Distribution, Cellular Localization, and Ontogeny of Preprothyrotropin-Releasing Hormone-(160-169) (Ps4)-Binding Sites in the Rat Pituitary
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0013-7227/98/$03.00/0 Vol. 139, No. 3
Endocrinology Printed in U.S.A.
Copyright © 1998 by The Endocrine Society
Distribution, Cellular Localization, and Ontogeny of
Preprothyrotropin-Releasing Hormone-(160 –169)
(Ps4)-Binding Sites in the Rat Pituitary*
KARINE VALENTIJN†, FRANCK VANDENBULCKE, ESTER PIEK‡,
JEAN-CLAUDE BEAUVILLAIN, AND HUBERT VAUDRY
European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular
Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale (INSERM U-413),
Unité Affilieé au Centre National de la Recherche Scientifique (UA CNRS), University of Rouen
(K.V., E.P., H.V.), 76821 Mont-Saint-Aignan; and the Laboratory of Neuroendocrinology and Neuronal
Pathology, INSERM U-422 (F.V., J.C.B.), 59045 Lille, France
ABSTRACT cytochemical analysis using antibodies against the different an-
The rat TRH precursor contains five copies of TRH separated by terior pituitary hormones indicated that the cells possessing [125I-
connecting peptides. Previous studies have shown that the de- Tyr0]Ps4-binding sites did not correspond to TSH-, PRL-, GH-,
capeptide prepro-TRH (160 –169; Ps4) potentiates the effect of TRH ACTH-, or LH-secreting cells. In contrast, cells expressing Ps4
on TSH secretion. In the present study, we have characterized Ps4 receptors were immunoreactive for the S-100 protein, a marker of
receptors in the rat pituitary by in vitro autoradiography using folliculo-stellate cells. During postnatal development, a 4-fold in-
[125I-Tyr0]Ps4 as a radioligand, and we have investigated the evo- crease in the concentration of [125I-Tyr0]Ps4-binding sites occurred
lution of receptor density during ontogenesis. Incubation of rat from birth to weaning in the pituitary, with a marked and transient
pituitary slices with [125I-Tyr0]Ps4 revealed intense binding in the increase at the time of weaning. Thereafter, the density of sites
anterior lobe and virtually no binding in the neurointermediate declined gradually until day 60. In conclusion, the present study
lobe. Biochemical characterization of the Ps4-binding sites sug- shows that folliculo-stellate cells express [125I-Tyr0]Ps4-binding
gested the existence of a single class of sites exhibiting high affinity sites in the anterior pituitary, and that these sites are develop-
for [Tyr0]Ps4 (IC50 5 8.3 6 1.2 nM) and a much lower affinity for mentally regulated. The present data suggest that the potentiating
Ps4 (IC50 5 9.3 6 1.2 mM). Emulsion-coated cytoautoradiography effect of Ps4 on TRH-induced TSH secretion is mediated by folli-
performed on cultured anterior pituitary cells showed that only culo-stellate cells. (Endocrinology 139: 1306 –1313, 1998)
26% of the cells possessed [125I-Tyr0]Ps4-binding sites. Immuno-
T HE RAT TRH precursor contains five copies of the TRH
progenitor sequence Gln-His-Pro-Gly, each flanked by
paired basic amino acids and linked by connecting peptides
quently revealed the existence of a dense accumulation of
Ps4-containing nerve terminals in the external zone of the
median eminence (4). In vitro perifusion studies have shown
(1). Several studies have been undertaken to investigate the that the release of Ps4 by mediobasal hypothalamic slices is
possible functions of these cryptic peptides. Most of these triggered by potassium-induced depolarization through a
studies have been focused on the fourth connecting peptide, calcium-dependent process (4, 5). Concurrently, physiolog-
termed Ps4, a decapeptide (Ser-Phe-Pro-Trp-Met-Glu-Ser- ical experiments have demonstrated that Ps4 potentiates the
Asp-Val-Thr) linking the third and fourth copies of the TRH effect of TRH on TSH release from the rat pituitary gland (6,
progenitor. It was first demonstrated that posttranslational 7). The potentiating action of Ps4 on TRH-induced TSH re-
processing of pro-TRH generates authentic Ps4 in the olfac- lease is mediated by activation of voltage-dependent calcium
tory lobe, hypothalamus, and spinal cord (2) and in the channels and involves a pertussis toxin-sensitive G protein
pancreas (3). Immunocytochemical studies have subse- (8). Ps4 also acts in synergy with TRH in the dorsal motor
nucleus of the vagus to potentiate gastric acid secretion (9).
These data indicated that one of the connecting peptides
Received August 25, 1997. generated during processing of rat pro-TRH can modulate
Address all correspondence and requests for reprints to: Dr. H.
Vaudry, European Institute for Peptide Research (IFRMP 23), Labora- the action of TRH in the pituitary gland and in discrete brain
tory of Cellular and Molecular Neuroendocrinology, Institut National de nuclei. In support of this idea, the occurrence of specific
la Santé et de la Recherche Médicale (INSERM U-413), Unité Affiliée au binding sites for Ps4 has been demonstrated in the anterior
Centre National de la Recherche Scientifique (UA CNRS), University of pituitary (6, 10, 11) as well as in the brain and various pe-
Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry@
univ-rouen.fr. ripheral organs (12). In addition, it has been shown that
* This work was supported by grants from INSERM (U-413 and another cryptic peptide derived from the TRH precursor,
U-422), the Lille-Amiens-Rouen-Caen (LARC) network, and the Conseil prepro-TRH-(178 –199) or Ps5 (1, 13, 14), may inhibit both
Régional de Haute-Normandie. ACTH (15–17) and GH secretion (18, 19).
† Recipient of a fellowship from the Ministère de l’Education Natio-
nale, de l’Enseignement Supérieur et de la Recherche.
The aim of the present study was to determine by auto-
‡ Recipient of a fellowship from the European Union (ERASMUS radiography the type of pituitary cells that express Ps4 re-
program). ceptor and to investigate the variations in the concentrations
1306
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of these binding sites during postnatal development by tissues were incubated with 0.004% deoxyribonuclease for 3 min and
means of quantitative autoradiography. with 0.1% soybean trypsin inhibitor for 10 min. The digested tissues
were incubated with culture medium containing 2 mm EDTA for 5 min
and 1 mm EDTA for 15 min, washed three times with 1 mm EDTA, and
Materials and Methods disaggregated by gentle aspiration through a siliconized Pasteur pipette
Animals with a flame-polished tip. Dispersed cells were centrifuged at 300 3 g
Wistar rats (Dépré, St. Doulchard, France) were maintained under and washed twice. The pellet was resuspended in culture medium
controlled conditions of temperature (24 6 1 C) under an established supplemented with 5% FBS and 1% antimycotic-antibiotic solution. Pi-
photoperiod (lights on from 0700 –1900 h), with food pellets and water tuitary cells were cultured in 24-well culture plates (2 3 106 cells/well)
ad libitum. Adult male rats were used as pituitary donors for cell cultures. on poly-l-lysine-coated coverslips and kept at 37 C in a CO2-air incu-
For developmental studies, pituitaries from male and female rat pups bator for 15 h. The cells were then rinsed three times with 50 mm
were collected at various postnatal stages, from birth (day 0) to day 60. Tris-HCl buffer containing 0.1% BSA and 0.01% bacitracin, dried under
The pups were weaned on day 21. Rats were killed by decapitation, and a cold air stream, and kept frozen until use.
the pituitaries were placed in a drop of embedding medium (Tissue Tek,
Leica, France) and frozen on dry ice. When the animals were younger Binding studies
than 8 days, the whole heads were snap-frozen in isopentane. Tissues
were kept at 280 C until used for autoradiographic studies. Animal Cultured cells were washed twice with 50 mm Tris-HCl buffer and
manipulations were performed according to the recommendations of incubated for 120 min at room temperature in the same buffer containing
the French ethical committee and under the supervision of authorized [125I-Tyr0]Ps4. In saturation experiments, the cells were incubated with
investigators. 12–1565 pm of the radioligand, and nonspecific binding was determined
by adding 1026 m [Tyr0]Ps4 to the incubation buffer. In competition
Chemicals and antibodies experiments, the cells were incubated with 400 pm of the radioligand in
the presence of increasing concentrations (10211-1026 m) of [Tyr0]Ps4.
Ps4 and [Tyr0]Ps4 were obtained from Neosystem (Strasbourg, The coverslips were gently removed from the culture wells and rinsed
France). BSA was purchased from Boehringer Mannheim (Mannheim, six times in cold Tris-HCl buffer. The radioactivity was then counted on
Germany). Na125I (IMS-30) was obtained from Dositek, (Saclay, France). a g-counter (model 1277, LKB, Rockville, MD).
DMEM, Ham’s F-12 medium, and the antimycotic-antibiotic solution
were purchased from Life Technologies (Cergy Pontoise, France). FBS
was provided by Biosys (Compiegne, France). The LM1 nuclear emul- Cytoautoradiography
sion was obtained from Amersham (Les Ulis, France). All other chem- Cultured pituitary cells were incubated with 400 pm [125I-Tyr0]Ps4 as
icals were purchased from Sigma Chemical Co. (St. Louis, MO). Radio- described above. Nonspecific binding was determined by adding 1026
iodinated [Tyr0]Ps4 was prepared by the chloramine-T method (20) as m [Tyr0]Ps4 to the incubation buffer. The cells were then dried under
previously described (4). The iodinated peptide was purified by reverse vacuum in the presence of paraformaldehyde vapor for 24 h. The cov-
phase HPLC on an Orpegen RP-7s-300 column (0.5 3 15 cm) using a erslips were dipped into Kodak NTB-2 liquid emulsion (Eastman Kodak,
Gilson liquid chromatograph (model 811, Gilson, Oberlin, OH) with a Rochester, NY) diluted 1:1 with distilled water at 40 C, as previously
0 – 60% linear gradient of acetonitrile in 0.1% trifluoroacetic acid. Mono- described (24). After 4 days of exposure, the emulsion was developed,
iodinated [Tyr0]Ps4 eluted at 33% acetonitrile. The specific radioactivity and the autoradiographic preparations were counterstained with tolu-
of the tracer was approximately 2000 Ci/mmol. idine blue.
Polyclonal antibodies against TSH, PRL, GH, LHb, and ACTH were
generous gifts from Dr. Y. Tillet (INRA, Nouzilly, France). The specificity
of these antibodies has been previously described (21, 22). Control ex- Autoradiography combined with immunocytochemistry on
periments were performed by preabsorbing the antisera with the ho- semithin consecutive sections
mologous antigens. Polyclonal antibodies against the bovine S-100 pro-
tein were obtained from Dako (Glostrup, Denmark). Adult rat pituitaries were fixed for 10 min by immersion in 1%
glutaraldehyde in 0.1 m phosphate buffer (pH 7.4). The pituitaries were
Autoradiographic studies sliced in a Vibratome (Leica, Heidelberg, Germany) (80-mm thick sec-
tions). Four slices per pituitary were collected and immediately incu-
Whole heads or pituitaries were sliced on a cryostat (Frigocut, bated for 10 min in 50 mm Tris-HCl buffer containing 0.1% BSA. Three
Reichert-Jung, Nussloch, Germany) at 20 mm in the frontal plane. Tissue Vibratome slices per pituitary were incubated at room temperature with
slices were thaw-mounted on gelatin-coated slides, dehydrated under 400 pm [125I-Tyr0]Ps4. The other tissue slices were incubated under the
vacuum, and kept frozen until use. All incubation steps were performed same conditions in the presence of 1026 m [Tyr0]Ps4 to determine non-
at room temperature in 50 mm Tris-HCl buffer (pH 7.4) containing 0.1% specific binding. After incubation, all slices were rinsed in three con-
BSA and 0.01% bacitracin. Tissue slices were preincubated in the buffer secutive baths (5 min each) of cold Tris-HCl buffer and postfixed in an
for 10 min and then incubated for 120 min with 6.5 pm [125I-Tyr0]Ps4. To ice-cold solution of 4% glutaraldehyde in phosphate buffer for 30 min.
visualize nonspecific binding, adjacent slices were incubated under the Then, slices were postfixed in a solution of 1% OsO4 in phosphate buffer
same conditions in the presence of 1026 m [Tyr0]Ps4. For competition for 20 min, dehydrated, and embedded in Araldite (Fluka, Buchs,
studies, slices were incubated in the presence of increasing concentra- Switzerland).
tions (10211-1024 m) of Ps4 or [Tyr0]Ps4. Finally, the slices were washed Semithin sections (1.5-mm thick) were cut from each embedded Vi-
four times for 30 sec each time in cold buffer and dried under a cold air bratome slice, put on glass slides, and coated by dipping in Amersham
stream. The sections were apposed onto 3H-Hyperfilm (Amersham) for LM1 nuclear emulsion. Adjacent semithin sections obtained just before
4 days. After exposure, the slices were stained with cresyl violet. Quan- and after this latter section were collected on other glass slides and used
tification of the autoradiograms was performed with a BIO 500 com- for immunocytochemistry. The sections used for autoradiography were
puter-assisted image analyzer (Biocom, Les Ulis, France) using a stan- exposed for 4 – 6 weeks at 4 C. Semithin autoradiographs were devel-
dard curve derived from coexposed 125I-containing brain paste oped in D19 (Kodak), fixed in 30% sodium thiosulfate, rinsed in distilled
standards (23). water, and slightly counterstained in 0.1% azur blue.
On the adjacent sections used for immunocytochemistry, Araldite
Cell culture had to be removed by using sodium methoxide according to the method
of Mayor et al. (25). The sections were treated with 10% hydrogen
The pituitary glands from 10 adult male rats were placed into culture peroxide for 8 min and incubated overnight with one of the primary
medium (75% DMEM and 25% Ham’s F-12) containing 0.3% BSA and antisera (against TSH, LHb, PRL, GH, ACTH, or S-100) diluted 1:200,
1% antimycotic-antibiotic solution. The anterior lobes were cut into eight at 20 C. Then, the sections were incubated for 2 h with donkey antirabbit
pieces, and the pituitary fragments were enzymatically dissociated at 37 IgG conjugated with horseradish peroxidase (diluted 1:200). Peroxidase
C for 15 min with a solution of 0.25% trypsin in culture medium. The was visualized with H2O2 and 3,39-diaminobenzidine tetrahydrochloride.
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Vol 139 • No 3
The semithin autoradiographs were observed first, and the positive
cells were photographed. Then, the adjacent immunostained sections
were analyzed to find the same area and the same cells.
Results
Distribution of [125I-Tyr0]Ps4-binding sites in the adult
rat pituitary
The occurrence of [125I-Tyr0]Ps4-binding sites was visual-
ized by autoradiography on pituitary sections (Fig. 1A).
Counterstaining of the tissue slices with cresyl violet (Fig. 1B)
revealed that the autoradiographic reaction was particularly
intense in the anterior lobe, whereas the intermediate and
neural lobes were virtually unlabeled. In the presence of 1026
m [Tyr0]Ps4, the labeling was totally suppressed (Fig. 1C).
Quantification of the autoradiograms from serial sections (20
mm in thickness) of five pituitaries revealed that the density FIG. 2. Rostro-caudal variations in the concentration of [125I-
of [125I-Tyr0]Ps4-binding sites was constant throughout the Tyr0]Ps4-binding sites in sections of the anterior lobe of the adult male
anterior lobe (Fig. 2). Sections at any antero-posterior levels rat pituitary. The figure is representative of five independent exper-
of the pituitary were, therefore, used for subsequent studies. iments. [125I-Tyr0]Ps4-binding sites were evenly distributed through-
Incubation of pituitary slices with 6.5 pm [125I-Tyr0]Ps4 in out the adenohypophysis.
the presence of increasing concentrations of [Tyr0]Ps4 (10211-
1027 m) or Ps4 (1029-1024 m) resulted in a dose-dependent
inhibition of labeling (Fig. 3). The IC50 values for [Tyr0]Ps4
and Ps4 were 8.3 6 1.2 nm (n 5 6) and 9.3 6 1.2 mm (n 5 4),
respectively. The binding of [125I-Tyr0]Ps4 was not displaced
by TRH, prepro-TRH-(178 –199), a-melanotropin, b-endor-
FIG. 3. Typical competition of [Tyr0]Ps4 (F) and Ps4 (E) for [125I-
Tyr0]Ps4 binding to distal pituitary slices. Each point represents the
mean of quadruplicate determinations. Typical autoradiograms used
for quantification of binding are shown at different concentrations of
competitor. The figure is representative of four and six independent
experiments for Ps4 and [Tyr0]Ps4, respectively. B0, Bound radioli-
gand in the absence of competitor; B, bound radioligand in the pres-
ence of graded concentrations of competitors.
phin, somatostatin, vasoactive intestinal polypeptide, sub-
stance P, CRH, or GH-releasing hormone (data not shown).
Characterization of [125I-Tyr0]Ps4-binding sites on cultured
pituitary cells
Saturation studies revealed the presence of high affinity
[125I-Tyr0]Ps4-binding sites on cultured pituitary cells (Fig.
FIG. 1. Autoradiographic localization of [125I-Tyr0]Ps4-binding sites 4). Scatchard plot analysis (Fig. 4, inset) indicated that the
in frontal plane sections of adult male rat pituitary. A, Autoradiogram radioligand interacts with a single class of binding sites with
showing the distribution of [125I-Tyr0]Ps4-binding sites. Note the in- a Kd value of 0.16 6 0.012 nm and a binding capacity of
tensity of labeling in the anterior lobe. B, Cresyl violet staining of the 3561 6 601 sites/cell (n 5 4).
section shown in A. AL, Anterior lobe; IL, intermediate lobe; NL,
neural lobe. C, Autoradiogram of the adjacent section to that shown Competition studies were carried out using [Tyr0]Ps4 as a
in A and B, incubated with [125I-Tyr0]Ps4 in the presence of 1026 M competitor. The competition curve was monophasic, with an
[Tyr0]Ps4. IC50 value of 1.5 6 0.5 nm (n 5 4; Fig. 5).
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FIG. 4. Typical saturation curve of [125I-Tyr0]Ps4 binding to cultured
anterior pituitary cells. Total binding (‚) and nonspecific binding
measured in the presence of 1026 M [Tyr0]Ps4 (ƒ) were directly quan-
tified in a g-counter. Specific binding (F) was calculated as the dif-
ference between total and nonspecific binding. Each point represents
the mean of duplicates. Inset, Corresponding Scatchard plot. The
figure is representative of four independent experiments. B, Bound
radioligand; F, free radioligand.
FIG. 6. Visualization by cytoautoradiography of [125I-Tyr0]Ps4-bind-
ing sites on cultured anterior pituitary cells. A and B, Cells incubated
with [125I-Tyr0]Ps4 alone. Filled arrowheads point to intensely la-
FIG. 5. Typical competition curve of [125I-Tyr0]Ps4 binding to cul-
beled cells. Open arrows point to cells that are virtually devoid of
tured rat anterior pituitary cells. Each point represents the mean of
binding sites. C and D, Cells incubated in the same conditions as those
duplicates. The figure is representative of four independent experi-
in A and B, except that 1026 M [Tyr0]Ps4 was added. A and C, Bright-
ments. B0, Bound radioligand in the absence of competitor; B, bound
field photomicrographs of pituitary cells stained with toluidine blue.
radioligand in the presence of graded concentrations of [Tyr0]Ps4.
B and D, Darkfield photomicrographs of the areas shown in A and C,
respectively. Bar 5 15 mm.
Localization of [125I-Tyr0]Ps4-binding sites on cultured
pituitary cells
chemistry on consecutive semithin sections. However, in
The cellular distribution of [125I-Tyr0]Ps4-binding sites these conditions, immunoreaction with the antibodies
was studied on emulsion-coated cultured cells. Cytoautora- against the bovine S-100 protein was relatively weak. Com-
diographic labeling revealed that 26% of pituitary cells (n 5 parison of homologous fields on adjacent sections clearly
238 of 922) possessed a high density of silver grains (Fig. 6, demonstrated that the cells bearing [125I-Tyr0]Ps4-binding
A and B). In the presence of 1026 m [Tyr0]Ps4, labeling was sites (i.e. cells decorated with silver grains) were not immu-
totally abolished. nolabeled with antibodies against TSH (Fig. 7, a and b), PRL
(Fig. 7, c and d), GH (Fig. 7, e and f), LHb, or ACTH (not
Immunocytochemical identification of pituitary cells shown). Despite the difficulty mentioned above, many cells
expressing [125I-Tyr0]Ps4-binding sites exhibiting silver grains were unambiguously immunola-
Postfixation of Vibratome pituitary slices with 4% glutar- beled with the antibodies against the bovine S-100 protein
aldehyde made it possible to localize [125I-Tyr0]Ps4 by au- (Fig. 7, g and h). Control sections incubated with [125I-
toradiography and hypophysial hormones by immunocyto- Tyr0]Ps4 in the presence of 1026 m [Tyr0]Ps4 exhibited only
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Vol 139 • No 3
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occurrence of specific binding sites for [125I-Tyr0]Ps4 in the
anterior lobe of the rat pituitary.
Autoradiographic labeling of frontal sections of the rat
pituitary showed the occurrence of a high density of specific
[125I-Tyr0]Ps4-binding sites in the anterior lobe and virtually
no labeling in the neurointermediate lobe. As [125I-Tyr0]Ps4
binding was evenly distributed in the anterior pituitary, dis-
placement curves could be performed on tissue slices by
quantitative autoradiography. The [Tyr0]Ps4 analog ap-
peared to be several orders of magnitude more potent than
the native peptide in displacing [125I-Tyr0]Ps4 from its bind-
ing sites. Consistent with this observation, previous studies
have shown that a dipeptidyl aminopeptidase cleaves the
Phe2-Pro3 bond of Ps4, leading to the formation of the inac-
tive peptide Ps4 (3–10), whereas the [Tyr0]Ps4 analog is re-
sistant to enzymatic degradation (6). Moreover, addition of
a Tyr residue at the N-terminus of Ps4 has been shown to
increase the binding affinity of the peptide (10, 11). Charac-
terization of [125I-Tyr0]Ps4-binding sites on cultured anterior
pituitary cells revealed that the binding was specific and
FIG. 8. Evolution of the concentration of [125I-Tyr0]Ps4-binding sites
in the rat pituitary during postnatal development. A, Typical auto-
saturable. The biochemical characteristics of the binding sites
radiograms of pituitary slices at different ages. B, Concentrations of determined on cultured cells (this study) were very similar
[125I-Tyr0]Ps4-binding sites at various ages from birth to adulthood. to those previously reported using membrane-enriched
Each point represents the mean of 3–10 animals. preparations of whole pituitaries (6) with Kd values of 0.18
and 0.16 nm, respectively.
a few silver grains scattered throughout the sections (not The first clue suggesting that [125I-Tyr0]Ps4-binding sites
shown). may not be borne by TSH cells was provided by cytoauto-
radiographic studies on cultured pituitary cells. A high den-
Concentration of [125I-Tyr0]Ps4-binding sites in the sity of silver grains was observed on a subset of large cells,
pituitary during development which, in the primary culture, represented about one fourth
of the entire pituitary cell population. The morphological
The evolution of the concentration of [125I-Tyr0]Ps4-bind- characteristics and the proportion of labeled cells made it
ing sites in the rat pituitary during postnatal development is unlikely that these cells could correspond to TSH cells, the
shown in Fig. 8. The concentration of binding sites was rel- latter being the smallest cells of the anterior pituitary and
atively low at birth (2.57 6 0.40 fmol/mg protein) and in- representing less that 10% of all pituitary cells (29).
creased slowly until day 15 (4.66 6 0.68 fmol/mg protein). To identify unambiguously the type of cells expressing
Then, a 2-fold increase in the density of recognition sites was [125I-Tyr0]Ps4-binding sites, histoautoradiographic localiza-
observed on day 22, i.e. just after weaning. After day 30, the tion of the recognition sites and immunocytochemical iden-
concentration of binding sites decreased gradually until tification of the different pituitary cell types were performed
adulthood. In 60-day-old rats, the concentration of binding on adjacent semithin sections. Accumulation of silver grains
sites was identical to that measured on day 15. was never observed on cells immunolabeled with antibodies
against TSH, PRL, GH, LHb, or ACTH. In contrast, cells
Discussion exhibiting [125I-Tyr0]Ps4-binding sites were immunoreactive
Several lines of evidence indicate that the pro-TRH- for the S-100 protein, a specific marker of folliculo-stellate
derived peptide Ps4 may regulate the activity of anterior cells (30, 31). In agreement with this finding, it has been
pituitary cells. 1) The presence of immunoreactive Ps4 has recently demonstrated that glioma cells, which also express
been detected within nerve terminals in the external zone of the S-100 protein, possess specific binding sites for [125I-
the median eminence (4). 2) Authentic Ps4 is released from Tyr0]Ps4 (32).
hypothalamic neurons in vitro (4, 5), and substantial amounts The occurrence of a high density of Ps4-binding sites on
of Ps4 are found in the hypophysial portal blood (26). 3) folliculo-stellate cells and the absence of recognition sites on
Synthetic Ps4 has been shown to potentiate TRH-induced TSH cells suggest that the potentiating effect of Ps4 on TRH-
TSH release (6, 8) and to increase TSH and PRL gene ex- evoked TSH secretion previously reported (6 – 8) involves the
pression (27, 28). The present study now demonstrates the contribution of folliculo-stellate cells. In support of this idea,
FIG. 7. Autoradiographic localization of [125I-Tyr0]Ps4-binding sites and immunocytochemical identification of pituitary cells on semithin
sections (1.5 mm of thickness) of the pituitary gland. Vibratome-cut pituitary slices were incubated with 400 pM [125I-Tyr0]Ps4, and semithin
sections were prepared. Consecutive semithin sections were either dipped in nuclear emulsion (a, c, e, and g) or immunolabeled with antisera
against TSH (b), PRL (d), GH (f), or the S-100 protein (h). Capillaries (arrowheads) made it possible to recognize homologous fields. Cells
decorated with silver grains were immunostained only with the antibodies against the S-100 protein (arrows). Magnification: A–F, 3150; G
and H, 3200.
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Vol 139 • No 3
the potentiating action of Ps4 has been observed on rat pi- postnatal development can be accounted for at least in part
tuitary fragments, a model that preserves the cytoarchitec- by the proliferation of folliculo-stellate cells. However, the
ture of the tissue and thus allows paracrine communication rapid fluctuations in receptor density observed at the time of
between the various pituitary cells types (6, 8). In addition, weaning indicate that the number of receptors per folliculo-
it has been previously demonstrated that folliculo-stellate stellate cells can also be regulated.
cells play a role in the regulation of pituitary cell activity (33). In conclusion, the present data have shown that in the rat
In particular, it has been shown that folliculo-stellate cells pituitary, Ps4-binding sites are expressed in folliculo-stellate
modulate the responses of GH and PRL cells to various cells, which probably mediate the potentiating effect of
neuroendocrine factors (34, 35). Concurrently, it has been Ps4 on TRH-induced TSH release. The concentration of Ps4-
reported that the inhibitory effect of interferon-g on pituitary binding sites increases during the postnatal period, suggest-
hormone secretion is mediated through folliculo-stellate cells ing that Ps4 plays a role in the regulation of the pituitary as
(36). It has also been found that the stimulatory effect of early as the hypothalamo-hypophysial complex has become
PACAP on pituitary hormone and cytokine secretion may functional.
involve folliculo-stellate cells (37, 38). Finally, it has been
proposed that in the pars intermedia of the toad Xenopus Acknowledgments
laevis, folliculo-stellate cells are involved in the inhibitory
The authors thank Dr. Denis Tranchand Bunel for iodination of
effect of neuropeptide Y on aMSH release (39). The fact that [Tyr0]Ps4, and Mrs. Sabrina Mancel for typing the manuscript.
[125I-Tyr0]Ps4-binding sites are exclusively borne by folli-
culo-stellate cells suggests that the regulation of TSH secre-
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