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Department of Cell Biology, University of Córdoba (J.L.G.d.A., M.M.M., R.M.V.M., F.G.N.), Córdoba, Spain; and European Institute for Peptide Research, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 23, Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, Unité Affiliée au Centre National de la Recherche Scientifique, University of Rouen (I.L., M.C.T., H.V.), Mont-Saint-Aignan, France
Address all correspondence and requests for reprints to: Dr. Francisco Gracia-Navarro, Department of Cell Biology, Faculty of Sciences, Avda. San Alberto Magno s/n, University of Córdoba, E-14004 Córdoba, Spain. E-mail: bc1grnaf{at}uco.es
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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MSH, microfluorimetric measurement of
the cytosolic free calcium concentration
([Ca2+]i), and in situ
hybridization for quantification of POMC messenger RNA (mRNA). Under
basal conditions, LD melanotrope cells showed higher secretory
activity, acetylation rate, [Ca2+]i, and POMC
mRNA content compared to HD cells. Incubation of the cells with 100
nM TRH for 2 h induced a more pronounced activation of
MSH secretion, [Ca2+]i mobilization, and
POMC mRNA accumulation in LD than in HD melanotrope cells. Conversely,
TRH increased the rate of acetylation of MSH in HD cells, but did
not affect acetylation in LD cells. Taken together, these results
demonstrate that the frog intermediate lobe is composed of two subsets
of endocrine cells with distinct biochemical and functional
characteristics. The coexistence of two cell subpopulations in the frog
pars intermedia is consistent with the idea of a cell secretory cycle,
in which each melanotrope subset represents a specific state of
cellular activity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The intermediate lobe of the rat pituitary is composed of two cell
types, i.e. melanotrope and corticotrope cells (15), which
both synthesize the precursor protein POMC (16). The intermediate lobe
of amphibians, which consists of a single endocrine cell type, the
melanotrope cells (17), represents a valuable model in which to
investigate the phenomenon of cell heterogeneity. Posttranslational
processing of POMC in amphibian melanotrope cells generates several
biologically active peptides, including MSH and ß-endorphin (18, 19). The hormone MSH, which causes dispersion of the pigment melanin
in dermal melanophores, plays a pivotal role in the control of
background color adaptation (20). N-Terminal acetylation of MSH is
an important processing event that increases the melanotropic activity
of the peptide (21). In anuran amphibians, the major intracellular form
of the peptide is des-N-acetyl MSH,
and the acetylation reaction to generate MSH is associated with the
secretory process (22, 23, 24). It has also been demonstrated that
N-acetylation is physiologically
regulated by background adaptation (25) or hypothalamic neurohormones
(26, 27).
The secretory activity of frog melanotrope cells is regulated by
multiple factors, including classical neurotransmitters and
neuropeptides (see Ref. 28 for review). In particular, TRH is a potent
stimulator of MSH secretion from amphibian intermediate lobe
(29, 30, 31). The effect of TRH on melanotrope cells is mediated through
activation of phospholipase C and mobilization of intracellular calcium
stores (32).
Heterogeneity of rat melanotrope cells has previously been reported
with respect to secretory activity (33) and POMC gene expression (34).
Recently, two subpopulations of melanotrope cells have been isolated in
the intermediate lobe of Rana ridibunda after separation of
dispersed cells with a continuous Percoll density gradient (35). These
melanotrope cell subsets, referred to as high density (HD) and low
density (LD) cells, differ in both secretory granule content and
spontaneous MSH secretion; HD cells have a more granulated cytoplasm
and a substantially lower secretory activity than LD cells.
The aim of the present study was to compare the biochemical and
physiological characteristics of HD and LD cells. For each
subpopulation, we investigated the secretory activity, the cytosolic
calcium concentration ([Ca2+]i) and the POMC
messenger RNA (mRNA) density under basal and TRH-stimulated conditions.
