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Endocrinology Vol. 138, No. 11 5013-5018
Copyright © 1997 by The Endocrine Society

ARTICLES

Immunocytochemical Localization of the NPY/PYY Y1 Receptor in the Developing Pancreas1

Malene Jackerott and Lars-Inge Larsson

Department of Molecular Cell Biology, Statens Seruminstitut, Artillerivej 5, Building 81, DK-2300 Copenhagen S, Denmark

Address all correspondence and requests for reprints to: Professor L.-I. Larsson, DSc, Department of Molecular Cell Biology, Statens Seruminstitut, Artillerivej 5, Building 81, DK-2300 Copenhagen S, Denmark. E-mail: Larsson{at}biobase.dk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neuropeptide Y, peptide YY, and pancreatic polypeptide are structurally related peptides that are considered to play a role in the regulation of pancreatic secretion and blood flow. Several receptor subtypes for these peptides have been identified, and the Y1, Y2, Y4/PP1, Y5, and Y5/PP2/Y2b receptors are cloned. We have prepared polyclonal peptide antibodies that recognize the Y1 receptor and now report on its localization in the adult and developing rat pancreas. In the adult pancreas, Y1 receptors were detected both in some centroacinar and intralobular duct cells and in endothelial cells. In the developing pancreas (E12.5–E16.5), Y1 receptor immunoreactivity was observed in numerous nonendocrine epithelial cells. These cells occurred in the immediate vicinity of peptide YY-positive endocrine cells. At E16.5, a fraction of these Y1 receptor-containing cells co-stored amylase. One day later, Y1 receptor immunoreactivity became restricted to pancreatic duct-like cells that occurred in close proximity to peptide YY cells. In fetal rats, intense Y1 receptor staining was also observed in endothelial cells. These observations, together with the finding of early pancreatic peptide YY expression, suggest that peptide YY produced by fetal endocrine cells may exert an action on exocrine cells, duct cells and endothelial cells during development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NEUROPEPTIDE Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) constitute a family of 36 amino acid-peptides, termed the PP family (1). NPY is widely distributed in the central and peripheral nervous system and belongs to the most abundant neuropeptides. In the pancreas, NPY has been localized both to insulin-producing islet cells and to nerve fibers (2, 3). PYY represents a hormone present in endocrine cells of the lower intestine, stomach, and pancreas (4, 5, 6), whereas PP is expressed in peripherally situated islet cells (7). In the developing pancreas, PYY is one of the earliest expressed hormones and is present in all the different early hormone-producing cell types (3, 8). Following birth, the expression of PYY in the pancreas rapidly declines (9).

All three peptides of the PP family have been shown to inhibit exocrine pancreatic secretion (1). In addition, NPY and PYY both exhibit inhibitory effects on insulin secretion (10, 11) and induce vasoconstriction in the pancreas and intestines (12). Recently, several PP-family receptor subtypes have been cloned. These all contain seven transmembrane domains and belong to the G protein-coupled superfamily of receptors. The first receptor to be cloned was the Y1 receptor (Y1-R) (13, 14, 15, 16). Subsequently, the Y2 receptor (17, 18, 19), the Y4/PP1 receptor (20, 21), the Y5 receptor (22, 23), and the Y5/PP2/Y2b receptor (24, 25, 26) were cloned. All these receptors differ in binding properties and tissue distribution and share low sequence identity. The Y1-R binds NPY and PYY as well as the Y1-R-selective analog [Leu31, Pro34]NPY with similar affinities (15, 16) and is expressed in the brain and in several peripheral organs (13, 27).

Binding sites for the PP-family of peptides have previously been localized to vascular smooth muscle cells in the pancreas by receptor autoradiographic technique (28). Using the Y1-R agonist [Leu31, Pro34]NPY, these binding sites were classified as representing the Y1-R subtype. In light of the recent cloning of new subtypes of PP-family receptors, binding studies like the above have, however, to be reevaluated, because [Leu31, Pro34]NPY also binds to the Y4/PP1, Y5 and Y5/PP2/Y2b receptors.

