Evidence for vasopressin production in the human gastrointestinal system

Peptides, Vol. 12, pp. 1051-1056. © Pergamon Press plc, 1991. Printed in the U.S.A.

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Evidence for Vasopressin Production in the Human Gastrointestinal System A N D R E W S. F R I E D M A N N , * V I N C E N T A. M E M O L I t A N D W I L L I A M G. N O R T H *

*Department of Physiology and "~Department of Pathology, Dartmouth Medical School, Hanover, NH 03756 Received 9 M a y 1991 FRIEDMANN, A. S., V. A. MEMOLI AND W. G. NORTH. Evidencefor vasopressinproduction in the human gastrointestinal system. PEPTIDES 12(5) 1051-1056, 1991.--In the present study we performed immunohistochemical examination of segments from the human gastrointestinal system for the presence of cells containing vasopressin (VP) and vasopressin-associated human neurophysin (VP-HNP). VP immunoreactivity was found in crypt cells of the stomach and small intestine, and in mononuclear cells within the lamina propria and submucosa. VP-HNP was demonstrated in the crypt and lamina propria regions of the small intestine, and was colocalized with vasopressin in crypt cells. This colocalization indicates local vasopressin synthesis by these cells and raises the possibility that they may perform an endocrine or exocrine function in the human gastrointestinal system. Vasopressin

Neurophysin

Gastrointestinal system

Immunohistochemistry

Nonneuronal synthesis

by routine light microscopy and any tissue with abnormal histology was excluded from the study. Human pituitaries were obtained at autopsy (4-12 hours postmortem). Two different fixation procedures, one involving overnight immersion in acetone (22), and the other a 3--4-hour immersion in Bouin's reagent (14), were employed to examine the effects of tissue preparation on staining. Duplicate samples were processed using either procedure. The acetone was cleared with washes of methyl benzoate and xylene and the Bouin's reagent removed through ascending concentrations of ethanol. Tissues were then embedded in molten (60°C) paraffin, cut into 4 micron sections, and mounted on silane-coated slides.

THE nonapeptide vasopressin (VP) is classically recognized as a product of magnocellular neurons in the anterior hypothalamus. Synthesized as part of a larger precursor molecule containing an associated neurophysin, it is transported down the axons of these neurons to the posterior pituitary (16). The peptide is released, along with equimolar amounts of its neurophysin, into the blood. Since 1985, several investigators have reported that cells other than hypothalamic neurons produce VP (3). These studies indicate VP synthesis occurs in the ovaries, testes, adrenals, nonhypothalamic regions of the central nervous system, the anterior pituitary, and the thymus. Peptides such as somatostatin, neurotensin, and thyrotropin releasing-hormone were originally found to be synthesized in central neurons but are now known to also be produced by gut endocrine cells (26). S~inchez-Franco and coworkers found immunohistochemical evidence for the presence of VP in neurons of the cat small and large intestine (21). Using radioimmunoassay for the peptide, they also detected VP in the stomach and intestine of rats. Aravich and coworkers claimed that both VP and its associated neurophysin could be demonstrated as present in putative plasma cells and in nerve fibers of rat duodenum using immunofluoresence (1). In this study, a specific monoclonal antibody to VP and specific antibodies to VP-HNP were employed to determine if immunoreactive (ir) VP (ir-VP) and immunoreactive vasopressin-associated human neurophysin (ir-VP-HNP) were present in the human gastrointestinal system and, if so, to define their localization.

Antibodies A mouse monoclonal antibody to VP was produced using hybridoma technology. Vasopressin was coupled to bovine thyroglobulin and injected into BALB C mice (24). Six weeks following a booster injection and the demonstration of high titer antisera in these mice, spleen cells were harvested and hybridized with ceils from a mouse NS1 myeloma cell line. Isolated hybridoma cells were cloned and screened for the presence of VP antibodies. One positive clone (DEN1) was grown in culture and employed to generate ascites preparations containing the antibody. The polyclonal antibodies to vasopressin-associated human neurophysin (VP-HNP) were generated in a rabbit to a purified preparation of protein coupled to thyroglobulin. The characteristics and specificity of these antibodies (Abbie) have been described in earlier publications (9, 17, 18).

METHOD

Tissue Small segments representing each region of the human gastrointestinal tract were obtained from surgical sections at the Mary Hitchcock Medical Center. Care was taken to obtain tissue samples from normal organs or from normal areas of organs with localized disease processes. All specimens were examined

Immunohistochemistry Tissues fixed in acetone or in Bouin's reagent were stained for VP or VP-HNP using the avidin-biotin complex (ABC) procedure (11). Prior to staining, the paraffin was removed by three

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FRIEDMANN, MEMOLI AND NORTH

5-minute washes with xylene. The Bouin's-fixed sections were rehydrated through washing in descending concentrations of ethanol and the sections prepared by the AMeX (acetone, methyl benzoate, xylene) method were washed in acetone. All slides were then immersed in phosphate-buffered saline (PBS) at room temperature (2 × 3 minute washes). To determine the effects of enzymatic digestion on antigen detection, some tissues fixed in Bouin's reagent were treated with a trypsin preparation from porcine pancreas (Behring Diagnostics, potency of 28,305 NFU/ml diluted 1:40 with PBS) for 10 minutes at room temperature (8). The trypsin was removed by a one-minute wash with 95% ethanol followed by a 10-minute wash with PBS. To block out nonspecific reactions, all sections were then incubated with 3% normal serum (horse for VP and goat for VP-HNP) for 20 minutes at 37°C. Incubations with the mouse monoclonal antibody to VP (ascites dilutions 1:300 to 1:800) were carried out overnight at 4°C. The rabbit polyclonal antibody to VP-HNP was applied to slides (dilutions of antiserum 1:500 to 1:1000) for 30 minutes at 37°C. After washing in PBS (2 x 3 minutes), biotinylated secondary antibody (either horse anti-mouse IgG or goat anti-rabbit IgG) was applied at a concentration of 20 ixg/ml for 30 minutes at 37°C. Nonbound secondary antibody was removed with PBS (2 x 3 minute washes) and the activity of endogenous peroxidase then blocked by immersing the slides in 3% hydrogen peroxide dissolved in absolute methanol (25). Incubation with the avidin-peroxidase complex at 25 txg/ml was carried out at 37°C for 30 minutes. Following a wash in PBS (2 x 3 minutes), the visualization of bound complex was achieved by adding 3,3'-diaminobenzidine (0.2 mg/ml dissolved in PBS and 0.03% hydrogen peroxide) for 2-5 minutes. The slides were then counterstained with hemotoxylin, dehydrated and coverslipped. Normal sera, biotinylated antibodies, and the avidin-peroxidase complex were all obtained from Vector Laboratories, Burlingame, CA. Antibody specificity was determined by the positive identification of the axonal terminals of VP neurons in human posterior pituitary with the VP- and VP-HNP-directed antibodies, the lack of staining in sections incubated with diluted normal serum instead of primary antibody, and the attenuation of staining after the preincubation of antibody preparations with VP. Sections were examined by light microscopy using an Olympus BH2BHYU microscope. RESULTS

Vasopressin The monoclonal antibody to VP gave positive immunostaining of vasopressin neuronal endings of the posterior pituitary. Immunohistochemical evaluation of tissues from the gastrointestinal tract employing this antibody revealed cells in the stomach and small intestine that were immunoreactive for VP (Table 1). The ability to detect ir-VP was not affected by the type of fixative used nor by the predigestion of tissues with trypsin. In sections of the stomach body, duodenum, jejunum, and ileum, VP-

positive cells were located in the crypt, lamina propria, and submucosa (Figs. la, 2, and 3) . The morphology and location of immunoreactive cells demonstrate those in the crypts to be epithelial ceils, and those in the submucosa and lamina propria to be mononuclear cells. Many of these mononuclear cells had the qualities of plasma cells (Fig. 2), while others displayed characteristics inconsistent with this cell type (Fig. 3). In some epithelial cells, the stain was contained within processes that extended to the lumen of the crypt (Fig. 1a). Examination of nerve plexuses in the submucosa and muscularis externa failed to reveal immunoreactivity for VP. No cells containing ir-VP were found in the large intestine. Staining was blocked by the substitution of primary antibody with diluted normal serum and, in selected sections, by the preincubation of primary antibody with soluble antigen.

Vasopressin-Associated Human Neurophysin The specificity of the antibodies to VP-HNP was demonstrated by positive identification of neurophysin in human posterior pituitary. No staining was found in sections incubated with diluted normal serum instead of the antibodies to VP-HNP. These antibodies stained cells throughout the small intestine (Table 2). This staining, unlike that in the pituitary, was variable with regard to the type of fixative used. Staining of cells in the small intestine was observed only in specimens that had been fixed by the AMeX method (Fig. lb). Neurophysin-positive cells were found in the crypts and lamina propria for all regions of the small intestine. The morphology of immunoreactive cells in the crypts was consistent with that of epithelial cells, while staining in the lamina propria was confined to mononuclear cells that had characteristics similar to those that stained positive for VP. As with VP reactivity, reactivity due to VP-HNP was not found in neurons of the small intestine nor in any cells of the large intestine. Positive staining of cells in the body of the stomach was at best questionable with antibodies to VP-HNP, and this only in AMeX-prepared tissue.

Colocalization of Vasopressin and Vasopressin-Associated Human Neurophysin Cells containing both VP and VP-HNP were demonstrated throughout the mucosal layer of the small intestine and no cells stained for either substance in colon. Cells in the intestinal submucosa stained only for VP. ir-VP and ir-VP-HNP were colocalized to the same crypt cells of the duodenum when adjacent sections of tissue were stained with antibody preparations for each substance (Fig. la, lb). DISCUSSION

The data presented here demonstrate that cells containing ir-VP are scattered throughout regions of the human stomach and small intestine, but are apparently absent from the neurons and neuronal extensions of the gastrointestinal system and from all

FACING PAGE FIG. 1. Colocalization of (a) vasopressin and (b) vasopressin-associated neurophysin immunoreactivity to a single crypt cell of the human duodenum. Immunohistochemical staining performed using an avidin-biotin complex (ABC) procedure on adjacent 4 p,m sections. A mouse monoclonal antibody to vasopressin and rabbit polyclonal antibodies to vasopressin-associated human neurophysin were employed and visualization was achieved by the peroxidase conversion of 3,3'-diaminobenzidine to a brown precipitate. Each preparation was counterstained with hemotoxylin ( x 893). FIG. 2. Vasopressin immunoreactivity in mononuclear cells within the body of the human stomach. These cells in the lamina propria have large granular nuclei characteristic of plasma cells. Similar cells were found to contain vasopressin-associated neurophysin irnmunoreactivity ( x 893).

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FIG. la.

FIG. lb

FIG. 2

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FRIEDMANN, MEMOLI AND NORTH

FIG. 3. Vasopressin immunoreactivity in a submucosal cell of the human ileum. The morphology of this cell is inconsistent with that of a plasma cell ( x 893). layers of the large intestine. In the stomach and small intestine, immunoreactivity for VP was found in endocrine-like epithelial cells of the crypts as well as in mononuclear cells within the lamina propria and submucosa. From their location and general appearance, it is possible that these mononuclear cells within the lamina propria and submucosa were mast cells. Findings in the present study are contrary to those of S~chez-Franco et al. in the cat (21), who reported that VP immunoreactivity was present in both small and large intestine and was confined to neuronal processes. Our data are also contrary to those obtained in a study by Aravich and coworkers on rat duodenum (1). These authors localized ir-VP exclusively to putative plasma cells of the lamina propria and to neuronal processes. While some cells immunoreactive for VP in our study might have been plasma cells, others displayed a morphology clearly inconsistent with this cell type. The recent work of Galin et al. (10) showing that lymphocytes can synthesize a number of peptides and studies by Preibisz et al. (19) demonstrating that human platelets have a high capacity to bind VP support the expectation that cells of hemopoietic origin in the gastrointestinal tract could stain positively for ir-VP. Vasopressin produced by monocytes in the lamina propria could act together with corticotropin releasing'hormone (CRH) to enhance stress-related release of opiocortin from lymphocytes, and thus have an action similar to that on corticotrophic cells in the pituitary. However, the location and structure of VP-positive cells in the crypts and submucosa of all segments of the stomach and small intestine suggests these cells are structurally intrinsic to the gastroenteropancreatic (GEP) system and fulfill possible endocrine, exocrine, or paracrine functions. The presence of VP in cells of the crypts and lamina propria

of the duodenum, jejunum, and ileum is not likely to be due to uptake of the peptide by these cells, even though Jan,(tky et al. (13) have demonstrated an uptake of plasma-borne 3H-vasopressin by the intestine. This is because VP-HNP was also localized to similar cell types in all these regions and, in fact, was colocalized with VP to crypt cells of the duodenum by immunochemical staining of serial sections. This provides strong support for the local synthesis of VP by these cells, as VP-HNP represents a major portion of the precursor to VP and is coproduced with the peptide in hypothalamic neurons. Colocalization of VP and VP-HNP to cells within the lamina propria was also observed, but in most cases the small volume of these cells prevented them from being evaluated in serial sections. While failure to demonstrate the presence of ir-VP-HNP in VP-positive cells in the intestinal submucosa might indicate the presence of VP results from selective uptake, it is also possible that synthesis of the peptide by these cells does occur but through a processing that differs from that found in hypothalamic neurons. The possibility that VP synthesis in the cells of the gastrointestinal tract differs from that found in hypothalamic neurons is supported by our finding that immunohistochemical detection of ir-VP-HNP in the gut is dependent on whether the tissue was fixed in Bouin's solution or by the AMeX procedure. Fixation in Bouin's solution is reported to alter the antigenicity of some proteins through cross-linking, while the AMeX method minimizes antigen modification and produces immunohistochemical results similar to those found with the use of frozen sections (22). The inability to detect neurophysin in sections from stomach and small intestine fixed in Bouin's reagent indicates that this fixative reduces the antigenicity of gut ir-VP-HNP. This sensitivity to Bouin's reagent is not seen with staining of VP-

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TABLE 1

TABLE 2 DISTRIBUTIONOF VASOPRESSIN-ASSOCIATEDNEUROPHYSININ DIFFERENTREGIONSOF THE GASTROINTESTINALSYSTEM

DISTRIBUTIONOF VASOPRESSININ DIFFERENTREGIONS OF THE GASTROINTESTINALSYSTEM Fixation Method* Region Stomach (body) Duodenum Jejunum Ileum Colon

Fixation Method*

Bouin's

AMeX

Bouin's (Trypsin)

+t + + + -

na:~ + + + -

+ + + + -

Region Stomach (body) Duodenum Jejunum Ileum Colon

Bouin's

AMeX

- t -

?~: + + + -

Bouin's (Trypsin)

*Tissue fixation performed in Bouin's reagent and in acetone (AMeX procedure). Some tissue sections were predigested with trypsin. tPositive (+) staining and an absence of staining ( - ) obtained using the avidin-biotin complex procedure and a mouse monoclonal antibody to vasopressin. ~Not attempted.

*Tissue fixation performed in Bouin's reagent and in acetone (AMeX procedure). Some tissue sections were predigested with trypsin. tPositive (+) staining and an absence of staining ( - ) obtained using the avidin-biotin complex procedure and a mouse monoclonal antibody to vasopressin. ~:Uncertain.

HNP in the posterior pituitary using our antibodies, and raises the possibility for structural differences between the neurophysin produced by hypothalamic neurons and that produced by intestinal cells. Since VP detection was not dependent on the type of fixative used, nor on predigestion with trypsin, it is likely that the sensitivity found for VP-HNP is not due to a general loss of antigen. Differences in posttranscriptional or posttranslational processing associated with VP synthesis in the gut could lead to altered forms of the neurophysin molecule. In hypothalamic neurons, the message for VP is produced by the removal of two introns from the transcribed VP gene (16). Translation of this message results in a VP precursor that is packaged into secretory vesicles and enzymatically cleaved to yield VP, VP-HNP, and a glycopeptide. Studies since 1984 indicating that the Brattleboro rat produces VP in nonhypothalamic cells (3) support the view that different types of processing in the synthesis of VP may exist. The Brattleboro rat is incapable of producing a significant amount of hypothalamic VP because of a single base deletion in the VP gene (23). However, VP production by nonhypothalamic cells is apparently not prevented by this genetic defect. Alternate methods of pre- and posttranslational processing of expressed message for peptides are exemplified by growth hormone, gastrin, cholecystokinin, and vasoactive intestinal peptide (VIP) (4,5). In the case of VIP, a peptide, histidine isoleucine (PHI), is cleaved from the VIP precursor in the intestine, while in the stomach a C-terminally extended form of PHI, not recognized by antibodies directed against the C-terminal portion of the molecule, is produced. In a similar manner, it is conceivable that the neurophysin produced in the stomach may also be extended at the C terminus and therefore poorly recognized by the antibodies used in this study. The enzymes involved in posttranslational processing of the VP precursor are pH dependent (16). An alteration in the pH within secretory granules of gastrointestinal cells may change these enzymatic activities and result in the production of a larger form of the neurophysin.

Synthesis of VP by cells of the gastrointestinal system would suggest the peptide has some physiological role in the processes of digestion. A number of effects on digestive function have already been reported for intravenously administered VP. These include the inhibition of gastrin-induced acid and pepsinogen release, the inhibition of secretin-induced pancreatic secretion, the reduction of submucosal blood flow, and the modulation of salt and water absorption by the intestine (2, 6, 7, 12, 15, 20). In the present study, ir-VP and ir-VP-HNP were found colocalized within processes of intestinal crypt cells that extended to the lumen and this supports an exocrine or paracrine function for VP at these sites. While a general exocrine role is unlikely because the peptide is rapidly degraded by interlumenal enzymes, actions on neighboring cells within the crypts might be possible if these cells possess the appropriate receptors. In this respect, it is of interest that VP immunoreactivity was often observed not only within certain crypt cells, but also on the lumenal surface of adjacent cells. Basolateral juxtapositioning of the staining could imply a possible endocrine role for the peptide produced by these cells. Submucosally produced VP could conceivably influence regional blood flow within the intestine. While immunohistochemical identification of the VP-containing mononuclear cells within the lamina propria and submucosa was not performed, some had a morphology consistent with that of mast cells. As noted earlier, VP is taken up by cells of hemopoietic origin. Alternatively, histamine is a product of mast cells and has been reported to cause secretion of chloride into the gastrointestinal lumen. If VP is also produced and released from mast cells, it could serve to alter salt and water balance by modulating the actions of histamine. ACKNOWLEDGEMENTS We thank Susan Gagnon, Maudine Waterman and Karlya Wheeler for technical assistance and Peter Serry for help in obtaining surgical specimens. This work was, in part, supported by Public Health Service Grants CA 19613, CA 46551, and AG 07771.

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