTNOTES
 
* This work was supported by a grant from Eli Lilly International Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Anatomisches Institut der Universität München, Biedersteiner Strasse 29, 80802 München, Germany. Tel.: 49-89-4140-3150; Fax: 49-89-397035; E-mail: Mayerhofer{at}lrz.uni-muenchen.de.

1 The abbreviations used are: GABA, {gamma}-aminobutyric acid; CACA, cis-4-aminocrotonic acid; GH, growth hormone; TSH, thyroid-stimulating hormore; RT, reverse transcription. Back


    ACKNOWLEDGMENTS
 
We thank all our colleagues, in particular Barbara Zschiesche, Andreas Mauermayer, and Marlies Rauchfuss. We are grateful to Dr. Ralf Enz for the generous gift of anti-GABA-C antiserum and to Prof. Enrico Cherubini and Dr. Frédéric Didelon for expert advice. Access to the PALM® Robot-MicroBeam device for the experiments of laser microdissection was made possible by Dr. Viktor Meineke, Munich, Germany.



    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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