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Endocrinology Vol. 139, No. 3 1306-1313
Copyright © 1998 by The Endocrine Society

ARTICLES

Distribution, Cellular Localization, and Ontogeny of Preprothyrotropin-Releasing Hormone-(160–169) (Ps4)-Binding Sites in the Rat Pituitary1

Karine Valentijn2, Franck Vandenbulcke, Ester Piek3, 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

Address all correspondence and requests for reprints to: Dr. H. 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 Affiliée au Centre National de la Recherche Scientifique (UA CNRS), University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry{at}univ-rouen.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The rat TRH precursor contains five copies of TRH separated by connecting peptides. Previous studies have shown that the decapeptide prepro-TRH (160–169; Ps4) potentiates the effect of TRH on TSH secretion. In the present study, we have characterized Ps4 receptors in the rat pituitary by in vitro autoradiography using [125I-Tyr0]Ps4 as a radioligand, and we have investigated the evolution of receptor density during ontogenesis. Incubation of rat pituitary slices with [125I-Tyr0]Ps4 revealed intense binding in the anterior lobe and virtually no binding in the neurointermediate lobe. Biochemical characterization of the Ps4-binding sites suggested the existence of a single class of sites exhibiting high affinity for [Tyr0]Ps4 (IC50 = 8.3 ± 1.2 nM) and a much lower affinity for Ps4 (IC50 = 9.3 ± 1.2 µM). Emulsion-coated cytoautoradiography performed on cultured anterior pituitary cells showed that only 26% of the cells possessed [125I-Tyr0]Ps4-binding sites. Immunocytochemical analysis using antibodies against the different anterior pituitary hormones indicated that the cells possessing [125I-Tyr0]Ps4-binding sites did not correspond to TSH-, PRL-, GH-, ACTH-, or LH-secreting cells. In contrast, cells expressing Ps4 receptors were immunoreactive for the S-100 protein, a marker of folliculo-stellate cells. During postnatal development, a 4-fold increase in the concentration of [125I-Tyr0]Ps4-binding sites occurred from birth to weaning in the pituitary, with a marked and transient increase at the time of weaning. Thereafter, the density of sites declined gradually until day 60. In conclusion, the present study shows that folliculo-stellate cells express [125I-Tyr0]Ps4-binding sites in the anterior pituitary, and that these sites are developmentally regulated. The present data suggest that the potentiating effect of Ps4 on TRH-induced TSH secretion is mediated by folliculo-stellate cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE 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 (1). Several studies have been undertaken to investigate the possible functions of these cryptic peptides. Most of these studies have been focused on the fourth connecting peptide, termed Ps4, a decapeptide (Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-Val-Thr) linking the third and fourth copies of the TRH progenitor. It was first demonstrated that posttranslational processing of pro-TRH generates authentic Ps4 in the olfactory lobe, hypothalamus, and spinal cord (2) and in the pancreas (3). Immunocytochemical studies have subsequently 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 that the release of Ps4 by mediobasal hypothalamic slices is triggered by potassium-induced depolarization through a calcium-dependent process (4, 5). Concurrently, physiological experiments have demonstrated that Ps4 potentiates the effect of TRH on TSH release from the rat pituitary gland (6, 7). The potentiating action of Ps4 on TRH-induced TSH release is mediated by activation of voltage-dependent calcium channels and involves a pertussis toxin-sensitive G protein (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 generated during processing of rat pro-TRH can modulate the action of TRH in the pituitary gland and in discrete brain nuclei. In support of this idea, the occurrence of specific binding sites for Ps4 has been demonstrated in the anterior pituitary (6, 10, 11) as well as in the brain and various peripheral organs (12). In addition, it has been shown that another cryptic peptide derived from the TRH precursor, prepro-TRH-(178–199) or Ps5 (1, 13, 14), may inhibit both ACTH (15, 16, 17) and GH secretion (18, 19).

The aim of the present study was to determine by autoradiography the type of pituitary cells that express Ps4 receptor and to investigate the variations in the concentrations of these binding sites during postnatal development by means of quantitative autoradiography.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Wistar rats (Dépré, St. Doulchard, France) were maintained under controlled conditions of temperature (24 ± 1 C) under an established photoperiod (lights on from 0700–1900 h), with food pellets and water ad libitum. Adult male rats were used as pituitary donors for cell cultures. For developmental studies, pituitaries from male and female rat pups were collected at various postnatal stages, from birth (day 0) to day 60. The pups were weaned on day 21. Rats were killed by decapitation, and the pituitaries were placed in a drop of embedding medium (Tissue Tek, Leica, France) and frozen on dry ice. When the animals were younger than 8 days, the whole heads were snap-frozen in isopentane. Tissues were kept at -80 C until used for autoradiographic studies. Animal manipulations were performed according to the recommendations of the French ethical committee and under the supervision of authorized investigators.

Chemicals and antibodies
Ps4 and [Tyr0]Ps4 were obtained from Neosystem (Strasbourg, France). BSA was purchased from Boehringer Mannheim (Mannheim, Germany). Na125I (IMS-30) was obtained from Dositek, (Saclay, France). 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 emulsion was obtained from Amersham (Les Ulis, France). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Radioiodinated [Tyr0]Ps4 was prepared by the chloramine-T method (20) as previously described (4). The iodinated peptide was purified by reverse phase HPLC on an Orpegen RP-7s-300 column (0.5 x 15 cm) using a Gilson liquid chromatograph (model 811, Gilson, Oberlin, OH) with a 0–60% linear gradient of acetonitrile in 0.1% trifluoroacetic acid. Monoiodinated [Tyr0]Ps4 eluted at 33% acetonitrile. The specific radioactivity of the tracer was approximately 2000 Ci/mmol.

Polyclonal antibodies against TSH, PRL, GH, LHß, and ACTH were generous gifts from Dr. Y. Tillet (INRA, Nouzilly, France). The specificity of these antibodies has been previously described (21, 22). Control experiments were performed by preabsorbing the antisera with the homologous antigens. Polyclonal antibodies against the bovine S-100 protein were obtained from Dako (Glostrup, Denmark).

Autoradiographic studies
Whole heads or pituitaries were sliced on a cryostat (Frigocut, Reichert-Jung, Nussloch, Germany) at 20 µm in the frontal plane. Tissue slices were thaw-mounted on gelatin-coated slides, dehydrated under vacuum, and kept frozen until use. All incubation steps were performed at room temperature in 50 mM Tris-HCl buffer (pH 7.4) containing 0.1% BSA and 0.01% bacitracin. Tissue slices were preincubated in the buffer for 10 min and then incubated for 120 min with 6.5 pM [125I-Tyr0]Ps4. To visualize nonspecific binding, adjacent slices were incubated under the same conditions in the presence of 10-6 M [Tyr0]Ps4. For competition studies, slices were incubated in the presence of increasing concentrations (10-11-10-4 M) of Ps4 or [Tyr0]Ps4. Finally, the slices were washed four times for 30 sec each time in cold buffer and dried under a cold air stream. The sections were apposed onto 3H-Hyperfilm (Amersham) for 4 days. After exposure, the slices were stained with cresyl violet. Quantification of the autoradiograms was performed with a BIO 500 computer-assisted image analyzer (Biocom, Les Ulis, France) using a standard curve derived from coexposed 125I-containing brain paste standards (23).

Cell culture
The pituitary glands from 10 adult male rats were placed into culture medium (75% DMEM and 25% Ham’s F-12) containing 0.3% BSA and 1% antimycotic-antibiotic solution. The anterior lobes were cut into eight pieces, and the pituitary fragments were enzymatically dissociated at 37 C for 15 min with a solution of 0.25% trypsin in culture medium. The tissues were incubated with 0.004% deoxyribonuclease for 3 min and 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 disaggregated by gentle aspiration through a siliconized Pasteur pipette with a flame-polished tip. Dispersed cells were centrifuged at 300 x g and washed twice. The pellet was resuspended in culture medium supplemented with 5% FBS and 1% antimycotic-antibiotic solution. Pituitary cells were cultured in 24-well culture plates (2 x 106 cells/well) on poly-L-lysine-coated coverslips and kept at 37 C in a CO2-air incubator for 15 h. The cells were then rinsed three times with 50 mM Tris-HCl buffer containing 0.1% BSA and 0.01% bacitracin, dried under a cold air stream, and kept frozen until use.

Binding studies
Cultured cells were washed twice with 50 mM Tris-HCl buffer and incubated for 120 min at room temperature in the same buffer containing [125I-Tyr0]Ps4. In saturation experiments, the cells were incubated with 12–1565 pM of the radioligand, and nonspecific binding was determined by adding 10-6 M [Tyr0]Ps4 to the incubation buffer. In competition experiments, the cells were incubated with 400 pM of the radioligand in the presence of increasing concentrations (10-11-10-6 M) of [Tyr0]Ps4. The coverslips were gently removed from the culture wells and rinsed six times in cold Tris-HCl buffer. The radioactivity was then counted on a {gamma}-counter (model 1277, LKB, Rockville, MD).

Cytoautoradiography
Cultured pituitary cells were incubated with 400 pM [125I-Tyr0]Ps4 as described above. Nonspecific binding was determined by adding 10-6 M [Tyr0]Ps4 to the incubation buffer. The cells were then dried under vacuum in the presence of paraformaldehyde vapor for 24 h. The coverslips were dipped into Kodak NTB-2 liquid emulsion (Eastman Kodak, Rochester, NY) diluted 1:1 with distilled water at 40 C, as previously described (24). After 4 days of exposure, the emulsion was developed, and the autoradiographic preparations were counterstained with toluidine blue.

Autoradiography combined with immunocytochemistry on semithin consecutive sections
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 sliced in a Vibratome (Leica, Heidelberg, Germany) (80-µm thick sections). Four slices per pituitary were collected and immediately incubated for 10 min in 50 mM Tris-HCl buffer containing 0.1% BSA. Three Vibratome slices per pituitary were incubated at room temperature with 400 pM [125I-Tyr0]Ps4. The other tissue slices were incubated under the same conditions in the presence of 10-6 M [Tyr0]Ps4 to determine nonspecific binding. After incubation, all slices were rinsed in three consecutive baths (5 min each) of cold Tris-HCl buffer and postfixed in an ice-cold solution of 4% glutaraldehyde in phosphate buffer for 30 min. Then, slices were postfixed in a solution of 1% OsO4 in phosphate buffer for 20 min, dehydrated, and embedded in Araldite (Fluka, Buchs, Switzerland).

Semithin sections (1.5-µm thick) were cut from each embedded Vibratome slice, put on glass slides, and coated by dipping in Amersham LM1 nuclear emulsion. Adjacent semithin sections obtained just before and after this latter section were collected on other glass slides and used for immunocytochemistry. The sections used for autoradiography were exposed for 4–6 weeks at 4 C. Semithin autoradiographs were developed in D19 (Kodak), fixed in 30% sodium thiosulfate, rinsed in distilled water, and slightly counterstained in 0.1% azur blue.

On the adjacent sections used for immunocytochemistry, Araldite had to be removed by using sodium methoxide according to the method of Mayor et al. (25). The sections were treated with 10% hydrogen peroxide for 8 min and incubated overnight with one of the primary antisera (against TSH, LHß, PRL, GH, ACTH, or S-100) diluted 1:200, at 20 C. Then, the sections were incubated for 2 h with donkey antirabbit IgG conjugated with horseradish peroxidase (diluted 1:200). Peroxidase was visualized with H2O2 and 3,3'-diaminobenzidine tetrahydrochloride.

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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution of [125I-Tyr0]Ps4-binding sites in the adult rat pituitary
The occurrence of [125I-Tyr0]Ps4-binding sites was visualized by autoradiography on pituitary sections (Fig. 1AGo). Counterstaining of the tissue slices with cresyl violet (Fig. 1BGo) 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 10-6 M [Tyr0]Ps4, the labeling was totally suppressed (Fig. 1CGo). Quantification of the autoradiograms from serial sections (20 µm in thickness) of five pituitaries revealed that the density of [125I-Tyr0]Ps4-binding sites was constant throughout the anterior lobe (Fig. 2Go). Sections at any antero-posterior levels of the pituitary were, therefore, used for subsequent studies.



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  		Figure 1. Autoradiographic localization of [125I-Tyr0]Ps4-binding sites in frontal plane sections of adult male rat pituitary. A, Autoradiogram showing the distribution of [125I-Tyr0]Ps4-binding sites. Note the intensity of labeling in the anterior lobe. B, Cresyl violet staining of the section shown in A. AL, Anterior lobe; IL, intermediate lobe; NL, neural lobe. C, Autoradiogram of the adjacent section to that shown in A and B, incubated with [125I-Tyr0]Ps4 in the presence of 10-6 M [Tyr0]Ps4.

 


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  		Figure 2. Rostro-caudal variations in the concentration of [125I-Tyr0]Ps4-binding sites in sections of the anterior lobe of the adult male rat pituitary. The figure is representative of five independent experiments. [125I-Tyr0]Ps4-binding sites were evenly distributed throughout the adenohypophysis.

 
Incubation of pituitary slices with 6.5 pM [125I-Tyr0]Ps4 in the presence of increasing concentrations of [Tyr0]Ps4 (10-11-10-7 M) or Ps4 (10-9-10-4 M) resulted in a dose-dependent inhibition of labeling (Fig. 3Go). The IC50 values for [Tyr0]Ps4 and Ps4 were 8.3 ± 1.2 nM (n = 6) and 9.3 ± 1.2 µM (n = 4), respectively. The binding of [125I-Tyr0]Ps4 was not displaced by TRH, prepro-TRH-(178–199), {alpha}-melanotropin, ß-endorphin, somatostatin, vasoactive intestinal polypeptide, substance P, CRH, or GH-releasing hormone (data not shown).



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  		Figure 3. Typical competition of [Tyr0]Ps4 (•) and Ps4 ({circ}) 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 radioligand in the absence of competitor; B, bound radioligand in the presence of graded concentrations of competitors.

 
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. 4Go). Scatchard plot analysis (Fig. 4Go, inset) indicated that the radioligand interacts with a single class of binding sites with a Kd value of 0.16 ± 0.012 nM and a binding capacity of 3561 ± 601 sites/cell (n = 4).



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  		Figure 4. Typical saturation curve of [125I-Tyr0]Ps4 binding to cultured anterior pituitary cells. Total binding ({triangleup}) and nonspecific binding measured in the presence of 10-6 M [Tyr0]Ps4 ({triangledown}) were directly quantified in a {gamma}-counter. Specific binding (•) was calculated as the difference 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.

 
Competition studies were carried out using [Tyr0]Ps4 as a competitor. The competition curve was monophasic, with an IC50 value of 1.5 ± 0.5 nM (n = 4; Fig. 5Go).



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  		Figure 5. Typical competition curve of [125I-Tyr0]Ps4 binding to cultured rat anterior pituitary cells. Each point represents the mean of duplicates. The figure is representative of four independent experiments. B0, Bound radioligand in the absence of competitor; B, bound radioligand in the presence of graded concentrations of [Tyr0]Ps4.

 
Localization of [125I-Tyr0]Ps4-binding sites on cultured pituitary cells
The cellular distribution of [125I-Tyr0]Ps4-binding sites was studied on emulsion-coated cultured cells. Cytoautoradiographic labeling revealed that 26% of pituitary cells (n = 238 of 922) possessed a high density of silver grains (Fig. 6Go, A and B). In the presence of 10-6 M [Tyr0]Ps4, labeling was totally abolished.



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  		Figure 6. Visualization by cytoautoradiography of [125I-Tyr0]Ps4-binding sites on cultured anterior pituitary cells. A and B, Cells incubated with [125I-Tyr0]Ps4 alone. Filled arrowheads point to intensely labeled cells. Open arrows point to cells that are virtually devoid of binding sites. C and D, Cells incubated in the same conditions as those in A and B, except that 10-6 M [Tyr0]Ps4 was added. A and C, Brightfield photomicrographs of pituitary cells stained with toluidine blue. B and D, Darkfield photomicrographs of the areas shown in A and C, respectively. Bar = 15 µm.

 
Immunocytochemical identification of pituitary cells expressing [125I-Tyr0]Ps4-binding sites
Postfixation of Vibratome pituitary slices with 4% glutaraldehyde made it possible to localize [125I-Tyr0]Ps4 by autoradiography and hypophysial hormones by immunocytochemistry on consecutive semithin sections. However, in these conditions, immunoreaction with the antibodies against the bovine S-100 protein was relatively weak. Comparison of homologous fields on adjacent sections clearly demonstrated that the cells bearing [125I-Tyr0]Ps4-binding sites (i.e. cells decorated with silver grains) were not immunolabeled with antibodies against TSH (Fig. 7Go, a and b), PRL (Fig. 7Go, c and d), GH (Fig. 7Go, e and f), LHß, or ACTH (not shown). Despite the difficulty mentioned above, many cells exhibiting silver grains were unambiguously immunolabeled with the antibodies against the bovine S-100 protein (Fig. 7Go, g and h). Control sections incubated with [125I-Tyr0]Ps4 in the presence of 10-6 M [Tyr0]Ps4 exhibited only a few silver grains scattered throughout the sections (not shown).



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  		Figure 7. Autoradiographic localization of [125I-Tyr0]Ps4-binding sites and immunocytochemical identification of pituitary cells on semithin sections (1.5 µm 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, x150; G and H, x200.

 
Concentration of [125I-Tyr0]Ps4-binding sites in the pituitary during development
The evolution of the concentration of [125I-Tyr0]Ps4-binding sites in the rat pituitary during postnatal development is shown in Fig. 8Go. The concentration of binding sites was relatively low at birth (2.57 ± 0.40 fmol/mg protein) and increased slowly until day 15 (4.66 ± 0.68 fmol/mg protein). Then, a 2-fold increase in the density of recognition sites was observed on day 22, i.e. just after weaning. After day 30, the concentration of binding sites decreased gradually until adulthood. In 60-day-old rats, the concentration of binding sites was identical to that measured on day 15.



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  		Figure 8. Evolution of the concentration of [125I-Tyr0]Ps4-binding sites in the rat pituitary during postnatal development. A, Typical autoradiograms of pituitary slices at different ages. B, Concentrations of [125I-Tyr0]Ps4-binding sites at various ages from birth to adulthood. Each point represents the mean of 3–10 animals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several lines of evidence indicate that the pro-TRH-derived peptide Ps4 may regulate the activity of anterior pituitary cells. 1) The presence of immunoreactive Ps4 has been detected within nerve terminals in the external zone of the median eminence (4). 2) Authentic Ps4 is released from hypothalamic neurons in vitro (4, 5), and substantial amounts of Ps4 are found in the hypophysial portal blood (26). 3) Synthetic Ps4 has been shown to potentiate TRH-induced TSH release (6, 8) and to increase TSH and PRL gene expression (27, 28). The present study now demonstrates the 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, displacement curves could be performed on tissue slices by quantitative autoradiography. The [Tyr0]Ps4 analog appeared to be several orders of magnitude more potent than the native peptide in displacing [125I-Tyr0]Ps4 from its binding 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 inactive peptide Ps4 (3, 4, 5, 6, 7, 8, 9, 10), whereas the [Tyr0]Ps4 analog is resistant 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). Characterization of [125I-Tyr0]Ps4-binding sites on cultured anterior pituitary cells revealed that the binding was specific and saturable. The biochemical characteristics of the binding sites determined on cultured cells (this study) were very similar to those previously reported using membrane-enriched preparations of whole pituitaries (6) with Kd values of 0.18 and 0.16 nM, respectively.

The first clue suggesting that [125I-Tyr0]Ps4-binding sites may not be borne by TSH cells was provided by cytoautoradiographic studies on cultured pituitary cells. A high density of silver grains was observed on a subset of large cells, which, in the primary culture, represented about one fourth of the entire pituitary cell population. The morphological characteristics and the proportion of labeled cells made it unlikely that these cells could correspond to TSH cells, the latter being the smallest cells of the anterior pituitary and representing less that 10% of all pituitary cells (29).

To identify unambiguously the type of cells expressing [125I-Tyr0]Ps4-binding sites, histoautoradiographic localization of the recognition sites and immunocytochemical identification of the different pituitary cell types were performed on adjacent semithin sections. Accumulation of silver grains was never observed on cells immunolabeled with antibodies against TSH, PRL, GH, LHß, or ACTH. In contrast, cells exhibiting [125I-Tyr0]Ps4-binding sites were immunoreactive for the S-100 protein, a specific marker of folliculo-stellate cells (30, 31). In agreement with this finding, it has been recently demonstrated that glioma cells, which also express the S-100 protein, possess specific binding sites for [125I-Tyr0]Ps4 (32).

The occurrence of a high density of Ps4-binding sites on folliculo-stellate cells and the absence of recognition sites on TSH cells suggest that the potentiating effect of Ps4 on TRH-evoked TSH secretion previously reported (6, 7, 8) involves the contribution of folliculo-stellate cells. In support of this idea, the potentiating action of Ps4 has been observed on rat pituitary fragments, a model that preserves the cytoarchitecture of the tissue and thus allows paracrine communication between the various pituitary cells types (6, 8). In addition, it has been previously demonstrated that folliculo-stellate cells play a role in the regulation of pituitary cell activity (33). In particular, it has been shown that folliculo-stellate cells modulate the responses of GH and PRL cells to various neuroendocrine factors (34, 35). Concurrently, it has been reported that the inhibitory effect of interferon-{gamma} on pituitary hormone secretion is mediated through folliculo-stellate cells (36). It has also been found that the stimulatory effect of PACAP on pituitary hormone and cytokine secretion may involve folliculo-stellate cells (37, 38). Finally, it has been proposed that in the pars intermedia of the toad Xenopus laevis, folliculo-stellate cells are involved in the inhibitory effect of neuropeptide Y on {alpha}MSH release (39). The fact that [125I-Tyr0]Ps4-binding sites are exclusively borne by folliculo-stellate cells suggests that the regulation of TSH secretion by TRH and Ps4 may be far more complex than previously thought. In particular, the proportion of TSH cells and folliculo-stellate cells may be independently regulated, allowing greater fine-tuning of TRH-induced TSH secretion by Ps4.

The mechanism by which Ps4, acting on folliculo-stellate cells, can potentiate TRH-induced TSH release is currently unknown. It is conceivable that Ps4 can modulate the secretion of a paracrine factor responsible for enhancement of the response of TSH cells to TRH as proposed by Denef and co-workers (33, 34, 35, 36). Alternatively, folliculo-stellate cells may mediate the effect of Ps4 on TSH release by means of factors diffusing through gap junctions (40).

This study has demonstrated that [125I-Tyr0]Ps4-binding sites are detectable early after birth and that the concentration of sites increases gradually during the first 3 postnatal weeks. Previous studies have shown that TRH receptors are also expressed at birth and that their density increases postnatally (41, 42). Concurrently, the number of TRH-producing neurons in the paraventricular nucleus increases during the first week of life (43, 44). However, in rats, the capillary loops of the portal blood system penetrate the median eminence only at the end of the first postnatal week (45), and TRH-containing nerve endings make contact with the portal blood vessels only 2 days after birth (46). In fact, the hypothalamic control of the pituitary takes place only during the second postnatal week (47, 48). It thus appears that the increase in the concentration of [125I-Tyr0]Ps4 is concomitant with the development of the hypothalamo-pituitary-thyroid axis in rats. A transient reduction of [125I-Tyr0]Ps4-binding sites occurred just after weaning, suggesting that separation of the pups from their mother causes hormonal, metabolic, and/or behavioral alterations that affect the expression of the receptors.

Developmental studies have shown the occurrence of folliculo-stellate cells in the rat pituitary as early as embryonic day 20 (49), although the S-100 protein can only be detected after postnatal day 5 (49, 50). The number of folliculo-stellate cells increases markedly from days 10–40 and then reaches a plateau (50). These data suggest that the increase in the density of [125I-Tyr0]Ps4-binding sites during postnatal development can be accounted for at least in part by the proliferation of folliculo-stellate cells. However, the rapid fluctuations in receptor density observed at the time of weaning indicate that the number of receptors per folliculo-stellate cells can also be regulated.

In conclusion, the present data have shown that in the rat pituitary, Ps4-binding sites are expressed in folliculo-stellate cells, which probably mediate the potentiating effect of Ps4 on TRH-induced TSH release. The concentration of Ps4-binding sites increases during the postnatal period, suggesting that Ps4 plays a role in the regulation of the pituitary as early as the hypothalamo-hypophysial complex has become functional.


   Acknowledgments
 
The authors thank Dr. Denis Tranchand Bunel for iodination of [Tyr0]Ps4, and Mrs. Sabrina Mancel for typing the manuscript.


   Footnotes
 
1 This work was supported by grants from INSERM (U-413 and U-422), the Lille-Amiens-Rouen-Caen (LARC) network, and the Conseil Régional de Haute-Normandie. Back

2 Recipient of a fellowship from the Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche. Back

3 Recipient of a fellowship from the European Union (ERASMUS program). Back

Received August 25, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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