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3-Melanocyte-Stimulating Hormone Detected through Intracellular Ca2+ Responses in Immature Rat Pituitary Constitute a Fraction of All Main Pituitary Cell Types, but Mostly Express Multiple Hormone Phenotypes at the Messenger Ribonucleic Acid Level. Refractoriness to Melanocortin-3 Receptor Blockade in the Lacto-Somatotroph Lineage1
Laboratories of Cell Pharmacology and Physiology (G.C.), University of Leuven Medical School, Campus Gasthuisberg (O & N), B-3000 Leuven, Belgium
Address all correspondence and requests for reprints to: Prof. Carl Denef, Laboratory of Cell Pharmacology, University of Leuven Medical School, Campus Gasthuisberg (O & N), B-3000 Leuven, Belgium. E-mail: carl.denef{at}med.kuleuven.ac.be
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Abstract |
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3-MSH has recently been shown to be a biologically active peptide in
the rat anterior pituitary. It induces a sustained rise in
intracellular free calcium levels ([Ca2+]i)
in a relatively small population of immature pituitary cells. The
present study was intended to identify the target cells of this peptide
and to discern the signal-transducing melanocortin (MC) receptor. In
dispersed pituitary cells from 14-day-old rats, increasing doses of
3-MSH (0.1, 1, and 10 nM) evoked a sustained oscillating
[Ca2+]i rise in an increasing number of cells
(up to 14.5%). Within the responsive cells, 53% showed GH
immunoreactivity (-ir), 12% showed PRL-ir, 2% showed TSHß-ir, 5%
showed LHß-ir, and 10% showed ACTH-ir, whereas 18% did not express
any hormone-ir to a detectable level. As assessed by single cell RT-PCR
for the presence of pituitary hormone messenger RNA (mRNA), 26% of the
3-MSH-responsive cells contained only GH mRNA, 5% contained only
PRL mRNA, and 4% contained only TSHß mRNA. Twenty-two percent
contained mRNA of GH, PRL, and TSHß in various dual or triple
combinations. About 24% of the 3-MSH-responsive cells expressed
POMC mRNA, mostly together with other mRNAs, i.e. with
GH mRNA and/or PRL mRNA or with mRNA of GH, PRL, and TSHß. Eighteen
percent of the responsive cells expressed LHß, all of them together
with mRNA of GH, PRL, and TSHß in various combinations. The absence
of hormone mRNA was found in less than 1% of the responsive cells. In
cells chosen at random (representative of the total pituitary cell
population), the proportion of cells expressing two or multiple hormone
mRNAs was twice as low as that in the 3-MSH-responsive population,
whereas the proportion of cells expressing a single hormone mRNA was
twice as high (about two thirds of all cells). Moreover, unlike in the
3-MSH-responsive cell population, randomly chosen cells were found
that coexpressed POMC mRNA with LHß mRNA.
The effect of 3-MSH on [Ca2+]i was blocked
by the MC-3 receptor antagonist SHU9119 (used up to a 1000-fold excess)
in 46% or less of the responsive cells. SHU9119 failed to block the
[Ca2+]i response to 3-MSH in PRL-, GH-,
and TSHß-ir cells, but it did block the response in most ACTH-ir
cells and in cells expressing no hormone to a detectable level. Single
cell RT-PCR revealed that expression of MC-3 receptor mRNA was detected
in only 16% of 3-MSH-responsive cells.
The present data suggest that the target cells of 3-MSH in terms of
[Ca2+]i responses in the immature rat
pituitary constitute subpopulations of all main pituitary cell types,
including nonhormonal (or low expression hormonal) cells. However, in
contrast to the total pituitary cell population, most of these cells
display multilineage gene activation at the mRNA level,
i.e. express mRNA of GH, PRL, TSHß, POMC, and LHß in
dual, triple, or quadruple combinations. Although 3-MSH may act
through the MC-3 receptor in a portion of these cells, most of these
cells (mainly in the lacto-somatotroph lineage) may transduce the
signal through another receptor or through an MC-3 receptor with
unconventional binding characteristics.
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Introduction |
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3-MSH AND THE C-terminally truncated
derivatives 1- and 2-MSH, are peptides that can be generated
from the N-terminal fragment of POMC (POMC1–74)
(1). These peptides exert similar as well as divergent effects in the
brain, adrenal gland, kidney, and cardiovascular system (reviewed in
Refs. 2, 3). We recently showed that 3-MSH is biologically active
in the anterior pituitary of immature rats. It has a mitogenic action
on lactotrophs, somatotrophs, and thyrotrophs (4). Moreover, it induces
a sustained oscillating rise in the intracellular free Ca2+
concentration ([Ca2+]i) in an unidentified
small proportion (
14%) of the cells (5). The peculiarity of this
response is that the magnitude and the frequency of
[Ca2+]i oscillations does not change with the
concentrations of 3-MSH applied, indicating that the cells respond
in an all or none manner. However, increasing the concentration results
in a significant rise in the percentage of cells showing
[Ca2+]i responses to the peptide, suggesting
that the dose determines the number of cells recruited to respond or
that the threshold of response differs according to cell type. The
latter characteristic is interesting in view of recent findings that
signaling molecules during vertebrate development, such as members of
the transforming growth factor-ß family, act by establishing a
concentration gradient from the site of production and that distinct
threshold concentrations are sufficient to pattern distinct cell types
and activate different genes (6, 7, 8, 9).
The candidate receptor mediating the actions of 3-MSH is the
melanocortin-3 (MC-3) receptor, as this receptor is the only one among
the five MC receptors known today that mediates the actions of 3-MSH
in low nM doses (10, 11, 12). However, several observations on
the action of -MSH peptides in the cardiovascular system suggest the
existence of other receptors for which these peptides have high
affinity (3). Although MC-3 receptor messenger RNA (mRNA) is present in
the anterior pituitary of immature rats (5), we found that the MC-3
receptor antagonist SHU9119 (13) blocked the effect of 3-MSH on
[Ca2+]i in less than 50% of the responsive
cells (5), suggesting that more than one 3-MSH receptor may exist in
the pituitary: a typical MC-3 receptor and either a variant of the
latter or a hitherto unidentified 3-MSH receptor.
In view of the above data, the present investigation was intended to
identify the cell type(s) displaying [Ca2+]i
responses to 3-MSH and to determine which of the responsive cell
types are sensitive and which are refractory to blockade by SHU9119.
The cells showing [Ca2+]i responses to
3-MSH were visualized by fluo-3 imaging, and the hormone phenotypes
expressed were identified at the protein level by means of
immunostaining and at the mRNA level by single cell RT-PCR. The latter
technique has been used for the simultaneous detection of different
mRNA species within a single cell (14, 15) in complex tissues, such as
brain (16), hemopoietic system (17), and pituitary gland (18).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3-MSH was obtained from Peninsula Laboratories, Inc., Europe (Merseyside, UK).
Ac-Nle4-cyclo[Asp5,(D-Nal
(2)7,Lys10]
-MSH-(4–10)-NH2
(SHU9119) (13) was purchased from Neosystem Laboratoire (Strasbourg,
France). Peptides were prepared as stock solution (0.1 mM)
in 0.1% crystalline BSA and kept at -25 C. All reagents were
analytical grade.
Animals
Female Wistar rats, 12–15 days old, were obtained from the
University Animal Breeding Facility (Heverlee, Belgium). They were
killed by decapitation.
Pituitary cell dissociation and short term culture
Whole pituitaries were cut into small tissue blocks and
enzymatically dispersed into single cells as previously described
(19, 20, 21). For Ca2+ imaging, dispersed cells were seeded on
a Lab-Tek chamber slide (1.8 cm2/chamber; 500,000
cells/chamber; Nunc, Roskilde, Denmark). For photometric
Ca2+ recording followed by single cell RT-PCR, dispersed
cells were seeded on glass coverslips (Prochilab, Bordeaux, France).
The coverslips (Bellco, Vineland, NJ) used for identification by
immunostaining after Ca2+ imaging were photoengraved with a
numbered/lettered grid to enable accurate location of the
Ca2+-recorded cells. In all cases, Lab-Tek and glass
coverslips were coated with poly-L-ornithine (0.001%;
Sigma Chemical Co., St. Louis, MO). Cells were maintained
in serum-free defined medium (19, 20, 21) containing 0.5% BSA at 37 C in a
humidified atmosphere (1.5% CO2-98.5% air), and
[Ca2+]i changes in response to 3-MSH were
tested within 24 or 48 h of culture.
Detection of
[Ca2+]i changes in
response to 3-MSH
[Ca2+]i was recorded in individual
cells either by fluo-3 video imaging, allowing the simultaneous study
of 20–40 cells/field (immunostaining studies), or by conventional
photometric microspectrofluorometry (patch-clamp single cell RT-PCR
studies). The culture medium was replaced with HBSS containing 1.3
mM Ca2+, pH 7.3. Cells were then incubated for
20 min at 37 C with 10 µM fluo-3 acetoxymethyl ester
(fluo-3/AM) and 0.02% pluronic F127 in HBSS. After loading, cells were
washed with and kept in HBSS during recordings. Recording of the
fluorescence was performed at room temperature. The dye was excited at
488 nm, and the emitted light was recorded at 530 nm.
Details of fluo-3 video imaging, including the criteria used to determine whether a given cell type was responding, were previously described (5, 21). As the lack of emission or excitation spectral shift of fluo-3 upon Ca2+ binding makes it difficult to calibrate fluorescence signals in terms of precise absolute values of [Ca2+]i, all fluorescence measurements (expressed in arbitrary units) are values normalized to the function of the basal fluorescence intensities after background correction. It is then possible to extrapolate [Ca2+]i from fluo-3 fluorescence changes and to consider that an increase in fluo-3 fluorescence corresponds to an increase in [Ca2+]i and a decrease of fluorescence, to a decrease of [Ca2+]i.
For the photometric microspectrofluorometry in the patch-clamp single cell RT-PCR studies, the glass coverslips holding the cultured cells were sealed in a hollowed plastic petri dish, which was placed on the microscope stage equipped for epifluorescence (x40 oil immersion objective, NA 1.3, Nikon, Melville, NY). Emitted fluorescence was measured from a field slightly larger than the cell under study and detected by a photomultiplier tube (Hamamatsu type R928, Hamamatsu Photonics, Hamamatsu City, Japan) at 535 ± 35 nm. Fluorescence signals were digitized on a PC and analyzed off-line with Axotape Software (Version 2.0.2, Axon Instruments, Inc., Foster City, CA). Fluorescence signals are expressed as ratios (F/F0) of the fluorescence during a response (F) relative to the resting fluorescence before stimulation (F0).
Test substances (3-MSH, SHU9119), diluted in recording medium
(HBSS), were applied to the recorded cells by perfusion in the case of
video imaging and by low pressure ejection from micropipettes (3- to
5-µm tip diameter) positioned approximately 40 µm from the cell in
the case of the photometric technique.
As previously described (21), 53% of the pituitary cells displayed
spontaneous [Ca2+]i oscillations with
variable frequency and amplitude and therefore were called
spontaneously oscillating cells. Cells not showing these oscillations
were called nonoscillating cells. Both oscillating and nonoscillating
cells were included in the present study. Only a small number of cells
that were spontaneously nonoscillating during 1 min of examination
started to show some spontaneously oscillating
[Ca2+]i transients during a further 2-min
examination; this number was 8 cells of 347 cells registered (2.3%).
There was no additional effect of application of the vehicle only; 10
of 483 registered cells (2.07%) became spontaneously oscillating. As
among the 3-MSH-responsive cells there were about twice as much
spontaneously oscillating as spontaneously nonoscillating cells (5),
and as data for responding cells included both of these cell
populations, the proportional number of potentially false positive
responders to 3-MSH would be about 0.7%, which was within
experimental variation.
Identification of cells responsive to 3-MSH, in the absence and
presence of SHU9119, by triple immunofluorescence of pituitary
hormones
The accurate location of all pituitary cells for which
[Ca2+]i had been recorded
(3-MSH-responsive and nonresponsive cells) was ascertained by the
numbered and lettered grid pattern on the coverslip. In this way, all
[Ca2+]i-recorded cells could be caught again
after the immunostaining step to determine their hormone
immunoreactivity (-ir) content.
The hormone content of the cells was identified as previously described (21, 22). Briefly, cells were fixed in Zamboni fluid (4% paraformaldehyde and 15% saturated picric acid in phosphate buffer, pH 7.6). In a first series, triple immunostaining was performed using rabbit antiserum against rat PRL (rabbit anti-rPRL-IC5, diluted 1:6,000), monkey antiserum against rat GH (monkey anti-rGH-IC1, diluted 1:6,000), and guinea pig antiserum against rat TSHß (guinea pig anti-rTSHß-IC, diluted 1:4,000), all provided by the NIDDK through the National Hormone and Pituitary Program. Cells were incubated with all antisera overnight at 4 C. The secondary antibodies, Cy3-labeled goat antirabbit IgG(H+L) (diluted 1:200; from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; orange-red fluorescence), fluorescein isothiocyanate (FITC)-labeled goat antimonkey IgG (diluted 1:150; from Nordic, Tilburg, The Netherlands; green fluorescence), and 7-amino-4-methylcoumarin-3-acetic acid (AMCA)-labeled antiguinea pig IgG(H+L) (diluted 1:100; from Jackson ImmunoResearch Laboratories, Inc.; blue fluorescence), were applied for 2 h at room temperature. In a second series, monkey anti-rGH antiserum, rabbit antiserum against human ACTH (diluted 1:10,000), and guinea pig antiserum against rLH-ß (diluted 1:4,000), all provided by the NIDDK through the National Hormone and Pituitary Program, were simultaneously applied, followed by incubation with the above-mentioned second antibodies. The immunoreactive cells were visualized under a Leica Corp. microscope equipped for transmitted light fluorescence (Leica Corp., Wetzlar, Germany) using I3, UVA, and N2.1 filters for FITC, AMCA, and Cy3 fluorescence, respectively. Omission of the first antibody abolished staining. Replacing the first antibody with nonimmune rabbit or guinea pig serum did not result in any fluorescent staining with the FITC-, AMCA-, or Cy3-labeled antibodies, all three applied with each of the primary nonimmune sera. For each hormone staining, positive fluorescent cells were only seen with the homologous fluorescent-labeled second antibody and not with the heterologous second antibodies. For example, positive staining with rabbit antirat PRL antiserum was obtained with Cy3-labeled goat antirabbit IgG, but not with FITC-labeled goat anti-monkey IgG or AMCA-labeled anti-guinea pig IgG.
Single cell
[Ca2+]i measurements
and single cell RT-PCR of hormone, MC-3 receptor, and L19
mRNAs
Single cell RT-PCR was performed using protocols similar to
those previously described (23, 24). Details in the present study are
as follows.
Detection and harvest of cytoplasm of cells showing
[Ca2+]i changes in
response to 10 nM 3-MSH. The cells
responsive to 3-MSH were detected on the basis of an increase in
fluo-3 fluorescence reflecting an increase in
[Ca2+]i increase as described above. The
average time between the application of 3-MSH and response of the
cells was 28.4 ± 1.87 sec (mean ± SEM;
calculated for 92 cells). In case a change in fluo-3 fluorescence was
noted, the cytoplasm of the responsive cell was collected using the
patch-clamp technique. Patch pipettes were pulled from heated (200 C,
overnight) borosilicate glass tubing. When filled with the internal
pipette solution (10 µl 140 mM KCl, 2 mM
MgCl2, 1.1 mM EGTA, and 10 mM
HEPES, pH 7.25), the patch pipettes showed an electrical resistance of
2–5 M
. A high resistance seal was formed between the patch pipette
and the cell membrane (25). After establishing a whole cell patch-clamp
configuration, the cell contents were aspirated under visual control.
The pipette content (8 µl) was ejected into an
Eppendorf microtube without touching the tube wall,
immediately frozen, and kept at -80 C until processed. For each cell
patch-clamped and harvested, a new pipette was used.
RT reaction. The RT reaction for each cell was performed on the harvested cytoplasm using random hexamer primers without prior RNA purification. Each individual cell cytoplasm was adjusted to 8.8 µl with internal pipette solution, then heated to 65 C for 10 min to break up secondary mRNA structures and placed on ice for 5 min or more. The RT reaction was performed in a final volume of 20 µl for each cell cytoplasm using the following final reaction conditions: 10 mM Tris-HCl (pH 8.3); 56 mM KCl; 3.8 mM MgCl2; 0.5 mM each of deoxy (d)-ATP, dCTP, dGTP, and dTTP (Perkin-Elmer Corp., Foster City, CA); 3 µM random hexamer oligonucleotides (Perkin-Elmer Corp.); 50 U murine leukemia virus (MuLV) reverse transcriptase (Perkin-Elmer Corp.); and 20 U ribonuclease inhibitor (Perkin-Elmer Corp.). The reaction mixture was incubated for 10 min at 20 C to allow hybridization of the primers to the RNA. The RT was performed at 42 C for 20 min, followed by a 5-min incubation at 95 C, then chilled on ice and stored at -20 C until PCR amplification. In the case of RT of MC-3 receptor mRNA, cell cytoplasm was divided into a portion on which the RT reaction was performed and a control portion on genomic DNA without RT (MuLV reverse transcriptase and ribonuclease inhibitor omitted), as MC receptor genes are intronless.
