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0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society Vol. 141, No. 5 Printed in U.S.A. Molecular and Electrophysiological Evidence for a GABAC Receptor in Thyrotropin-Secreting Cells* ERIC BOUE-GRABOT†, 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 ABSTRACT 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 -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 1- and 2-subunits were expressed at similar levels in the rat retina, 1 messenger RNA (mRNA) was enriched, relative to 2 mRNA in the rat pituitary. Northern blot experiments also showed that, in the pituitary, 1 and 2 mRNAs are shorter in size than those expressed in the retina. The use of a subunit-specific antibody revealed colocalization of 1-subunit and anti-TSH labeling on rat pituitary sections. TSH guinea pig pituitary cells were also labeled with a -subunit antiserum. Moreover, whole-cell patch clamp on single guinea pig TSH cells showed that GABA induced a bicucullineinsensitive 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. (Endocrinology 141: 1627– 1632, 2000) T when recombinant rat 1- and 2-subunits were coexpressed in oocytes (11). These results, combined with studies demonstrating expression of -subunits in rat bipolar cells (12, 13), suggested that retinal GABAC receptors in rats were heteromeric and composed of at least 1- and 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 1and 2-subunits in restricted brain domains that may contain functional GABAC receptors (16). So far, however, a clear correlation between the expression of -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 (17–20). 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 1-subunit in this tissue (22). Because GABAC receptors in the rat retina are believed to be composed of 1and 2-subunits, in the present study we have further examined the expression of -subunits in the anterior pituitary by combining RT-PCR and Northern blot. We also report the immunodetection of the -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. HE MAJOR INHIBITORY neurotransmitter, ␥-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 ligandgated 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 (␣1– 6, 1–3, ␥1–3, ␦, and ⑀) (2– 4). By contrast, GABAC receptors, identified in bovine, rat, and perch retinas (5–7), are neither sensitive to bicuculline nor modulated by benzodiazepines, barbiturates, and steroids. Human -subunits, cloned from human retina libraries and expressed in oocytes, form homooligomeric chloride channels that share many pharmacological properties with retinal GABAC receptors (8 – 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 Received October 12, 1999. 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: [email protected]. * This work has been supported by Centre National de la Recherche Scientifique, the University of Bordeaux 2, and the Conseil Regional Aquitaine. † Present address: Institut neurologique de Montréal, Universite McGill, Montréal H3A 2B4, Canada. 1627 1628 GABAC RECEPTOR IN THYROTROPIN-SECRETING CELLS Materials and Methods RNA isolation, complementary DNA (cDNA) probes, and Northern blot analysis In the absence of any information on guinea pig -subunit gene sequences, these experiments were done on rats. These methods, using tissues from adult Wistar rats, have been reported extensively (16). The 1⫹2 cDNA probe derived from a conserved sequence in the presumed extracellular domain. The 1 and 2 cDNA probes were chosen from the 1 and 2 variable intracellular domain, respectively. High-stringency hybridization (Saline-Sodium-Phosphate-EDTA 0.1⫻, 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 1–3-subunits. PCR experiments and amplification product analyses were conducted as described (16); 1 l 35 S 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-m, horizontal sections were collected. Polyclonal antisera directed against 1-subunit-specific peptide (16), -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), antiACTH (24), and anti-rPRL (25) (h, human; r, rat). Pituitary sections were incubated first for 18 h at room temperature with purified 1 antiserum (diluted 1:200) or with 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 peroxidaselinked 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 -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 1 antiserum (50 g/ml) with the peptide antigen 1N (100 g/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 Endo • 2000 Vol 141 • No 5 (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 ⫻ 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 ⫻ 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 ⫻1000, exhibited conspicuously large round cells (diameter ⱖ 25 m). 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⍀ 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 Expression of -subunit messenger RNAs (mRNAs) in pituitary by Northern blot and RT-PCR analysis To examine the expression of -subunit mRNAs, Northern blots with poly(A)⫹-selected RNA extracted from retina and pituitary were hybridized with probes derived from -subunit cDNAs. The 1⫹2 probe (Fig. 1A), derived from a conserved sequence among -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 1-specific probe (Fig. 1B), only the 5- and 4.5-kb mRNAs were detected in eye and pituitary, respectively. In the same way, a 2-specific probe (Fig. 1C) revealed the 2.4-kb mRNA in the retina, and the 2- and 1-kb mRNAs in the pituitary. The products of the 1-subunit gene (5 and 4.5 kb), as of the 2 FIG. 1. Northern blot analysis of rat 1 and 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 1 and 2 (A), or exclusively from 1 (B) and 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. GABAC RECEPTOR IN THYROTROPIN-SECRETING CELLS 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 2 signal in the pituitary was very faint, suggesting a low expression level. The 1-kb band, revealed by the 1⫹2 and 2 probes in the pituitary, may reflect an alternative splicing of the 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. 1D), indicated that the expression levels of -subunit genes were within the same range in the retina and the pituitary. We further determined, by RT-PCR, which -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. 2A), from conserved regions among 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 1 transcript should produce 164- and 166-bp fragments. In the same way, BglII restriction enzyme should produce 137- and 193-bp fragments from 2 cDNA, and XbaI restriction enzyme should produce 51- and 279-bp fragments from 3 cDNA (Fig. 2A). 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 1, 2, and 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 3-specific restriction enzyme XbaI. These results indicate that, in the anterior pituitary, the 1 mRNA FIG. 2. Autoradiogram showing restriction analysis of RT-PCR products of GABA receptor 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 1–3-subunit RT-PCR products were digested with EcoRI (E), BglII (B), and XbaI (X) restriction enzymes specific for 1-, 2-, and 3-subunits, respectively. Restriction DNA fragments from 1-, 2-, and 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 1- and 2-subunit RT-PCR products were digested with EcoRI (E) and BglII (B) restriction enzymes specific for 1- and 2-subunits, respectively. DNA restriction fragments from 1- and 2-subunits are indicated on the left of the gel, and the sizes of fragments are shown on the right. The ratio of 2 to 1 cDNA products from four independent reactions is shown at the bottom of B. 1629 level was higher than that of 2 mRNA and that the 3 subunit gene was not expressed (Fig. 2). Because rhoB primer is degenerate and we wished to examine more closely the relative levels of 1 and 2 mRNAs in the anterior pituitary, we used a rhoC primer (Fig. 2B) from the 1 and 2 nucleotidic sequences (rhoC does not amplify 3-subunit mRNA). The 1- and 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 1 transcript should produce 166- and 101-bp fragments, and BglII restriction hydrolysis of the PCR product from 2 cDNA should produce 137- and 130-bp fragments. Figure 2B showed that, in the retina and anterior pituitary, the ratios of 2- vs. 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 1 mRNA level was considerably higher than that of 2 mRNA. Localization of GABAC receptor 1-subunit in the pituitary Horizontal sections of rat pituitary were immunostained with an antibody against the 1-subunit (16). Figure 3 shows micrographs of the rat pituitary. As shown in Fig. 3A, immunoreactivity was found on anterior lobe cells. No significant immunolabeling was observed in the neurointermediate lobe. To identify 1-expressing cells, among the various types of adenohypophyseal cells, successive staining experiments were performed using first the antiserum to the 1-subunit and then antisera to the various pituitary hormones (see Materials and Methods). In sections examined, all cells that displayed 1-subunit immunoreactivity (Fig. 3B1) were also stained with anti-TSH antibodies after the elution of 1antibodies (Fig. 3B2). However, 20% of anti-TSH-labeled cells showed little or no immunoreaction to the 1-subunit (37/ 200), indicating that some thyrotrophs may not express the 1-subunit. On the other hand, 1-subunit immunoreactivity (Fig. 3C1) was never colocalized with FSH (Fig. 3C2), ACTH, and PRL (not shown) staining, demonstrating that gonadotrophs, corticotrophs, and lactotrophs are devoid of 1-subunit immunoreactivity. Because the 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 1subunit that recognizes the 1-, 2-, and 3-subunits (13). The pattern of labeling observed (Fig. 4B) was consistent with TSH cells (Fig. 4A) within the guinea pig pituitary, strongly suggesting that, in both species, 1 is expressed in thyrotrophs. 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 (ⱕ2%), 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 1630 GABAC RECEPTOR IN THYROTROPIN-SECRETING CELLS FIG. 3. Localization of 1-subunit and TSH or FSH by successive immunohistochemical staining on horizontal sections of rat pituitary. A, Immunodetection of 1-subunit protein. B1, B2 and C1, C2, Successive localization on the same section of 2 antigens. B, Section incubated first with 1-specific antibodies (B1) and then with antiTSH (B2), after decolorizing and elution of 1-antibodies. Arrows mark cells stained by both antibodies, displaying similar labeling pattern. C, Section incubated successively with 1-specific antibodies (C1) and then with FSH hormone antibodies (C2). Arrows show a nonoverlapping distribution of 1-subunit and FSH. IL, intermediate lobe. Calibration bars: A, 100 m; B and C, 50 m. 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. 5A, application of GABA (10 m) to such a large cell evoked an inward desensitizing whole-cell current that was insensitive to bicuculline (100 m). 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 Endo • 2000 Vol 141 • No 5 FIG. 4. Immunoreactivity for TSH and -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 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 m; C, 50 m. 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. 5B). Unlike the bicuculline-sensitive currents that did not run down quickly, the bicuculline-insensitive currents ran down in less than 3 min (Fig. 5C). In both cases, however, the GABA-activated current was not inhibited by the GABAB inhibitor saclofen (10 m; not shown). Furthermore, the current-voltage relationship of the bicuculline-insensitive current (Fig. 5D) revealed a mean measured reversal potential (⫺0.56 ⫾ 1.7 mV; n ⫽ 5) close to the chloride Nernst potential. GABAC RECEPTOR IN THYROTROPIN-SECRETING CELLS 1631 Discussion FIG. 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 M 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 1subunit cDNA at a holding potential of ⫺60 mV. 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. 5E) transfected with rat 1-subunit cDNA (16). We demonstrate here, for the first time outside the retina, the presence of a 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 -subunit mRNAs and GABAC functional behavior suggest different GABAC receptors in the pituitary and retina. Northern blot analyses revealed that -subunit mRNAs were expressed in the pituitary in sufficiently high amounts to be detected by Northern blot. The slight difference in mRNA sizes of 1 and 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 1-subunit (32). The functional alternatively spliced 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 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 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 -subunit mRNAs in the rat pituitary and retina confirmed that 1 was the main GABAC receptor subunit expressed in the pituitary, whereas 1 and 2 were equally expressed in the retina. Taken together, our experiments clearly showed a tissue-specific expression of 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 1-antibody. We also showed that all the rat pituitary cells labeled by the 1-antibody were labeled by an anti-TSH antibody, demonstrating cell-specific expression of the 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 receptormediated 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 1632 GABAC RECEPTOR IN THYROTROPIN-SECRETING CELLS their subunit composition (36, 37). It can be hypothesized that pituitary GABAC receptors contain an alternative 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 rundown of the bicuculline-insensitive GABA-gated Cl⫺ current expressed in thyrotrophs also suggest that such channels may have been overlooked in the brain. Because -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 bicucullineinsensitive GABA-gated Cl⫺ channel and the presence of -subunits, it would be reasonable to propose the presence of a 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 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 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. We would like to thank B. 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