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Endocrinology, doi:10.1210/en.2003-1413
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Endocrinology Vol. 145, No. 5 2402-2411
Copyright © 2004 by The Endocrine Society

An Intrinsic {gamma}-Aminobutyric Acid (GABA)ergic System in the Adrenal Cortex: Findings from Human and Rat Adrenal Glands and the NCI-H295R Cell Line

Klaus Metzeler, Agnes Agoston and Manfred Gratzl

Anatomisches Institut, Universität München, 80802 München, Germany

Address all correspondence and requests for reprints to: Dr. Manfred Gratzl, Anatomisches Institut der Universität München, Biedersteiner Strasse 29, 80802 München, Germany. E-mail: gratzl{at}LMU.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
{gamma}-Aminobutyric acid (GABA), a major neurotransmitter in the central nervous system, also acts as a paracrine or autocrine signaling molecule in endocrine tissues such as the pancreatic islets, adenohypophysis, and testis. In the present study, we describe local GABA production and functional GABAB receptors in the adrenal cortex, possibly forming an auto- or paracrine GABAergic system. Using immunohistochemistry and RT-PCR, we localized the GABA-synthesizing enzyme glutamate decarboxylase 67 and the vesicular GABA transporter in steroid-producing cells of the human and rat adrenal cortex. Immunocytochemistry, Western blots, and RT-PCR experiments demonstrated the presence of glutamate decarboxylase 67 in the human adrenocortical cell line NCI-H295R. Measurements of glutamate decarboxylase activity confirmed that, in these cells and in rat adrenals, glutamate is decarboxylated to form GABA. In addition, we found expression of the GABAB(1a), GABAB(1e), and GABAB(2) subunits of the heterodimeric GABAB receptor in NCI-H295R cells as shown by RT-PCR. GABAB(1a) and its truncated splice variant GABAB(1e) were also found in human and rat adrenal glands. Immunostaining for the GABAB(2) subunit revealed its presence in the human and rat adrenal cortex and in NCI-H295R cells. The GABAB receptors we identified were functional because the GABAB agonist baclofen inhibited T-type Ca2+ currents in whole-cell patch clamp experiments on NCI-H295R cells. This effect was blocked by pertussis toxin. Furthermore, the {alpha}2-, {alpha}3-, ß2-, ß3- {gamma}2-, and {epsilon}-subunits of the GABAA receptor were detected in this cell line by RT-PCR. Hence, we conclude that GABA is synthesized and stored by steroid-producing cells of the adrenal cortex and may influence these cells in a paracrine or autocrine manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADRENAL STEROIDOGENESIS IS tightly regulated via several pathways. In addition to humoral control by the circulating hormones ACTH and angiotensin II, a wide variety of other hormones, neuropeptides, neurotransmitters, and cytokines participate in the control of adrenocortical function. These substances originate from the adrenal medulla, neurons, vascular cells, and immune cells (1). Recently, there was a report that {gamma}-aminobutyric acid (GABA) influences steroidogenesis in the rat adrenal cortex in vivo (2). The origin of adrenocortical GABA and the mechanism of its action, however, remained unclear.

An as-yet-unexplored possibility is that adrenocortical cells themselves may secrete factors that modulate their own function, forming an autocrine or paracrine regulatory loop. This concept is supported by numerous studies showing that adrenocortical cells, although being of mesodermal origin, possess markers of neuroendocrine differentiation. Their presence was first noticed in adrenocortical neoplasias (3, 4, 5) but subsequently also in the normal adrenal cortex. Specifically, synaptobrevin 2 (also termed vesicle-associated membrane protein 2), neuron-specific enolase, and the neural cell adhesion molecules are present in steroid-producing adrenocortical cells (6, 7, 8, 9). The synaptic vesicle membrane protein synaptophysin is expressed by the adrenocortical tumor cell line NCI-H295R (9). All those findings raise the question whether these elements of neuroendocrine differentiation are linked to the local production and secretion of neurotransmitters that could play a role in the regulation of adrenal function. Para- or autocrine GABAergic signaling pathways have been described in several other endocrine organs such as the pancreatic islets, adenohypophysis, ovary, and testis (10, 11, 12, 13, 14, 15).

The proteins that are responsible for GABA synthesis and storage can serve as traits to identify sites of GABA production in neuronal and endocrine tissues. The enzyme glutamate decarboxylase (GAD) catalyzes the formation of GABA from glutamic acid. There are two functional isoforms of this enzyme, GAD 65 and GAD 67, which are encoded by separate genes (16) and show different patterns of expression both in the central nervous system and in the periphery (17). The vesicular inhibitory amino acid transporter (VIAAT) mediates the uptake of GABA into secretory vesicles. Therefore, localization of this transporter is another way to identify GABA-producing cells (18). In its target cells, GABA exerts its regulatory actions via ionotropic GABAA and GABAC and metabotropic GABAB receptors. All three types of GABA receptors are expressed in peripheral organs including the intestine, adenohypophysis, pancreatic islets, heart, ovary, and testis (13, 14, 15, 19, 20, 21, 22).