PCR amplification. The PCR primers used to amplify the
RT-generated pituitary hormone, MC-3 receptor, and ribosomal protein
L19 complementary DNAs (cDNAs) were designed on the basis of
established GenBank sequences with commercially available software
(Genejockey II, Biosoft, Cambridge, UK). Primers were
synthesized by Pharmacia Biotech (Uppsala, Sweden). The
primer sequences, GenBank accession numbers of the template sequence
with positions of the primers, and expected sizes of the amplified
fragments are summarized in Table 1
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-subunit (GSU), and ribosomal protein
L19 (all in independent amplification reactions), PCR was performed by
adding 3 µl RT template to a mixture of (final concentrations): 50
mM KCl, 10 mM Tris-HCl (pH 8.3), 4.5
mM MgCl2, 200 µM of each dNTP,
sense and antisense primer (1 µM of each), and 1 U
AmpliTaq Gold (Perkin Elmer Corp.) in a final volume of 25
µl. For PCR of MC-3 receptor cDNA, 5 µl reverse transcribed (+RT)
and nonreverse transcribed (-RT) template was used in a final volume
of 25 µl. The conditions of DNA amplification were the same for all
pairs of primers and included an initial denaturation step of 10 min at
95 C (which also activates the Gold variant of the Taq
polymerase); 45 cycles of 20 sec at 95 C, 20 sec at 60 C, and 30 sec at
72 C; and finally 7 min at 72 C. The nested PCR for MC-3 receptor cDNA
was performed using 2 µl of the first PCR product as template under
the same reaction conditions, except for the final concentration of
MgCl2 (1.5 mM), and the number of cycles (=
40). Half of the PCR samples were electrophoresed in a 2% agarose gel
= stained with ethidium bromide (EtBr; 0.5 µg/ml).
Sensitivity of the RT-PCR. To assess the sensitivity of our
RT-PCR method, standards of POMC and MC-3 receptor cDNA and of POMC
mRNA were prepared. MC-3 receptor cDNA was cloned by RT-PCR from total
rat pituitary RNA using as 5'-primer
5'-CGCAAGCTTCGCCGATAACCATGAACTCT-3' containing a HindIII
restriction site, and as 3'-primer 5'-CGCGGATCCCTCGGGGTTCCTAGCCCAC-3'
containing a BamHI site (5). The 1012-bp amplified fragment
was inserted into the HindIII and BamHI cloning
sites of pcDNA3 (Invitrogen, Groningen, The Netherlands).
Plasmid DNA was propagated, purified, and digested with
HindIII and BamHI, and the MC-3 receptor cDNA
fragment was eluted from agarose gel with the Geneclean II Kit (Vista,
CA). POMC cDNA was obtained by EcoRI digestion of pBSrPOMC
(prPAE, provided by Dr. R. Mains, Johns Hopkins University, Baltimore,
MD), followed by gel purification of the 907-bp fragment (QIAquick Gel
Extraction Kit, QIAGEN, Valencia, CA). POMC mRNA was
prepared by in vitro transcription of linearized pBSrPOMC
with T7 RNA polymerase. RNA-free pBSrPOMC was prepared with the
QIAGEN Plasmid Kit, linearized with BamHI,
ethanol precipitated, and used for in vitro transcription
during 3 h at 37 C in the following reaction conditions: 4 µg
linearized DNA template, 1 x transcription buffer, 40 U T7 RNA
polymerase, 1 mM of each rNTP (all from Roche Molecular Biochemicals, Brussels, Belgium), and 20 U
ribonuclease inhibitor (Perkin Elmer Corp.). Full-length
transcripts (985 bp) were purified from a nondenaturing 1% agarose gel
by means of the QIAquick Gel Extraction Kit (QIAGEN) under
ribonuclease-free conditions. Residual DNA template was removed by
treatment with ribonuclease-free deoxyribonuclease I (Life Technologies, Inc., Gaithersburg, MD). The concentrations of the
cDNA and mRNA standards were measured by spectrophotometry (absorbance
at 260 nm) and/or by spectrofluorometry using the PicoGreen double
stranded DNA and RiboGreen RNA quantitation dyes from
Molecular Probes, Inc. (Eugene, OR). PCR, as performed in
the present study, detected 2–3 copies of POMC cDNA and 2–8 molecules
of MC-3 receptor cDNA. With the RT-PCR method as described, we were
able to detect 5–8 copies of POMC mRNA, meaning a 50% efficiency of
the RT reaction. Taking into account that only part of the RT mixture
of the cell lysate is used for amplification, the detection limit of
our RT-PCR method is judged to be in the range of 50 copies of
mRNA/cell for both POMC and MC-3 receptor (supposing that RT efficiency
is 50%). However, cellular environment and secondary and tertiary
structures of RNA as present in the cell cytoplasm may impose
additional constraints.
Controls. As a positive control for the RT-PCR, total pituitary RNA was used. Total RNA from pituitary cells of 14-day-old rats was isolated by the guanidium thiocyanate-phenol-chloroform extraction procedure (26). After a deoxyribonuclease I (Life Technologies, Inc.) treatment to eliminate genomic DNA, 5 µg total RNA were reverse transcribed into cDNA at 42 C using random hexamer primers (Perkin Elmer Corp.) and MuLV reverse transcriptase (Perkin Elmer Corp.) in a 20-µl final volume, followed by PCR, both as described above.
RT and PCR were performed following procedures minimizing the chances
of cross-contamination (27). To rule out contamination between reagents
or individual PCR amplifications, the following controls were performed
in each experiment. First, for every two to four cells harvested, a
pipette control was included, i.e. the patch-clamp pipette
was held for a while in the close vicinity of a cell, and after
withdrawal, the pipette content was ejected in the test tube and
subjected to RT-PCR as described for cell cytoplasm. Second,
contamination from extraneous sources was checked by replacing the
cellular template with water for every set of cells analyzed by RT-PCR.
Experiments were accepted only if all control lanes were negative. On
only one occasion, in a set of seven cells, was there a positive RT-PCR
signal in a pipette control, and these cells were excluded from the
study. Another control consisted of aspirating HBSS medium in which the
cells had been incubated. GH and PRL mRNA signals were not detected in
any of these control samples. Furthermore, amplification of genomic DNA
was checked using primers that flanked a sequence containing at least
one intron or was ruled out using primers that spanned the intron (see
Table 1
). As mentioned above, for detection of MC-3 receptor mRNA
(intronless gene) the PCR was performed on -RT and +RT cytoplasm.
Reproducibility of the amplification reactions was verified on 25 cell cytoplasm samples. The same PCR products were found in both reactions.
Restriction analysis of RT-PCR products. To confirm the
identity of the amplified products, restriction analysis was carried
out. After electrophoresis, PCR products were gel purified using the
Geneclean II kit [BIO 101, Inc. (Westburg, The Netherlands), or
Polylab N.V. (Antwerpen, Belgium)] and used as a template for a second
PCR with the same set of primers. The final reaction volume of 100 µl
contained 1–3 µl gel-purified DNA, 50 mM KCl, 10
mM Tris-HCl (pH 8.3), 1.5 mM MgCl2,
100 µM of each dNTP, 1 µM upstream primer,
1 µM downstream primer, and 1 U AmpliTaq Gold. After 10
min at 95 C; 35 cycles of 20 sec at 95 C, 20 sec at 60 C, and 30 sec at
72 C; and a final step of 7 min at 72 C for elongation, the PCR product
was ethanol precipitated and resuspended in 50 µl H2O.
Eight-microliter aliquots were digested by appropriate restriction
enzymes (all from Roche Molecular Biochemicals, Brussels,
Belgium) as summarized in Table 1. Digested products were analyzed by
electrophoresis in EtBr-stained 2% agarose gel.
Data analysis
All experiments were repeated several times on different
pituitaries and different cultures obtained from different animals.
Where necessary, values were expressed as the mean ±
SEM of several independent experiments and compared by
ANOVA with post-hoc Scheffe’s multiple comparison tests. To
analyze the differences in the number of cells responsive to 3-MSH
in the absence and presence of SHU9119, the 
2 test
or Fisher’s exact test was used. These tests were also used to compare
the number of single hormone mRNA and multiple hormone mRNA containing
cells in the 3-MSH-responsive population with those in the total
pituitary cell population.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3-MSH alone and in combination with the MC-3 receptor
antagonist SHU9119 on
[Ca2+]i 3-MSH at doses of 0.1,
1 and 10 nM caused a sustained oscillating increase in
[Ca2+]i in, respectively, 4.8 ± 2.9%,
10.1 ± 1.1%, and 14.5 ± 0.6% of the analyzed cells (Fig. 1A). A 50- or 100-nM dose did
not further increase the number of responsive cells. In fact, at 100
nM this number was lower again (8.1 ± 0.6%; data not
shown). The 10-nM dose was therefore used in experiments
aimed to identify the target cells of 3-MSH. A representative
recording of the fluo-3 fluorescence changes induced by applying 10
nM 3-MSH is shown in Fig. 1B.
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also shows the effect of MC-3 receptor blockade by SHU9119 on
the [Ca2+]i responses. As previously shown
(5), SHU9119 had to be given 30 min before 3-MSH, because on its own
it showed a temporary stimulatory effect on
[Ca2+]i [most likely through its agonist
action at the MC-5 receptor (13)], and this effect had disappeared
after 30 min. It was also shown that pretreatment with SHU9119 did not
desensitize the cells for later response to 3-MSH (5). When
pituitary cells were pretreated for 30 min with the MC-3 receptor
antagonist SHU9119 (0.1 and 1 µM) and then tested for
[Ca2+]i responses to 1 and 10 nM
3-MSH, still in the presence of the same concentration of the
antagonist, the percentage of cells showing
[Ca2+]i responses significantly decreased.
However, even at 1 µM (a 100- or 1000-fold excess
compared with the dose of 3-MSH), SHU9119 was effective in only 45%
(1 nM 3-MSH) and 46% (10 nM 3-MSH) of
the cells (Fig. 1A). Interestingly, SHU9119 was ineffective in blocking
the effect of 0.1 nM 3-MSH. Consistent with the
observations of others (13), we have found that a 100-fold excess of
SHU9119 over 3-MSH completely blocks the biological action of
3-MSH at the MC-3 receptor, as assessed in a HEK293 cell line stably
transfected with the MC-3 receptor (unpublished data).
Identification of cells responsive to 3-MSH in the absence and
presence of SHU9119
Cells were cultured on coverslips marked with numbered demarcation
lines, allowing accurate localization of the cells to be identified
after the [Ca2+]i recordings were performed
in several microscopic fields. In a first run, cells responsive to 10
nM 3-MSH were analyzed simultaneously for their contents
of PRL, GH, and TSHß by immunofluorescent staining. In a second run,
cells responsive to 10 nM 3-MSH were analyzed
simultaneously for their contents of GH, ACTH, and LHß by
immunostaining. This was done for three independent cell preparations.
Then, the number of ACTH-ir and LHß-ir cells within the
immunonegative cells of the first immunostaining runs were calculated
from the data obtained in the respective second immunostaining runs,
and the number of PRL-ir and TSHß-ir cells within the immunonegative
cells of the second immunostaining runs were calculated from the data
obtained in the respective first immunostaining runs. The numbers of
identified cells within each cell type category obtained in the
different runs as well as the calculated numbers were then combined and
expressed as percentage of total 3-MSH-responsive cells. An example
of the triple immunofluorescent staining is shown in Fig. 2.
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3-MSH. Figure 3 shows that about half of the cells
responsive to 3-MSH in the absence of SHU9119 were GH-ir, but PRL-,
TSHß-, LHß-, and ACTH-ir cells were also found. About 18% of the
3-MSH-responsive cells were not immunoreactive to either of the
antisera applied. From Fig. 3 it is clear that SHU9119 markedly
decreased the proportions of ACTH-ir, LHß-ir, and immunonegative
cells that remained responsive to 3-MSH, but did not lower the
percentages of GH-, PRL-, and TSHß-ir cells that remained responsive.
Statistical analysis (performed on absolute numbers) showed that the
distribution of 3-MSH-responsive cells among the different cell type
categories was significantly changed by SHU9119 (by overall
2 test, P < 0.001), but that for
individual cell types statistical significance was reached only for
ACTH-ir cells (by Fisher’s exact test, P = 0.0237) and
immunonegative cells (by Fisher’s exact test, P =
0.0013). For LHß-ir cells, Fisher’s exact test was not significant
(P = 0.07).
|
3-MSH within each cell type category was expressed as a
percentage of the total number of pituitary cells recorded (Fig. 4).
|
In all the immunostaining runs the number of immunoreactive and
immunonegative cells not responsive to 3-MSH in the microscopic
fields studied was also counted. This allowed calculation of the
proportion of cells in each cell type category that responded to
3-MSH as well as the proportional distribution of the various
pituitary cell types in the total pituitary cell population. It was
found that 23% of the GH-ir cells, 10% of the PRL-ir cells, 10% of
the TSHß-ir cells, 8% of the ACTH-ir cells, 7% of the LHß-ir
cells, and 11% of the immunonegative cells responded to 3-MSH (in
the absence of SHU9119). Figure 3 also shows the proportional
distribution of the various pituitary cell types in the total pituitary
cell population. The proportion of GH-ir cells in the
3-MSH-responsive population was significantly higher than that in
the total pituitary cell population (by Fisher’s exact test, using
absolute numbers, P = 0.0001), indicating preference of
3-MSH for target GH-ir cells.
Detection of hormone mRNAs and MC-3 receptor mRNA in cells
responsive to 3-MSH by single cell RT-PCR
To identify cells responsive to 3-MSH at the mRNA level, the
technique of [Ca2+]i measurements was
combined with single cell RT-PCR. In initial experiments, mRNA of
pituitary hormones, of the MC-3 receptor, and of the ribosomal protein
L19 were amplified from a RNA extract of the entire pituitary of
14-day-old rats using RT-PCR. Amplification conditions were optimized,
yielding single bands of the predicted size for each primer set (see
also Table 1). Next, a similar analysis was performed on cytoplasm
harvested from single pituitary cells responsive to 10 nM
3-MSH. Examples of results obtained after single cell RT-PCR and
agarose gel electrophoresis are shown in Figs. 5 and 6.
Digestion with appropriate restriction enzymes yielded DNA fragments of
the expected sizes (Fig. 7 and Table 1),
confirming the identities of the products amplified from single cell
cDNA.
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|
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A total of 152 cells showing an increase in
[Ca2+]i upon application of 10 nM
3-MSH were analyzed. All cells were tested for the presence of mRNA
of GH, PRL, TSHß, and POMC. In 17 cells none of the above mRNAs could
be detected. However, all of these cells except 1 were also negative
for the mRNA of the ribosomal protein L19. These 16 L19-negative cells
were considered false negative cells, possibly due to failure of
cytoplasm aspiration by the patch pipette, inadequacy of the RT
reaction, or degradation of RNA. There was not sufficient cytoplasmic
material to test all envisaged mRNAs on all cells. Therefore, 69 cells
were tested for the presence of MC-3 receptor mRNA, and another 53 were
tested for the presence of mRNA of LHß. All LHß-positive cells were
also tested for GSU mRNA. Figure 8
shows the distribution of the different hormone mRNAs as a percentage
of the total 3-MSH-responsive cells, not including the L19-negative
cells. In 26% of the responsive cells only GH mRNA was found. Only a
small number of cells contained only PRL mRNA, only TSHß mRNA, or
only POMC mRNA. In contrast, a considerable number of cells contained
the mRNAs of GH, PRL, TSHß, and POMC in dual, triple, and even
quadruple combinations. Of all of the cells tested (136 cells), there
was only 1 that did not contain 1 or more of these mRNAs. LHß mRNA
was always found together with mRNA of GH and/or PRL or TSHß,
amounting to 18% of the 3-MSH-responsive cells. These cells also
contained GSU mRNA (see also Fig. 6).
|
also shows the distribution of the different hormone mRNAs in
cells patched at random (representing the total pituitary cell
population). It is clear that the proportion of cells containing one
mRNA in the total pituitary cell population is twice as high as that in
the 3-MSH-responsive population, whereas in the latter, the
proportion of cells containing transcripts of two or more hormone mRNAs
was twice as high as that in the total population (by 2
test, P = 0.0008). Moreover, in the randomly chosen
cells there were cells that coexpressed POMC mRNA with LHß mRNA,
whereas these cells were not found in the 3-MSH-responsive cell
population. These results were not affected by the time in culture, as postexamination of the mRNA distributions in the cells grouped according to time in culture (24 or 48 h) gave essentially the same results (data not shown).
Figure 9 shows the distribution of MC-3
receptor mRNA among 3-MSH-responsive cells. Because the MC-3
receptor gene has no introns, PCR was run with prior RT reaction (+RT)
and without prior RT reaction (-RT). A PCR product was never obtained
in the -RT samples, indicating that chromosomal DNA is not amplified
in our test conditions. That chromosomal DNA is not readily amplified
under single cell RT-PCR conditions is consistent with observations by
others (15). In the +RT samples, MC-3 receptor mRNA was found in only a
small part of the 3-MSH-responsive cells (12 of 69 cells; examples
are shown in Fig. 5). It was found coexpressed with GH mRNA alone or in
combination with mRNA of PRL, TSHß, and POMC and in 1 cell not
expressing either of these hormone mRNAs (but positive for L19 mRNA).
When the MC-3 receptor mRNA-containing cells were examined as a
proportion of all responsive cells expressing GH, PRL, or POMC mRNA, it
was found that MC-3 receptor mRNA was detectable in 11 of 57 cells
expressing GH mRNA (19%), in 5 of 23 cells expressing PRL mRNA (22%),
and in 2 of 22 cells expressing POMC mRNA (9%). As MC-3 receptor mRNA
expression was observed in only 16% of the 3-MSH-responsive cells,
we wanted to exclude the possibility that a single amplification run
would have missed detection of MC-3 receptor mRNA due to very low
expression levels in some cells. A seminested PCR on the first PCR
product of all 69 cells was performed. The first PCR product of cells
positive for MC-3 receptor mRNA was diluted 100-fold, whereas material
in negative lanes was used as such (as described in Materials and
Methods). An amplified and clearly detectable fragment of the
expected size (183 bp) was only observed in the cells already positive
for MC-3 receptor mRNA in the first PCR and not in cells previously
negative (data not shown). The detection limit of the whole method was
estimated to be in the range of 50 copies of MC-3 receptor mRNA/cell
(see Materials and Methods; data not shown).
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|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
3-MSH in
the rat pituitary by determining cellular hormone phenotypes expressed
at both the protein and mRNA levels in cells showing
[Ca2+]i transients upon exposure to this
peptide. Immunocytochemistry revealed that a relatively small
proportion (7–23%) of all main pituitary cell types showed a
[Ca2+]i response to 3-MSH, including cells
that did not express hormone to a level detectable by light microscopy.
However, some preference of 3-MSH to target GH-ir cells was found. A
clearly unexpected picture emerged when phenotypes were detected at the
hormone mRNA level by single cell RT-PCR. About 39% of the
3-MSH-responsive cells contained one of the specific hormone mRNAs
defining each of the main pituitary cell types, whereas 61% expressed
mRNAs of GH, PRL, TSHß, POMC, and LHß in dual, triple, and even
quadruple combinations within the same cell. In contrast, in the
overall pituitary cell population the proportion of cells coexpressing
two or more hormone mRNAs was almost twice as low as that in the
3-MSH-responsive population. Moreover, cells coexpressing POMC and
LHß were not found in the 3-MSH-responsive population, although
these cells were present in the overall pituitary cell population.
Thus, when examining hormone phenotypes at either the protein or the
mRNA level, the target cells of 3-MSH are not randomly distributed
among the various pituitary cell types and subtypes.
The differential distribution of hormone mRNAs in the
3-MSH-responsive population compared with that in the total cell
population furthermore excludes that the presence of multiple hormone
mRNAs in the same cell is due to very low "illegitimate" gene
transcription, reported to occur in probably every cell type in the
organism (28, 29, 30). Moreover, these extreme low copy number
"illegitimate" mRNAs have been detected in extremely sensitive PCR
conditions, far exceeding those used in the present study (30).
Clearly, the presence of multiple hormone mRNAs preferentially in most
of the 3-MSH responsive cells appears to be of biological
relevance.
To our knowledge, coexpression of mRNA for GH, PRL, and TSHß and of POMC mRNA or LHß mRNA with mRNA of GH, PRL, and/or TSHß in multiple combinations in normal pituitary cells has not been reported previously. However, dual combinations within the same cell of PRL, GH, and TSHß has been reported previously in both normal and adenomatous pituitary cells (31, 32, 33, 34, 35, 36, 37, 38). Some extremely rare human pituitary adenomas produce GH and ACTH or GH, PRL, and ACTH and store these hormones in the same secretory granules (39, 40). In some GH-secreting adenomas, mRNA of POMC and LHß have been observed (41). Childs et al. (42) have shown ACTH-ir in a considerable number of gonadotrophs during early postnatal life in the rat and in a low percentage of gonadotrophs in adult rat pituitary (43) as well as in a small subpopulation of cells containing TSHß (44).
The present findings uncover novel aspects in pituitary cell lineage
specification. The main pituitary cell types are defined on the basis
of the expression of a specific hormone and its corresponding mRNA, and
it has become clear that this is determined by various lineage-specific
transcription factors (reviewed in Ref. 45). The majority of the
3-MSH-responsive cells express various pituitary hormone mRNAs in
dual, triple, or quadruple combinations. These cells apparently express
multilineage phenotypes, raising intriguing questions for future
research. Do these cells coexpress the transcription factors typical
for the different lineages or (in addition) other factors allowing
multilineage gene expression? How do these cells relate to single
lineage cells expressing only one pituitary hormone gene? Do
multilineage gene-expressing cells already exist during embryonic life
or do they appear later on in life? The discovery in the present study
of an agonist, 3-MSH, preferentially targeting these cells may open
new perspectives for defining the functional significance of pituitary
cells displaying multilineage gene expression.
Another intriguing finding was that the number of cells expressing a
particular pituitary hormone mRNA (alone or in combination with other
hormone mRNAs) is always higher than the number of cells expressing the
hormone protein as determined by immunodetection, whereas coexpression
of different hormones could not be detected. For example, some 47% of
the 3-MSH-responsive cells contained PRL mRNA, but only 12%
contained PRL protein. On the other hand, in a considerable number of
cells responsive to 3-MSH, no hormone could be detected, whereas
only 1 of 136 3-MSH-responsive cells did not contain 1 or more of
the pituitary hormone mRNAs. Taken together, these data suggest that a
significant part of the nonhormone- as well as the hormone-containing
cells represent cells containing multiple hormone mRNAs but none or
only one of the corresponding hormone proteins in a detectable amount.
It is possible that light microscopic detection of immunostained
hormone underestimates coexpression, but if so, one hormone seems to be
stored in a much higher amount than the other in a given cell type.
These multiple mRNA-containing cells may be progenitor cells or
"reserve" cells waiting for a stimulus to terminally differentiate
(express single or multiple hormone proteins at a higher level)
depending on developmental or other physiological needs. Some data in
the literature are consistent with this view. Cosecretion of LH, FSH,
and GH from gonadotrophs at proestrus or of TSH and ACTH during cold
stress has been reported by Childs et al. (44, 46). There
have been reports of Pit-1-expressing cells that do not yet express
PRL, GH, or TSH (37, 47, 48). TSHß-ir adenomas have been reported
that express GH and PRL mRNA in the same cells, but a part of these
cells is immunonegative for GH and PRL (38). GH-secreting adenomas
exist that show PRL-ir but no GH-ir, whereas mRNAs for both GH and PRL
in the same cells are found (49).
We previously (and more extensively in the present study) showed that
treatment with SHU9119, a potent competitive MC-3 receptor antagonist
(13), blocked [Ca2+]i responses in only 46%
or less of the cells responsive to 3-MSH even at a 1000-fold molar
excess, suggesting that in most of the responsive cells, 3-MSH
transmits its signal through a receptor different from the MC-3
receptor (the only known receptor with high affinity for this peptide).
We previously (5) excluded that the latter receptor is the MC-4 or MC-5
receptor. To further test the above hypothesis we explored whether the
receptor blocked by SHU9119 and the one not blocked by this compound
were differentially distributed among the different target cells. It
was found that the 3-MSH receptor not blocked by SHU9119 is located
in PRL-ir, GH-ir, and TSHß-ir cells, whereas the receptor blocked by
SHU9119 is present in ACTH-ir cells and in cells that do not detectably
express hormone. Thus, this differential distribution of a
SHU9119-blockable and a SHU9119-nonblockable receptor strengthens our
proposal (5) that a 3-MSH receptor may exist in the pituitary that
is at least functionally different from the cloned MC-3 receptor tested
to date in transfected cell lines. Current investigations in our
laboratory support the latter hypothesis. We found that 3-MSH
potently (effective from 0.001 nM) increases
[Ca2+]i in the PRL- and GH-secreting
GH3 cell line, that this effect is not affected by SHU9119,
and that RT-PCR performed on a RNA extract from this cell line could
not reveal a signal for the presence of MC-3 receptor mRNA (Langouche,
L., M. Roudbaraki, and C. Denef, unpublished observations in three
independent experiments). In further support of limited involvement of
the MC-3 receptor in most of the 3-MSH-induced
[Ca2+]i responses in the normal pituitary
cells was the finding that MC-3 receptor mRNA was detectable in only
16% of the cells responsive to 3-MSH. However, MC-3 receptor mRNA
was detectable in only 9% of the cells expressing POMC mRNA. This is
not consistent with the above interpretation that in most ACTH-ir cells
the response to 3-MSH was blocked by SHU9119. There are several
possible explanations for this discrepancy. The absence of a PCR signal
may arise from a statistical sampling error in the case of low
abundance messages (50). We cannot exclude that the expression of MC-3
receptor mRNA in part of the ACTH-ir cells is too low to be detectable;
the detection limit of our method is in the range of 50 MC-3 receptor
mRNA molecules/cell. Another possible explanation is that ACTH-ir cells
that express the MC-3 receptor on their surface might no longer contain
detectable levels of MC-3 receptor mRNA, as is, for example, the case
in many CD34-expressing bone marrow stem cells (51). However, within
the scope of the present investigation and regardless of the above
tentative explanations, the potential biological relevance of the
findings is that there seem to exist 3-MSH receptors that are
blockable and others that are not blockable by SHU9119 and that they
are differentially distributed according to cell type. An additional
peculiar characteristic of the [Ca2+]i
response to 3-MSH is that there is a graded recruitment of
responding cells with increasing doses of 3-MSH. Very tempting
issues for future research are whether different subtypes of cells are
recruited at different peptide doses.
In conclusion, the present findings show that the target cells of
3-MSH as detected on the basis of [Ca2+]i
changes in the immature rat pituitary constitute specific
subpopulations within each main pituitary cell type category, including
nonhormone-containing cells (or low expression hormonal cells), the
majority of which contain transcripts of the GH, PRL, and TSHß genes
together with the POMC or LHß gene in various combinations. The data
reveal the existence of cells displaying multilineage gene activation.
Because [Ca2+]i responses are not blocked by
SHU9119 in GH-, PRL-, and TSHß-ir cells and most of these cells do
not seem to express the MC-3 receptor at a substantial level, it is
suggested that most of the 3-MSH effects in these cells are mediated
by a hitherto unknown receptor type or at least by a MC-3 receptor with
ligand-binding characteristics different from those of the cloned MC-3
receptor examined to date in transfected cell lines.
|
Acknowledgments |
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|
Footnotes |
|---|
2 Supported by a fellowship from the Flemish "Instituut voor
bevordering van het wetenschappelijk onderzoek in de industrie"
(I.W.T.).
Received February 22, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
3-MSH and
anti-POMC1–76 antibodies on DNA replication in lactotrophs in
aggregate cell cultures of immature rat pituitary. J
Neuroendocrinol 9:627–637[CrossRef][Medline]
3-MSH-induced changes in
intracellular Ca2+ concentration? J Neuroendocrinol 11:171–179[CrossRef][Medline]
-melanotropin analogues of
Ac-Nle4-cyclo[Asp5,D-Phe7,Lys10]-melanocyte-stimulating
hormone-(4–10)-NH2 with bulky aromatic amino acids at position 7 show
high antagonist potency and selectivity at specific melanocortin
receptors. J Med Chem 38:3454–3461[CrossRef][Medline]
-conotoxin GVIA lacking the class D1
subunit. Neuroreport 8:485–489[Medline]
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