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Cellular Localization of Orexin Receptors in Human Pituitary*

Departments of Morphological Sciences (M.B., T.G.-C., R.G., A.V.-B., A.B.), Physiology (M.L., R.S., C.D.), and Medicine (F.C.), School of Medicine–University Clinical Hospital, University of Santiago de Compostela, E-15782 Santiago de Compostela, Spain; and Centre National de la Recherche Scientifique UMR 5578 (G.M.), Université Claude Bernard-Lyon 1, Villeurbane, France

Address correspondence and requests for reprints to: Prof. Tomás García-Caballero, Department of Morphological Sciences, School of Medicine, University of Santiago de Compostela, San Francisco, E-15782 Santiago de Compostela, Spain. E-mail: cmhisihq{at}usc.es

	Abstract

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Orexins-A and -B are hypothalamic peptides derived from a precursor called prepro-orexin and relationated with the stimulation of food intake. They act on G protein receptors named orexin receptor 1 (OX₁R) and orexin receptor 2 (OX₂R), respectively. In the present study, we used RT-PCR and immunohistochemical techniques to detect the presence of OX₁R and OX₂R in human pituitary. A band of the expected size for both OX₁R and OX₂R was shown in human pituitary by RT-PCR. The cellular localization of OX₁R and OX₂R was carried out using histological techniques. By consecutive sections we demonstrated that OX₁R was present in acidophil, diffusely distributed cells, which represent the half of the total adenohypophysis cell population. As was expected, these cells were shown to coexpress GH. OX₂R was found in the pars intermedia and in clusters of basophil cells of the anterior pituitary, which coexpress ACTH. These results were confirmed by double immunofluorescence techniques. We also found focal positivity in axon terminals of neurohypophysis, more intense for OX₂R than for OX₁R. In conclusion, these results demonstrated for the first time that OX₁R and OX₂R were expressed by somatotrope and corticotrope cells, respectively.

	Introduction

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OREXINS ARE HYPOTHALAMIC peptides that arise by proteolysis from a 130 amino acid common precursor (prepro-orexin) that is mainly synthesized in neurons within and around the lateral and posterior hypothalamus (1, 2). The orexin-containing neurons project to multiple neuronal systems (3). Orexin-A and -B are 33 and 28 residue peptides, respectively, that show a 46% sequence homology and are very similar in rats and humans (orexin-A is identical in both species, and orexin-B only differs in 2 residues). Orexins act on G protein previously orphan receptors named orexin receptor 1 (OX₁R) and orexin receptor 2 (OX₂R). OX₁R is selective for orexin-A, whereas OX₂R binds both orexin-A and -B (2). Orexin receptors have a broad distribution in the brain (4). Orexins, after the Greek word "orexis," which means appetite, have a physiological role in the stimulation of food intake (2). The wide distribution of orexin fibers and orexin receptors indicate that orexins may play important physiological roles other than feeding, such as modulation of macrophage functions (5); regulation of blood pressure, neuroendocrine system, body temperature, and sleep-waking cycle (3, 6, 7, 8); stimulation of gastric acid secretion (9); and involvement in the stress reaction in rats (10). Recently, hybridization signals for orexin receptors were demonstrated in rat pituitary (primarily in the intermediate and anterior lobes; Ref. 11). To define a possible direct regulatory role of orexins on pituitary cell function, we have sought to characterize the expression of both orexin receptors in the human adenohypophysis as well as to identify the cell types that express these proteins. To this end, we used a combination of different techniques such as RT-PCR and immunohistochemistry.

	Materials and Methods

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Tissues

Human pituitaries from seven individuals [four males and three females; mean age, 64.7 yr (range, 46–85)] were obtained from recent autopsies (<24 h) performed at the Pathology Department (Prof. J. Forteza) of the University Clinical Hospital (Santiago de Compostela, Spain). None of the patients had evidence or history of endocrine disease.

RNA isolation

Pituitary (adenohypophysis and posterior lobe) and hypothalamic samples were frozen immediately in dried ice. Total RNA was isolated using Trizol Reagent (Life Technologies, Inc., Rockville, MD). For RNA integrity analysis, 5 µg total RNA were resolved in a 1% agarose gel and the RNA was visualized by ethidium bromide fluorescence in a digital imaging system (Molecular Analyst; Bio-Rad Laboratories, Inc., Richmond, CA). Pituitary RNA shows a good preservation state (Fig. 1A).

View larger version (42K): [in this window] [in a new window]   Figure 1. A, RNA integrity of a human pituitary sample. The typical two-band pattern of 28S and 18S ribosomal RNA (rRNA) and the absence of a smear confirms the good preservation of the sample. B, Detection of OX1R and OX2R expression by RT-PCR. In both figures amplification of hypoxanthine-guanine phosphoribosyltransferase (HPRT) is also shown. Lane 1, human hypothalamus; lane 2, human pituitary; lane 3, RT- (without RT) of human hypothalamus; lane 4, RT- (without RT) of human pituitary; lane 5, PCR- (without Taq polymerase); lane M, 100-bp molecular weight marker.

The RT reaction was carried out in a volume of 30 µL containing 3 µg total RNA and incubated at 37 C for 60 min. The enzyme was inactivated by heating at 95 C for 5 min.

We amplified 9 µL of the RT product. We have designed the following sets of primers: OX₁R, 5'-TGAAGTGAAGCAGATGCGTGC-3' and 5'-TGGTGACGCTGGTGAGCA-3' (the size of the product is 412 bp); OX₂R, 5'-AGCAGATCCGAGCCAGAAGG-3' and 5'-GGCTGCTGGGAGTGTGCTTA-3 (the size of the product is 425 bp).

The temperatures and times used were 38 cycles at 94 C for 1 min, 67 C for 1 min, 72 C for 1 min, with a final extension step at 72 C for 10 min.

We used hypoxanthine-guanine phosphoribosyltransferase as a housekeeping gene, amplified as described above using the following primers: 5'-CAGCCCTGGCGTCGTGATTA-3' and 5'-GCAAGACGTTCAGTCCTGTC-3' (the size of the PCR product is 138 bp).

Amplifications were carried out in a DNA thermal cycler (Eppendorf, Hamburg, Germany). Each RNA sample was retrotranscribed and amplified simultaneously with a retrotranscriptase-free control to discard possible contamination with DNA. The amplified products were resolved in a 2% agarose gel, and the DNA was visualized by ethidium bromide fluorescence in a digital imaging system (Molecular Analyst; Bio-Rad Laboratories, Inc.).

Immunohistochemistry

The pituitary samples were immediately immersion fixed in 10% buffered formalin for 24 h, dehydrated, and embedded in paraffin by a standard procedure. Serial sections 4 µm thick were mounted on 3-aminopropyl-triethoxysilane-coated slides and deparaffinized before hematoxylin and eosin staining or immunohistochemical techniques. Rabbit polyclonal antisera anti-OX₁R and anti-OX₂R (Alpha Diagnostic, San Antonio, TX) were used. Both antisera have been previously validated for immunohistochemistry (12). Antigen retrieval was carried out by pressure cooker unmasking for 2 min in 0.01 M sodium citrate buffer (pH 6.0). Polyclonal antisera anti-GH (Dakopatts, Glostrup, Denmark) and anti-ACTH (BioGenex Laboratories, Inc. San Ramon, CA) were also used without unmasking procedure. The streptavidin-biotin complex immunohistochemical method was used, and the sections were consecutively incubated in: 1) polyclonal antibodies anti-OX₁R at a dilution of 1:200 for 1 h, anti-OX₂R at 1:100 overnight, anti-GH at 1:2000 for 1 h, or anti-ACTH at 1:100 for 1 h; 2) 3% hydrogen peroxide for 10 min (Merck, Darmstadt, Germany) to block endogenous peroxidase; 3) biotinylated goat antibodies to mouse/rabbit immunoglobulins (Duet kit; Dakopatts) at a dilution of 1:100 for 30 min; 4) streptavidin-biotin-peroxidase complex (Duet kit; Dakopatts), prepared according to the protocol provided by the manufacturer, for 30 min; and 5) 3,3'-diamino-benzidine-tetrahydrochloride solution prepared by dissolving one 3,3'-diamino-benzidine-tetrahydrochloride-buffer tablet (Merck) in 10 mL distilled water for 10 min. Between steps, the sections were washed twice for 5 min with PBS [0.01 mol/L phosphate buffer (pH 7.4) containing 0.15 mol/L NaCl] and, after step 5, with distilled water. All dilutions were made in PBS. This buffer was added with 0.1% BSA (Sigma, St. Louis, MO) for dilution of the primary antisera (step 1) and with 1.5% normal goat serum (Dakopatts) for the biotinylated antibodies (step 3). No counterstaining was done.

Controls for specificity of immunohistochemistry included incubation with the primary antibodies preabsorbed overnight at 4 C with the immunogen peptides (OX₁R and OX₂R, 10 nmol/mL; Alpha Diagnostic) and using alternately PBS in place of one of the other incubation steps.

Double labeling

Double labeling experiments were carried out on paraffin sections processed as described. Primary antibodies against OX₁R (rabbit polyclonal diluted at 1:25; Alpha Diagnostic) and GH (mouse monoclonal diluted at 1:200; Chemicon International, Temecula, CA) were mixed and applied to the slides and incubated overnight. After two washes in PBS, incubation with specie-specific secondary antibody was done: goat antirabbit coupled with tetramethylrhodamine isothiocyanate (Sigma, St. Louis, MO) diluted at 1:25 and goat antimouse coupled with fluorescein isothiocyanate (FICT; Sigma) diluted at 1:40 in PBS with 10% of normal goat serum (Dakopatts). Antirabbit OX₂R (Alpha Diagnostic) was labeled with Cy3 Fluor Kit (Amershan Pharmacia, Little Chalfont, BuckingHamshire, UK), and antihuman ACTH obtained from the National Institute of Diabetes, Digestive and Kidney Diseases was labeled with FICT Fluor Kit (Amershan Pharmacia). Fifty microliters of each antibody were applied to the slides and incubated overnight individually. After washing in distilled water, slides were mounted with Immuno-Fluore mounting medium (ICN, Aurora, Ohio). The sections were observed and photographed by using a Provis AX70 microscope (Olympus Corp., Tokyo, Japan).

	Results

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RT-PCR

RT-PCR of human adenohypophysis complementary DNA yielded a band of the expected size for both OX₁R and OX₂R (Fig. 1B).

Immunohistochemistry

Intense immunoreactivity for orexin receptors was demonstrated in the human adenohypophysis. By serial sections, immunoreactivity for OX₁R was demonstrated in acidophil cells (Fig. 2, A and B), whereas positivity for OX₂R was found in basophil cells (Fig. 2, C and D). The OX₁R immunostaining was more intense than that of OX₂R. OX₁R expression was found in 50% of all adenohypophysal cells that were diffusely distributed. The staining properties (acidophilia), percentage, and distribution of the cells led us to identify them as somatotropes, a fact that was demonstrated by immunostaining of consecutive sections for OX₁R and GH (Fig. 2, E and F). Positivity for OX₂R was very intense in the pars intermedia (Fig. 2, C and D) and in focal clusters of basophil cells of the anterior pituitary (Fig. 2G). As expected for these characteristics, OX₂R-positive cells were shown to coexpress ACTH (Fig. 2, G and H). At high magnification, immunostaining was also found in scarce axon terminals of the neurohypophysis, and in this localization the signal intensity was more markedly for OX₂R (Fig. 2I) than for OX₁R (data not shown). No immunostaining was observed either when any essential step of the reaction was omitted, or when the primary antisera were preabsorbed with their homologous antigens (Fig. 2, J and K).

