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Endocrinology Vol. 141, No. 5 1627-1632
Copyright © 2000 by The Endocrine Society

ARTICLES

Molecular and Electrophysiological Evidence for a GABAC Receptor in Thyrotropin-Secreting Cells1

Eric Boue-Grabot2, Anne Taupignon, Gérard Tramu and Maurice Garret

Laboratoire de Neurophysiologie Centre National de la Recherche Scientifique-Unité Mixte de Recherche 5543 (E.B.-G., A.T., M.G.), Université Victor Segalen Bordeaux 2, 33076 Bordeaux Cedex, France and Laboratoire de Neurocytochimie Fonctionnelle (G.T.), Centre National de la Recherche Scientifique-Unité Mixte de Recherche 5807, Université de Bordeaux 1, 33405 Talence Cedex, France

Address all correspondence and requests for reprints to: M. Garret, Laboratoire de Neurophysiologie, Unité Mixte de Recherche 5543, Université de Bordeaux 2, 146 rue Léo-Saignat 33076 Bordeaux cedex, France. E-mail: maurice.garret{at}umr5543.u-bordeaux2.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the pituitary, GABA regulates the release of several hormones via different receptors. GABAC receptors are heterooligomers that differ from GABAA receptors in that they contain {rho}-subunits and are insensitive to bicuculline. However, molecular and functional evidence for the presence of GABAC receptors outside the retina has yet to be established. The present work was performed on guinea pig and rat pituitaries. Both Northern blot and RT-PCR analysis showed that, although {rho}1- and {rho}2-subunits were expressed at similar levels in the rat retina, {rho}1 messenger RNA (mRNA) was enriched, relative to {rho}2 mRNA in the rat pituitary. Northern blot experiments also showed that, in the pituitary, {rho}1 and {rho}2 mRNAs are shorter in size than those expressed in the retina. The use of a subunit-specific antibody revealed colocalization of {rho}1-subunit and anti-TSH labeling on rat pituitary sections. TSH guinea pig pituitary cells were also labeled with a {rho}-subunit antiserum. Moreover, whole-cell patch clamp on single guinea pig TSH cells showed that GABA induced a bicuculline- insensitive Cl- current. In contrast to the Cl- current generated by GABAC receptors in the retina, the bicuculline-insensitive Cl- currents in TSH cells quickly desensitized. These results suggest that a novel GABAC receptor may regulate TSH secretion and that the structure and/or biochemical regulation of this pituitary receptor is different from that found in the retina.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MAJOR INHIBITORY neurotransmitter, {gamma}-aminobutyric acid (GABA), activates three pharmacologically and structurally different classes of GABA receptors: GABAA, GABAB, and GABAC receptors. GABAB receptors are coupled to G protein, activated selectively by baclofen, inhibited by saclofen, and insensitive to bicuculline. GABAA and GABAC receptors are both ligand-gated Cl- channels selectively activated by muscimol. GABAA receptors are antagonized by bicuculline and have allosteric binding sites for pharmacologically important drugs (for review, see Ref. 1). They are formed by differential assembly of multiple subunits ({alpha}1–6, ß1–3, {gamma}1–3, {delta}, and {epsilon}) (2, 3, 4). By contrast, GABAC receptors, identified in bovine, rat, and perch retinas (5, 6, 7), are neither sensitive to bicuculline nor modulated by benzodiazepines, barbiturates, and steroids. Human {rho}-subunits, cloned from human retina libraries and expressed in oocytes, form homooligomeric chloride channels that share many pharmacological properties with retinal GABAC receptors (8, 9, 10). In the rat retina, bicuculline-insensitive GABA-gated Cl- channels, localized in bipolar cells, are not inhibited by picrotoxin (6). The native response was mimicked only when recombinant rat {rho}1- and {rho}2-subunits were coexpressed in oocytes (11). These results, combined with studies demonstrating expression of {rho}-subunits in rat bipolar cells (12, 13), suggested that retinal GABAC receptors in rats were heteromeric and composed of at least {rho}1- and {rho}2-subunits. Bicuculline-insensitive GABA effects have also been reported in various parts of vertebrate brains (14, 15), and we have previously reported the expression of {rho}1- and {rho}2-subunits in restricted brain domains that may contain functional GABAC receptors (16). So far, however, a clear correlation between the expression of {rho}-subunits and functional bicuculline-insensitive GABA-gated Cl- channels has only been demonstrated in retina cells (6, 12, 13).

