Original Papers Digestion 13: 325-333 (1975)
Effect o f Secretin on Intestinal Monosaccharide Absorption and Net Water Movement in the Rat R. Böhmer and K. Rommel Department of Clinical Chemistry, University of Ulm, Ulm
Key Words. Galactose • Glucose • Intestinal absorption • Intestinal mucosa • Phenol phthaleins ■Rats • Secretin Abstract. Repeated intravenous injections of 2.0 clinical units (CU) secretin per kg body weight showed no effect on jejunal glucose absorption and net water movement in perfusion studies in vivo. The in vitro uptake of galactose and 3-O-mcthylglucose was not altered by a secretin load given 12 min before sacrifice. The values for Km and Vmax were identical after secretin and in a control group. The conflicting results of different authors concerning the effect of secretin on net water movement and solute absorption may be due to differences in experimental techniques, different, mostly pharmacological doses of the hormone, and a diverse response of heterogeneous species.
The effects of secretin on pancreatic secretion are well known (9, 11), but there are conflicting results about the influence of secretin on intestinal net water movement and solute absorption (1—3, 8, 14-16, 18, 19). The present investigation has been performed in order to study the influ ence of repeated intravenous injections of secretin on the intestinal net water movement and glucose absorption in the rat small intestine in vivo and on the galactose and 3-O-methylglucose uptake in vitro. The last mentioned monosac charides are either poorly or not metabolized in the intestinal epithelium cell and are absorbed by the same active transport system as glucose.
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Received: July 12. 1975; accepted: August 27. 1975.
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Materials and Methods Animals Female specific pathogen-frcc (SPF) Wislar rats, strain F'W 49 (Thomae, Biberach. FRG), weight 173.5 201.5 g (mean t SD = 187.0 t 6.5), nutrition with Altromin standard diet, were randomized. The intestinal flora consisted of Lactobacillus acidophilus (ATCC 11506); Lactobacillus bifidus (isolated from human baby feces); Streptococcus lactis: Streptococcus fecalis (ATCC 10541); Bacteroides symbiosus (= fusobacterium symbiosum) (ATCC 14940), and Escherichia coli (isolated from rats). The experiments were performed after 12 15 h of fasting with water ad libitum. Chemicals D(+)-glucose and phenol red have been obtained from Fluka AG, Buchs, Switzerland; 3-O-methylglucose from Calbiochem AG, Luzern, Switzerland; D-galactose from Serva, Heidelberg, FRG; toluol p.A. and 2,5-diphenyloxazol (PPO) from Merck, Darmstadt, FRG; soluene-350 from Packard Instrument, Zürich, Switzerland; D-galactose-MC and 3-0methyl-£)-glucose-MC from the Radiochemical Centre, Amersham, England, and GIHsecretin from the Karolinska Institutet, Stockholm, Sweden. The secretin preparations were taken from a batch which was used for routine secretin-cholecystokinin tests in man and which was shown to be active by stimulating pancreatic secretion. Krebs-Henseleit buffer according to Semenza et al. (23). Perfusion Technique The animals were anesthetized with sodium pentobarbitone (Nembutal®), 60 mg/kg body weight i.p., and were infused intravenously with Ringer solution (1.9ml/h) as a volume replacement. Catheters were tied into the lumen of the jejunum at 20 and 45 cm distal to the pylorus, and this segment was perfused at a constant rate of 12 ml/h for 170 min. The perfusate was collected in 10-min aliquots. The first three samples (0 30 min after starting the experiment) were rejected. The perfusion fluid (305 mosm/l, pH 5.5) consisted of: glucose 30 mmol/l, Na 147 mmol/1, K 4.0 mmol/l, Ca 2.25 mmol/1, Cl 155 mmol/l and phenol red 0.02 g/1 as a nonabsorbable marker. 60. 90 and 120 min after the beginning of the perfusion 2.0 clinical units (CU) secretin per kg body weight, diluted in 0.5 ml Ringer solution, were injected into the tail vein. In a control group equal volumes of Ringer solution without secretin were injected.
