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

Peptides, Vol. 19, No. 8, pp. 1417–1423, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/98 $19.00 1 .00

PII S0196-5485(98)00082-5

Effect of Sorbin Derivatives on Cholera Toxininduced Intestinal Secretion in Rat In Vivo F. MARQUET,* A. BOTELLA,† L. BUENO,† D. PANSU* AND M. DESCROIX–VAGNE*1 *INSERM,Unite´ 45, Hoˆpital E Herriot, 69437 Lyon cedex 3, France †INRA, Laboratoire de Pharmacologie-Toxicologie, 180 chemin de Tournefeuille, 31300 Toulouse, France Received 13 January 1998; Accepted 28 April 1998 MARQUET, F., A. BOTELLA, L. BUENO, D. PANSU AND M. DESCROIX–VAGNE. Effect of sorbin derivatives on cholera toxin-induced intestinal secretion in rat in vivo. PEPTIDES 19(8) 1417–1423, 1998.—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, Na1 and K1, in the jejunum and at the same time the net flux of water and Cl2 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. © 1998 Elsevier Science Inc. Sorbin Intestine Jejunum Duodenum Secretion Bicarbonate Cholera toxin 5-HT PGE2.

Inhibition

Sodium

Chloride

METHOD

SORBIN is a new peptide isolated from pig proximal intestine (22). It has been localized in enterochromaffin endocrine cells of the digestive tract from the antrum to the ileum in pig, to the colon in man and in pancreatic insulinsecreting b cells (2). The presence of sorbin in the enteric nervous system allows one to classify it as a neuropeptide and its presence in some carcinoid tumors gives it a place in pathology, which deserves additional studies (1). The main activity of sorbin which led to its characterization and its nomenclature by Grossman (22) was the increase of intestinal absorption in basal conditions (6) and the decrease of stimulated intestinal secretion (10,13). The present study was undertaken to determine the effect of C-terminal-sorbin derivatives (Table 1) on the intestinal hypersecretion induced by intraluminal administration of cholera toxin locally instilled or distally placed in the intestine using two complementary techniques, the continuously perfused jejunum and the jejunal ligated loop in anesthetized rats in vivo.

Animals Approximately 72 male Wistar rats weighing 250 to 300 g (from Janvier S.A., Le Genest St Isle, France) were fasted 24 h before the experiments with water ad libitum and were used in groups of six for infusion studies; the first periods, which were discarded, were used to eliminate the residual intestinal contents. The rats were anesthetized for several hours with urethane [3 g/kg per intraperitoneal (IP) injection]. About 170 male Sprague–Dawley rats (from Iffa-Credo, St Germain sur l’Arbresle, France), were fasted 48 h with water ad libitum and used for ligated loop studies to avoid a disturbing intestinal washing. The rats were anesthetized for a short period of time with an IP injection of Nembutal® (sodium pentobarbital, 55 mg/kg). The experimental procedures were in agreement with the laws of the European Union (user numbers 1895, 187 & 191).

1 Requests for reprints should be addressed to M. Descroix–Vagne, INSERM Unite´ 45, Pavillon H Bis, Hoˆpital Edouard Herriot, 69437 Lyon Cedex 03, France. E-mail: [email protected]

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MARQUET ET AL. TABLE 1 AMINO-ACID COMPOSITION OF SORBIN DERIVATIVES C20-sorbin Tyr.Glu.Pro.Gly.Lys.Ser.Ser.Ile.Leu.Gln.His.Glu.Arg.Pro.Val.Thr.Lys.Pro.Gln.Ala-NH2 C10-sorbin His.Glu.Arg.Pro.Val.Thr.Lys.Pro.Gln.Ala-NH2 D7-sorbin Pro.Val.Thr.Lys.Pro.Gln.DAla-NH2

Jejunal Infusion Technique

Duodenal and Jejunal Ligated Loop Technique

A xypho-umbilical laparotomy was performed on each rat to expose the jejunal portion of the small intestine. A 15-cm segment of the proximal jejunum was isolated, its proximal part was cannulated and rinsed with Tyrode’s solution maintained at 37°C, to remove all the luminal contents. The segment was ligated and cannulated distally for intraluminal infusion and was then replaced in the abdominal cavity. The incision was sutured and rats were placed on a hot pad for the duration of experiments in order to stabilize body temperature at 37°C (5). Net water flux was measured by adding to the perfusion solution 14C-labeled polyethylene glycol (PEG 4000, NEN France, as non-absorbed marker) at a concentration of 185 kBq/ml. The catheter attached to the proximal end of the isolated intestinal segment was connected to a perfusion pump and Tyrode’s solution was perfused at a rate of 12 or 6 ml/h for 2 h, the time required to reach the equilibrium of water absorption rate. Cholera toxin (Sigma Chemical Company, St. Louis, MO) was added to the Tyrode’s solution and perfused at a rate of 16 mg/kg for 1 h. At the end of this period, a subcutaneous or intraduodenal injection of water (controls) or of the different peptides was performed under intraloop perfusion of Tyrode’s solution without cholera toxin for 5 h. The catheter attached to the distal end of the jejunum was connected to a fraction collector which collected 15-min fractions of the perfusate. Radioactivity in the perfusate was determined by liquid scintillimetry and net water flux for each 15-min interval was calculated using the following formula, derived from that proposed by Sladen and Dawson (20):

The protocol of ligated loops in situ which has been previously described (6), is explained in detail in the present study with the modification used for the administration of cholera toxin. After the median laparotomy, jejunum was first luminally injected with a syringe and a needle, with 1 ml of water with or without 16 mg/ml of cholera toxin at about 4 cm below the Treitz ligament, with a finger pinch to avoid a reflux. Then, the jejunum was gently replaced in the abdomen which was sutured. One hour after cholera toxin administration, the abdominal wall was reopened, two ligated loops, duodenal and jejunal, were realized in each rat. For the duodenum, the choledoco-pancreatic duct was ligated and the loop was delimited between two ligatures, the first one at the level of the pylorus, and the second at the level of the Treitz ligament, making the duodenal loop about 8 cm long (about 1 g wet weight). The proximal jejunal loop was delimited between 2 ligatures about 4 cm below the Treitz ligament and was 10 cm long (about 1 g wet weight). After injection with a syringe and a needle in each segment of one ml of test solution, a third ligature on each loop was tightened to close the puncture. Then the abdominal wall was sutured. The animals were kept at 25°C for one hour. The rats were perfused intravenously (IV) with 9 g/l NaCl saline solution with or without the C20 peptide at a rate of 3 ml/h, for 2 h, immediately after cholera toxin injection and during one hour after the loop constitution. The test solution, which was selected to minimize basal water absorption, was characterized by a low saline concentration (Na1 80 mM, K1 5.2 mM, Ca21 1.2 mM, Cl2 77.6 mM, HCO2 3 10 mM, pH 8.2). It was made iso-osmotic with 136 mM of mannitol and contained 5 g/l of PEG 4000 and 1.01 kBq/ml of 3H-PEG 4000 (NEN, France) used as nonabsorbed marker. One hour after the loop injection, the rats were sacrificed. The loop content was collected, weighed, centrifuged and tested. Blood was collected for plasma prostaglandin E2 (PGE2) and 5-hydroxytryptamine (5-HT) determination. The percentage of 3H-PEG 4000 recovery was determined by liquid scintillimetry (Packard 1600CA Tricarb). When it was lower than 70%, the sample was discarded. Bicarbonate measurement was made by acido-alcalimetric method as described by Preshaw and Grossman (18). Chloride content

net flux in ml/cm-h 5 @1 2 ~DPMs/DPMe!# 3 ~P/L! where DPMs is activity of 14C in the standard (2 first hour periods), DPMe is activity of the samples (next periods), P is perfusion rate (12 ml/h for C20 and C10, 6 ml/h for D7), and L is the length of the perfused jejunal segment in centimeters (cm). Positive values indicate net water absorption and negative values net water secretion.

SORBIN ON CHOLERA TOXIN SECRETION

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was monitored by potentiometric determination (8). Sodium and potassium were determined by flame photometry. PGE2 was determined in both plasma and fluids from duodenum and jejunum using the radioimmunoassay described and commercialized as Biotrak™ (Amersham, Les Ulis, France). The sensitivity of the PGE2 RIA was 10 pg/ml and the interassay variation was 10%. 5-HT was measured in both plasma and fluids from duodenum and jejunum with a reverse phase liquid chromatography and amperometric detection using Clinrep® from Merck (Precision Instruments, Marseille, France).The intra-assay and the inter-assay variations were respectively 3% and 4%. The linear range for 5-HT was 1 to 1000 ng/ml. Sorbin Derivatives All peptides were synthesized (Neosystem, France) according to the entire sorbin sequence (22) with the C-terminal part (Table 1), as eicosapeptide-amide (C20, MW 2265), decapeptide-amide (C10, MW 1161), or heptapeptideDala7-amide (D7, MW 739, BN 52080, Institut Henri Beaufour; Reference 11). For subcutaneous injections, the doses of C20 were 0.2, 2, and 20 mg/kg (0.088, 0.88, and 8.8 nmol/kg, respectively), for C10 were 0.1, 1, and 10 mg/kg (0.086, 0.86, and 8.6 nmol/kg, respectively), and for D7 were 0.1, 1, and 10 mg/kg (0.135, 1.35, and 13.5 nmol/kg, respectively). For intra-duodenal administration, C20 was given at the dose 200 mg/kg (88.4 nmol) and C10 and D7 at the dose of 100 mg/kg (respectively 86.1 and 135.3 nmol). For IV continuous perfusion, C20 was given at a dose of 20 mg/kg-h (8.84 nmol). Histological Control At the end of the experiment, a histologic control was performed to verify that the dose of cholera toxin was not damaging the intestinal mucosa. Statistical Analysis The results are expressed as means 6 SEM. The treated groups were compared to controls using analysis of variance and Student’s t-test with the common variance, only when F was statistically significant. To test the validity of repeated t-test, the means were also verified with Q student’s test 5% range (21). RESULTS Infused Jejunal Loop During jejunal infusion with Tyrode’s solution, cholera toxin induced a sharp decrease of jejunal absorption which was already different from control values one hour after cholera perfusion was stopped (p , 0.05). A complete reversal from absorption to secretion (negative values) was observed in 2 to 4 h after cholera toxin perfusion (Fig. 1). The hypersecretion was accompanied by a massive loss of mucus.

FIG. 1. Dose-response to SC injection of C20, C10, and D7 (C-terminal sequence of sorbin) on jejunal water hypersecretion induced by cholera toxin in anesthetized rats. Each point represents the mean 6 SEM for six rats. Each individual hourly value was obtained from four consecutive 15-min values from 21 to 6 h after starting of jejunal cholera toxin infusion (CT) (16 mg/kg-h, during 1 h). C, control, cholera toxin treated alone; numbers on curves represent the dose in mg/kg. The perfusion rate was 12 ml/h for C20 and C10 and only 6 ml/h for D7, explaining the differences in control values. Positive values represent absorption, negative values represent secretion. Statistical significance was calculated by analysis of variance followed by Student’s t-test with comparisons to the cholera toxin group alone (C): *p , 0.05; **p , 0.01.

C-terminal sorbin peptides given by SC. route significantly increased absorption or decreased secretion according to the doses (Fig. 1). The smallest active fragment of

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MARQUET ET AL.

Ligated Jejunal Loop Using the ligated loop technique with a hypo-ionic solution, jejunum was already secreting in basal condition (Fig. 3). Cholera toxin placed in the jejunum for 1 h significantly increased, in the next hour, the secretion of water, Na1, Cl2 and bicarbonate (Fig. 3). Administered IV, C20 decreased significantly water (14.6%) and Na1 (14.9%) net fluxes (Fig. 3), whereas Cl2 and bicarbonate were not significantly affected. K1 net flux, which was increased by cholera toxin (from 0.54 6 0.12 to 3.41 6 0.28 mEq/h), was also significantly decreased (p , 0.05) by C20 sorbin (2.55 6 0.23 mEq/h).

FIG. 2. Effects of intra-duodenal injection of C20, C10, and D7 (200, 100, and 100 mg/kg, respectively) on jejunal water hypersecretion induced by cholera toxin in anesthetized rats. The perfusion rate was 12 ml/h for all sorbin derivatives. Each point represents the mean 6 SEM for six rats. Each individual hourly value was obtained from four consecutive 15-min values from 21 to 6 h after starting of jejunal cholera toxin infusion (CT) (16 mg/kg-h, during 1 h). C, control, cholera toxin treated alone. Positive values represent absorption, negative values represent secretion. Statistical significance was calculated by analysis of variance followed by Student’s t test with comparisons to the cholera toxin group alone (C): *p , 0.05; **p , 0.01.

sorbin was the modified heptapeptide-amide (which statistically increased basal water absorption from 45 6 14 ml/ h-cm to 64 6 13 ml/h-cm (p , 0.05), 42% increase, at the dose of 1 mg/kg (1.35 nmol; data not shown). Five hours after the SC injection, the inhibitory effect was still significant for all sorbin peptides. The inhibition induced by C20 was 21, 40, and 84% of cholera toxin stimulation, respectively for the 3 doses. C10 administered in the same range of nmol doses gave an inhibition of only 1, 22, and 31%. The inhibition produced by D7, at doses 1.5 higher, was 11, 29 and 62%. The 50% inhibitory dose was 3.5 for C20, 9.8 for D7 and only 15.7 nmol/kg for C10. The loss of mucus in sorbin derivative treated rats was obviously less than in control animals, even if the quantification of mucus secretion was not performed. C-terminal sorbin peptides given by intraduodenal injection decreased the cholera toxin-induced hypersecretion (Fig. 2), but the inhibitory effect required a longer period of time to attain a statistically significant difference from controls. The inhibition was 90% and 62% for equimolar doses of C20 and C10, while that induced by D7 at a dose 1.5 higher, was 56%. The mucus loss was also less abundant.

Duodenal Ligated Loop Duodenal loops also secreted in basal condition with the hypo-ionic test solution. Intrajejunal administration of cholera toxin significantly increased water, Na1, Cl2 and 1 HCO2 fluxes 3 net fluxes (Fig. 3) in the duodenum. K increased significantly (p , 0.001) from 1.15 6 0.18 to 5.16 6 0.44 mEq/h. Administered IV, C20 decreased significantly the secretion of water (11.3%) and Cl2 (12.6%). The decrease of Na1 and bicarbonate fluxes did not reach the statistical significance. PGE2 Release Cholera toxin increased significantly PGE2 release in jejunal and duodenal fluids (p , 0.05) (Table 2). This increase was 0.71 ng/h in the jejunum and only 0.43 ng/h in the duodenum. In basal condition, PGE2 release was already higher in the jejunal than in the duodenal loop contents. The PGE2 release in both intestinal segments was only slightly decreased by C20 but did not differ anymore statistically from control values. In plasma, PGE2 concentrations after cholera toxin stimulation did not differ significantly from controls. 5-HT Release No change in 5-HT concentration in plasma nor in duodenal and jejunal secretions was observed in our experimental conditions (Table 2). In order to control our technique of 5-HT determination, 20 rats received by IP injection, either saline or 100 mg/kg of 5-hydroxy-L-tryptophan (12) 30 min after administration, plasma 5-HT raised from 23.3 6 2.6 ng/ml to 105.9 6 19.5 ng/ml (t 5 4.2, p , 0.001) DISCUSSION Sorbin is a large peptide isolated from the upper part of the intestine (22) but its physiological role remains largely unknown. Recent studies showing its localization in the endocrine cells of the digestive tract, intestine and pancreas, as well as in the enteric nervous system (2,1) raises a new interest on this peptide which might be a neuropeptide

SORBIN ON CHOLERA TOXIN SECRETION

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FIG. 3. Effect of IV infusion of C20-sorbin (20 mg.kg21 z h21) on jejunal (top) and duodenal (bottom) water and electrolyte hypersecretion induced by intra-jejunal injection of cholera toxin (16 mg per rat). Values are means 6 SEM: positive values represent absorption, negative values represent secretion. Statistical significance calculated by analysis of variance followed by Student’s t-test is expressed as: ***p , 0.001 for comparison between control group (n 5 55) and cholera toxin group (n 5 58), as **p , 0.05 for comparison between cholera toxin group and C20 group (n 5 55).

regulating gastrointestinal functions since it increases intestinal absorption in basal conditions (6) and has an inhibitory effect on stimulated water and electrolyte secretion mainly in duodenum and ileum (10,13). More knowledge has to be gained for this new molecule which might be involved in pathological states, since some carcinoid tumors contain sorbin in association with 5-HT (1,2). In this study, we showed that synthetic derivatives of sorbin are able to inhibit jejunal secretion induced by cholera toxin, in a dose dependent manner. Their effects were observed using two different in vivo techniques: open infused and ligated loops, reflecting physiological (normal transit) and pathological

(fluid stasis) situations respectively. The smallest active fragment of sorbin is a heptapeptide modified to increase its plasma stability (9,14,15). The inhibitory effect on cholera toxin-induced secretion was obtained after SC or IV injection, as well as after intraduodenal administration, showing a good resistance of the peptide to intestinal proteolysis, since the oral active dose was only ten times higher than that required for systemic route. The inhibitory effect was shown in jejunum, which was directly stimulated by intraluminal cholera toxin, as well as in the duodenum indirectly stimulated. These data are in favor of an indirect effect of sorbin acting on a mediator released by cholera toxin. In order to

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MARQUET ET AL. TABLE 2 5-HT AND PGE2 RELEASE IN PLASMA AND IN FLUIDS FROM DUODENAL AND JEJUNAL LIGATED LOOPS IN SITU 5-HT Release Plasma

Controls CT CT 1 C20

PGE2 Release

Duodenum

Jejunum

Plasma

Duodenum

Jejunum

ng/ml

n

ng/h

n

ng/h

n

ng/ml

n

nh/h

n

ng/h

n

272.14 6 56.92 188.95 6 32.17 172.97 6 32.76

23 24 32

17.40 6 2.04 18.54 6 1.58 18.19 6 1.09

23 24 32

41.35 6 5.04 33.80 6 3.48 39.22 6 3.52

23 24 32

1.19 6 0.22 1.04 6 0.14 0.92 6 0.13

17 16 20

1.42 6 0.11 1.85 6 0.11 1.71 6 0.14

50 54 48

2.12 6 0.19 2.84 6 0.27 2.59 6 0.19

50 54 49

CT: Cholera Toxin; PGE2: Prostaglandin E2. The data are the means 6 SEM obtained in controls (n 5 number of rats), after intrajejunal administration of 16 mg/kg of cholera toxin with or wtihout C20 (C-terminal sequence of sorbin) intravenously administered at the dose of 20 mg/kg-h (8.84 nmol/kg-h). The data were compared by analysis of variance and Student t-test (*p , 0.05 between control and traited groups).

evaluate the impact of sorbin on two of these transmitters (17,4) elicited by cholera toxin, we have measured luminal and blood levels of 5-HT (16) and PGE2 (11). We chose to estimate them in the first phase of the stimulation, one hour after the end of the intraluminal administration of the toxin, in the jejunum. To sensitize the secretory response, we infused in ligated loop an hypo-ionic test solution which allows an equilibrium between absorption and secretion in basal conditions (7). The increased release of PGE2 in fluids obtained from jejunum and duodenum is in agreement with a previous report showing such an increase with a similar dose during the first hour of cholera toxin stimulation (4). The absence of stimulation of 5-HT release that we observed fits also with the data of Beubler (4) that did not show a time course for 5-HT as for PGE2 but only a stimulation after a 4-h period. Moreover, the dose required to induce a significant release of 5-HT into the luminal fluid in rabbit is 20 times higher than that inducing water secretion (17). So we can speculate that 5-HT, which is as expected released in plasma after 5-hydroxy-L-tryptophan administration, is not directly

involved in the first period of cholera toxin stimulation of water and electrolyte secretion. The early stimulation of PGE2 release simultaneously with the increase of jejunal and duodenal secretion is in part responsible for the response of the duodenum when the cholera toxin is administered in the jejunum. The small reduction of cholera toxin-induced PGE2 release, elicited by C20-sorbin, suggests that PGE2 might be a target for sorbin effect, a hypothesis which deserves complementary studies, using specific inhibitor of PGE2 synthesis as indomethacin. PGE2 is known as a potent stimulant of Cl2 secretion from intestinal and colonic crypts (23,19) and has a second effect of strongly inhibiting the electroneutral absorption on the villous epithelial cell (3). Furthermore, a relationship between endogenous sorbin and 5-HT is suggested by their co-localization in enterochromaffin cells (2). ACKNOWLEDGEMENTS The authors acknowledge the technical assistance of G. Jourdan, L. Escoffier and F. Abou El Fadil Nicol and the English translation of John Carew.

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