GABA is also active in the pituitary, where it modulates the release of several hormones via A-, B-, and C-type GABA receptors (17, 18, 19, 20). We and others have previously demonstrated the expression of different GABAA receptor subunits in the anterior lobe of the rat pituitary (21, 22). Our RT-PCR experiments have also shown the expression of the {rho}1-subunit in this tissue (22). Because GABAC receptors in the rat retina are believed to be composed of {rho}1- and {rho}2-subunits, in the present study we have further examined the expression of {rho}-subunits in the anterior pituitary by combining RT-PCR and Northern blot. We also report the immunodetection of the {rho}-subunits in rat and guinea pig TSH cells. For technical reasons, the electrophysiological characterization of the GABA-induced current was conducted on guinea-pig pituitary cells. This analysis revealed the presence of a bicuculline-insensitive GABA-gated chloride current in individual thyrotroph cells. Our results suggest that this current is mediated by a novel GABAC receptor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
RNA isolation, complementary DNA (cDNA) probes, and Northern blot analysis
In the absence of any information on guinea pig {rho}-subunit gene sequences, these experiments were done on rats. These methods, using tissues from adult Wistar rats, have been reported extensively (16). The {rho}1+2 cDNA probe derived from a conserved sequence in the presumed extracellular domain. The {rho}1 and {rho}2 cDNA probes were chosen from the {rho}1 and {rho}2 variable intracellular domain, respectively. High-stringency hybridization (Saline-Sodium-Phosphate-EDTA 0.1x, 65 C) was carried out; then the filters were exposed to x-ray films for 1–10 days.

PCR analysis
The primers used for gene expression analysis were selected from domains conserved among the {rho}1–3-subunits. PCR experiments and amplification product analyses were conducted as described (16); 1 µl 35S deoxycycidine triphosphate (800 Ci/mmol, 10 mCi/ml, Amersham Pharmacia Biotech, Saclay, France) was also added to label the PCR product. Negative control experiments were run with water instead of cDNA, and with RNA samples treated like RT-PCR template (except that reverse transcriptase was omitted). No amplification products were found in the control experiments (not shown). The 35S-labeled PCR products were separated on an 8% acrylamide gel and vacuum dried. Radiolabeling was detected by exposure to x-ray films or analyzed on a Molecular Dynamics, Inc. Phosphorimager system (Saclay, France).

Immunohistochemistry
Adult Wistar rats (300 g) and guinea pigs (600 g) were overdosed with pentobarbital and immediately perfused transcardially with 150 ml saline solution. Pituitaries were rapidly dissected out, fixed for 1 h by immersion in a solution containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer, and soaked overnight at 4 C in phosphate buffer containing 20% sucrose. Then the pituitary was frozen and cut on a cryostat. Regularly spaced, 10-µm, horizontal sections were collected. Polyclonal antisera directed against {rho}1-subunit-specific peptide (16), {rho}-subunits (Enz et al., 1996), and pituitary hormones (TSH, FSH, ACTH, and PRL) raised in rabbits were used as primary antibodies. The antihormone antisera are extensively used, and their specificity has been evaluated in previous studies: anti-hTSH and anti-hFSH (23), anti- ACTH (24), and anti-rPRL (25) (h, human; r, rat).

Pituitary sections were incubated first for 18 h at room temperature with purified {rho}1 antiserum (diluted 1:200) or with {rho} antiserum (1 : 100) in 0.01 M veronal buffer (VB) containing 0.2% Triton X-100. Sections were then incubated for 2 h at room temperature in goat antirabbit peroxidase-linked IgGs (1:200; Jackson ImmunoResearch Laboratories, Inc.). The peroxidatic activity was revealed using 4 chloro-1 naphthol chromogen. It was not possible to identify rat GH- and guinea pig TSH-producing cells after immunodetection of {rho}-subunits, probably because of alteration of the antigens during the elution procedure.

The first staining was photographed, then the blue reaction products were removed by immersion in acetone. Antibodies were then eluted by gentle stirring in a mixture made of 1 vol of 2.5% KMnO4, 1 vol of 5% H2SO4, and 200 vol of distilled H2O for 1 min (26). Sections were washed in VB before immunodetection of the second antigen: sections were incubated for 2 h with anti-rPRL (1:400), anti-hTSH (1:1000), anti-ACTH (1:500), or anti-hFSH (1:500). After washing with VB, goat antirabbit peroxidase-linked IgGs (1:150, Jackson ImmunoResearch Laboratories, Inc., Asnières, France) was applied for 1 h. Reaction products were formed with 3,3' diaminobenzidine tetra HCL, then photographed.

No immunoreactivity was observed when elution efficiency was controlled by incubation of sections with normal rabbit serum instead of hormone antibodies or when specificity controls were performed by preincubation of the purified {rho}1 antiserum (50 µg/ml) with the peptide antigen {rho}1N (100 µg/ml, not shown).