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Incubation o f Pieces 12 min after an intravenous injection of 5 CU secretin per animal the small intestine was removed under pentobarbitone anesthesia (60 mg/kg body weight). The segment 20 45 cm distal to the pylorus was everted, divided into 10 pieces and then processed with the tissue-holding apparatus technique described by Semenza (22). This technique excludes the uptake of the substrate from the serosal side. In a control group the animals received an intravenous injection of Ringer solution instead of secretin. The pieces were randomized and incubated for 5 min at 37 °C in the following incubation solutions: Krebs-Henseleit buffer (pH 7.4) containing D-galactose-1JC, 0.1 /iCi/ml. and increasing galactose concentrations (4, 8, 12, 16 or 20 mmol/l); Krebs-Hcnseleit-buffer (pH 7.4) containing 3-0-methylglucose-MC. 0.1 uCi/ml, and increasing 3-O-methylglucose concentrations (4, 8, 12. 16 or 20 mmol/l). The buffer substrate solutions were continuously gassed with 0 ; + CO, (95:5). At the end of the incubation procedure the tissue-holding apparatus was removed from the incu-
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bation medium and the tissue rinsed with ice-cold buffer. Then the samples were stamped out, once more rinsed with cold buffer and freeze-dried. Methods o f Determination and Calculation (i) glucose was determined with hexokinase/gIucose-6-phosphate dehydrogenase (21), phenol red according to the method described by Henry el al. (12). Glucose absorption was calculated as follows: A = 2-(Gp —
GS 'MP
MS
where A = glucose absorption Oumol/lO min); Gp = glucose concentration in the test solu tion; Gg = glucose concentration in the sample; Mp = marker (phenol red) concentration in the original test solution; Mg = marker concentration in the sample. The net water movement was calculated by the phenol red concentration in the sample compared to the concentration in the test solution which was taken as 100 %. (ii) The freeze-dried mucosal pieces were weighed and then dissolved in 1 mi soluene-350 for 12 h at 37 °C. After total dissolution, 10 ml scintillation fluid (12.5 g PPO in 2,500 ml toluol) were added and the counts were measured in the scintillation counter. In the galactose as well as in the 3-O-methylglucose experiments, control samples containing 0.1 ml of the original incubation fluids (with increasing substrate concentrations) in 1 ml soluene were counted, too. The uptake of galactose and 3-O-mcthylglucose was calculated as follows: .. _ cpm/mg d.w.-102 -|S | cpm/0.1 ml i.f.-4-5 ' where U = monosaccharide uptake Oimol/ml tissue water/min); cpm/mg d.w. = cpm/mg tissue dry weight; cpm/0.1 ml i.f. = cpm/0.1 ml original incubation fluid; |S | = substrate concentration in the original incubation fluid; factor 4 is used to convert dry weight to total tissue water (5); factor 5 corrects the incubation time (5 min) to 1 min; factor 10: consists of two components: (1) conversion of cpm/mg to cpm/ml in the numerator (X 1,000), and (2) conversion of the volume of the incubation fluid (0.1 ml) in the control samples to 1 ml in the denominator (X 10). Double reciprocal plots were constructed and Km and Vmax evaluated in both galac tose and 3-O-methylglucose experiments. (iii) The statistical evaluation was performed with the calculation of the linear regres sion lines and the two-way analysis of variance.
The net water movement, expressed as phenol red concentration, is shown in figure 1. The phenol red concentration amounted to 106-109% (m eant SD = 107.37+ 1.04%) in the course of the control experiments and to 105— 110% (m ea n t SD= 107.31 ± 1.34%) in the secretin group. No significant difference between the control group and the secretin group (p > 0.05) could be proved. There was no increasing or decreasing tendency to be seen during the course of the perfusion. These data express a net water movement from the lumen to the tissue of about 7 % of the applied volume.
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Results
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Fig. 1. Net water movement (mean ± SD) after repeated intravenous injections of 2.0 CU secretin/kg body weight. • = Control group (n = 14); * = secretin group (n = 12).
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Fig. 2. Glucose absorption (mean ± SD) after repeated intravenous injections of 2.0 CU secretin/kg body weight. • = Control group (n = 14); * = secretin group (n = 12).
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1
mM galactose
The absorption of glucose (fig. 2) decreased with increasing perfusion time in both groups. This slope was significantly different from zero; between con trols and the secretin group there was no significant difference. The regression coefficient was 0.386 in the secretin group and 0.410 in the control group, that means that glucose absorption was diminished about 0.386 juntol/10 min after secretin and 0.410/tm ol/10 min in control rats. The mean value of the glucose absorption in all samples in the control group (x ± SD = 24.5 ± 7.7 /rmoi/10 min) was not significantly different from that after secretin (x ± SD = 23.4 ± 7.3 /umol/10 min; p > 0.05). The galactose uptake is shown in figure 3. Km was 14.8 mmol/1 in the control group without secretin and 15.4 mmol/1 after 5.0 CU secretin. Vmax was 4.33 jumol/ml tissue water/min in the controls and 4.23 /umol/ml tissue water/ min after secretin. The galactose uptake showed no significant difference between the control group and the secretin group (p > 0.05).
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Fig. 3. Galactose uptake after 5.0 CU secretin (double reciprocal plot), x------- X Control group; o ------ o = secretin group.
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1 mM 3 - 0 -M G
Fig. 4. 3-O-Methylglucose (3-O-MG) uptake after 5.0 CU secretin (double reciprocal plot), x-------X = Control group; o-— -o = secretin group.
The incubation with 3-0-methylglucose brought about similar results (fig. 4). Km was 16.5 and 19.1 mmol/1 in the control and secretin groups, respec tively; Vmax was 3.48 mmol/ml tissue water/min in the control group and 3.66 /rmol/ml tissue water/min after secretin. The uptake of 3-O-methylglucose was not significantly altered by secretin (p > 0.05).
This study was designed to demonstrate the effect of repeated intravenous injections of 2.0 CU secretin/kg body weight on glucose absorption and net water movement in the proximal jejunum of the rat. In the control group as well
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Discussion
331
as in the secretin group there was a significant diminution of the glucose absorp tion in the course of 140 min perfusion time. Repeated intravenous secretin injections of 2.0 CU/kg body weight did not alter the glucose absorption (fig. 2). The net water movement expressed as changes in the phenol red concentra tion was the same in the control group and after secretin without shift in the course of the experiment (fig. 1). Errors in the calculation of net water move ment by secretin-stimulated output of bile or pancreatic juice were avoided by tying off the lumen of the small intestine at the beginning of the perfused loop. The uptake experiments were performed with galactose and 3-O-methylglucose because these monosaccharides are transported by the same active transport system as glucose, but are only poorly (galactose) or not (3-O-methylglucose) metabolized by the epithelium cell of the small intestine. The obtained data for Km and Vmax showed no significant differences after 5.0 CU secretin compared to control animals (fig. 3,4). The data presented in this paper are in accordance with our earlier results in the rat (2) and with those of other investigators who found no effect of secretin on intestinal net water movement and glucose absorption in the dog (1, 3). Hubei (15) proved a reduction of jejunal sodium and water absorption and an increase of chloride secretion in the jejunum of the rat after an intravenous injection of 100 or 200 U/kg secretin (BOOTS or GIH). In the ileum, secretin reduced the absorption of chloride and water (15). A comparison of the present results with those of Hubei (15) is impossible because this author used a glucose-free perfusion fluid and applied much higher doses of secretin. In the human jejunum Mekhjian et al. (16) found a secretion of water and sodium during the intravenous infusion of 4 U GIH-secretin/kg/h while Hicks and Turnberg (14) reported a reduction of tire sodium, potassium, chloride and water absorption in the proximal jejunum during 2 U GIH-secretin/kg/h. In the mid jejunum, secretin did not alter the electrolyte and water transport (14). Moritz et al. (19) proved a dose-dependent inhibitory effect of GlH-secretin on the absorption of water, sodium, potassium and chloride. 2 U secretin/kg/h pro duced a significantly greater fluid accumulation than 1 U/kg/h. In contrast to this, Modigliani et al. (18) did not find any effect by BOOTS-secretin (3 U/kg/h) on the hydro-electrolytic transport across the small intestine in man. Dollinger et al. (8) reported that 1 U GIH-secretin/kg/h had no effect on the jejunal net movement of water, sodium, chloride and glucose in man, whereas 2 U/kg/h had a significantly inhibitory effect. The dose-dependent inhibitory effect of secretin on the intestinal motility (4, 7, 10, 13) with increased flow rate (14) and decreased intraluminal pressure (20) may play a part in explaining the varying results regarding the effect of secretin on intestinal sugar absorption. An increased flow rate seems to increase glucose absorption (6), while a decreased intraluminal pressure causes an inhibi tion of water and glucose absorption (17). This secretin effect on the intestinal
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motility may be more or less pronounced depending on the employed dose, species-specific behavior and the perfusion rate. The conflicting data pertaining the effect of secretin on the intestinal ab sorption and net water movement may be due to differences in experimental techniques, different, mostly pharmacological doses of the hormone and a diverse response of heterogeneous species.
1 Barbezat, G.O. and Grossman, M.I.: Intestinal secretion: stimulation by peptides. Science, N.Y.174: 4 2 2 -424 (1971). 2 Böhmer, R. und Rommel, K.: Einfluss von Sekretin und Pankreozymin auf die Lac tosedigestion und die Wassersorption der Ratte. Z. klin. Chem. klin. Biochem. 4: 403-405 (1970). 3 Bynum, T.E.; Jakobson, F.D., and Johnson, I . . R Gastrin inhibition of intestinal absorption in dogs. Gastroenterology 61: 858-862 (1971). 4 Chey, W.Y.: l.orber, S.H.: Kusakcioglu, O., and Hendricks, J.: Effect of secretin and pancreozymin-cholecystokinin on motor function of stomach and duodenum. Fed. Proc. Fed. Am. Socs exp. Biol. 26: 383 (1967). 5 Crane, R.K. and Mandelstam, P.: The active transport of sugars by various preparations of hamster intestine. Biochim. biophys. Acta-fJ: 460-476 (1960). 6 Dawson, A.M. and McMichael, 11.B.: The effect of flow rate on glucose absorption demonstrated by perfusion studies in rat jejunum in vivo. J. Physiol., Lond. 196: 32P-33P (1968). 7 Dollinger, H.C.: Berz, R.: Raptis, S.: Uexkiill, Th. von, and Goebell, H.: Effects of secretin and cholecystokinin on motor activity of human jejunum. A radiotelemetering study of jejunal motility during secretin and cholecystokinin intravenous infusion. Digestion 12: 9 16(1975). 8 Dollinger, H.C.; Rommel, K.: Raptis, S., and Goebell, 11.: Effect of pentagastrin, secre tin and cholecystokinin-pancreozymin on water, electrolyte and glucose absorption in the human jejunum. 5th World Congr. Gastroenterology, Mexico 1974. Abstracts, p. 274 (1974). 9 Forell, M.M. und Stahlheber, II.: Über die Wirkung von Sekretin und Pankreozymin auf die exkretorische Pankreasfunktion und ihre diagnostische Anwendungsmöglich keit. Klin. Wschr. 14: 675-679 (1964). 10 Gutierrez, Chey, W. Y.: Dinoso, V., and l.orber, S.H.: Effect of intestinal hormones on motor function of the small bowel and sigmoid colon in man. Gastroenterology 60: 672(1971). 11 Harper, A.A. and Raper, H.S.: Pancreozymin, a stimulant of the secretion of pancreatic enzymes in extracts of the small intestine. J. Physiol., Lond. 102: 115-125 (1943). 12 Henry, R.J.; Cannon, D.C., and Winkelman, J.W. (ed.): Clinical chemistry; 2nd ed., pp. 1550-1551 (Harper & Row, Hagerstown 1974). 13 Hermon-Taylor, J. and Code, C.F.: Effect of secretin on small bowel myoelectric activity of conscious healthy dogs. Am. J. dig. Dis. 15: 545- 550 (1970). 14 Hicks, T. and Tumberg, L.A.: The influence of secretin on ion transport in the human jejunum. Gut 14: 485-490 (1973). 15 Hubei, K.A.: Effects of secretin and glucagon on intestinal transport of ions and water in the rat. Proc. Soc. exp. Biol. Med. 139: 656-658 (1972).
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References
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Dr. R. Böhmer. Abteilung für Klinische Chemie, Universität Ulm, Steinhövelstrassc 9. 0 -7 9 0 0 Ulm (FRG)
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16 Mekhjian, H.; King, D.: Sanzenbacher, I.., and Zollinger, R.: Glucagon and secretin inhibit water and electrolyte transport in the human jejunum. Gastroenterology 62: 782(1972). 17 Mirkovitch, V.: Menge, H., and Robinson, J.W.L.: The effect of intraluminal hydro static pressure on intestinal absorption in vivo. Expericntia 30: 912 913(1974). 18 Modigliani, R.: Huet, P.M.: Rambaud, J.C., and Bernier, J.J.: Effect of secretin upon movements of water and electrolytes across the small intestine in man. Revue eur. F.tud. clin. biol. 16: 361-364 (1971). 19 Moritz, M.: Finkeistein, G.: Meshkinpour, II.: Fingerut, J., and l.orber, 3.1... Effect of secretin and cholccystokinin on the transport of electrolyte and water in human jejunum. Gastroenterology 64: 76-80 (1973). 20 Ramirez, M. and Farrar, J.T.: The effect of secretin and cholecystokinin-pancreozymin on the intraluminal pressure of the jejunum in the unanesthetized dog. Am. J. dig. Dis. 15: 539-544 (1970). 21 Schmidt, F.H.: Die enzymatische Bestimmung von Glucose and Fructose neben einander. Klin. Wschr. 39: 1244 1247(1961). 22 Semenza, G.: Studies on intestinal sucrase and sugar transport. VII. A method for measuring intestinal uptake. The absorption of the anomeric forms of some mono saccharides. Biochim. biophys. Acta 173: 104-112 (1969). 23 Semenza, G.: Bircher, J.: Miilhaupt, E.: Koide, T.: Pfenninger, E.: Marthaler, Th.: Gmünder, U., and Haemmerli, U.P.: Arbutin absorption in human small intestine. A simple procedure for the determination of active sugar uptake in peroral biopsy specimens. Clin. chim. Acta 25: 213-219 (1969).
