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Neuroendocrine -Aminobutyric Acid (GABA): Functional Differences in GABA_A Versus GABA_B Receptor Inhibition of the Melanotrope Cell of Xenopus laevis¹

Department of Cellular Animal Physiology, Nijmegen Institute for Neurosciences, Subfaculty of Biology, University of Nijmegen, Nijmegen, The Netherlands

Address all correspondence and requests for reprints to: Dr. B. G. Jenks, Department of Cellular Animal Physiology, Nijmegen Institute for Neurosciences, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands. E-mail: Jenks{at}sci.kun.nl

	Abstract

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The melanotrope cell of Xenopus laevis is innervated by nerve terminals that contain, among other transmitter substances, the neurotransmitter -aminobutyric acid (GABA). Postsynaptically the melanotrope cell possess both GABA_A and GABA_B receptors. Activation of either receptor type leads to an inhibition of MSH release from the cell. The present study concerns the functional significance of the existence of two types of GABA receptors on the melanotrope regarding two questions: 1) do the different receptor types have different effects on the melanotrope? and 2) can the endogenous ligand GABA differentially activate these receptors? Concerning the first question, we have tested the hypothesis that the GABA_A receptor (a chloride ion channel) and the GABA_B receptor (a G protein-coupled receptor negatively linked to adenylyl cyclase) may have differential effects on the sensitivity of the cell to stimulation by cAMP-dependent mechanisms. We show that treatments with either isoguvacine (GABA_A agonist) or baclofen (GABA_B agonist) inhibit intracellular Ca²⁺ oscillations and peptide secretion from melanotrope cells. Treatments known to increase intracellular cAMP in the melanotrope (e.g. use of the peptide sauvagine or the cAMP analog 8-bromo-cAMP) completely overcame the inhibition induced by baclofen, but not that caused by isoguvacine. We conclude that the GABA_A and GABA_B receptors have different effects on the Xenopus melanotrope cell by differentially affecting the sensitivity the cell shows to stimulation by cAMP-dependent mechanisms. Concerning possible differential activation of the receptor types, we found that we could use a membrane potential probe (from the bis-oxonol family) to differentiate between GABA_A and GABA_B receptor activation. Using this probe we showed that low GABA concentrations (<10^-7 M) give a response indicative of the GABA_B receptor, whereas at high GABA concentrations (>10^-7 M), the GABA_A receptor response predominates. We, therefore, conclude that GABA can differentially activate the two types of GABA receptors on the Xenopus melanotrope cell.

	Introduction

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THE MELANOTROPE cell of the pituitary intermediate lobe synthesizes the multifunctional precursor protein POMC (1, 2), which is processed to yield several bioactive peptides, including MSH. In amphibians, this cell is involved in the regulation of pigment distribution in skin melanophores during the process of background adaptation (3, 4). In Xenopus laevis, secretion of POMC peptides by the melanotrope cell is under complex stimulatory and inhibitory control. The amphibian CRH-related peptide sauvagine and TRH stimulate secretion (5, 6). These neuropeptides act as neurohormones, being released from nerve terminals in the pars nervosa. The melanotrope cell is directly inhibited by the classical neurotransmitters dopamine and -aminobutyric acid (GABA) (7) and by neuropeptide Y (NPY) (8). These three inhibitors coexist in nerve terminals synaptically contacting the melanotrope. Within the synapses, GABA is confined to small electron-lucent vesicles, whereas dopamine and NPY are colocalized in electron-dense vesicles (9, 10). Postsynaptically it has been shown that NPY inhibits through a Y₁ receptor (10), dopamine through a D₂-like receptor (12), and GABA through two distinct types of receptors, the GABA_A receptor and the GABA_B receptor (13, 14).

The intracellular messenger cAMP is important in regulating MSH secretion from Xenopus melanotropes (15, 16, 17). Sauvagine stimulates secretion through a stimulation of the production of cAMP, whereas NPY, dopamine, and the GABA_B receptor agonist baclofen inhibit production of the cyclic nucleotide. It has been shown that Ca²⁺ oscillations within the melanotrope cell, initiated through opening of N-type voltage-operated Ca²⁺ channels on the membrane, are probably the driving force for secretion (18, 19, 20, 21). cAMP seems to be important in regulating the frequency of these oscillations. Factors that increase cAMP production increase the frequency of oscillations, whereas factors that inhibit cAMP production block Ca²⁺ oscillations (18, 21). Previous studies have attempted to find a rationale for the complexities in the regulation of the secretory process of the Xenopus melanotrope cell. In particular, attention has been given to the actions of the colocalized inhibitory neurotransmitters, dopamine, NPY, and GABA. These transmitter substances were equally effective in inhibiting the secretory process, and no evidence has been found for cooperative interaction among the inhibitors (22, 23).

The present study focuses on the GABAergic component of the Xenopus intermediate lobe neuroendocrine interface, namely the postsynaptic presence of both the GABA_A receptor (forming a Cl^- channel) and the GABA_B receptor (negatively linked to adenylyl cyclase). If the presence of two different postsynaptic receptors for the same endogenous ligand is to have functional significance, it can be expected that 1) the two receptor types have different effects on the cell, and 2) the endogenous ligand can differentially activate the receptors. In our study of the GABA_A and GABA_B receptors we have addressed both of these aspects. First, in view of the importance of adenylyl cyclase and cAMP in the regulation of the secretory process of the melanotrope, we examined whether GABA_A or GABA_B receptor activation could differentially affect the melanotrope cell sensitivity to stimulation by cAMP-dependent mechanisms. To this end we examined intracellular Ca²⁺ dynamics and secretion of immunoreactive and radiolabeled peptides from melanotropes treated with specific GABA_A and GABA_B receptor agonists (isoguvacine and baclofen, respectively). Under these conditions, we determined the response of the cells to treatments with 1) the cell-permeable cAMP analog 8-bromo-cAMP (8Br-cAMP), 2) the neuropeptide sauvagine, and 3) the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Furthermore, using a membrane potential probe, we were able to differentiate between GABA_A and GABA_B receptor activation of the melanotrope cell and have used this probe to determine whether GABA can differentially activate GABA_A or GABA_B receptors. We show that there is a concentration-dependent differential activation of the two GABA receptor types and that these receptors, in turn, differentially affect the sensitivity of the melanotrope to stimulation by cAMP-dependent mechanisms.

	Materials and Methods

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Animals
Xenopus laevis were bred in our aquatic facility (Department of Animal Physiology, Nijmegen, The Netherlands). Young-adult animals were maintained in black plastic containers for 3 weeks before the experiments, under constant illumination at 22 C. The animals were black-adapted to induce a high level of MSH synthesis and secretion by the melanotrope cells.

Superfusion of neurointermediate lobes
Freshly dissected neurointermediate lobes were rinsed in Ringer’s solution containing 112 mM NaCl, 2 mM KCl, 2 mM CaCl₂, 15 mM HEPES (Calbiochem, La Jolla, CA), 0.3 mg/ml BSA (Sigma, St. Louis, MO), 2 mg glucose, and 1 mg/ml ascorbic acid (pH 7.4; carbogen-aerated) and placed individually on a filter in a 10 µl superfusion chamber. Ringer’s solution was pumped at a rate of 1.5 ml/h, and 7.5-min fractions were collected and stored at -20 C until assayed for MSH using a RIA. Factors tested for their effects on MSH secretion were introduced into the superfusion medium, either alone or in various combinations. Factors tested were baclofen (Ciba-Geigy, Basel, Switzerland), isoguvacine (Cambridge Research Biomedicals Ltd., Cambridge, United Kingdom), sauvagine (Bachem, Bubendorf, Switzerland), IBMX (Sigma), and 8Br-cAMP (Sigma). Concentrations and administration protocols are explained in Results.

RIA for MSH
The MSH assay was performed as described previously, using an antiserum raised and characterized in our laboratory (24). The antiserum has equal affinity for the acetylated and nonacetylated forms of MSH, and cross-reactivity with ACTH is less than 0.01%. Bound and free antibodies were separated by polyethylene glycol-albumin precipitation. The detection limit is 2.5 pg MSH/50 µl sample. All superfusion fractions were assayed in duplicate.

Preparation of cultured radiolabeled melanotropes
Isolation and culture of melanotrope cells were performed as described previously (11). In short, after perfusing the animal with Xenopus Ringer’s solution to remove blood cells, neurointermediate lobes were dissected and incubated for 45 min in Ringer’s solution without CaCl₂, to which 0.25% (wt/vol) trypsin (Life Technologies, Renfrewshore, UK) had been added. Cells were subsequently dispersed in Leibovitz L15 medium, which had been adjusted to Xenopus blood osmolarity (L15/MilliQ ratio = 2:1) and contained 10% FCS (Life Technologies). Dispersion was accomplished by gentle trituration of the lobes with a siliconized Pasteur’s pipette. After washing, the cells of 1.5 lobe equivalents were suspended in 100 µl lysine-free L15 medium (+10% FCS) containing 75 µCi [³H]lysine (80 Ci/mM Amersham). The cell suspension was placed on a coverslip coated with poly-L-lysine (Sigma, St. Louis, MO; >300 kDa) to which the cells were allowed to attach for 24 h. Then, 2 ml lysine-free L15 medium (+10% FCS) were added, and cells were cultured for 2 days.

Combined analysis of [³H]peptide secretion and intracellular Ca²⁺[Ca²⁺]_i dynamics
The coverslip containing the cultured cells was placed in a Leiden chamber and washed 3 times with Ringer’s solution. Then, the cells were incubated for 30 min in 1 ml Ringer’s solution containing 8 µM of the Ca²⁺ indicator fura red/AM (Molecular Probes, Eugene, OR) and 1 µM pluronic F127 (Molecular Probes). Subsequently, the chamber was rinsed 3 times and superfused with Ringer’s solution (flow rate, 1 ml/min) for 30 min before the experiment was initiated. The fluorescence intensities of individual melanotropes were analyzed with a Bio-Rad MRC-600 confocal laser scanning microscope by fast photon counting. On-line measurements for up to 15 individual melanotropes were obtained using a time-course ratiometric software package (TCSM, Bio-Rad). Measurements were analyzed according to the method of Koopman et al. (25). Briefly, the data collected first were inverted, so that an increase in fluorescence signal reflects an increase in [Ca²⁺]_i (for fura red a high [Ca²⁺]_i gives a low fluorescent signal and vice versa). A linear fit line to the base of the signal was calculated, and the data of the signal line were divided by the values of the linear fit line, resulting in a new line with the data now normalized to 1. This procedure compensates for photobleaching, and because of the normalization, the average of the relative [Ca²⁺]_i signals of various cells can be calculated even when the cells have different probe-loading characteristics (and thus different absolute fluorescence signal intensities).

During the period that on-line fluorescence intensity measurements were being made, 1-min fractions of superfusate were collected to measure the release of radiolabeled peptides. To each fraction of superfusate, 1 ml scintillation fluid (Optiphase HiSafe, Wallac, Loughborough, UK) was added and the amount of radioactivity in each fraction was determined in a liquid scintillation counter. HPLC analysis shows that approximately 30% of the radioactivity in the superfusate is unincorporated [³H]lysine, and of the incorporated radiolabel, over 90% represented radiolabeled POMC-derived peptides (our unpublished observations).

Analysis of bis-(1,3-dibutyl barbiturate)trimethine oxonol (bis-oxonol) uptake and release (membrane potential probe)
Melanotropes were prepared as described above (except they were not radiolabeled) and brought into superfusion in a Leiden chamber placed on the confocal microscope. The cells were superfused with amphibian Ringer’s solution containing bis-(1,3-dibutyl barbiturate) trimethine oxonol (Molecular Probes) at a final concentration of 90 nM. This member of the bis-oxonol family functions as a slow membrane potential probe, with depolarization leading to uptake of the probe (thus leading to higher cell fluorescence), and hyperpolarization causing loss of the probe from the cell (26, 27). Using Bio-Rad filter block combination BHS and the TCMS software (Bio-Rad Laboratories, Richmond, CA), the fluorescent signal of up to 15 cells was followed in each experiment; measurements were made at 6-sec intervals. The appropriateness of the probe to reflect membrane depolarizations was tested by examining the effects of various concentrations of K⁺ on the fluorescent signal. Then, the effects of pulses of isoguvacine, baclofen, and GABA on the fluorescent signal were determined. The substances were dissolved in Ringer’s solution containing the bis-oxonol and introduced by superfusion.

Calculations and statistics
Values are shown as the average of several experiments ± SEM. The percent inhibition of MSH or POMC-derived peptide secretion in response to treatments with GABA receptor agonists was calculated from the average secretion in three fractions immediately before administration of the agonist, with the average of the three fractions displaying maximum inhibition (peak values) induced by the agonists. The percent stimulation of secretion by treatments that increase intracellular cAMP were calculated from the average secretion in the two fractions displaying maximum stimulation induced by the treatment relative to the 100% basal secretion. Where appropriate, percent stimulation and inhibition values were compared for statistical significance using Student’s paired t test ( = 5%).

In experiments with the membrane potential probe, the effect of each GABA pulse on the fluorescence signal for each cell was expressed as the percent change, calculated by taking the difference between the average fluorescence signal of 10 values immediately before a pulse of GABA and the average signal of the highest (or lowest) 10 values during a GABA pulse and expressing this difference as a percentage of the former.

	Results

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Effects of GABA receptor agonists and 8Br-cAMP on MSH release from neurointermediate lobes
To compare the way in which the GABA receptor agonists inhibit MSH release from neurointermediate lobes, the agonists were tested at concentrations that evoke strong inhibitions (60–80% below basal release level). Our previous studies (13, 14) have shown that for neurointermediate lobe tissue, these concentrations are 10^-5 M for baclofen and 10^-4 M for isoguvacine. When a 15-min pulse with 6 mM 8Br-cAMP was given to lobes in which MSH secretion had been inhibited by baclofen (Fig. 1a), secretion returned to 96 ± 7% of the control level (defined as 100%). It has been our experience that secretion from freshly dissected whole neurointermediate lobes is difficult to stimulate above basal values, reflected in the present data by the failure of 8Br-cAMP to stimulate above the 100% basal level under baclofen-inhibited conditions (Fig. 1a). Possibly under basal conditions such lobes are releasing at or near there maximum rate. When lobes inhibited by isoguvacine were treated with 8Br-cAMP (Fig. 1b), secretion returned to 76 ± 8% of the control level, a value significantly lower than that achieved under baclofen inhibition. Baclofen produced 65 ± 3% peak value inhibition when given alone, but when given in combination with 6 mM 8Br-cAMP, the reduction was significantly weaker (25 ± 3% inhibition; Fig. 1c). Isoguvacine produced 46 ± 4% peak value inhibition when given alone, and this inhibition did not significantly differ when this treatment was given in combination with 8Br-cAMP (44 ± 8%; Fig. 1d). 8Br-cAMP alone had a slow and weak stimulatory action (Fig. 1, c and d).

View larger version (49K): [in this window] [in a new window]   Figure 1. Effects of GABA receptor agonists and 8Br-cAMP on MSH release from superfused neurointermediate lobes of Xenopus laevis. a and b, Effect of 6 mM 8Br-cAMP on MSH release from superfused neurointermediate lobe inhibited by 10-5 M baclofen (GABAB receptor agonist) or 10-4 M isoguvacine (GABAA receptor agonist); c and d, effect of 10-5 M baclofen or 10-4 M isoguvacine alone or in combination with 6 mM 8Br-cAMP on MSH release from superfused neurointermediate lobes. The durations of treatments are indicated by horizontal bars/dotted lines or shaded bars. Fractions were collected every 7.5 min, and MSH was determined by RIA. Basal (control) release was calculated from the three fractions preceding the first treatment. These values were 1907 ± 47 (a), 1755 ± 32 (b), 2024 ± 65 (c), and 1892 ± 44 (d) pg MSH/100 µl sample of superfusate. Values are the averages of four experiments, and vertical lines represent -SEM.

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of 10-4 M IBMX on inhibition of MSH release induced by 10-5 M baclofen and 10-4 M isoguvacine. Superfusion experiments were conducted with control (C) and IBMX-treated lobes. Fractions were collected every 7.5 min, 15-min pulses of baclofen and isoguvacine were given, and peak inhibition was calculated. Values are the average of four experiments. *, Significant reduction in the degree of inhibition achieved relative to the control value (P < 0.05).

View larger version (49K): [in this window] [in a new window]   Figure 3. Effect of 1 mM 8Br-cAMP and 10-6 M baclofen or 10-5 M isoguvacine on intracellular Ca2+ dynamics and secretion from cultured melanotrope cells. The relative [Ca2+]i of individual melanotrope cells is given in a and d, the average relative Ca2+ signal of a number of cells from the two experimental protocols is shown in b and e, and the release of radiolabel from the cultured cell preparations used in the Ca2+ imaging is given in c and f. For each cell preparation, the average radiolabel in the three fractions immediately before administration of 8Br-cAMP was defined as 100% of basal release, and the amount of label in all other fractions was expressed relative to these values (100% values were 650, 732, 1296, and 788 cpm for the preparations used the baclofen experiments and 967, 1747, 2090, and 1152 cpm for the preparations used in the isoguvacine experiments). Shown in c and f is the average (-SEM) percent basal release of the four preparations. Fractions in which 8Br-cAMP and the GABA receptor agonists were administered are indicated by horizontal bars and dotted lines.

In experiments with isoguvacine, 8Br-cAMP treatment also increased the frequency of Ca²⁺ oscillations (an example is shown in Fig. 3d), the average Ca²⁺ signal (Fig. 3e), and the rate of secretion (Fig. 3f). Under conditions of 8Br-cAMP stimulation, isoguvacine induced a Ca²⁺ transient in all cells simultaneously (e.g. Fig. 3d), a phenomenon that was reflected in the average Ca²⁺ signal as a very dominant peak shortly after adding isoguvacine (Fig. 3e). Subsequently, Ca²⁺ oscillations were suppressed during the entire period of isoguvacine treatment (e.g. Fig. 3d), which was reflected in a decreased average [Ca²⁺]_i signal (Fig. 3e). There was an increase in secretion at the start of isoguvacine treatment, followed by a strong and sustained decrease for the remainder of the treatment period (Fig. 3f). After isoguvacine treatment, the level of secretion increased, but did not fully return to the 8Br-cAMP-stimulated level.

Effects of GABA receptor agonists and 8Br-cAMP on secretion from cultured melanotropes
The experiments involving [Ca²⁺]_i measurements cannot be extended for long periods due to the loss of fluorescence signal as a result of photobleaching. To confirm that baclofen induces a weaker inhibition than isoguvacine of melanotrope cell secretory activity under 8Br-cAMP, a long duration secretory study was carried out in which pulses of GABA receptor agonist were given before, during, and after application of 8Br-cAMP so that the experiments had internal control pulses (Fig. 4). The pulses with baclofen resulted in 65 ± 5%, 12 ± 6%, and 52 ± 7% inhibitions, respectively. The inhibition induced by baclofen in combination with 8Br-cAMP (second pulse) was significantly attenuated compared to the control pulses with baclofen alone. The inhibitions induced by the three pulses of isoguvacine were 46 ± 5%, 47 ± 6% and 40 ± 7%, respectively. The inhibition induced by isoguvacine in combination with 8Br-cAMP (second pulse) was not significantly different from the control pulses. The transitory stimulation of secretion at the beginning of isoguvacine treatment was again evident (Fig. 4b).

View larger version (28K): [in this window] [in a new window]   Figure 4. Effects of 10-6 M baclofen (a) or 10-5 M isoguvacine (b) alone or in the presence of 1 mM 8Br-cAMP on the secretion of radiolabel from cultured cells. Basal release was determined as the average radiolabel in the three fractions before the first pulse of GABA receptor agonist and was set at 100%; these values were 567, 890, 1343, and 760 cpm for the cell preparations in a and 788, 1192, 1098, and 837 cpm for the cell preparations used in b. Those fractions in which 8Br-cAMP and GABA receptor agonists were administered are indicated by the horizontal bars/dotted lines and shaded bars, respectively.

View larger version (23K): [in this window] [in a new window]   Figure 5. Effects of 10-5 M sauvagine on secretion from radiolabeled melanotropes during inhibition by 10-6 M baclofen (a) and 10-5 M isoguvacine (b). Basal release was calculated from the average radiolabel in the three fractions preceding the first pulse of receptor agonist and was set at 100%. These values were 998, 847, 643, and 1098 cpm for the experiment shown in a and 1234, 992, 998, and 776 for the experiment shown in b. Fractions in which sauvagine and GABA agonists were administered are indicated by horizontal bars/dotted lines and shaded bars, respectively.

View larger version (33K): [in this window] [in a new window]   Figure 6. The effect of high [K+] (a), baclofen (b), and isoguvacine (c) on the fluorescence intensity of cells superfused in the presence of the membrane potential probe bis-oxonol. In each case, profiles from four cells are shown. The shaded bars indicate fractions in which pulses with high K+ or GABA receptor agonists were administered, and the concentrations of K+, baclofen, and isoguvacine are indicated (the effects of a single pulse of 10-5 M isoguvacine is shown in b, and those of a single pulse of 10-6 M baclofen are shown in c. The osmolarity of the superfusion medium during pulses with high K+ was kept constant by adjusting the NaCl concentration.

Isoguvacine caused an increase in fluorescence intensity in all cells studied (n = 37 in 4 cell preparations); the response appeared to be all or nothing, rather than showing clear dose-dependent characteristics. Profiles for 4 isoguvacine-treated cells are shown in Fig. 6c. Each experiment with isoguvacine was terminated with a single pulse of baclofen at 10^-6 M; this pulse induced a decrease in fluorescence in all cells studied (e.g. Fig. 6c).

Low concentrations of GABA caused a decrease in cell fluorescence, whereas high concentrations caused an increase in cell fluorescence. Examples of this behavior are shown for 4 cells in an experiment in which 11 cells were recorded (Fig. 7a). All 11 cells showed a decrease in fluorescence at 10^-8 M GABA. At 10^-6 M GABA, 1 of the 11 cells still showed a decrease in fluorescence (top profile, Fig. 7a). The rest of the cells showed an increase in fluorescence at this GABA concentration, which was particularly strong and long lasting for 1 of the cells (bottom profile); the remaining 9 cells showed responses that were very similar, 2 example profiles of which are presented in Fig. 7a (2 middle profiles). At 10^-4 M GABA, all cells showed an increase in fluorescence. The average change in fluorescence intensity at various GABA concentrations for 56 cells from 8 cell preparations is shown in Fig. 7b. Although the GABA concentration at which the switch was made between a cell responding with a fluorescence intensity decrease to an increase varied from cell to cell, even within the same cell preparation (e.g. Fig. 7a), usually at 10^-6 M GABA most cells in a given preparation had made the switch from a hyperpolarizing to a depolarizing response (Fig. 7b).

View larger version (29K): [in this window] [in a new window]   Figure 7. Effects of GABA on the fluorescence intensity of cells superfused in the presence of bis-oxonol. In a, the profiles for 4 cells from a single experiment are shown. Shaded bars indicate where pulses of GABA were given, and the concentrations of GABA are indicated. In b, average changes in the fluorescence intensity of membrane potential probe induced by GABA are shown. Data were averaged from 8 experiments (±SEM) involving recordings from 56 cells.

View larger version (40K): [in this window] [in a new window]   Figure 8. Effects of GABA on secretion of radiolabel from cultured cells. The results shown are from a single cell preparation. The experiment was repeated four times with similar results. Shaded bars indicate when pulses of GABA were administered, and the concentrations of GABA are indicated.

	Discussion

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Previous studies have established that activation of adenylyl cyclase stimulates MSH secretion from melanotrope cells of Xenopus laevis and that inhibition of the production of cAMP inhibits this secretion (28). We now show differential effects of the inhibitory GABAergic receptor mechanisms on the stimulation induced by 1) the cell-permeable analog 8Br-cAMP; 2) treatment with IBMX, which inhibits phosphodiesterase activity and is known to increase intracellular cAMP levels in Xenopus melanotropes (16, 17); and 3) treatment with sauvagine, a peptide that stimulates adenylyl cyclase in Xenopus melanotropes (15, 16). The results show that GABA_B receptor inhibition can be largely overcome by cAMP or treatments known to increase intracellular cAMP levels, whereas the inhibition induced by the GABA_A receptor can be only partially overcome by such treatments. The fact that the differential actions were seen in experiments conducted with isolated cultured melanotropes establishes that these actions reflect actions of the melanotrope cells themselves rather than complex presynaptic mechanisms or the involvement of other cell types, such as folliculo-stellate cells, which are known to be present in intermediate lobe tissue (29). The observation that secretion of immunoreactive MSH from freshly dissected neurointermediate lobe tissue displayed the same behavior, with respect to the differential actions of the GABAergic receptor mechanisms, argues that this is a physiological phenomenon rather than an artifact of isolation of the melanotrope from the intact tissue and culture in vitro.

The mechanism underlying the differential actions of GABA_A and GABA_B receptors remains to be fully elucidated. Previous studies have shown that Xenopus melanotrope cells display spontaneous Ca²⁺ oscillations, which are initiated through the action of an N-type Ca²⁺ channel (20). Factors that stimulate secretion increase the frequency of the Ca²⁺ oscillations, whereas factors inhibiting secretion abolish the oscillations (18, 21). These observations have given rise to the idea that neurotransmitters control the rate of secretion through regulation, directly or indirectly, of the activity of N-type Ca²⁺ channels. The fact that 8Br-cAMP increases oscillation frequency, and GABA receptor activation decreases this frequency is in full agreement with the above concept. The GABA_B receptor is a G protein-coupled receptor that acts by inhibiting adenylyl cyclase activity and/or activating K⁺ channels (30). As the inhibition of Ca²⁺ oscillations via the GABA_B receptor can be largely overcome by cAMP treatment, it seems that in the Xenopus melanotrope, the GABA_B receptor mainly exerts its function by inhibiting adenylyl cyclase activity. It has been reported that the rat GABA_B receptor can be desensitized by a cAMP-dependent protein kinase (31). Such desensitization could be an alternative explanation for our observations.

Concerning the mechanism of the GABA_A receptor, this receptor is a member of a ligand-gated Cl^- ion channel superfamily in which subunit composition determines the main conductance state of the channel. The direction of the ion flow depends on the electrochemical driving force, which is determined by the resting membrane potential and the Cl^- gradient. In the Xenopus melanotrope, the initial Ca²⁺ spike seen upon GABA_A receptor activation probably reflects an outward flux of Cl^- ions leading to activation of the N-type Ca²⁺ channel (short term regulation), and the maintained depolarized state subsequently inactivates the Ca²⁺ channel (30) (long term regulation). The fact that the single Ca²⁺ spike occurred simultaneously in all cells is reflected in the secretory data showing a short increase in the release of radiolabeled peptides before secretion becomes inhibited (biphasic effect). This spike of secretion was not visible in the secretory studies with intact neurointermediate lobes, probably because the time resolution in these studies was less than that in the studies with cultured cells. There are indication that some members of the GABA_A receptor family can be desensitized by a cAMP-dependent mechanism (32, 33, 34). This does not appear to be the case for the GABA_A receptor of Xenopus, at least in the short term, because this receptor is able to exert its full inhibitory action in the presence of 8Br-cAMP.

Having established differential effects of GABA_A and GABA_B receptors on Ca²⁺ dynamics and peptide secretion from melanotrope cells of Xenopus laevis, we next considered possible differential activation of these two receptor types. The observation that GABA_A receptor activation can cause opening of voltage-operated Ca²⁺ channels indicates that activation of this receptor can, at least in vitro, depolarize the melanotrope plasma membrane, a phenomenon also observed in other cells (35, 36, 37, 38, 39). Therefore, we examined the use of a membrane potential probe of the bis-oxonol family to differentiate between GABA_A and GABA_B receptor activation. Indeed, GABA_A receptor activation gave a depolarizing response (uptake of probe), whereas GABA_B receptor activation gave a hyperpolarizing response (loss of probe), which established the probe as a useful tool to assess the relative contribution of each receptor type when the endogenous ligand GABA was administered to the cells. The results revealed that GABA differentially activates the two receptors types on the Xenopus melanotrope; low concentrations of GABA (10^-10-10^-7 M) caused decreased fluorescence, indicative of GABA_B receptor action, whereas only at high GABA concentrations (usually >10^-7 M) were depolarizing responses indicative of GABA_A receptor activation observed. Although presumably both GABA_A and GABA_B receptors are activated at high GABA concentrations, the results indicate that the GABA_A receptor can completely dominate or override GABA_B receptor effects on membrane potential. At the level of secretion, low concentrations of GABA produced only inhibition, indicating the exclusive involvement of GABA_B receptors. The transient stimulation of secretion seen at higher GABA concentrations most likely reflects the action of the GABA_A receptor on Ca²⁺ channels noted earlier. The inhibition of secretion at high GABA concentrations could be through either GABA_A or GABA_B receptor action, although from the results with the membrane potential probe, we suggest that the GABA_A receptor plays a dominant role.

There are a number of cell types that possess both GABA_A and GABA_B receptors (40, 41, 42, 43). In general, it would seem that GABA_A receptor mechanisms are more sensitive than GABA_B receptors mechanisms to activation by GABA (44, 45). Interestingly, in our model the metabotropic mechanism seems to be more sensitive than the ionotropic mechanism to activation by GABA. There are two major factors determining cell sensitivity to a ligand: the number of receptors and the affinity of the receptors for the ligand. Although the underlying mechanism for the differences in sensitivity of the Xenopus melanotrope cell to the two GABA receptor types remains to be determined, it may be that there are relatively more GABA_B receptors than GABA_A receptors on the membrane. Our single cell analysis indicates that every melanotrope cell possesses GABA_A as well as GABA_B receptors, but there may be some heterogeneity among the melanotrope cells in the relative number of the two GABA receptor types. This was reflected in the differences among individual cells in the GABA concentration that was capable of inducing a switch from the GABA_B to the GABA_A receptor response.

In conclusion, we show 1) that the GABA_A and GABA_B receptors differentially affect the sensitivity of the Xenopus melanotrope cell to stimulation by cAMP-dependent signal transduction mechanisms, and 2) that these two receptor types can be differentially activated, depending on the concentration of GABA (Fig. 9). GABA regulates the secretory activity of the melanotrope cell of Xenopus through release from nerve terminals that make synaptic contact with the melanotropes. Our results indicate that with low levels of GABA release from these terminals, the cells would be inhibited preferentially via the GABA_B receptor. Under these circumstances, the cell would be sensitive to neurotransmitters that stimulate adenylyl cyclase, such as sauvagine. As the rate of GABA release increases, however, the GABA_A receptor would play an increasingly important role in the inhibition exerted by GABA, making the cells progressively less responsive to neurotransmitters that stimulate adenylyl cyclase.

View larger version (30K): [in this window] [in a new window]   Figure 9. Model for the differential action of GABA on the melanotrope cell of Xenopus laevis. Low concentrations of GABA inhibit primarily through the GABAB receptor. Under these circumstances, the inhibition can be largely overcome by receptors activating adenylyl cyclase, such as those activated by the peptide neurotransmitter sauvagine. At high GABA concentrations, the GABAA receptor is activated. Under these circumstances, receptors activating adenylyl cyclase are only partially effective in overcoming the inhibited state of the cell.

	Acknowledgments

 
The authors thank Peter Cruijsen and Alvaro Quero Pena for technical assistance, and Ron Engels for the animal care.

	Footnotes

 
¹ This work was supported by a Nederlandse organisatie voor Wetenschappelijk Onderzoek/Medische Wetenschappen-INSERM exchange grant, a grant from the European Community (EU HCM, ERBCHRXCT920017), and the Erasmus Program of the European Community.

Received June 10, 1996.

	References

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