GAD, GABAA, and GABAB receptors are present in whole rat adrenal glands (14, 17, 22, 23). GAD activity was also found in isolated bovine chromaffin cells (24, 25), and catecholamine release by these cells can be modulated via GABAA and GABAB receptors (25, 26). We chose to investigate the adrenal cortex as a possible additional source of adrenal GABA. In the present study, we show that the enzymes for GABA production and storage are present in the human and rat adrenal cortex and that the human adrenocortical cell line NCI-H295R expresses GABAA and GABAB receptor subunits, including the truncated GABAB(1e) splice variant. Furthermore, we present evidence that NCI-H295R cells possess functional GABAB receptors because stimulation with baclofen inhibits the activity of T-type Ca2+ channels via Gi/o proteins. These results point toward an auto- or paracrine role of GABA in the regulation of adrenocortical function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human and rat tissue samples
Paraffin-embedded and frozen normal human adrenals were obtained from a tissue archive as reported previously (27). Male and female Sprague Dawley rats were bred at the Technische Universität München following the institutional animal care guidelines. Adult animals were painlessly killed under ether anesthesia. Their adrenal glands and cerebella were removed and either immediately frozen in liquid nitrogen for subsequent protein and RNA isolation or fixed in 4% paraformaldehyde or Bouin’s fixative and embedded in paraffin.

Cell culture
NCI-H295R cells (termed H295R hereafter) were obtained from the American Type Culture Collection (Manassas, VA) and cultured in DMEM/Ham’s F-12 medium containing sodium selenite (5 ng/ml), insulin (10 µg/ml), transferrin (5.5 µg/ml), ethanolamine (2 µg/ml), albumin (1 mg/ml), linoleic acid (9 µg/ml), and HEPES (15 mM) (all from Sigma, Taufkirchen, Germany) and supplemented with 3% of NU-Serum I (BD Biosciences, Bedford, MA). Cells were kept at 37 C in a humidified atmosphere containing air and carbon dioxide (95%/5% vol/vol).

Antibodies
A monoclonal antibody directed against rat aldosterone synthase and polyclonal rabbit antiserum to GAD were purchased from Chemicon International, Inc. (Temecula, CA). The aldosterone synthase antibody served as a specific marker for the mineralocorticoid-producing cells of the zona glomerulosa (28). The anti-GAD antiserum was raised against a synthetic decapeptide from the C terminus of rat GAD 65, yet it recognized human and rat GAD 65 and GAD 67. Antiserum to the VIAAT was a generous gift from Bruno Gasnier (Institut de Biologie Physico-Chimique, Paris, France) (18). Polyclonal antiserum to the GABAB(2) receptor subunit was obtained courtesy of Graham Disney and Fiona Marshall (GlaxoWellcome Research and Development, Stevenage, UK) (29). Biotin-, peroxidase- and fluorescein isothiocyanate (FITC)-labeled goat antirabbit antibodies and biotin-coupled goat anti-mouse antibody were purchased from Dianova (Hamburg, Germany).

Immunostaining
H295R cells were fixed in 4% paraformaldehyde for 30 min, and immunostaining was performed as described previously (30). After blocking with 2% goat normal serum, incubation with the primary antibody (diluted 1:1000 in potassium PBS; pH 7.4) was carried out overnight at 4 C. The cells were subsequently washed and incubated for 1 h at room temperature with FITC-coupled goat antirabbit IgG (1:200). Stainings were visualized using a TCS SP2 confocal microscope (Leica, Heidelberg, Germany) or an Axiovert fluorescence microscope (Zeiss, Oberkochen, Germany). Controls were performed by omitting the first antibody or replacing it with purified nonimmune rabbit IgG (1:10,000).

Slices of human and rat adrenal tissues were deparaffinized before immunostaining, treated with PBS containing 8% of H2O2 and 1% of methanol to block endogenous peroxidase activity, and heated in a microwave oven in 10 mM sodium citrate buffer (pH 6.0) for antigen retrieval. They were blocked in PBS containing 5% normal goat serum and incubated overnight at 4 C with the primary antibodies (anti-GAD, 1:1000; anti-VIAAT, 1:4000; anti-GABAB(2), 1:1000; antialdosterone synthase, 1:20). After washing, the slices were probed with the appropriate biotin-coupled secondary antibodies (1:500) for 2 h, and the staining was visualized using the avidin-biotin complex method (Vectastain Elite Kit, Vector Laboratories, Burlingame, CA) and diaminobenzidine as the chromogenic substrate. The slices were examined using an Axioplan microscope (Zeiss). In all experiments, we performed controls omitting the first antibody or using nonimmune serum of the same species instead. For VIAAT, we also performed adsorption controls by preincubating the first antibody with the immunogenic fusion peptide (1 ng/µl) for 20 min before use.

SDS-PAGE and Western blotting
H295R cells were washed with PBS and lysed in sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 10% saccharose, and 2% sodium dodecyl sulfate. Frozen tissue samples of rat adrenals and cerebella were thawed and homogenized in the same buffer with a rotor-stator homogenizer. Protein content was determined using the Dc protein assay (Bio-Rad, Hercules, CA). After adding 10% of ß-mercaptoethanol, 5–30 µg of protein per lane was loaded on 15% polyacrylamide gels and separated electrophoretically. Proteins were then blotted to nitrocellulose membranes and probed with anti-GAD primary antibody (1:1000, overnight at 4 C) followed by a peroxidase-coupled goat antirabbit IgG secondary antibody (1:5000). Immunoreactive bands were visualized with enhanced chemiluminescence reagent on Hyperfilm ECL films (both from Amersham, Uppsala, Sweden).

