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Endocrinology Vol. 141, No. 3 1083-1092
Copyright © 2000 by The Endocrine Society

ARTICLES

Subunit Composition and Pharmacological Characterization of {gamma}-Aminobutyric Acid Type A Receptors in Frog Pituitary Melanotrophs1

Estelle Louiset, Ruth McKernan, Werner Sieghart and Hubert Vaudry

European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, Centre National de la Recherche Scientifique, University of Rouen (E.L., H.V.), 76821 Mont-Saint-Aignan, France; the Department of Biochemistry and Molecular Biology, Merck, Sharp, and Dohme Research Laboratories, Terlings Park (R.M.K.), Harlow, Essex, United Kingdom CM20 2QR; and the Section of Biochemical Psychiatry, University Clinic for Psychiatry (W.S.), A-1090 Vienna, Austria

Address all correspondence and requests for reprints to: Dr. E. Louiset, European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, UA CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The frog pars intermedia is composed of a single population of endocrine cells directly innervated by {gamma}-aminobutyric acid (GABA)ergic nerve terminals. We have previously shown that GABA, acting through GABAA receptors, modulates both the electrical and secretory activities of frog pituitary melanotrophs. The aim of the present study was to take advantage of the frog melanotroph model to determine the relationship between the subunit composition and the pharmacological properties of native GABAA receptors. Immunohistochemical labeling revealed that in situ and in cell culture, frog melanotrophs were intensely stained with {alpha}2-, {alpha}3-, {gamma}2-, and {gamma}3-subunit antisera and weakly stained with a {gamma}1-subunit antiserum. Melanotrophs were also immunolabeled with a monoclonal antibody to the ß2/ß3-subunit. In contrast, frog melanotrophs were not immunoreactive for the {alpha}1-, {alpha}5-, and {alpha}6-isoforms. The effects of allosteric modulators of the GABAA receptor on GABA-activated chloride current were tested using the patch-clamp technique. Among the ligands acting at the benzodiazepine-binding site, clonazepam (EC50, 5 x 10-9 M), diazepam (EC50 , 10-8 M), zolpidem (EC50, 3 x 10-8 M), and ß-carboline-3-carboxylic acid methyl ester (EC50, 10-6 M) were found to potentiate the whole cell GABA-evoked current in a dose-dependent manner. Methyl-6,7-dimethoxy-4-ethyl-ß-carboline-3-carboxylate (IC50, 3 x 10-5 M) inhibited the current, whereas Ro15–4513 had no effect. Among the ligands acting at other modulatory sites, etomidate (EC50, 2 x 10-6 M) enhanced the GABA-evoked current, whereas 4'-chlorodiazepam (IC50, 4 x 10-7 M), ZnCl2 (IC50, >5 x 10-5 M), and furosemide (IC50, >3 x 10-4 M) depressed the response to GABA. PK 11195 did not affect the GABA-evoked current or its inhibition by 4'-chlorodiazepam. The results indicate that the native GABAA receptors in frog melanotrophs are formed by combinations of {alpha}2-, {alpha}3-, ß2/3-, {gamma}1-, {gamma}2-, and {gamma}3-subunits. The data also demonstrate that clonazepam is the most potent, and zolpidem is the most efficient positive modulator of the native receptors. Among the inhibitors, 4'-chlorodiazepam is the most potent, whereas ZnCl2 is the most efficient negative modulator of the GABAA receptors. The present study provides the first correlation between subunit composition and the functional properties of native GABAA receptors in nontumoral endocrine cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE {gamma}-AMINOBUTYRIC acidA (GABAA) receptor is a heterooligomeric protein complex that forms a ligand-gated chloride channel. Molecular cloning of the GABAA receptor subunits has revealed the existence of at least 20 distinct isoforms belonging to 7 subfamilies, i.e. {alpha}1–6, ß1–4, {gamma}1–3, {delta}, {rho}1–3, {epsilon}, {pi}, and {theta} (1, 2). The diversity of the subunits suggests the existence of a large number of pentameric subunit combinations (1). For instance, coexpression of {alpha}-, ß-, and {gamma}-subunits, which represent the minimal associations giving rise to fully functional recombinant GABAA receptors (3), would yield theoretically more than 10,000 different pentameric combinations (4).

The GABAA receptor function is allosterically modulated by a variety of endogenous factors and drugs, including benzodiazepines (BZDs), ß-carbolines, imidazopyridines, anesthetics, barbiturates, steroids, ethanol, and zinc (5). The effects of these agents on recombinant GABAA receptors are determined by the nature of the {alpha}-, ß-, and {gamma}-subunits forming the receptor complex (6, 7, 8, 9, 10). In the central nervous system, cell-specific expression of the different subunits (11) gives rise to a wide variety of native GABAA receptor subtypes (12, 13, 14). Thus, the biochemical and pharmacological characterization of GABAA receptors in the brain has been hampered by the heterogeneity of the cell types and the diversity of the GABAA receptor subunits expressed by nerve cells (15, 16, 17).

