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Endocrinology Vol. 142, No. 7 3060-3067
Copyright © 2001 by The Endocrine Society

ARTICLES

Melanotrope Cell Plasticity: A Key Mechanism for the Physiological Adaptation to Background Color Changes1

Rafael Vazquez-Martinez, Juan R. Peinado, Jose L. Gonzalez de Aguilar, Laurence Desrues, Marie Christine Tonon, Hubert Vaudry, Francisco Gracia-Navarro and Maria M. Malagon

Department of Cell Biology, University of Córdoba (R.V.M., J.R.P., J.L.G.d.A., F.G.N., M.M.M.), 14071-Córdoba, Spain; and European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, UA CNRS, University of Rouen (L.D., 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, Edificio C-6, 3a Planta, Campus Universitario de Rabanales, University of Córdoba, E-14071 Córdoba, Spain. E-mail: fgracia{at}uco.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The intermediate lobe of the pituitary secretes the melanotropic hormone {alpha}-MSH, which in amphibians plays a crucial role in skin color adaptation. It has been previously demonstrated that, in the frog Rana ridibunda, the intermediate lobe is composed of two distinct subpopulations of melanotrope cells that can be separated in vitro by using Percoll density gradients. These two melanotrope cell subsets, referred to as high-density (HD) and low-density (LD) cells, differ in their ultrastructural characteristics as well as in their biosynthetic and secretory activity. However, the specific, physiological role of the heterogeneity displayed by melanotrope cells remains elusive. In the present study, we investigated the effects of background color adaptation on melanotrope cell subpopulations. We found that adaptation of frogs to dark or white environment did not modify either the overall number of cells per intermediate lobe or the apoptotic and proliferation rates of melanotrope cells. On the other hand, adaptation of the animals to a white background significantly increased the proportion of hormone-storage HD cells and caused a concomitant decrease in that of LD cells (which exhibit higher levels of {alpha}-MSH release and POMC messenger RNA than HD cells). Conversely, after black-background adaptation the proportion of LD cells was markedly increased, suggesting that interconversion of HD cells to LD cells occurs during physiological activation of the intermediate lobe. In addition, black-background adaptation also enhanced {alpha}-MSH release by both cell subpopulations and increased inositol phosphate production in LD cells. These data indicate that, in frog, the proportions of the two melanotrope cell subsets undergo marked modifications during skin color adaptation, likely reflecting the occurrence of a secretory cell cycle whose dynamics are highly correlated to the hormonal demand imposed by the environment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MELANOTROPE CELLS of the intermediate lobe of the pituitary synthesize the multifunctional precursor protein POMC, which generates, through proteolytic cleavage, several biologically active peptides including {alpha}-MSH (1, 2, 3). In mammals, the hormonal role of {alpha}-MSH remains obscure so that it is not possible to investigate the regulation of melanotrope cell activity in physiological conditions. In contrast, in poikilotherm vertebrates, especially in amphibians, {alpha}-MSH plays a crucial role in the process of skin color adaptation, and thus the activity of melanotrope cells can be easily manipulated by placing the animals on white or dark background (4). For instance, in the toad Xenopus laevis, it has been shown that changes of the color of the background modifies not only the rate of {alpha}-MSH secretion (5, 6) but also the level of POMC gene expression (7, 8, 9), the ultrastructure of melanotrope cells (10, 11), the turnover of polyphosphoinositide (12), and the acetylation rate of POMC-derived peptides (13).

We have previously demonstrated that, in the frog Rana ridibunda, the intermediate lobe of the pituitary is composed of two subpopulations of melanotrope cells that can be separated, in a Percoll gradient, according to their sedimentation velocity (14). These two melanotrope cell subsets, referred to as high-density (HD) and low-density (LD) cells, differ in their morphological, biochemical, and functional characteristics (14, 15, 16, 17). In particular, LD melanotrope cells display a higher basal secretory activity, acetylation rate, and POMC messenger RNA (mRNA) content than HD cells (15). In addition, TRH induces a more efficient stimulation of {alpha}-MSH secretion and POMC gene expression in LD cells than in HD cells (15), and dopamine negatively regulates the secretory and biosynthetic activity of LD, but not of HD cells (16).

Cell heterogeneity is not only restricted to melanotrope cells, but appears to be a common feature among the endocrine cell types of the anterior pituitary gland (see Ref. 18) for review). Nevertheless, the melanotrope cells, which are the only endocrine cell type composing the amphibian intermediate lobe, constitute a particularly suited model in which to investigate the biological significance of cell heterogeneity because they play a very specific physiological role, whose macromolecular manifestation is the relatively rapid change of skin color pigmentation. Using this experimental model, we have examined the influence of background color adaptation on the proportion and endocrine activity of the two melanotrope subpopulations that compose the frog intermediate lobe. The results reported herein suggest that melanotrope cell subsets represent different phases of a putative secretory cycle and provide compelling evidence that subpopulations may convert into their counterparts depending on the hormonal demand of the animal.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male frogs (Rana ridibunda) of about 40 g body weight were purchased from a commercial supplier (Couétard, Saint-Hilaire de Riez, France). Animals were maintained under running water at constant temperature (8 C) with a 12-h light, 12-h dark cycle, for at least 1 week before the background adaptation experiments started. Skin color adaptation was performed by keeping the animals under constant illumination on either a black or a white background for 3 weeks. The frogs were killed by decapitation between 0800 and 0900 h, and the neurointermediate lobes were dissected under a microscope. Animal manipulations were performed according to the recommendations of the local ethical committees at our institutions and under the supervision of authorized investigators.

Reagents
Collagenase type V, trypsin type I, BSA, Leibovitz culture medium, antibiotic-antimycotic solution, 4'-6-diamidino-2-phenylindole (DAPI), 5-bromo-2'-deoxyuridine (BrdU), anti-BrdU monoclonal antibody, biotinylated goat antimouse IgG, diaminobenzidine tetrahydrochloride (DAB), Triton X-100, formamide, dextran sulfate, polyvinylpyrrolidone, salmon sperm DNA (DNA disodium salts, Type III), polyadenylic acid, levamisole, and isobutylmethylxanthine (IBMX) were purchased from Sigma-Aldrich Corp. Inc. (St Louis, MO). Percoll and Ficoll type 400 were obtained from Pharmacia LKB (Uppsala, Sweden). FBS was from Sera-Lab Ltd. (Crawley Down, UK). DNA labeling and detection kits, proteinase K, and yeast transfer RNA were from Roche Molecular Biochemicals (Mannheim, Germany). The cAMP RIA and myo-[3H]inositol (100 Ci/mmol) were from Amersham International (Versailles, France). Anion-exchange resin AG1-X8 (100–200 mesh; formate form) was from Bio-Rad Laboratories, Inc. (Richmond, CA).

Isolation of melanotrope cells
Isolated melanotrope cells were obtained using a dispersion protocol as previously described (14). Briefly, for each experiment, 40 neurointermediate lobes from animals in each adaptation group were collected and enzymatically dissociated by incubation at 26 C for 45 min in culture medium containing 0.2% (wt/vol) collagenase type V, and 0.2% (wt/vol) trypsin type I. The culture medium consisted in Leibovitz medium 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, pH 7.4. The dissociation was continued in the same medium supplemented with 2 mM EDTA for 5 min, and with 1 mM EDTA for 5 min. The tissues were then mechanically dispersed using a siliconized Pasteur pipette until a homogeneous cellular suspension was obtained. After each step, the cellular suspension was centrifuged at 50 x g for 5 min. The total number of cells and the cellular viability were determined by the Trypan blue exclusion test in a Neubauer chamber.

Determination of apoptosis
To elucidate whether background color adaptation induces melanotrope cell death, the apoptotic rate of intermediate lobe cells from both black- and white-adapted frogs was determined. Specifically, animals were maintained under a black or a white background for 2 days, 10 days or 3 weeks and killed thereafter. In addition, groups of normal nonadapted frogs were also used. After the frogs were killed, the intermediate lobes were enzymatically and mechanically dispersed as described above, and isolated cells were plated at a density of 30,000 cells/50 µl on 35-mm Petri dishes. Cells were incubated at 26 C in 2 ml culture medium supplemented with 10% FBS for 2 h. Thereafter, cells were fixed in the Petri dishes with ethanol:acetic acid (3, 1) for 3 min at room temperature, rinsed with distilled water 3 times for 2 min each, and then nuclei were stained with 4'-6-diamidino-2-phenylindole (DAPI). Samples were mounted in glycerol:PBS (1, 1) and fluorescence was visualized in a Universal microscope (Carl Zeiss, Oberkochen, Germany) using a Fluar X40 objective in the epifluorescence mode (Nikon Corp., Tokyo, Japan) and a stabilized UV light source. Quantitative evaluation of apoptotic cells by DAPI staining was performed by counting at least 400 cells, randomly selected in two to three Petri dishes per adaptation group and experiment.

Measurement of cell proliferation
After a 2-day, 10-day, or 3-week adaptation period, isolated intermediate lobe cells from both black-adapted, white-adapted, and normal nonadapted frogs were plated on 35-mm Petri dishes (30,000 cells/50 µl) and incubated with 10-6 M BrdU in serum-free medium for 2 h at 26 C. Cells were then rinsed with 0.01 M PBS (pH 7.4). Subsequently, cells were fixed in the culture dishes overnight with Bouin’s fixative and thoroughly rinsed with distilled water and PBS until removal of any trace of fixative. The BrdU-incorporating cells were identified immunocytochemically following the protocol described by Avivi et al. (19) with the corresponding modifications. Briefly, cells were sequentially incubated with 5% H2O2 for 30 min, 2 N HCl for 30 min at 37 C, 0.01% trypsin type I in PBS for 5 min at 37 C, and 1% BSA in PBS for 30 min. After several washes in PBS, cells were incubated with anti-BrdU monoclonal antibody (1:1,000) overnight at 4 C in a humid chamber. Cells were then successively incubated first with biotinylated goat antimouse IgG (1:1,000) for 1 h at room temperature, and second with avidin-biotin-peroxidase complex for 45 min at room temperature. The antisera and avidin-biotin-peroxidase complex were diluted in PBS containing 0.5% BSA. Finally, cell-bound peroxidase activity was revealed with DAB (0.56 mM DAB, 0.04% H2O2 in 50 mM acetate buffer, pH 6.0). The specificity of the immunoreaction was checked by omission of the primary antiserum. An additional positive control was carried out using the human mielocytic HL60 cell type in exponential growth phase. All controls were processed concurrently with melanotrope culture samples using identical protocols.

The immunostaining was visualized under a light microscope using a X40 objective lens. The proliferation rate was calculated on a minimum of two Petri dishes per adaptation group and experiment by examining, at least, 600 cells per dish in randomly selected microscopic fields.

Separation of melanotrope cells by density gradients
A hyperbolic density gradient of Percoll was prepared as previously described (14) by mixing 6 ml of a 50% Percoll solution with 3 ml of a 15% Percoll solution at a rate of 0.25 ml/min. A 250-µl sample of the cellular suspension (1–2 x 106 cells) obtained after dispersion of intermediate lobes from frogs adapted to either a black- or a white-background for 3 weeks was carefully loaded on the top of the gradient. This was centrifuged at 3,000 x g for 25 min (4 C) and 9 fractions (1 ml each) were collected manually. Each fraction was washed with Leibovitz medium by centrifugation (750 x g for 10 min at 4 C). After removal of the supernatants, pellets were resuspended in fresh medium, and cell viability and recovery percentages were determined. Cells recovered from fraction 1 (bottom of gradient) and fractions 5 to 7 constituted the HD and LD melanotrope cell subpopulations, respectively.

Cell culture
Aliquots of 40,000 cells from LD and HD cell subsets were plated on 35-mm Petri dishes and incubated at 26 C in 2 ml culture medium supplemented with 10% FBS for 48 h. Thereafter, culture medium samples were collected to quantify the concentration of {alpha}-MSH-related peptides by using a double-antibody RIA method described elsewhere (20). After removal of the culture media, cells in the Petri dishes were processed for quantification of intracellular {alpha}-MSH content by RIA (14), or for determination of POMC mRNA levels by in situ hybridization.

In situ hybridization of POMC mRNA
The in situ hybridization procedure was performed as described in detail previously (15, 16). The probe used was the EcoRI 1184-bp insert of frog POMC complementary DNA subcloned into pGEM-3Zf (21), which was digoxigenin-labeled by random priming using a Digoxigenin DNA labeling kit. Three culture dishes were processed per experiment for in situ hybridization, and each experiment was repeated at least three times. Briefly, after removal of the culture medium, cells were rinsed with 0.01 M PBS (pH 7.4) and fixed in the Petri dishes with 4% paraformaldehyde in PBS for 15 min at room temperature. Thereafter, cells were sequentially incubated with 1% Triton X-100, 5 µg/ml proteinase K, and postfixed in 4% paraformaldehyde before incubation with the hybridization mixture. Hybridization buffer [50% deionized formamide, 5 x Denhardt’s solution (1% Ficoll type I, 1% polyvinylpyrrolidone, 1% BSA), 5 x SSPE (0.75 M NaCl, 0.05 M NaH2PO4, 5 mM EDTA, pH 7.4), 4% dextran sulfate, 0.1% SDS, 250 µg/ml heat-denatured salmon sperm DNA, 200 µg/ml yeast transfer RNA, and 2 µg/ml polyriboadenosine] containing the digoxigenin-labeled probe at 35 ng/200 µl was placed in the culture dishes. After overnight hybridization in a humid chamber at 37 C, cells were subsequently rinsed with 2 x 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 A (100 mM Tris-HCl, 150 mM NaCl, pH 7.5) and sequentially incubated with 10 mM levamisole in buffer B (100 mM Tris-HCl, 150 mM NaCl, 50 mM MgCl2, pH 9.5), 1% blocking reagent, and with the alkaline phosphatase-labeled anti-digoxigenin F(ab) fragment. 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, 4.5 µl/ml nitroblue tetrazolium salt, 0.24 mg/ml levamisole in buffer B). Finally, Petri dishes were mounted in buffer A:glycerol (1:1).

Densitometric quantification of intracellular POMC mRNA content in single HD and LD melanotrope cells was performed using an image analysis system. Petri dishes bearing the cells were placed in the stage of a Universal microscope (Zeiss, Oberkochen, Germany) equipped with a stabilized light source. Regions of interest were selected randomly with a x40 objective and captured by a CCTV camera (Sony, Japan), and digitized images were transferred to a computer. The optical density of stained cells was assessed by the software package for Image Analysis Visilog (Version 4.1; Noesis, France), which calculated the intracellular POMC mRNA content in single cells in terms of integrated optical density (IOD). For each experiment, at least 40 POMC mRNA-positive cells per culture dish were analyzed. An average IOD value of nonstained cells was calculated in each dish ("blank" value) and subtracted to the IOD value of single positive cells in that dish. To avoid variations on the IOD due to factors such as probe labeling, illumination, or focusing, samples from the same experimental set were simultaneously hybridized and measured within the same session.

Determination of cAMP concentration
After a 48-h culture, LD cells from black and white background-adapted frogs were incubated at 26 C for 30 min in 1 ml Leibovitz medium containing 0.1 mM IBMX to inhibit phosphodiesterase.

The reaction was stopped by adding ice-cold 20% (wt/vol) trichloroacetic acid (TCA). After homogenization by sonication (Vibra Cell, Sonics and Materials, Danbury, CT), the proteins were removed by centrifugation (13,000 x g for 4 min at 4 C) and the supernatants washed three times in 1 ml water-saturated diethylether, dried, and reconstituted in cAMP RIA buffer (0.05 M sodium acetate, pH 5.8). cAMP concentration was determined in each sample using a cAMP RIA commercial kit.

Polyphosphoinositide metabolism
The concentrations of phosphoinositols and phosphoinositides were measured as previously described (22). Briefly, after 48 h of culture, LD cells from black and white background-adapted frogs were incubated at 26 C for 4, 8, and 24 h in 1 ml modified Leibovitz medium (0.5 mM glucose, 0.4 mM CaCl2), supplemented with myo-[3H]inositol (200 mCi/mmol). Thereafter, cells were rinsed 6 times with modified Leibovitz medium supplemented with 2 mM inositol. The reaction was stopped with ice-cold 20% TCA and cells were homogenized by sonication. After centrifugation (13,000 x g for 10 min at 4 C), the supernatant containing the phosphoinositols was washed 3 times with water-saturated diethylether, neutralized with 1 M NaHCO3, and stored at -20 C until analysis. Polyphosphoinositides were extracted from the pellet by 200 µl chloroform/methanol (2:1, vol/vol) and the amount of radioactivity associated with the inositol-labeled lipids determined by counting in a 1217 Rackbeta spectrometer (63% efficiency).

Tritiated phosphoinositols were separated by anion-exchange chromatography (AG1-X8 resin). Free inositol and inositol mono-, bis-, and trisphosphates (IP1, IP2, and IP3) were sequentially eluted with 10 x 2 ml distilled water and with 20 x 2 ml each of the solutions of 0.2 M, 0.45 M, and 0.8 M ammonium formate in formic acid (0.1 M). Scintillation fluid (6 ml) was added to each fraction and the radioactivity determined by counting.

Statistical analysis
Data are expressed as mean ± SEM of the number of experiments indicated in each figure. Differences in cell distribution were statistically analyzed by the {chi}-square test. A one-way ANOVA (for apoptotic and proliferation rates, and cAMP production) or a two-way ANOVA (for intracellular and secreted {alpha}-MSH, POMC mRNA levels, and inositol phosphate production), followed by a post hoc Duncan’s test for comparison of multiple variables, were applied to determine statistically significant differences between groups of data. Statistical analysis was assessed by 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
 
Separation of intermediate lobe cells after background adaptation
After dispersion of intermediate lobes from black- and white-adapted frogs, the cell yields were similar (74,750 ± 8,655 and 69,271 ± 6,208 cells/intermediate lobe, respectively; n = 10). Cellular viability was also alike in both groups of animals (94.1 ± 1.2% and 96.8 ± 0.7% for black- and white-adapted frogs, respectively; n = 10).

Effect of background adaptation on apoptosis and proliferation of intermediate lobe cells
The percentage of apoptotic cells in animals undergoing background color adaptation was evaluated by staining cells with DAPI, a fluorescent DNA dye which visualizes the nuclear condensation and the chromatin fragmentation characteristic of apoptosis. Irrespective of the duration of the adaptation process, intermediate lobe cells from both black- and white-adapted frogs showed similar apoptotic rates, which were both comparable to the values obtained from normal nonadapted frogs (Table 1Go). Similarly, quantification of BrdU-incorporating cells yielded similar proliferation rate values in nonadapted animals and in animals adapted to different background color conditions for both short-term (2 days) or long-term (either 10 days or 3 weeks) adaptation periods, and only the proliferation index of intermediate lobe cells from frogs adapted to a white background decreased significantly between 10 days and 3 weeks (Table 1Go).


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  		Table 1. Apoptotic and proliferation rates of intermediate lobe cells from black-, white-, and normal non-adapted frogs

 
Isolation of melanotrope cell subpopulations after background adaptation
In animals adapted to either a black or a white background for 3 weeks, the cell recoveries after separation in the Percoll density gradient were similar (42.2 ± 1.8% and 42.6 ± 7.0%, respectively; n = 4), and the viability of the cells recovered in each fraction was always over 85%. In contrast, the profiles of distribution of cells from black and white background-adapted frogs were significantly different ({chi}-square test, P < 0.001) (Fig. 1Go). Specifically, the proportion of HD cells from black background-adapted frogs was 4-fold lower (P < 0.01) than that from white background-adapted animals, and, reciprocally, the proportion of LD cells from black background-adapted frogs was significantly higher (P < 0.001) than that from white background-adapted animals (Fig. 1Go).



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  		Figure 1. Distribution of frog (Rana ridibunda) intermediate lobe cells through a Percoll density gradient. The animals were adapted to a black (solid bars; n = 6) or a white background (open bars; n = 5) for three weeks. The data are the mean ± SEM. {chi}-square test, P < 0.001. The specific percentage of cells recovered in the HD and LD fractions from each adaptation group are represented in the inset. *, P < 0.01 and **, P < 0.001 vs. black-adapted animals; #, P < 0.05 and ##, P < 0.001 vs. the HD subpopulation (two-way ANOVA followed by a post hoc Duncan’s test).

 
Effect of background adaptation on intracellular {alpha}-MSH concentration and {alpha}-MSH release
HD cells from both black- and white-adapted frogs exhibited more intracellular {alpha}-MSH than LD cells (P < 0.05; n = 3). Moreover, HD and LD subpopulations from black-adapted frogs contained the same amount of {alpha}-MSH than their corresponding counterparts from white-adapted animals (Fig. 2AGo). Regarding the secretory activity, after 48 h in culture, basal {alpha}-MSH release from HD cells was significantly lower (P < 0.001; n = 5) than that from LD cells in both black and white background-adapted frogs (Fig. 2BGo). In addition, black-background adaptation appears to augment hormonal release in both HD and LD cells. Specifically, increases in {alpha}-MSH secretion accounted for by 48.5% in HD cells and 30.6% in LD cells, respectively (Fig. 2BGo; P < 0.001; n = 5).



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  		Figure 2. Quantification of intracellular {alpha}-MSH concentration (A) and {alpha}-MSH release (B) in the HD and LD subpopulations derived from black- (solid bars) and white-adapted animals (open bars) during a 48-h culture period. Results represent the mean ± SEM of three and five independent experiments for intracellular hormonal content and hormonal release, respectively. *, P < 0.001 vs. black-adapted intermediate lobes; #, P < 0.05 and ##, P < 0.001 vs. HD subpopulation (two-way ANOVA followed by a post hoc Duncan’s test).

 
Effect of background adaptation on POMC mRNA level
The percentages of POMC mRNA-positive cells from black- and white-adapted frogs were similar in the HD subpopulation (71.0 ± 4.4% and 68.8 ± 5.8%, respectively; n = 3) and in the LD subpopulation (83.7 ± 3.2% and 84.9 ± 1.7%, respectively; n = 3). Densitometric quantification of POMC mRNA at the single-cell level revealed that LD cells from both black- and white-adapted frogs contained a higher amount of POMC transcripts than HD cells (Fig. 3Go; P < 0.05). However, LD melanotrope cells from white-adapted frogs contained a lower level of POMC mRNA content (P < 0.01) than those from black-adapted animals. In contrast, no differences in POMC mRNA level were observed between HD melanotrope cells from either black- or white-adapted frogs.



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  		Figure 3. Effect of background adaptation on POMC mRNA content in HD and LD subpopulations from black- (solid bars) and white-adapted animals (open bars). The data represent the mean ± SEM of three independent experiments. *, P < 0.01 vs. black-adapted animals; #, P < 0.05 and ##, P < 0.001 vs. the HD subpopulation (two-way ANOVA followed by a post hoc Duncan’s test).

 
Effect of background adaptation on cAMP production
Inasmuch as cAMP is an important intracellular intermediate in the regulation of {alpha}-MSH secretion, we investigated whether cAMP production is involved in the changes of the secretory activity of LD cells induced by background adaptation. We found that intracellular cAMP concentration in LD cells was similar after adaptation of animals to black or white background (2,951 ± 617 fmol/105 cells, and 3,100 ± 637 fmol/105 cells, respectively; n = 3). This indicates that cAMP does not appear to play any crucial role in the regulation of melanotrope secretory activity in response to background adaptation.

Effect of background adaptation on polyphosphoinositide turnover
The rate of incorporation of [3H]inositol into phospholipids and the rate of hydrolysis of polyphosphoinositides were determined in LD cells from black and white background-adapted animals after incubation of the cells with [3H]inositol for various periods of time (Fig. 4Go). The amount of radioactive inositol incorporated into phospholipids (Fig. 4AGo) was higher in LD cells from black- than from white-adapted animals only after 8 h of incubation (P < 0.05; n = 3). In contrast, the percentage of labeled inositol phosphates was at least 2-fold higher in cells derived from black- than from white-adapted animals at each time point tested (Fig. 4BGo; P < 0.05).



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  		Figure 4. Time-course of the effect of background adaptation on the incorporation of [3H]inositol in (A) polyphosphoinositides and (B) inositol phosphates in LD melanotrope cells derived from black- (solid bars) and white-adapted frogs (open bars). Incubation with [3H]inositol started at t=0. The data represent the mean ± SEM of three independent experiments. *, P < 0.05 and **, P < 0.01 vs. corresponding values in black-adapted animals (two-way ANOVA followed by a post hoc Duncan’s test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is now ample evidence that various pituitary cell types are composed of discrete subpopulations that differ in their morphofunctional characteristics (see Ref. 18 for review). Cell heterogeneity was first observed on the entire pituitary cell population (23, 24) and subsequently demonstrated on isolated cell subsets obtained by means of cell separation methods (25, 26, 27, 28, 29, 30, 31). Although these studies have established the basic features characterizing cell heterogeneity, the physiological significance and cellular mechanisms underlying this phenomenon still remain unclear. In the present report, we demonstrate the biological relevance of cell heterogeneity as a mechanism of cellular adaptation that enables the animal to respond in a balanced and flexible way to external stimuli.

Previous studies have demonstrated that the melanotrope cell population from the intermediate lobe of Rana ridibunda is composed of two cell subsets that can be separated by centrifugation in a Percoll density gradient (14). Besides their distinct ultrastructural characteristics, these two melanotrope cell subpopulations exhibit distinct spontaneous activity and differential responsiveness to hypothalamic regulators (14, 15, 16, 17). Thus, the scarcely granulated LD melanotrope cells display high basal POMC mRNA content and {alpha}-MSH release and are highly responsive to both TRH stimulation and dopamine inhibition. In contrast, HD melanotrope cells appear as a hormone storage cell subset, because they show low basal biosynthetic and secretory activities, and little or no response to TRH and dopamine, respectively. The present results demonstrate that, as in normal nonadapted frogs (14), the intermediate lobe of both black- and white-background adapted animals consists of typical LD and HD cells. Thus, the basic cellular features that characterize each melanotrope subset ({alpha}-MSH release, intracellular hormonal concentration, and POMC mRNA levels) remained essentially unchanged during the adaptation process. However, adaptation to different background color conditions resulted in significant changes in the relative proportions of the two cell subsets. Specifically, the LD subpopulation largely predominated in animals placed on a black background, whereas similar proportions of the two cell subsets were found in animals placed on a white background. In agreement with these data, electron microscopic examination of the intermediate lobe of the toad Xenopus laevis has previously shown ultrastructural changes of melanotrope cells in response to background adaptation (10). This latter study has revealed that Xenopus intermediate lobe is also composed of two subtypes of melanotrope cells, referred to as type I and type II, which are morphologically analogous to the LD and HD subpopulations in Rana ridibunda, respectively (10, 14). In black background-adapted toads, the vast majority of melanotrope cells were ultrastructurally identified as type I cells (corresponding to frog LD cells), whereas in white background-adapted toads, type II cells (corresponding to frog HD cells) predominated (10). It appears therefore that, in both frogs and toads, the proportions of melanotrope cell subtypes vary according to background color conditions.

In addition, our data demonstrate that, in frog, the response of the melanotrope cell population to background color adaptation is achieved not only by the control of the number of highly active LD cells and hormone storage HD cells, but also by the concurrent regulation of their intrinsic {alpha}-MSH synthesizing and secretory capacity. Thus, the subpopulations of melanotrope cells from black-adapted frogs were more active than those from white-adapted animals. This effect was much more evident in the LD subpopulation, both in terms of hormone synthesis and secretion. In addition, LD cells from black-adapted frogs also exhibit a higher level of inositol phosphate formation than those from white-adapted animals. These results are in agreement with those reported previously in this same species (22, 32, 33), and suggest the direct involvement of receptor-regulated phosphoinositide turnover in the control of the secretory activity of melanotrope cells during background adaptation. Moreover, comparable results on inositol phosphate metabolism have been obtained in toads adapted to different environmental colors, thus confirming the importance of the inositide pathway in the regulation of background adaptation in amphibians (12). On the other hand, our data on intracellular cAMP concentration in LD cells from both black and white background-adapted animals suggest that the adenylate cyclase activity does not play an important role in the regulation of {alpha}-MSH production in response to background color changes.

The physiologically dependent changes in the proportions of the subsets of amphibian melanotrope cells have been also shown to occur for rat and porcine gonadotrope (34, 35, 36, 37, 38) and somatotrope cells (31, 39, 40, 41), and rat lactotrope cells (42, 43). As suggested by Takahashi for rat lactotrope cells (44), such changes could be accounted for by two different mechanisms: 1) each cell subpopulation derives from proliferation of separated, undifferentiated cells, and 2) each cell subpopulation represents a different maturation phase of the same cell type, and thereby cells from one subpopulation can convert into the other subpopulation and vice versa. In the present study, we provide compelling evidence that in the frog intermediate lobe, interconversion of HD and LD melanotrope cells is the most likely mechanism to explain the changes in the proportions of the cell subsets that occur during the adaptation process. Thus, despite the marked adaptation-related changes in the percentages of HD and LD cells, the total number of cells per intermediate lobe was not affected by the environmental color. Under these conditions, the decrease in the number of cells separated in a subpopulation and the concurrent increase in that of the other subpopulation, observed during adaptation of the animals to either a black or a white background, could be exclusively accounted for by the concomitant occurrence of mitosis in a melanotrope cell subset and cell death in its counterpart. However, our present data demonstrate that neither the proliferation rate nor the apoptotic index of frog intermediate lobe cells was modified during the adaptation of the animals to either a black or a white background. In addition, both black- and white-adapted animals exhibited similar low proliferation and apoptotic indexes, which were also comparable to the values observed in normal nonadapted frogs. Taken together, our present findings strongly suggest that in the frog, melanotrope HD cells convert into LD cells and vice versa through a mechanism that would contribute to precisely adjust the amount of {alpha}-MSH produced by the intermediate lobe and, hence, the degree of skin pigmentation in response to changes in the physiological conditions. Thus, during black background adaptation, for which a high amount of {alpha}-MSH is required, the hormone storage HD cells would convert into highly active secretory and biosynthetic LD cells. In contrast, LD cells would convert into HD cells in conditions of lower hormonal demand, like those imposed by white background adaptation. Furthermore, in view of our results, we propose that the conversion of cells from a subpopulation into its counterpart would take the form of a secretory cell cycle (each subpopulation representing a phase of the cycle) by which cells would acquire different degrees of cellular activity and the animal would finely tune its hormonal response to changes in the physiological milieu. The original idea of such secretory cell cycle was already proposed more than 60 yr ago by Severinghaus (45), and in its simplest interpretation it would refer to the transition of cells from a phase or subpopulation to the other one by a loss or increase in granule content. However, these and our previous findings, which demonstrate the critical differences between the two melanotrope cell subpopulations, strongly suggest that the conversion of LD into HD cells and vice versa, i.e. the secretory cell cycle, does not only consist in a mere process of granule replenishment or hormone release, but it also involves substantial changes in POMC expression and processing, secretion and acetylation rate of {alpha}-MSH, cell responsiveness to regulatory factors, etc. Moreover, these changes occur in strict correspondence to variations in the external inputs reaching the animal, thus indicating that the conversion of hormone-storage HD cells into highly active LD cells and vice versa must be regulated. In this scenario, the hypothalamus, through the release of specific stimulatory (i.e. TRH) or inhibitory (dopamine) signals, could play a pivotal role in the regulation of the dynamics of the melanotrope cell secretory cycle.

In conclusion, our results indicate that, in frog, the heterogeneity of the melanotrope cell population is closely interrelated to the hormonal requirements imposed by the background adaptation process. The fact that changes in the proportion of a particular subpopulation correlates with skin pigmentation is indicative of a dynamic process by which melanotrope cells acquire different degrees of activity, that likely correspond to different phases of a secretory cell cycle.


   Footnotes
 
1 This study was supported by Dirección General de Investigación Científica y Técnica (Grant PB 97-0454) and the Institut National de la Santé et de la Recherche Médicale (U413). Presented, in part, at the 19th Conference of European Comparative Endocrinologists, Nijmegen, The Netherlands, 1998. Back

Received November 28, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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