In the present study, we have raised an antibody against a synthetic peptide from the extracellular region of the rat Y1-R to study its localization in the rat pancreas. Because the developing rat pancreas contains numerous PYY immunoreactive cells (3, 8), the study was further extended to the developing pancreas.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Production of antisera
An N-terminal peptide (amino acids 20–31) of the rat Y1-R (GenBank accession number Z11504) was synthesized (Ross-Petersen, Hørsholm, Denmark), coupled to keyhole limpet hemocyanin (Boehringer Mannheim, Mannheim, Germany) using glutaraldehyde and used for immunizing rabbits sc. This peptide represented a region of the Y1-R with no homology to other cloned NPY receptor subtypes, indicating that cross-reactivity of the antisera to NPY receptor subtypes different from Y1-R is unlikely. Sera were obtained after repeated boosters (Jackerott and Larsson, to be published). By Western blot analysis of colonic muscle proteins, the antisera showed to recognize a protein of 70 kDa corresponding to previously published sizes of Y1-R (Jackerott and Larsson, to be published). To remove antibodies directed against keyhole limpet hemocyanin, antisera, diluted 1:1000 in Tris-buffered saline, were absorbed overnight at 4 C with 200 µg/ml keyhole limpet hemocyanin coupled to CNBr-activated sepharose beads (Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s instructions.

Animals and tissue processing
Wistar rats were mated overnight; noon of the day when the vaginal plug was discovered was considered as day 0.5 of gestation (E0.5). Pregnant female rats were killed with carbon dioxide asphyxiation, and the decapitated fetuses were fixed overnight in 4% paraformaldehyde dissolved in 0.1 M sodium phosphate buffer, pH 7.4. With embryos older than E16.5, the abdominal cavity was opened before fixation to facilitate penetration of the fixative solution. Rat embryos were examined from gestational day E12.5 to E20.5. Two-day-old postnatal rats were similarly immersion-fixed before dissection. Adult rats were intracardially perfused first with saline and then with the fixative followed by incubation overnight of pancreatic specimen in the fixative. The splenic part of the pancreas or, for embryos younger than E16.5, the gastrointestinal region containing the pancreatic primordia were dissected out, cryoprotected overnight in 30% sucrose, mounted in tissue-tek (Miles Inc., Elkhart, IN) and frozen in n-hexane (Merck, Damstedt, Germany) cooled by liquid nitrogen.

Immunocytochemistry
Five-micrometer cryostat sections were pretreated for 30 min with methanol containing 0.03% H2O2 to inhibit endogenous peroxidase activity (29). Following incubation for 30 min in 10% normal goat serum, the sections were exposed overnight at 4 C to rabbit Y1-R antisera (no. 307 diluted 1:4000, no. 308 diluted 1:8000). Subsequently, biotin-labeled goat antirabbit Ig (Dako A/S, Glostrup, Denmark) was applied. The biotin-labeled sites were visualized by horseradish peroxidase-catalyzed deposition of fluorochrome-conjugated tyramide using the Renaissance TSA-Direct kit (DuPont NEN, Boston, MA), which involved application of horseradish peroxidase-conjugated streptavidin followed by deposition of either tetramethyl rhodamine-tyramide or fluorescein-tyramide according to the manufacturer’s instructions. This principle for biotin amplification (30) has been noted to enhance the immunocytochemical signal (31).

Double immunocytochemistry for Y1-R together with PYY or amylase was performed by visualizing the Y1-R immunoreactive sites by deposition of tetramethyl rhodamine- or fluorescein-tyramide as described above, followed by incubation for 30 min in 10% normal serum from the species donating the secondary antiserum and exposure overnight at 4 C to polyclonal rabbit antiserum to either rat/porcine PYY (Milab, Malmö, Sweden) diluted 1:800 or human {alpha}-amylase (Sigma, St. Louis, MO) diluted 1:1000. Finally, the site of antigen-antibody reaction was revealed with AMCA-conjugated goat antirabbit Ig (Dako A/S) or TX red-labeled donkey antirabbit Ig (Jackson ImmunoResearch Laboratories). While this study was in progress, a report describing this method for double immunocytochemistry of antigens situated in different compartments appeared (32). Thus, as reported by Hunyady et al. (32), the tyramide deposits shield the first cycle of primary and secondary antisera against reaction with the antisera applied in the subsequent staining cycle. In addition, we have found that the antigenicity of the second epitope is not impaired by the deposition of tyramide (Jackerott and Larsson, to be published). All secondary antibodies were preabsorbed overnight with 10 µl/ml normal rat serum. Controls included absorptions of the Y1-R antisera with 100 µg/ml of the synthetic Y1-R peptide. Specimens were examined by fluorescent epiillumination using selective and aligned FITC, TX red and AMCA filters together with a triple FITC/TX red/AMCA filter.

RT-PCR analysis of Y1-R
Total cellular RNA was isolated from adult rat cerebral cortex and from pancreatic tissue from E16.5 rat embryos by acid guanidinium thiocyanate/phenol/chloroform extraction (33) using the TRIzol Reagent (GIBCO BRL, Gaithersburg, MD). Following reverse transcription (RT) of total RNA to complementary DNA (cDNA) using Superscript II Reverse Transcriptase and random hexamer primers (GIBCO BRL), cDNA was amplified by PCR using primers based on the published sequence of the rat Y1 receptor: forward: 5'-AAA TGT ATC ACT TGC GGC GTT CA-3' and reverse: 5'-GCG ACC ACG ATG GAG AGC AG-3'. These primers anneal to exon 2 and 3, respectively, of the Y1-R gene, thereby resulting in PCR-products of different sizes depending on whether genomic DNA or cDNA is amplified. Control RT-PCR was performed without RT on total RNA from cerebral cortex. Standard PCR was performed in 50 µl reaction volume using AmpliTaq polymerase (Perkin Elmer, Nieuwerkerk, The Netherlands) under the following conditions: initial denaturation at 94 C for 5 min, followed by 35 cycles of denaturation at 94 C for 45 sec, annealing at 55 C for 1 min and extension at 72 C for 1.5 min, and finally extension at 72 C for 10 min. The amplified products were separated by electrophoresis on a 1.5% agarose gel. The 258-bp PCR product resulting from amplification of embryonic pancreatic cDNA was reamplified under the same conditions. Following precipitation of DNA, the latter PCR reaction and the primary PCR reaction amplified from cerebral cortex cDNA were digested with PstI or RsaI, and the fragments were separated by electrophoresis on a 2.5% agarose gel.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Localization of Y1-R in the adult and neonatal rat pancreas
In the adult pancreas, immunoreactivity for Y1-R was detected in blood vessels (Fig. 1AGo) and in some centroacinar and intralobular duct cells (Fig. 1BGo) using either of the two Y1-R antisera. The Y1-R no. 308 antiserum stained endothelial cells (Fig. 1AGo), whereas a general staining of blood vessels was observed with the Y1-R no. 307 antiserum. Following preabsorption with the peptide used to raise the antisera, the reactivity of the Y1-R no. 308 antiserum was totally eliminated, whereas the Y1-R no. 307 antiserum was still able to diffusely stain blood vessels, but not duct cells. Thus, the diffuse staining of blood vessels obtained with Y1-R no. 307 was considered artifactual.



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  		Figure 1. Immunocytochemical localization of Y1-R in adult and neonatal pancreas and in fetal stomach. A and B, Adult rat pancreas immunostained for Y1-R reveals the existence of Y1-R in endothelial cells (A) and in some duct cells (B; arrows). C and D, Neonatal (P2) rat pancreas double stained for Y1-R (red fluorescence; C) and amylase (blue fluorescence; D) and photographed in single red (C) and double exposure (D) shows the localization of Y1-R immunoreactivity in pancreatic ducts (arrows) connecting the amylase-positive acinar cells. E, Section through fetal rat stomach at E12.5 demonstrates the presence of Y1-R immunoreactivity in most of the epithelial cells. Bar, 30 µm.

 
A similar localization of Y1-R-immunoreactivity was detected in the neonatal (P2) pancreas. Double immunofluorescence for Y1-R and amylase clearly illustrated the structure of the exocrine tissue with amylase-positive acinar cells connected by Y1-R-containing duct cells (Fig. 1Go, C–D).

Localization of Y1-R in the fetal rat pancreas
At the earliest stages studied (E12.5 to E16.5), Y1-R-immunoreactivity was detected in numerous epithelial cells in the pancreas. At E12.5 and E14.5, the Y1-R antisera further specifically stained the epithelium of the stomach and duodenum together with scattered cells present in the wall of the stomach (Fig. 1EGo). From E15.5 to E16.5, the intensity of the Y1-R-staining of the pancreatic epithelial cells increased (Fig. 2Go). At E17.5, however, the pattern shifted, so that the Y1-R immunoreactivity became restricted to the pancreatic duct cells. In addition, the intensity of the staining of these cells was much weaker than the intensity of the cellular staining observed one day earlier. This remarkable change in Y1-R-immunoreactivity from E16.5 to E17.5 was observed in fetuses from two different litters on each stage.



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  		Figure 2. Sections through fetal rat pancreas at E15.5 (A–C) and E16.5 (D–F) double immunostained for Y1-R (red fluorescence; A+D) and PYY (blue fluorescence; B + E) and photographed in single red (A + D), single blue (B + E) and double exposure (C + F). Note the presence of Y1-R in endothelial cells of a blood vessel (arrows) (A–C) and in PYY-negative epithelial cells close to PYY-positive cells (A–F). Also note that the intensity of the Y1-R staining in the epithelial cells increases from E15.5 to E16.5. G–I, Section through E16.5 rat pancreas double stained for Y1-R (green fluorescence; G) and amylase (red fluorescence; H) and photographed in single green (G), single red (H) and double exposure (I). In double exposure, coexisting red and green fluorescence colors produce a yellowcolor. Note that a fraction of the Y1-R-positive epithelial cells are also amylase positive. Bar, 30 µm.

 
Double immunofluorescence showed that most of the PYY-positive endocrine cells present between E12.5 and E16.5 were situated in clusters surrounded by Y1-R-containing cells (Fig. 2Go, A–C). Other PYY immunoreactive cells were scattered in the pancreatic epithelium and intermingled with cells labeled for Y1-R (Fig. 2Go, D–F). At E17.5, when the pattern changed, PYY immunoreactive endocrine cells occurred in close proximity to Y1-R-positive cells in the pancreatic ducts. Y1-R immunoreactivity was also detected in a few endocrine cells storing PYY using either of the two receptor antisera. However, following preabsorption of the antisera with the peptide antigen, Y1-R-positive endocrine cells were still observed, indicating that the Y1-R-reactivity in endocrine cells was nonspecific.

By double immunofluorescence, a fraction of the Y1-R-positive epithelial cells present at E15.5 and E16.5 was found to contain amylase. At E16.5, all amylase-positive cells displayed Y1-R-immunoreactivity (Fig. 2Go, G–I). However, in many of these, the intensity of the Y1-R-staining was weaker than in amylase-negative epithelial cells. From E16.5 to E17.5, more amylase-positive cells appeared and now displayed strong amylase immunoreactivity localized to zymogen granules. At this stage, a typical acinar structure was also established. None of the mature zymogen (acinar) cells co-stored Y1-R, whereas duct-like cells connecting the acini displayed weak Y1-R-immunoreactivity. The localization of Y1-R was similar at E18.5 and E20.5.

In the fetal pancreas, immunoreactive Y1-R was also detected in endothelial cells of developing blood vessels. The intensity of this staining was much stronger in embryonic than in adult endothelial cells (Fig. 2Go, A–C).

Expression of Y1-R messenger RNA (mRNA) in the fetal rat pancreas
The expression of Y1-R mRNA in the embryonic pancreas was also studied by RT-PCR. Cerebral cortex, known to express Y1-R, was used as a positive control. RT-PCR on cerebral cortex RNA resulted in a correctly sized product of approximately 260 bp. In addition, a weaker band of approximately 360 bp was present; this band corresponds in size to the product expected from genomic DNA (14). When the RT enzyme was omitted from the reaction, only the band of approximately 360 bp was observed, indicating that the fragment of approximately 260 bp descends from cDNA. RT-PCR on E16.5 pancreatic RNA resulted in the same two fragments together with a third unknown fragment of approximately 470 bp. Digestion of the approximately 260 bp PCR-product from either sample with PstI resulted in two fragments of approximately 100 and approximately 160 bp, whereas RsaI digested the product into approximately 200 and 60 bp fragments (Fig. 3Go). These restriction fragment sizes correspond to those expected from the correct Y1-R PCR product.



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  		Figure 3. Expression of Y1-R mRNAs analyzed by RT-PCR. Following one round of PCR on cerebral cortex cDNA (lanes 1–3) or two rounds of PCR on E16.5 pancreas cDNA using Y1-R primers (lanes 4–6), the PCR products were separated on a 2.5% agarose gel, either directly (lanes 1 and 4) or after digestion with PstI (lanes 2 and 5) or with RsaI (lanes 3 and 6). PstI digests the 258-bp product into two fragments of 159 bp and 99 bp, whereas the RsaI-digestion results into fragments of 203 bp and 55 bp, thereby confirming that the PCR products arise from Y1-R cDNA. M, Molecular weight marker indicated in bp to the left. Arrow, 258-bp fragment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study describes the localization of Y1-R in the fetal and adult pancreas using immunocytochemistry. In addition, the presence of Y1-R mRNA in the fetal pancreas was demonstrated by RT-PCR. In the adult pancreas, immunoreactive Y1-R was detected in endothelial cells. A previous study has also localized Y1-R to pancreatic blood vessels using receptor autoradiography (28). In this study, most of the binding sites for NPY were situated in the vascular smooth muscle cells, whereas the endothelial cells displayed fewer binding sites. As the NPY analog, used to characterize the receptor subtype, has been shown to bind to several of the cloned receptor subtypes, the binding sites in the smooth muscle cells may belong to another receptor subtype than Y1-R. However, by in situ hybridization, Wharton et al. (27) have localized Y1-R mRNA to the intima as well as the media of myocardial, colonic, and renal blood vessels. From the figure included by Wharton et al., it would appear that highest concentration of silver grains were localized over the intima. Our data, hence, indicate that NPY/PYY may act on Y1 receptors present on endothelial cells. This concurs with a previous study by Daly et al. (34) demonstrating that NPY-induced vasoconstriction is dependent on an intact endothelium. However, subsequent studies have described that the NPY-induced vascular effects are mediated through an endothelium-independent mechanism (35, 36), indicating that NPY/PYY may have additional/alternative effects on the endothelium.

In addition to the endothelial cells, Y1-R immunoreactivity was detected in some centroacinar and duct cells in the adult pancreas. The centroacinar cells are thought to be responsible for the fluid and electrolyte secretion from the pancreatic acini (37). The presence of Y1-R on these cells suggests that the inhibitory effects of NPY/PYY on the pancreatic secretion of bicarbonate is mediated, at least in part, by a direct effect on the centroacinar cells. The possibility that a paracrine signal from the centroacinar cells could act as a mediator for the PYY-induced inhibitory effects on protein secretion from the adjacent acinar cells could moreover be entertained. Such a hypothesis concurs with studies demonstrating that PYY-induced inhibition of pancreatic protein (and bicarbonate) secretion is independent of extrinsic pancreatic innervation (38, 39, 40). One of these studies moreover indicated that the PYY-induced effects on the exocrine secretion is mediated by Y1-R (40). However, another study claimed that locally expressed Y2 receptors are involved (41), whereas still others have suggested that changes in pancreatic blood flow or neural control mechanisms mediate the inhibitory actions of PYY (42, 43, 44). Together, these data indicate that NPY/PYY may inhibit exocrine secretion by more than one mechanism. These potential multiple mechanisms may be paralleled by expression of Y1-R and other receptor subtypes at multiple sites.

We were able to establish a developmental sequence in the localization pattern of Y1-R. Thus, until E17.5, numerous nonendocrine epithelial cells contained immunoreactive Y1-R and some of these cells co-stored amylase. At the stage in development when the amylase cells began to display distinct zymogen granules and became organized into acinar structures, the amylase cells displayed no Y1-R immunoreactivity. Instead, immunoreactive Y1-R was present in pancreatic duct-like cells connecting the secretory acini. Several other markers have been detected in the majority of the epithelial cells in the early pancreas, including Glut-2 (45), STF-1/PDX-1 (46), Trk-A (47), FA-1 (48) and CK20 (49). Most of these markers later appeared in islet cells, whereas CK20, like Y1-R, was subsequently restricted to duct cells. These observations suggest that early epithelial cells expressing these markers may have the capacity to differentiate into endocrine, exocrine as well as duct cells.

The close proximity of PYY and Y1-R immunoreactive cells further suggests that the endocrine cells may influence the surrounding epithelial cells through paracrine secretion of PYY. The developmental importance of such a paracrine interaction may not be restricted to the pancreatic epithelium, as Y1-R immunoreactivity was also detected in the epithelium of the fetal stomach and duodenum in the present study. In the adult rat, PYY cells are also present in the gastric and colonic mucosa (4). In addition, Upchurch et al. (50) recently demonstrated that similar to the fetal islet cells, all the different colonic endocrine cell types express PYY during fetal development. Accordingly, it is possible that PYY and Y1-R interactions may play a role in the development of the gastroenteropancreatic epithelium.

In summary, our current data demonstrate the localization of Y1-R immunoreactivity to centroacinar and intralobular duct cells and to endothelial cells in the adult pancreas, suggesting that these sites may mediate the NPY/PYY-induced inhibitory effects on the vasoconstriction and exocrine pancreatic secretion. The presence of a large number of PYY-producing cells close to Y1-R-positive epithelial and endothelial cells in the developing pancreas suggest that PYY may be involved in the development of exocrine cells, duct cells and blood vessels.


   Footnotes
 
1 This study was supported by a grant from the Danish National Research Fund (Center for Gene Regulation and Plasticity in the Neuroendocrine Network) and the Danish Biotechnology Program (Center for Medical Biotechnology). Back

Received March 21, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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E. Bertelli and M. Bendayan
Association between Endocrine Pancreas and Ductal System. More than an Epiphenomenon of Endocrine Differentiation and Development?
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