In endocrine cells, diverse {alpha}-, ß-, and {gamma}-subunits are also expressed (18, 19, 20, 21, 22, 23), suggesting the existence of multiple GABAA receptor subtypes. A few electrophysiological studies aimed at characterizing GABAA receptors have been performed on tumoral cells derived from endocrine tissues (24, 25). However, to date, no attempt has been made to correlate the subunit composition with the electrophysiological and pharmacological properties of native GABAA receptors in nontumoral endocrine cells.

The pars intermedia of the frog pituitary is composed of a single population of endocrine cells, the melanotrophs, that are directly innervated by GABAergic nerve terminals (26, 27, 28). These cells secrete the melanotropic peptide, {alpha}MSH, a hormone that plays a key role in the process of skin color adaptation in amphibians. The effects of GABA on the secretory and electrical activities of frog melanotrophs are mediated through GABAA receptors (29, 30, 31) and modulated by BZDs (32, 33), neuroactive steroids (34, 35), and the octadecaneuropeptide ODN (32). Frog melanotrophs thus represent a very suitable model in which to investigate the subunit composition and the pharmacological characteristics of native GABAA receptors expressed by nontumoral endocrine cells. In the present study we have identified by immunohistochemistry the different {alpha}-, ß-, and {gamma}-subunits of the GABAA receptor in the pituitary gland of the frog Rana ridibunda, using specific antibodies against the various isoforms. We have taken advantage of the presence of a homogeneous population of melanotrophs in the frog pars intermedia to correlate the subunit composition of the native GABAA receptors with the potency and efficacy of a series of compounds modulating the GABA-activated chloride current.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male frogs, Rana ridibunda, were obtained from a commercial source (Couétard, Saint-Hilaire de Riez, France). Frogs were housed in a temperature-controlled room (8 ± 1 C) under an established photoperiod of 12 h light/day (lights on from 0600–1800 h). The animals had free access to running water. They were maintained in these conditions for 1 week before use. Animal manipulations were performed according to the recommendations of the French ethical committee and under the supervision of authorized investigators.

Reagents and test substances
Leibovitz culture medium (L15), BSA (fraction V), GABA, ß-carboline-3-carboxylic acid methyl ester (ß-CCM), and 1-[2-chlorophenyl]-N-methyl-N-[1-methylpropyl]-3-isoquinolinecarboxamide (PK 11195) were purchased from Sigma (St. Louis, MO). The antibiotic-antimycotic solution (0.1 mg/ml kanamycin, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml fungizone) and the FBS were supplied by BioWhittaker, Inc. (Walkersville, MD). The N-terminal nonapeptide of the {alpha}1-subunit was obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA), and the C-terminal nonapeptides of the {alpha}2- and {alpha}3-subunits were purchased from Multiple Peptide Systems (San Diego, CA). Diazepam and clonazepam were provided by Hoffmann-La Roche (Basel, Switzerland). 4'-Chlorodiazepam (Ro 5–4864) was supplied by Fluka (Buchs, Switzerland). Ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]-(1,4)benzodiazepine-3-carboxylate (Ro 15–4513) and methyl-6,7-dimethoxy-4-ethyl-ß-carboline-3-carboxylate (DMCM) were obtained from RBI (Natick, MA). Furosemide was obtained from ICN Biomedicals, Inc. (Aurora, OH). Etomidate (Hypnomidate) was provided by Janssen Pharmaceuticals-Cilag (Issy-les-Moulineaux, France). Zolpidem was purchased from Synthelabo-Recherche (Bagneux, France).

Tissue sections
Ten frogs were anesthetized by immersion in 3-aminobenzoic acid ethyl ester (4 mM) in carbonate buffer. The animals were transcardially perfused with 60 ml 0.1 M PBS, pH 7.3, followed by 60 ml MacLean’s fixative. The brains with the attached pituitaries were quickly removed and postfixed overnight in the same fixative. The tissues were stored for 12 h in PBS containing 15% and 30% sucrose successively. The brains were embedded in Jung Tissue Freezing Medium (Leica Corp., Nussloch, Germany) and frozen at -25 C. Sagittal sections were cut at 10 µm with a cryomicrotome (Jung Frigocut 2800 E, Leica Corp.). A total of 40–50 sections were collected per brain.