Guinea-pig pituitary cell culture and patch clamp recording
For studies on isolated thyrotrophs, pituitaries from 250–350-g female guinea pigs were used. The anterior pituitary was first separated from the posterior pituitary. It was further dissected to isolate the rostral (anteromedian and lateral) part of the pars distalis known, in guinea pigs, to contain most thyrotrophs (27). The cell dissociation method was adapted from that described (19). Triturating took place in calcium- and magnesium-free Hanks’ medium containing 2 mM EGTA. Yield was 5–8 x 105 cells per gland. Cells were harvested by gentle centrifugation and plated onto glass coverslips in 35-mm Petri dishes at a density of 2.5 x 105 cells/dish. Experiments were performed on days 1 and 2.

Guinea pig thyrotrophs have been described as voluminous cells, always much bigger than any other cells (27). Indeed, coverslips viewed at x1000, exhibited conspicuously large round cells (diameter >= 25 µm). We selected such cells for our whole-cell patch clamp experiments. In some experiments, cell type identification was confirmed, after recording on scored coverslips, by immunocytochemical labeling using our anti-hTSH antibody (not shown). The patch amplifier was a RK-300 unit (Biologic, Claix, France). The bath medium (38 C) contained Hanks’ saline buffered solution with 10 mM HEPES. To test the dependency of the reversal potential of GABA-induced currents on external Cl-, a low Cl- Hanks’ saline was used. It contained 45 mM Cl- instead of 149.7 mM Cl-, and NaCl was replaced by Na methanesulfonate. Pipettes, coated with sylgard and fire-polished, had an average resistance of 3 M{Omega} when filled with (in mM): KCl, 120; HEPES, 10; EGTA, 11; ATP-Mg, 2; GTP-Na, 0.4; 280–300 mosmol/liter; pH 7.25. The expected ECL was -2 mV or (with the low Cl- Hanks’ medium) +29 mV. Drugs were applied to isolated cells from double-barreled pipettes by pneumatic ejection.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of {rho}-subunit messenger RNAs (mRNAs) in pituitary by Northern blot and RT-PCR analysis
To examine the expression of {rho}-subunit mRNAs, Northern blots with poly(A)+-selected RNA extracted from retina and pituitary were hybridized with probes derived from {rho}-subunit cDNAs. The {rho}1+2 probe (Fig. 1AGo), derived from a conserved sequence among {rho}-subunits, demonstrated hybridization to mRNAs in retina (5 and 2.4 kb) and pituitary (4.5, 2, and 1 kb). When the same membrane was hybridized with a {rho}1-specific probe (Fig. 1BGo), only the 5- and 4.5-kb mRNAs were detected in eye and pituitary, respectively. In the same way, a {rho}2-specific probe (Fig. 1CGo) revealed the 2.4-kb mRNA in the retina, and the 2- and 1-kb mRNAs in the pituitary. The products of the {rho}1-subunit gene (5 and 4.5 kb), as of the {rho}2 gene (2.4 and 2 kb), may be attributable to alternative splicing or alternative polyadenylation between retina and pituitary. It should be noted that the 2-kb {rho}2 signal in the pituitary was very faint, suggesting a low expression level. The 1-kb band, revealed by the {rho}1+2 and {rho}2 probes in the pituitary, may reflect an alternative splicing of the {rho}2 gene product unlikely to encode a functional GABA receptor subunit (approximately 450 amino acids). Correction for the variation in sample loading, using the ubiquitous G3PDH probe (Fig. 1DGo), indicated that the expression levels of {rho}-subunit genes were within the same range in the retina and the pituitary.



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  		Figure 1. Northern blot analysis of rat {rho}1 and {rho}2-subunit expression. Poly A+ RNA was extracted from the retina (R) and pituitary (P). Ten micrograms of RNAs were loaded on the gel, electrophoresed, transferred, and subjected to RNA hybridization analysis. Northern blot membranes were hybridized, stripped, and reprobed with successive probes derived from a region common to {rho}1 and {rho}2 (A), or exclusively from {rho}1 (B) and {rho}2 (C) GABA receptor subunits, respectively. A glyceraldehyde 3-phosphate dehydrogenase probe was used to control for variations in sample loading (D). Estimated sizes for each band shown on the right were determined using an RNA ladder.

 
We further determined, by RT-PCR, which {rho}-subunits were present in the anterior pituitary, using primers previously validated in experiments with RNA extracted from the retina and brain (16). We used a set of primers, rhoA and rhoB (Fig. 2AGo), from conserved regions among {rho}1–3-subunits. An aliquot of each PCR amplification product (330 bp) was digested with the appropriate restriction enzyme: EcoRI restriction hydrolysis of the PCR product obtained with {rho}1 transcript should produce 164- and 166-bp fragments. In the same way, BglII restriction enzyme should produce 137- and 193-bp fragments from {rho}2 cDNA, and XbaI restriction enzyme should produce 51- and 279-bp fragments from {rho}3 cDNA (Fig. 2AGo). Amplification, using retina and antepituitary cDNA, yielded a band of the expected size, 330 bp. The PCR product from the retina, verified by restriction pattern analysis, contained {rho}1, {rho}2, and {rho}3-subunits, as reported (8, 28, 29). The PCR products obtained from the anterior pituitary were digested with EcoRI and BglII restriction enzymes but not with the {rho}3-specific restriction enzyme XbaI. These results indicate that, in the anterior pituitary, the {rho}1 mRNA level was higher than that of {rho}2 mRNA and that the {rho}3 subunit gene was not expressed (Fig. 2Go).