Effect o f Secretin on Intestinal Monosaccharide Absorption and Net Water Movement in the Rat R. Böhmer and K. Rommel Department of Clinical Chemistry, University of Ulm, Ulm
Key Words. Galactose • Glucose • Intestinal absorption • Intestinal mucosa • Phenol phthaleins ■Rats • Secretin Abstract. Repeated intravenous injections of 2.0 clinical units (CU) secretin per kg body weight showed no effect on jejunal glucose absorption and net water movement in perfusion studies in vivo. The in vitro uptake of galactose and 3-O-mcthylglucose was not altered by a secretin load given 12 min before sacrifice. The values for Km and Vmax were identical after secretin and in a control group. The conflicting results of different authors concerning the effect of secretin on net water movement and solute absorption may be due to differences in experimental techniques, different, mostly pharmacological doses of the hormone, and a diverse response of heterogeneous species.
The effects of secretin on pancreatic secretion are well known (9, 11), but there are conflicting results about the influence of secretin on intestinal net water movement and solute absorption (1—3, 8, 14-16, 18, 19). The present investigation has been performed in order to study the influ ence of repeated intravenous injections of secretin on the intestinal net water movement and glucose absorption in the rat small intestine in vivo and on the galactose and 3-O-methylglucose uptake in vitro. The last mentioned monosac charides are either poorly or not metabolized in the intestinal epithelium cell and are absorbed by the same active transport system as glucose.
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Received: July 12. 1975; accepted: August 27. 1975.
Böhmer/Rommel
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Materials and Methods Animals Female specific pathogen-frcc (SPF) Wislar rats, strain F'W 49 (Thomae, Biberach. FRG), weight 173.5 201.5 g (mean t SD = 187.0 t 6.5), nutrition with Altromin standard diet, were randomized. The intestinal flora consisted of Lactobacillus acidophilus (ATCC 11506); Lactobacillus bifidus (isolated from human baby feces); Streptococcus lactis: Streptococcus fecalis (ATCC 10541); Bacteroides symbiosus (= fusobacterium symbiosum) (ATCC 14940), and Escherichia coli (isolated from rats). The experiments were performed after 12 15 h of fasting with water ad libitum. Chemicals D(+)-glucose and phenol red have been obtained from Fluka AG, Buchs, Switzerland; 3-O-methylglucose from Calbiochem AG, Luzern, Switzerland; D-galactose from Serva, Heidelberg, FRG; toluol p.A. and 2,5-diphenyloxazol (PPO) from Merck, Darmstadt, FRG; soluene-350 from Packard Instrument, Zürich, Switzerland; D-galactose-MC and 3-0methyl-£)-glucose-MC from the Radiochemical Centre, Amersham, England, and GIHsecretin from the Karolinska Institutet, Stockholm, Sweden. The secretin preparations were taken from a batch which was used for routine secretin-cholecystokinin tests in man and which was shown to be active by stimulating pancreatic secretion. Krebs-Henseleit buffer according to Semenza et al. (23). Perfusion Technique The animals were anesthetized with sodium pentobarbitone (Nembutal®), 60 mg/kg body weight i.p., and were infused intravenously with Ringer solution (1.9ml/h) as a volume replacement. Catheters were tied into the lumen of the jejunum at 20 and 45 cm distal to the pylorus, and this segment was perfused at a constant rate of 12 ml/h for 170 min. The perfusate was collected in 10-min aliquots. The first three samples (0 30 min after starting the experiment) were rejected. The perfusion fluid (305 mosm/l, pH 5.5) consisted of: glucose 30 mmol/l, Na 147 mmol/1, K 4.0 mmol/l, Ca 2.25 mmol/1, Cl 155 mmol/l and phenol red 0.02 g/1 as a nonabsorbable marker. 60. 90 and 120 min after the beginning of the perfusion 2.0 clinical units (CU) secretin per kg body weight, diluted in 0.5 ml Ringer solution, were injected into the tail vein. In a control group equal volumes of Ringer solution without secretin were injected.
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Incubation o f Pieces 12 min after an intravenous injection of 5 CU secretin per animal the small intestine was removed under pentobarbitone anesthesia (60 mg/kg body weight). The segment 20 45 cm distal to the pylorus was everted, divided into 10 pieces and then processed with the tissue-holding apparatus technique described by Semenza (22). This technique excludes the uptake of the substrate from the serosal side. In a control group the animals received an intravenous injection of Ringer solution instead of secretin. The pieces were randomized and incubated for 5 min at 37 °C in the following incubation solutions: Krebs-Henseleit buffer (pH 7.4) containing D-galactose-1JC, 0.1 /iCi/ml. and increasing galactose concentrations (4, 8, 12, 16 or 20 mmol/l); Krebs-Hcnseleit-buffer (pH 7.4) containing 3-0-methylglucose-MC. 0.1 uCi/ml, and increasing 3-O-methylglucose concentrations (4, 8, 12. 16 or 20 mmol/l). The buffer substrate solutions were continuously gassed with 0 ; + CO, (95:5). At the end of the incubation procedure the tissue-holding apparatus was removed from the incu-
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bation medium and the tissue rinsed with ice-cold buffer. Then the samples were stamped out, once more rinsed with cold buffer and freeze-dried. Methods o f Determination and Calculation (i) glucose was determined with hexokinase/gIucose-6-phosphate dehydrogenase (21), phenol red according to the method described by Henry el al. (12). Glucose absorption was calculated as follows: A = 2-(Gp —
GS 'MP
MS
where A = glucose absorption Oumol/lO min); Gp = glucose concentration in the test solu tion; Gg = glucose concentration in the sample; Mp = marker (phenol red) concentration in the original test solution; Mg = marker concentration in the sample. The net water movement was calculated by the phenol red concentration in the sample compared to the concentration in the test solution which was taken as 100 %. (ii) The freeze-dried mucosal pieces were weighed and then dissolved in 1 mi soluene-350 for 12 h at 37 °C. After total dissolution, 10 ml scintillation fluid (12.5 g PPO in 2,500 ml toluol) were added and the counts were measured in the scintillation counter. In the galactose as well as in the 3-O-methylglucose experiments, control samples containing 0.1 ml of the original incubation fluids (with increasing substrate concentrations) in 1 ml soluene were counted, too. The uptake of galactose and 3-O-mcthylglucose was calculated as follows: .. _ cpm/mg d.w.-102 -|S | cpm/0.1 ml i.f.-4-5 ' where U = monosaccharide uptake Oimol/ml tissue water/min); cpm/mg d.w. = cpm/mg tissue dry weight; cpm/0.1 ml i.f. = cpm/0.1 ml original incubation fluid; |S | = substrate concentration in the original incubation fluid; factor 4 is used to convert dry weight to total tissue water (5); factor 5 corrects the incubation time (5 min) to 1 min; factor 10: consists of two components: (1) conversion of cpm/mg to cpm/ml in the numerator (X 1,000), and (2) conversion of the volume of the incubation fluid (0.1 ml) in the control samples to 1 ml in the denominator (X 10). Double reciprocal plots were constructed and Km and Vmax evaluated in both galac tose and 3-O-methylglucose experiments. (iii) The statistical evaluation was performed with the calculation of the linear regres sion lines and the two-way analysis of variance.
The net water movement, expressed as phenol red concentration, is shown in figure 1. The phenol red concentration amounted to 106-109% (m eant SD = 107.37+ 1.04%) in the course of the control experiments and to 105— 110% (m ea n t SD= 107.31 ± 1.34%) in the secretin group. No significant difference between the control group and the secretin group (p > 0.05) could be proved. There was no increasing or decreasing tendency to be seen during the course of the perfusion. These data express a net water movement from the lumen to the tissue of about 7 % of the applied volume.
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Results
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Fig. 1. Net water movement (mean ± SD) after repeated intravenous injections of 2.0 CU secretin/kg body weight. • = Control group (n = 14); * = secretin group (n = 12).
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Fig. 2. Glucose absorption (mean ± SD) after repeated intravenous injections of 2.0 CU secretin/kg body weight. • = Control group (n = 14); * = secretin group (n = 12).
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1
mM galactose
The absorption of glucose (fig. 2) decreased with increasing perfusion time in both groups. This slope was significantly different from zero; between con trols and the secretin group there was no significant difference. The regression coefficient was 0.386 in the secretin group and 0.410 in the control group, that means that glucose absorption was diminished about 0.386 juntol/10 min after secretin and 0.410/tm ol/10 min in control rats. The mean value of the glucose absorption in all samples in the control group (x ± SD = 24.5 ± 7.7 /rmoi/10 min) was not significantly different from that after secretin (x ± SD = 23.4 ± 7.3 /umol/10 min; p > 0.05). The galactose uptake is shown in figure 3. Km was 14.8 mmol/1 in the control group without secretin and 15.4 mmol/1 after 5.0 CU secretin. Vmax was 4.33 jumol/ml tissue water/min in the controls and 4.23 /umol/ml tissue water/ min after secretin. The galactose uptake showed no significant difference between the control group and the secretin group (p > 0.05).
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Fig. 3. Galactose uptake after 5.0 CU secretin (double reciprocal plot), x------- X Control group; o ------ o = secretin group.
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1 mM 3 - 0 -M G
Fig. 4. 3-O-Methylglucose (3-O-MG) uptake after 5.0 CU secretin (double reciprocal plot), x-------X = Control group; o-— -o = secretin group.
The incubation with 3-0-methylglucose brought about similar results (fig. 4). Km was 16.5 and 19.1 mmol/1 in the control and secretin groups, respec tively; Vmax was 3.48 mmol/ml tissue water/min in the control group and 3.66 /rmol/ml tissue water/min after secretin. The uptake of 3-O-methylglucose was not significantly altered by secretin (p > 0.05).
This study was designed to demonstrate the effect of repeated intravenous injections of 2.0 CU secretin/kg body weight on glucose absorption and net water movement in the proximal jejunum of the rat. In the control group as well
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Discussion
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as in the secretin group there was a significant diminution of the glucose absorp tion in the course of 140 min perfusion time. Repeated intravenous secretin injections of 2.0 CU/kg body weight did not alter the glucose absorption (fig. 2). The net water movement expressed as changes in the phenol red concentra tion was the same in the control group and after secretin without shift in the course of the experiment (fig. 1). Errors in the calculation of net water move ment by secretin-stimulated output of bile or pancreatic juice were avoided by tying off the lumen of the small intestine at the beginning of the perfused loop. The uptake experiments were performed with galactose and 3-O-methylglucose because these monosaccharides are transported by the same active transport system as glucose, but are only poorly (galactose) or not (3-O-methylglucose) metabolized by the epithelium cell of the small intestine. The obtained data for Km and Vmax showed no significant differences after 5.0 CU secretin compared to control animals (fig. 3,4). The data presented in this paper are in accordance with our earlier results in the rat (2) and with those of other investigators who found no effect of secretin on intestinal net water movement and glucose absorption in the dog (1, 3). Hubei (15) proved a reduction of jejunal sodium and water absorption and an increase of chloride secretion in the jejunum of the rat after an intravenous injection of 100 or 200 U/kg secretin (BOOTS or GIH). In the ileum, secretin reduced the absorption of chloride and water (15). A comparison of the present results with those of Hubei (15) is impossible because this author used a glucose-free perfusion fluid and applied much higher doses of secretin. In the human jejunum Mekhjian et al. (16) found a secretion of water and sodium during the intravenous infusion of 4 U GIH-secretin/kg/h while Hicks and Turnberg (14) reported a reduction of tire sodium, potassium, chloride and water absorption in the proximal jejunum during 2 U GIH-secretin/kg/h. In the mid jejunum, secretin did not alter the electrolyte and water transport (14). Moritz et al. (19) proved a dose-dependent inhibitory effect of GlH-secretin on the absorption of water, sodium, potassium and chloride. 2 U secretin/kg/h pro duced a significantly greater fluid accumulation than 1 U/kg/h. In contrast to this, Modigliani et al. (18) did not find any effect by BOOTS-secretin (3 U/kg/h) on the hydro-electrolytic transport across the small intestine in man. Dollinger et al. (8) reported that 1 U GIH-secretin/kg/h had no effect on the jejunal net movement of water, sodium, chloride and glucose in man, whereas 2 U/kg/h had a significantly inhibitory effect. The dose-dependent inhibitory effect of secretin on the intestinal motility (4, 7, 10, 13) with increased flow rate (14) and decreased intraluminal pressure (20) may play a part in explaining the varying results regarding the effect of secretin on intestinal sugar absorption. An increased flow rate seems to increase glucose absorption (6), while a decreased intraluminal pressure causes an inhibi tion of water and glucose absorption (17). This secretin effect on the intestinal
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motility may be more or less pronounced depending on the employed dose, species-specific behavior and the perfusion rate. The conflicting data pertaining the effect of secretin on the intestinal ab sorption and net water movement may be due to differences in experimental techniques, different, mostly pharmacological doses of the hormone and a diverse response of heterogeneous species.
1 Barbezat, G.O. and Grossman, M.I.: Intestinal secretion: stimulation by peptides. Science, N.Y.174: 4 2 2 -424 (1971). 2 Böhmer, R. und Rommel, K.: Einfluss von Sekretin und Pankreozymin auf die Lac tosedigestion und die Wassersorption der Ratte. Z. klin. Chem. klin. Biochem. 4: 403-405 (1970). 3 Bynum, T.E.; Jakobson, F.D., and Johnson, I . . R Gastrin inhibition of intestinal absorption in dogs. Gastroenterology 61: 858-862 (1971). 4 Chey, W.Y.: l.orber, S.H.: Kusakcioglu, O., and Hendricks, J.: Effect of secretin and pancreozymin-cholecystokinin on motor function of stomach and duodenum. Fed. Proc. Fed. Am. Socs exp. Biol. 26: 383 (1967). 5 Crane, R.K. and Mandelstam, P.: The active transport of sugars by various preparations of hamster intestine. Biochim. biophys. Acta-fJ: 460-476 (1960). 6 Dawson, A.M. and McMichael, 11.B.: The effect of flow rate on glucose absorption demonstrated by perfusion studies in rat jejunum in vivo. J. Physiol., Lond. 196: 32P-33P (1968). 7 Dollinger, H.C.: Berz, R.: Raptis, S.: Uexkiill, Th. von, and Goebell, H.: Effects of secretin and cholecystokinin on motor activity of human jejunum. A radiotelemetering study of jejunal motility during secretin and cholecystokinin intravenous infusion. Digestion 12: 9 16(1975). 8 Dollinger, H.C.; Rommel, K.: Raptis, S., and Goebell, 11.: Effect of pentagastrin, secre tin and cholecystokinin-pancreozymin on water, electrolyte and glucose absorption in the human jejunum. 5th World Congr. Gastroenterology, Mexico 1974. Abstracts, p. 274 (1974). 9 Forell, M.M. und Stahlheber, II.: Über die Wirkung von Sekretin und Pankreozymin auf die exkretorische Pankreasfunktion und ihre diagnostische Anwendungsmöglich keit. Klin. Wschr. 14: 675-679 (1964). 10 Gutierrez, Chey, W. Y.: Dinoso, V., and l.orber, S.H.: Effect of intestinal hormones on motor function of the small bowel and sigmoid colon in man. Gastroenterology 60: 672(1971). 11 Harper, A.A. and Raper, H.S.: Pancreozymin, a stimulant of the secretion of pancreatic enzymes in extracts of the small intestine. J. Physiol., Lond. 102: 115-125 (1943). 12 Henry, R.J.; Cannon, D.C., and Winkelman, J.W. (ed.): Clinical chemistry; 2nd ed., pp. 1550-1551 (Harper & Row, Hagerstown 1974). 13 Hermon-Taylor, J. and Code, C.F.: Effect of secretin on small bowel myoelectric activity of conscious healthy dogs. Am. J. dig. Dis. 15: 545- 550 (1970). 14 Hicks, T. and Tumberg, L.A.: The influence of secretin on ion transport in the human jejunum. Gut 14: 485-490 (1973). 15 Hubei, K.A.: Effects of secretin and glucagon on intestinal transport of ions and water in the rat. Proc. Soc. exp. Biol. Med. 139: 656-658 (1972).
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Dr. R. Böhmer. Abteilung für Klinische Chemie, Universität Ulm, Steinhövelstrassc 9. 0 -7 9 0 0 Ulm (FRG)
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16 Mekhjian, H.; King, D.: Sanzenbacher, I.., and Zollinger, R.: Glucagon and secretin inhibit water and electrolyte transport in the human jejunum. Gastroenterology 62: 782(1972). 17 Mirkovitch, V.: Menge, H., and Robinson, J.W.L.: The effect of intraluminal hydro static pressure on intestinal absorption in vivo. Expericntia 30: 912 913(1974). 18 Modigliani, R.: Huet, P.M.: Rambaud, J.C., and Bernier, J.J.: Effect of secretin upon movements of water and electrolytes across the small intestine in man. Revue eur. F.tud. clin. biol. 16: 361-364 (1971). 19 Moritz, M.: Finkeistein, G.: Meshkinpour, II.: Fingerut, J., and l.orber, 3.1... Effect of secretin and cholccystokinin on the transport of electrolyte and water in human jejunum. Gastroenterology 64: 76-80 (1973). 20 Ramirez, M. and Farrar, J.T.: The effect of secretin and cholecystokinin-pancreozymin on the intraluminal pressure of the jejunum in the unanesthetized dog. Am. J. dig. Dis. 15: 539-544 (1970). 21 Schmidt, F.H.: Die enzymatische Bestimmung von Glucose and Fructose neben einander. Klin. Wschr. 39: 1244 1247(1961). 22 Semenza, G.: Studies on intestinal sucrase and sugar transport. VII. A method for measuring intestinal uptake. The absorption of the anomeric forms of some mono saccharides. Biochim. biophys. Acta 173: 104-112 (1969). 23 Semenza, G.: Bircher, J.: Miilhaupt, E.: Koide, T.: Pfenninger, E.: Marthaler, Th.: Gmünder, U., and Haemmerli, U.P.: Arbutin absorption in human small intestine. A simple procedure for the determination of active sugar uptake in peroral biopsy specimens. Clin. chim. Acta 25: 213-219 (1969).
