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).

Mannitol enhances intracellular calcium diffusion in the rat ileum--a hypothesis.

The addition of 92 or 136 mM mannitol to a modified saline solution that contained 1.25 mM Ca2+ led to a mannitol concentration-dependent increase in the amount of calcium absorbed in 1 h from 8 cm long ileal loops prepared from fasted male Sprague-Dawley rats, with body weights of 190 +/- 10 g. It is argued that this mannitol-enhanced movement of calcium out of the loop cannot have utilized the paracellular pathway, inasmuch as the luminal calcium concentration of the mannitol instillate decreased during the experiment, with a negative calcium gradient between luminal and body fluids. Instead it is proposed that uncomplexed mannitol and the uncharged calcium complex of mannitol entered the ileal cells. The uncomplexed intracellular mannitol would bind additional calcium that had crossed the brush border down its gradient. The increase in total intracellular calcium will raise the effective intracellular gradient and thereby amplify intracellular calcium diffusion. This in turn increases calcium absorption.

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.

Development of intracellular calcium measurement by time-resolved photon-counting fluorescence.

Calcium green I, a ratiometric probe based on fluorescence lifetime measurements, was used to monitor intracellular calcium activity ([Ca2+]i) in RINm5F cells using a time-resolved fluorescence confocal microscope. The probe affinity constant has been recalibrated in single cells using ionomycin as a calcium ionophore and ethylenebis(oxyethylenenitrilo)tetraacetic acid as a calcium buffer; Kd was found to equal 150 nmol/L. The kinetics of ionomycin equilibration showed that the calcium release from calcium stores occurs before equilibration with extracellular calcium. The response to the muscarinic agonist carbachol, measured on 17 cells receiving three consecutive applications was characterized both by a [Ca2+]i peak lasting 50 s without any trailing plateau and by desensitization with a 30% decrease in the response. The dose-dependent response was obtained for a carbachol concentration from 5 mumol/L to 0.5 mmol/L. The ability of our set-up to obtain a value every 10 ms enabled us to record asynchronous spikes of [Ca2+]i in the RINm5F cells. The spikes, lasting less than 1 s, are significantly bigger than the noise, and they are not observed in the colonic HT29 cells.

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.

Nutritional aspects of calcium absorption.

The amount of calcium absorbed in the intestine depends on habitual calcium intake. When intake is low, active transcellular calcium transport in the duodenum is upregulated and a larger proportion of calcium is absorbed by the active process than by the passive paracellular process that prevails in the jejunum and ileum. Bioavailability of the calcium source-digestibility and solubilization-plays a role under conditions of low calcium intake but is relatively unimportant when calcium intakes are high (e.g. >800 mg/d in people). Vitamin D intake is a second factor, as active calcium transport is directly and proportionally dependent on the presence in the intestinal cell of calbindin D9k, the biosynthesis of which is totally vitamin D dependent. Passive absorption in jejunum and ileum is the major absorptive process when calcium intake is adequate or high. Passive calcium absorption is a complicated function of solubility in the distal small intestine, the length of sojourn of the chyme in a given intestinal segment, and the rate of paracellular diffusion from lumen to lymph and blood. Calcium that reaches the large intestine undergoes absorption there by both active and passive processes. Probably no more than 10% of total calcium absorption takes place in the large intestine, whether calcium intake is low or high. Calcium absorption by the large bowel can assume nutritional importance under conditions of significant small bowel resection.

[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.

Oxyntomodulin reduces hydromineral transport through rat small intestine.

Glicentin (GLIC) and oxyntomodulin (OXM) are released from the ileum and colon during digestion. Both hormones reduce fluid and proton secretion in the stomach. The luminal concentration of sodium and chloride underlying the nutrient absorption, the effect of OXM on electrolyte transport through the small intestine, was assessed in vivo using ligated loops and in vitro using Ussing chambers. In vivo, a zero transport state, estimated by the net water, chloride, and sodium fluxes, was observed when an 80 mM NaCl normoosmolar solution (274 mosm) was administered intraluminally. Active secretion was observed with hyperosmotic challenge (474 mosm). The amplitude of this active secretion increased 2.5- to 3-fold when an electrogenic challenge (NaCl 40 mM) was substituted to the hyperosmotic one. OXM (800 fmol/ml plasma) did not modify the basal transport in the duodenum or in the jejunum (t = 45 min). When active secretion was induced by the hyperosmotic challenge, OXM (200 fmol/ml plasma) had no effect on duodenal or jejunal transport (t = 50 min). When active secretion was induced by an electrogenic challenge, OXM (300 fmol/ml plasma) preferentially reduced the hydromineral transport in jejunum. In vitro, OXM also induced a reduction in the ion transport towards the jejunal lumen (EC50 = 20 pM), the amplitude of which depended upon the integrity of the tetrodotoxin-sensitive neurons. In conclusion, OXM was able to reduce the large secretion induced in rat jejunum in vivo by an electrogenic gradient. In vitro, the antisecretory effect of OXM was partly mediated by the neurons present in the intrajejunal wall.

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.

1,25-Dihydroxycholecalciferol regulates rat intestinal calbindin D9k posttranscriptionally.

To determine whether calbindin D9k (CaBP) is subject to posttranscriptional control, 6-wk-old Sprague Dawley-derived rats were fed one of three purified diets, 1.5% Ca and 3.0% Ca, mostly as carbonate, and 2.9% Ca, mostly as gluconate. Two weeks later, 5-cm segments of duodenum, jejunum, ileum, cecum and colon were obtained and analyzed for CaBP and CaBP-mRNA. Analysis of the steady-state distribution of CaBP-mRNA and of CaBP revealed a statistically significant (r = 0.95; P < 0.01) linear relationship between CaBP-mRNA and CaBP. When, however, animals that had been fed the 1.5% Ca diet received by intrajugular injection 1.2 nmol 1,25-dihydroxycholecalciferol [1.25-(OH)2-D3] and their CaBP-mRNA and CaBP were analyzed as a function of time after 1,25-(OH)2-D3 administration, the kinetic response of the two molecules differed. The CaBP-mRNA increased linearly by approximately 68% for 4 h after administration and then declined over the next 6 h to a concentration below the preinjection value. Thus, appearance and disappearance of CaBP-mRNA approximated 17% x h(-1). The CaBP, however, increased steeply to 80% above preinjection concentration until 2 h postinjection, i.e., at a rate of 40% x h(-1). Thereafter, CaBP decreased to 35% above the preinjection value between 5 and 10 h postinjection (2.5% x h(-1)). These findings are consistent with a 1,25-(OH)2-D3-mediated posttranscriptional regulation of CaBP concentrations, because the 1,25-(OH)2-D3-mediated increase in CaBP-mRNA is not reflected in an immediately changed CaBP level.

Calcium solubility, intestinal sojourn time and paracellular permeability codetermine passive calcium absorption in rats.

To investigate the nonsaturable, paracellular pathway of intestinal Ca absorption, the luminal contents of 12-cm segments of the intestine of 8-wk-old male Sprague-Dawley rats were analyzed for pH, sojourn time and soluble and insoluble Ca over a 24-h period. The rats had been fed one of two high Ca diets for 2 wk: 1.5% Ca (diet group 3a) and 3.1% (diet group 5a). The pH of the small intestine increased from < 6.6 to > 8.0 from duodenum to ileum; transit time increased from 2.5 min in the duodenum to 58 min in the distal ileum, with the entire ileum accounting on the average for 74% of the transit time of 3 h. The amount of Ca solubilized throughout the intestine was 32 +/- 3.3 mumol in diet group 3a and 53 +/- 5.3 mumol in diet group 5a, i.e., 2.7% and 2.0% of the total luminal Ca. Because absorption by diet group 3a was 1.45 +/- 0.23 mmol/d and that by diet group 5a was 2.50 +/- 0.18 mmol/d, the amounts absorbed were 45.3 and 47.1 times greater than present in the lumen in soluble form at any one time. Thus, over a 24-h period, an average of 3.2% (46.2/1440) of the soluble Ca present in the lumen at any time was absorbed per min. Calculations involving the gradient between luminal and plasma Ca show that the rate of Ca diffusion from lumen to blood is < 2% of what it would be if the paracellular path were unrestricted. Thus, intestinal sojourn time, Ca solubility and mucosal permeability to Ca are factors that determine the rate of passive Ca absorption.

[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.

Increased chemocytotoxicity to colon cancer cells by shock wave-induced cavitation.

BACKGROUND/AIMS: Cavitation has been shown to hinder colon cancer cell proliferation in vitro. This study aimed at investigating the interest of combining cavitation and cytotoxic drugs in vitro. METHODS: HT-29 cells were exposed in suspension to cavitation (shock waves plus bubbles) before 5-fluorouracil (FUra) administration. Cytotoxicity was studied by means of clonogenic survival, cell proliferation by [3H]deoxyuridine ([3H]dUdR) incorporation, and influence of the treatments on the cell cycle by cytofluorimetry; the effects of cavitation on RNA incorporation of FUra, cell permeability, and activity of thymidilate synthetase (TS) were also studied. RESULTS: A preliminary exposure to cavitation (as compared with FUra alone) induced decreased colony formation (by up to 2 log in certain conditions) and colony size. Cavitation alone induced increased incorporation of [3H]dUdR during 48 hours and stimulated TS activity, but in the presence of FUra, the concentration of the drug that causes 50% inhibition of control cell growth for [3H]dUdR incorporation was reduced by up to 1 log, and TS inhibition was increased after cavitation as compared with FUra alone. RNA incorporation of [14C]FUra was increased by cavitation, as a consequence of altered cell permeability rather than a direct RNA effect. Seventy-two hours after treatment, cavitation plus FUra decreased by more than 50% the S-phase fraction and also inhibited mitosis. CONCLUSIONS: Submitting HT-29 cells to cavitation before treatment by FUra significantly increases the effects of the drug. The action of both agents appears to be partially synergistic with a cycle specificity.

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.