JOE
HOME HELP CONTACT US SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Journal of Endocrinology (2006) 191, 121-128       DOI: 10.1677/joe.1.06903
© 2006 Society for Endocrinology
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schulz, S.
Right arrow Articles by Schulz, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schulz, S.
Right arrow Articles by Schulz, S.

Immunocytochemical identification of low-affinity NTS2 neurotensin receptors in parietal cells of human gastric mucosa

Stefan Schulz1, Christoph Röcken2,5, Matthias P A Ebert3 and Solveig Schulz4

1 Departments of Pharmacology and Toxicology
2 Pathology, Otto-von-Guericke-University, Leipzieger Strasse 44, 39120 Magdeburg, Germany
3 Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 81675 München, Germany
4 Obstetrics and Gynecology, Otto-von-Guericke-University, Leipziger Strasse 44, 39120 Magdeburg, Germany
5 Department of Pathology, Charité – Universitätsmedizin Berlin, Schumannstrasse 20/21, 10117 Berlin, Germany

(Requests for offprints should be addressed to S Schulz; Email: stefan.schulz{at}medizin.uni-magdeburg.de)


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The biological effects of neurotensin (NT) are mediated by two distinct G protein-coupled receptors, NTS1 and NTS2. Although it is well established that neurotensin inhibits gastric acid secretion in man, the plasma membrane receptor mediating these effects has not been visualized yet. We developed and characterized a novel antipeptide antibody to the carboxy-terminal region of the human NTS2 receptor. The cellular and subcellular distribution of NTS2 receptors was evaluated in various human gastrointestinal tissues. Specificity of the antiserum was demonstrated by (1) detection of a broadband migrating at Mr 90 000–100 000 in Western blots of membranes from NTS2-expressing tissues; (2) cell-surface staining of NTS2-transfected cells; (3) translocation of NTS2 receptor immunostaining after agonist exposure; and (4) abolition of tissue immunostaining by preadsorbtion of the antibody with its immunizing peptide. In the gastrointestinal tract, NTS2 receptor immunoreactivity was highly abundant in parietal cells of the gastric mucosa, in neuroendocrine cells of the stomach small and large intestine, and in cells of the exocrine pancreas. NTS2 receptors were clearly located in the plasma membrane and uniformly present on nearly all target cells. The presence of NTS2 receptors was rarely detected in human tumors. This is the first localization of NTS2 receptors in human formalin-fixed, paraffin-embedded tissues at the cellular level. The abundant expression of low-affinity NTS2 receptors on the plasma membrane of human parietal cells provides a morphological substrate for the direct inhibition of gastric acid secretion observed after i.v. administration of neurotensin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neurotensin (NT) is a tridecapeptide originally isolated from calf hypothalamus (Carraway & Leeman 1973). Like many other neuropeptides, it fulfils a dual function as neurotransmitter or neuromodulator in the central nervous system and as a local hormone in the periphery, mainly the gastrointestinal tract (Vincent et al. 1999, Kitabgi 2002, Rozengurt et al. 2002). In the brain, NT modulates dopaminergic transmission in the nigrostriatal and mesocortilimbic pathways as well as hormone secretion from the anterior pituitary (Vincent et al. 1999). It also exerts potent hypothermic and analgesic effects (Sarret et al. 2005). In the periphery, NT is synthesized and secreted by endocrine-like N cells predominantly in the small intestine (Reinecke 1985). NT is thought to act as a hormonal regulator of postprandial gastrointestinal functions since plasma levels of neurotensin are elevated after a meal, and i.v. or central administration of this peptide causes inhibition of gastric acid secretion (Blackburn et al. 1980, Holst Pedersen et al. 1986, Mogard et al. 1987, Karinch et al. 1998, Xing et al. 1998). NT also alters motility in the stomach, small intestine and colon, stimulates secretion in the pancreas and biliary tract, and causes Cl–secretion from human colonic mucosa (Trimble et al. 1987, Gullo et al. 1992, Chey & Chang 2001). In addition, NT has been shown to stimulate growth of normal intestinal mucosa in vivo as well as various tumor cell lines in vitro, including those originating from the pancreas, prostate, brain, and lung (Bozou et al. 1986, Sethi et al. 1992, Ishizuka et al. 1993, Seethalakshmi et al. 1997, Herzig et al. 1999, Guha et al. 2002, 2003, Thomas et al. 2003, Zhao et al. 2004). Conversely, tumor cell proliferation can be inhibited by the neurotensin receptor antagonist, SR48692 (Iwase et al. 1997, Herzig et al. 1999, Moody et al. 2001).

NT mediates its biological effects through interaction with three receptor subtypes, referred to as NTS1, NTS2, and NTS3 (Vincent et al. 1999, Kitabgi 2002). Two of these, NTS1 and NTS2, correspond to seven transmembrane domain G protein-coupled receptors, whereas the third, NTS3, is a single transmembrane domain sorting receptor that is predominantly associated with vesicular organelles and shares 100% homology with gp95/sortilin (Vincent et al. 1999, Kitabgi 2002). NTS1 and NTS2 receptors can be distinguished pharmacologically by their high affinity (NTS1) versus low affinity (NTS2) for NT. The low-affinity (nanomolar range) NTS2 receptor differs from the high-affinity (subnanomolar range) NTS1 receptor not only by its tenfold lower affinity for NT, but also by its selective recognition of levocabastine, a nonpeptide histamine H1 receptor antagonist (Gendron et al. 2004). NT receptors have been identified in various primary human tumors, for example in most meningiomas and Ewing’s sarcomas, three quarters of ductal pancreatic carcinomas, and at a moderately lower incidence in astrocytomas, medullobalstomas, medullary thyroid cancers, and small cell lung cancer (Przedborski et al. 1991, Reubi et al. 1998, 1999a,, Reubi et al. b,c, Wang et al. 2000). These neoplasms display predominantly NTS1 receptors characterized by their low affinity for levocabastine (Przedborski et al. 1991, Reubi et al. 1998, 1999a,, Reubi et al. b,c, Wang et al. 2000).

Despite the large number of studies describing various NT effects on gastrointestinal functions, the cellular sites of neurotensin receptors in human tissues still need to be completely elucidated. In the present study, we have generated and characterized antibodies directed to the carboxy-terminal tail of the NTS2 receptor. We have also developed an immunohistochemical protocol that allows efficient detection of this receptor in formalin-fixed, paraffin-embedded human tissues. The generation of this novel antibody enabled us to determine the cellular and subcellular distribution of NTS2 receptor proteins in a variety of human gastrointestinal tissues.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients, tumors, and tissue preparation

Seventy-one tumor specimens were retrieved from the archives of the Department of Pathology, Otto-von-Guericke University Magdeburg, Germany. At the time of tumor removal patients had not received chemotherapy. All tissue specimens had been fixed in formalin and embedded in paraffin. The following tumors were investigated: colorectal adenocarcinoma (n = 5), ductal pancreatic adenocarcinoma (n = 5), ductal invasive breast carcinoma (n = 4), ovarian carcinoma (n = 10), prostate cancer (n = 4), thyroid carcinoma (n = 6), carcinoid (n = 15), pancreatic insulinoma (n = 8), growth hormone-producing pituitary adenoma (n = 4), pheochromocytoma (n = 2), glioblastoma (n = 4), and meningioma (n = 4).

Several of the neuroendocrine tumors contained adjacent normal tissue, which was also analyzed. In addition, several fresh tumor specimens were immediately frozen in liquid N2 and stored at –70 °C until Western blot analysis. The following tumors were investigated: ductal pancreatic adenocarcinoma (n = 4) and ovarian carcinoma (n = 4).

Generation and purification of antipeptide antibodies

Polyclonal antisera were generated against the carboxy-terminal tails of the neurotensin receptor subtypes, NTS1 and NTS2. The identity of the peptides is given in Table 1Go. Peptides, NTS1 (398–418) and NTS2 (390–410), were synthesized, purified, and coupled to keyhole limpet hemocyanin as described (Schulz et al. 2000). The conjugates were mixed in the ratio of 1:1 with Freund’s adjuvant and injected into groups of three rabbits each; 9034–9036 for NTS1 and 9037–9039 for NTS2 antisera production. Animals were injected at 4-week intervals, and serum was obtained 2 weeks after the start of immunization with the second injection. The specificity of the antisera as well as possible cross-reactivity with other NTS receptor subtypes was initially tested using immunodotblot analysis as described (Schulz et al. 2000). For subsequent analysis, antibodies were affinity-purified against their immunizing peptides using the Sulfo-Link coupling gel according to the manufacturer’s instructions (Pierce, Rockford, IL, USA).


View this table:
[in this window]
[in a new window]
  		Table 1 Amino acid sequences of carboxy-terminal regions of human neurotensin receptors
 
Immunocytochemistry

Plasmids encoding NTS1 or NTS2 were kindly provided by Dr David Shire (Sanofi-Synthelabo, Labège, France). Human embryonic kidney 293 (HEK-293) cells were stably transfected with either NTS1 or NTS2. Cells were grown on coverslips overnight and either not exposed or exposed to 1 µM NT (Bachem, Weil am Rhein, Germany). The cells were then fixed and incubated with 1 µg/ml anti-NTS1 (9036) or anti-NTS2 (9039) antibodies followed by cyanin 3.18-conjugated secondary antibodies (Amersham). Specimens were mounted and examined using a Leica TCS-NT laser scanning confocal microscope as described (Pfeiffer et al. 2001, Schulz et al. 2004).

Western blot analysis

The membranes were prepared from stably transfected HEK-293 cells as well as fresh tumor specimens. Cells and tissues were lysed in homogenization buffer (5 mM EDTA, 3 mM EGTA, 250 mM sucrose, 10 mM Tris–HCl, pH 7.6 containing 1 mM phenylmethylsulfonylfluoride, 1 µM pepstatin A, 10 µg/ml leupeptin, and 2 µg/ml aprotinin), and membranes were pelleted at 20 000 g for 30 min at 4 ° C. Membranes were then dissolved in lysis buffer (150 mM NaCl, 5 mM EDTA, 3 mM EGTA, 20 mM Hepes, pH 7.4 containing 4 mg/ml dodecyl- ß-maltoside, and proteinase inhibitors as above) and incubated with 150 µl wheat germ lectin agarose beads (Amersham) for 90 min at 4 ° C. Beads were washed five times in lysis buffer, and adsorbed glycoproteins were eluted with SDS-sample buffer for 20 min at 60 ° C. Samples were then subjected to 8% SDS-PAGE and immunoblotted onto nitrocellulose. Blots were incubated with 1 µg/ml anti-NTS1 (9036) or anti-NTS2 (9039) antibodies followed by peroxidase-conjugated secondary antibodies and enhanced chemiluminescence detection (Amersham). For adsorption controls, antisera were preincubated with 10 µg/ml of their cognate peptides for 2 h at room temperature.

Immunohistochemistry

Paraffin sections of 7 µm were cut and floated on positively charged slides and immunohistochemically stained as described (Mundschenk et al. 2003, Schulz et al. 2004). Briefly, sections were dewaxed, microwaved in 10 mM citric acid (pH 6.0) for 20 min at 600 W and subsequently incubated with 2 µg/ml anti-NTS2 (9039) antibodies over- night at 4 ° C. Staining of primary antibody was detected using biotinylated goat anti-rabbit IgG followed by an incubation with avidin-biotinylated peroxidase solution. Tissue was then rinsed and stained with 3,3'-diaminobenzidine-glucose oxidase for 15 min. Cell nuclei were lightly counterstained with hematoxylin. For immunohistochemical controls, the primary antibody was either omitted, replaced by preimmune sera or adsorbed with several concentrations ranging from 1 to 10 µg/ml homologous or heterologous peptides for 2 h at room temperature. A tumor known to stain positively was included in each batch of staining as a positive control.

Tissue microarrays

A tissue microarray was generated from paraffin blocks obtained from a total of 171 partial or complete gastrectomy specimens. Six tissue cylinders, each 0.6 mm in diameter, were randomly punched from the marked tumor regions of the donor tissue blocks and placed into recipient blocks using a precision instrument (Beecher Instruments, Silver Spring, MD, USA). Paraffin sections were cut from each recipient block and stained with hematoxylin and eosin to confirm successful transfer of tumor tissue: a mean number of 5.0 ± 1.4 core cylinders from each patient was found to include tumor tissue. Immunoreactions were visualized via an avidin–biotin complex, using the Vectastain ABC alkaline phosphatase kit, with Fast Red/Naphthol Mx as chromogen. Specimens were counterstained with hematoxylin. A tumor was categorized as positive when at least four out of six core cylinders stained for NTS2.

Assessment of staining patterns

All slides were evaluated by two independent investigators. The presence or absence of staining and the depth of color along with the number of cells showing a positive reaction and whether the staining was localized to the plasma membrane or not, were noted. Tumors were only categorized as positive when they exhibited a moderate to strong plasma membrane and/or cytoplasmic staining in the majority of tumor cells was easily visible with a low-power objective.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of NTS2 receptor antibodies

Specificity of the antisera was monitored using Western blot analysis. When membrane preparations from stable transfected cells were electrophoretically separated and blotted onto nitrocellulose, the antisera 9036 (anti-NTS1) and 9039 (anti-NTS2) revealed broad bands in cells transfected with their cognate neurotensin receptor subtype (Fig. 1Go). In HEK-293 cells stably expressing NTS1, the anti-NTS1 antibody (9036) detected a band migrating at Mr 55 000 and a second broadband migrating at Mr 80 000–120 000 suggesting that the NTS1 receptor exists in differently glycosylated forms in HEK-293 cells (Fig. 1AGo). In cells stably expressing NTS2, the anti-NTS2 antibody (9039) detected a major band migrating at Mr 55 000–60 000 and a second faint band migrating at Mr 90 000 suggesting that the NTS2 receptor does not undergo extensive glycosylation in these cells (Fig. 1BGo). These results indicate that both NTS1- and NTS2-expressing cells contain high levels of receptor protein and the antibodies selectively detected their cognate receptor and did not cross-react. Antisera were further characterized using immunofluorescent staining of transfected cells. When HEK-293 cells stably expressing NTS1 or NTS2 were stained with anti-NTS1 (9036) or anti-NTS2 (9039) antibodies, prominent immunofluorescence localized at the level of the plasma membrane was detected (Fig. 2A and CGo). After incubation with NT, NTS1- and NTS2-immunoreactivity (ir) was translocated from the plasma membrane into the cytosol, indicating that both NTS1 and NTS2 were rapidly endocytosed in an agonist-dependent manner (Fig. 2B and DGo). Next, the neurotensin-receptor antisera were tested for possible cross-reactivity with other proteins present in human tissues. When membrane preparations from a human ductal pancreatic adenocarcinoma were electrophoretically separated and blotted onto nitrocellulose, the anti-NTS1 antibody (9036) detected a broadband migrating at Mr 90 000–110 000 (Fig. 3AGo). When membrane preparations from a human ovarian carcinoma were electrophoretically separated and blotted onto nitrocellulose, the anti-NTS2 antibody (9039) detected a broadband migrating at Mr 80 000–100 000 (Fig. 3BGo). These findings suggest that both NTS1 and NTS2 exist in highly glycosylated forms in human tissues. Immunoreactive bands for each antiserum were completely abolished by preadsorbtion with 10 µg/ml of their immunizing peptides (Fig. 3Go). When the anti-NT1 antibodies (9034–9036) were subjected to immunohistochemical staining of human tissues, all the three antisera exhibited a strong nuclear staining in a number of tissues and were therefore not further characterized.


Figure 1
View larger version (41K):
[in this window]
[in a new window]
  		Figure 1 Western blot analysis of the specificity of anti-NTS1 and anti-NTS2 antibodies. Membrane preparations from HEK-293 cells stably transfected to express either NTS1 or NTS2 were separated on 8% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes. Membranes were then incubated with affinity-purified anti-NTS1 (9036) (A) or anti-NTS2 (9039) (B) antibodies at a concentration of 1 µg/ml. Blots were developed using enhanced chemiluminescence. Two additional experiments gave similar results. Ordinate, migration of protein molecular weight markers (Mr x 10–3).

 

Figure 2
View larger version (61K):
[in this window]
[in a new window]
  		Figure 2 Characterization of anti-NTS1 and anti-NTS2 antibodies by immunofluorescent staining of transfected cells. (A) and (B) HEK- 293 cells stably transfected to express NTS1 were either not exposed (A) or exposed to 1 µM NT for 30 min (B) subsequently fixed and immunofluorescently stained with anti-NTS1 (9036) antibody. (C) and (D) HEK-293 cells stably transfected to express NTS2 were either not exposed (C) or exposed to 1 µM NT for 30 min (D), subsequently fixed and immunofluorescently stained with anti-NTS2 (9039) antibody. Note that in untreated cells, prominent immunofluorescence was localized at the level of the plasma membrane and that exposure to NT induced a rapid translocation of both NTS1 and NTS2 receptors from the plasma membrane into the cytosol. Representative results from one of the three independent experiments are shown. NT, neurotensin. Scale bar, 20 µm.

 

Figure 3
View larger version (50K):
[in this window]
[in a new window]
  		Figure 3 Western blot analysis of the specificity of anti-NTS1 and anti-NTS2 antibodies in human tumors. (A) Membrane preparations from a ductal pancreatic adenocarcinoma were separated on 8% SDS-polyacrylamide gels, blotted onto nitrocellulose and incubated with 1 µg/ml anti-NTS1 antibody (9036) in the absence (– ) or presence ( + ) of 10 µg/ml peptide antigen. (B) Membrane preparations from an ovarian carcinoma were separated on 8% SDS-polyacrylamide gels, blotted onto nitrocellulose and incubated with 1 µg/ml anti-NTS2 antibody (9039) in the absence (– ) or presence ( + ) of 10 µg/ml peptide antigen. Blots were developed using enhanced chemiluminescence. Representative results from one of the three independent experiments are shown. Ordinate, migration of protein molecular weight markers (Mr x 10– 3).

 
NTS2 receptor immunohistochemical staining in normal and neoplastic human tissues

The anti-NTS2 (9039) antibody was then subjected to immunohistochemical staining of a variety of human tissues. Initial experiments showed that heat-induced epitope retrieval is required for efficient immunohistochemical staining of paraffin-embedded tissue (not shown). Many neuroendocrine tumors contained adjacent noncancerous tissue which enabled us to analyze the distribution of NTS2 receptors in several parts of the normal gastrointestinal tract. Prominent localizations of NT2 receptors in the stomach, intestine, and pancreas are shown in Fig. 4A–FGo. The highest densities of immunoreactive NTS2 receptors were observed in the basal portion of the gastric mucosa (Fig. 4A and BGo). NTS2 receptor immunoreactivity was predominantly confined to the plasma membrane of a subpopulation of cells in the gastric mucosa, which according to their size, appearance, and distribution most likely represent parietal cells (Fig. 4B and CGo). Interestingly, immunoreactive NTS2 receptors decorated mostly the basal and lateral but not the apical part of the plasma membrane of these cells (Fig. 4B and CGo). This immunostaining was completely abolished by preadsorbtion of the antibody with 10 µg/ml of its immunizing peptide (Fig. 4BGo, inset). We have previously reported that the cholecystokinin (CCK)1 receptor is predominantly confined to the plasma membrane of gastric chief cells (Schulz et al. 2005). Staining of adjacent sections of gastric mucosa with anti-NTS2 and anti-CCK1 antibodies revealed that immunoreactive NTS2 and CCK1 receptors clearly decorate distinct cell populations (Fig. 4CGo and C' ). These findings suggest that NTS2 receptors are almost exclusively confined to gastric parietal cells. In the small and large intestine, NTS2 receptor immunoreactivity was selectively localized to neuroendocrine cells (Fig. 4DGo). In neuroendocrine cells, immunoreactive NTS2 receptors were confined to the plasma membrane and cytoplasmic vesicles (Fig. 4DGo). Immunoreactive NTS2 receptors were also found in the plasma membrane of cells in the exocrine pancreas (Fig. 4EGo).


Figure 4
View larger version (96K):
[in this window]
[in a new window]
  		Figure 4 NTS2 immunohistochemical staining in human normal and neoplastic tissues. Upper panel, NTS2 immunohistochemical staining in gastric mucosa (A, B), comparison of NTS2 and CCK1 immunohistochemical staining in adjacent sections of gastric mucosa (C, C' ). Lower panel, NTS2 immunohistochemical staining in large intestine (D), exocrine pancreas (E), and pancreatic insulinoma (F). Sections were dewaxed, microwaved in citric acid, and incubated with affinity-purified anti-NTS2 antibody (9039) at a concentration of 2 µg/ml. Sections were then sequentially treated with biotinylated anti-rabbit IgG and AB peroxidase solution. Sections were then developed with 3,3'-diaminobenzidine as chromogen (brown), and cell nuclei were lightly counterstained with hematoxylin (blue). Note that in the normal gastrointestinal tract, NTS2 receptor immunoreactivity was predominantly detected at the plasma membrane of parietal cells in the gastric mucosa as well as cells of the exocrine pancreas. In contrast, NTS2 receptor immunoreactivity was localized to the plasma membrane and the cytoplasmic vesicles in neuroendocrine cells of the small and large intestine and in pancreatic insulinoma cells. For adsorption controls, primary antibody was incubated with the 10 µg/ml of the peptide used for immunizations. Representative results from one of the three independent experiments are shown. Inset in (B), peptide adsorption control. Scale bars, A = 100 µm; B, D, E, F = 50 µm; C, C' = 10 µm.

 
The anti-NTS2 antibody (9039) was then subjected to immunohistochemical staining of seventy-one human tumors. The presence of NTS2 receptors was rarely detected in human tumors. As shown in Fig. 4Go F, NTS2 immunoreactivity detected in human tumor cells was often confined to the plasma membrane as well as the cytoplasmic vesicles (Fig. 4FGo). Immunoreactive NTS2 receptors were found in four out of ten ovarian carcinomas, two out of five pancreatic ductal adenocarcinomas, two out of five colorectal adenocarcinomas, and one out of four breast cancers. In contrast, NTS2 receptors were not detected in prostate (n = 4) or thyroid cancer (n = 6). Immunoreactive NTS2 receptors were also observed in two out of eight pancreatic insulinomas but not in other neuroendocrine malignancies such as carcinoid (n = 15), growth hormone-producing pituitary adenoma (n = 4), or pheochromocytoma (n = 2). NTS2 receptors were also not detected in meningiomas (n = 4). An abundant expression of NTS2 receptors was evident in three out of four glioblastoma samples; however, in each of these cases immunoreactive NTS2 receptors were clearly confined to the contaminating nerve tissue and not to the tumor cells (not shown). Given the abundant expression of NTS2 in normal gastric mucosa, we examined the presence of immunoreactive NTS2 receptors in 171 gastric cancer patients using tissue microarrays (Fig. 5Go). NTS2-ir was found as cytoplasmic and/or membranous staining in 24 (14%) gastric cancers. No correlation was found between expression of NTS2 in tumor cells and tumor type (intestinal versus diffuse), local tumor growth (T-category), nodal (N-category) or distant metastases (M-category), overall tumor stage, or patient survival.


Figure 5
View larger version (82K):
[in this window]
[in a new window]
  		Figure 5 NTS2 immunohistochemical staining in a tissue microarray of human gastric cancer patients containing neoplastic and adjacent normal tissues. (A) Immunohistochemical staining in gastric mucosa, (B) and (C) immunohistochemical staining in intestinal type gastric cancer. Sections were dewaxed, microwaved in citric acid, and incubated with affinity-purified anti-NTS2 antibody (9039) at a concentration of 2 µg/ml. Sections were then sequentially treated with biotinylated anti-rabbit IgG and AB alkaline phosphatase solution. Sections were then developed with Fast Red/Naphthol Mx as chromogen (red), and cell nuclei were counterstained with hematoxylin (blue). Note that NTS2 receptor immunoreactivity was detected in normal gastric mucosa (A) and in the gastric cancer shown in (B) but not in (C). All samples were contained within the same tissue microarray. Scale bar, A = B = C = 100 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well established that NT acts as a hormonal regulator of gastrointestinal functions; however, the precise cellular and subcellular sites of its plasma membrane receptors in the human gastrointestinal tract have not been identified. We therefore generated antibodies that exert selective specificity for the human NTS2 receptor. We show that the cytoplasmic tail of this receptor can serve as an epitope for the generation of antisera that effectively stain formalin-fixed, paraffin-embedded human tissues. Several lines of evidence indicate that this antibody specifically detects its targeted neurotensin receptor and does not cross-react. Firstly, in Western blots of membranes from transfected cells, the anti-NTS2 antibody detected a band migrating at Mr 55 000–60 000 only in NTS2- but not in NTS1-expressing cells. Secondly, the anti-NTS2 antibody revealed prominent cell surface staining of NTS2-transfected cells. This immunostaining translocated from the cell surface into the cytosol after agonist exposure indicating rapid endocytosis of the NTS2 receptor. Thirdly, in Western blots of membranes from receptor-expressing tissues, the anti-NTS2 antibody detected a broadband migrating at Mr 80 000–100 000, which corresponds to a highly glycosylated form of the receptor. Fourthly, tissue immunostaining of the anti-NTS2 antiserum was completely abolished by preadsorbtion with homologous but not heterologous peptides. Finally, it should be noted that three out of three NTS2 antisera gave similar results.

Earlier studies have clearly established that i.v. administration of neurotensin directly inhibits gastric acid secretion and stimulates exocrine pancreas secretion in man (Blackburn et al. 1980, Holst Pedersen et al. 1986, Gullo 1987, Mogard et al. 1987, Trimble et al. 1987, Gullo et al. 1992, Chey & Chang 2001). NT is synthesized and secreted by endocrine-like N cells predominantly in the small intestine (Reinecke 1985). NT is also present in nerve fibers innervating the gastrointestinal tract and the pancreas (Reinecke 1985). The generation of the anti-NTS2 antibody (9039) enabled us to visualize NTS2 receptors in the plasma membrane of gastric parietal cells and pancreatic acinar cells, suggesting that the neurotensin-induced effects on gastric acid and exocrine pancreas secretion are mediated by the low-affinity NTS2 receptor. These NTS2 receptors could be targeted by NT released from gastrointestinal and pancreatic nerve fibers or NT secreted from endocrine-like N cells, thereby modulating the cessation of increased gastric acid secretion after food ingestion. The NTS2 receptor also decorates numerous neuroendocrine cells in the stomach, small and large intestine, suggesting that this receptor may function as an autoreceptor modulating the release of neurotensin. This mayexplain the high levels of internalized receptors detected in these cells. NT also alters motility in the stomach, small intestine, and colon indicating additional functions of other neurotensin receptors in the gastrointestinal tract (Rettenbacher & Reubi 2001).

High numbers of neurotensin receptors have been detected in various primary human tumors, such as meningiomas, Ewing’s sarcomas, ductal pancreatic carcinomas, astrocytomas, medullobalstomas, medullary thyroid cancers, and small cell lung cancer (Przedborski et al. 1991, Reubi et al. 1998, 1999a,, Reubi et al. b,c, Wang et al. 2000). In the present study, both NTS1 and NTS2 were detected by Western blot in membrane preparations of human tumor samples. However, immunohistochemical staining of our panel of 71 human tumor specimens revealed that low-affinity NTS2 receptors are very rarely detected in human tumors. Immunoreactive NTS2 receptors were only found in a minor subset of ovarian, pancreatic, colonic, breast, and gastric carcinomas. Nevertheless, this is consistent with previous findings because the majority of neurotensin-binding sites present in primary human tumors correspond to high-affinity NTS1 receptors (Przedborski et al. 1991, Reubi et al. 1998, 1999a,, Reubi et al. b,c, Wang et al. 2000).

In conclusion, we have generated and extensively characterized anti-NTS2 antibodies. Using these antibodies, we provide the first demonstration of NTS2 receptors in human formalin-fixed and paraffin-embedded tissues. The localization of NTS2 receptors in gastric parietal cells, pancreatic acinar cells, and neuroendocrine cells strongly suggests a physiological role for this receptor in the postprandial regulation of gastric acid and exocrine pancreas secretion by neurotensin. These findings identify the low-affinity NTS2 receptor as a potential pharmacological target for the inhibition of gastric acid secretion.


   Acknowledgements
 
We thank Beate Peter and Dana Mayer for skilful technical assistance, and Dr David Shire for plasmids encoding NTS1 and NTS2. This work was supported by a grant from the Rudolf Bartling Stiftung (to C R). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Blackburn AM, Fletcher DR, Bloom SR, Christofides ND, Long RG, Fitzpatrick ML & Baron JH 1980 Effect of neurotensin on gastric function in man. Lancet 1 987–989.[CrossRef][Web of Science][Medline]

Bozou JC, Amar S, Vincent JP & Kitabgi P 1986 Neurotensin-mediated inhibition of cyclic AMP formation in neuroblastoma N1E115 cells: involvement of the inhibitory GTP-binding component of adenylate cyclase. Molecular Pharmacology 29 489–496.[Abstract]

Carraway R & Leeman SE 1973 The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami. Journal of Biological Chemistry 248 6854–6861.[Abstract/Free Full Text]

Chey WY & Chang T 2001 Neural hormonal regulation of exocrine pancreatic secretion. Pancreatology 1 320–335.[CrossRef][Web of Science][Medline]

Gendron L, Perron A, Payet MD, Gallo-Payet N, Sarret P & Beaudet A 2004 Low-affinity neurotensin receptor (NTS2) signaling: internalization-dependent activation of extracellular signal-regulated kinases 1/2. Molecular Pharmacology 66 1421–1430.[Abstract/Free Full Text]

Guha S, Rey O & Rozengurt E 2002 Neurotensin induces protein kinase C-dependent protein kinase D activation and DNA synthesis in human pancreatic carcinoma cell line PANC-1. Cancer Research 62 1632–1640.[Abstract/Free Full Text]

Guha S, Lunn JA, Santiskulvong C & Rozengurt E 2003 Neurotensin stimulates protein kinase C-dependent mitogenic signaling in human pancreatic carcinoma cell line PANC-1. Cancer Research 63 2379–2387.[Abstract/Free Full Text]

Gullo L 1987 The effect of neurotensin on pure pancreatic secretion in man. Scandinavian Journal of Gastroenterology 22 343–348.[Web of Science][Medline]

Gullo L, De Giorgio R, Corinaldesi R & Barbara L 1992 Neurotensin: a physiological regulator of exocrine pancreatic secretion? Italian Journal of Gastroenterology 24 347–351.

Herzig MC, Chapman WG, Sheridan A, Rake JB & Woynarowski JM 1999 Neurotensin receptor-mediated inhibition of pancreatic cancer cell growth by the neurotensin antagonist SR 48692. Anticancer Research 19 213–219.[Web of Science][Medline]

Holst Pedersen J, Skov Olsen P & Kirkegaard P 1986 Effect of neurotensin and neurotensin fragments on gastric acid secretion in man. Regulatory Peptides 15 77–86.[CrossRef][Web of Science][Medline]

Ishizuka J, Townsend CM Jr & Thompson JC 1993 Neurotensin regulates growth of human pancreatic cancer. Annals of Surgery 217 439–445. Discussion 446.[Web of Science][Medline]

Iwase K, Evers BM, Hellmich MR, Kim HJ, Higashide S, Gully D, Thompson JC & Townsend CM Jr 1997 Inhibition of neurotensin-induced pancreatic carcinoma growth by a nonpeptide neurotensin receptor antagonist, SR48692. Cancer 79 1787–1793.[CrossRef][Web of Science][Medline]

Karinch AM, Schmidt GL & Kauffman GL Jr 1998 Pretreatment with SR48692 has different effects on central neurotensin-induced gastric mucosal defense and inhibition of gastric acid secretion in rats. Brain Research 810 123–129.[CrossRef][Web of Science][Medline]

Kitabgi P 2002 Targeting neurotensin receptors with agonists and antagonists for therapeutic purposes. Current Opinion in Drug Discovery and Development 5 764–776.

Mogard MH, Maxwell V, Sytnik B & Walsh JH 1987 Regulation of gastric acid secretion by neurotensin in man. Evidence against a hormonal role. Journal of Clinical Investigation 80 1064–1067.[Web of Science][Medline]

Moody TW, Chiles J, Casibang M, Moody E, Chan D & Davis TP 2001 SR48692 is a neurotensin receptor antagonist which inhibits the growth of small cell lung cancer cells. Peptides 22 109–115.[CrossRef][Web of Science][Medline]

Mundschenk J, Unger N, Schulz S, Hollt V, Steinke R & Lehnert H 2003 Somatostatin receptor subtypes in human pheochromocytoma: subcellular expression pattern and functional relevance for octreotide scintigraphy. Journal of Clinical Endocrinology and Metabolism 88 5150–5157.[Abstract/Free Full Text]

Pfeiffer M, Koch T, Schroder H, Klutzny M, Kirscht S, Kreienkamp HJ, Hollt V & Schulz S 2001 Homo- and heterodimerization of somatostatin receptor subtypes. Inactivation of sst(3) receptor function by heterodimerization with sst(2A). Journal of Biological Chemistry 276 14027–14036.[Abstract/Free Full Text]

Przedborski S, Levivier M & Cadet JL 1991 Neurotensin receptors in human meningiomas. Annuals of Neurology 30 650–654.

Reinecke M 1985 Neurotensin. Immunohistochemical localization in central and peripheral nervous system and in endocrine cells and its functional role as neurotransmitter and endocrine hormone. Progress in Histochemistry and Cytochemistry 16 1–172.[Web of Science][Medline]

Rettenbacher M & Reubi JC 2001 Localization and characterization of neuropeptide receptors in human colon. Naunyn-Schmiedeberg’s Archives of Pharmacology 364 291–304.[CrossRef][Web of Science][Medline]

Reubi JC, Waser B, Friess H, Buchler M & Laissue J 1998 Neurotensin receptors: a new marker for human ductal pancreatic adenocarcinoma. Gut 42 546–550.[Abstract/Free Full Text]

Reubi JC, Waser B, Schaer JC & Laissue JA 1999a Neurotensin receptors in human neoplasms: high incidence in Ewing’s sarcomas. International Journal of Cancer 82 213–218.[CrossRef][Web of Science][Medline]

Reubi JC, Waser B, Schmassmann A & Laissue JA 1999b Receptor autoradiographic evaluation of cholecystokinin, neurotensin, somatostatin and vasoactive intestinal peptide receptors in gastro-intestinal adenocarcinoma samples: where are they really located? International Journal of Cancer 81 376–386.[CrossRef][Web of Science][Medline]

Reubi JC, Zimmermann A, Jonas S, Waser B, Neuhaus P, Laderach U & Wiedenmann B 1999c Regulatory peptide receptors in human hepatocellular carcinomas. Gut 45 766–774.[Abstract/Free Full Text]

Rozengurt E, Guha S & Sinnett-Smith J 2002 Gastrointestinal peptide signalling in health and disease. European Journal of Surgery Supplement 23–38.

Sarret P, Esdaile MJ, Perron A, Martinez J, Stroh T & Beaudet A 2005 Potent spinal analgesia elicited through stimulation of NTS2 neurotensin receptors. Journal of Neuroscience 25 8188–8196.[Abstract/Free Full Text]

Schulz S, Pauli SU, Handel M, Dietzmann K, Firsching R & Hollt V 2000 Immunohistochemical determination of five somatostatin receptors in meningioma reveals frequent overexpression of somatostatin receptor subtype sst2A. Clinical Cancer Research 6 1865–1874.[Abstract/Free Full Text]

Schulz S, Rocken C, Mawrin C, Weise W & Hollt V 2004 Immunocytochemical identification of VPAC1, VPAC2, and PAC1 receptors in normal and neoplastic human tissues with subtype-specific antibodies. Clinical Cancer Research 10 8235–8242.[Abstract/Free Full Text]

Schulz S, Rocken C & Mawrin C 2005 Immunohistochemical localization of CCK1 cholecystokinin receptors in normal and neoplastic human tissues. Journal of Clinical Endocrinology and Metabolism 90 6149–6155.[Abstract/Free Full Text]

Seethalakshmi L, Mitra SP, Dobner PR, Menon M & Carraway RE 1997 Neurotensin receptor expression in prostate cancer cell line and growth effect of NT at physiological concentrations. Prostate 31 183–192.[CrossRef][Web of Science][Medline]

Sethi T, Langdon S, Smyth J & Rozengurt E 1992 Growth of small cell lung cancer cells: stimulation by multiple neuropeptides and inhibition by broad spectrum antagonists in vitro and in vivo. Cancer Research 52 2737s–2742s.[Abstract/Free Full Text]

Thomas RP, Hellmich MR, Townsend CM Jr & Evers BM 2003 Role of gastrointestinal hormones in the proliferation of normal and neoplastic tissues. Endocrine Reviews 24 571–599.[Abstract/Free Full Text]

Trimble ER, Shaw C, Bruzzone R, Gjinovci A & Buchanan KD 1987 Direct stimulation of enzyme secretion from rat exocrine pancreas by neurotensin and its naturally occurring fragments. Gastroenterology 92 699–703.[Web of Science][Medline]

Vincent JP, Mazella J & Kitabgi P 1999 Neurotensin and neurotensin receptors. Trends in Pharmacological Science 20 302–309.[CrossRef][Medline]

Wang L, Friess H, Zhu Z, Graber H, Zimmermann A, Korc M, Reubi JC & Buchler MW 2000 Neurotensin receptor-1 mRNA analysis in normal pancreas and pancreatic disease. Clinical Cancer Research 6 566–571.[Abstract/Free Full Text]

Xing L, Karinch AM & Kauffman GL Jr 1998 Mesolimbic expression of neurotensin and neurotensin receptor during stress-induced gastric mucosal injury. American Journal of Physiolgoy 274 R38–R45.

Zhao D, Zhan Y, Koon HW, Zeng H, Keates S, Moyer MP & Pothoulakis C 2004 Metalloproteinase-dependent transforming growth factor-alpha release mediates neurotensin-stimulated MAP kinase activation in human colonic epithelial cells. Journal of Biological Chemistry 279 43547–43554.[Abstract/Free Full Text]

Received 3 April 2006
Received in final form 2 June 2006
Accepted 21 June 2006
Made available online as an Accepted Preprint 14 July 2006





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schulz, S.
Right arrow Articles by Schulz, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schulz, S.
Right arrow Articles by Schulz, S.

HOME HELP CONTACT US SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS