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Endocrinology Vol. 140, No. 1 159-164
Copyright © 1999 by The Endocrine Society

ARTICLES

Differential Effects of Dopamine on Two Frog Melanotrope Cell Subpopulations1

José L. González de Aguilar, María M. Malagón, Rafael M. Vázquez-Martínez, Antonio J. Martínez-Fuentes, Marie Christine Tonon, Hubert Vaudry and Francisco Gracia-Navarro

Department of Cell Biology, University of Córdoba (J.L.G.d.A., M.M.M., R.M.V.M., A.J.M.F., F.G.N.), 14004-Córdoba, Spain; and European Institute for Peptide Research, Institut Federatif de Recherches Multidisciplinaires sur les Peptides no. 23, Laboratory of Cellular and Molecular Neuroendocrinology (Institut National de la Santé et de la Recherche Médicale U413), Unité Affiliée au Centre National de la Recherche Scientifique (UA CNRS), University of Rouen (M.C.T., H.V.), 76821-Mont-Saint-Aignan, France

Address all correspondence and requests for reprints to: Dr. Francisco Gracia-Navarro, Department of Cell Biology, Avda. San Alberto Magno s/n, University of Córdoba, E-14004 Córdoba, Spain. E-mail: bc1grnaf{at}uco.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The frog intermediate lobe consists of a single endocrine cell type, the melanotrope cells, which are under the tonic inhibitory control of dopamine. Separation of dispersed pars intermedia cells in a Percoll density gradient has revealed the existence of two melanotrope cell subpopulations, referred to as high-density (HD) and low-density (LD) cells. The aim of the present study was to investigate the effects of dopamine on each of these melanotrope cell subsets. Increasing doses of dopamine, ranging from 10-9–10-6 M, inhibited the release of {alpha}-melanocyte-stimulating hormone ({alpha}-MSH) in LD (but not in HD) melanotrope cells. In addition, dopamine provoked a significant reduction of the rate of acetylation of {alpha}-MSH in LD cells but not in HD cells. Similarly, dopamine significantly decreased the accumulation of POMC messenger RNA in LD cells, whereas it did not affect POMC gene expression in the HD melanotrope subset. On the other hand, microfluorimetric studies revealed that dopamine induced a significant reduction of KCl-stimulated cytosolic free calcium concentration in both LD and HD cells. The present study provides additional evidence for functional heterogeneity of melanotrope cells in the frog pars intermedia. Because dopamine plays a pivotal role in the regulation of {alpha}-MSH secretion, these data suggest the involvement of cell heterogeneity in the physiological process of background color adaptation in amphibians.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE pars intermedia of the pituitary is a relatively simple endocrine gland that is well suited to investigation of the functional significance of cell heterogeneity. In both mammals and amphibians, the intermediate lobe is mainly composed of a unique endocrine cell type, the melanotrope cells (1, 2). Posttranslational processing of POMC in melanotrope cells generates several peptide hormones, including {alpha}-melanocyte-stimulating hormone ({alpha}-MSH) and ß-endorphin (3, 4). In amphibians, {alpha}-MSH causes dispersion of the pigment melanin in dermal melanophores and, thus, plays a pivotal role in the process of background color adaptation (5). In frog and toad, the storage form of {alpha}-MSH is des-N{alpha}-acetyl {alpha}-MSH; and acetylation reaction, which confers the melanotropic activity to the peptide (6), occurs during exocytosis (7, 8). Consequently, neuroendocrine factors that control the release of {alpha}-MSH may also regulate the N{alpha}-acetylation and, therefore, the biological potency of the peptide (9, 10, 11).

The activity of the amphibian pars intermedia is regulated by a number of aminergic and peptidergic neuroendocrine factors (12, 13). In particular, dopamine is a potent inhibitor of frog (14) and toad melanotrope cells (15). In the frog Rana ridibunda, dopamine (acting through a D2 receptor subtype) inhibits both the metabolic (16) and electrical activity (17) of melanotrope cells. Interestingly, in anuran amphibians, dopamine causes concomitant inhibition of the acetylation and release of {alpha}-MSH-related peptides (9, 10).

Previous studies have shown the existence of (sub)populations of PRL cells, with respect to their responses to dopamine. Boockfor and Frawley (18) have reported that PRL cells, located in different regions of the anterior pituitary of lactating rats, exhibit distinct responses to dopamine. Separation of PRL cells, by density gradient centrifugation, has revealed the occurrence of subpopulations of PRL cells exhibiting differential electrophysiological responses to dopamine (19). In addition, dopamine exerts differential effects on PRL gene expression in the various subsets of PRL cells (20).

In the intermediate lobe, cell heterogeneity also has been reported. Early studies by Chronwall et al. (21) revealed a heterogeneous distribution of POMC messenger RNA (mRNA) in the rat intermediate lobe. Similarly, {alpha}-MSH secretory activity (22) and dopamine D2 receptor isoform expression (23) have been also shown to be heterogeneous among rat melanotrope cells. In line with these findings, we have recently isolated two subpopulations of melanotrope cells from the intermediate lobe of the frog Rana ridibunda by centrifugation of dispersed cells in a Percoll density gradient (24). Functional studies have shown that the low-density (LD) cells possess a higher secretory activity, acetylation rate, cytosolic free calcium concentration ([Ca2+]i), and POMC mRNA content than the high-density (HD) cells (25). In addition, the LD melanotrope cells exhibit stronger responses to TRH (25), a major stimulator of {alpha}-MSH secretion in amphibians (26, 27).

The aim of the present study was to investigate the effects of dopamine on the secretory activity, the acetylation rate, the [Ca2+]i, and the POMC mRNA content in LD and HD cells from the frog pars intermedia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male frogs (Rana ridibunda) of about 40 g BW (Couétard, Saint-Hilaire de Riez, France) were maintained under running water at 8 C on a 12-h light, 12-h dark regimen for at least 1 week before death. The animals were killed by decapitation, and the neurointermediate lobes were carefully dissected under a microscope. All animal manipulations were performed according to the recommendations of the local ethical committees and under the supervision of authorized investigators.

Preparation of melanotrope cell subpopulations
A homogeneous cellular suspension was prepared by enzymatic treatment, followed by mechanical dissociation with a flame-polished Pasteur pipette, as previously reported (24). Dispersed cells were suspended in Leibovitz culture medium (Sigma Chemical Co., St. Louis, MO), diluted 2:3 (to adjust to Rana ridibunda osmolality), and supplemented with 1 mM glucose, 0.4 mM CaCl2, and 1% (vol/vol) antibiotic-antimycotic solution (Sigma Chemical Co.), pH 7.4. Cell viability, as estimated by the Trypan blue exclusion test (28), was 88%.

Separation of melanotrope cell subpopulations was performed as previously described (24). Briefly, dispersed cells (2.2 x 106 cells in 250 µl culture medium) were layered on a 9-ml density gradient of Percoll (Pharmacia LKB, Uppsala, Sweden). The density distribution of the gradient spanned hyperbolically from 1.027–1.072 g/ml. After centrifugation (3000 x g for 25 min, 4 C), 9 fractions, of 1 ml each, were collected by hand. Two melanotrope cell subpopulations were selected and referred to as HD (fraction 1) and LD (fractions 4 and 5) cells, respectively (24). HD and LD melanotrope cells represented, respectively, 20.8% and 52.1% of total cells recovered from the gradient.

Culture of melanotrope cell subpopulations
After separation, melanotrope cells from the HD and LD subpopulations were plated at a density of 30,000 cells/well in 35-mm Petri dishes and incubated at 26 C in 2 ml culture medium supplemented with 10% FBS (Sera-Lab Ltd., Crawley Down, UK). After 48 h, the medium was removed, and cells were preincubated in 1 ml serum-free culture medium for 2 h. Then, cells were incubated for another 2 h with 1 ml culture medium, in the absence or presence of increasing doses of dopamine (Sigma Chemical Co.) ranging from 10-9 to 10-6 M. Medium samples were collected and centrifuged at 60 x g for 5 min, and supernatants were stored at -20 C until hormone assay. After culture, cells were processed for in situ hybridization. To analyze the effect of dopamine on [Ca2+]i, cells were plated on microgrid coverslips (Eppendorf, Netheler, Germany) at a density of 5,000 cells/well and incubated in 2 ml culture medium supplemented with 10% FBS for 3–5 days.

HPLC
The amounts of nonacetylated and acetylated forms of {alpha}-MSH released by HD and LD melanotrope cells were determined by reversed-phase HPLC analysis combined with RIA detection. Incubation media were prepurified on Sep-Pak cartridges (Alltech Europe, Laarne, Belgium) and subjected to HPLC analysis on a Lichrosorb RP-18 column (0.46 x 25 cm; Merck, Paris, France). The mobile phase consisted of a gradient established with 0.1% trifluoroacetic acid (vol/vol, pH 2.4) and a mixture of acetonitrile/methanol (80:20, vol/vol). The column was equilibrated with 25% acetonitrile/methanol and eluted at a flow rate of 1 ml/min using the gradient shown (see Fig. 2Go). The following synthetic standards were analyzed using the same gradient: des-N{alpha}-acetyl {alpha}-MSH, {alpha}-MSH and diacetyl {alpha}-MSH, and their respective sulfoxide derivatives. The oxidized forms of the peptides were obtained by treating the synthetic standards with hydrogen peroxide, as previously described (29). Fractions were collected every 1 min and dried by vacuum centrifugation until hormone assay.



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  		Figure 2. Reversed-phase HPLC analysis of {alpha}-MSH immunoreactive peptides released by each cultured frog melanotrope cell subpopulation. The culture media from HD cells (A and C) and LD cells (B and D), cultured in the absence (A and B) or in the presence (C and D) of 10-6 M dopamine, were prepurified on Sep-Pak C-18 cartridges and chromatographed on a Lichrosorb RP-18 column. One-milliliter fractions were collected and assayed for {alpha}-MSH-like immunoreactivity ({alpha}-MSH-LI). The arrows indicate the elution time of the synthetic standards. The dotted line on graph D shows the concentration of acetonitrile/methanol (80/20) in the eluting solvent. Sdes{alpha} (I), Sulfoxide derivative of des-N{alpha}-acetyl {alpha}-MSH; S{alpha} (II), sulfoxide derivative of {alpha}-MSH; Sdi{alpha} (III), sulfoxide derivative of diacetyl {alpha}-MSH; des{alpha} (IV), des-N{alpha}-acetyl {alpha}-MSH; {alpha} (V), {alpha}-MSH; di{alpha} (VI), diacetyl {alpha}-MSH.

 
{alpha}-MSH RIA
{alpha}-MSH concentrations in the culture media and HPLC fractions were measured using a double-antibody RIA method (30). The antiserum exhibited full cross-reactivity with des-N{alpha}-acetyl {alpha}-MSH, {alpha}-MSH and diacetyl {alpha}-MSH, and their sulfoxide derivatives. Cross-reactivity with other POMC-derived peptides, including ACTH and ß-LPH, was lower than 0.1%. The detection limit of the assay was 3 pg/tube, and the intra- and interassay coefficients of variation were 5% and 13%, respectively.

In situ hybridization of POMC mRNA
The in situ hybridization procedure was performed as previously described (25). The probe was the EcoRI 1184-bp insert of frog POMC complementary DNA subcloned into pGEM-3Zf (31), which was digoxigenin-labeled by random priming using a Digoxigenin DNA labeling kit (Boehringer Mannheim, Mannheim, Germany). Briefly, after removal of the culture medium, cells were rinsed with 0.01 M PBS (pH 7.2) and were fixed in the Petri dishes with 4% (wt/vol) paraformaldehyde for 15 min at room temperature. Thereafter, cells were sequentially passed through 1% (vol/vol) Triton X-100, 5 µg/ml proteinase K (Boehringer Mannheim) and were postfixed in 4% paraformaldehyde before addition of the hybridization mixture. Hybridization solution [50% (vol/vol) deionized formamide (Sigma Chemical Co.), 5 x SSPE (0.75 M NaCl, 0.05 M NaH2PO4·H2O, 5 mM EDTA, pH 7.4), 4% (vol/vol) dextran sulfate (Sigma Chemical Co.), 5 x Denhardt’s solution (100 mg Ficoll type 400; Pharmacia LKB), 100 mg polyvinylpyrrolidone (Sigma Chemical Co.), 100 mg BSA (Sigma Chemical Co.) in 100 ml H2O), 0.1% (vol/vol) SDS (pH 7.2), 200 µg/ml yeast transfer RNA (Boehringer Mannheim), 250 µg/ml heat-denatured salmon sperm DNA (Sigma Chemical Co.), and 2 µg/ml poly-A (Sigma Chemical Co.)], containing the digoxigenin-labeled probe at 35 ng/200 µl, was placed in the Petri dishes. After overnight hybridization in a humid chamber at 37 C, cells were subsequently rinsed with 2 x saline-sodium citrate (SSC; 0.3 M NaCl, 0.03 M sodium citrate, pH 7.4), 1 x SSC, and 0.5 x SSC. Then, cells were washed in buffer 1 (100 mM Tris-HCl, 150 mM NaCl, pH 7.5) and sequentially incubated with 10 mM levamisole (Sigma Chemical Co.) in buffer 2 (100 mM Tris-HCl, 150 mM NaCl, 50 mM MgCl2, pH 9.5), with 1% (wt/vol) blocking reagent (Boehringer Mannheim), and with the alkaline phosphatase-labeled antidigoxigenin F(ab) fragment (Boehringer Mannheim). Cell-bound alkaline phosphatase activity was visualized by incubating the cells with the color solution [3.5 µl/ml 5-bromo-4-chloro-3-indolyl phosphate (Boehringer Mannheim), 4.5 µl/ml nitroblue tetrazolium salt (Boehringer Mannheim), 0.24 mg/ml levamisole in buffer 2]. Finally, Petri dishes were mounted in buffer 1 plus glycerol (1:1).

POMC mRNA quantification was accomplished on a Pentium computer equipped with digitizer boards and the software package for Image Analysis Visilog (V. 4.1; Noesis, France) connected by a CCTV camera (Sony, Tokyo, Japan) to an Universal microscope (Zeiss, Oberkochen, Germany). A 40 x objective and a stabilized light source were used. Before measuring each set of cells, Köhler alignment of the light path was carried out to ensure homogeneous illumination. The staining intensity (OD) and the area of each individual cell (30 cells randomly selected per dish) were measured to calculate the integrated OD. This parameter was correlated with the amount of POMC mRNA into the cell. Background was evaluated on nonstained cells (5 cells per dish) and subtracted. To avoid within-experiment variations, control and treated cells were hybridized and measured together on the same set.

Measurement of cytosolic free calcium concentration
Cultured cells were incubated with 5 µM indo-1 acetoxymethylester and 0.02% (wt/vol) Pluronic F127 (both from Molecular Probes, Inc., Eugene, OR) in culture medium for 30 min at 22 C in the dark. The cells were washed twice with fresh medium, and [Ca2+]i was monitored by a dual-wavelength microfluorimetry technique, as previously described (32). The microfluorimeter was constructed from an inverted microscope equipped for epifluorescence with a fluor 40 x objective (Nikon Corporation, Tokyo, Japan). The fluorescence emission of indo-1, induced by excitation at 355 nm, was recorded at two wavelengths (405 nm and 485 nm) by separate photometers. After conversion of photon currents to voltage signals, both 405-nm and 485-nm signals, as well as the 405/485 ratio (R), were simultaneously recorded by a software FASTINCA 1.03 (Nikon). R was calibrated to express [Ca2+]i using the formula established by Grynkiewicz et al. (33): [Ca2+]i = Kd x ß x [(R - Rmin)/(Rmax - R)]. Rmin is the 405/485 ratio obtained after incubation of cells with 2 mM EGTA and 10 µM A-23187 (Molecular Probes, Inc.) for 30 min at 22 C in the dark (Ca2+-free conditions). Rmax is the 405/485 ratio obtained after incubation of cells with 2 mM CaCl2 and 10 µM A-23187 for 30 min at 22 C in the dark (Ca2+-saturated conditions). ß is the fluorescence ratio between the signal at 485 nm in Ca2+-free medium and the signal at 485 nm in Ca2+-saturated medium. The averaged values of Rmin, Rmax, and ß were 0.102 ± 0.001, 0.713 ± 0.018, and 3.677 ± 0.246 (n = 90), respectively. The dissociation constant (Kd) of indo-1 was 250 nM. To determine the effect of dopamine on [Ca2+]i, cells were prestimulated with a depolarizing pulse of KCl (56 mM; 2 sec) before administration of a 100-fold concentrated solution of dopamine for 10 sec. All substances were administered in the vicinity of the cells by a pressure ejection system.

Statistical analysis
Data are expressed as mean ± SEM. Differences between groups were statistically analyzed either by Student’s t test, or by two-way ANOVA followed by the post hoc Duncan’s test. Statistical analyzes were carried out with the program Statistica for Windows (Statsoft, Inc., Tulsa, OK). Differences were considered to be significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The rate of secretion of {alpha}-MSH-related peptides was 2.4-fold higher in LD cells than in HD cells (P < 0.05; Fig. 1Go). Incubation of the cells with increasing doses of dopamine (10-9–10-6 M) caused a significant decrease of {alpha}-MSH release in LD cells. In contrast, none of the assayed doses of dopamine affected {alpha}-MSH release from HD cells (Fig. 1Go).



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  		Figure 1. Effect of dopamine on {alpha}-MSH release from each cultured frog melanotrope cell subpopulation. HD (solid bars) and LD cells (hatched bars) were incubated in the absence (C) or in the presence of increasing doses of dopamine for 2 h. The data represent the mean (± SEM) of, at least, seven independent experiments. *, P < 0.05; **, P < 0.01 (vs. corresponding control). #, P < 0.05 (vs. control HD cells; all determined by ANOVA followed by the post hoc Duncan’s test).

 
HPLC analysis of {alpha}-MSH-related peptides, combined with RIA detection, revealed the occurrence of six immunoreactive forms of the peptide in culture media from LD and HD cells (Fig. 2Go). The retention times of these {alpha}-MSH-immunoreactive peptides corresponded exactly to those of the sulfoxide derivatives of des-N{alpha}-acetyl {alpha}-MSH (24 min), {alpha}-MSH (28 min), and diacetyl {alpha}-MSH (33 min), and to those of des-N{alpha}-acetyl {alpha}-MSH (35 min), {alpha}-MSH (43 min), and diacetyl {alpha}-MSH (54 min). In HD cells, the amount of acetylated peptides released in basal conditions was slightly lower than that of nonacetylated peptides (Fig. 3Go). Conversely, in LD cells, the amount of acetylated peptides released spontaneously was significantly higher than that of nonacetylated peptides (P < 0.01; Fig. 3Go). To determine the effect of dopamine on the acetylation rate, we used the most effective dose to inhibit {alpha}-MSH release, i.e. 10-6 M (see Fig. 1Go). Incubation of HD cells with dopamine did not produce any significant change on the absolute amount and the proportions of nonacetylated and acetylated peptides (Fig. 2Go, A and C; Fig. 3Go). Conversely, dopamine caused a marked reduction of the absolute amounts of all {alpha}-MSH-immunoreactive peptides released by LD cells (Fig. 2Go, B and D). Moreover, in LD cells, dopamine provoked a more pronounced inhibition of the secretion of acetylated forms (65.8%) than nonacetylated forms of {alpha}-MSH (53.3%) (Fig. 3Go). In particular, in LD cells, dopamine inhibited by 77.7% the amount of diacetyl {alpha}-MSH and its sulfoxide derivative.



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  		Figure 3. Comparison of the concentration of nonacetylated and acetylated forms of {alpha}-MSH released by each cultured frog melanotrope cell subpopulation. HD (solid bars) and LD cells (hatched bars) were incubated in the absence (C) or in the presence of 10-6 M dopamine (DA) for 2 h. The culture medium was analyzed by HPLC, and the {alpha}-MSH-immunoreactive peptides were quantified by RIA. The nonacetylated form (DES) corresponds to des-N{alpha}-acetyl {alpha}-MSH and its sulfoxide derivative. The acetylated form (AC) corresponds to {alpha}-MSH and its sulfoxide derivative, as well as diacetyl {alpha}-MSH and its sulfoxide derivative. The data represent the mean (± SEM) of three independent experiments. *, P < 0.001 (vs. corresponding control). #, P < 0.01 (vs. corresponding DES; all determined by ANOVA, followed by the post hoc Duncan’s test).

 
Quantitative in situ hybridization of POMC mRNA showed that, in basal conditions, the mRNA level was significantly lower (P < 0.05) in HD cells than in LD cells (Fig. 4Go). Incubation of both melanotrope cell subpopulations with increasing doses of dopamine (10-9–10-6 M) significantly inhibited the mean POMC mRNA levels in LD cells, whereas it had no effect in HD cells (Fig. 4Go).



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  		Figure 4. Quantification of POMC mRNA in each cultured frog melanotrope cell subpopulation. The figure shows the POMC mRNA content in HD (solid bars) and LD cells (hatched bars) in the absence (C) or in the presence of increasing doses of dopamine for 2 h. The data represent the mean (± SEM) of, at least, three independent experiments and are expressed in arbitrary units (a.u.) *, P < 0.05; **, P < 0.001 (vs. corresponding control). #, P < 0.05 (vs. control HD cells; all determined by ANOVA, followed by the post hoc Duncan’s test).

 
In resting conditions, the [Ca2+]i was significantly lower (P < 0.05) in HD cells than in LD cells (Fig. 5Go; Table 1Go). Ejection of a short pulse of dopamine (10-4 M; 10 sec) in the vicinity of the cells did not affect the basal [Ca2+]i in either of the cell subpopulations (data not shown). Thus, the effect of dopamine on [Ca2+]i was assayed after prestimulation with a depolarizing pulse of KCl (56 mM; 2 sec). Exposure of the cells to KCl induced an immediate and transient increase in [Ca2+]i in both HD and LD cells (Fig. 5Go). The amplitude of the [Ca2+]i response to a KCl pulse was significantly lower (P < 0.05) in HD cells than in LD cells (Table 1Go). Application of dopamine during the KCl-evoked stimulation of [Ca2+]i provoked an abrupt and transient decrease of the [Ca2+]i response in both cell subpopulations (Fig. 5Go). Quantitative analysis revealed that dopamine could totally suppress the [Ca2+]i response induced by KCl in HD and LD cells (Table 1Go).



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  		Figure 5. Effect of dopamine on [Ca2+]i in cultured frog melanotrope cells from each subpopulation. The figures represent typical recordings from an HD cell (A) and a LD cell (B). The arrows indicate the onset of KCl (56 mM; 2 sec) or dopamine (DA; 10-4 M; 10 sec) application.

 

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  		Table 1. Quantitative analysis of the effect of dopamine on [Ca2+]i in HD and LD melanotrope cells

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Separation of dispersed frog pars intermedia cells in a Percoll density gradient has recently revealed the existence of two melanotrope cell subpopulations (24). The present study now demonstrates that each frog melanotrope cell subset exhibits distinct physiological responses to dopamine.

Dopamine induced a marked inhibition of {alpha}-MSH secretion in cultured LD cells, whatever the doses tested. Conversely, {alpha}-MSH secretion from HD cells was not affected, even by dopamine at a concentration of 10-6 M. Inasmuch as LD cells are more abundant and secrete more {alpha}-MSH than HD cells, the present data are compatible with the global inhibitory effect of dopamine on whole amphibian pars intermedia (14, 34). Moreover, because the spontaneous secretory activity is much higher in LD cells than in HD cells (24, and this study), our results indicate that dopamine inhibits selectively hormone release from melanotrope cells displaying an intense secretory activity. Interestingly, in the rat intermediate lobe, two types of melanotrope cells with distinct ultrastructural characteristics (21, 35) have been shown to be differentially affected by in vivo treatment with dopaminergic drugs. Specifically, administration of the dopamine agonist bromocriptine reduced the number of melanotrope cells displaying ultrastructural features of biosynthetically active cells (35). In line with these findings, it has been also demonstrated that, in the lactotrope cell population, dopamine inhibits preferentially the release of PRL from lactotrope cells with high secretory activity (36, 37).

Acetylation of the N-terminal serine residue markedly increases the melanotropic activity of {alpha}-MSH (6). Because, in amphibians, melanotrope cells release both acetylated and nonacetylated forms of {alpha}-MSH (7, 8), neuroendocrine factors regulating melanotrope cell activity may control simultaneously the quantity of {alpha}-MSH-related peptides and the proportion of the acetylated forms of {alpha}-MSH (9). The present study has demonstrated that dopamine does not affect the secretion of {alpha}-MSH-immunoreactive peptides from HD cells but reduces the release of all molecular forms of {alpha}-MSH in LD cells. In the latter cell subpopulation, dopamine caused a more pronounced inhibition of the secretion of acetylated forms. It thus seems that, in LD cells, dopamine reduces the global release of {alpha}-MSH-related peptides and inhibits predominantly the secretion of the biologically active acetylated form. These data suggest that, in amphibians, dopamine-induced inhibition of LD cells must play a crucial role during skin color adaptation from a dark to a white or pale environment. In support of this hypothesis, it has been reported that dopamine reduces the ratio of acetylated vs. nonacetylated forms of {alpha}-MSH released from black background-adapted toads but had virtually no effect on the proportion of acetylated peptides released from white background adapted animals (10).

Previous reports have shown that dopamine D2 receptor agonists decrease POMC biosynthesis and POMC mRNA accumulation in the intermediate lobe of mammals (21, 38, 39) and amphibians (40, 41, 42). The present study has shown that dopamine did not affect the mean POMC mRNA levels in HD cells but significantly reduced the amount of transcripts in LD cells in a dose-dependent manner very similar to that found in hormone release. As a consequence, POMC mRNA levels in LD cells, after dopamine treatment, resembled those observed in HD cells, thus reducing the differences in transcript content found between HD and LD cells under control conditions. Accordingly, in vivo bromocriptine administration has been shown to diminish the heterogeneity in POMC mRNA content in rat melanotrope cells (21). Taken together, our findings add compelling evidence for functional heterogeneity of frog melanotrope cells in response to dopamine, and they strongly support the existence of melanotrope cells that are unresponsive to the negative control exerted by dopamine in the amphibian pars intermedia.

In the toad Xenopus laevis, melanotrope cells display spontaneous Ca2+ oscillations (43), which are abrogated by dopamine (44). Conversely, in the frog Rana ridibunda, dopamine has no effect on the [Ca2+]i baseline (16). Electrophysiological studies have previously shown that, in frog melanotrope cells, dopamine blocks voltage-dependent L- and N-like calcium currents (17, 45). In agreement with this finding, we now show that dopamine occludes the [Ca2+]i response evoked by a depolarizing pulse of KCl. The ability of dopamine to inhibit the KCl-induced [Ca2+]i rise in HD and LD cells indicates that both melanotrope cell subpopulations possess functional dopamine receptors. Thus, the lack of effect of dopamine on {alpha}-MSH acetylation and release, and POMC mRNA accumulation in HD cells, cannot be accounted for by dopamine receptor deficiency.

We have previously shown that LD melanotrope cells are scarcely granulated and exhibit a high secretory activity, whereas HD melanotrope cells are heavily granulated and display a lower secretory capacity (24, 25). In the present study, the fact that dopamine negatively regulates the secretory and biosynthetic activity of LD, but not of HD melanotrope cells, strongly suggests that morphological and functional heterogeneity of melanotrope cells plays an important physiological role in the control of {alpha}-MSH production by the frog intermediate lobe.


   Acknowledgments
 
We are indebted to Drs. R. H. Andreatta and V. Rasetti (Ciba-Geigy, Basel, Switzerland) for their generous gift of {alpha}-MSH standards.


   Footnotes
 
1 Presented, in part, at the 18th Conference of European Comparative Endocrinologists, Rouen, France, 1996. This study was supported by Dirección General de Investigación Científica y Técnica (Grant PB 94–0451-CO2–01), the Institut National de la Santé et de la Recherche Médicale (U413), European Union Human Capital and Mobility Program (Contract ERBCHRXCT920017), and the Spain-France Exchange Program (310B). Back

Received April 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wheatherhead B 1983 The pars intermedia of the pituitary gland. In: Navarabraham V, Harrison RJ (eds) Progress in Anatomy. Cambridge University Press, New York, vol 3:1
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  7. Martens GJM, Jenks BG, van Overbeeke AP 1981 N-{alpha}-acetylation is linked to {alpha}-MSH release from pars intermedia of the amphibian pituitary gland. Nature 294:558–559[CrossRef][Medline]
  8. Vaudry H, Jenks BG, van Overbeeke AP 1983 The frog pars intermedia contains only the non-acetylated form of {alpha}-MSH. Acetylation to generate {alpha}-MSH occurs during the release process. Life Sci 33:97–100
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