In addition, we compared the rates of acetylation of MSH in HD and
LD melanotrope cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents and test substances
Collagenase type V, trypsin type I, BSA, Leibovitz culture
medium, and antibiotic-antimycotic solution were purchased from Sigma
Chemical Co. (St. Louis, MO). Indo-1 acetoxymethylester, pluronic F127,
and the calcium ionophore A-23187 were obtained from Molecular Probes
(Eugene, OR). TRH was purchased from PREM (Zyma Farmacéutica,
Barcelona, Spain). Percoll was obtained from Pharmacia LKB (Uppsala,
Sweden). FBS was obtained from Sera-Lab (Crawley Down, UK).
Isolation and separation of melanotrope cells
Isolated melanotrope cells were obtained using a dispersion
protocol described previously (35). Briefly, 40 neurointermediate lobes
were collected for each experiment and enzymatically dissociated by
incubation at 26 C in culture medium containing 0.2% (wt/vol)
collagenase type V and 0.2% (wt/vol) trypsin type I for 45 min. The
culture medium consisted of Leibovitz culture 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. Then, the tissues
were mechanically dispersed using a siliconized Pasteur pipette until a
homogeneous cellular suspension was obtained. Cell viability, as
determined by the trypan blue exclusion test (36), was 89%.
A density gradient of Percoll was prepared as previously described (35). A hyperbolic gradient was obtained 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 (2.2 x 106 cells) was carefully loaded on top of the gradient. After centrifugation (3,000 x g for 25 min; 4 C), nine fractions of 1 ml each were collected manually. Cells contained in fractions 1 (bottom of the gradient) and 4 plus 5 contained the HD and LD cell subpopulations, respectively. HD melanotrope cells represented 20.8% of the total cells recovered from the gradient, whereas LD cells represented 52.1%.
Culture of melanotrope cell subpopulations
Primary culture of each melanotrope cell subset was performed to
evaluate their functional characteristics under both basal and
TRH-stimulated conditions. Aliquots of 30,000 cells were plated on
35-mm petri dishes and incubated at 26 C in 2 ml culture medium
supplemented with 10% FBS. After 48 h, the medium was removed,
and the cells were preincubated in 1 ml serum-free culture medium for
2 h. The cells were incubated for another 2 h with 1 ml
medium in the absence or presence of 100 nM TRH. Medium
samples were collected and centrifuged at 60 x g for 5
min, and the supernatants were stored at -20 C until hormone assay.
After culture, cells were processed for in situ
hybridization. To determine the [Ca2+]i,
cells were plated on microgrid coverslips (Eppendorf, Netheler,
Germany) at a density of 5000 cells/well and incubated in 2 ml culture
medium supplemented with 10% FBS for 3–5 days until
[Ca2+]i measurement.
HPLC
Incubation media were first prepurified on Sep-Pak cartridges
(Alltech Europe, Laarne, Belgium). Each sample was then subjected to
reverse phase HPLC analysis on Lichrosorb RP-18 column (0.46 x 25
cm; Merck, Paris, France) using 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 in Fig. 2
. The following synthetic standards
were analyzed using the same gradient:
des-N-acetyl MSH, MSH, and
diacetyl MSH, as well as their respective sulfoxide derivatives.
Oxidation of the synthetic standards was performed using hydrogen
peroxide as described previously (37). Fractions were collected every 1
min and dried in a Speed-Vac concentrator (Savant, Hicksville, NY)
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MSH RIAMSH-related peptides in the culture
media and HPLC fractions were measured using a double antibody RIA
method described previously (38). The sensitivity of the assay was 3
pg/tube. The MSH antiserum exhibited full cross-reactivity with
des-N-acetyl MSH, MSH, and
diacetyl MSH. The cross-reactivity of the antiserum with other
POMC-derived peptides was lower than 0.1%.
Measurement of cytosolic calcium concentration
Cultured cells were first incubated with 5 µM
indo-1 acetoxymethylester and 0.02% (vol/vol) Pluronic F127 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 system constructed from an inverted
microscope equipped with a fluor x40 objective in the epifluorescence
mode (Nikon Corp., Tokyo, Japan). The fluorescence emission of indo-1
induced by excitation at 355 nm was recorded at two wavelengths (405
and 485 nm) by separate photometers as previously described (39). After
conversion of photon currents to voltage signals, both 405- and 485-nm
signals and the 405/485 ratio were continuously monitored by a software
FASTINCA 1.03 (Nikon Corp.). [Ca2+]i was
calculated from the formula established by Grynkiewicz et
al. (40): [Ca2+]i = Kd x
ß x [(R - Rmin)/(Rmax - R)], where
Kd is the dissociation constant for indo-1 (250
nM), ß is the fluorescence ratio between the signal at
485 nm in calcium-free medium and the signal at 485 nm in
calcium-saturated medium, R is the 405/485 ratio of any unknown calcium
concentration, Rmin is the 405/485 ratio obtained after
incubation of cells with 2 mM EGTA and 10 µM
A-23187 for 30 min at 22 C in culture medium in the dark (calcium-free
conditions), and 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 culture medium in the
dark (calcium-saturated conditions). The averaged values were:
Rmin = 0.102 ± 0.001, Rmax = 0.713
± 0.018, and ß = 3.677 ± 0.246 (n = 90). The effect of
TRH on [Ca2+]i was tested by administering
pulses of 10 µM TRH for 2 sec in the vicinity of the
cells by means of a pressure ejection system.
In situ hybridization of POMC mRNA
Quantification of POMC mRNA was performed by in situ
hybridization using a nonradioactive procedure, as previously described
(41). After removal of the culture medium, cells were rinsed with 0.01
M PBS (pH 7.2) and fixed in petri dishes with 4%
paraformaldehyde in PBS for 15 min at room temperature. After three
washes with PBS, cells were dehydrated in graded ethanol and stored at
-80 C until use. Before hybridization, cells were thawed, hydrated,
and treated with 1% (vol/vol) Triton X-100 in PBS for 10 min. After
two washes in PBS, cells were incubated with 5 µg/ml proteinase K
(Boehringer Mannheim, Mannheim, Germany) in PBS for 15 min at 37 C. The
enzymatic activity was stopped with 2 mg/ml glycine in PBS. Cells were
washed in PBS, postfixed with 4% paraformaldehyde in PBS for 5 min,
washed again, and dehydrated in graded ethanol. Cells were
prehybridized for 2 h at 37 C in a solution containing 50%
(vol/vol) deionized formamide (Sigma), 5 x SSPE (20 x SSPE
is 3 M NaCl, 0.2 M
NaH2PO4·H2O, and 0.02
M EDTA, pH 7.4), 4% (vol/vol) dextran sulfate (stock 40%,
wt/vol; Sigma), 5 x Denhardt’s solution [50 x Denhardt’s
solution is 1% (wt/vol) Ficoll type 400 (Pharmacia LKB), 1% (wt/vol)
polyvinylpyrrolidone (Sigma), 1% (wt/vol) BSA, 0.1% (vol/vol) SDS
(stock 10% (wt/vol); pH 7.2), 200 µg/ml yeast transfer RNA
(Boehringer Mannheim), 250 µg/ml heat-denatured salmon sperm DNA
(Sigma), and 2 µg/ml polyadenylic acid (Sigma). The probe used in
this study was the EcoRI 1184-bp insert of frog POMC
complementary DNA subcloned into pGEM-3Zf (42). The fragment was
purified by agarose gel electrophoresis and then digoxigenin-labeled by
random priming using a digoxigenin DNA labeling kit (Boehringer
Mannheim). Cells were covered with 200 µl heat-denatured labeled POMC
DNA probe (35 ng/dish) in the hybridization solution. Hybridization was
performed in a humid chamber for 16 h at 37 C. The dishes were
subsequently rinsed with 2 x SSC (20 x SSC is 3
M NaCl and 0.3 M sodium citrate, pH 7.4) for 90
min, 1 x SSC for 90 min, 0.5 x SSC for 30 min at 37 C, and
0.5 x SSC for 30 min. Cells were washed in buffer 1 (100
mM Tris-HCl and 150 mM NaCl, pH 7.5) and
incubated with 10 mM levamisole (Sigma) in buffer 2 (100
mM Tris-HCl, 150 mM NaCl, and 50 mM
MgCl2, pH 9.5) for 1 h. Then, cells were washed twice
with buffer 1 and treated with 1% (wt/vol) blocking reagent
(Boehringer Mannheim) for 30 min. Thereafter, cells were incubated with
the alkaline phosphatase-labeled antidigoxigenin F(ab) fragment
(Boehringer Mannheim) diluted 1:500 in buffer 1 for 4 h. After
successive washes in buffers 1 and 2, 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 (Boehringer Mannheim), and 0.24 mg/ml
levamisole in buffer 2] for 16 h in the dark. The color reaction
was stopped by rinsing the plates with buffer 1. Finally, they were
mounted in buffer 1 plus glycerol (1:1).
Control staining included 1) pretreatment of dishes with 200 µg/ml pancreatic ribonuclease A and 150 U/ml ribonuclease T (Sigma) in 2 x SSC for 1 h at 37 C, 2) omission of the probe, and 3) omission of the antidigoxigenin serum. As an additional control, the probe was tested in a cell type (human monocyte THP-1 cells) that does not express POMC. All controls were processed concurrently with samples using the same protocol.
POMC mRNA quantification was accomplished on a Dasher 386SX computer (Data General, Westboro, MA) equipped with IMAGO software for image analysis (SIVA Research Group, University of Córdoba, Córdoba, Spain) and connected by a CCTV camera (Hitachi, Tokyo, Japan) to a light microscope (Zeiss, Oberkochen, Germany). A x40 objective and a stabilized light source were used. Before measuring each set of cells, Köhler focus was carried out to ensure an even and homogeneous illumination. The staining intensity (optical density) and the area of each individual cell (50 cells randomly selected/dish) were measured to calculate the integrated optical density. This parameter was correlated with the amount of POMC mRNA in the cell.
Background was evaluated for nonstained cells (10 cells/dish) and subtracted. To avoid within-experiment variations, control and treated cells were hybridized and measured simultaneously.
Statistical analysis
Data were expressed as the mean ± SE of the
number of experiments indicated in each figure. The statistical
analysis was preceded by a test for the joint assessment of normality
(Kolmogorov-Smirnov test). Student’s t test or Mann-Whitney
rank sum test were applied depending on whether the distribution of
data was parametric or nonparametric, respectively. Mathematical
processing and statistical analysis were performed with the software
SIGMAPLOT 5.01 and SIGMASTAT 1.02 (Jandel Scientific, Corte Madera,
CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MSH secreted during 2 h by HD cells
under basal conditions (19.4 ± 3.0 ng/100,000 cells; n = 4)
was significantly less than that secreted by LD cells (96.0 ±
13.8 ng/100,000 cells; n = 4; P < 0.001).
Incubation of the cells with 100 nM TRH provoked a
significant stimulation of MSH secretion in the two melanotrope cell
subsets (Fig. 1). The relative increase in MSH
release was 2-fold higher in LD cells (+307%) than in HD cells
(+141%).
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MSH-immunoreactive peptides secreted by the
melanotrope cell subpopulationsMSH in culture media
of LD and HD cells (Fig. 2
). Peaks I and II exhibited
the same retention time as the sulfoxide derivatives of
des-N-acetyl MSH and MSH,
respectively, whereas peaks III and IV coeluted with
des-N-acetyl MSH and MSH,
respectively. A minor component (peak V) coeluting with diacetyl MSH
was also resolved in culture medium of LD cells. The sulfoxide form of
diacetyl MSH (retention time, 35 min) was not detected. Therefore,
peaks I and III represent the nonacetylated forms of MSH, whereas
peaks II, IV, and V represent the acetylated forms.
Under basal conditions, the proportions of
des-N-acetyl MSH and acetylated forms
of MSH secreted by HD cells were not significantly different.
Conversely, the amount of acetylated forms of MSH secreted by LD
cells was significantly higher than that of
des-N-acetyl MSH (Fig. 3). Specifically, the percentage of acetylated forms
secreted by the two melanotrope cell subsets was 38.3% in HD cells and
57.9% in LD cells. Incubation of HD cells with 100 nM TRH
produced a significant increase in the proportion of acetylated forms
of MSH released by the cells (P < 0.05; Fig. 3). In
contrast, although TRH provoked a significant increase in the total
amount of MSH released from LD cells (P < 0.001),
the proportions of nonacetylated and acetylated forms of MSH
secreted under these conditions were similar (Fig. 3).
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). The
percentages of cells responsive to TRH were similar in the two cell
subsets, i.e. 62.3% in HD and 66.1% in LD cells.
Sequential administration of TRH provoked a substantial attenuation of
the Ca2+ response in both cell subpopulations (Fig. 4).
Quantitative analyses of different parameters calculated from such
experiments are summarized in Table 1. The maximal
[Ca2+]i achieved after the first stimulation
was significantly higher in LD cells than in HD cells
(P < 0.01). In contrast, the percentage of
[Ca2+]i increment after the first ejection of
TRH was significantly higher in HD cells than in LD cells
(P < 0.01). The lag between the administration of TRH
and the maximum [Ca2+]i response was not
significantly different in the two cell subpopulations. In both cell
subsets, the lag period was significantly shorter after the second and
third pulses than after the first pulse of TRH (P <
0.01).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MSH acetylation rates,
[Ca2+]i patterns, and POMC mRNA contents.
Functional heterogeneity of melanotrope cell subpopulations under
basal conditions
Separation of pituitary cells by density gradients has previously
demonstrated the existence of cell subpopulations exhibiting
differential secretory activity. For instance, in aging rats (43) and
prepubertal pigs (11), HD somatrope cells were found to secrete higher
amounts of GH than LD cells. Reverse hemolytic plaque assay and
immunoblotting techniques have also shown that individual pituitary
cells belonging to the same phenotype may differ in their secretory
activity (2, 8, 44). The present study indicates that the amount of
MSH secreted by LD melanotrope cells during a 2-h incubation is 5
times higher than that secreted by HD cells. In agreement with these
data, studies conducted on rat lactotrope cells (which exhibit a number
of functional similarities with amphibian melanotrope cells) have shown
that LD cells secrete more PRL than HD cells (9).
It is well established that the N-terminal acetylation of MSH is an
important posttranslational event that determines the biological
activity of the peptide (21). As in amphibians, acetylation of MSH
occurs just before or during the exocytotic process (22, 23), we have
investigated the acetylation activity in each melanotrope cell
subpopulation. HPLC analysis of conditioned culture medium confirmed
that LD cells have a higher secretory rate than HD cells and revealed
that HD cells secrete a lower proportion of acetylated forms of MSH
than LD cells. These findings indicate that the two melanotrope cell
subsets exhibit distinct secretory patterns; LD cells have a higher
secretory and acetylation activity than HD cells. As the amount of
biologically active (i.e. acetylated) forms of MSH
released by LD cells is 8 times higher than that released by HD cells,
our data indicate that LD cells must play a predominant role in the
maintenance of skin pigmentation.
The relationship between [Ca2+]i and
secretory activity of melanotrope cells is well documented in mammals
(45) and amphibians (46, 47). We thus compared the resting
[Ca2+]i in the two subpopulations of frog
melanotrope cells. Our data revealed that
[Ca2+]i was higher in LD than in HD cells,
indicating that the elevated secretory rate of the LD cell subset can
be accounted for by a high basal [Ca2+]i.
Studies conducted in Xenopus laevis melanotrope cells have
shown that the N-type calcium channel blocker 
-conotoxin inhibits
spontaneous Ca2+ oscillations, whereas the L-type calcium
channel blocker nifedipine has no effect (48), indicating that N-type
voltage-operated Ca2+ channels are responsible for
maintenance of basal [Ca2+]i. In frog
melanotrope cells, both N- and L-type Ca2+ channels have
been characterized (49, 50). Whether the higher
[Ca2+]i observed in LD cells can be ascribed
to an increased density in N-type Ca2+ channels awaits
further investigations.
A number of studies have demonstrated a correlation between hormone
secretion and gene transcription in endocrine cells. For instance, in
rats, LD lactotrope cells exhibit both a high rate of PRL secretion and
a high concentration of PRL mRNA (9). Consistent with this concept, the
present study revealed that frog LD melanotrope cells contain a higher
amount of POMC transcripts than HD cells. We have previously shown that
LD melanotrope cells are less granulated and contain a lower
concentration of MSH than HD cells (35). Taken together, these
observations suggest that LD cells display a high transcriptional
activity, which contributes to replenish their POMC store and
compensate their elevated secretory rate. In contrast, HD melanotrope
cells, with a lower secretory capacity, would store the synthesized
hormone in secretory granules and release it at a much lower rate.
Effect of TRH on the melanotrope cell subpopulations
It has previously been shown that TRH is a potent MSH-releasing
factor in frogs (29, 30, 51, 52) and toads (31). We thus compared the
responses of the two melanotrope cell subsets to TRH. The present data
indicate that TRH was more potent in stimulating MSH release from LD
than HD cells. These data are consistent with other reports showing
variations in the responsiveness of rat lactotrope cells to TRH
(53, 54, 55).
In amphibians, acetylation of MSH is a regulated process that can be
modulated by dopamine (26, 27). In fish, TRH administration
specifically enhances the release of diacetyl MSH (56).
Paradoxically, the effect of TRH on acetylation of MSH has never
been investigated in amphibians. The present study demonstrated that
TRH causes an increase in the rate of acetylation of MSH in HD cells
without affecting the proportion of acetylated MSH in LD cells.
These data suggest that TRH stimulation causes recruitment of the
quiescent HD cells that are stimulated to produce predominantly the
biologically active, i.e. acetylated form, of MSH.
The mechanism of action of TRH has been investigated in detail in GH3 cells (57, 58, 59). Activation of TRH receptors causes stimulation of phospholipase C, leading to calcium mobilization from intracellular stores. In amphibian melanotrope cells, TRH also activates polyphosphoinositide metabolism (32). We now show that TRH produces a marked increase in [Ca2+]i in the two melanotrope cell subpopulations. Repeated administration of TRH provoked an attenuation of the response, which can be ascribed to conversion of the receptor from a high to a low affinity state (60) probably due to receptor phosphorylation (61). Interestingly, the maximum [Ca2+]i evoked by TRH activation was observed in LD cells. It has been shown that in rat melanotrope cells, a certain [Ca2+]i threshold is required to trigger exocytosis (45). Therefore, the greater responsiveness of LD melanotrope cells to TRH may be accounted for at least in part by the higher efficacy of TRH to induce calcium mobilization in the LD cell subset.
Finally, we found that TRH caused a significant increase in POMC mRNA
content in LD cells, but not in HD cells. The ability of TRH to
activate POMC gene transcription is consonant with the more pronounced
effect of TRH on the secretory capacity of the LD cell subpopulation.
In summary, our data indicate that the predominant effect of TRH on
MSH release from frog LD melanotrope cells is associated with a
stronger effect on [Ca2+]i elevation and an
increase in POMC mRNA level in the LD cell subpopulation.
Physiological relevance of the existence of melanotrope cell
subpopulations
The phenomenon of cell heterogeneity has been described for
various types of pituitary cells (12, 35, 43, 62), but the
physiological relevance of this phenomenon has been difficult to
interpret due to the complexity of the regulation and the wide range of
functions controlled by each endocrine cell type. In amphibians, it is
reasonable to assume that the heterogeneity of melanotrope cells must
be directly related to their main physiological role, that is the
regulation of skin pigmentation (20). Numerous studies have reported
the effects of background color adaptation on the activity of pars
intermedia cells in the toad Xenopus laevis. In particular,
it has been shown that melanotrope cells from black background-adapted
animals exhibit a high biosynthetic and secretory activity (63). It has
also been reported that in the medium of perifused neurointermediate
lobes from dark-adapted toads, the proportion of acetylated MSH is
much higher than that in the medium from white-adapted animals (25).
Similarly, the POMC gene is actively expressed during adaptation of
Xenopus laevis to a black background (64). These
observations strongly suggest that LD cells, which exhibit an intense
secretory activity, a high acetylation rate, and an elevated POMC mRNA
content, are actually responsible for the process of skin color
adaptation. In support of this hypothesis, De Rijk et al.
(65) reported that pars intermedia cells from black-adapted
Xenopus laevis are scarcely granulated and possess a
developed rough endoplasmic reticulum; in other words, they have the
same morphological characteristics as Rana ridibunda LD
cells (35).
The heterogeneity of melanotrope cells in the frog pars intermedia is
consistent with the idea of a cell secretory cycle by which the cells
may regulate MSH secretion in a flexible and balanced way. The
relative importance of each subpopulation may then depend on the amount
of hormone required in a particular physiological state. Therefore,
further investigations are needed to elucidate the implication of cell
heterogeneity in the process of skin color adaptation.
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Acknowledgments |
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MSH
standards, and to Dr. Ch. López-Pedrera (Centro de
Investigación Biomédica, Hospital Universitario Reina
Sofía, Córdoba, Spain) for providing human monocyte THP-1
cells. |
Footnotes |
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Received September 9, 1996.
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  J. R. Peinado, R. Vazquez-Martinez, D. Cruz-Garcia, A. Ruiz-Navarro, Y. Anouar, M. C. Tonon, H. Vaudry, F. Gracia-Navarro, J. P. Castano, and M. M. Malagon Differential Expression and Processing of Chromogranin A and Secretogranin II in Relation to the Secretory Status of Endocrine Cells Endocrinology, March 1, 2006; 147(3): 1408 - 1418. [Abstract] [Full Text] [PDF]
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 R. Vazquez-Martinez, J. P. Castano, M. C. Tonon, H. Vaudry, F. Gracia-Navarro, and M. M. Malagon Melanotrope secretory cycle is regulated by physiological inputs via the hypothalamus Am J Physiol Endocrinol Metab, November 1, 2003; 285(5): E1039 - E1046. [Abstract] [Full Text] [PDF]
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L. Galas, M.-C. Tonon, D. Beaujean, R. Fredriksson, D. Larhammar, I. Lihrmann, S. Jegou, A. Fournier, N. Chartrel, and H. Vaudry Neuropeptide Y Inhibits Spontaneous {alpha}-Melanocyte-Stimulating Hormone ({alpha}-MSH) Release via a Y5 Receptor and Suppresses Thyrotropin-Releasing Hormone-Induced {alpha}-MSH Secretion via a Y1 Receptor in Frog Melanotrope Cells Endocrinology, May 1, 2002; 143(5): 1686 - 1694. [Abstract] [Full Text] [PDF]
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R. Vazquez-Martinez, J. R. Peinado, J. L. Gonzalez de Aguilar, L. Desrues, M. C. Tonon, H. Vaudry, F. Gracia-Navarro, and M. M. Malagon Melanotrope Cell Plasticity: A Key Mechanism for the Physiological Adaptation to Background Color Changes Endocrinology, July 1, 2001; 142(7): 3060 - 3067. [Abstract] [Full Text] [PDF]
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L. Galas, M. Lamacz, M. Garnier, E. W. Roubos, M.-C. Tonon, and H. Vaudry Involvement of Protein Kinase C and Protein Tyrosine Kinase in Thyrotropin-Releasing Hormone-Induced Stimulation of {alpha}-Melanocyte-Stimulating Hormone Secretion in Frog Melanotrope Cells Endocrinology, July 1, 1999; 140(7): 3264 - 3272. [Abstract] [Full Text]
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J. L. González de Aguilar, M. M. Malagón, R. M. Vázquez-Martínez, A. J. Martínez-Fuentes, M. C. Tonon, H. Vaudry, and F. Gracia-Navarro Differential Effects of Dopamine on Two Frog Melanotrope Cell Subpopulations Endocrinology, January 1, 1999; 140(1): 159 - 164. [Abstract] [Full Text]
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M. Garnier, M. Lamacz, L. Galas, S. Lenglet, M.-C. Tonon, and H. Vaudry Pharmacological and Functional Characterization of Muscarinic Receptors in the Frog Pars Intermedia Endocrinology, August 1, 1998; 139(8): 3525 - 3533. [Abstract] [Full Text] [PDF]
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R. Hnasko, S. Khurana, N. Shackleford, R. Steinmetz, M. J. Low, and N. Ben-Jonathan Two Distinct Pituitary Cell Lines from Mouse Intermediate Lobe Tumors: A Cell that Produces Prolactin-Regulating Factor and a Melanotroph Endocrinology, December 1, 1997; 138(12): 5589 - 5596. [Abstract] [Full Text] [PDF]
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