RNA isolation, reverse transcription, PCR
Total RNA was isolated from frozen human and rat adrenals and H295R cells using the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. One microgram of total RNA was treated with RQ1 DNase (Promega, Mannheim, Germany) to eliminate possible contamination by genomic DNA. Reverse transcription using 15-mer polydeoxythymidine or random hexamer primers and Moloney murine leukemia virus-reverse transcriptase (Stratagene, La Jolla, CA) was performed as described previously (19). In control reactions, no RNA sample was added to the reverse transcription reaction mix, which subsequently was used for PCR amplification.

PCR using Taq polymerase (Promega) was carried out for 35–40 cycles, each consisting of 30 sec at 94 C, 60 sec at 55 C, and 60 sec at 72 C. If necessary, the product of this first PCR was purified using the MinElute kit (Qiagen), and 1 µl of the eluate was used as template for a second PCR with nested primers. The gene-specific primers are described in Tables 1Go and 2Go. All primer pairs, except those for the GABAA receptor subunits {alpha}1 and {alpha}2, were chosen to span at least one intron of the genomic sequence to detect any contamination with genomic DNA (11, 16, 31, 32, 33). For the GABAB(1) receptor subunit, we used primers spanning exon 11 and therefore distinguishing between cDNA encoding the GABAB(1e) subunit, which lacks this exon, and the full-length GABAB(1a) subunit (23). A second primer pair was designed with the 3' primer corresponding to a sequence within exon 11 and therefore only recognizing GABAB(1a) but not the truncated subunit. In all experiments, the identity of the amplified cDNA was confirmed by direct sequencing using one of the PCR oligonucleotide primers (Agowa, Berlin, Germany). For the identification of the GABAB(1) receptor subunit splice variants (1a) and (1e), the different PCR products were cut out of the agarose gel and cleaned up using the QIAquick Gel Extraction kit (Qiagen) before sequencing them.


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  		TABLE 1. Primers used for PCR analysis of GAD, VIAAT, and GABAB receptor subunit expression

 

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  		TABLE 2. Primers used for PCR analysis of GABAA receptor subunit expression in NCI-H295R cells

 
GAD activity measurement
Determination of GAD activity by measuring the production of radiolabeled carbon dioxide from 14C-glutamate was performed as described previously (15, 34). In brief, cells or tissue samples were homogenized in 60 mM potassium phosphate buffer, pH 7.1, containing 0.5% Triton X-100, 1 mM 2-aminoethyl-isothiouronium bromide, and 1 mM phenylmethanesulfonyl fluoride (all from Sigma), centrifuged, and the supernatants were used in the assay. The assay was performed in a total reaction volume of 60 µl, containing 20 µl of sample and 0.1 mM EDTA, 0.5% Triton X-100, 0.1 mM dithiothreitol, 0.05 mM pyridoxal phosphate, 9 mM L-glutamate, 3.3 µCi/ml 14C-glutamate, and 30 mM potassium phosphate buffer. The reaction mix was incubated for 1 h at 37 C and then stopped by adding 100 µl of 10% trichloroacetic acid per vial. The mixture was then incubated for another hour, the released CO2 was absorbed on benzethonium hydroxide-drenched filter disks, and bound radioactivity was determined using a Tri-Carb 2100 liquid scintillation counter (Packard, Meriden, CT). The values obtained were normalized to protein content. Activities measured in rat adrenal glands (n = 6) and H295R cells (n = 18) were expressed as percent of the activity in rat cerebellum (n = 6). Samples of H295R cells (n = 3), rat adrenal glands (n = 3), and rat cerebellum (n = 5) that were heated to 95 C for 10 min served as negative controls. Statistic analyses were performed using GraphPad Prism 3.02 (GraphPad Software, San Diego, CA). GAD activities in heated and untreated samples were compared using a two-tailed Mann-Whitney test.

Electrophysiology
Whole-cell patch-clamp recordings were performed on H295R cells with an EPC-9 amplifier and Pulse 8 software (HEKA, Lambrecht, Germany) at room temperature (20–22 C). Ca2+ currents were measured using an extracellular solution containing (in mM) tetraethylammonium chloride (117), BaCl2 (20), sucrose (30), HEPES (10), MgCl2 (2), and glucose (5) (pH 7.45, adjusted with CsOH). Glass pipettes (GB 150–8P, Science Products GMBH, Hofheim, Germany) were filled with solution containing (in mM) CsCl (120), tetraethylammonium chloride-Cl (20), CaCl2 (1), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5), HEPES (10), MgCl2 (2), Mg-ATP (4), and Na-GTP (0.2) (pH 7.2, adjusted with CsOH) and had a resistance of 2.5–4.0 M{Omega}. The current output was filtered at 5 kHz with a four-pole Bessel filter, and data were acquired at 50-µsec intervals (20 kHz). Ca2+ currents were elicited by step depolarizations from a holding potential of –80 or –90 mV up to +50 mV with 10-mV increments and step intervals of 4 sec. For each cell, the membrane resistance and capacitance were determined immediately after the establishment of the whole-cell configuration. Baclofen (50 mM, dissolved in 50 mM NaOH), nifedipine (dissolved in dimethylsulfoxide, 10 mM), and NiSO4 (dissolved in water, 100 mM) stock solutions were diluted into the external recording solution to the appropriate concentrations. To test whether Gi/o proteins were involved in the observed effects of GABAB receptor activation, H295R cells were cultured in the presence of 200 ng/ml pertussis toxin (PTX, stock dissolved in water, 100 µg/ml) for 12 h and subsequently used for patch-clamp experiments. All reagents were obtained from Sigma.

The voltage dependence of activation of Ca2+ currents was characterized from the current-voltage relationship (I-V curve). Peak current amplitudes were normalized to the maximum peak current (I/Imax), averaged, and plotted, and data points were fitted with a Boltzmann equation: I/Imax = 1/{1 + exp[(V – V50]/k)} using GraphPad Prism 3.02. V50 is the voltage at which I is half of Imax, and k indicates the slope of the relationship between channel activation/inactivation and membrane potential. To quantify the time course of activation ({tau}act) and inactivation ({tau}inact), traces were fitted by a single exponential equation. Data are expressed as means ± SEM in the text and in the graphs. The changes were analyzed statistically using the paired Student’s t test with the help of the SigmaPlot 8.0 software package (Wavemetrics, Lake Oswego, OR).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of VIAAT, GAD, and GABAB(2) in the adrenal cortex
We localized the vesicular GABA transporter (VIAAT) in the rat adrenal cortex by immunohistochemistry. Immunoreactivity was strongest in the outermost layer of the cortex, whereas a weaker staining was observed throughout the other regions of the cortex. No staining was observed in the medulla except for single cells showing weak immunoreactivity (Fig. 1Go, A and B). We did not investigate further whether these were chromaffin cells or dispersed adrenocortical, neural, or other cells located within the medulla. Preincubation of the primary antibody with the immunogenic peptide almost completely abolished VIAAT immunostaining (Fig. 1AGo, inset). To check for the presence of GAD in the rat adrenal gland, we performed immunostainings of adrenal tissue sections using a polyclonal antiserum that recognizes both GAD isoforms (GAD 65 and GAD 67). These experiments showed that GAD immunoreactivity was distributed similarly to that for the VIAAT, i.e. it was localized predominantly in the outer region of the rat adrenal cortex (Fig. 1DGo). Consecutive sections stained with antibodies against VIAAT, GAD, and aldosterone synthase revealed that VIAAT and GAD immunoreactivities were especially strong in, but not limited to, the mineralocorticoid-producing zona glomerulosa (Fig. 1Go, E–G).



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  		FIG. 1. Immunohistochemical localization of VIAAT, GAD, and GABAB receptors in the rat adrenal cortex. A, In the rat adrenal gland, the vesicular GABA transporter (VIAAT) is predominantly located in the outermost region of the cortex, but a weaker staining can also be observed in the other cortical zones. Bar, 200 µm; g., zona glomerulosa; f., zona fasciculata; r., zona reticularis of the adrenal cortex; m., adrenal medulla. Inset, Preincubation of the anti-VIAAT antibody with the immunogenic peptide almost completely abolished the staining on a consecutive section. B, the region outlined in panel A is shown at a higher magnification (bar, 50 µm). C, Immunostaining for the GABAB(2) receptor subunit shows its presence in the outer region of the adrenal cortex as well as in adrenomedullary cells. Bar, 200 µm. D, GAD immunoreactivity in the rat adrenal cortex, which also is strongest in the outer region of the cortex (bar, 100 µm). Consecutive sections of a rat adrenal gland were stained for VIAAT (E), GAD (F), and aldosterone synthase (G), revealing that mineralocorticoid-producing zona glomerulosa cells exhibit strong GAD and VIAAT immunoreactivity (bars, 50 µm).

 
In human adrenals, in contrast, VIAAT (Fig. 2AGo) and GAD (Fig. 2CGo) immunostainings were equally intense in all zones of the cortex.



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  		FIG. 2. Immunohistochemical localization of VIAAT, GAD, and GABAB receptors in the human adrenal cortex. A, VIAAT immunostaining in a human adrenal gland; B, corresponding preadsorption control. In comparison with the rat adrenal, the staining in the human cortex is more homogeneous. C, The same is true for GAD, which can also be detected in all zones of the human adrenal cortex. D, Similar to rat adrenals, the GABAB(2) subunit is expressed in the outer region of the human adrenal cortex as well as in the medulla (all bars, 200 µm; g., zona glomerulosa; f., zona fasciculata; r., zona reticularis of the adrenal cortex; m., adrenal medulla).

 
We also used a polyclonal antiserum against the GABAB(2) receptor subunit. In both human (Fig. 2DGo) and rat (Fig. 1CGo) adrenals, immunoreactivity was seen in the outer region of the adrenal cortex and in the medulla.

GAD 67 is the predominant GAD isoform in the adrenal
We subsequently verified these results at the mRNA level. RT-PCR experiments revealed that GAD 67 and VIAAT are expressed in human and rat adrenals (Figs. 3AGo and 4Go). Using nested RT-PCR, we also found GAD 65 mRNA in the adrenals of both species (Fig. 3BGo). However, Western blot experiments using protein from whole rat adrenal glands and a polyclonal GAD antiserum revealed only one immunoreactive band, corresponding to GAD 67 (Fig. 3CGo). In samples from rat cerebellum, the antibody detected both GAD 65 and GAD 67.



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  		FIG. 3. GAD expression in human and rat adrenals and in H295R cells. A, We detected GAD 67 mRNA in H295R cells, in the human adrenal, and in rat adrenal glands in RT-PCR experiments. B, By using nested PCR, GAD 65 mRNA also was detected in human and rat adrenals but not in H295R cells. C, Western blotting using anti-GAD antiserum confirmed that GAD 67 is the predominant GAD isoform expressed in the rat adrenal gland and in H295R cells. Rat cerebellum (cereb.) served as a positive control.

 


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  		FIG. 4. VIAAT, the vesicular GABA transporter, is expressed in the human and rat adrenal. RT-PCR results demonstrate the presence of VIAAT mRNA in the rat adrenal gland. In the human adrenal, VIAAT mRNA was identified using a nested RT-PCR approach.

 
GAD is present and active in NCI-H295R cells
We performed immunocytochemistry on the adrenocortical tumor cell line H295R, and we observed a strong cytoplasmic staining with an anti-GAD antiserum (Fig. 5AGo). To identify which GAD isoforms are expressed in H295R cells, we also performed Western blots using the same antiserum. In these experiments, we only detected GAD 67 but not GAD 65 (Fig. 3CGo). RT-PCR experiments confirmed these results, showing that mRNA for GAD 67 is present in H295R cells (Fig. 3AGo), whereas GAD 65 could not be detected, even when a nested PCR approach was used (Fig. 3BGo).



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  		FIG. 5. H295R cells express GAD and GABAB(2 ). H295R cells were immunostained with polyclonal anti-GAD antiserum (A, bar, 40 µm) or an antiserum against the GABAB(2) receptor subunit (B, bar, 20 µm) and FITC-labeled secondary antibody. The insets show controls where the primary antibody was omitted (A) or replaced with nonimmune purified rabbit IgG (B).

 
GAD 67 was shown to be active in H295R cells as well as in rat adrenals by measuring its catalytic activity in an in vitro assay. Lysates of H295R cells exhibited 3.54 ± 0.33% (mean ± SEM) of the activity found in rat cerebellum, and in lysates of rat adrenal glands, GAD activity was 3.10 ± 1.39% compared with rat cerebellum. Boiling almost completely abolished the activity in H295R lysates to 0.03 ± 0.03% (P = 0.008 for the difference from untreated samples), in adrenal samples to 0.38 ± 0.19% (P = 0.024), and in cerebellum to 0.08 ± 0.02% (P = 0.004) of untreated cerebellum samples.

H295R cells express GABAB and GABAA receptor subunits
We investigated the presence of the GABAB(2) subunit on H295R cells by immunocytochemistry. In H295R cells, we observed intense immunostaining, particularly on cells growing in clusters (Fig. 5BGo). Control experiments using nonimmune IgG were negative (Fig. 5BGo, inset).

These findings were confirmed by RT-PCR experiments. In H295R cells, we first showed expression of the GABAB(2) receptor subunit (Fig. 6AGo). Because a variety of splice variants of the GABAB(1) subunit exist, we then used primers designed to distinguish between the GABAB(1a) and GABAB(1e) subunits. We observed two different PCR products with sizes of 571 and 421 bp in samples from H295R cells, rat adrenal glands, and, as reported previously, in human adrenal glands (23) (Fig. 6BGo). Purification and sequencing of these two products proved that they corresponded to the GABAB(1a) and GABAB(1e) splice variants. In rat cerebellum, which was used as a positive control, only GABAB(1a) was present. A second primer pair that is specific for GABAB(1a) and does not recognize GABAB(1e) was also used (Fig. 6AGo), and it confirmed the presence of mRNA for the full-length (1a) subunit in H295R cells.



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  		FIG. 6. GABAB receptor subunit expression in the H295R cell line and the adrenal gland. A, Two GABAB receptor subunits, GABAB(1a) and GABAB(2), shown by RT-PCR in H295R cells. B, Expression of both the GABAB(1a) subunit and its truncated splice variant GABAB(1e) in H295R cells and human and rat adrenal glands. Expected product sizes were 571 bp for the (1a) variant and 421 bp for the (1e) variant. Rat cerebellum cDNA (cereb.) showed exclusive expression of GABAB(1a).

 
Furthermore, we investigated the presence of various subunits of the GABAA receptor in H295R cells by RT-PCR. We found mRNA for the GABAA receptor subunits {alpha}2, {alpha}3, ß2, ß3, {gamma}2, and {epsilon} (Fig. 7Go), whereas, in contrast, mRNA for the {alpha}1- and ß1-subunits could not be detected (data not shown).



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  		FIG. 7. Expression of GABAA receptor subunits in H295R cells. mRNA for the GABAA receptor subunits {alpha}2, {alpha}3, ß2, ß3, {gamma}2, and {epsilon} was detected in RT-PCR experiments (+ are reactions with H295R cDNA and – are the corresponding negative controls).

 
GABAB receptor stimulation decreases T-type Ca2+ currents in H295R cells
In whole-cell patch-clamp experiments, we investigated the properties of Ca2+ currents in H295R cells (Fig. 8Go). Examining the Ca2+ current-voltage (I-V) relationship, we found three different subpopulations of cells, according to the properties of their recorded Ca2+ currents: group T cells (8 of 68) exclusively showing low voltage-activated (LVA) T-type Ca2+ currents, group L cells (11 of 68) showing only high voltage-activated (HVA) L-type Ca2+ currents, and finally, group L/T cells (49 of 68) showing mainly L- but also T-type Ca2+ currents. From a holding potential of –80 mV, depolarizing steps activated both T-type and L-type Ca2+ currents which, as can be seen in Fig. 8AGo, have partially overlapping voltage dependencies. In group T cells, the T-type current became detectable at around –50 mV and peaked at –20 mV (Fig. 8BGo). This current was insensitive to nifedipine (4 µM) and showed high sensitivity to Ni2+ (100 µM Ni2+ completely blocked this current, data not shown). On average, the peak current density at –20 mV was –7.6 ± 1.2 pA/pF in group T cells (n = 8). In group L cells, the threshold potential for the L-type current was around –40 mV, and it peaked at +10 mV. The average mean current amplitude at +10 mV was –7.8 ± 1.5 pA/pF (n = 4).



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  		FIG. 8. Effect of a GABAB agonist (200 µM baclofen) on Ca2+ currents in H295R cells. Cells were grouped according to the types of Ca2+ currents they expressed (A, group L/T; B, group T; C, group L). A, Left side, peak current-density voltage relationship (I-V curve) of Ca2+ currents in group L/T cells (step protocols), under control conditions ({square}), and in the presence of 200 µM baclofen ({triangleup}, n = 6). Note that the inhibition of inward current by 200 µM baclofen is more evident at negative test potentials (–30 to 0 mV). Right side, Ramp currents were examined using 2000 msec voltage ramps from –110 to +80 mV (0.095 mV/msec). Representative current traces are shown between –80 and +50 mV, in control ({bullet}), after 4 µM nifedipine ({blacksquare}), and after 4 µM nifedipine + 200 µM Baclofen ({blacktriangleup}) application. B, Group T cells. Left side, peak current-density voltage relationship (I-V curve) before ({square}) and after 200 µM baclofen application ({triangleup}, n = 4). Right side, T-type Ca2+ current traces were elicited at different test potentials (–40, –20, 0, +20 mV) in control ({square}) and in the presence of 200 µM baclofen ({triangleup}). C, Group L cells. Left side, Mean current-density voltage relationship before ({square}) and after 200 µM baclofen application ({triangleup}, n = 4). Mean currents were measured as an average at 80–95% of step protocols. Right side, Representative L-type Ca2+ current traces measured at different test potentials (–40, –20, 0, +10 mV) in control ({square}) and in the presence of 200 µM baclofen ({triangleup}). Data are shown as means ± SEM; the horizontal calibration bars are 100 msec, and the vertical calibration bars are 100 pA. *, Effects were considered significant (P < 0.05).

 
To assess the influence of GABAB receptor activation on Ca2+ currents, 200 µM baclofen (a specific GABAB receptor agonist) was applied. As shown in Fig. 8AGo, in cells showing both T- and L-type Ca2+ channels, the effect of baclofen on Ca2+ currents was stronger at negative test potentials. Indeed, baclofen decreased the peak current amplitude by 23.7 ± 6.1% at –20 mV (P = 0.006) and by 17.3 ± 5.0% at +10 mV (P = 0.027; n = 6). On the basis of these findings, we hypothesized that baclofen selectively inhibits the T-type Ca2+ current. To adequately test this hypothesis, we blocked the L-type current with 4 µM nifedipine before and during baclofen application. As shown in Fig. 8AGo (right side), baclofen attenuated the nifedipine-insensitive T-type Ca2+ current. Additionally, we performed experiments with baclofen on group T and group L cells, respectively, in which only one type of Ca2+ current was detected. In group T cells, short (60-sec) application of 200 µM baclofen significantly reduced (–33.4 ± 9.7%) the T-type current amplitude at the peak of the I-V curve (n = 4; P = 0.024; Fig. 8BGo). In this group, neither the half-maximal voltage (V1/2) and slope of activation (V1/2 = –44.0 ± 1.7 mV, k = 5.4 ± 0.5 mV vs. V1/2 = –42.2 ± 0.9 mV, k = 6.3 ± 0.6 mV) nor the activation and inactivation kinetics ({tau}act = 2.1 ± 0.5 msec, {tau}inact = 14.6 ± 1.9 msec vs. {tau}act = 1.9 ± 0.4 msec, {tau}inact = 14.5 ± 1.7 msec) were affected by 200 µM baclofen. Similar to our observations in group L/T cells, there was no influence of baclofen administration on the L-type current in group L cells (Fig. 8CGo). The mean current density of the L-type current did not change significantly at any voltages. These observations show that only T-type Ca2+ currents are influenced by GABAB receptor activation in H295R cells. When the cells were preincubated with PTX, subsequent baclofen application (200 µM) had no significant effect on the peak current density of the T-type current (at –20 mV): control, –3.6 ± 0.3 pA/pF; baclofen, –3.6 ± 0.3 pA/pF (P = 0.8; n = 5, data not shown). Similarly, there was no effect on the biophysical parameters (V1/2 = –43.5 ± 1.7 mV, k = 6.2 ± 0.5 mV vs. V1/2 = –45.0 ± 2.3 mV, k = 5.9 ± 0.5 mV; and {tau}act = 2.4 ± 0.3 msec, {tau}inact = 15.5 ± 1.3 msec vs. {tau}act = 2.1 ± 0.2 msec, {tau}inact = 15.4 ± 1.7 msec).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
By combining molecular, pharmacological, and electrophysiological approaches, we demonstrated the presence of a local GABAergic system in the human and rat adrenal cortex and in a human adrenocortical cell line (NCI-H295R). The H295R cell line, which originates from a human adrenocortical carcinoma, is well established as a model system for the study of adrenocortical function because it can produce all major adrenal steroids (i.e. glucocorticoids, mineralocorticoids, and androgens) in response to appropriate stimuli (35). This is the first report on GABA production and the presence of functional GABAB receptors modulating Ca2+ currents in adrenocortical cells.

The GABA-producing enzyme GAD and the vesicular transporter for GABA, VIAAT, are crucial elements of GABAergic signaling systems. We used expression of GAD and VIAAT as markers of GABA-producing cells in the adrenal gland and showed that both components are present in the human and rat adrenal cortex. These findings are in line with the observation that a variety of neuroendocrine markers are located in the adrenal cortex (6, 7, 8, 9). Our finding that GAD 67, but not GAD 65, is the predominant GAD isoform in the adrenal is consistent with an earlier report (17). GAD 65 mRNA was detected in human and rat adrenals only after using nested RT-PCR, and we could not detect GAD 65 protein in rat tissue by Western blot. This finding may reflect a low level of GAD 65 expression in adrenocortical or adrenomedullary cells. Due to the high sensitivity of nested RT-PCR, the positive result for GAD 65 could also be due to the presence of other cell types, like neurons, in the tissue specimen used for RNA preparation, or it may result from illegitimate transcription.

There is a third form of glutamate decarboxylase, GAD 25, which results from alternative splicing of GAD 67 mRNA. It encodes a 25-kDa protein lacking enzymatic activity. This splice variant has been described previously in human adrenal cortex, testis, and pancreatic islets (36). Our results obtained from RT-PCR and Western blotting, however, clearly showed that GAD 67 is expressed by adrenocortical cells as well. Additionally, we demonstrated that GAD is active in H295R cells and in rat adrenal glands. The GAD activity measured in the adrenal is low compared with the cerebellum, yet it is similar to the activity found in other endocrine tissues such as the adenohypophysis or testis in which local GABAergic systems are already known (12, 15). Apparently, there are differences in the distribution of GAD and VIAAT between the human and rat adrenal: in rat, both seem to be located primarily in the outer region of the adrenal cortex, whereas they can be detected in all zones of the human cortex. We did not examine this finding further in our study. Taken together, our results from human and rat adrenals and H295R cells provide evidence that adrenocortical cells are able to produce and store GABA for subsequent release.

Our finding that GABA is produced in the adrenal cortex raises the question of its site of action. There are at least two possible targets for adrenocortical GABA. On the one hand, morphological studies showed that adrenocortical and medullary cells are in close interaction with each other (37), and that the blood flow in the adrenal is directed from the cortex toward the medulla (38). Therefore, GABA produced within the cortex may act on receptors located on adrenomedullary cells. Several studies have demonstrated that GABA can modulate catecholamine release from chromaffin cells via activation of GABAA and GABAB receptors (25, 26, 39), and our own results confirm that GABAB(2) immunoreactivity can be observed in the human and rat adrenal medulla. Thus, our results could point toward a GABAergic pathway in which GABA released from the adrenal cortex affects medullary chromaffin cells in an endocrine manner.

During our study of adrenal tissue samples and the adrenocortical cell line NCI-H295R, we found another target for adrenocortical GABA. We located GABA receptors on the cortical cells themselves, suggesting a para- or autocrine signaling pathway. We detected mRNA for several GABAA receptor subunits in NCI-H295R cells by RT-PCR. The subunits {alpha}1–{alpha}3, ß1–ß3, {gamma}1, {gamma}2, and {epsilon} have been found in whole rat adrenals before (22). Because other studies have shown that only a subset of these subunits is also present in the adrenal medulla, Akinci and Schofield (22) conclude that the adrenal cortex might also express GABAA receptor subunits. Our RT-PCR results support this hypothesis, and the subunit composition we found in H295R cells would be sufficient to allow the assembly of pentameric GABAA receptors (40).

However, in the present study, we did not examine further whether the presence of GABAA receptor mRNA leads to the formation of functional ionotropic receptors. Instead, we focused on metabotropic GABAB receptors. We detected GABAB(2) immunoreactivity in the human and rat adrenal cortex and in H295R cells. At the mRNA level, we found expression of the GABAB(1a), GABAB(1e), and GABAB(2) receptor subunits in H295R cells. Although the existence of splice variants of the GABAB(2) subunit is still a matter of discussion, at least seven variants have been described for GABAB(1) (32, 41, 42). The GABAB(1e) subunit, which we identified in H295R cells and rat adrenals, encodes a truncated protein lacking the transmembrane and intracellular domains, and it is known to be present in a variety of peripheral human tissues, including the adrenal gland (23). Our study is the first report on the expression of this truncated subunit in a nontransfected human cell line and in the rat adrenal.

GABAB(1a) and GABAB(2) subunits are known to heterodimerize to form functional GABAB receptors (29). Activation of GABAB receptors typically leads to inhibition of adenylyl cyclase activity, opening of G protein-activated inwardly rectifying K+ channels, and inhibition of voltage-gated Ca2+ currents (42). In contrast, it was previously described that heterodimeric receptors consisting of GABAB(1e) and GABAB(2) subunits expressed in HEK293 cells do not bind the GABAB antagonist CGP 54626A and are not able to activate G protein-activated inwardly rectifying K+ channels or inhibit adenylyl cyclase activity, yet it was not examined whether they retain the ability to inhibit Ca2+ currents (23). The functional relevance of the GABAB(1e) splice variant still remains unresolved. One possible role for this truncated subunit, as suggested by Schwarz et al. (23), could be that GABAB(1a) and GABAB(1e) compete for heterodimerization with GABAB(2). Thus, GABAB(1e) may be involved in regulating the formation of functional GABAB receptors.

We chose to further investigate the effect of GABAB receptor agonists on Ca2+ currents in H295R cells because the regulation of intracellular Ca2+ levels is known to play a crucial role in the control of adrenocortical function (43). In human, rat, and bovine zona glomerulosa cells, the presence of L- and T-type Ca2+ channels has been demonstrated (44, 45). Pharmacological studies have shown that T-type channel activity is linked to aldosterone production, possibly by inducing calcium influx from the extracellular space via the endoplasmic reticulum to the mitochondria (46, 47).

Although Ca2+ currents via HVA and LVA channels are primarily regulated by membrane potential, they can be influenced by G protein-coupled receptors (48, 49). Modulation of HVA (N-, P/Q-, and, in some cases, L-type) Ca2+ currents by GABAB receptors is well documented in neurons (50, 51, 52, 53). Regarding the effect of GABA on LVA (T-type) Ca2+ currents, the studies are controversial. Inhibition of T-type Ca2+ channels after GABAB receptor activation has been observed in rat sensory neurons and lamprey spinal neurons (48, 49, 54), whereas other studies found no such effect (53). This could be explained by the fact that there are three isotypes of T-type Ca2+ channel {alpha}1-subunits ({alpha}1G, {alpha}1H, and {alpha}1I), which display diverse biophysical and pharmacological properties and may be regulated differentially (55). A recent report suggests that a specific interaction between the G protein ß2{gamma}2 subunit and the {alpha}1H T-type channel causes a voltage-independent inhibition of the Ca2+ current (56). Interestingly, this {alpha}1H Ca2+ channel is present in the rat and bovine zona glomerulosa (45), and H295R cells are known to possess functional {alpha}1H as well as {alpha}1C and {alpha}1D (L-type) channels (57). At least under certain culture conditions, they may also express {alpha}1G T-type channels (47). However, in our study, we only found evidence for {alpha}1H, because the T-type calcium currents we recorded were highly sensitive to Ni2+ (55). Moreover, our results show that not all types of Ca2+ currents can be detected simultaneously in every single cell. Rather, there are different cell populations: although a minority of cells show either only L-type or only T-type current, the majority express both types with predominance of the L-type current. This observation is in agreement with a previous report on primary cultured human zona glomerulosa cells (44).

In our experiments, we found that, in H295R cells, treatment with the GABAB agonist baclofen led to an inhibition of T-type Ca2+ currents. Due to their influence on calcium currents, GABAB receptors might participate in the control of intracellular Ca2+ levels in adrenocortical cells. The H295R cell line expresses GABAB(1a) and GABAB(2) subunits, suggesting that GABAB(1a)/GABAB(2) heterodimers may be formed, which could well explain the effect on Ca2+ currents we observed. On the other hand, a potential influence of GABAB(1e)/GABAB(2) dimers on Ca2+ channels has not been investigated yet, and we cannot rule out this possibility on the basis of our results. Most GABAB receptor effects are mediated via Gi/o proteins that can be inactivated by PTX (42). Indeed, the baclofen effect on T-type currents in H295R cells was completely blocked by PTX pretreatment, indicating that a PTX-sensitive G protein is involved.

The physiological role of adrenocortical GABA receptors and of GABAergic modulation of T-type Ca2+ currents must be studied further. Because the distribution of GAD and VIAAT is different in the human adrenal cortex as compared with the rat adrenal, it will be important to study whether the function of adrenocortical GABA also differs between these two species. Recently, in vivo experiments in rats subjected to stress showed that baclofen administration can affect blood corticosterone concentrations (2). Thus, GABAB receptors may be part of an autocrine or paracrine pathway involved in the regulation of adrenocortical function. In summary, our study provides evidence for a so-far-unknown local GABAergic system in the adrenal cortex. Our results should be seen in conjunction with similar, well-documented findings in other endocrine tissues such as the adenohypophysis, pancreas, ovary, and testis (10, 11, 12, 13, 14, 15). In this broader context, our work supports the concept that GABA, originally described as a neurotransmitter in the central nervous system, in fact is a widespread regulatory signaling molecule in various peripheral organs and a local transmitter in the adrenal cortex.


   Acknowledgments
 
We thank all of our colleagues, especially Annette Krieger, Romi Rämsch, Marlies Rauchfuss, Barbara Zschiesche, and Andreas Mauermayer for expert technical assistance and thank Dr. Katia Gamel-Didelon, Pia Körner, Dr. Lars Kunz, and Professor Artur Mayerhofer for helpful discussions and critical reading of the manuscript.


   Footnotes
 
This work was supported by grants from Deutsche Forschungsgemeinschaft (Graduiertenkolleg 333: Biology of Human Diseases) to K.M. and A.A.

Abbreviations: FITC, Fluorescein isothiocyanate; GABA, {gamma}-aminobutyric acid; GAD, glutamate decarboxylase; HVA, high voltage-activated; LVA, low voltage-activated; PTX, pertussis toxin; {tau}act, time constant of activation; {tau}inact, time constant of inactivation; VIAAT, vesicular inhibitory amino acid transporter.

Received October 21, 2003.

Accepted for publication January 9, 2004.


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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals