Expression of Orexin A and its Receptor 1 in the Bovine Urethroprostatic Complex.код для вставкиСкачать
THE ANATOMICAL RECORD 291:169–174 (2008) Expression of Orexin A and Its Receptor 1 in the Bovine Urethroprostatic Complex FINIZIA RUSSO,1 LUIGI MICHELE PAVONE,1,2 SIMONA TAFURI,1,2 LUIGI AVALLONE,1 NORMA STAIANO,1,2 AND ALFREDO VITTORIA1* 1 Department of Biological Structures, Functions, and Technologies, University of Naples Federico II, Naples, Italy 2 Department of Biochemistry and Medical Biotechnologies, University of Naples Federico II, Naples, Italy ABSTRACT Orexin A (oxA) and orexin B are recently discovered peptides derived from the proteolytic cleavage of the common precursor prepro-orexin. They bind two G protein-coupled receptors, deﬁned orexin 1 (ox1R) and orexin 2 receptor. Both peptides are highly expressed in the lateral hypothalamic area of the brain and are involved in the regulation of many functions of the body, the best investigated of which is food intake. Recent data described the presence of orexins in peripheral organs such as the adrenal glands, stomach, bowel, pancreas, and testis. Here, we report the detection of oxA and ox1R in the exocrine and endocrine cytotypes of the cattle urethroprostatic complex by using immunohistochemistry. The expression of prepro-orexin and ox1R mRNA transcripts in the prostatic tissue was assessed by reverse-transcriptase polymerase chain reaction, while the presence of both the proteins in the tissue was conﬁrmed by Western blotting analysis. Our ﬁndings provide the ﬁrst evidence for the presence of oxA and ox1R in the urethroprostatic complex of the cattle and demonstrate that both proteins are locally synthesized, thus suggesting a role for oxA on both physiological and pathological functioning of the complex. Anat Rec, 291:169–174, 2008. Ó 2008 Wiley-Liss, Inc. Key words: orexin a; orexin 1 receptor; urethroprostatic complex; cattle Orexin A (oxA) and orexin B (oxB) are two peptides discovered in 1998 in the lateral hypothalamus of the rat (de Lecea et al., 1998; Sakurai et al., 1998). OxA is a 33 amino acid peptide with N-terminal pyroglutamyl residue and two intrachain disulphide bonds, whereas OxB is a linear peptide composed of 28 amino acids. They both derive from a common 130 amino acid precursor peptide, prepro-orexin, by proteolytic cleavage. Orexins exert their actions by binding and activating two different G protein-coupled receptors termed as orexin 1 (ox1R) and orexin 2 (ox2R) receptor. The binding properties of these receptors are partially different: ox1R is highly selective for oxA, whereas ox2R shows similar afﬁnity for both the peptides (Sakurai et al., 1998). Although orexins are primarily expressed in the lateral hypothalamus, the brain area controlling food intake, orexigenic ﬁbers project toward multiple cerebral Ó 2008 WILEY-LISS, INC. regions (Peyron et al., 1998). Thus, in addition to a key role in food intake (Sakurai, 1999), the involvement of orexins in the central control of additional biological functions has been established. They have been shown to regulate arterial blood pressure and heart rate Grant sponsor: MIUR, Rome, Italy; Grant numbers: PRIN 2005/2006; FISR 2005. *Correspondence to: Alfredo Vittoria, Department of Biological Structures, Functions, and Technologies, University of Naples Federico II, Via F. Delpino n.1, 80137 Naples, Italy. Fax: 39-081-2536097. E-mail: firstname.lastname@example.org Received 22 September 2007; Accepted 14 November 2007 DOI 10.1002/ar.20641 Published online in Wiley InterScience (www.interscience. wiley.com). 170 RUSSO ET AL. (Shirasaka et al., 1999), sleep/wake cycle (Piper et al., 2000), sexual behavior and arousal (Gulia et al., 2003), water assumption (Kunii et al., 1999), and plasma corticosterone levels (Kuru et al., 2000). Moreover, the orexin system has been demonstrated to be closely associated with the pathogenesis of narcolepsy in humans and mice (Chemelli et al., 1999; Peyron et al., 2000). Recent studies demonstrated that both the orexins and their receptors are also expressed in peripheral organs belonging to the gastrointestinal (Kirchgessner and Liu, 1999; Ehrström et al., 2005) and genital (Karteris et al., 2004; Barreiro et al., 2005) tracts. In particular, the presence of oxA has been described in a cellular population of the digestive mucosa whose elements were deﬁned APUD (amine precursor uptake and decarboxylation) or neuroendocrine (NE) cells (Pearse, 1977) or paraneurons (Fujita et al., 1988). Such cells are scattered throughout the exocrine epithelia, synthesize biogenic amines, and contain an acidic protein, chromogranin A (chr A), which is considered to be their own marker (Deftos, 1991). The role of oxA produced by these cells in several mammals has been related to the regulation of gut motility, blood ﬂow, and epithelial secretion (Kirchgessner and Liu, 1999; Ehrström et al., 2005). The expression of oxA in the genital tract has been demonstrated in the rat testis, where the peptide appears to play a role in steroidogenesis (Barreiro et al., 2005). In addition, the expression of mRNAs encoding for the prepro-orexin and the cognate receptors (ox1R and/or ox2R) has been reported in the testis, penis, epididymis, and seminal vesicles of several mammals (Jöhren et al., 2001; Karteris et al., 2004; Barreiro et al., 2005; Zhang et al., 2005), and of the fowl (Ohkubo et al., 2003). In this study, the presence of oxA and ox1R has been investigated in the urethroprostatic complex of the cattle by means of immunohistochemistry. Furthermore, the expression of prepro-orexin and ox1R in this tissue has been established by reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting analyses. MATERIALS AND METHODS Antibodies and Chemicals Horseradish peroxidase conjugated anti-rabbit and anti-goat IgG were purchased from Sigma Chemical Co. (St. Louis, MO); goat polyclonal anti-oxA (sc-8070), and anti-ox1R (sc-8073) antibodies and their respective blocking peptides (sc-8070 P for oxA, and sc-8073 P for ox1R) from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit polyclonal anti–prepro-orexin antibody (AB3096) and its blocking peptide (AG774) from Chemicon International, Inc. (Temecula, CA); rabbit polyclonal anti–chr A antibody (20086) from ImmunoStar Inc. (Hudson, WI); biotinylated secondary antibodies and avidin–biotin complex were from Vector Laboratories (Burlingame, CA); Triazol from Invitrogen (Milano, Italia). The primers for bovine prepro-orexin and ox1R were provided by Primm (Milano, Italia), and the kit for PCR and RT-PCR by Promega (Milano, Italia). Tissue Sampling The prostate of cattle is entirely contained in the lamina propria of the urethral mucosa just caudally to the collicular zone. The gland of the adult bull is 2–3 cm long, 0.4–0.8 cm thick, and it almost entirely surrounds the lining epithelium of the urethra from which it is separated by a thin layer of connective tissue. Transversely cut samples of postcollicular urethra were collected from 9 adult, healthy, noncastrated cattle in a local slaughterhouse, soon after the death. The fragments containing both the urethral and prostatic epithelia were longitudinally divided in two parts, which were processed for immunohistochemistry and biochemical analyses, respectively. A portion of samples was ﬁxed in Bouin’s ﬂuid for 24 hr, dehydrated through ascending ethanol and embedded in Paraplast Plus. The second portion was brought in the laboratory in an ice-cold bath, and observed by a light stereomicroscope to separate the lining epithelium from the bulk of the prostate. This latter was frozen in liquid nitrogen, until use. Immunohistochemistry Sections (5 mm-thick) were cut by a microtome, collected on slides, and stained by the immunohistochemical avidin–biotin technique. In the speciﬁc step, polyclonal antibodies raised against chr A, oxA, and ox1R were used. The ﬁrst antibody was diluted 1:4,000 and the others 1:200; all were applied on sections overnight at 48C. The other components of the immunological reaction were contained in the Vectastain Elite ABC kit (PK6101 rabbit; PK6105 goat) from Vector Laboratories Inc. The ﬁnal staining was performed using a solution of 330 -diaminobenzidine (DAB) on the sections for 2–10 min. Sometimes, an antigen unmasking procedure preceded the immunohistochemical reaction and was carried out by dipping the sections in 0.01 M sodium citrate buffer, pH 6.0, and heating them in a microwave oven for 10 min at 750 W. Controls were obtained by substituting the primary antisera with PBS or normal serum in the speciﬁc step, or alternatively, by absorbing each primary antiserum with an excess of the relative peptide (100 mg of peptide/ml of diluted antiserum). Such controls were always negative. The preparations were observed by a Nikon E 600 light microscope, and microphotographs were taken using a Coolpix 8400 Nikon digital camera. Homogenate Preparation The prostates were homogenized by an Ultraturrax L407 at 48C with 5 ml/1.5 g tissue of buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 10 mM NaF, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P40, 1 mM phenylmethylsulfonyl ﬂuoride, 0.1 U/ml aprotinin, 10 mg/ml leupeptin, and 1 mM Na3VO4. Homogenates were centrifuged at 15,000 3 g for 10 min at 48C. Supernatants were divided into small aliquots, and stored at 2808C until used. The amount of total proteins in each sample was determined by the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA). Western Blotting Analysis Homogenate samples containing equal amount of proteins were boiled for 5 min in SDS buffer (50 mM TrisHCl, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromphenol blue, and 5% m-mercaptoethanol), and run on a 12.5% SDS/polyacrylamide gel. After electrophoresis, the pro- OREXIN A DETECTION IN THE URETHROPROSTATIC COMPLEX 171 Fig. 1. Neuroendocrine (a–d) and exocrine (e,f) cells of the cattle urethral (a–c) and prostatic (d–f) epithelium. a: Differently shaped, chr A–containing cells scattered in the lining epithelium of a mucosal crypt. b,c: OxA-containing cells showing linear, opposite polar (b) and irregular (c) course of their cytoplasmic extensions. d: Chr A–containing cell of a glandular acinus whose irregular cytoplasmic extension is clearly oriented toward the basal membrane. e,f: OxA- (e) and ox1R- (f) immunopositive, granular material contained in the cytoplasm of exocrine cells of the prostatic parenchyme. Avidin–biotin immunohistochemical technique, 3-30 -diaminobenzidine (DAB) staining. Scale bars 5 10 mm. teins were transferred to nitrocellulose using a Mini trans-blot apparatus (Bio-Rad Laboratories) according to the manufacturer’s instructions. Membranes were blocked for 1 hr at room temperature with TBS-T buffer (150 mM NaCl, 20 mM Tris HCl, pH 7.4, 0.1% Tween 20) containing 5% milk. The blots were incubated overnight with polyclonal antibodies against prepro-orexin or ox1R, both diluted 1:1,000 in TBS-T containing 2.5% milk. After the incubation, the membranes were washed three times with TBS-T and incubated for 1 hr with horseradish peroxidase conjugated anti-(goat IgG) Ig diluted 1:3,000 in TBS-T containing 2.5% milk. The proteins were visualized by enhanced chemiluminescence (ECL) (Amersham, Little Chalfont, UK). To ensure speciﬁcity, preabsorption of prepro-orexin or ox1R antibodies with their relative control peptides (AG774 and sc-8073 P, respectively) was performed before Western blotting. verse primer 50 -CTTGCCCAGCGTGAGGAT-30 for prepro-orexin; forward primer 50 -AGGCTGCGGTCATGGA AT-30 and reverse primer 50 -TTCCTGACCAGGGCTGAC30 for ox1R. The PCR products were separated on a 2% agarose gel and visualized by ethidium bromide using a 1-kb DNA ladder to estimate the band sizes. As a negative control for all reactions, distilled water was used in place of cDNA. RNA Extraction and RT-PCR Analysis Total RNA was extracted from cattle prostate sample by using Triazol solution. The RNA was re-suspended in 50 ml of diethyl pyrocarbonate treated water, and stored at 2808C until used. Synthesis of cDNAs for the detection of prepro-orexin and ox1R mRNAs was performed by using a reverse transcription system (Promega, Madison, WI). The following speciﬁc primers were used: forward primer 50 -ATCCTTCCTCTACAAAGGTCTCC-30 and re- RESULTS Localization of oxA and ox1R in the Cattle Urethroprostatic Complex: Immunohistochemical Analysis Chr A–containing NE cells appeared to be scattered in the stratiﬁed epithelium of the postcollicular portion of the urethra (Fig. 1a). These cells showed a focal distribution being grouped in small clusters of 10–30 elements each. Their shape was round, elongate, or irregular. The elongate cells were clearly bipolar, and showed the opposite extremities oriented toward the urethral lumen and the basal membrane. The irregular cells showed narrow cytoplasmic extensions, often curved in their course, oriented toward the neighboring exocrine cells. A small subpopulation of the NE urethral cells contained oxA and were ascribed, as far as the shape, to the elongate or bipolar type (Fig. 1b,c). These cells were much less numerous than the chr A-containing cells and were 172 RUSSO ET AL. Fig. 2. Expression of prepro-orexin and ox1R mRNAs and the proteins in the prostrate of cattle. A: Reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting analyses for preproorexin expression. B: RT-PCR and Western blotting analyses for ox1R expression. The blots on the left indicate the presence in the prostate of mRNA transcripts of 200 and 300 bp for prepro-orexin (upper blot) and ox1R (lower blot), respectively (lane 2); lane 1 corresponds to the DNA ladder and lane 3 to the negative control. The aspeciﬁc bands at the basis of lanes 2 and 3 corresponding to the primer dimers are probably due to the low temperature used in the PCR run. The blots on the right indicate the presence in the prostate of the two proteins, prepro-orexin with a molecular mass of 16 kDa (upper blot) and ox1R with a molecular mass of 50 kDa (lower blot). The arrows on the right indicate the bands corresponding to the proteins. Similar results were obtained from four separate experiments of identical design. found in six subjects out of nine. Their cytoplasm appeared completely ﬁlled by ﬁne positive granules also present in the two opposite extremities. No positivity for ox1R was observed in the urethral epithelium. The chr A–containing NE cells of the cattle prostate were somewhat rare, isolated and scattered in the exocrine epithelium (Fig. 1d). They were similar in shape to the analogous urethral cells, and no positivity for oxA and ox1R was observed. On the contrary, positivity for both peptides was found in the exocrine epithelium of the prostate in all examined subjects (Fig. 1e,f). OxA- or ox1R-containing cells were grouped in clusters composed of dozens up to few hundred of elements scattered in the glandular parenchyma. When these cells were observed in a series of consecutive sections, they showed distributions roughly overlapping to each other. In both cases, the intensity of the staining varied from light to bright. of 200 bp for the prepro-orexin (Fig. 2A, blot on the left) and of 300 bp for ox1R (Fig. 2B, blot on the left) in all tested samples. The presence of prepro-orexin and ox1R in the cattle prostate was conﬁrmed by immunoblotting, using, respectively, a rabbit polyclonal antibody raised against a 17 amino acid peptide mapping near the C-terminus of mouse prepro-orexin, and a goat polyclonal antibody raised against a peptide mapping near the Cterminus of ox1R of rat origin. The detected preproorexin showed a molecular mass of 16 kDa (Fig. 2A, blot on the right), while ox1R a molecular mass of 50 kDa (Fig. 2B, blot on the right). The speciﬁcity of the response was conﬁrmed by preincubation of the preproorexin and ox1R antibodies with their respective blocking peptides. There was no expression of prepro-orexin and ox1R in these preparations, whereas the presence of the proteins was detected in a mouse brain homogenate that was used as positive control (data not shown). Expression of Prepro-orexin and ox1R in the Cattle Prostate: Biochemical Analysis The expression of prepro-orexin and ox1R mRNAs in the prostate was analyzed by RT-PCR. This analysis resulted in the ampliﬁcation of speciﬁc DNA fragments DISCUSSION The NE cells scattered in the glandular and lining epithelia of the body are considered as receptosecretory cells able to receive mechanical and/or chemical stimuli OREXIN A DETECTION IN THE URETHROPROSTATIC COMPLEX by their apical (luminal) extremity and to respond producing and releasing biologically active substances (amines or peptidic hormones) through the opposite-polar extremity oriented toward the neighboring exocrine cells or the subepithelial capillaries (Fujita et al., 1988). In particular, the NE cells scattered in the urethral epithelium have been widely accepted as a source of hormones in the genital tract and the collicular zone where they are particularly numerous as a focal structure under this point of view (Hanyu et al., 1987; Vittoria et al., 1990, 1992). The NE cells of the cattle and sheep urethroprostatic complex have been found to contain chr A, serotonin, somatostatin, and enkephalin (Vittoria et al., 1990; Arrighi et al., 2004). The results of our immunohistochemical study on the urethroprostatic complex of cattle demonstrated the presence of oxA, but not of ox1R, in a small subpopulation of NE urethral cells. It seems possible to hypothesize that the NE cells of the cattle urethra producing oxA release the peptide toward the neighboring exocrine cells and/or the subepithelial blood capillaries, in conformity with a paracrine or endocrine modality of secretion, respectively. Targets of oxA in the mammalian genital tract are the seminal vesicles, penis, epididymis, and testis (Ohkubo et al., 2003; Karteris et al., 2004; Barreiro et al., 2005; Zhang et al., 2005) in which the presence of orexin receptors and/or their respective mRNAs has been described. Moreover, immunohistochemistry revealed the localization of oxA only in two testicular cytotypes of the rat genital tract, namely the Leydig cells and the spermatocytes (Barreiro et al., 2005). Orexin-containing NE cells have been described in the gastrointestinal tract and pancreas of several mammals, including humans (Kirchgessner and Liu, 1999; Ehrström et al., 2005). They are localized in the stomach, bowel, and pancreatic islets in which they co-store, respectively, gastrin, serotonin and insulin, or pancreatic polypeptide. The hypothesized functions exerted by the orexins produced by these cells include the activation of both intrinsic and extrinsic primary afferent neurons, stimulation of intestinal secretion after a meal, and modulation of the insulin effects on food intake and/or glucose metabolism. Our immunohistochemical analysis showed the presence of both oxA and ox1R in the exocrine epithelium of the cattle prostate. The expression of genes encoding prepro-orexin and ox1R analyzed by RT-PCR demonstrated the presence of mRNA transcripts of both these proteins in the prostate tissue of cattle. In addition, the expression of the two proteins was conﬁrmed by Western blotting analysis. The apparent molecular masses of prepro-orexin (16 kDa) and ox1R (50 kDa) as assessed in our study is comparable with those found for the proteins localized in other tissues of different mammalian species including humans (Karteris et al., 2004). The small differences observed in the molecular mass of ox1R (50–55 kDa) is thought to be due mainly to the extent of glycosylation. The presence of both oxA and the relative receptor 1 in the prostate of another ruminant species, the water buffalo Bubalus bubalis, has been found just recently by our research group (unpublished ﬁnding). The lonely report concerning oxA and a glandular parenchyma in the cattle indicates that the peptide stimulates the synthesis of catecholamines from the adrenal 173 glands through the protein kinase C-mediated tyrosine hydroxylase activation (Kawada et al., 2003). On the other hand, studies on in vitro cultured cells showed that orexins modulate the growth of rat adrenocortical cells, by exerting both proliferogenic and antiproliferogenic effects mediated by ox1R and ox2R, respectively (Spinazzi et al., 2005). Conversely, orexins acting at native or recombinant ox1R in colon cancer and neuroblastoma cells have been shown to suppress cell growth by inducing apoptosis (Rouet-Benzineb et al., 2004). On the basis of these results, we postulate that the cattle urethroprostatic complex may be a source of oxA that could be either used locally or spread out toward neighboring targets, thus playing a critical role in both physiological and pathological states of the genital tract. In conclusion, the data presented in this study provide the ﬁrst evidence for the presence of oxA and ox1R in the urethroprostatic complex of a mammalian species, although further studies are needed to assess the role exerted by them. ACKNOWLEDGMENT We thank Mr. Antonio Calamo for technical assistance. LITERATURE CITED Arrighi S, Cremonesi F, Bosi G, Domeneghini C. 2004. Endocrineparacrine cells of the male urogenital apparatus: a comparative histochemical and immunohistochemical study in some domestic ungulates. 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