Clinical Science (1992) 83, 277-280 (Printed in Great Britain)
277
Rapid Communication
Pancreatic secretion of lysosomal enzymes stimulated by intraduodenal instillation of a liquid meal in rabbits T. HIRANO, T. MANABE, A.
K. SALUJA* and M. L. STEER*
First Department of Surgery. Faculty of Medicine, Kyoto University, Kyoto, japan, and *Department of Surgery. Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts, U.S.A. (Received 9 June 1992; accepted 3 July 1992)
1. Studies have been performed to determine the effect of intraduodenal food on pancreatic secretion of lysosomal enzymes. 2. Intraduodenal instillation of a liquid meal (3g/kg body weight; 15.3% protein, 19.7% fat, 59.7% carbohydrate) caused significant increases in pancreatic juice volume and pancreatic secretion of amylase and protein compared with basal values for 2 h after instillation in anaesthetized rabbits. 3. Intraduodenal instillation of a liquid meal also caused significant increases in pancreatic secretion of three lysosomal enzymes (cathepsin B, N-fi-acetylgalactosaminidase and N-acetyl-b-glucosaminidase) compared with basal values for 2 h after instillation. 4. In addition, there were significant correlations between cathepsin B secretion and amylase secretion (r=0.7764, P< 0.001) and between cathepsin B secretion and protein secretion (r=0.6216, P< 0.001), both in basal conditions and in response to the liquid meal. 5. These results are evidence for the localization of lysosomal enzymes in the secretory granules-zymogen granules in normal acinar cells, and also indicate that the pancreatic secretion of lysosomal enzymes is gut-hormone-regulated.
responsible for the pancreatic secretion of lysosomal enzymes in normal physiological states, such as in response to food intake. In this study, we report the stimulation by food intake of lysosomal enzyme secretion from the exocrine pancreas.
METH6DS Animals
Six New Zealand White rabbits, weighing about 2.5 kg, were obtained from Shizuoka Experimental Animal Supply (Shizuoka, Japan). They were kept in light-dark cycle regulated (light: 05.0G17.00 hours) and air-conditioned (23 rt 3°C) animal quarters in our institute. They were given free access to tap water and food (Oriental Rodent Chow, Tokyo, Japan), and were allowed to acclimatize to the standard laboratory conditions for at least 4 days. The rabbits were maintained throughout the study in accordance with the guidelines of the Committee on Animal Care of Kyoto University. Chemicals
INTRODUCTION
There have been several reports on the secretory profiles of lysosomal enzymes in cell lines [l-31, and it has been suggested that there may be a physiological role for lysosomal enzymes in biological fluids [4]. Most of these studies have been performed using non-polarized cell types and the results of these studies have indicated that, under most conditions, lysosomal enzymes are secreted in a constitutive manner [5, 61. However, little is known about the mechanisms
Isocal Meal (15.3% protein, 19.7% fat, 59.7% carbohydrate) was purchased from Mead Johnson (Evansville, IN, U.S.A.). Benzyloxycarbonyl-arginylarginine-P-naphthylamide was obtained from AG (Budendorf, Bachem Feinchemikalien Switzerland) and P-naphthylamine was purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.). 4-Methylumbelliferyl substrates for N-acetyl-Pglucosaminidase and N-P-acetylgalactosaminidase were purchased from Sigma Chemical Co. All other reagents were of the highest purity commercially available.
Key words: N-P-acetylgalaaoraminidase, N-acetyl-8-glucosaminidase, cathepsin 6, lysosomal enzymes, pancreatic juice. Correspondence: Dr Teuuya Hirano, First Department of Surgery, Faculty of Medicine, Kyoto University, 62-10 Shirhigatani-Teranomaecho, Sakyoku, Kyoto 606, Japan.
270
T. Hirano et al.
Animal preparation
Assays
After a 16 h fast, rabbits were anaesthetized with an intravenous injection of pentobarbital (30mg/kg initially, supplemented by periodic doses of lOmg/kg) and a cannula (PE-50; Clay Adams, Parsippany, NJ, U.S.A.) was introduced into the inferior vena cava via the right femoral vein for the venous line. After laparotomy, the pylorus was ligated and a drainage gastrostomy cannula (PE-70) was positioned. The pancreatic duct was cannulated (PE-50) extraduodenally, adjacent to the duodenum, and another cannula (PE-50) was positioned in the descending portion of the duodenum just distal to the pylorus. 1 After placement and exteriorization of various ' catheters, the abdominal wound was closed. The core temperature of the rabbits was maintained using a heating pad and overhead lamps, and during the experiments rabbits were infused with intravenous heparinized saline (150 mmol/l NaCI) at a rate of 1.58ml/h. After a 30min stabilization, the first l'h pancreatic juice fraction was collected as a basal value (B) and then liquid meal (Isocal Meal, 3g/kg body weight in 15ml of water) was instilled into the duodenum through the duodenostomy catheter over 15min using an infusion pump. After a basal sample collection, three further 1 h collections of pancreatic juice (D1, D, and D3) were obtained during and after the instillation of the liquid meal. For each fraction, protein concentration and the activities of the enzymes amylase, cathepsin By N-P-acetylgalactosaminidase and N-acetyl-Pglucosaminidase were measured. Protein secretion was expressed in mgh-'kg-' and all enzyme secretions were expressed in units h-lkg-'.
Amylase activity was measured by the method of Irie et al. [7], with blue starch (Shionogi Amylase A-Test; Shionogi, Osaka, Japan) as the substrate. One unit of activity was defined as that which liberated l m g of maltose from the substrate/min at 30°C. Cathepsin B activity was measured by the with method of McDonald & Ellis [S] benzyloxycarbonyl-arginyl-arginine-P-naphthylamide as the substrate, and one unit of activity was defied as that which liberated 1 nmol of P-naphthylaminel min at 37°C. The activities of N-/I-acetylgalactosaminidase (EC 3.2.1.53) and N-acetyl-Pglucosaminidase (EC 3.2.1.30) were measured by the method of Peters et al. [9]. One unit of these two enzymes was defined as that which hydroIysed 1nmol of substrate/min. Protein concentration was measured by the method of Lowry et al. [lo] with BSA as the standard. Statistical analysis
The results are reported as means )SEM for n determinations. The significance of changes was evaluated by analysis of variance and the Tukey procedure, and a P value of less than 0.05 was considered to be significant. RESULTS
Intraduodenal instillation of a liquid meal resulted in the stimulation of pancreatic juice secretion (By 0.124+0.010; D,, 0.273+0.018; D,, 0.230 )0.014; D,, 0.147 & 0.012 ml h - kg - l), and this increase (two- to three-fold compared with the
* T
ir*
** T
B
D,
Dz D,
Fig. I. Secretion of pancreatic juice (a), amylase (b) and protein (c) from the rabbit exocrine pancreas stimulated by intraduodenal instillation of a liquid meal. There were six rabbits in this experiment, and each rabbit produced one basal fraction (B) amd three post-stimulation fractions (DI, Dz and D,) each collected over I h. Values are means +SEH. Statistical significance: *P
277
Rapid Communication
Pancreatic secretion of lysosomal enzymes stimulated by intraduodenal instillation of a liquid meal in rabbits T. HIRANO, T. MANABE, A.
K. SALUJA* and M. L. STEER*
First Department of Surgery. Faculty of Medicine, Kyoto University, Kyoto, japan, and *Department of Surgery. Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts, U.S.A. (Received 9 June 1992; accepted 3 July 1992)
1. Studies have been performed to determine the effect of intraduodenal food on pancreatic secretion of lysosomal enzymes. 2. Intraduodenal instillation of a liquid meal (3g/kg body weight; 15.3% protein, 19.7% fat, 59.7% carbohydrate) caused significant increases in pancreatic juice volume and pancreatic secretion of amylase and protein compared with basal values for 2 h after instillation in anaesthetized rabbits. 3. Intraduodenal instillation of a liquid meal also caused significant increases in pancreatic secretion of three lysosomal enzymes (cathepsin B, N-fi-acetylgalactosaminidase and N-acetyl-b-glucosaminidase) compared with basal values for 2 h after instillation. 4. In addition, there were significant correlations between cathepsin B secretion and amylase secretion (r=0.7764, P< 0.001) and between cathepsin B secretion and protein secretion (r=0.6216, P< 0.001), both in basal conditions and in response to the liquid meal. 5. These results are evidence for the localization of lysosomal enzymes in the secretory granules-zymogen granules in normal acinar cells, and also indicate that the pancreatic secretion of lysosomal enzymes is gut-hormone-regulated.
responsible for the pancreatic secretion of lysosomal enzymes in normal physiological states, such as in response to food intake. In this study, we report the stimulation by food intake of lysosomal enzyme secretion from the exocrine pancreas.
METH6DS Animals
Six New Zealand White rabbits, weighing about 2.5 kg, were obtained from Shizuoka Experimental Animal Supply (Shizuoka, Japan). They were kept in light-dark cycle regulated (light: 05.0G17.00 hours) and air-conditioned (23 rt 3°C) animal quarters in our institute. They were given free access to tap water and food (Oriental Rodent Chow, Tokyo, Japan), and were allowed to acclimatize to the standard laboratory conditions for at least 4 days. The rabbits were maintained throughout the study in accordance with the guidelines of the Committee on Animal Care of Kyoto University. Chemicals
INTRODUCTION
There have been several reports on the secretory profiles of lysosomal enzymes in cell lines [l-31, and it has been suggested that there may be a physiological role for lysosomal enzymes in biological fluids [4]. Most of these studies have been performed using non-polarized cell types and the results of these studies have indicated that, under most conditions, lysosomal enzymes are secreted in a constitutive manner [5, 61. However, little is known about the mechanisms
Isocal Meal (15.3% protein, 19.7% fat, 59.7% carbohydrate) was purchased from Mead Johnson (Evansville, IN, U.S.A.). Benzyloxycarbonyl-arginylarginine-P-naphthylamide was obtained from AG (Budendorf, Bachem Feinchemikalien Switzerland) and P-naphthylamine was purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.). 4-Methylumbelliferyl substrates for N-acetyl-Pglucosaminidase and N-P-acetylgalactosaminidase were purchased from Sigma Chemical Co. All other reagents were of the highest purity commercially available.
Key words: N-P-acetylgalaaoraminidase, N-acetyl-8-glucosaminidase, cathepsin 6, lysosomal enzymes, pancreatic juice. Correspondence: Dr Teuuya Hirano, First Department of Surgery, Faculty of Medicine, Kyoto University, 62-10 Shirhigatani-Teranomaecho, Sakyoku, Kyoto 606, Japan.
270
T. Hirano et al.
Animal preparation
Assays
After a 16 h fast, rabbits were anaesthetized with an intravenous injection of pentobarbital (30mg/kg initially, supplemented by periodic doses of lOmg/kg) and a cannula (PE-50; Clay Adams, Parsippany, NJ, U.S.A.) was introduced into the inferior vena cava via the right femoral vein for the venous line. After laparotomy, the pylorus was ligated and a drainage gastrostomy cannula (PE-70) was positioned. The pancreatic duct was cannulated (PE-50) extraduodenally, adjacent to the duodenum, and another cannula (PE-50) was positioned in the descending portion of the duodenum just distal to the pylorus. 1 After placement and exteriorization of various ' catheters, the abdominal wound was closed. The core temperature of the rabbits was maintained using a heating pad and overhead lamps, and during the experiments rabbits were infused with intravenous heparinized saline (150 mmol/l NaCI) at a rate of 1.58ml/h. After a 30min stabilization, the first l'h pancreatic juice fraction was collected as a basal value (B) and then liquid meal (Isocal Meal, 3g/kg body weight in 15ml of water) was instilled into the duodenum through the duodenostomy catheter over 15min using an infusion pump. After a basal sample collection, three further 1 h collections of pancreatic juice (D1, D, and D3) were obtained during and after the instillation of the liquid meal. For each fraction, protein concentration and the activities of the enzymes amylase, cathepsin By N-P-acetylgalactosaminidase and N-acetyl-Pglucosaminidase were measured. Protein secretion was expressed in mgh-'kg-' and all enzyme secretions were expressed in units h-lkg-'.
Amylase activity was measured by the method of Irie et al. [7], with blue starch (Shionogi Amylase A-Test; Shionogi, Osaka, Japan) as the substrate. One unit of activity was defined as that which liberated l m g of maltose from the substrate/min at 30°C. Cathepsin B activity was measured by the with method of McDonald & Ellis [S] benzyloxycarbonyl-arginyl-arginine-P-naphthylamide as the substrate, and one unit of activity was defied as that which liberated 1 nmol of P-naphthylaminel min at 37°C. The activities of N-/I-acetylgalactosaminidase (EC 3.2.1.53) and N-acetyl-Pglucosaminidase (EC 3.2.1.30) were measured by the method of Peters et al. [9]. One unit of these two enzymes was defined as that which hydroIysed 1nmol of substrate/min. Protein concentration was measured by the method of Lowry et al. [lo] with BSA as the standard. Statistical analysis
The results are reported as means )SEM for n determinations. The significance of changes was evaluated by analysis of variance and the Tukey procedure, and a P value of less than 0.05 was considered to be significant. RESULTS
Intraduodenal instillation of a liquid meal resulted in the stimulation of pancreatic juice secretion (By 0.124+0.010; D,, 0.273+0.018; D,, 0.230 )0.014; D,, 0.147 & 0.012 ml h - kg - l), and this increase (two- to three-fold compared with the
* T
ir*
** T
B
D,
Dz D,
Fig. I. Secretion of pancreatic juice (a), amylase (b) and protein (c) from the rabbit exocrine pancreas stimulated by intraduodenal instillation of a liquid meal. There were six rabbits in this experiment, and each rabbit produced one basal fraction (B) amd three post-stimulation fractions (DI, Dz and D,) each collected over I h. Values are means +SEH. Statistical significance: *P
