1Department of Cell Biology, University of Cordoba, 14014-Cordoba, Spain; and 2European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale U413, Unité Associée Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France
Submitted 23 May 2003 ; accepted in final form 16 July 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
dopamine; melanotrope cell heterogeneity; neuropeptide Y; secretory cycle; thyrotropin-releasing hormone
These results have led our group to propose that, as suggested for other endocrine cell types (58, 16, 35, 36), the morphologically and functionally distinct sub-types or subpopulations of cells that comprise the total population of melanotropes are, in fact, the reflection of different phases that occur at a given point in time within a dynamic and complex secretory cycle, which would operate, albeit with diverse specific features, in any endocrine cell type. Nevertheless, the mechanisms that govern the acquisition of a particular state of cellular activity and therefore determine the conversion of cells from a subpopulation to the other remain to be elucidated. Inasmuch as in amphibians melanotrope cell activity is directly regulated by the hypothalamus (for review see Ref. 12), it is conceivable that hypothalamic neurohormones modulate the timing of this secretory cycle. Accordingly, the aim of the present study has been to ascertain whether known hypothalamic regulators of the frog intermediate lobe, such as the inhibitors dopamine (12, 15, 17, 42) and neuropeptide Y (NPY) (3, 1215, 17, 34, 41) and the stimulator thyrotropin-releasing hormone (TRH) (12, 18, 28, 43), are able to induce the interconversion of cells from the two frog melanotrope subpopulations as well as to analyze the relationship between the control of the dynamics of the melanotrope secretory cycle and background color adaptation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals. Adult male frogs (R. ridibunda) of 40 g body wt were purchased from a commercial supplier (J. R. Garcia Jardor, Mosqueiro, Ourense, Spain). Animals were maintained under running water at a constant temperature (8°C) with a 12:12-h light-dark cycle for
1 wk before experiments started. Skin color adaptation was performed by keeping the animals under constant illumination on either a black or a white background for 3 wk. The frogs were killed by decapitation between 8:00 and 9:00 AM, and the neurointermediate lobes were dissected under a microscope. Animal manipulations were performed according to the recommendations of the local ethics committees at our institutions and under the supervision of authorized investigators.
Isolation of melanotrope cells. Isolated melanotrope cells were obtained using a dispersion protocol as previously described (23). Briefly, for each experiment, 3040 neurointermediate lobes from either black or white background-adapted animals were collected and enzymatically dissociated by incubation at 26°C for 45 min in culture medium [Leibovitz medium diluted 2:3 (to adjust to R. ridibunda osmolality) and supplemented with 1 mM glucose, 0.4 mM CaCl2, and 1% antibiotic antimycotic solution] containing 0.2% collagenase type V and 0.2% trypsin type I. Then, cells were incubated sequentially in the same medium supplemented with 2 and 1 mM EDTA for 5 min each. Afterward, cells were mechanically dispersed using a siliconized Pasteur pipette until a homogeneous cellular suspension was obtained. The total number of cells and the cellular viability were determined by the Trypan blue exclusion test in a Neubauer chamber.
Cell culture. Dispersed melanotrope cells from black- or white-adapted animals were plated at a density of 300,000 cells/200 µl culture medium into six-well culture plates and allowed to attach to the plate for 60 min. Subsequently, cell cultures received 1,800 µl/well culture medium, supplemented with 10% FBS and 0.1% gentamicin sulfate, containing the corresponding test substances. Specifically, cultures from black-adapted frogs were incubated with 104 M apomorphine or 107 M NPY, and those from white-adapted frogs with 105 M TRH, at 26°C for 72 h. As controls, cells from either black- or white-adapted frogs were cultured in medium alone under the same incubation conditions as those treated with the corresponding factors. In all experimental groups, treatment was renewed daily.
For the determination of the apoptotic and proliferation rates of melanotropes in culture, 40,000-cell aliquots from the initial cell suspension, obtained after the dispersion of intermediate lobes from either black- or white-adapted frogs, were plated in 35-mm plastic dishes. These cultures received the same experimental treatments as those described above.
Separation of melanotrope subpopulations. After the 72-h culture period in the presence or absence of the corresponding test substance, cells were detached from the plates by sequential incubation in Leibovitz medium containing 2 mM EDTA for 20 min and 1 mM EDTA plus 0.05% trypsin for 15 min. Thereafter, the medium containing the cells was centrifuged at 750 g for 5 min, and the pellets were resuspended in 1 ml of Leibovitz medium. Cells recovered from the wells that had received the same experimental treatment were pooled together and washed again with Leibovitz medium by centrifugation (750 g for 10 min at 4°C). The cellular recovery rate and viability were determined for each experimental group.
A 250-µl sample of the cellular suspension from each experimental group (500,000 cells) was carefully loaded on the top of a Percoll density gradient prepared as described previously (23). In brief, the hyperbolic gradient of Percoll was prepared 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. After centrifugation (3,000 g for 25 min at 4°C), nine fractions (1 ml each) were collected manually and centrifuged (750 g for 10 min at 4°C), and pellets were resuspended in 200 µl of fresh medium. The viability and recovery percentage of each fraction were determined for each experimental group. Cells in fraction 1 (bottom of the gradient) contained the HD, or storage, melanotrope cell subpopulation, whereas secretory, or LD, melanotropes separated in fractions 57 (2123, 39, 40).
Determination of apoptosis. As indicated, melanotrope cells were cultured in 35-mm dishes and exposed to 104 M apomorphine or 107 M NPY (for cultures from black-adapted frogs) or to 105 M TRH (for cultures from white-adapted animals). After the 72-h treatment period, medium was removed, and cells were fixed with ethanol-acetic acid (3:1), as described previously for the same cell model (39). Briefly, nuclei were stained with 4'-6-diamidino-2-phenylindole (DAPI) to reveal the nuclear condensation and the chromatin fragmentation characteristic of apoptosis (11), and fluorescence was visualized in a Universal microscope (Zeiss, Jena, Germany) with a Fluar x40 objective in the epifluorescence mode (Nikon, Tokyo, Japan). Quantitative evaluation of apoptotic cells by DAPI staining was performed by counting 200 cells randomly selected in at least two plates per treatment group and experiment.
Measurement of cell proliferation. The proliferation index of melanotropes was evaluated after the 72-h culture period in the absence or presence of the test substances as described previously (39). After removal of the medium, cells were exposed to 106 M BrdU for 2 h at 26°C, rinsed with 0.01 M PBS (pH 7.2), and then fixed in the culture dishes overnight with Bouin's fixative. The BrdU-incorporating cells were identified immunocytochemically after sequential incubation in 5% H2O2 (30 min, room temperature), 2 N HCl (30 min, 37°C), 0.01% trypsin type I in PBS (5 min, 37°C), 1% BSA in PBS (30 min, room temperature), and an anti-BrdU monoclonal antibody (overnight, 4°C). Finally, cells were incubated with biotinylated goat anti-mouse IgG and avidin-biotin peroxidase complex (1 h each, room temperature), and cell-bound peroxidase activity was revealed with DAB. The specificity of the immunoreaction was checked by omission of the primary antiserum. All controls were processed concurrently with melanotrope culture samples by using identical protocols.
The immunostaining was visualized under a light microscope fitted with a x40 objective, and the proliferation rate was calculated on at least two plates per treatment group and experiment. In each plate, microscopic fields were randomly selected and at least 600 cells were examined.
Statistical analysis. Data are expressed as means ± SE of the number of experiments indicated in each figure. Differences in cell distribution were statistically analyzed by the 2-test. Two-way (for percentages of melanotrope cells) or one-way (for apoptotic and proliferation rates) analyses of variance (ANOVA), followed by a post hoc Duncan's test for multiple comparisons, were applied to compare experimental treatments. Statistical analyses were assessed with the software Statistica for Windows (Statsoft, Tulsa, OK). Differences were considered to be significant at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Results on the recovery of cells from the cultures by EDTA-trypsin treatment after treatment of melanotropes with the different test substances are shown in Table 1. None of the treatments affected the recovery rate of cells from the plates except for apomorphine-treated cultures, which exhibited a significantly lower value than that of the corresponding control cultures. In contrast, subsequent separation of the resulting cells by density gradients did not affect the recovery of cells, which was similar in all the experimental groups (36.5 ± 6.1, 49.1 ± 9.7, 33.2 ± 9.1, 57.8 ± 14.5, and 48.8 ± 20.3% for black-background adaptation control, apomorphine-treated cells, NPY-treated cells, white-background adaptation control, and TRH-treated cells, respectively).
|
Effects of apomorphine and NPY on cultured melanotrope cells from black background-adapted frogs. Separation in Percoll density gradients of the total population of melanotrope cells from black-adapted animals after the 72-h period under control conditions yielded a bimodal distribution of cells through the gradient (Fig. 1A). Specifically, melanotropes were separated into two distinct zones of the gradient, fractions 1 and 57, which corresponded to the subpopulations of hormone-storage (HD) and secretory (LD) melanotropes, respectively, which have been previously characterized in detail (2123, 39, 40). Cells separated in these fractions accounted for 77.2% of the total number of cells recovered from the gradient, among which the number of secretory cells was approximately sixfold higher than that of storage cells (Fig. 1B).
|
Exposure of dispersed intermediate lobe cells from black background-adapted frogs for 3 days to the dopamine receptor agonist apomorphine caused a significant modification of the sedimentation characteristics of the cells within the density gradient, which resulted in marked changes in the relative proportions of cells separated in each fraction (2-test, P < 0.05; Fig. 1A). As shown in Fig. 1B, apomorphine treatment reduced by almost 20% the proportion of secretory cells with respect to control values, whereas it evoked a concomitant increase in the percentage of storage melanotropes, which was twofold higher than that found under control conditions (Fig. 1B).
Similar to what was observed for apomorphine, long-term in vitro exposure of melanotrope cells from black-adapted frogs to NPY resulted in a shift of the cell population to the heavy fractions of the gradient (2-test, P < 0.05; Fig. 2A). Indeed, NPY increased by 24.3% the proportion of storage melanotropes and decreased by 18.5% that of secretory melanotropes compared with the percentages obtained for the corresponding cell subpopulations from control cultures (Fig. 2B).
|
We also assessed the rates of proliferation and apoptosis in cultures of melanotropes from black background-adapted animals exposed to medium alone or to the inhibitors apomorphine or NPY (Table 1). This revealed that neither apomorphine nor NPY modified the apoptotic rate of melanotropes in culture compared with control values. Similarly, no differences were observed in the proliferation index between control and NPY-treated cultures, and although apomorphine-treated cells exhibited a lower BrdU-labeling index than control cells, this difference did not reach statistical significance.
Effects of TRH on cultured melanotrope cells from white background-adapted frogs. The distributions of both control and TRH-treated cells obtained from white background-adapted animals in the density gradient are illustrated in Fig. 3A. After a 72-h culture period, melanotropes from both experimental groups essentially separated into the HD and LD fractions (fractions 1 and 57, respectively). Actually, the overall percentage of cells contained in both cell subsets for control and TRH-treated cultures represented 85.2 and 89.5%, respectively, of the total number of cells recovered from the gradient. However, the proportion of melanotropes separated in each density fraction was significantly different for each experimental group (2-test, P < 0.05; Fig. 3A). Thus, whereas TRH increased the number of secretory cells (16.1%), it concurrently decreased that of storage melanotropes (11.8%) compared with the corresponding values obtained from cells cultured in medium alone. In contrast to this effect of TRH on the balance between the two melanotrope cell subtypes, the tripeptide did not alter the rate of apoptosis or proliferation in melanotropes from white-adapted frogs in culture (Table 1).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Involvement of dopamine in the neuroendocrine regulation of skin color adaptation in amphibians is supported by earlier studies demonstrating that agents that induce catecholamine depletion in fibers reaching the intermediate lobe caused skin darkening (for review see Ref. 12). Accordingly, our first set of studies was aimed at elucidating the effects of the major catecholaminergic inhibitor dopamine on melanotrope cells from animals displaying high plasma -MSH levels, that is, black background-adapted frogs. Thus long-term in vitro treatment of intermediate lobe cells from these animals, which essentially correspond to secretory, or LD melanotropes (39, 40), caused a significant modification of the sedimentation characteristics of the cells within the density gradient with respect to untreated controls, which resulted in marked changes in the relative proportions of the two cell subtypes. Specifically, apomorphine reduced the percentage of secretory cells and evoked a concomitant increase in the percentage of storage cells. Inasmuch as these changes are reminiscent of those observed during adaptation of the animals in vivo to a white background (39, 40), our results strongly suggest that an important mechanism employed by dopamine to regulate the adaptation process would be inducing secretory melanotropes to acquire the hormone storage phenotype.
Because percentages of the two subtypes of melanotropes in the intermediate lobe are relative and depend on the total number of cells of the gland, we assessed the rates of proliferation and apoptosis in the cell cultures to ascertain whether apomorphine-induced changes resulted from true conversions of cells from one subpopulation (LD) into its counterpart (HD) or, alternatively, from an increase in proliferation of storage cells and/or a concomitant disappearance of secretory cells. This assessment revealed that, in fact, the BrdU-labeling index in apomorphine-treated cultures was slightly, albeit not significantly, lower than that observed in the corresponding controls. This indicates that the net increase in the number of storage melanotropes induced by apomorphine cannot be accounted for by an enhanced rate of cellular proliferation and, therefore, that the cells that separate in this subpopulation after apomorphine treatment do not derive from mitosis of preexisting cells. In support of these findings, it has been shown that dopamine or its agonists inhibit proliferation of rat melanotropes both in vivo and in vitro (9, 19, 20), as well as that of other pituitary cell types such as lactotropes (1, 30). Actually, dopamine and its agonists are commonly used in prolactinoma therapy to reduce tumor size because of their well-known antiproliferative (32) and cytotoxic (33) properties, and we consequently expected apomorphine to increase apoptosis in our cultures. However, the apoptotic rate was similar in control and apomorphine-treated cultures. Nevertheless, at this point, we should introduce the caveat that assessment of apoptosis might have been slightly underestimated if cells that progressed into later stages of apoptosis detached from the culture dish and were thus undetectable, as suggested by the lower cell recovery found in apomorphine-treated cultures. This caution notwithstanding, it must be emphasized that even a selective disappearance of secretory melanotropes caused by apomorphine could not account for the changes observed. Therefore, when viewed as a whole, our results strongly suggest that storage melanotropes appear as a result of the apomorphine-induced conversion of secretory cells.
We also tested the effect of long-term in vitro exposure of melanotrope cells from black-adapted frogs to the naturally occurring hypothalamic inhibitor NPY (4). Results from these experiments showed that NPY caused a marked decrease in the number of secretory cells and a concurrent increase in that of storage cells without altering the rate of either cellular proliferation or apoptosis. These findings indicate that, as found for apomorphine, NPY is able to induce the transition of melanotrope cells from a highly active secretory physiological condition to a storage state. Interestingly, the relative proportions of the two subtypes of melanotropes after culture of intermediate lobe cells from black-adapted frogs in the presence of either apomorphine or NPY were strikingly similar to those observed in intermediate lobes of white-adapted frogs (39, 40). These results support the idea that these inhibitors are able to induce in vitro the same cellular changes that occur in vivo during white-background adaptation. Moreover, they strongly suggest that dopamine and NPY play a pivotal role during this physiological process. Consistent with this notion, quantitative immunocytochemistry and in situ hybridization studies of the hypothalamus from black background-adapted Xenopus have shown that the levels of NPY mRNA and NPY immunoreactivity are very low in the ventrolateral part of the suprachiasmatic nucleus, where NPY-producing neurons controlling intermediate lobe function are located (25, 26, 37). In contrast, high mRNA levels and NPY immunoreactivity are found in this hypothalamic region in white-adapted toads (25, 26).
Having established that hypothalamic inhibitors can mimic in vitro the cellular changes that occur during white-background adaptation, we next asked whether TRH, the major hypothalamic stimulator for amphibian melanotrope cells (12, 18, 28, 43), could induce the cellular changes observed during adaptation of frogs to a black background [i.e., conversion of storage cells into secretory cells (39, 40)]. Indeed, in the melanotrope population of white-adapted frogs, which comprises similar proportions of both cell subtypes (39, 40), long-term in vitro exposure to TRH markedly increased the number of secretory melanotropes, whereas it concurrently decreased that of hormone storage cells. Inasmuch as TRH did not alter apoptosis or proliferation in these cultures, such changes in proportions are necessarily the result of the conversion of storage cells into active melanotropes. Moreover, the percentages of both types of melanotropes observed after treatment of intermediate lobe cells from white-adapted animals with TRH were virtually identical to those found in animals physiologically adapted to a black background (39, 40). Therefore, our results demonstrate that TRH can mimic in vitro the effects caused by black-background adaptation on frog melanotrope cell subpopulations and thereby strongly support the involvement of the tripeptide in the adaptation process.
The concept that the hypothalamus can control the composition of the population of a given pituitary cell type, i.e., the balance between its distinct subpopulations, raises two important questions. The first relates to the possible physiological relevance of such regulatory mechanism. The present results, together with those reported previously on amphibian melanotropes, may provide a good example in that regard. As previously demonstrated (2123, 38), frog storage melanotropes are heavily granulated and contain high intracellular -MSH levels, although they show low levels of hormone biosynthesis and release under basal culture conditions. More importantly, storage melanotropes also exhibit a limited response, in terms of both POMC expression and
-MSH release, to in vitro short-term treatment with TRH (21, 23). In addition, among the three major secretoinhibitors of the frog intermediate lobe (34), dopamine, GABA, and NPY, only high concentrations of the last are able to reduce the already low secretory activity of hormone storage melanotropes (22, 38). In clear contrast, secretory melanotropes in culture are highly responsive in the short term to TRH stimulation (21) as well as to the hypothalamic inhibitors dopamine, GABA, and NPY (22, 38). In view of these and our present findings, it seems reasonable to suggest that the hypothalamus controls plasma
-MSH levels during the process of adaptation to background color by regulating the total activity of the intermediate lobe through two different but interrelated mechanisms: 1) in the short term, by acutely controlling hormone synthesis and release, and 2) in the long term, by converting highly secretory cells into hormone storage cells and vice versa. This process is graphically illustrated in Fig. 4. Thus, during white-background adaptation, dopamine and NPY would reduce
-MSH levels in the short term by inhibiting its release from the highly responsive, predominantly secretory, or LD subtype in black-adapted frogs. At the same time, dopamine and NPY would enable a more effective long-term reduction of
-MSH production by causing a lasting conversion of highly active and responsive secretory melanotropes into poorly responsive hormone storage cells. Conversely, during adaptation from a white to a black background, the required high
-MSH levels would be achieved primarily by TRH in the short term through stimulation of
-MSH release from highly responsive melanotropes. Concurrently, TRH would also induce a long-term conversion of the hormone storage cells into their active counterparts. In sum, this system would provide the hypothalamus an additional, long-lasting mechanism to finely regulate the activity of the intermediate lobe in an effective and economical manner.
|
The second question that derives from our findings is two pronged: what form does the conversion of cells from one subpopulation into the other cell subtype take; and what precise factors and events underlie this regulatory process? Regarding the first part of the question, we and others have previously suggested that heterogeneity within pituitary cell types, such as melanotropes (39), somatotropes (16), gonadotropes (35, 36) and lactotropes (27), is in fact the reflection of cells undergoing a secretory cycle. This cellular process would comprise the transition of cells from one phase or subpopulation to the other one due not only to a loss or increase in granule content but also to changes in key functional features of the cells. In the case of melanotropes, such changes would include selective responsiveness to regulatory factors as well as the differential regulation of the synthesis, maturation, acetylation rate, and secretion of -MSH along with variations in other important components of the secretory pathway (24, 29). Regarding the second part of the question, the precise regulatory mechanisms that underlie the melanotrope cell-secretory cycle await elucidation. One can envision a number of points at which the activity and responsiveness of melanotrope cells can be regulated. This might include from transcription factors to signaling enzymes and proteins as well as membrane receptors. To give just one example, bromocriptine has been shown to regulate the type of G proteins (31) and dopamine receptor isoforms (10) expressed in rat melanotrope cells. Future studies should aim at ascertaining the points at which this regulation is primarily exerted and at determining whether the secretory cycle is a general mechanism for the control of endocrine cell activity.
![]() |
DISCLOSURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |