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Expression of Orexin A and its Receptor 1 in the Bovine Urethroprostatic Complex.

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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: avittori@unina.it
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.
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