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  		Figure 2. Autoradiogram showing restriction analysis of RT-PCR products of GABA receptor {rho}1–3-subunits. A schematic representation of the PCR primers and restriction enzyme sites used in the study is shown at the top of each autoradiogram. Experiments were performed on rat retina (Rt) and rat anterior pituitary tissue (AP). A, Restriction analysis of GABA receptor {rho}1–3-subunit RT-PCR products were digested with EcoRI (E), BglII (B), and XbaI (X) restriction enzymes specific for {rho}1-, {rho}2-, and {rho}3-subunits, respectively. Restriction DNA fragments from {rho}1-, {rho}2-, and {rho}3-subunits are indicated on the left of the gel, and the sizes of fragments are shown on the right. B, Restriction analysis of GABA receptor {rho}1- and {rho}2-subunit RT-PCR products were digested with EcoRI (E) and BglII (B) restriction enzymes specific for {rho}1- and {rho}2-subunits, respectively. DNA restriction fragments from {rho}1- and {rho}2-subunits are indicated on the left of the gel, and the sizes of fragments are shown on the right. The ratio of {rho}2 to {rho}1 cDNA products from four independent reactions is shown at the bottom of B.

 
Because rhoB primer is degenerate and we wished to examine more closely the relative levels of {rho}1 and {rho}2 mRNAs in the anterior pituitary, we used a rhoC primer (Fig. 2BGo) from the {rho}1 and {rho}2 nucleotidic sequences (rhoC does not amplify {rho}3-subunit mRNA). The {rho}1- and {rho}2-subunit cDNA sequences have a 100% match to rhoA and rhoC oligonucleotide primers and compete for primer binding and amplification (for review, see Ref. 30). PCR fragments were identified by restriction analysis: EcoRI restriction hydrolysis of the PCR product obtained with {rho}1 transcript should produce 166- and 101-bp fragments, and BglII restriction hydrolysis of the PCR product from {rho}2 cDNA should produce 137- and 130-bp fragments. Figure 2BGo showed that, in the retina and anterior pituitary, the ratios of {rho}2- vs. {rho}1-mRNA were different. In conclusion, the comparison of both tissues, by Northern blot and RT-PCR analyses, indicated that, in the rat pituitary, the {rho}1 mRNA level was considerably higher than that of {rho}2 mRNA.

Localization of GABAC receptor {rho}1-subunit in the pituitary
Horizontal sections of rat pituitary were immunostained with an antibody against the {rho}1-subunit (16). Figure 3Go shows micrographs of the rat pituitary. As shown in Fig. 3AGo, immunoreactivity was found on anterior lobe cells. No significant immunolabeling was observed in the neurointermediate lobe.



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  		Figure 3. Localization of {rho}1-subunit and TSH or FSH by successive immunohistochemical staining on horizontal sections of rat pituitary. A, Immunodetection of {rho}1-subunit protein. B1, B2 and C1, C2, Successive localization on the same section of 2 antigens. B, Section incubated first with {rho}1-specific antibodies (B1) and then with anti-TSH (B2), after decolorizing and elution of {rho}1-antibodies. Arrows mark cells stained by both antibodies, displaying similar labeling pattern. C, Section incubated successively with {rho}1-specific antibodies (C1) and then with FSH hormone antibodies (C2). Arrows show a nonoverlapping distribution of {rho}1-subunit and FSH. IL, intermediate lobe. Calibration bars: A, 100 µm; B and C, 50 µm.

 
To identify {rho}1-expressing cells, among the various types of adenohypophyseal cells, successive staining experiments were performed using first the antiserum to the {rho}1-subunit and then antisera to the various pituitary hormones (see Materials and Methods). In sections examined, all cells that displayed {rho}1-subunit immunoreactivity (Fig. 3BGo1) were also stained with anti-TSH antibodies after the elution of {rho}1-antibodies (Fig. 3BGo2). However, 20% of anti-TSH-labeled cells showed little or no immunoreaction to the {rho}1-subunit (37/200), indicating that some thyrotrophs may not express the {rho}1-subunit. On the other hand, {rho}1-subunit immunoreactivity (Fig. 3CGo1) was never colocalized with FSH (Fig. 3CGo2), ACTH, and PRL (not shown) staining, demonstrating that gonadotrophs, corticotrophs, and lactotrophs are devoid of {rho}1-subunit immunoreactivity. Because the {rho}1-antiserum is directed against the N-terminal peptide (15 amino acids) that varies from one species to another, we used an antiserum directed against the N-terminal region (positions 16–171) of the {rho}1-subunit that recognizes the {rho}1-, {rho}2-, and {rho}3-subunits (13). The pattern of {rho} labeling observed (Fig. 4BGo) was consistent with TSH cells (Fig. 4AGo) within the guinea pig pituitary, strongly suggesting that, in both species, {rho}1 is expressed in thyrotrophs.