View larger version (82K): [in this window] [in a new window]   Figure 2. A and B, Serial sections of human anterior pituitary stained with hematoxylin and eosin (A) and immunostained for OX1R (B). Only eosinophil cells are positive for OX1R (some of them are identified by arrows) (magnification, x90). C and D, Serial sections of human pituitary stained with hematoxylin and eosin (C) and immunostained for OX2R (D). Immunoreactivity for OX2R was only found in basophil cells of the pars intermedia, being negative the eosinophil cells of the anterior pituitary (AP) as well as the posterior pituitary (PP) (magnification, x20). E and F, Serial sections of human anterior pituitary immunostained for OX1R (E) and GH (F). Note that the same cells are positive in both pictures (magnification, x90). G and H, Serial sections of human anterior pituitary immunostained for OX2R (G) and ACTH (H). The same cell clusters show immunoreactivity for both antisera (magnification, x90). I, Immunoreactivity for OX2R in the human neurohypophysis. High magnification led us to observe that some axons presented positivity (magnification, x130). J and K, Negative controls for OX1R (J) and OX2R (K) in human adenohypophysis. No signal was detected when the antisera were preabsorbed with the homologous antigens (Nomarsky differential interference contrast; magnification, x130). L–N, Double immunofluorescence for OX1R and GH. OX1R-expressing cells are identified by tetramethylrhodamine isothiocyanate immunofluorescence (L) and GH-positive cells by FICT immunofluorescence (M). Colocalization of both OX1R and GH in the same cells is illustrated by the yellow color obtained using the double exposure method of photomicrograpy (N; magnification, x225). O–Q, Double immunofluorescence for OX2R and ACTH. All OX2R-displaying cells (Cy3 immunofluorescence in O) are also positive for ACTH (FICT immunofluorescence in P). Coexpression is confirmed by the double exposed micrography (yellow in Q; magnification, x250).

To confirm the previous results, double immunofluorescence techniques were used. Two-color immunofluorescence demonstrated that all OX₁R-positive cells were also GH positive (Fig. 2, L–N) and that all OX₂R-positive cells were also ACTH positive (Fig. 2, O–Q).

	Discussion

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Orexins are synthesized in neurons of the lateral and posterior hypothalamus and have a central role in feeding regulation. However, the demonstration of a wide projection of orexin nerve fibers in the brain suggest complex roles of the peptides in autonomic and neuroendocrine control (3, 7), and the broad central distribution of orexin receptors may indicate additional as yet undefined physiological functions for orexins (4).

The present study describes for the first time the expression of orexin receptors in human pituitary. Recently, orexin receptor messenger RNAs were demonstrated in rat pituitary by in situ hybridization techniques. Intense signals were shown in the intermediate and anterior lobes, and weak signals in the posterior lobe (11). These authors also demonstrated that in the rat anterior lobe the expression of OX₁R was more intense than that of OX₂R. These results are in agreement with our findings in human pituitary where the cells immunostained for OX₁R were much more numerous than the cells expressing OX₂R. Immunohistochemistry performed in serial sections and double immunofluorescence techniques allowed us to demonstrate that all OX₁R-expressing cells were somatotropes and that all OX₂R-expressing cells were corticotropes. To confirm the previous results, double immunofluorescence techniques were used. As in the study by Date et al. (11), we also found a weak expression of orexin receptors in the neurohypophysis. However, these authors showed that the signal intensity of OX₁R messenger RNA in the intermediate lobe of the rat was as strong as that of OX₂R (11), and in human pituitary we found that basophil cells characteristic of the pars intermedia only express OX₂R. Recently, it was demonstrated that the plasma concentrations of ACTH and corticosterone were significantly increased after central administration (13) or sc injection (14) of orexin-A. On the other hand, central administration of orexin-A seems to inhibit in vivo GH secretion in the rat (our unpublished observations). Although it has been proposed that these effects may result from the release of hypothalamic factors (13), our data open up the possibility that some of the neuroendocrine actions of these peptides could be exerted directly at pituitary level. In summary, the present findings provide the first evidence that orexin receptors are expressed in the human pituitary gland. Whereas somatotrope cells seem to express OX₁R, positive immunostaining for OX₂R was found in corticotropes. It remains to be established whether alteration in orexin receptors gene expression could lead to alterations in human pituitary cell proliferation and/or hormone secretion.

	Acknowledgments

 
We thank the expert technical assistance of D. Fernández-Roel.

Received August 9, 2000.

Revised November 21, 2000.

Accepted December 2, 2000.

	References

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Blanco, M. Articles by Beiras, A. Search for Related Content PubMed PubMed Citation Articles by Blanco, M. Articles by Beiras, A.