Effect of sorbin on electrolyte transport in rat and human intestine

Bruno Eto1, Michel Boisset1, Bertrand Griesmar2, and Jehan-François Desjeux1,2

1 Conservatoire National des Arts et Métiers, Laboratoire de Biologie, 75141 Paris 03; and 2 Unité de Recherche sur les Fonctions Intestinales, le Métabolisme et la Nutrition, Institut National de la Santé et de la Recherche Médicale, Unité 290, Hôpital Saint-Lazare, 75010 Paris, France


    ABSTRACT
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Stimulating water absorption in the colon represents an important target to reduce stool output in secretory diarrhea. Recently, a 153-amino-acid peptide was isolated from porcine upper small intestine and purified, taking into account the increase of water absorption in guinea pig gallbladder. Accordingly, this peptide was named sorbin. The aim of the present study was to determine if the COOH-terminal heptapeptide of sorbin (C7-sorbin) participates in the regulation of electrolyte transport in the colon. Different regions (from duodenum to colon) of stripped intestinal mucosa from rats or humans were mounted in Ussing chambers to measure the changes in short-circuit current (Delta Isc) and net 22Na and 36Cl fluxes (JNanet and JClnet) after serosal exposure of 10-7 to 10-3 M C7-sorbin. In fasted rat intestine, C7-sorbin (10-4 M) induced an immediate reduction in Isc in the distal ileum and proximal and distal colon but not in the duodenum and jejunum. In the colon, Isc reduction and JNanet and JClnet stimulation were dose dependent (EC50 = 2 × 10-5 M). At 10-3 M, maximal effect was observed (Delta Isc = -1.14 ± 0.05, Delta JNanet = +4.97 ± 1.38, and Delta JClnet = +9.25 ± 1.44 µeq · h-1 · cm-2). C7-sorbin (10-3 M) inhibited the increase in Isc induced by a series of 10 secretory agents such as secretin, vasoactive intestinal peptide, PGE2, and serotonin. In HT-29-Cl19A cells, C7-sorbin induced an increase in Isc, with a maximal effect at 10-3 M (Delta Isc = 0.29 ± 0.10 µeq · h-1 · cm-2). In human intestine, a dose-dependent decrease in Isc was observed in right and sigmoid colons in basal and stimulated conditions (EC50 congruent  10-5 M; at 10-4 M, Delta Isc = -2.66 ± 0.17 µeq · h-1 · cm-2) but not in the jejunum. The results indicate that C7-sorbin stimulated NaCl neutral absorption and inhibited electrogenic Cl- in rat and human intestinal epithelia. In addition, the antisecretory effect was essentially observed in the distal part of both rat and human intestine and the magnitude of the proabsorptive effect was directly related to the magnitude of the previously induced secretion.

sodium chloride absorption; ion transport; colon


    INTRODUCTION
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Abstract
Introduction
Materials and methods
Results
Discussion
References

THE GASTROINTESTINAL TRACT IS uniquely designed to control water and electrolyte economy of the body (for recent review, see Ref. 10). In one day, the gastrointestinal tract must deal with a minimum of 8-9 liters of water from ingested meals and drinks and digestive secretions. Much of this volume is absorbed in the small intestine secondary to nutrient and electrolyte absorption, leaving 1-2 liters that enter the large intestine. Here, 90% of the remaining water is absorbed, yielding an average daily stool volume of 100-200 ml.

In diarrheal diseases, the increase in stool volume is the consequence of a situation in which the volumes presented to the colon exceed its large reserve capacity. Thus, in secretory diarrhea such as cholera, diarrhea occurs when the large volume produced by the secretory effect of the toxins from the vibrio in the small intestine overwhelms the absorptive capacity of the colon (for recent review, see Ref. 11). At a constant secretion rate in the small intestine, the extent of water and electrolyte losses in stools are directly related to the absorptive capacity of the colon. Hence, the severity of dehydration in cholera could be attributed in part to the decrease in colonic absorptive capacity (36). In addition, the severity of viral infection in young piglets, before 3 days of age, appears to be the consequence of a lack of stimulation of colonic absorption in response to the increased volume presented to the colon from the small intestine (1). In contrast to 3-day-old piglets, the colons of 3-wk-old infected piglets had increased fluid absorption that was about six times over controls; this compensatory response prevented diarrhea in these older animals.

The epithelial cells lining the gastrointestinal tract are the key players in the absorptive and secretory events. Electrolytes are actively transported from lumen to blood or in the opposite direction as a result of the activity of the Na+-K+-ATPase and asymmetric distribution of specific transporters or channels, between luminal and basolateral membranes of the enterocytes. Regulation of intestinal electrolyte absorption or secretion occurs via intracellular messengers that are modulated by various external stimuli, including neuromediators. Several of them, including peptide YY (PYY) (9, 20), somatostatin (5, 16, 28), ANG (7, 17), enkephalin (16, 24, 27), and norepinephrine (28), reduce intestinal secretion in physiological and pathological conditions. However, the role of these hormones in stimulating colonic absorption as a response to increased secretion is ill defined.

Sorbin is a peptide of 153 amino acids that was isolated and purified, taking into account the increase in water absorption, from porcine upper small intestine in guinea pig gallbladder (32, 37). In the rat, the COOH-terminal heptapeptide of the natural molecule was found to be the minimal biologically active fragment. The synthetic modified fragment [Pro-Val-Thr-Lys-Pro-Gln-(D-Ala)-NH2 (C7-sorbin)] was found to increase water and electrolyte absorption in the rat duodenum and to decrease intestinal secretion induced by vasoactive intestinal peptide (VIP) perfusion (7, 23, 29). In addition, it reduced water secretion induced by cholera toxin in the rat (J. Fioramonti and L. Bueno, personal communication). Because sorbin has been selected and identified based on its effect on water absorption in the gallbladder, its main activity might be the stimulation of an NaCl neutral absorptive process (18, 21). In addition, if sorbin is mainly active on NaCl absorption, it may be predicted that its effect would be observed in the ileum and the colon where the neutral NaCl transport is primarily located (6).

Thus the aim of the present study was to determine if C7-sorbin participates in the regulation of electrolyte transport in the colon in basal and stimulated secretion conditions. The results indicate that C7-sorbin stimulated NaCl neutral absorption and inhibited electrogenic Cl- secretion in the rat colon. In addition, the antisecretory effect was essentially observed in the distal part of rat and human intestine. The magnitude of the proabsorptive effect was directly related to the magnitude of the previously induced secretion.


    MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Chemicals

Bumetanide, 5-hydroxytryptamine (serotonin, 5-HT), PGE2, clonidine, amiloride, TTX, naloxone, and BSA (99% fatty acid free) were purchased from Sigma (St. Quentin-Fallavier, France). Gastrin, VIP, porcine motilin, peptide histidine isoleucine, secretin, helodermin, substance P, rat alpha -atrial natriuretic factor-(1---28), neurotensin, ANG I, PYY, and somatostatin (somatotropin release-inhibiting factor, SRIF), at least 95% pure, were obtained from Neosystem (Strasbourg, France). H-sorbin (C7-sorbin) was a kind gift from Institut Henri Beaufour (Les Ulis, France). Scintillation fluid (Aquasafe 300+) was purchased from BAI (Elancourt, France). H36Cl (0.2 M, specific activity 0.34 MBq/mg Cl-) was from Amersham (Les Ulis, France). 22NaCl (specific activity 23.38 MBq/mg Na+) was from DuPont NEN (Les Ulis, France). All other chemicals were analytic grade reagents.

Peptide Solutions

Stock solutions (10-2 M) of C7-sorbin were made with 0.25% (wt/vol) BSA in ultrapure water, stored at -25°C, and thawed immediately before use. Working solutions were prepared extemporaneously at 4°C by serial dilution of stock solutions to achieve final concentrations of 10-7 to 10-3 M when added to serosal medium. All the other solutions of secretory agents were prepared as described above and used at 2 × 10-7 and 10-6 M.

Preparation of Tissues

Mature male Sprague-Dawley rats weighing 180-250 g were obtained from Iffa Credo (St. Germain s/Arbresle, France) and housed in individual cages and fed standard laboratory chow (UAR, Villemoisson s/Orge, France) until used in these studies. Food was withdrawn 18 h before the experiments, but animals had free access to drinking water.

For electrophysiology studies, animals were killed by cervical dislocation, and segments of intestine from fasted animals were removed and rinsed free of intestinal content by flushing with ice-cold Ringer solution. The animals' stomachs were found to be empty. Tissues were stripped off the muscular layer, opened along the mesenteric border, and mounted as flat sheets between the two halves of acrylic Ussing chambers, as previously described (20, 35).

Human tissues were obtained at surgery from digestive cancer patients. Healthy pieces were stored in ice-cold Ringer medium until used (close to 1 h after the tissue was removed). Tissues were prepared as described above and mounted in Ussing chambers.

Short-Circuit Current Studies in Ussing Chambers

The isotonic Ringer solution used throughout the experiments contained (in mM) 115 NaCl, 25 NaHCO3, 1.2 MgCl2, 1.2 CaCl2, 2.4 K2HPO4, and 0.4 KH2PO4. The pH was 7.40 at 37°C when bubbled with the 95% O2-5% CO2 mixture used to circulate the chamber fluid. The Cl--free solution was made by replacing NaCl with sodium isethionate and CaCl2 and MgCl2 with the sulfate salts. The standard Na+-free solution was prepared by replacing NaCl with choline chloride. The osmolarity of the solution was checked by an osmometer and adjusted close to 300 mosM by the addition of mannitol.

The spontaneous transmural electrical potential difference, reflecting the asymmetry of electrical charges between the luminal and serosal membranes, was measured via 3 M KCl solution in 4% (wt/vol) agar bridges. These bridges were placed on both sides of the tissue and adapted to calomel half-cells, linked to a high-impedance voltmeter. Potential difference was short-circuited throughout the experiment by a short-circuit current (Isc) via 3 M KCl in agar bridges placed in each reservoir, adapted to Ag-AgCl electrodes in relation with an automatic voltage-clamp system (DVC 1000, World Precision Instruments, Sarasota, FL). Delivered Isc, corrected for fluid resistance, was recorded continuously on a computer. Isc represents the sum of the net ion fluxes transported across the epithelium in the absence of an electrochemical gradient (mainly Na+, Cl-, and HCO-3). Every 30 s, the tissue was automatically clamped at +1 mV for 3 s to calculate transepithelial electrical conductance, according to Ohm's law. Peptides were added to the serosal medium after stabilization of Isc.

Effect of C7-Sorbin on Isc

C7-Sorbin was used at both single and cumulative doses. In the single dose experiments, one piece of tissue per chamber was exposed to only one given concentration that ranged from 10-7 to 10-3 M of C7-sorbin, until a new steady state for Isc was obtained. In cumulative dose experiments, one piece of tissue per chamber was successively exposed to increasing doses of C7-sorbin up to the maximal effect of peptide within a 20-min time period (i.e., from 10-7 to 10-3 M). After the maximal decrease in Isc was obtained, 5 × 10-5 M bumetanide was added to serosal fluid to measure residual electrogenic Cl- secretion (20).

Effect of C7-Sorbin on Na+ and Cl- Fluxes

At steady state of the electrical parameters, tissues were paired according to their conductance value (±20%). C7-sorbin was then introduced at 10-3 M into the serosal medium. Next, 74 kBq (2 µCi) of 36Cl- or 37 kBq (1 µCi) of 22Na+ were introduced into the mucosal or the serosal bath of paired tissues. A 1-ml sample of the serosal or mucosal fluid was withdrawn at 15-min intervals for 60 min and replaced by 1 ml of Ringer solution at 37°C. Scintillation fluid (4 ml) was then added to the samples, which were counted for 10 min. The effects of C7-sorbin on unidirectional mucosal-to-serosal and serosal-to-mucosal Cl- and Na+ fluxes were determined during the steady state of transport (30-60 min). The net Cl- and Na+ fluxes were the differences between the opposite unidirectional fluxes obtained on paired tissues. (For comparison, Isc was expressed both in µA/cm2 and in µeq · h-1 · cm-2 and the fluxes of Na+ and Cl- were expressed in µeq · h-1 · cm-2.)

Effect of C7-Sorbin on Secretion Induced by Intestinal Peptides

In this experiment, one piece of tissue per chamber was exposed to 2 × 10-7 M of each secretory agent until a new steady state for Isc was obtained. Then, 10-3 M C7-sorbin was added to the serosal medium.

Statistics

Results are reported as means ± SE. All determinations were performed in pieces of tissue (n) from at least seven rats or three humans. ANOVA was performed according to the general linear model procedure, and comparison of means was by the least-square difference test of the SAS package (SAS Institute, Cary, NC).


    RESULTS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Effect of C7-Sorbin in Rat Intestine

Effect of C7-sorbin on Isc. Typical recordings of the effect of C7-sorbin on Isc in rat colon are presented in Fig. 1. In basal condition, addition of 10-3 M C7-sorbin in the serosal compartment was followed by an immediate and steady decrease in Isc (Fig. 1A). A similar effect was observed (Fig. 1B) after stimulation of Isc by 10-4 M 5-HT. Further addition of 5 × 10-5 M bumetanide in the serosal solution did not decrease the current, strongly suggesting that C7-sorbin entirely inhibited electrogenic Cl- secretion. Previous treatment with naloxone (10-6 M) or TTX (2 × 10-6 M) did not alter the response of colon to C7-sorbin.


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Fig. 1.   Typical recording of the effect of sorbin analog (C7-sorbin) on short-circuit current (Isc). A: basal condition. B: condition after stimulation by serosal 10-4 M 5-hydroxytryptamine (5-HT). BUM, bumetanide.

Effect of C7-sorbin in different segments of rat intestine. A small antisecretory effect of C7-sorbin was found in both duodenum (Delta Isc = -2.67 ± 0.67 µA/cm2) and jejunum (Delta Isc = -5.27 ± 0.94 µA/cm2), as shown in Fig. 2. At 10-4 M, the effect of C7-sorbin was more marked in the ileum (Delta Isc -8.16 ± 3.37 µA/cm2, P < 0.05) and even more so in the colon (Delta Isc = -17.69 ± 2.82 µA/cm2, P < 0.001).


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Fig. 2.   Variation in Isc induced by C7-sorbin (10-4) in rat intestine. Solid bars and vertical bars represent means and SE, respectively, of 6-7 tissues from 5 rats. * P < 0.05; *** P < 0.001. DUO, duodenum; JEJ, jejunum; ILE, ileum; COL, colon.

Dose response of C7-sorbin on Isc. In rat colon, C7-sorbin induced a dose-dependent reduction in Isc in basal condition. The apparent threshold was at 10-6 M, the EC50 value close to 2 × 10-5 M, and maximal response was at 10-3 M (Fig. 3). Almost identical results were obtained when the tissue was stimulated by 10-4 M 5-HT before C7-sorbin addition (not shown).


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Fig. 3.   Concentration response of C7-sorbin on basal Isc in fasted rat proximal and distal colons. Results are expressed as percentage of the effect of bumetanide on Isc. Solid circles and vertical bars represent means and SE, respectively, of 8-15 tissues from 14 rats.

Effect of C7-sorbin on Cl- and Na+ fluxes in rat colon. The simplest explanation for the reduction in Isc is an inhibition of electrogenic Cl- secretion linked to a stimulation of neutral NaCl absorption that cannot be directly measured by Isc. Therefore, fluxes were measured by use of 22Na+ and 36Cl-. Figure 4 shows that C7-sorbin reduced Isc and stimulated Na+ and Cl- absorption in a dose-dependent manner. C7-sorbin also induced a dose-dependent decrease in tissue conductance together with an decrease in unidirectional fluxes from serosa to mucosa (Table 1). In jejunum, however, small but significant effects of C7-sorbin on Isc and net Cl- flux were only noticed at 10-3 M (Table 2).


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Fig. 4.   Effect of various concentrations of C7-sorbin on Isc, net Na+ flux (JNanet), and net Cl- flux (JClnet) in rat proximal and distal colons. Means and SE of 16 colonic segments from 8 rats are given. Negative results denote secretion. * Different from control at P < 0.05; ** different from control at P < 0.01; *** different from control at P < 0.001. See MATERIALS AND METHODS for details.

                              
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Table 1.   Effect of C7-sorbin on unidirectional isotopic Na+ and Cl- fluxes in colonic epithelium in fasted rat


                              
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Table 2.   Effect of C7-sorbin on short-circuit current, conductance, and transepithelial Cl- fluxes in the jejunum of fasted rats

To confirm the results obtained by Na+ and Cl- fluxes in the colon, the effect of C7-sorbin was assessed on Isc using modified Ringer solution (see MATERIALS AND METHODS). When Na+ was replaced by choline, response to 10-3 M C7-sorbin was completely abolished (Delta Isc = 0.67 ± 2.35 µA/cm2 vs. Delta Isc = -19.70 ± 4.50 µA/cm2, P < 0.05). Replacement of Cl- by isethionate and sulfate in the medium also almost completely inhibited Isc reduction (Delta Isc = -1.0 ± 0.50 µA/cm2 vs. Delta Isc = -19.70 ± 4.50 µA/cm2, P < 0.05). These results suggest that the C7-sorbin-induced decrease in Isc could be interpreted as a reduction in electrogenic Cl- secretion together with a stimulation of neutral NaCl absorption.

Effect of C7-sorbin on secretion induced by secretory agents in rat colon. Several secretory agents present in the intestinal mucosa can stimulate Cl- secretion, including 5-HT, PGE2, and VIP. Thus a series of these compounds were first added to the serosal side of stripped colonic mucosa, followed by serosal addition of 10-3 M C7-sorbin. After a peak, Isc returned to a steady value greater than the basal value; further addition of C7-sorbin elicited a decrease in Isc until a new steady state was reached. For most tested compounds, subsequent addition of C7-sorbin was followed by a significant decrease in Isc. The magnitude of the Isc decrease after C7-sorbin was inversely related to the magnitude of Isc stimulated by secretory agents. The relationship between the steady-state Isc obtained after stimulation by secretory agents and the decrease in Isc elicited by further addition of C7-sorbin is presented in Fig. 5.


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Fig. 5.   Inhibition of Isc stimulation in response to secretory agents by 10-3 M C7-sorbin in rat proximal and distal colon. Circles and vertical bars represent means and SE of 4 tissues from 4 rats. The following concentrations were used: 2 × 10-7 M vasoactive intestinal peptide (VIP), 5 × 10-7 M ANG II (ANG), 10-6 M motilin (MOT), 10-6 M substance P (SP), 10-6 M gastrin (GAS), 10-7 M neurotensin (NT), 2 × 10-7 M secretin (Sec), 10-6 M atrial natriuretic factor-(1---28) (ANF), 2 × 10-6 M PGE2, 10-4 M 5-HT, 2 × 10-7 M peptide histidine isoleucine (PHI), and 2 × 10-7 M helodermin (Helod). Magnitude of Isc decrease after 10-3 M C7-sorbin (on the ordinate axis) is plotted against magnitude of secretory agent-induced Isc (on the abscissa axis). See MATERIALS AND METHODS for details.

Comparison of different antisecretory agents in rat colon. The dose responses of four antisecretory agents on Isc were compared in rat colon; EC50 of SRIF was obtained at lower concentrations than that of other antisecretory agents such as PYY, C7-sorbin, and clonidine in the following sequence: SRIF > PYY > C7-sorbin > clonidine. When we compared the maximal effect of these antisecretory agents, the effect of C7-sorbin was similar to that obtained with SRIF, whereas the lowest maximal effect was obtained with clonidine (Fig. 6).


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Fig. 6.   Comparison of dose-response profiles of various antisecretory agents on Isc in rat colon. Squares and circles represent mean percent decrease in Isc induced by bumetanide (7 tissues from 4 rats). Clo, clonidine; PYY, peptide YY; SRIF, somatostatin (somatotropin release-inhibiting factor). See MATERIALS AND METHODS for details.

Effect of C7-Sorbin in HT-29 Cell Monolayers

The effect of C7-sorbin was assessed in vitro on human colonic cell line HT-29-Cl19A at concentrations ranging from 10-6 to 10-3 M (Fig. 7). C7-sorbin did not alter Isc at concentrations up to 10-5 M. At 10-4 M and 10-3 M, C7-sorbin induced an increase in Isc (Delta Isc = +0.29 ± 0.10 µeq · h-1 · cm-2 at 10-3 M vs. 0.0 ± 0.01 µeq · h-1 · cm-2 at 10-5 M; P < 0.05, n = 13) without modification of conductance. The increase in Isc was not suppressed by pretreatment of cells with 10-4 M amiloride.


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Fig. 7.   C7-sorbin-induced changes in Isc and conductance in HT-29-Cl19A cell monolayer (typical recording). Vertical lines represent conductance of the monolayer. Amil, 10-4 M amiloride (mucosal); Bum, 5 × 10-5 M bumetanide (serosal). See MATERIALS AND METHODS for details.

Effect of C7-Sorbin on Human Colon

Effect of C7-sorbin on different segments of human intestine. The effect of C7-sorbin was tested on different segments of human intestine (Fig. 8). C7-sorbin dose dependently reduced basal Isc in the colon (right colon and sigmoid), as shown in Table 3. Addition of bumetanide did not further modify the Isc. In the jejunum, C7-sorbin had no effect on Isc.


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Fig. 8.   Variations in Isc induced by cumulative doses of C7-sorbin in different segments of human intestine (typical recordings). RC, right colon; SM, distal sigmoid colon; MJ, medium jejunum. Bum(s), 5 × 10-5 M serosal bumetanide; Amil(m), 10-4 M mucosal amiloride. See MATERIALS AND METHODS for details.

                              
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Table 3.   Effect of cumulative doses of C7-sorbin on Isc at basal state in different segments of human intestine

Effect of C7-sorbin on human colon after stimulation of Isc by VIP. The effect of C7-sorbin was assessed in human colon after stimulation of tissue by VIP (Fig. 9). After Isc stimulation by 2 × 10-7 M VIP, C7-sorbin reduced secretion in human colon. The threshold for the C7-sorbin effect was 10-5 M, but significant reduction in Isc was observed at concentrations over 10-5 M. 


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Fig. 9.   Typical recordings of effect of C7-sorbin on Isc in human colon (sigmoid) after prestimulation by VIP. A: VIP, C7-sorbin, and bumetanide (5 × 10-5 M) were given in the serosal compartment, whereas amiloride (10-4 M) was given in the mucosal compartment. B: control (5 × 10-5 M bumetanide in serosal compartment). See MATERIALS AND METHODS for details.


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Our study clearly suggested that C7-sorbin stimulated NaCl neutral absorption and inhibited electrogenic Cl- secretion in the rat colon. In addition, the antisecretory and proabsorptive effects were essentially observed in the ileum and colon, in rat and human intestine. The magnitude of the proabsorptive effect was directly related to the magnitude of the previously induced secretion.

The proabsorptive effect is constitutive of the peptide sorbin, which was selected from extracts from porcine mucosa on the basis of a capacity to stimulate absorption of water in guinea pig gallbladder (32, 37). The COOH-terminal heptapeptide has mainly been tested for its effect on water and electrolyte absorption in vivo in intestinal loops in the rat (7, 23, 29). It was found that C7-sorbin induced absorption of water and electrolytes in duodenum and ileum. In addition, C7-sorbin decreased the VIP-stimulated secretion of water (37%), Na+ (30%), Cl- (63%), and HCO-3 (25%) (29). These effects were greater than those produced by equimolar doses of neuropeptide Y, SRIF, and metenkephalinamide in rat ileum ligated loops (29). In our study using isolated fragments of intestine mounted in Ussing chambers, we have confirmed the antisecretory and proabsorptive effects of C7-sorbin in the intestine of rats.

We also have extended the previous observations in terms of site and mechanism of action and relationship with the secretory status of the intestine. The present results clearly indicate that the C7-sorbin effect displays an aboral gradient: the decrease in Isc was hardly observed in the duodenum and jejunum, and in the ileum it was 46% lower than that observed in the colon. The present results might explain why large numbers of animals were necessary to demonstrate an effect of sorbin in the small intestine (7, 23, 29). The proabsorptive effect of sorbin in the colon is not unique, as several other agents have been found to display similar effects, including PYY (9, 20), somatostatin (5, 16, 28), ANG (7, 17), enkephalin (16, 24, 27), and norepinephrine (28). However, the present study clearly indicates that the main effect of sorbin is located in the distal part of the rat and human intestine.

The aboral gradient for the effect of sorbin on electrolyte transport may be due to several factors, including the distribution for the sorbin receptors and effectors. Very little is known about the receptors. In recent experiments, no specific binding for sorbin was found in basolateral membranes of intestinal epithelial cells (M. Laburthe and T. Voisin, personal communication). In terms of cellular effectors, the effect of sorbin on stimulating water absorption in the gallbladder pointed to the possibility of an effect on a neutral NaCl absorptive process (18, 21, 37). We thus measured on the same pieces of tissue the electrical parameters and the Na+ and Cl- isotopic transepithelial fluxes in absence of a transepithelial electrochemical gradient. Also, because the coefficients of variation are commonly much greater for the isotopic fluxes than for the electrical parameters, the determinations were made at four different concentrations of C7-sorbin, i.e., on 16 adjacent pieces of colon or jejunum in the same experiment. The results confirmed that the colon is the main target for sorbin, whereas isotopic fluxes were not significantly modified in the jejunum. In the colon, a dose-dependent stimulation of the neutral NaCl absorption together with a smaller reduction in electrogenic Cl- secretion was observed. The results obtained after substitution of Na+ or Cl- indicate that Na+ and Cl- are required for the effect of C7-sorbin. In addition, comparison of the ionic unidirectional fluxes with the transepithelial conductance further strengthened the possibility that sorbin stimulates electrolyte absorption (transepithelial conductance expressed in mS/cm2 is similar to the sum of the diffusional ionic fluxes measured in the absence of an electrochemical gradient and expressed in µeq · h-1 · cm-2) (13). Thus the change in conductance could be compared with the change in the unidirectional ionic fluxes. In the present experiments, conductance decreased with increasing sorbin concentrations; concurrently, the unidirectional ionic fluxes from serosa to mucosa decreased, suggesting a reduction in transepithelial ionic diffusion, whereas the flux in mucosa to serosa was not significantly altered. This may indicate a decrease in the diffusional flux compensated for by an increase in the nondiffusional flux from mucosa to serosa compatible with a stimulation of a neutral NaCl absorptive flux. Although it was beyond the scope of these experiments to identify the molecular structures supporting the NaCl absorption and the Cl- secretion, the literature suggests that Na+/H+ exchanger (NHE3) (38) and cystic fibrosis transmembrane conductance regulator (2, 22) transporters are good candidates for membrane effectors that support the proabsorptive and antisecretory effects of sorbin.

Interestingly, we observed a similar aboral distribution in the human intestine. This may indicate a similar distribution for the ionic transporters involved in the effect of sorbin in the rat and human intestine. Thus sorbin extracted from porcine intestine is also active on rat and human intestine. However, these results do not indicate the presence of sorbin or its receptors in the human intestine. When we measured the effect of C7-sorbin on HT-29-Cl19A, an intestinal epithelial cell line derived from a human colonic cancer patient, it came as a surprise that the Isc did not decrease but increased at a relatively high C7-sorbin concentration (10-4 M). This may indicate that sorbin does not act directly on the intestinal epithelial cells, but, like the many mediators located in the lamina propria, sorbin may interact with different cell types that control electrolyte transport by the epithelial cells (3, 8, 10, 12). However, neither naloxone nor TTX altered the C7-sorbin effect.

One of the most intriguing results is the effect of sorbin after secretion was stimulated: the intensity of the effect of sorbin appeared to be directly related to the intensity of the secretion previously induced by a variety of agents (Fig. 5). The physiological consequence of a regulatory link between absorption and secretion is obvious when considering the movement of water and electrolytes in the intestine after a meal (14, 34). During a meal, a large quantity of water enters the intestinal lumen as a result of the volume of the meal and the meal-induced secretion by the salivary glands, stomach, pancreas, biliary system, and intestine. Several hormones participate in the meal-induced secretion (30). Water and electrolytes thus secreted in the intestinal lumen are reabsorbed by the intestine, and the stimulation of intestinal absorption must be quantitatively adjusted to the amount secreted; the mechanism of meal-stimulated absorption involves the nutrients of the meal that stimulate electrolyte absorption by different mechanisms (4, 11, 14, 15, 25). In addition, several hormones display antisecretory and proabsorptive effects (5, 7, 9, 16, 17, 20, 24, 28). PYY acting in the upper part of the intestine is secreted in the blood from colonic cells at the beginning of the meal (19). However, it does not seem to display antisecretory effects until 1 h after the beginning of the meal (19). What is striking about sorbin is the link between the intensity of secretion and the intensity of the sorbin response. This suggests that if sorbin is a natural constituent of the human intestine it may play an important role in the economy of water and electrolyte handling. In addition, in diarrheal diseases, the symptoms may be reduced by stimulating colonic absorption in response to secretion. This has already been suggested for human cholera (36) and viral infection in piglets (1). In addition, sorbin has been found to display a beneficial effect in cholera toxin-induced diarrhea in the rat (J. Fioramonti and L. Bueno, personal communication).

At the cellular level, stimulated Cl- secretion has been found to be associated with a stimulated glucose and Na+ absorption (31). In cAMP-induced Cl- secretion, it has been suggested that the number of Na+-glucose transporter (SGLT1) molecules responsible for the glucose-Na+ cotransport is increased in the luminal membrane (26). In addition, it has been reported that methylated casein, an antidiarrheal drug, exhibits antisecretory activity only in cholera-treated rabbit ileum and has no effect in control conditions (33). However, we have no direct indication that a similar mechanism exists to explain the sorbin effect.

Finally, for the following reasons, the present results suggest that C7-sorbin is a good candidate for use in the reduction of water and electrolyte losses in diarrheal diseases: 1) it is mainly active in the distal part of the intestine; 2) its proabsorptive effect is apparent after intestinal secretion has been stimulated; 3) the intensity of its proabsorptive effect is grossly proportional to the intensity of previously induced secretion by a variety of mediators; 4) the size of the heptapeptide makes it feasible for synthesis in industrial environments; 5) there is no evidence of tachyphilaxie; the dose-response curves were similar when cumulative doses were given to the same tissue and when one dose was given to one tissue; and 6) the results of the dose-effect relationship may point to a limitation; apparently, in this in vitro system, the EC50 is one order of magnitude higher than other peptides, such as PYY and SRIF, that have antidiarrheal properties. However, the maximum effect appeared to be essentially the same as that of the most active peptides.


    ACKNOWLEDGEMENTS

This study was partly supported by the Institut Henri Beaufour, Avenue du Canada, Les Ulis, France.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: M. Boisset, Laboratoire de Biologie, Conservatoire National des Arts et Métiers, 2, rue Conté, 75141 Paris 03, France.

Received 25 February 1998; accepted in final form 14 September 1998.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

1.   Argenzio, R. A., H. W. Moon, L. J. Kemeny, and S. C. Whipp. Colonic compensation in transmissible gastroenteritis of swine. Gastroenterology 86: 1501-1509, 1984[Medline].

2.   Bajnath, R. B., K. Dekker, H. R. De Jonge, and J. A. Groot. Chloride secretion induced by phorbol dibutyrate and forskolin in the human colonic carcinoma cell line HT-29Cl.19A is regulated by different mechanisms. Pflügers Arch. 430: 705-712, 1995[Medline].

3.   Beubler, E., A. Schirgi-Degen, and R. Gamse. Inhibition of 5-hydroxytryptamine- and enterotoxin-induced fluid secretion by 5-HT receptor antagonists in the rat jejunum. Eur. J. Pharmacol. 248: 157-162, 1993[Medline].

4.   Binder, H. J., and P. Mehta. Short-chain fatty acids stimulate active sodium and chloride absorption in vitro in the rat distal colon. Gastroenterology 96: 989-996, 1989[Medline].

5.   Botella, A., F. Vabre, J. Fioramonti, F. Thomas, and L. Bueno. In vivo inhibitory effect of lanreotide (BIM 23014), a new somatostatin analog, on prostaglandin- and cholera toxin-stimulated intestinal fluid in the rat. Peptides 14: 297-301, 1993[Medline].

6.   Chang, E. B., and M. C. Rao. Intestinal water and electrolyte transport. Mechanisms of physiological and adaptative responses. In: Physiology of the Gastrointestinal Tract, edited by L. R. Johnson. New York: Raven, 1994, p. 2027-2081.

7.   Charpin, G., A. R. Chikh-Issa, H. Guignard, G. Jourdan, C. Dumas, D. Pansu, and M. Descroix-Vagne. Effect of sorbin on duodenal absorption of water and electrolytes in the rat. Gastroenterology 103: 1568-1573, 1992[Medline].

8.   Cooke, H. J. Neuro-modulation of ion secretion by inflammatory mediators. Ann. NY Acad. Sci. 664: 346-352, 1992[Medline].

9.   Cox, H. M., A. W. Cuthbert, R. Hakanson, and C. Wahlestedt. The effect of neuropeptide Y and peptide YY on electrogenic ion transport in rat intestinal epithelia. J. Physiol. (Lond.) 398: 65-80, 1988[Abstract].

10.   Crowe, S. E., and D. W. Powell. Fluid and electrolyte transport during enteric infections. In: Infections of the Gastrointestinal Tract, edited by M. J. Blaser, P. D. Smith, J. I. Ravdin, H. B. Greenberg, and R. L. Guerrant. New York: Raven, 1995, p. 107-141.

11.   Desjeux, J.-F., A. Briend, and J. D. Butzner. Oral rehydration solution in the year 2000: pathophysiology, efficacy and effectiveness. Baillère's Clin. Gastroenterol. 11: 509-527, 1997[Medline].

12.   Desjeux, J.-F., and M. Heyman. The acute infectious diarrhoeas as diseases of the intestinal mucosa. J. Diarrhoeal Dis. Res. 15: 224-231, 1997[Medline].

13.   Desjeux, J.-F., Y. H. Tai, and P. F. Curran. Characteristics of sodium flux from serosa to mucosa in rabbit ileum. J. Gen. Physiol. 64: 274-292, 1974[Abstract/Free Full Text].

14.   Desjeux, J.-F., C. Tannenbaum, Y. H. Tai, and P. F. Curran. Effects of sugars and amino acids on sodium movement across small intestine. Am. J. Dis. Child. 131: 331-340, 1977.

15.   Desjeux, J.-F., E. Turk, and E. M. Wright. Congenital selective Na+ D-glucose cotransport defects leading to renal glycosuria and congenital selective intestinal malabsorption of glucose and galactose. In: The Metabolic and Molecular Bases of Inherited Disease, edited by C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle. New York: McGraw Hill, 1995, p. 3563-3580.

16.   Dobbins, J. W., K. Dharmsathaphorn, L. Racusen, and H. J. Binder. The effect of somatostatin and enkephalin on ion transport in the intestine. Ann. NY Acad. Sci. 372: 594-612, 1981[Abstract].

17.   Dolman, D., and C. J. Edmonds. The effect of aldosterone and the renin-angiotensin system on sodium, potassium and chloride transport by proximal and distal rat colon in vivo. J. Physiol. (Lond.) 250: 597-611, 1975[Abstract].

18.   Donowitz, M., Y. H. Tai, and N. Asarkof. Effect of serotonin on active electrolyte transport in rabbit ileum, gallbladder, and colon. Am. J. Physiol. 239 (Gastrointest. Liver Physiol. 2): G463-G472, 1980[Abstract/Free Full Text].

19.   Eto, B., M. Boisset, M. Anini, T. Voisin, and J.-F. Desjeux. Comparison of the antisecretory effect of the endogenous forms of peptide YY on fed and fasted rat jejunum. Peptides 18: 1249-1255, 1997[Medline].

20.   Eto, B., M. Boisset, P. Eden, A. Balasubramaniam, and J.-F. Desjeux. Effects of peptide YY and its analogues on chloride ion secretion in fed and fasted rat jejunum. Peptides 16: 1403-1409, 1995[Medline].

21.   Frizzell, R. A., and K. Heintze. Transport functions of the gallbladder. Int. Rev. Physiol. 21: 221-247, 1980[Medline].

22.   Frizzell, R. A., G. Rechkemmer, and R. L. Shoemaker. Altered regulation of airway epithelial cell chloride channels in cystic fibrosis. Science 233: 558-560, 1986[Medline].

23.   Grishina, O., G. Charpin, F. Marquet, D. Pansu, and M. Descroix-Vagne. Effect of C-terminal derivatives of sorbin on duodenal ion transports in rats. Gastroenterol. Clin. Biol. 19: 487-493, 1995[Medline].

24.   Hautefeuille, M., V. Brantl, A. M. Dumontier, and J.-F. Desjeux. In vitro effects of beta -casomorphins on ion transport in rabbit ileum. Am. J. Physiol. 250 (Gastrointest. Liver Physiol. 13): G92-G97, 1986[Medline].

25.  Hediger, M. A., Y. Kanai, G. You, and S. Nussberger. Mammalian ion-coupled solute transporters. J. Physiol. (Lond.) 482, Suppl.: 7S-17S, 1995.

26.   Hirsch, J. R., D. D. F. Loo, and E. M. Wright. Regulation of Na+/glucose cotransporter expression by protein kinases in Xenopus laevis oocytes. J. Biol. Chem. 271: 14740-14746, 1996[Abstract/Free Full Text].

27.   Kachur, J. F., R. J. Miller, and M. Field. Control of guinea pig intestinal electrolyte secretion by a delta-opiate receptor. Proc. Natl. Acad. Sci. USA 77: 2753-2756, 1980[Abstract].

28.   Keast, J. R., J. B. Furness, and M. Costa. Effects of noradrenaline and somatostatin on basal and stimulated mucosal ion transport in the guinea-pig small intestine. Naunyn Schmiedebergs Arch. Pharmacol. 333: 393-399, 1986[Medline].

29.   Marquet, F., O. Grishina, D. Pansu, and M. Descroix-Vagne. Effect of C-terminal derivatives of sorbin on ileal ion transport stimulated by VIP in rats. Gastroenterol. Clin. Biol. 18: 702-707, 1994[Medline].

30.   Miazza, B., R. Palma, J. R. Lachance, J. A. Chayvialle, P. P. Jonard, and R. Modigliani. Jejunal secretory effect of intraduodenal food in humans. A comparison of mixed nutrients, proteins, lipids, and carbohydrates. Gastroenterology 88: 1215-1222, 1985[Medline].

31.   Nath, S. K., M. Rautureau, M. Heyman, H. Reggio, A. L'helgoualc'h, and J.-F. Desjeux. Emergence of Na+-glucose cotransport in an epithelial secretory cell line sensitive to cholera toxin. Am. J. Physiol. 256 (Gastrointest. Liver Physiol. 19): G335-G341, 1989[Abstract/Free Full Text].

32.   Pansu, D., M. Vagne, A. Bosshard, and V. Mutt. Sorbin, a peptide contained in porcine upper small intestine which induces the absorption of water and sodium in the rat duodenum. Scand. J. Gastroenterol. 16: 193-199, 1981[Medline].

33.   Peyrot, M., J.-F. Desjeux, A. B. Mansour, A. M. Dumontier, M. Hautefeuille, and D. Tome. Anticholeraic effect of methylated casein in rat jejunum. Pediatr. Res. 18: 1075-1079, 1984[Abstract].

34.   Phillips, S. F. Diarrhea: a current view of the pathophysiology. Gastroenterology 63: 495-518, 1972[Medline].

35.   Powell, D. W., H. J. Binder, and P. F. Curran. Electrolyte secretion by the guinea pig ileum in vitro. Am. J. Physiol. 223: 531-537, 1972[Medline].

36.   Speelman, P., T. Butler, I. Kabir, A. Ali, and J. Banwell. Colonic dysfunction during cholera infection. Gastroenterology 91: 1164-1170, 1986[Medline].

37.   Vagne-Descroix, M., D. Pansu, H. Jornvall, M. Carlquist, H. Guignard, G. Jourdan, A. Desvigne, M. Collinet, C. Caillet, and V. Mutt. Isolation and characterisation of porcine sorbin. Eur. J. Biochem. 201: 53-59, 1991[Abstract].

38.   Yun, C. H., C. M. Tse, S. K. Nath, S. A. Levine, S. R. Brant, and M. Donowitz. Mammalian Na+/H+ exchanger gene family: structure and function studies. Am. J. Physiol. 269 (Gastrointest. Liver Physiol. 32): G1-G11, 1995[Abstract/Free Full Text].


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