Digestion 15: 329-337 (1977)
/w-s/Vw-Isolated P erfused R at Pancreas: a New M ethod for Pharm acological S tudies o f th e E xocrine P an creas1 R. Tournut, A. Estival, N. Vaysse, J.P. Pascal and A. Ribet2 Groupe de Recherche de Biologie et Pathologie digestive, Inserm U-151, Toulouse
Key Words. Bicarbonates • Gastrointestinal hormones • Pancreas • Pancreatic juice • Proteins • Rats Abstract. A method of in-situ-isolated perfused rat pancreas is described. In situ per fusion of the isolated rat pancreas allows a faster set-up, thanks to a simpler operating procedure. The sensitivity of the isolated rat pancreas has been tested with secretin and cholecystokinin. The pancreas responds to doses of secretin as low as 0.005 CU/kg-h. Such a highly sensitive model might prove to be useful for the bioassay of secretin and the study of the effects of pancreatic secretagogues.
The study of the effects of drugs on the exocrine pancreas would ideally require the isolation of drug receptors. Practically, the isolated perfused organ provides a good compromise between this ideal need and technical frontiers: anatomy is preserved including pancreatic parenchyma, blood vessels and excre tory ducts. The method also allows for total recovery of the secreted juice without contamination. The method has already been used in dogs (1,13, 14, 18), cats (3) and rats (15, 19, 23); the above techniques require an accurate operating procedure, a sophisticated apparatus, and, in some cases, the use of expensive animals. Con-
1 This work was supported by grants No. CRL 750317 from the Inserm and No. 7570912 from the DGRST. 2 The authors are very grateful to Dr. Bales and Ms. M.J. Bastie, for ultrastructural findings and to Ms. C. Pichon for technical assistance.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/29/2018 1:58:09 AM
Received: June 30, 1976; accepted: October 26, 1976.
Tournul /Estival /Vaysse ¡Pascal/Ribet
330
versely, in rats, a method o f m-s/fw-isolated perfused pancreas could allow a faster set-up, the use of cheaper animals, and, possibly, experiments in series. Our aim in this work was: (1) to simplify the operating procedure described by Kantio (15) and Ueda (19) in their ex-v/Vo-isolated perfused rat pancreas meth ods and (2) to demonstrate its validity as a model for the study of the effects of pancreatic secretagogues, such as duodenal hormones.
Operative Procedure. Male Wistar rats (300 g mean body weight) were anesthetized by intraperitoneal injection of Pentobarbital® (50 mg/kg). After laparotomy, the pancreas was separated from the spleen, stomach and intestine after ligation of the splenic coronary, superior mesenteric arteries and celiac trunk beyond their pancreatic division. The other branches of the abdominal aorta, the renal veins and the inferior vena cava were also tied. The aorta was cannulated for arterial infusion under the origin of the superior mesenteric artery. The choledochus was dissected away from the portal vein and then ligated above the highest pancreatic lobules. The main pancreatic duct was cannulated with a polyvinyl-chloride catheter 1 mm OD). The perfusion medium was drained through the portal vein and was not recircu lated. Perfusion. The standard perfusion medium was composed as follows (mEq/1): Na* 144.5, CP 133.2, K* 5.80, Ca**5, Mg** 1.5, H, PO; 1.10, SO;’, HCOj 20; bovine albumin 1%c, glucose 1 g/liter, and 10 cm3 of 'Solute hypertonique d’acidcs amines®’ (Roger Bellon) were added in order to achieve a 24 mM concentration of tryptophan as suggested by Campagne and Gruber (2). A 95% O, 5% COa gas mixture was bubbled through the perfusion fluid reservoir. From the reservoir the fluid entered a heat-exchange coil in order to maintain the temperature of the pancreas at 38 ± 0.1 °C. The fluid was delivered to the arterial cannula by a Watson-Marlow (M.H.R.E. Watson-Marlow Ltd., Marlow, GB) flow inducer at a constant pressure of 40 mm Hg. Biopsies for histological control were performed at the end of each experiment before perfusion was discontinued. Juice collection. The pancreatic juice was collected continuously in 10-mI samples. Bicarbonate total proteins and lipase concentrations were measured in all samples. Bicarbon ate concentration was measured by means of an automated micromethod assaying the decoloration of an alcaline solution of phenolphtalcin by carbon dioxide liberated by an acid reagent. Total protein concentrations were measured by a modified version of the method of Lowry et al. (17). Lipase activity in the juice was measured titrimetrically (10). Experimental procedures. Experiments were started after the 30-min period necessary for stabilization of the system. Basal secretion was collected for 1 h. The stimulants were then infused in the arterial line at a constant rate: (l)CCK-PZ G1H was infused at graded doses from 0.5 to 32 Ivy dog units (IU)/kg-h (n > 4 for each dose) and (2) G1H pure natural secretin was infused at doses ranging from 0.005 to 50 clinical units (CU)/kg-h (n > 4 for each dose). Calculations. From data obtained with various dosages of drugs assayed, the DowdRiggs linear transformation (9) of the Michaelis-Menten equation (response versus response/ dose) was used to determine the calculated maximal response (CMR) and the dose required for half-maximal response (Ds0).
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/29/2018 1:58:09 AM
Methods
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/29/2018 1:58:09 AM
Fig. 1. Ultrastructural appearance of rat pancreas 60 min after starting the experiment. Morphology of pancreatic acinar cells remained unchanged and cytoplasmic components were preserved (a). Note the large edematous area and the integrity of vascular walls (b). N = Nuclei; RER = rough endoplasmic reticulum; M = mitochondria; Z = zymogen granules; L = acinar lumen. X 3,000.
Tournut /Estival /Vaysse /Pascal/Ribel
332
Fig. 2. An example of the variations in volumes (-------), bicarbonates (..........) and protein (------- ) secretions throughout one experiment.
Validity o f the method. Histological controls performed in 20 experiments showed interstitial edemas with preservation of acinar, ductal and vascular archi tecture (fig. 1). Figure 2 displays a typical secretion pattern during an experi ment, showing that after stimulation the juice becomes richer in bicarbonate and proteins. Figure 3 demonstrates a very higlUy significant correlation between volumes and bicarbonate outputs, and between total protein concentration and lipase activity in the juice. The sensitivity of the isolated perfused rat pancreas was demonstrated by a pancreatic response to doses as low as 0.005 CU/kg-h of secretin. With such a dose of secretin, the observed response was statistically different from basal secretion (t = 2.59; p < 0.02). A good linear relationship was found between the doses of secretin on the one hand and the volume of juice and the bicarbonate outputs on the other (fig. 4). Correlations were found for CCK-PZ but only for doses ranging from 0.5 to 8 IU/kg-h (fig. 5). The great sensitivity to secretin allowed us to calculate CMR and Dso of the preparation. For volumes CMR is 3.34 /zl/min and Dso 0.35 CU/kg-h of secretin. When bicarbonate outputs were considered, CMR was found to be 132 X 10"6 mEq/min and Dso 0.52 CU/kg-h of secretin. As secretin has a molecular weight of 3,055 we write that a Dso of 0.35 CU/kg-h equals 48.5 pAf/kg-h.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/29/2018 1:58:09 AM
Results
/n-siru-lsolated Perfused Rat Pancreas
333
Volume flow, pl/min
Fig. 3. a Relationship between protein concentration and lipase activity of the juice, n = Number of experiments; r = correlation coefficient, b Relationship between volume flow and bicarbonate output, n = Number of experiments; r = correlation coefficient.
The juice collected from our preparation was rich in proteins and bicarbon ates and resembles pancreatic juices collected from in vivo (15) or ex vivo preparations (23). Responses to a maximal stimulation showed a peak response followed by a decrease even though the stimulant was continuously infused. This pattern compares to that observed by Folsch and Wormsley (12) in anesthetized, hypothermic rats. Reproducibility is demonstrated by fair dose-response rela tionship. Sensitivity to secretin is 3 times smaller than that observed in the conscious dog (4): D50 of secretin in dogs is 7 pAi/kg*h, i.e. 0.06 CU/kg-h and
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/29/2018 1:58:09 AM
Comments
Tournut/Estival/Vaysse/Pascal/Ribet
334
Secretin, CU/kg h
D50 of secretin with our method is about 7 times greater, this may be ascribed to the fact that secretin was never recirculated. When maximal secretory rates were considered, we found that the isolated perfused rat pancreas response compares to responses observed in anesthetized rats (5,12), conscious rats (6, 15, 22) and uremic rats (11). The technique used with conscious rats (6, 22) allows basal secretory rates up to 10 ql/min, that is 50 times more than the basal secretory rate in our experiments. However, the maximum secretory rate is about 12)al/min in conscious rats (20% increase), and 3 ¿/1/min in our preparation (1,500% increase).
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/29/2018 1:58:09 AM
Fig. 4. Relationship between the dose of secretin and the volume flow (a) and the bicarbonate output (b). n = Number of experiments; r = correlation coefficient. Vertical bars indicate standard error of the mean.
/n-sifu-I sola ted Perfused Rat Pancreas
335
Fig. 5. Relationship between the dose of CCK-PZ and the volume flow (a) and the protein output (b). n = Number of experiments; r= coefficient of correlation. Vertical bars indicate standard error of the mean.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/29/2018 1:58:09 AM
Levels of protein secretion appeared to be higher in isolated rat pancreas than in techniques using anesthetized rats (5, 8, 12, 15) but smaller than in conscious rats (6 ,7 ,2 2 ). An important advantage of this technique is that the effects of anesthesia, and the influence of nerves and other organs are excluded. Furthermore, an open circuit eliminates all the hormones and by-products which might be released by the preparation itself; so stimulation can only be exogenous and cannot be recirculated: this might explain a greater Dso of secretin with ou; preparation than that calculated by Debas and Grossman (4) in conscious dogs. Other prepa
Tournut/Estival/ Vaysse/Pascal/R ibet
336
rations (15, 19, 23) derived from the same principle offer the same advantages but are more time-consuming and complicated for the surgical preparation. The great sensitivity of the isolated perfused rat pancreas might prove useful in the bioassay of secretin (16). Its sensitivity might even be enhanced by addi tion of theophyllin into the perfusion fluid, as suggested by Scratcherd (20) and Scratcherd et al (21). In conclusion, the method of isolated rat pancreas per fused in situ appears to be suitable and highly sensitive for the study of the effects of gastrointestinal hormones and related peptides upon the exocrine of pancreatic secretagogues.
References
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/29/2018 1:58:09 AM
1 Augier, D Boucard, J.P.: Pascal, J.P.; Ribet, A„ and Vaysse, N.: Relationship between blood flow and secretion in the isolated perfused canine pancreas. J. Physiol., Lond. 221: 55-69 (1972). 2 Campagne, R.N. and Gruber, M.: Aminoacid and energy requirement of protein syn thesis in rat pancreatic tissu in vitro. Biochim. biophys. Acta 55: 353-360 (1962). 3 Case, R.M.; Harper, A.A., and Scratcherd, T.: Water and electrolyte secretion by the perfused pancreas of the cat. J. Physiol., Lond. 196: 133-149 (1968). 4 Debas, H.T. and Grossman, M.I.: Pure cholecystokinin: pancreatic protein and bicar bonate response. Digestion 9: 469-481 (1973). 5 Debray, C ; Tour, J. de la: Vaille, C.; Roze, C. et Souchard, M.: Contribution à l’étude de la sécrétion biliaire et pancréatique externe chez le rat. J. Physiol., Paris 54: 459499 (1962). 6 De Smul, A.; De Waele, B.; Wissocq, P., and Kickens, R.: Exogenous and endogenous secretion stimulation in the conscious rat. Digestion 11 : 39-50 (1974). 7 De Waele, B.; De Smul, A.; Wissocq, P. et Kickens, R.: La sécrétion pancréatique chez le rat. Influence de l’intervention chirurgicale, de la narcose, de l’hypothermie et de la dérivation du suc gastrique ou du suc pancréatique. Biol. Gastro-Enterol. 1: 253-263 (1974). 8 Dockray, G.J.: The action of secretin, cholecystokinin-pancreozymin and caerulein on pancreatic secretion in the rat. J. Physiol., Lond. 225: 679-692 (1972). 9 Dowd, J.E. and Riggs, D.S.: A comparison of estimate of Michaelis-Menten kinetic constants from various linear transformations. J. biol. Chem. 240: 863-869 (1965). 10 Figarella, C.; Souville, C; Quel, O.; Michel, R. et Sarles, 11. : Problèmes posés par le dosage des enzymes pancréatiques dans le suc duodénal. Vie méd. 47: 493-500 (1966). 11 Fleisher, K. ànd Kasper, H. : Exocrine pancreatic function in uraemic rats. Acta hepato-gastro-enter. 21: 398-403 (1974). 12 Folsch, U.R. and Wormsley, K.G.: Pancreatic enzyme response to secretin and chole cystokinin in the rat. J. Physiol., Lond. 234: 79-94 (1973). 13 Grenier, J.F.; Gillet, M.; Kachelhoffer, J.: Wong, P.; Barth, A.M.; Randrianarivo, P.; Stocbner, P.; Weiss, A.G.; Moody, A.J.; Fries, A.V. et Sundby, F.: Circulation extracorporelle du massif duodéno-pancréatique chez le chien. Technique, fonctionnement, intérêt. Annls Chir. thorac. cardiovasc. 8: 349-360 (1969). 14 Hermon Taylor, J.: A technique for perfusion of the isolated canine pancreas: response to secretin and gastrin. Gastroenterology 55: 488-501 (1968).
/n-sf'fu-Isolated Perfused Rat Pancreas 15 16 17 18
19
20 21
22 23
337
Kanno, T.: Calcium-dependent amylase release and electrophysiological measurements in the cells of the pancreas. J. Physiol., Lond. 226: 353-371 (1972). Love, J.W.: A method for the assay of secretin using rats. Q. J1 exp. Physiol. 42: 279-284 (1957). Lowry, O.H.; Rosebrough, N.J.; Farr, A.L., and Randall, R.J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193: 265-275 (1951). Ribet, A.: Pascal, J.P.; Frexinos, J.; Vaysse, N.; Boucard, J.P.; Vaysse, P. et Laval, J.: Le pancréas en circulation extra-corporelle: méthodologie, technique chirurgicale, con trôle biologique et ultrastructural. Sci. méd. 5: 529-539 (1971). Saunders, R.M. and Moser, C.A.: Changes in vascular resistances induced by prosta glandins E2 and F2 in the isolated rat pancreas. Archs int. Pharmacodyn. Thér. 191: 86-92 (1972). Scratcherd, T.: A sensitive method for the biological assay of secretin. Abstract. Gut 12: 861-862 (1971). Scratcherd, T.; Case, R.M., and Smith, P.A.: A sensitive method for the biological assay of secretin and substances with secretin-like activity in tissue and biological fluids. Scand. J. Gastroenterol. 10: 821-828 (1975). Shaw, H.M. and Heath, T.J. : Basal and post-prandial pancreatic secretion in rats. Q. J1 exp. Physiol. 58: 335-343 (1973). Veda, N.: An improved method for perfusion of the isolated rat pancreas. J. Physiol., Lond. 250: 4 -5 P (1975).
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/29/2018 1:58:09 AM
Dr. R. Tournut. Groupe de Biologie et Pathologie digestive, Inserm U-151, Bâtiment L3, CHU Rangueil, Chemin du Vallon, F -31400 Toulouse (F'rance)
/w-s/Vw-Isolated P erfused R at Pancreas: a New M ethod for Pharm acological S tudies o f th e E xocrine P an creas1 R. Tournut, A. Estival, N. Vaysse, J.P. Pascal and A. Ribet2 Groupe de Recherche de Biologie et Pathologie digestive, Inserm U-151, Toulouse
Key Words. Bicarbonates • Gastrointestinal hormones • Pancreas • Pancreatic juice • Proteins • Rats Abstract. A method of in-situ-isolated perfused rat pancreas is described. In situ per fusion of the isolated rat pancreas allows a faster set-up, thanks to a simpler operating procedure. The sensitivity of the isolated rat pancreas has been tested with secretin and cholecystokinin. The pancreas responds to doses of secretin as low as 0.005 CU/kg-h. Such a highly sensitive model might prove to be useful for the bioassay of secretin and the study of the effects of pancreatic secretagogues.
The study of the effects of drugs on the exocrine pancreas would ideally require the isolation of drug receptors. Practically, the isolated perfused organ provides a good compromise between this ideal need and technical frontiers: anatomy is preserved including pancreatic parenchyma, blood vessels and excre tory ducts. The method also allows for total recovery of the secreted juice without contamination. The method has already been used in dogs (1,13, 14, 18), cats (3) and rats (15, 19, 23); the above techniques require an accurate operating procedure, a sophisticated apparatus, and, in some cases, the use of expensive animals. Con-
1 This work was supported by grants No. CRL 750317 from the Inserm and No. 7570912 from the DGRST. 2 The authors are very grateful to Dr. Bales and Ms. M.J. Bastie, for ultrastructural findings and to Ms. C. Pichon for technical assistance.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/29/2018 1:58:09 AM
Received: June 30, 1976; accepted: October 26, 1976.
Tournul /Estival /Vaysse ¡Pascal/Ribet
330
versely, in rats, a method o f m-s/fw-isolated perfused pancreas could allow a faster set-up, the use of cheaper animals, and, possibly, experiments in series. Our aim in this work was: (1) to simplify the operating procedure described by Kantio (15) and Ueda (19) in their ex-v/Vo-isolated perfused rat pancreas meth ods and (2) to demonstrate its validity as a model for the study of the effects of pancreatic secretagogues, such as duodenal hormones.
Operative Procedure. Male Wistar rats (300 g mean body weight) were anesthetized by intraperitoneal injection of Pentobarbital® (50 mg/kg). After laparotomy, the pancreas was separated from the spleen, stomach and intestine after ligation of the splenic coronary, superior mesenteric arteries and celiac trunk beyond their pancreatic division. The other branches of the abdominal aorta, the renal veins and the inferior vena cava were also tied. The aorta was cannulated for arterial infusion under the origin of the superior mesenteric artery. The choledochus was dissected away from the portal vein and then ligated above the highest pancreatic lobules. The main pancreatic duct was cannulated with a polyvinyl-chloride catheter 1 mm OD). The perfusion medium was drained through the portal vein and was not recircu lated. Perfusion. The standard perfusion medium was composed as follows (mEq/1): Na* 144.5, CP 133.2, K* 5.80, Ca**5, Mg** 1.5, H, PO; 1.10, SO;’, HCOj 20; bovine albumin 1%c, glucose 1 g/liter, and 10 cm3 of 'Solute hypertonique d’acidcs amines®’ (Roger Bellon) were added in order to achieve a 24 mM concentration of tryptophan as suggested by Campagne and Gruber (2). A 95% O, 5% COa gas mixture was bubbled through the perfusion fluid reservoir. From the reservoir the fluid entered a heat-exchange coil in order to maintain the temperature of the pancreas at 38 ± 0.1 °C. The fluid was delivered to the arterial cannula by a Watson-Marlow (M.H.R.E. Watson-Marlow Ltd., Marlow, GB) flow inducer at a constant pressure of 40 mm Hg. Biopsies for histological control were performed at the end of each experiment before perfusion was discontinued. Juice collection. The pancreatic juice was collected continuously in 10-mI samples. Bicarbonate total proteins and lipase concentrations were measured in all samples. Bicarbon ate concentration was measured by means of an automated micromethod assaying the decoloration of an alcaline solution of phenolphtalcin by carbon dioxide liberated by an acid reagent. Total protein concentrations were measured by a modified version of the method of Lowry et al. (17). Lipase activity in the juice was measured titrimetrically (10). Experimental procedures. Experiments were started after the 30-min period necessary for stabilization of the system. Basal secretion was collected for 1 h. The stimulants were then infused in the arterial line at a constant rate: (l)CCK-PZ G1H was infused at graded doses from 0.5 to 32 Ivy dog units (IU)/kg-h (n > 4 for each dose) and (2) G1H pure natural secretin was infused at doses ranging from 0.005 to 50 clinical units (CU)/kg-h (n > 4 for each dose). Calculations. From data obtained with various dosages of drugs assayed, the DowdRiggs linear transformation (9) of the Michaelis-Menten equation (response versus response/ dose) was used to determine the calculated maximal response (CMR) and the dose required for half-maximal response (Ds0).
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/29/2018 1:58:09 AM
Methods
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/29/2018 1:58:09 AM
Fig. 1. Ultrastructural appearance of rat pancreas 60 min after starting the experiment. Morphology of pancreatic acinar cells remained unchanged and cytoplasmic components were preserved (a). Note the large edematous area and the integrity of vascular walls (b). N = Nuclei; RER = rough endoplasmic reticulum; M = mitochondria; Z = zymogen granules; L = acinar lumen. X 3,000.
Tournut /Estival /Vaysse /Pascal/Ribel
332
Fig. 2. An example of the variations in volumes (-------), bicarbonates (..........) and protein (------- ) secretions throughout one experiment.
Validity o f the method. Histological controls performed in 20 experiments showed interstitial edemas with preservation of acinar, ductal and vascular archi tecture (fig. 1). Figure 2 displays a typical secretion pattern during an experi ment, showing that after stimulation the juice becomes richer in bicarbonate and proteins. Figure 3 demonstrates a very higlUy significant correlation between volumes and bicarbonate outputs, and between total protein concentration and lipase activity in the juice. The sensitivity of the isolated perfused rat pancreas was demonstrated by a pancreatic response to doses as low as 0.005 CU/kg-h of secretin. With such a dose of secretin, the observed response was statistically different from basal secretion (t = 2.59; p < 0.02). A good linear relationship was found between the doses of secretin on the one hand and the volume of juice and the bicarbonate outputs on the other (fig. 4). Correlations were found for CCK-PZ but only for doses ranging from 0.5 to 8 IU/kg-h (fig. 5). The great sensitivity to secretin allowed us to calculate CMR and Dso of the preparation. For volumes CMR is 3.34 /zl/min and Dso 0.35 CU/kg-h of secretin. When bicarbonate outputs were considered, CMR was found to be 132 X 10"6 mEq/min and Dso 0.52 CU/kg-h of secretin. As secretin has a molecular weight of 3,055 we write that a Dso of 0.35 CU/kg-h equals 48.5 pAf/kg-h.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/29/2018 1:58:09 AM
Results
/n-siru-lsolated Perfused Rat Pancreas
333
Volume flow, pl/min
Fig. 3. a Relationship between protein concentration and lipase activity of the juice, n = Number of experiments; r = correlation coefficient, b Relationship between volume flow and bicarbonate output, n = Number of experiments; r = correlation coefficient.
The juice collected from our preparation was rich in proteins and bicarbon ates and resembles pancreatic juices collected from in vivo (15) or ex vivo preparations (23). Responses to a maximal stimulation showed a peak response followed by a decrease even though the stimulant was continuously infused. This pattern compares to that observed by Folsch and Wormsley (12) in anesthetized, hypothermic rats. Reproducibility is demonstrated by fair dose-response rela tionship. Sensitivity to secretin is 3 times smaller than that observed in the conscious dog (4): D50 of secretin in dogs is 7 pAi/kg*h, i.e. 0.06 CU/kg-h and
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/29/2018 1:58:09 AM
Comments
Tournut/Estival/Vaysse/Pascal/Ribet
334
Secretin, CU/kg h
D50 of secretin with our method is about 7 times greater, this may be ascribed to the fact that secretin was never recirculated. When maximal secretory rates were considered, we found that the isolated perfused rat pancreas response compares to responses observed in anesthetized rats (5,12), conscious rats (6, 15, 22) and uremic rats (11). The technique used with conscious rats (6, 22) allows basal secretory rates up to 10 ql/min, that is 50 times more than the basal secretory rate in our experiments. However, the maximum secretory rate is about 12)al/min in conscious rats (20% increase), and 3 ¿/1/min in our preparation (1,500% increase).
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/29/2018 1:58:09 AM
Fig. 4. Relationship between the dose of secretin and the volume flow (a) and the bicarbonate output (b). n = Number of experiments; r = correlation coefficient. Vertical bars indicate standard error of the mean.
/n-sifu-I sola ted Perfused Rat Pancreas
335
Fig. 5. Relationship between the dose of CCK-PZ and the volume flow (a) and the protein output (b). n = Number of experiments; r= coefficient of correlation. Vertical bars indicate standard error of the mean.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/29/2018 1:58:09 AM
Levels of protein secretion appeared to be higher in isolated rat pancreas than in techniques using anesthetized rats (5, 8, 12, 15) but smaller than in conscious rats (6 ,7 ,2 2 ). An important advantage of this technique is that the effects of anesthesia, and the influence of nerves and other organs are excluded. Furthermore, an open circuit eliminates all the hormones and by-products which might be released by the preparation itself; so stimulation can only be exogenous and cannot be recirculated: this might explain a greater Dso of secretin with ou; preparation than that calculated by Debas and Grossman (4) in conscious dogs. Other prepa
Tournut/Estival/ Vaysse/Pascal/R ibet
336
rations (15, 19, 23) derived from the same principle offer the same advantages but are more time-consuming and complicated for the surgical preparation. The great sensitivity of the isolated perfused rat pancreas might prove useful in the bioassay of secretin (16). Its sensitivity might even be enhanced by addi tion of theophyllin into the perfusion fluid, as suggested by Scratcherd (20) and Scratcherd et al (21). In conclusion, the method of isolated rat pancreas per fused in situ appears to be suitable and highly sensitive for the study of the effects of gastrointestinal hormones and related peptides upon the exocrine of pancreatic secretagogues.
References
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/29/2018 1:58:09 AM
1 Augier, D Boucard, J.P.: Pascal, J.P.; Ribet, A„ and Vaysse, N.: Relationship between blood flow and secretion in the isolated perfused canine pancreas. J. Physiol., Lond. 221: 55-69 (1972). 2 Campagne, R.N. and Gruber, M.: Aminoacid and energy requirement of protein syn thesis in rat pancreatic tissu in vitro. Biochim. biophys. Acta 55: 353-360 (1962). 3 Case, R.M.; Harper, A.A., and Scratcherd, T.: Water and electrolyte secretion by the perfused pancreas of the cat. J. Physiol., Lond. 196: 133-149 (1968). 4 Debas, H.T. and Grossman, M.I.: Pure cholecystokinin: pancreatic protein and bicar bonate response. Digestion 9: 469-481 (1973). 5 Debray, C ; Tour, J. de la: Vaille, C.; Roze, C. et Souchard, M.: Contribution à l’étude de la sécrétion biliaire et pancréatique externe chez le rat. J. Physiol., Paris 54: 459499 (1962). 6 De Smul, A.; De Waele, B.; Wissocq, P., and Kickens, R.: Exogenous and endogenous secretion stimulation in the conscious rat. Digestion 11 : 39-50 (1974). 7 De Waele, B.; De Smul, A.; Wissocq, P. et Kickens, R.: La sécrétion pancréatique chez le rat. Influence de l’intervention chirurgicale, de la narcose, de l’hypothermie et de la dérivation du suc gastrique ou du suc pancréatique. Biol. Gastro-Enterol. 1: 253-263 (1974). 8 Dockray, G.J.: The action of secretin, cholecystokinin-pancreozymin and caerulein on pancreatic secretion in the rat. J. Physiol., Lond. 225: 679-692 (1972). 9 Dowd, J.E. and Riggs, D.S.: A comparison of estimate of Michaelis-Menten kinetic constants from various linear transformations. J. biol. Chem. 240: 863-869 (1965). 10 Figarella, C.; Souville, C; Quel, O.; Michel, R. et Sarles, 11. : Problèmes posés par le dosage des enzymes pancréatiques dans le suc duodénal. Vie méd. 47: 493-500 (1966). 11 Fleisher, K. ànd Kasper, H. : Exocrine pancreatic function in uraemic rats. Acta hepato-gastro-enter. 21: 398-403 (1974). 12 Folsch, U.R. and Wormsley, K.G.: Pancreatic enzyme response to secretin and chole cystokinin in the rat. J. Physiol., Lond. 234: 79-94 (1973). 13 Grenier, J.F.; Gillet, M.; Kachelhoffer, J.: Wong, P.; Barth, A.M.; Randrianarivo, P.; Stocbner, P.; Weiss, A.G.; Moody, A.J.; Fries, A.V. et Sundby, F.: Circulation extracorporelle du massif duodéno-pancréatique chez le chien. Technique, fonctionnement, intérêt. Annls Chir. thorac. cardiovasc. 8: 349-360 (1969). 14 Hermon Taylor, J.: A technique for perfusion of the isolated canine pancreas: response to secretin and gastrin. Gastroenterology 55: 488-501 (1968).
/n-sf'fu-Isolated Perfused Rat Pancreas 15 16 17 18
19
20 21
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Dr. R. Tournut. Groupe de Biologie et Pathologie digestive, Inserm U-151, Bâtiment L3, CHU Rangueil, Chemin du Vallon, F -31400 Toulouse (F'rance)
