Coding region of the sorbin gene in different species.

The coding region of 153 amino-acid sorbin, isolated from porcine intestine has been cloned and sequenced in pig, human and rat. The coding region includes 459 bases comprising the 5' region of 24 bases, the middle region named "sorbin-like sequence" (25-432) and the 3' region (433-459). The peptidic C-terminal segment presents the biological activity: absorption of water and electrolytes from the intestine and gall-bladder. The cDNA homology between the three species was 95%. Three forms of mRNA were found, two major forms (6.5 and 8 Kb) and one minor (4.5 Kb).

Inhibitory effect of sorbin on pepsin secretion in conscious cats and rabbits.

Sorbin, a 153 amino acid polypeptide isolated from porcine upper small intestine and its shortest synthetic derivative, the C-terminal heptapeptide (C7-sorbin), substituted by D alaninamide in the last position (D7-sorbin), have proabsorptive and antisecretory effect in the different parts of the intestine. We showed that labeled C7-sorbin accumulated not only in the enterocytes and the enteric nervous system but also in the gastric chief cells in the rat. The chief cell secretion of pepsin was then studied in two other species, the cat and the rabbit, simultaneously with the acid secretion of parietal cells. Lipase secretion was studied in the rabbit because lipase is exclusively secreted by the upper cells of the fundic glands, which do not secrete pepsin. The animals were equipped with a gastric fistula, fully innervated, and a Heidenhain pouch, vagally denervated, during a continuous perfusion of pentagastrin (PG) 2 microg/kg. h and vasoactive intestinal peptide (VIP) 4 microg/kg. h. D7-sorbin (100 pmol/kg. h) inhibited cat and rabbit pepsin secretion from the innervated gastric fistula secretion and from the cat denervated Heidenhain pouc secretion, but was without effect on acid secretion and lipase secretion. These data indicate that the inhibitory effect of sorbin is specific on chief cells because the acid parietal cell secretion in both species and lipase upper cell secretion of the fundic glands, in the rabbit, are not implicated.

Presence of sorbin in human digestive tract and endocrine digestive tumours.

BACKGROUND: Sorbin, a 153 amino acid peptide isolated from porcine intestine, was localised by immunohistochemistry in endocrine cells of the intestinal mucosa and pancreas and in the enteric nervous system in the pig. AIMS: To identify sorbin cells in normal human digestive tissues and to explore the expression of sorbin in 37 digestive endocrine tumours: 14 intestinal carcinoid tumours and 23 endocrine pancreatic tumours including six insulinomas. METHODS: Two polyclonal antibodies against the C-terminal and the N-terminal sequences of porcine sorbin raised in rabbit were used to evaluate sorbin expression by immunohistochemistry. RESULTS: In the human digestive tract, sorbin, characterised by both C-terminal and N-terminal immunoreactivity, was found in enterochromaffin cells of the gastric and intestinal epithelium from the pyloric junction to the descending colon. C-Terminal sorbin immunoreactivity alone was found in plexii from the enteric nervous system and in some insulin-containing cells of normal pancreas. C-Terminal and N-terminal antibodies disclosed sorbin in five of 14 intestinal carcinoid tumours; C-terminal antibody alone disclosed a C-terminal sorbin peptide in two of six insulinomas and three of 17 endocrine pancreatic tumours. The presence of sorbin was not associated with a specific clinical syndrome. CONCLUSIONS: Sorbin is present in the digestive tract in several forms. It is expressed in some intestinal and pancreatic endocrine tumours.

Distant intestinal stimulation by cholera toxin in rat in vivo.

Cholera toxin (16 microg/rat) locally administered in the jejunum of anesthetized rats stimulated jejunal secretion and also distant duodenal secretion, as determined with the ligated loop technique. The release of prostaglandin E2 in both jejunal and duodenal secretions and in plasma was increased by cholera toxin, while the release of 5-hydroxytryptamine (5-HT) was unchanged in the early phase of secretion (2 h). The inhibitor of prostaglandin E2 release, indomethacin (10 mg/kg, s.c.), and the 5-HT3 subtype receptor antagonist, granisetron (30 microg/kg i.v.), inhibited the jejunal secretion but had no effect on distant duodenal secretion. However, indomethacin statistically significantly decreased prostaglandin E2 release in both jejunal and duodenal secretions as well as in plasma. The vasoactive intestinal peptide antagonist (VIP-(6-28), 1.2 nmol/100 g h) did not modify jejunal and duodenal secretions. Our study confirmed the local involvement of 5-HT and prostaglandin E2 in choleraic jejunal secretion but not in distant duodenal secretion.

[Effect of serotonin on unidirectional ion fluxes in rat intestine in vivo]. / Effet de la sérotonine sur les flux unidirectionnels des ions dans l'intestin de rat in vivo.

AIMS: A stimulating intestinal secretory effect is described in vitro and an inhibition with selective inhibitors of the different receptors of serotonin (5-HT), in vivo. But a direct effect, in vivo, in fully vascularized and innervated intestine has not yet been clearly evidenced. We studied the effect of 5-HT in anesthetized rats with ligated loops. This work, performed at 4 intestinal levels, allowed a comparison with the effects of a known stimulant of intestinal secretion, VIP, and a specific inhibitor of Na/H exchange, dimethylamiloride (DMA). RESULTS: 5-HT induced an inhibition of epithelial Na influx in agreement with the inhibition of Na/H exchanger, an inhibition of the influx of Cl, partially passive absorption following Na by paracellular route. A decrease of Na and Cl efflux was induced by 5-HT in duodenum, jejunum and ileum while in colon, a stimulation was obtained by intraluminal but not intravenous route. CONCLUSION: Even though 5-HT induced a liquid accumulation in all intestinal segments, the effect differed according to the intestinal level, either inhibition of absorption in the small intestine, or stimulation of secretion in the colon. The comparison of the effect of 5-HT with that of DMA shows that the inhibition of absorption is not only due to Na/H exchanger inhibition.

Effect of sorbin derivatives on cholera toxin-induced intestinal secretion in rat in vivo.

The effect of synthetic sorbin derivatives was determined on cholera toxin-stimulated jejunal secretion in anesthetized rats in vivo, using both perfused and ligated loop. An inhibitory effect on water secretion induced by cholera toxin was shown with C-terminal sorbin peptides: C20 (YEPGKSSILQHERPVTKPQA-amide), C10 and Dala 7 heptapeptide-amide of sorbin, given by subcutaneous (SC) or intraduodenal administration. When perfused intravenously, C20-sorbin inhibited the cholera-induced stimulation of net flux of water, Na+ and K+, in the jejunum and at the same time the net flux of water and Cl- in the duodenum, which was not in contact with the toxin. 5-hydroxytryptamine was not significantly changed in plasma or fluid. Prostaglandin E2 release in jejunal as well as duodenal fluid was significantly stimulated by cholera toxin, but was not significantly different from basal value after C20 administration.

5-(N,N-Dimethyl)-amiloride to discriminate the Unidirectional electrolyte transports in rat small intestine and proximal colon in vivo.

The effect of dimethyl-amiloride (DMA), a selective Na+/H+ exchange blocker, was studied on electrolyte net fluxes and unidirectional fluxes of Na and Cl at four levels of rat intestine in vivo in basal conditions. DMA was applied intraluminally at concentrations of 10(-4) and 10(-3) M in the model of ligated loops prepared from duodenum, proximal jejunum, distal ileum and ascending colon in fasted Sprague Dawley rats. Two iso-osmotic test solutions were used: (1) hypo-ionic: Na+ 80 mM and (2) iso-ionic: Na+ 148 mM, pH 8.2. 22Na was placed in the loop and 36Cl was given by intravenous route at the beginning of the experiment. Na+/H+ was calculated by two different means, one was based on pH variation following amiloride inhibition of Na influx, the other on the calculation of the passive Na transport. The quantitative evaluation shows that Na/H exchange largely contributes to the electroneutral absorption and luminal pH regulation. The exchanger activity decreases from duodenum, jejunum, ileum and colon where it is completed by K/H exchange to assure low colon luminal pH.

