Effects of substance P and vasoactive intestinal polypeptide on contractile activity and epithelial transport in the ferret jejunum.

Previous studies in the ferret demonstrated that vagal nerve stimulation induced an atropine-resistant water secretion. Substance P and vasoactive intestinal polypeptide are possible mediators of this secretory response. The objectives of this study were to investigate the in vivo effects of substance P and vasoactive intestinal polypeptide on the jejunal musculature and epithelium. Substance P caused an increase in jejunal motility, water secretion, and transmural potential difference. Cholinergic blockade did not affect the substance P-induced contractions, but did reduce the increase in transmural potential difference, suggesting an inhibition of water secretion. Vasoactive intestinal polypeptide abolished motor activity; however, it induced an increase in transmural potential difference that was atropine and tetrodotoxin resistant. By immunohistochemical methods, immunoreactive vasoactive intestinal polypeptide and immunoreactive substance P were localized to both nerve cell bodies and nerve fibers in the ferret intestine. Determination of intestinal concentrations of vasoactive intestinal polypeptide and substance P in the ferret showed concentrations of these two neuropeptides that were similar to those in human intestine and demonstrated much higher concentrations of these substances in the muscular layer than in the epithelial layer. Our data demonstrate that in the ferret substance P excites and vasoactive intestinal polypeptide inhibits jejunal motor activity. However, both peptides increase water secretion. Our results suggest that in response to vagal stimulation, neuronally released substance P or vasoactive intestinal polypeptide may participate in the atropine-resistant water secretion.

Similarities in intestinal humoral immunity in dermatitis herpetiformis without enteropathy and in coeliac disease.

Intestinal humoral immunity was examined in eight patients with dermatitis herpetiformis and normal jejunal histology (as determined by quantitative morphometry) on a gluten-containing diet. Jejunal aspirate was taken at the time of jejunal biopsy, and levels of total immunoglobulins (IgA, IgM, IgG) and specific antibody to gliadin and two other dietary proteins, betalactoglobulin and ovalbumin, were measured. The pattern of secretory immune responses in the dermatitis herpetiformis patients was similar to that in twenty-six patients with untreated coeliac disease--ie, higher than normal concentrations of IgA, IgM, and IgG and high levels of specific antibodies (IgA and IgM) to the three dietary proteins. Serum levels of IgA antigliadin were similar in the dermatitis herpetiformis and control (twenty-eight patients who underwent jejunal biopsy to exclude coeliac disease) groups, and serum levels of IgG antigliadin were intermediate between those of the control and coeliac disease groups. These findings suggest that investigation of gut humoral immunity may provide a diagnostic index of latent coeliac disease. The definition of coeliac disease as a permanent gluten-sensitive enteropathy may have to be revised if the proposed two-stage model is confirmed.

Canine neurotensin, neurotensin6-13 and neuromedin N: primary structures and receptor activity.

Canine neurotensin (NT) and neuromedin N (NMN) were isolated from extracts of ileal mucosa using radioimmunoassay for detection. The structures determined were consistent with those predicted by earlier cDNA work. The molar ratio of NT to NMN was ca. 7, suggesting that the NT/NMN precursor, which contains one copy of each peptide, undergoes complex posttranslational processing or that other NT-precursors lacking NMN exist. In addition to NT, small quantities of NT6-13 and NT2-13 were obtained. Native and synthetic preparations of these peptides were indistinguishable in a radioreceptor assay employing rat brain membranes and 125I-labeled NT; NT6-13 was ca. 8-times more potent than NT and NMN was about one-sixth as potent as NT. NT6-13 was also ca. 10 times more potent than NT in inhibiting spontaneous contractile activity in longitudinally-oriented smooth muscle strips of porcine jejunum. Preparations of intestinal N-cells as well as N-cell vesicles also appeared to contain NT2-13 and NT6-13; however, it is not yet clear whether these peptides are utilized physiologically or simply represent metabolites of NT. These results suggest that further work on the processing of NT precursor and on biologic abilities of partial sequences of NT could be fruitful.

Galanin binding sites in rat gastric and jejunal smooth muscle membrane preparations.

Receptors for galanin in membranes from the rat gastric and jejunal smooth muscle were studied using [125I] radioiodinated synthetic porcine galanin. Specific binding was time and temperature dependent. At 32 degrees C radioligand was degraded in the presence of smooth muscle membranes in a time-dependent manner. At optimal experimental conditions, the equilibrium binding analyses showed the presence of a single population of high affinity binding sites in both the rat stomach and jejunum (Kd value of 2.77 +/- 0.78 nM and 4.93 +/- 1.74 nM for stomach and jejunal smooth muscle membranes, respectively). The concentration of the high affinity binding sites was 58.19 +/- 11.04 and 32.36 +/- 5.68 fmol/mg protein, for gastric and jejunal preparations, respectively. Specific binding was completely inhibited by 10(-6) M of nonradioactive galanin; was 75% blocked by 1 microM of galanin(9-29); it was 10% blocked by 1 microM of galanin(15-29). Galanin(1-15) at a concentration of 1 microM was ineffective for inhibiting [125I]galanin binding. Deletion of four C-terminal amino acid residues from galanin(9-29) to give galanin(9-25) also resulted in almost complete loss of affinity. Radioiodinated galanin and N-terminally deleted fragments had receptor binding potency in the following order: galanin(1-29) greater than galanin(9-29) greater than galanin(15-29). We conclude that the C-terminal part of the galanin chain is important for the rat gastric and jejunal smooth muscle membrane receptor recognition and binding and that N-terminal amino acid sequences are probably not so important, since galanin(1-15) was not active but galanin(9-29) retained most of the receptor binding activity.

Characterization of the major form of cholecystokinin in human intestine: CCK-58.

Acid extracts of human intestines obtained from surgical samples or from organ donors contain cholecystokinin (CCK) immunoreactivity. From surgical samples, extracted and eluted quickly, greater than 75% of the CCK immunoreactivity eluted in the same region as purified canine CCK-58 during analytical reverse-phase high-pressure liquid chromatography (HPLC). A major portion of the CCK immunoreactivity from donor intestinal extracts also eluted in this region. This immunoreactivity has been purified from human intestinal extracts by a series of several reverse-phase and cation-exchange chromatographies. Amino acid and microsequence analysis showed that this immunoreactivity is human CCK-58. Tryptic digestion of purified human CCK-58 produced another immunoreactive form that eluted in the position of CCK-8 during analytical reverse-phase HPLC. The immunoreactivity of the trypsin-digested material was 2.6-fold higher than that of an identical sample of CCK-58 incubated without trypsin. Thus the carboxyl-terminal antibody used for radioimmunoassay cross-reacts greater than twofold less with human CCK-58. This diminished cross-reactivity would lead to an underestimation of the relative proportions of CCK-58 in tissue and plasma extracts. If CCK-58 is the major circulating form this diminished cross-reactivity would also lead to underestimations of the circulating levels of total CCK. Determination of human CCK-58 structure confirms that one of the major components of human CCK that expresses biological activity is CCK-58.

A combined TDDA-PVC pH and reference electrode for use in the upper small intestine.

Incorporation of a ligand into polyvinylchloride allows the production of ion selective electrodes that are soft-bodied and disposable. With features make them especially suitable for clinical, particularly gastroenterological, investigations. We report here on the construction of a combined reference and pH electrode suitable for use at jejunal biopsy. With this type of pH electrode, the pH of the mucosal surface of the jejunum in UK and in Indian subjects, without evidence of upper gastrointestinal disease, was about pH 6.0. This was almost identical to previous values measured using a separate reference electrode. Both the polymer electrode and a suitable data logger can be conveniently produced in the laboratory and compare favourably with commercially available systems, provided the range of pH likely to be encountered is within the operating range of the incorporated ligand.

Light microscopic localization of hepatic fatty acid binding protein mRNA in jejunal epithelia of rats using in situ hybridization, immunohistochemical, and autoradiographic techniques.

An in situ hybridization technique using a [35S]-labeled oligonucleotide probe was employed, in combination with immunohistochemistry and autoradiography, to examine gene expression for hepatic fatty acid binding protein (FABP) in the jejunal epithelia from both fed and fasted rats. In rats fed ad libitum, immunoreactivity and mRNA signal for FABP were localized to the absorptive epithelial cells lining the villus, whereas they were absent in the crypt epithelial cells. The level of FABP mRNA was relatively low in the tip of the villus, although FABP immunoreactivity remained high in this area. Animals fasted for 3 days exhibited a downward shift of the lower boundary of the FABP-expressing cell population into the middle portion of the crypt, in terms of the immunoreactivity and the mRNA signal. The proliferative cell compartment of the crypt, as revealed by [3H]-TdR incorporation, showed no substantial change in size between the fed and fasted states. The present results provided evidence that (a) during the differentiation and upward migration of the absorptive epithelial cells, the expression of FABP gene begins at the crypt-villus junction and declines before the cells reach the villus tip, and (b) fasting induces an earlier expression of the FABP gene in the maturing crypt epithelial cells.

Are the catabolic effects of tumor necrosis factor mediated by glucocorticoids?

The effect of tumor necrosis factor (TNF) on the hypothalamic-adrenal stress response was determined by infusion of TNF, 0, 2 x 10(5), and 4 x 10(5) U/kg per 24 hours, in parenterally fed male Wistar rats. Following infusions over 1 to 6 days, adrenal weight was increased with increasing dosage of TNF. Tumor necrosis factor at a dosage of 4 x 10(5) U/kg per 24 hours increased the plasma corticotropin level over the same period. In a further series of experiments the metabolic effects of TNF were compared with the effects of corticosterone, the predominant glucocorticoid in the rat. In comparison with controls, rats given corticosterone (75 mg subcutaneously) or TNF (2 x 10(5) U/kg per 24 hours) demonstrated decreased nitrogen balance and diminished carcass nitrogen content over a 6-day period. Tumor necrosis factor alone, however, induced a significant increase in liver nitrogen content and diminished jejunal mucosa DNA and protein levels in comparison with the control and corticosterone groups. Finally, adrenalectomized animals receiving basal corticosterone replacement were infused with TNF. Urinary nitrogen loss was significantly diminished in these animals compared with sham adrenalectomized controls, indicating that an intact adrenal stress response is necessary for the increased nitrogen loss following TNF infusion. Tumor necrosis factor may exert an important regulatory influence on the interorgan substrate flux that occurs during critical illness. The effects of TNF on skeletal muscle proteolysis can be simulated by adrenal glucocorticoid administration. The effects of this cytokine on visceral organs appear to be unique to TNF and cannot be reproduced by the administration of glucocorticoids alone.

Assessment of canine intestinal permeability, using 51Cr-labeled ethylenediaminetetraacetate.

The 51Cr-labeled EDTA was validated as a suitable permeability probe in dogs for measurement of passive, unmediated diffusion across intestinal mucosa via intercellular pathways. The 51Cr-labeled EDTA was stable in aqueous solution and did not bind to biologic tissue and fluids. After incubation of 51Cr-labeled EDTA in isolated jejunal loops, analytic subcellular fractionation of jejunal mucosa on reorientating sucrose-density gradients was performed, and no association of 51Cr-labeled EDTA with particulate intracellular organelles was detected. Intravenously administered 51Cr-labeled EDTA was rapidly and completely excreted in urine. Intestinal permeability to 51Cr-labeled EDTA after oral administration was assessed in healthy dogs. The percentage of the administered dose of 51Cr-labeled EDTA excreted in the urine in 24 hours ranged from 2.3 to 17.6% (median, 13%).

Biochemical changes in the jejunal mucosa of dogs with exocrine pancreatic insufficiency following pancreatic duct ligation.

The effects of exocrine pancreatic insufficiency on the small intestinal mucosa were examined in dogs following pancreatic duct ligation. There were no significant changes either in villus architecture or enterocyte height after duct ligation, but numbers of bacteria in duodenal juice increased then subsequently decreased following treatment with exogenous pancreatic enzymes. Pancreatic insufficiency resulted in a considerable increase in the proportion of microvillar membrane proteins of molecular mass over 200 kDa from 3.3 +/- 4 per cent (mean +/- SEM) to 13.6 +/- 7.2 per cent, and this decreased to 6.9 +/- 5.2 per cent following pancreatic enzyme supplementation. However, anticipated increases in activities of maltase and sucrase were not observed following duct ligation, and there was a reduction in lactase activity which was reversed by pancreatic supplementation. Activities of marker enzymes for the other subcellular organelles showed relatively minor or no changes throughout the study. These findings are consistent with a specific role for pancreatic enzymes in the post-translational processing of intestinal microvillar membrane proteins, and suggest that reduced degradation of brush border proteins in the absence of pancreatic secretions may be masked by quantitative and qualitative changes in the intestinal microflora.

Immunomicroscopic localization of the 10,000 molecular weight calcium-binding protein in the human enterocyte.

The cellular localization of the 10,000 molecular weight calcium-binding protein (CaBP or Calbindin-D) in the small intestinal epithelium of man was investigated by immunofluorescence and immunoelectron microscopy (immunogold staining). Indirect immunofluorescence on frozen sections showed intracellular staining in the enterocyte. The fluorescence was evenly distributed and no significant differences were observed between crypt and villus cells. No staining was found in goblet cells or in the submucosa. Correspondingly, immunogold labeled antibodies were scattered over the cytoplasm of the enterocyte. The terminal region appeared to be the most intensely decorated and the brush border region showed labeling above the background level. No labeling was associated with intracellular membranes or the basolateral membrane.

Isolation of glycoprotein D from herpes simplex virus type 1 by gel filtration high performance liquid chromatography.

Rabbit kidney (RK-13) and human jejunum and ileum (I-407) cells infected with herpes simplex virus type 1, strain F, were radiolabelled with [14C]glucosamine or [35S]methionine for 24 h. The cells were extracted with 1% Triton X-100 and the extracts were separated by gel filtration high performance liquid chromatography. Monoclonal antibody immunoprecipitation of the fractions collected from the column revealed a monomeric glycoprotein D (gD) of 52 - 56,000 molecular weight from RK-13 cells and two monomeric forms of gD, 54,000 and 58,000 molecular weight, from I-407 cells. Densitometry scanning of the autoradiograms from SDS-PAGE showed gD from the RK-13 host cells to be 98.7% pure with the [35S]methionine label and 97.0% pure with the [14C]glucosamine. On the other hand, gD from the I-407 host cells was only 78.6% with the [35S]methionine label and 96% pure with the [14C]glucosamine. This method could provide a means for the isolation of native gD for structural and immunological studies.

EGF- and TGF-alpha-like peptides in human fetal gut.

The presence of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) immunoreactivities in fetal human tissues was studied immunohistochemically at different gestational ages. EGF and TGF alpha immunoreactivities were detected from the 20th gestational wk. EGF immunoreactivity was limited to the small intestine, but TGF alpha immunoreactive cells were present in the colon also. According to radioreceptor assay, the intestine of a 19-wk-old human fetus contained 10 times more EGF receptor-binding substance than EGF, as measured by immunofluorometric assay. Chromatographic analysis suggests that TGF alpha-like peptides account for at least part of this activity, as so argues in favor of the presence of TGF alpha- and EGF-like peptides in the human fetal gut. Whether they are synthesized in the fetus is yet unknown.

Meal-induced peptide tyrosine tyrosine inhibition of pancreatic secretion in the rat.

In the present studies we examined the distribution, release, and biological actions of peptide tyrosine tyrosine (PYY) in the rat. The concentration and distribution of PYY was highest in the ileum and colon as determined by both radioimmunoassay of rat tissue extracts and immunocytochemistry. An ultrastructural comparison of rat and dog colonic PYY cells revealed a bipolar distribution of peptide-containing secretory granules in both species. Serum PYY and pancreatic exocrine secretory responses were monitored after presentation of a meal to meal-trained rats (n = 12). A significant increase in PYY concentrations was not observed until 120 min after meal presentation, a delayed response similar to that previously observed in the dog. PYY responses were also observed in rats after perfusion of the intestine at the level of the duodenum and ileum with an 80 mOsm micellar solution of sodium oleate. Duodenal instillations of the fatty acids resulted in a maximum PYY response after 120 min, whereas rats subject to ileal perfusion of fat exhibited maximum PYY release within the first hour. In other experiments, infusion of exogenous PYY at 100 pmol.kg-1.h-1, which reproduced plasma PYY levels observed after a meal and perfusion of the gut with fat, significantly inhibited CCK-stimulated bile pancreatic volume (P less than 0.02), protein (P less than 0.01), and amylase (P less than 0.01) output. These studies demonstrate a bipolar distribution of PYY-containing secretory granules in cells of the jejunal, ileal, and colonic mucosa, and show that PYY is released in response to a meal in amounts sufficient to inhibit cholecystokinin-stimulated pancreatic secretion. Evidence is presented that PYY may mediate the delayed inhibition of pancreatic secretion that is observed in the rat after ingestion of a meal.

The alpha 1-antitrypsin gene is expressed in a human intestinal epithelial cell line.

alpha 1-Antitrypsin (alpha 1-AT) is considered a typical plasma protein and a prototype of the serine proteinase inhibitor (serpin) family. It is synthesized in hepatocytes and, to a lesser extent, in macrophages. In this study we show that the alpha 1-AT gene is also expressed in human intestine and in a human colonic epithelial tumor cell line, Caco2. A single 1.6-kilobase alpha 1-AT-specific mRNA is present in jejunum and in Caco2 cells. It is identical in apparent size to that present in human hepatoma HepG2 cells but slightly smaller than that present in human macrophages, cells in which an alternative upstream transcriptional start site is used. Synthesis and secretion of alpha 1-AT in Caco2 cells is similar to that in HepG2 cells. It is synthesized as an approximately 52-kDa precursor polypeptide, converted to its mature, fully glycosylated 55-kDa form intracellularly, and the native protein is secreted with a half-time of 37 min. Functionally active alpha 1-AT is secreted into the basolateral and apical (luminal) fluid in pulse-chase labeling experiments of Caco2 cells cultured in polarized orientation on collagen-coated nitrocellulose membranes. Expression of alpha 1-AT in Caco2 enterocytes is not affected by soluble factors that regulate expression of alpha 1-AT in macrophages and hepatocytes. However, expression of alpha 1-AT increases markedly in Caco2 cells as they differentiate into enteric villous-type cells.

VIP receptors and content after bowel transplantation.

Advances in immunosuppressive therapy have renewed interest in small bowel transplantation. Little is known, however, about the functional capacity of transplanted intestine. To clarify the potential for normal function, we investigated whether elements of the enteric nervous system are preserved after denervation in our rat model of intestinal transplantation. We investigated whether VIP, a major peptide neurotransmitter of the enteric nervous system, and its receptors are preserved in the bowel after transplantation. In our model of transplantation, avascular fetal jejunum from term Fisher rats is transplanted to the subcutaneous tissues of host syngeneic rats. This "neogut" becomes vascularized and develops characteristics of native small bowel. VIP content was measured by RIA and the in situ distribution of VIP receptors was determined by the technique of receptor autoradiography. Neogut was studied 1 and 3 weeks after transplantation and compared with age-matched rat pup jejunum. Autoradiographs showed high silver grain density, representing VIP binding sites, in the mucosal layers of all tissues studied. VIP content in the transplanted bowel was comparable to that of native gut and showed a rise with developmental age similar to that of native gut. VIP levels (pmole/mg protein, x +/- SEM) were neogut 1 week, 0.26 +/- 0.14; jejunum 1 week, 0.25 +/- 0.07; neogut 3 weeks, 0.60 +/- 0.21; and jejunum 3 weeks, 0.69 +/- 0.16. These results show that VIP receptors and content are preserved in this model of transplantation. This suggests that the enteric nervous system and receptors for peptide neurotransmitters remain intact after transplantation and may retain the potential for regulatory function.

Evidence for regulation of peptide-YY release by the proximal gut.

Peptide-YY (PYY) is a novel enteric peptide that is structurally related to pancreatic polypeptide and neuropeptide-Y. The objectives of the present experiments were to characterize the following aspects of PYY metabolism: the distribution of PYY in the canine gastrointestinal tract, the release of PYY in response to oral ingestion of a mixed meal or intraduodenal (ID) administration of oleic acid, the effect of ileocolectomy on the release of PYY in response to ID administration of oleic acid when transit of chyme to the distal ileum and colon is prevented, the effect of interruption of intramural neural pathways of the small bowel on the release of PYY, and the effect of iv cholecystokinin on the release of PYY. The results of these experiments demonstrate that PYY immunoreactivity is distributed primarily in the terminal ileum, colon, and rectum. Circulating levels of PYY increase significantly (P less than 0.05) within 10-30 min after ingestion of a meal or to ID administration of a fatty acid. Complete interruption of the flow of chyme to the site of PYY-containing cells (i.e. ileum-colon) did not block the release of PYY; however, ileocolectomy abolished the release of PYY in response to ID administration of oleic acid. Severance of intramural neural pathways along the small bowel did not alter the release of PYY in response to an oral meal. Intravenous administration of graded doses of cholecystokinin stimulated the release of PYY in a dose-related manner. The results of these experiments indicate that the release of PYY from the distal ileum and colon is controlled, at least in part, by an extramural neural, endocrine, or a combination of both types of mechanisms which originate in the foregut.

GABA-immunoreactive cells in the rat gastrointestinal epithelium.

Frozen sections of the corpus ventriculi, antrum pyloricum, duodenum, jejunum, ileum and colon from animals perfusion fixed with glutaraldehyde were treated with an antiserum specific for glutaraldehyde-fixed GABA and processed by the peroxidase antiperoxidase method. Semi-thin plastic sections from the antrum pyloricum were treated similarly. Stained cells appeared in the epithelium of all segments examined except the corpus ventriculi. The highest density of cells was observed along the major curvature of the antrum pyloricum. Here they were located in the bottom half of the gastric glands. Many of the cells showed a process extending towards the glandular lumen. No significant staining in the epithelium appeared when the antiserum was preincubated with glutaraldehyde-GABA complexes, nor when the anti-GABA serum was exchanged with anti-glycine or preimmune serum. The present findings and previous physiological data suggest that GABA may play a role in gut endocrine regulation.