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  		Figure 4. Immunoreactivity for TSH and {rho}-subunits of the GABAC receptor on sagittal sections through guinea pig pituitary. A, Low magnification showing TSH-specific immunostaining of cells essentially localized in the rostroventral part of the anterior pituitary; B, neighboring section labeled with {rho} antibody; C, higher magnification of the microscopic field boxed in B, showing typical cytoplasmic staining of large cells; PS, pituitary stalk; calibration bars: A and B, 250 µm; C, 50 µm.

 
Whole-cell patch clamp recording.
In our primary cultures of normal male rat, TSH-positive cells after immunocytochemical staining accounted for only a very small proportion of cells (<=2%), in agreement with published data (31). Moreover, these cells had no specific morphological features, so they were difficult to identify among other cells under a light microscope. We therefore used guinea pig pituitaries, because: 1) the rostral part of the gland is especially rich in thyrotrophs (see Materials and Methods), thus making it possible to enrich the culture in TSH cells; and 2) these cells were easily identified by their size, much larger than that of any other cell type (27).

As shown in the example in Fig. 5AGo, application of GABA (10 µM) to such a large cell evoked an inward desensitizing whole-cell current that was insensitive to bicuculline (100 µM). It ran down quickly, as a further application of GABA evoked no current. In some experiments, cells were plated onto scored coverslips, and the coordinates of each recorded cell were registered. Cell types were then identified by immunocytochemical labeling using the anti-hTSH antibody. All the bicuculline-insensitive cells recorded were positive after anti-TSH labeling (not shown). In the same culture plates, smaller cells displayed a GABA-induced current that was completely and reversibly abolished by bicuculline (Fig. 5BGo). Unlike the bicuculline-sensitive currents that did not run down quickly, the bicuculline-insensitive currents ran down in less than 3 min (Fig. 5CGo). In both cases, however, the GABA-activated current was not inhibited by the GABAB inhibitor saclofen (10 µM; not shown). Furthermore, the current-voltage relationship of the bicuculline-insensitive current (Fig. 5DGo) revealed a mean measured reversal potential (-0.56 ± 1.7 mV; n = 5) close to the chloride Nernst potential. Furthermore, when the external Cl- was changed, the reversal potential changed as expected for a current carried by chloride ions. It shifted by + 26 ± 2 mV (n = 2) when external Cl- was lowered from 149.7 to 45 mM (not shown). This strongly suggested that GABA opened bicuculline-insensitive Cl- selective channels. Using the same recording method, this rapid desensitization of the bicuculline-insensitive current was not found in HEK-293 cells (Fig. 5EGo) transfected with rat {rho}1-subunit cDNA (16).



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  		Figure 5. Bicuculline-insensitive GABA-activated membrane currents are found in isolated guinea pig pituitary cells. Membrane currents, activated by 10 sec application of GABA or GABA plus bicuculline, were recorded from cells that were presumably thyrotrophs (A) or lactotrophs (B), at a holding potential of -60 mV, at various times (45, 90, and 135 sec for the first, second, and third traces, respectively), after formation of the whole-cell recording mode. Mean initial responses from bicuculline-insensitive cells were -125 ± 32 pA (mean cell capacitance: 24 ± 4 pF, n = 10). Those of bicuculline-sensitive cells were -213 ± 54 pA (mean cell capacitance: 8 ±1 pF, n = 10). Bars show the application periods of the drugs. C, Time-dependence of bicuculline-insensitive (squares) and bicuculline-sensitive (circles) GABA-activated membrane currents. To collate data from separate cells, currents were normalized to the initial response to 10 µM GABA, recorded in each cell after formation of the whole-cell recording mode, defined as 100%. All points are mean + SEM. D, Current voltage curve of a bicuculline-insensitive current obtained with a fast voltage ramp. Background current was subtracted. E, Membrane current activated by GABA and bicuculline in an HEK-293 cell transfected with {rho}1-subunit cDNA at a holding potential of -60 mV.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We demonstrate here, for the first time outside the retina, the presence of a {rho}1-subunit polypeptide restricted to one cell type in the endocrine pituitary. Moreover, we show that guinea pig TSH cells express a functional bicuculline-insensitive GABA-gated Cl- channel. Because it is now evident that the subunit composition of GABA-gated Cl- channels influences their pharmacological and electrophysiological properties (1), it is of interest that the expression profile of {rho}-subunit mRNAs and GABAC functional behavior suggest different GABAC receptors in the pituitary and retina.

Northern blot analyses revealed that {rho}-subunit mRNAs were expressed in the pituitary in sufficiently high amounts to be detected by Northern blot. The slight difference in mRNA sizes of {rho}1 and {rho}2 mRNAs in the retina and pituitary suggested tissue-specific RNA splicing. It would be worthwhile to screen a pituitary library to determine whether these alternative splicings result in alternative coding sequences, as reported for the retinal {rho}1-subunit (32). The functional alternatively spliced {rho}1-subunits described in that report did not show any apparent difference in channel behavior. The presence of a 1-kb mRNA, revealed by the {rho}2 probe in the pituitary, suggests an aberrantly spliced form, as reported for this subunit in the human retina, as well as for other GABAA receptor subunits (4, 8). However, an alternative spliced form of the {rho}2-subunit containing features common to members of the GABA-gated chloride channel family (i.e. a signal peptide, a Cys-Cys loop, and four transmembrane domains) cannot be completely ruled out. RT-PCR experiments used to compare the ratio of {rho}-subunit mRNAs in the rat pituitary and retina confirmed that {rho}1 was the main GABAC receptor subunit expressed in the pituitary, whereas {rho}1 and {rho}2 were equally expressed in the retina. Taken together, our experiments clearly showed a tissue-specific expression of {rho}- subunit genes in the retina and pituitary and suggest that the molecular composition of the GABAC receptor is different in these two tissues.

Effects of GABA on TSH and LH release via so-called bicuculline-insensitive GABAA receptors, have been reported (18, 20). We showed that the anti-FSH antibody well known as a gonadotroph marker (33) did not label cells stained by the {rho}1-antibody. We also showed that all the rat pituitary cells labeled by the {rho}1-antibody were labeled by an anti-TSH antibody, demonstrating cell-specific expression of the {rho}1 protein. Moreover, in single identified guinea-pig TSH-producing cells, GABA induced a bicuculline-insensitive Cl- current. The whole-cell GABA-activated membrane current displayed a rapid rising phase followed by a decrease (despite continued application of GABA) consistent with desensitization. Although GABAC currents in rat bipolar cells do not desensitize, desensitizing GABAC receptor- mediated currents have been reported in carp bipolar cells (34). The bicuculline-insensitive GABA-gated Cl- current in thyrotrophs also showed a rapid down-regulation of the whole-cell current. It is known that the rat retinal GABAC receptor is down-regulated by protein kinase C (35). It is also well established that the amplitude and desensitization kinetics response of GABAA receptor, like other ligand-gated channels, depend both on their phosphorylation states and their subunit composition (36, 37). It can be hypothesized that pituitary GABAC receptors contain an alternative {rho}1-subunit. In that case, the alternative sequence would be in the intracellular domain that is the target of protein kinases (36). It is also possible that TSH cells contain protein kinases not present in mammalian retinal bipolar cells. The rapid run-down of the bicuculline-insensitive GABA-gated Cl- current expressed in thyrotrophs also suggest that such channels may have been overlooked in the brain. Because {rho}-subunits may be differentially expressed in rats and guinea pigs, it will be necessary to find a method for enriching rat primary cultures in TSH cells. This will make it possible to fully characterize the pharmacology and correlate the electrophysiological properties of the pituitary GABAC receptor to its molecular structure.

If GABAC receptors can be defined by a bicuculline- insensitive GABA-gated Cl- channel and the presence of {rho}-subunits, it would be reasonable to propose the presence of a {rho}1-subunit-containing GABAC receptor in mammalian thyrotrophs. GABA modulates the pituitary hormone secretion by acting directly on the endocrine tissue that expresses several receptor subunits (21, 22). However, so far, the identity of the GABA receptor types expressed by any individual pituitary cell type is not known. The identification of a GABAC receptor in thyrotrophs may be physiologically relevant, because it may account for the modulation of TSH secretion (19). In this respect, it will be of interest to characterize the transduction pathway(s) linking the activation of GABAC receptors to the control of hormonal secretion.

In conclusion, we propose that, besides the retina, the pituitary also expresses a functional {rho}1-subunit-containing GABAC receptor. Our data provide the first evidence for the hypothesis (32, 38) that GABAC receptors may be more complex than previously thought, in terms of heterogeneity and modulation.


   Acknowledgments
 
We would like to thank B. Dufy for his support, P. Séguéla and N. Guerineau for comments on the manuscript, and Heinz Wässle for providing the antibody directed against {rho}-subunits.


   Footnotes
 
1 This work has been supported by Centre National de la Recherche Scientifique, the University of Bordeaux 2, and the Conseil Regional Aquitaine. Back

2 Present address: Institut neurologique de Montréal, Universite McGill, Montréal H3A 2B4, Canada. Back

Received October 12, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sieghart W 1995 Structure and pharmacology of gamma-aminobutyric acid A receptor subtypes. Pharmacol Rev 47:181–234[Medline]
  2. Macdonald RL, Olsen RW 1994 GABAA receptor channels. Annu Rev Neurosci 17:569–602[Medline]
  3. Davies PA, Hanna MC, Hales TG, Kirkness EF 1997 Insensitivity to anaesthetic agents conferred by a class of GABA(A) receptor subunit. Nature 385:820–823[CrossRef][Medline]
  4. Garret M, Bascles L, Boue-Grabot E, Sartor P, Charron G, Bloch B, Margolskee RF 1997 An mRNA encoding a putative GABA-gated chloride channel is expressed in the human cardiac conduction system. J Neurochem 68:1382–1389[Medline]
  5. Polenzani L, Woodward RM, Miledi R 1991 Expression of mammalian gamma-aminobutyric acid receptors with distinct pharmacology in Xenopus oocytes. Proc Natl Acad Sci USA 88:4318–4322[Abstract/Free Full Text]
  6. Feigenspan A, Wassle H, Bormann J 1993 Pharmacology of GABA receptor Cl- channels in rat retinal bipolar cells. Nature 361:159–162[CrossRef][Medline]
  7. Qian H, Dowling JE 1993 Novel GABA responses from rod-driven retinal horizontal cells. Nature 361:162–164[CrossRef][Medline]
  8. Cutting GR, Lu L, O’Hara BF, Kasch LM, Montrose-Rafizadeh C, Donovan DM, Shimada S, Antonarakis SE, Guggino WB, Uhl GR 1991 Cloning of the gamma-aminobutyric acid (GABA) {rho}1 cDNA: a GABA receptor subunit highly expressed in the retina. Proc Natl Acad Sci USA 88:2673–2677[Abstract/Free Full Text]
  9. Cutting GR, Curristin S, Zoghbi H, O’Hara B, Seldin MF, Uhl GR 1992 Identification of a putative gamma-aminobutyric acid (GABA) receptor subunit {rho}2 cDNA and colocalization of the genes encoding {rho}2 (GABRR2) and {rho}1 (GABRR1) to human chromosome 6q14–q21 and mouse chromosome 4. Genomics 12:801–806[CrossRef][Medline]
  10. Shimada S, Cutting G, Uhl GR 1992 Gamma-aminobutyric acid A or C receptor? Gamma-aminobutyric acid {rho}1 receptor RNA induces bicuculline-, barbiturate-, and benzodiazepine-insensitive gamma-aminobutyric acid responses in Xenopus oocytes. Mol Pharmacol 41:683–687[Abstract]
  11. Zhang D, Pan ZH, Zhang X, Brideau AD, Lipton SA 1995 Cloning of a gamma-aminobutyric acid type C receptor subunit in rat retina with a methionine residue critical for picrotoxinin channel block. Proc Natl Acad Sci USA 92:11756–11760[Abstract/Free Full Text]
  12. Enz R, Brandstatter JH, Hartveit E, Wassle H, Bormann J 1995 Expression of GABA receptor {rho}1- and rho2-subunits in the retina and brain of the rat. Eur J Neurosci 7:1495–1501[CrossRef][Medline]
  13. Enz R, Brandstatter JH, Wassle H, Bormann J 1996 Immunocytochemical localization of the GABAc receptor {rho}-subunits in the mammalian retina. J Neurosci 16:4479–4490[Abstract/Free Full Text]
  14. Arakawa T, Okada Y 1988 Excitatory and inhibitory action of GABA on synaptic transmission in slices of guinea pig superior colliculus. Eur J Pharmacol 158:217–224[CrossRef][Medline]
  15. Drew CA, Johnston GA 1992 Bicuculline- and baclofen-insensitive gamma-aminobutyric acid binding to rat cerebellar membranes. J Neurochem 58:1087–1092[CrossRef][Medline]
  16. Boue-Grabot E, Roudbaraki M, Bascles L, Tramu G, Bloch B, Garret M 1998 Expression of GABA receptor rho-subunits in rat brain. J Neurochem 70:899–907[Medline]
  17. Anderson R, Mitchell R 1986 Biphasic effect of GABAA receptor agonists on prolactin secretion: evidence for two types of GABAA receptor complex on lactotrophes. Eur J Pharmacol 124:1–9[CrossRef][Medline]
  18. Tapia-Arancibia L, Roussel JP, Astier H 1987 Evidence for a dual effect of gamma-aminobutyric acid on thyrotropin (TSH)-releasing hormone-induced TSH release from perifused rat pituitaries. Endocrinology 121:980–986[Abstract/Free Full Text]
  19. Lorsignol A, Taupignon A, Dufy B 1994 Short applications of gamma- aminobutyric acid increase intracellular calcium concentrations in single identified rat lactotrophs. Neuroendocrinology 60:389–399[Medline]
  20. Virmani MA, Stojilkovic SS, Catt KJ 1990 Stimulation of luteinizing hormone release by gamma-aminobutyric acid (GABA) agonists: mediation by GABAA-type receptors and activation of chloride and voltage-sensitive calcium channels. Endocrinology 126:2499–2505[Abstract/Free Full Text]
  21. Berman JA, Roberts JL, Pritchett DB 1994 Molecular and pharmacological characterization of GABAA receptors in the rat pituitary. J Neurochem 63:1948–1954[Medline]
  22. Boue-Grabot E, Dufy B, Garret M 1995 Molecular diversity of GABA-gated chloride channels in the rat anterior pituitary. Brain Res 704:125–129[CrossRef][Medline]
  23. Bello AR, Dubourg P, Kah O, Tramu G 1992 Identification of neurotensin-immunoreactive cells in the anterior pituitary of normal and castrated rats. A double immunocytochemical investigation at the light- and electron-microscopic levels. Neuroendocrinology 55:714–723[Medline]
  24. Covenas R, de Leon M, Narvaez JA, Aguirre JA, Tramu G, Gonzalez-Baron S 1997 ACTH/CLIP immunoreactivity in the cat brain stem. Peptides 18:965–970[CrossRef][Medline]
  25. Hemming FJ, Dubois MP, Dubois PM 1986 Somatotrophs and lactotrophs in the anterior pituitary of fetal and neonatal rats. Electron-microscopic immunocytochemical identification. Cell Tissue Res 245:457–460[Medline]
  26. Tramu G, Pillez A, Leonardelli J 1978 An efficient method of antibody elution for the successive or simultaneous localization of two antigens by immunocytochemistry. J Histochem Cytochem 26:322–324[Abstract]
  27. Beauvillain JC, Mazzuca M, Dubois MP 1976 The thyrotropic cells of the guinea pig pituitary. Electron microscopic study after characterization by immunocytochemical means. Cell Tissue Res 174:233–244[Medline]
  28. Ogurusu T, Taira H, Shingai R 1995 Identification of GABAA receptor subunits in rat retina: cloning of the rat GABAA receptor {rho}2-subunit cDNA. J Neurochem 65:964–968[Medline]
  29. Ogurusu T, Shingai R 1996 Cloning of a putative gamma-aminobutyric acid (GABA) receptor subunit {rho}3 cDNA. Biochim Biophys Acta 1305:15–18[Medline]
  30. Siebert PD, Larrick JW 1992 Competitive PCR. Nature 359:557–558[CrossRef][Medline]
  31. Ashworth R, Hinkle PM 1996 Thyrotropin-releasing hormone-induced intracellular calcium responses in individual rat lactotrophs and thyrotrophs. Endocrinology 137:5205–5212[Abstract]
  32. Martinez-Torres A, Vazquez AE, Panicker MM, Miledi R 1998 Cloning and functional expression of alternative spliced variants of the {rho}1 gamma- aminobutyrate receptor. Proc Natl Acad Sci USA 95:4019–4022[Abstract/Free Full Text]
  33. Inoue K, Kurosumi K 1984 Ultrastructural immunocytochemical localization of LH and FSH in the pituitary of the untreated male rat. Cell Tissue Res 235:77–83[Medline]
  34. Han MH, Li Y, Yang XL 1997 Desensitizing GABAC receptors on carp retinal bipolar cells. Neuroreport 8:1331–1335[Medline]
  35. Feigenspan A, Bormann J 1994 Modulation of GABAC receptors in rat retinal bipolar cells by protein kinase C. J Physiol (Lond) 481:325–330[Abstract/Free Full Text]
  36. Smart TG 1997 Regulation of excitatory and inhibitory neurotransmitter-gated ion channels by protein phosphorylation. Curr Opin Neurobiol 7:358–367[CrossRef][Medline]
  37. McDonald BJ, Amato A, Connolly CN, Benke D, Moss SJ, Smart TG 1998 Adjacent phosphorylation sites on GABAA receptor beta-subunits determine regulation by cAMP-dependent protein kinase. Nat Neurosci 1:23–28[CrossRef][Medline]
  38. Johnston GA 1996 GABAc receptors: relatively simple transmitter-gated ion channels? Trends Pharmacol Sci 17:319–323[CrossRef][Medline]



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