Cell culture
Cultures of pituitary melanotrophs were prepared as previously described (36). Briefly, neurointermediate lobes were dissociated by enzymatic and mechanical dispersion. The cells were plated at a density of 250,000 cells/ml in Leibovitz medium adjusted to Rana ridibunda osmolality (L15-water = 1:0.4) and supplemented with 10% FBS and antibiotic-antimycotic solution. Melanotrophs used for immunohistological and electrophysiological studies were plated on Supercell chambers and 35-mm culture dishes (CML, Nemours, France), respectively. The cells were cultured for 6–7 days at 22 C in a humidified atmosphere. Fifty Supercell chambers were prepared for immunocytochemical labeling, as follows. The culture medium was removed, the cells were rinsed with PBS, fixed for 30 min in 4% paraformaldehyde at room temperature, and rinsed three times with PBS.

Antibodies
Antisera were raised in rabbits against synthetic peptides derived from the rat {alpha}1-, {alpha}2-, and {alpha}3-subunits (37) or against fusion proteins encompassing a fragment of the rat {alpha}5- (38), {alpha}6- (39), {gamma}1-, and {gamma}3-subunits (40) and the bovine {gamma}2-subunit (40). The sequences of the peptides selected for immunization were specific for each receptor subunit. The mouse monoclonal antibody bd-17, recognizing both the ß2- and ß3-subunits, was purchased from Roche (Mannheim, Germany). All antisera were diluted in PBS supplemented with 1% BSA and 0.3% Triton X-100. The purified antibodies against the {alpha}1-, {alpha}2-, and {alpha}3-subunits were used at a concentration of 5 µg/ml. The antisera directed against the {alpha}5- and {alpha}6-subunits were used at a dilution of 1:200. The antisera against the {gamma}1-, {gamma}2-, and {gamma}3-subunits were used at a dilution of 1:100. The monoclonal antibody bd-17 was used at a concentration of 20 µg/ml.

Immunohistochemistry
Tissue sections or cultured cells were preincubated for 1 h at 20 C with normal goat serum diluted 1:50 in PBS supplemented with 1% BSA and 0.3% Triton X-100 and incubated overnight at 4 C with each primary antiserum. The tissue sections or cells were rinsed three times with PBS and incubated at room temperature for 2 h with fluorescein isothiocyanate-conjugated antirabbit or antimouse {gamma}-globulins (Coltag Laboratories, Burlingame, CA) diluted 1:100. The preparations were rinsed three times with PBS, mounted with PBS-glycerol (1:1), and examined using an Orthoplan microscope equipped with a Vario-Orthomat photographic system (Leica Corp.). Selected slices were also analyzed using a confocal laser scanning microscope (Leica Corp.).

The specificity of the immunoreactions with the {alpha}1-, {alpha}2-, and {alpha}3-subunits was verified by preincubating the antibodies with the different synthetic peptides (10-5 M) used for immunization for 2 h at room temperature. The staining was only abolished when the antisera were preincubated with their respective peptide antigen. The specificity of the {alpha}5-subunit immunoreaction was tested by replacing the antiserum with the preimmune serum from the same animal. Controls for specificity were also performed by substituting nonimmune serum for primary antisera and by replacing the monoclonal antibody to the ß2- and ß3-subunits by a monoclonal antibody to factor H (provided by Dr. M. Fontaine, INSERM U-519, Rouen, France).

Electrophysiological studies
Patch-clamp experiments were performed at room temperature in the whole cell voltage-clamp mode. The cells were continuously superfused at a constant flow rate (1 ml/min) with the extracellular saline solution containing 112 mM NaCl, 2 mM KCl, 2 mM CaCl2, and 15 mM HEPES-NaOH, pH 7.4. The patch pipettes were filled with the intracellular saline solution containing 100 mM potassium glutamate, 1 mM CaCl2, 2 mM MgCl2, 10 mM EGTA, 2 mM ATP, and 10 mM HEPES-KOH, pH 7.4, pCa 8. Currents were recorded at 0 mV from a patch-clamp amplifier (Axopatch 200A, Axon Instruments, Foster City, CA) and digitized at 10 kHz using a Digidata 1200 interface (Axon Instruments) connected to a personal computer and analyzed with the pClamp 6.0.2 software (Axon Instruments).

GABA was directly dissolved in the extracellular saline solution and applied in the vicinity of cultured melanotrophs by pneumatic pressure ejection from a micropipette. Benzodiazepines, ß-carbolines, zolpidem, and PK 11195 were dissolved as concentrated stock solutions in ethanol and extemporaneously diluted in the extracellular saline solution so that the final concentration of ethanol was less than 0.1%. ZnCl2 was dissolved in the extracellular saline solution. Furosemide was dissolved in dimethylsulfoxide and diluted in the extracellular saline solution (final dimethylsulfoxide concentration, <1%, vol/vol). Hypnomidate (preparation for injection containing 2 mg etomidate/ml) was diluted in the extracellular saline solution (final vehicle concentration, <=1%, vol/vol). Each drug solution was superfused for 2 min before and 1 min after the onset of a 10-sec GABA ejection. Control experiments have shown that none of the solvents has any effect on the spontaneous or GABA-evoked electrical activity of melanotrophs.

The relative current amplitude was calculated as I/Icontrol, where Icontrol and I are the maximum intensities of the GABA-induced current measured in the absence and presence of the modulatory agents, respectively. The relative current amplitudes were expressed as the mean ± SEM calculated from 4–14 independent experiments. Statistical analysis was performed using Student’s t test. To quantify the sensitivity of the receptors to the different drugs applied, the dose-response relationships were fitted using SigmaPlot 5.01 software (Jandel Scientific, Sausalito, CA) with the equation: f(x) = (Rmax - Rmin)/(1 + EC50/x)n + Rmin, where x is the drug concentration, Rmax is the maximum response, Rmin is the minimum response, EC50 is the drug concentration eliciting half-maximum effect, and n is the Hill coefficient.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification and localization of the GABAA receptor subunits in the frog pituitary
To identify the different subunits forming native GABAA receptors in frog melanotrophs, parasagittal sections of the pituitary were incubated with the antibodies to the {alpha}1-, {alpha}2-, {alpha}3-, {alpha}5-, {alpha}6-, ß2/ß3-, {gamma}1-, {gamma}2-, and {gamma}3-subunits of the GABAA receptor. The intermediate and neural lobes were devoid of {alpha}1-subunit-like immunoreactivity (LI). In contrast, the distal lobe was intensively labeled by the {alpha}1-subunit antibodies, notably in the rostroventral region facing the intermediate lobe and the median eminence (Fig. 1AGo). The intermediate lobe was densely loaded with {alpha}2-subunit immunoreactive material, whereas the distal and neural lobes were not labeled (Fig. 1BGo). The antiserum to the {alpha}3-subunit stained both the intermediate lobe and the dorsal region of the distal lobe (Fig. 1CGo). {alpha}5-Subunit-LI was distributed throughout the pituitary (Fig. 1DGo); the labeling was less intense in the intermediate lobe than in the neural lobe and the caudal part of the distal lobe (not shown). No {alpha}6-subunit-LI could be detected in any region of the pituitary (Fig. 1EGo), although the {alpha}6-subunit antiserum could specifically label a few cells in the cerebellum (not shown). The cellular distribution of the {alpha}2-, {alpha}3-, and {alpha}5-subunit immunoreactive material was studied in the intermediate lobe by confocal laser scanning microscopy. The {alpha}2- and {alpha}3-subunit-LI appeared to be localized in melanotrope cells (Fig. 1Go, F and G). In contrast, the {alpha}5-subunit-LI was not localized in endocrine cells, but was only present in the pituitary stalk and in scarce fibers innervating the intermediate lobe (Fig. 1HGo).



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  		Figure 1. Immunohistochemical localization of the {alpha}1-, {alpha}2-, {alpha}3-, {alpha}5-, and {alpha}6-subunits of the GABAA receptor on parasagittal sections of the frog pituitary. A, {alpha}1-Subunit-LI was observed throughout the pars distalis (PD), but in neither the pars intermedia (PI) or pars nervosa (PN). B, Intense {alpha}2-subunit-LI was observed in the PI, but not in the PD or PN. C, {alpha}3-Subunit-LI was observed in the PD and PI, but not in the PN. D, {alpha}5-Subunit-LI was observed in the PI and PN. E, {alpha}6-Subunit-LI was not detected in any pituitary region. F–H, Confocal laser scanning microscope analysis at the level of the PI showed that {alpha}2-subunit-LI (F) and {alpha}3-subunit-LI (G) are localized in melanotrophs, whereas {alpha}5-subunit-LI (H) is restricted to fibers innervating the PI (arrows). Intense {alpha}5-subunit-LI was observed in axons coursing along the pituitary stalk (PS). Scale bars: A–D, 50 µm; E–H, 10 µm.

 
All three lobes of the pituitary exhibited ß2/ß3-subunit-LI (Fig. 2AGo). However, the labeling was weak compared with that found in different brain areas, particularly in the granule cell layer of the cerebellar cortex (not shown). Faint {gamma}1-subunit-LI was detected in the intermediate and distal lobes (Fig. 2BGo). In contrast, intense {gamma}2-subunit-LI was observed throughout the pituitary. Specifically, numerous cells of the distal lobe and all melanotrophs were brightly fluorescent, whereas the neural lobe was densely innervated by immunoreactive axon terminals (Fig. 2CGo). The {gamma}3-subunit antiserum produced intense staining of the intermediate lobe (Fig. 2DGo). Examination of pituitary sections by confocal laser scanning microscopy showed that ß2/ß3-, {gamma}1-,{gamma}2-, and {gamma}3-LI were located in melanotrophs. A few {gamma}1- and {gamma}3-subunit immunoreactive fibers were also observed in the vicinity of melanotrophs (Fig. 2Go, E–H).