Sorbin in the porcine gastrointestinal tract and pancreas: an immunocytochemical analysis.

Sorbin is a 153-amino acid peptide that was initially discovered in the porcine duodenum. We have reported previously that this peptide regulates intestinal electrolyte transport and have described accumulation sites in the rat digestive tract. In the present study, we investigated the anatomical distribution and the site(s) of sorbin production in the porcine digestive tract using immunocytochemistry. The use of polyclonal antisera, which by cross-reaction studies were shown to be specific for different regions of the molecule, revealed a diversified distribution. Sorbin predominated in endocrine cells preferentially localized in the pyloric glands, duodenal crypts of Lieberkühn, and pancreatic islets; in the gastrointestinal tract, sorbin coexisted with Met-enkephalin or with substance P in a small fraction of serotonin-storing [enterochromaffin (ED)] cells, i.e. EC2 cells and EC1 cells, respectively; in the pancreas, sorbin coexisted with insulin in the beta-cells, also considered as serotonin-storing cells in the pig, and with EC cells in the exocrine pancreas. An enteric neuronal system containing sorbin was also reported. Our results demonstrate that sorbin is a component of the serotonin-storing cell type in the porcine gastrointestinal tract and pancreas, and suggest potential directions to investigate the functions of this new regulatory peptide.

Guanylin-, heat-stable enterotoxin of Escherichia coli- and vasoactive intestinal peptide-induced water and ion secretion in the rat intestine in vivo.

The heat-stable enterotoxin of Escherichia coli binds to an intestinal receptor, guanylyl cyclase-C, and produces cGMP to induce diarrhea. Guanylin is an endogenous ligand of this receptor. In the present in vivo study, the intestinal water and ion secretion induced by mucosal application of 2 nmol/ml guanylin or 5 or 10 units/ml heat-stable enterotoxin into closed loops was compared in the rat. The characteristics of secretion induced by cAMP following intravenous perfusion of 1.2 nmol/100 g per h vasoactive intestinal peptide were compared to those induced by cGMP. Unidirectional Na+ and Cl- fluxes were estimated by addition of 22Na into the loop and i.v. injection of 36Cl. Guanylin induced less water and ion secretion than that produced by heat-stable enterotoxin in the colon, confirming the results of in vitro studies, and also in duodenum and ileum. The cAMP- or cGMP-mediated response had a similar pattern, i.e., an inhibition of Na+ absorption and an increase in anion secretion.

Selection of cholesterol absorption inhibitors devoid of secondary intestinal effects.

The digestive tolerance of cholesterol absorption inhibitors, which requires a constant improvement, was the main purpose of this study. Given the known hypocholesterolemic and antiatherosclerotic properties of some steroid glycosides, we synthesized a series of sterol derivatives by coupling some phytosterols known to interact with sterol absorption and also to be poorly absorbed to a cationic group. The first derivative was a potent inhibitor of cholesterol absorption and a potent hypocholesterolemic agent in different animal models, but was responsible for severe gastro-intestinal side-effects. In order to control the tolerance of the newly synthesized compounds, cholesterol and taurocholate absorption were measured in the jejunum and in the ileum, respectively. The intestinal water and ionic transport and the estimation of histological changes in the intestinal mucosae were determined simultaneously. The in-situ isolated loop technique, in anaesthetized rats, allowed the simultaneous control of these three parameters which were used to select the best derivative, inhibitor of cholesterol absorption devoid of any deleterious effect, as seen via a three-dimensional representation. The results showed that it was possible to obtain a specific cholesterol absorption inhibitor without secretory and deleterious effects and suggested that the amphiphilic characteristics of the molecules were responsible for their deleterious effects on digestive tract.

Similarities in cellular expression and functions of melanin-concentrating hormone and atrial natriuretic factor in the rat digestive tract.

Melanin-concentrating hormone (MCH) is a cyclic peptide isolated first from salmon brain, then from rat and human hypothalamus. We have recently found expression of MCH messenger RNA and encoded peptides, e.g. MCH and neuropeptide-glutamic acid-isoleucine, within the rat gastrointestinal (GI) tract, but their cellular origin was unclear. Furthermore, similarities in the localization of rat atrial natriuretic factor (ANF) and rat MCH immunoreactivities within intestine suggested functional convergence. In the present study we determined first the presence and distribution of MCH messenger RNA and encoded peptides in the GI tract by combining in situ hybridization and immunohistochemical analysis. Our data revealed numerous MCH-containing cells located in the lamina propria and submucosa at both duodenal and colonic levels. Second, the localisation of MCH- and arginine vasopressin- or ANF-containing cells appears similar at the duodenal and colonic levels, respectively. Colocalization of MCH/neuropeptide-glutamic acid-isoleucine immunoreactivity (-IR) and catecholamine indicated that MCH-expressing cells are probably antigen-presenting cells forming part of the enterochromaffin cell system. Third, we performed reverse phase HPLC coupled to RIA to characterize MCH-like materials in different portions of the rat gut. Crude acidic extracts of rat intestine contained about 2-3 pmol/g tissue of MCH-IR, close to the values found in brain extracts. Reverse phase HPLC of MCH-IR in the GI tract revealed that only 10-30% of the immunoreactivity corresponded to mature MCH, whereas the rat brain contained 94% mature peptide. Finally, we compared the effect of MCH and ANF on water and electrolyte secretions at different levels of the GI tract by using the in situ ligated loop technique. Similar effects were noted for ANF and MCH; both stimulated water, Na, and K fluxes at the proximal colon level and increased Na and K fluxes in the duodenum. However, only ANF increased water and Cl fluxes in the duodenum and decreased bicarbonate secretion in the ileum, whereas MCH increased bicarbonate absorption in the jejunum. The dose required was 10 nmol/100 g.h for MCH, i.e. 10 times more than for the ANF. These studies strongly suggest that MCH produced by antigen-presenting cells of the lamina propria may have an important role, similar to that of ANF at the colonic level, in the physiology of the GI tract.

[Effect of C-terminal derivatives of sorbin on duodenal ion transports in rats]. / Effet des dérivés C-terminaux de la sorbine sur les flux ioniques duodénaux chez le rat.

OBJECTIVES AND METHODS: Synthetic derivatives of sorbin have been shown to inhibit VIP stimulated fluxes in the ileum in decreasing plasma-to-mucosa Na and Cl effluxes. The effect of this group of new peptides, without homology with any known peptides, was determined in rat duodenum where ion transport mechanisms differ. The improved technique of ligated loops in situ, was used, permitting the simultaneous measurement of net fluxes, influxes and effluxes for Na and Cl, in an integrated in vivo model. To determine the minimal active fragment of sorbin, synthetic C5, C7 and C20 peptides were tested and compared with known anti-secretor drugs such as loperamide, neuropeptide Y, somatostatine and metenkephalinamide. RESULTS: C7-sorbin was the minimal peptide able to decrease duodenal VIP-stimulated fluxes of water, Na and bicarbonate. It intervenes in increasing Na influx and more slightly Cl influx, which have been decreased by VIP. It does not modify much Na and Cl effluxes stimulated by VIP. Sorbin effect is in contrast with those of known antidiarrheic agents like somatostatine, loperamide, NPY and metenkephalinamide which chiefly decrease Cl efflux. CONCLUSIONS: Sorbin acts like an activator of absorption in the duodenum, in contrast to the other peptides or drugs and to its own anti-secretor effect in the ileum.

Pharmacokinetic, metabolic, and antidiarrheal properties of (D and L) heptapeptides of sorbin in rodent.

The C-terminal heptapeptide-amide (C7-sorbin) is the minimal biologically active fragment of sorbin inducing an increase in intestinal hydroelectrolytic absorption. An analogue (D7-sorbin), characterized by the replacement of the ultimate C-terminal amino acid L-alanine-amide by D-alanine-amide, was synthetized. For pharmacokinetic studies, D7-sorbin and C7-sorbin were tritium labeled. After IV injection, clearances were 10.6 and 30.2 ml-1 for D7-sorbin and C7-sorbin, respectively, and MRT were 34 and 18 min. After SC administration, Cmax attained 0.41% and 0.12% of the dose/ml, respectively. The IP route showed a 45-min delay before Cmax and a 100% bioavailability for both peptides. D7-sorbin was principally excreted in urine, as shown by balance study, and in part in intact form, as controlled by mass spectrometry. D7-sorbin induced a significant decrease of the VIP-induced ileal secretion, previously observed with C7-sorbin. The change of L-Ala to D-Ala increased the stability of the synthetic C-terminal peptide of sorbin whereas its biological activity, bioavailability, and route of elimination were unchanged.

Pharmacokinetics and organ distribution of the sorbin C-terminal peptides.

Sorbin is a 153 amino acid peptide isolated from porcine small intestine. The heptapeptide-amide is the minimal active site of the natural molecule. A comparison of the distribution of C-7 and C-20 sorbin, which have been shown to share the activity of sorbin in increasing intestinal absorption of electrolytes, was undertaken by radioimmunoassay, after perfusion of 200 micrograms/kg/h. A longer half-life in plasma was observed for C-20 sorbin than for C-7 sorbin, with a clearance rate of 18 +/- 4 ml/min/kg vs. 40.6 +/- 13.5 ml/min/kg and a distribution volume of 192 +/- 35 ml/kg vs. 286 +/- 123 ml/kg. The accumulation of tritiated C-7 sorbin was observed in enterocytes, serosal acini of the salivary glands, and fundus chief cells. The recovery of intact peptide in the intestine was 0.06% per gram of tissue. Eighteen percent of the peptide was detected in urine.

[Effect of C-terminal derivatives of sorbin on ileal ion transport stimulated by VIP in rats]. / Effet des dérivés C-terminaux de la sorbine sur les flux ioniques iléaux stimulés par le VIP chez le rat.

OBJECTIVES AND METHODS: Sorbin, a peptide isolated from porcine intestine and composed of 153 aminoacids, has been purified because its specific action is to increase water and ion absorption in the intestine and the gall bladder. We showed that synthetic peptides containing the amidated C-terminal part of sorbin had the same activity as the natural molecule in increasing duodenal absorption. In order to characterize the site of action of sorbin, the effect of two C-terminal derivatives were determined in ileal ligated loops in situ in anaesthetised rats, following VIP-induced water and electrolyte secretions. Their effect was compared to those of metenkephalinamide, NPY and somatostatin. Unidirectional fluxes were studied to analyze the mechanism of action of sorbin, by means of 22Na, administered into the intestinal loop, and 36Cl, injected into blood. RESULTS: Results show that C20-sorbin and C7-sorbin decreased the VIP-stimulated net flux of water (inhibition of 40 and 37%, respectively), Na (inhibition of 31 and 30%), C1 (inhibition of 80 and 63%) and HCO3 (inhibition of 15 and 25%). These effects are evidently greater than those produced by equimolar doses of NPY, somatostatin, and 32 times higher dose of metenkephalinamide. Sorbin acts as a potent anti-secretor, anti-VIP, in rat ileum.

[Comparison of hydroelectrolytic exchange characterized by calculation of saturable fluxes at three levels of the rat intestine]. / Comparaison des échanges hydroélectrolytiques caractérisés par le calcul des flux saturables à trois niveaux de l'intestin chez le rat.

Using the everted sac technique, which responded, as expected, to VIP by an increase of the secretion and to glucose by an increase of the absorption, we compared the water and electrolyte movements in the jejunum, ileum and colon in rats. Identical iso-osmolar test-solutions containing increasing NaCl concentrations, placed on the serosal and mucosal sides, allowed us to quantify fluxes in the absence of initial gradient. The measured net Na and Cl fluxes were dissociated into their two components, a passive flux from serosa to mucosa and a saturable flux from mucosa to serosa. The parameters of the saturable transport, calculated for each of the intestinal parts, showed the highest J max for the ileum (58.5 microEq.g-1.h-1 for Na and 52.8 microEq.g-1.h-1 for Cl) and the lowest Km for the colon that had the highest affinity for sodium and chloride (Km 11.1 mM for Na and 7.8 mM for Cl). These data confirm the functional difference between the three intestinal parts, with an active absorption of Na and active secretion of Cl in the jejunum, an apparent coupled Na and Cl absorption in the ileum and an active absorption with high affinity for both Na and Cl in the colon.

Effect of some gastrointestinal hormones on motor and electrical activity of the digestive tract in the conscious cat.

Three peptides structurally related to gastrin and known to be full agonists of antral motility in the conscious cat, pentagastrin (PG), cholecystokinin (CCK) and synthetic octapeptide of cholecystokinin (OP-CCK), were compared in relation to antral and duodenal electrical activity. They induced the same antral effect in eliciting an increase in the basal electrical rhythm (BER) and a short-lasting decrease in the frequency of the bursts of spikes. The electrical changes were correlated with lumen pressure changes measured in parallel, consisting of a decrease in the frequency of high-amplitude peaks and an increase in low-amplitude peaks. The additive effect of PG and CCK shows that the peptides are full agonists for antral electrical activity, as they are for antral motility and acid secretion. In contrast to the antrum, the three peptides increased the frequency of the duodenal spike bursts, CCK and OP-CCK decreased the BER frequency, while PG increased BER slightly. The increase in antral and duodenal BER obtained after a beef-liver meal, which produced a large endogenous gastrin release, suggests a major role for gastrin in antral motility induced by feeding, at least in the cat.

Interaction between pepsin and acid secretion during fundic perfusion in cat and rabbit.

1. The effect of perfusion at pH 5.5 of a Heidenhain pouch on acid and pepsin secretion was studied in cats and rabbits. 2. In basal conditions, acid secretion was not modified by the perfusion while pepsin secretion was increased in both species. 3. After a meal, the perfusion did not modify the stimulation of acid secretion in cats nor the absence of secretions in rabbit. Stimulation of pepsin secretion was the same with or without perfusion in both species. 4. In rabbit, in contrast to cat, pepsin and acid secretions were independent during the meal-induced phase of gastric secretion.

Post-prandial lipase, pepsin and acid secretion of a Heidenhain pouch in the rabbit.

The kinetics of the gastric secretion of lipase, pepsin and acid were studied after a meal in Heidenhain-pouch rabbits. After a 24-h fast, feeding immediately stimulated (< 15 min) lipase (x 4.1) and later on pepsin (x 1.8) output which reached respectively 16 and 47% of the output observed after pentagastrin stimulation (64 micrograms.kg-1.h-1 for 1 h), and which were significantly correlated. Lipase concentration was enhanced earlier and to a greater degree (x 7.3) than pepsin concentration (x 2.5). No stimulation of high basal acid secretion occurred. It was concluded that: 1) gastric lipase secretion is stimulated by feeding in the rabbit; 2) pepsin secretion is stimulated by feeding. The modalities of the secretion of lipase and pepsin are compatible with the existence of distinct secretory cells; 3) acid secretion is not stimulated by feeding. The decrease in acid secretion during the post-prandial phase favors a physiological role for lipase which is altered by low pH. The absence of acid stimulation by feeding in the rabbit, in contrast to other species, requires additional studies on the release of gastrointestinal hormones, namely gastrin, cholecystokinin and somatostatin.