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  		Figure 2. Immunohistochemical localization of the ß2/ß3-, {gamma}1-, {gamma}2-, and {gamma}3-subunits of the GABAA receptor on parasagittal sections of the frog pituitary. A, ß2/ß3-Subunit-LI was observed in the pars nervosa (PN), pars intermedia (PI), and pars distalis (PD). B, Weak {gamma}1-subunit-LI was observed in the PD and PI. C, {gamma}2-Subunit-LI was observed in the three lobes of the pituitary. D, Intense {gamma}3-subunit-LI was observed in the PI. E–H, Confocal laser scanning microscope analysis at the level of the PI showed that the ß2/ß3-subunit-LI (E), {gamma}1-subunit-LI (F), {gamma}2-subunit-LI (G), and {gamma}3-subunit-LI (H) are localized in melanotrophs. {gamma}1- and {gamma}3-subunit immunoreactive fibers are indicated by arrows. Scale bars: A–D, 50 µm; E–H, 10 µm.

 
The types of GABAA receptor subunits expressed by melanotrophs cultured for 6–7 days were also studied by immunocytochemistry. Cultured cells were not labeled with the antiserum against the {alpha}1-subunit (Fig. 3AGo). Cultured melanotrophs were intensely stained with the antibodies against the {alpha}2- and {alpha}3-subunits (Fig. 3Go, B and C), but not with the antibodies against the {alpha}5- and {alpha}6-subunits (Fig. 3Go, D and E). Cultured melanotrophs exhibited strong ß2/ß3-subunit-LI (Fig. 3FGo) and moderate {gamma}1-subunit-LI (Fig. 3GGo). Cultured cells were intensely labeled with the antibodies against the {gamma}2- and {gamma}3-subunits (Fig. 3Go, H–I).



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  		Figure 3. Immunocytochemical identification of {alpha}-, ß-, and {gamma}-subunits of the GABAA receptor in cultured frog melanotrophs. A, {alpha}1-Subunit-LI was not detected. B and C, Presence of {alpha}2-subunit-LI (B) and {alpha}3-subunit-LI (C). D and E, Absence of {alpha}5-subunit-LI (D) and {alpha}6-subunit-LI (E). F–I, Presence of ß2/ß3-subunit-LI (F), {gamma}1-subunit-LI (G), {gamma}2-subunit-LI (H), and {gamma}3-subunit-LI (I). Scale bars: A–H, 10 µm.

 
Effects of specific ligands of BZD binding sites of GABAA receptors on the GABA-induced current
The effects of graded concentrations (10-9–10-5 M) of diazepam and clonazepam, two BZD receptor agonists, were studied on the whole cell current evoked by a half-maximally effective dose of GABA (3 x 10-6 M). Clonazepam and diazepam induced a dose-dependent increase in the amplitude of the current elicited by GABA (Fig. 4Go, A and B). Half-maximal responses were observed at concentrations of 5 x 10-9 M for clonazepam and 10-8 M for diazepam; for both compounds, the maximum effect occurred at 10-6 M (Fig. 4DGo). In contrast, the inverse agonist of BZD-binding sites Ro 15–4513 (10-9–10-5 M) did not modify the current evoked by 3 x 10-6 M (not shown) or 10-5 M GABA (Fig. 4CGo). Exposure of melanotrophs to the ß-carboline DMCM (10-9 to 5 x 10-5 M) provoked a dose-dependent reduction of the current elicited by 10-5 M GABA (Fig. 5Go, A and D). Half-maximal and maximal inhibitions were obtained at concentrations of 3 x 10-5 and 10-4 M, respectively. Conversely, superfusion of melanotrophs with the ß-carboline ß-CCM (10-8 to 3 x 10-5 M) caused a modest potentiation of the current evoked by 3 x 10-6 M GABA (Fig. 5BGo). The imidazopyridine zolpidem (10-9–10-5 M) markedly potentiated the current induced by 3 x 10-6 M GABA (EC50 = 3 x 10-8 M; Fig. 5CGo). The efficacies and potencies of the different ligands acting at the BZD-binding sites are summarized in Table 1Go.



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  		Figure 4. Effect of BZDs on the GABA-evoked current in cultured frog melanotrophs. A–C, Ten-second pulses of 3 x 10-6 M GABA (A and B) or 10-5 M GABA (C) were ejected repeatedly at 2-min intervals (bars under the current traces). The pulses of GABA were administered in the absence (control) or presence of increasing concentrations (10-9–10-5 M) of diazepam (A), clonazepam (B), or Ro 15–4513 (C; bars above the current traces). D, Dose-response curves comparing the effects of graded concentrations of diazepam ({blacktriangledown}), clonazepam (•), and Ro 15–4513 ({blacksquare}) on the relative amplitude of the current evoked by GABA. The data represent the mean ± SEM calculated from a series of recordings (n = 4–12) similar to those presented in A–C.

 


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  		Figure 5. Effects of DMCM, ß-CCM, and zolpidem on the GABA-evoked current in cultured frog melanotrophs. A–C, Ten-second pulses of 10-5 M GABA (A) or 3 x 10-6 M GABA (B and C) were ejected repeatedly at 2-min intervals (bars under the current traces). The pulses of GABA were administered in the absence (control) or presence of increasing concentrations of DMCM (A; 10-9 to 5 x 10-5 M), ß-CCM (B; 10-8 to 3 x 10-5 M), or zolpidem (C; 10-9–10-5 M; bars above the current traces). D, Dose-response curves comparing the effects of graded concentrations of DMCM ({blacksquare}), ß-CCM ({blacktriangledown}), and zolpidem (•) on the relative amplitude of the current evoked by GABA. The data represent the mean ± SEM calculated from a series of recordings (n = 4–6) similar to those presented in A, B, and C.

 

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  		Table 1. Relative efficacy (maximal response) and potency (EC50) of the different agents tested for their modulatory effect on GABA-induced chloride current in frog melanotrophs

 
Effects of drugs acting at other sites of the GABAA receptor on the GABA-induced current
Application of the BZD 4'-chlorodiazepam (10-7 to 5 x 10-5 M) to melanotrophs reduced the current evoked by 10-5 M GABA in a dose-dependent manner (Fig. 6AGo). Half-maximum and maximum inhibitions were reached at concentrations of 4 x 10-7 and 10-4 M, respectively (Fig. 6CGo). The isoquinoline carboxamide derivative PK 11195 (5 x 10-5 M) did not modify the current elicited by 3 x 10-6 M GABA and did not affect the inhibition of the GABA-evoked current provoked by 10-5 M 4'-chlorodiazepam (Fig. 6BGo). Superfusion of melanotrophs with ZnCl2 (10-6–10-3 M) and the anthranilic acid derivative furosemide (10-7 to 3 x 10-3 M) gradually decreased the current induced by 10-5 M GABA (Fig. 7Go, A and B) with IC50 values of more than 5 x 10-5 M for ZnCl2 and more than 3 x 10-4 M for furosemide (Fig. 7DGo). In contrast, administration of the anesthetic etomidate (10-7–10-4 M) slightly increased the current induced by 3 x 10-6 M GABA with an EC50 of 2 x 10-6 M (Fig. 7Go, C and D). The efficacies and potencies of the different modulators of GABAA receptors are summarized in Table 1Go.



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  		Figure 6. Effects of 4'-chlorodiazepam and PK 11195 on the GABA-evoked current in cultured frog melanotrophs. A, Ten-second pulses of GABA (10-5 M) were ejected repeatedly at 2-min intervals (bars under the current traces). The pulses of GABA were administered in the absence (control) or presence of increasing concentrations of 4'-chlorodiazepam (10-7 to 5 x 10-5 M; bars above the current traces). B, Ten-second pulses of GABA (3 x 10-6 M) were ejected at 2-min intervals in the absence (control and wash) or presence of PK 11195 (5 x 10-5 M; open bars) and/or 4'-chlorodiazepam (10-5 M; hatched bars). C, Dose-response curve showing the effects of graded concentrations of 4'-chlorodiazepam ({blacksquare}) on the relative amplitude of the current evoked by GABA. The data represent the mean ± SEM calculated from a series of recordings (n = 14) similar to those presented in A.

 


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  		Figure 7. Effects of ZnCl2, furosemide, and etomidate on the GABA-evoked current in cultured frog melanotrophs. A–C, Ten-second pulses of 10-5 M GABA (A and B) or 3 x 10-6 M GABA (C) were ejected repeatedly at 2-min intervals (bars under the current traces). The pulses of GABA were administered in the absence (control) or presence of increasing concentrations of ZnCl2 (A; 10-6–10-3 M), furosemide (B; 10-7 to 3 x 10-3 M), or etomidate (C; 10-7–10-4 M; bars above the current traces). D, Dose-response curves comparing the effects of graded concentrations of ZnCl2 (•), furosemide ({blacktriangleup}), and etomidate ({blacksquare}) on the relative amplitude of the current evoked by GABA. The data represent the mean ± SEM calculated from a series of recordings (n = 5) similar to those presented in A, B, and C.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study has provided evidence for the presence of {alpha}2-, {alpha}3-, ß2/ß3-, {gamma}1-, {gamma}2-, and {gamma}3-subunit-like immunoreactivity in frog pituitary melanotrophs in situ and in vitro. This study has also demonstrated that the GABA-induced current is potentiated by clonazepam, diazepam, zolpidem, ß-CCM, and etomidate and is inhibited by DMCM, 4'-chlorodiazepam, ZnCl2, and furosemide.

The occurrence of {alpha}2- and {alpha}3-subunit-LI and the absence of {alpha}1-, {alpha}5-, and {alpha}6-immunoreactive material in the frog intermediate lobe and in cultured melanotrophs are in total agreement with previous RT-PCR and ribonuclease protection assay studies that have demonstrated the presence of {alpha}2- and {alpha}3-subunit, but not {alpha}1-, {alpha}5-, and {alpha}6-subunit, mRNAs in the rat pars intermedia (18, 19). The absence of {alpha}1, {alpha}5, and {alpha}6 immunoreactivities in frog melanotrophs cannot be ascribed to cross-species specificity, inasmuch as the {alpha}1- and {alpha}5-subunit antibodies produced intense staining in the distal and neural lobes, respectively, and the {alpha}6-subunit antibodies labeled the frog cerebellum. The lack of {alpha}1-subunit in the intermediate lobe contrasts with the situation in other endocrine glands, including the anterior lobe of the pituitary (Refs. 18, 19 and this study), pancreas (20), and adrenal medulla (22), which all express the {alpha}1-isoform.

The occurrence of ß2/ß3-subunit-LI in the frog pars intermedia is consistent with recent immunocytochemical observations made in the rat pituitary (23). The monoclonal antibody bd17 used in the present study has been previously applied to the localization of ß2/ß3-GABAA receptor subunits in various nonmammalian vertebrates (41), including amphibians (42) and fish (43, 44). In the frog pars intermedia, Western blot experiments using the bd17 antibody have recently shown the existence of two bands, exhibiting molecular masses of 50–60 kDa (31), that correspond to the molecular masses of the ß2- and ß3-subunits of the GABAA receptor of mammals (12).

The presence of {gamma}1- and {gamma}2-subunit-LI in frog melanotrophs is consonant with the expression of {gamma}1- and {gamma}2-subunit mRNAs in the rat intermediate lobe (18). To our knowledge the present study provides the first evidence for the expression of the {gamma}3-subunit in endocrine cells. Together, these observations indicate that the native GABAA receptors borne by frog melanotrophs are composed of {alpha}2-, {alpha}3-, ß2-, and/or ß3-, {gamma}1-, {gamma}2-, and {gamma}3-subunits. A random assembly of these subunits would result in a total of 63 GABAA receptor subtypes with an assumed stoichiometry of 2{alpha}1ß2{gamma} or 2{alpha}2ß1{gamma} (1, 5, 45). The number of subunit combinations is probably more restricted, because receptor immunoprecipitation followed by immunoblotting identification has clearly demonstrated that various associations, such as {alpha}2{alpha}3, {alpha}3ß2, and {gamma}1{gamma}2, do not exist in rat neuronal GABAA receptors (12, 13, 38). It has been also reported that {alpha}2ß2, {alpha}3{gamma}2, and {alpha}3{gamma}3 are rarely coassembled (12, 13). In contrast, the {alpha}2- or {alpha}3-subunit is preferentially associated with the ß3- and {gamma}2-subunits (12, 13). These observations suggest that eight prevalent GABAA receptor subtypes (2{alpha}22ß3{gamma}1/2/3, 2{alpha}22ß32{gamma}2/3, 2{alpha}3ß32{gamma}1, and 2{alpha}32ß3{gamma}1) could be expressed by frog melanotrophs.

Studies conducted with recombinant and immunopurified neuronal receptors have firmly established that the subunit composition determines the pharmacological profile of GABAA receptors. In particular, the presence of an {alpha}2- or {alpha}3-subunit confers high affinity to clonazepam and diazepam (3, 13), but much lower affinity to zolpidem (13, 46, 47). In contrast, GABAA receptors containing the {alpha}1-subunit exhibit high affinity for zolpidem (13, 46, 47, 48). In accord with the absence of the {alpha}1-subunit and the presence of the {alpha}2- and {alpha}3-subunits in frog melanotrophs, we found that clonazepam and diazepam were more potent than zolpidem in potentiating the GABA-evoked current. Similar observations have been reported on neurons isolated from the rat striatum (49), a brain region that expresses the {alpha}2-, {alpha}3-, and {alpha}5-subunits, but not the {alpha}1-subunit (11). Furosemide is a potent inhibitor of the activity of {alpha}6-containing GABAA receptors (7, 50). The absence of {alpha}6-subunit immunoreactivity in frog melanotrophs is consistent with the very low potency of furosemide to inhibit the GABA-evoked current. Reciprocally, 4'-chlorodiazepam, which does not bind to recombinant receptors containing the {alpha}6-isoform (51), strongly depressed the GABA-evoked current in frog melanotrophs. Taken together, these data indicate that the native GABAA receptors expressed by frog melanotrophs display a pharmacological profile similar to those of recombinant receptors that encompass the {alpha}2- and {alpha}3-subunits.

Previous electrophysiological studies have revealed that the ß-subunits play a crucial role in the modulatory effect of anesthetics (8, 52) and ß-carbolines (53) on recombinant receptors. It has been shown that the anesthetic etomidate is more potent on recombinant receptors containing a ß2- or ß3-subunit than on those possessing the ß1-isoform (8, 54). At high concentrations, ß-carbolines exhibiting a 3-carboxyl ester group, such as ß-CCM and DMCM, potentiate the action of GABA on recombinant receptors (55) by acting on the ß2- or ß3-subunit (53). The existence of ß2- and/or ß3-subunits in GABAA receptors of frog melanotrophs is congruent with the positive modulation exerted by etomidate and ß-CCM on the GABA-evoked current. In contrast, we found that DMCM, even at high concentrations, did not enhance the amplitude of the chloride current. The present data provide the first evidence for a correlation in native GABAA receptors between the presence of the ß2- and/or ß3-subunits and their sensitivity to etomidate and ß-CCM. They also demonstrate that, unlike recombinant receptors composed of ß2- or ß3-subunits, native GABAA receptors expressed in melanotrophs are differentially modulated by ß-CCM and DMCM.

The importance of the {gamma}-subunits in determining the sensitivity of GABAA receptors to allosteric modulators is clearly established. In particular, it has been demonstrated that the {gamma}2- and {gamma}3-subunits directly contribute to the formation of high affinity benzodiazepine-binding sites (3, 6, 47). It has also been shown that coexpression of {gamma}2-subunits with {alpha}1- and ß2-subunits markedly reduces the ability of Zn2+ to inhibit GABA-activated chloride currents (9). Consistent with these {gamma}2-subunit data, the present study has shown that GABAA receptors in frog melanotrophs, which contain {gamma}2- and {gamma}3-subunits, are sensitive to clonazepam, diazepam, and zolpidem. In addition, the sensitivity to Zn2+ of native GABAA receptors in melanotrophs (IC50, >5 x 10-5 M) was in the same range as that of recombinant {alpha}1ß2{gamma}2 receptors (5.1 x 10-5 M) but was more than 50-fold lower than that of recombinant {alpha}1ß2 receptors (0.9 x 10-6 M) (9). The observation that the benzodiazepine ligand Ro 15–4513 did not affect the GABA-evoked currents in frog melanotrophs is in agreement with the presence of the {gamma}2-subunit inasmuch as previous studies have shown that Ro 15–4513 has no effect on various recombinant GABAA receptors encompassing the {gamma}2-subunits (7, 56). Concurrently, it has been reported that, in {gamma}1-containing recombinant receptors, ß-carbolines may enhance, rather than reduce, the effect of GABA on chloride current (6, 45, 57). Thus, the occurrence of the {gamma}1-subunit, in addition to the {gamma}2- and {gamma}3-subunits, in frog GABAA receptors may explain why ß-CCM produced a modest stimulation (through interaction with the {gamma}1-subunit), whereas DMCM induced a dose-dependent inhibition (through interaction with the {gamma}2- and {gamma}3-subunits) of the GABA-evoked current. Taken together, these data demonstrate that the native GABAA receptors present in frog melanotrophs exhibit pharmacological properties similar to those described for recombinant receptors comprising {gamma}1-, {gamma}2-, and/or {gamma}3-subunits.

In conclusion, the present report shows that GABAA receptors borne by frog melanotrophs are formed by combinations of {alpha}2-, {alpha}3-, ß2/ß3-, {gamma}1-, {gamma}2-, and {gamma}3-subunits. The current evoked by GABA on cultured melanotrophs is potentiated by various compounds (order of potency: clonazepam>diazepam>zolpidem>ß-CCM>etomidate) and inhibited by other agents (order of potency: 4'chlorodiazepam>DMCM>ZnCl2>furosemide). The pars intermedia, which is composed of a homogeneous population of endocrine cells directly innervated by GABAergic fibers, appears to be a very suitable experimental model in which to study the regulation of native GABAA receptors in endocrine cells.


   Acknowledgments
 
We are grateful to Lionel Cazin for detailed discussions and help in the preparation of this manuscript. We thank Dr. J. Benavides (Synthelabo Recherche, Bagneux, France) for the generous gift of zolpidem. We are indebted to Dr. W. E. Haefely (Hoffman-La Roche) for providing diazepam and clonazepam. We appreciate the excellent technical assistance of Mrs. Catherine Buquet.


   Footnotes
 
1 This work was supported by grants from INSERM (U-413) and the Conseil Régional de Haute-Normandie. Back

Received November 24, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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