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Subcellular redistribution of lysosomal enzymes in experimental acute pancreatitis is associated with increased protease activation in the intraaceinar zymogen compartment
A376
AGA ABSTRACTS
CHANGES OF S E R U M A M Y L A S E AND SYMPTOMS IN ERCP INDUCED ACUTE PANCREATITIS. A.Mirmiran-Yazdy, S. Bank, CQ Pan, B. Stark, D i v i s i o n of Gastroenterology, Long Island Jewish Medical Center, New Hyde Park, NY, the L o n g Island Campus for Albert E i n s t e i n College of Medicine. Backqround: E n d o s c o p i c Retrograde C h o l a n g i o p a n c r e a t o g r a p h y (ERCP) induced acute p a n c r e a t i t i s has been reported with an incidence of 1% to 17% of patients having the procedure. P u b l i s h e d s t u d i e s have s h o w n that h y p e r a m y l a s e m i a occurs as early as two hours after ERCP. The duration, peak t i m e and level of amylase that are a s s o c i a t e d with symptoms are uncertain. Methods: Our study reviewed the r e c o r d s o f 20 patients who had ERCP induced acute pancreatitis. The data include serum amylase levels at 6, 12~ 24 hours and at the following 24 hour intervals until the levels returned to normal; symptoms of fever~ new onset or new p a t t e r n of a b d o m i n a l pain, nausea, vomiting, and white cell count after ERCP. Results: All patients had h y p e r a m y l a s e m i a (>300 u/L) 6 hours after ERCP. In 80% of patients amylase levels p e a k e d at 12 hours and 20% at 6 hours. H y p e r a m y l a s e m ~ a ( > 3 0 0 u/L) remained e l e v a t e d a t 48 hours then returned to normal at 72 to 96 hours. The clinical p r e s e n t a t i o n v a r i e d in different cases except the abdominal pain. Our study showed that during the first 6 hours all patients (i00%) had abdominal pain, 80% d e v e l o p e d fever, 50% had an e l e v a t e d white b l o o d cell count, and 35% d e v e l o p e d nausea and vomiting. Most symptoms were con- c u r r e n t with hyperamylasemia. In 6 0 % o f patients symptoms p e r s i s t e d after serum amylase returned to normal. Conclusion: In our study~ the symptoms of E R C P - i n d u c e d acute p a n c r e a t i t i s a s s o c i a t e d with hyperamylasemia (>300 u/L) d e v e l o p e d w i t h i n 6 hours after the procedure. Implication: Patients having ERCP should be m o n i t o r e d for at l e a s t 6 hours after the procedure. New onset or n e w p a t t e r n of abdominal pain and h y p e r a m y l a s e m i a (>300 u/L) strongly suggest ERCP ~ induced acute pancreatitis. A s y m p t o m a t i c p a t i e n t s with normal amylase are u n l i k e l y to develop p a n c r e a t i t i s after 6 hours.
• SUBCELLULAR REDISTRIBUTION OF LYSOSOMAL ENZYMES IN EXPERIMENTAL ACUTE PANCREATITIS IS ASSOCIATED WITH INCREASED PROTEASE ACTIVATION IN THE INTRAACINAR ZYMOGEN COMPARTMENT. K. Mithffer, C. Femandez-del Casfillo, D.W. Rattnerl A.L. Warshaw. Department of Surgery, Massachusetts General Hospital, Boston, MA.
Ectopic activation of pancreatic zymogens is believed to be a key event in the pathogenesis of acute pancreatitis. Intracellular redistribution of lysosomal enzymes into the zymogen compartment has been suggested as a potential mechanism for activation of inactive protease precursors and possible initiating event in acute pancreatitis. To investigate the pathophysiologic relevance of lysosomal enzyme redistribution f o r premature protease activation, rats were randomly infused with 0.9% NaC1 or caerulein at 0.25#g/kg/h or 5#g/kg/h for periods from 15min to 3 hrs (n > 4 at each time point). Early changes of serum amylase activity, pancreatic wet/dry weight ratio, concentration of trypsinogen activation peptide (TAP) in pancreatic tissue homogenates, and distribution of cathepsin B activity and TAP in the subcellular compartments after subcellular fractionation were investigated, RESULTS: No significant changes were noted in animals infused with saline or 0.25#g/kg/h caerulein. 5#g/kg/h caerulein increased pancreatic TAP content within 15 min (P < 0.05) while significant hyperamylasemia and pancreatic edema developed within 30 rain. All parameters increased continuously for 3 hrs. Activity of Cathepsin B in the zymogen compartment increased continuously over the 3h period (P<0.01). Concomitantly, increasing TAP generation was noted in the zymogen compartment (P<0.05) which correlated with the observed changes of Cathepsin B activity in the same compartment (r=0.87, P<0.001). CONCLUSIONS: Trypsinogen activation represents an early event in caerulein-induced acute pancreatitis. Redistribution of lysosomal enzymes into the subcellular zymogen compartment is associated with increased intracompartmental trypsinogen activation. This finding supports the relevance of the redistribution phenomenon in the early intraacinar changes in acute experimental pancreatitis.
GASTROENTEROLOGY, Vol. 108, No. 4
• DOSE DEPENDENCE OF ECTOPIC PROTEASE ACTIVATION AND PANCREATIC INJURY IN HYPERCALCEMIA-INDUCED EXPERIMENTAL PANCREATITIS. K. Mithffer, T.W. Frick, C. Femandez-del Castillo, K.B. Lewandrowski, D.W. Rattner. A.L. Warshaw. Departments of Surgery and Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA.
Clinical reports have shown the association of high calcium doses with the development of pancreatic injury after cardiopulmoaary bypass. Recent experimental studies demonstrated development of acute pancreatitis with increased intrap~creatic trypsmogen activation after administration of high quantities of calcium. In order to investigate if this effect of calcium is limited to high doses, rats randomly received bolus injections of 2ml 0.9% NaCI. or CaCI2 at 50mg/kg, 100mg/kg, 150mg/kg or 200mg/kg (n=5 each). Changes of serum [Ca2+], serum amylase activity, pancreatic Trypsinogen Activation Peptide (TAP), and pancreatic wet/dry weight ratio were then evaluated at lh following CaCI2 administration. RESULTS: All investigated parameters remained unchanged after saline infusion. Serum [Ca2+] increased in proportion to the amount of infused CaCI2
o°O 0
" P
s, P
~t.5
~o
15o 200 Calcium Dose ( m g / k g )
0
+ P
100 200 Calcium Dose ( m g / k g )
(P< 0.001, r=0.96). Similarly, serum amylase activity and pancreatic wet/dry weight ratio both increased in a dose dependent manner (Figures). While tissue TAP was not significantly elevated after 50 mg/kg CaCI2 (1044-22 nM/L. g), a significant rise of TAP to 236 4-37, 247 4-60, and 293_+72 nM/L • g (P < 0.05) was observed lh after infusion of 100. 150. and 200 mg/kg, respectively. CONCLUSIONS: These results indicate a direct relationship between the degree of hypercalcemia and severity of biochemical and morphologic damage in the pancreas after acute hypercalcemic challenges.
• NEUROHORMONAL REGULATION OF PANCREATIC EXOCRINE F U N C T I O N IN A N E W R A T M O D E L W I T H O U T GENE E X P R E S S I O N O F C H O L E C Y S T O K I N I N - A RECEPTOR. K. ' M. M a s u d a , 1'. K a w a n a m i , A. Funakosh'iT. t o f Clinical P h y s i o l o g y , Tokyo Metropolitan nstitute of Gerontology, Tokyo-173, National Kyushu Cancer Center, F u k u o k a - 8 1 5 W e have reported that Otsuka L o n g - E v a n s T o k u s h i m a Fatty (OLETF) rats revealed none or less e x p r e s s i o n of cholecystokinin (CCK)-A receptor gene in the pancreas, and that the pancreas of this strain of rats did not respond C C K stimulation (BBRC, 1994). In the present study, we examined w h e t h e r the p a n c r e a t i c responses to hormonal and neural stimulations except for CCK in OLETF rats were maintained. Methods: The rats were prepared with cannula draining bile and pancreatic juice, separately and with a duodenal cannula and a jugular v e i n cannula. In Some animals, an additional cannula was inserted into the left lateral ventricle. T h e p a n c r e a t i c r e s p o n s e s t o i n t r a v e n o u s injection o f neuromedin C, secretin and acetylcholine, to intraduodenal injection of capsaicin (013 mg/rat) which stimulated vagal afferent nerve, to cerebroventricular injection of TRH which stimulates vagal efferent nerve and to intragastric injection of liquid meal, were examined in vivo without anesthesia, and compared with t h e control ( L o n g - E v a n s - T o k u s h i m a = LETO) rats. Moreover, the gene expres.stons of CCK receptors in the small intestine were e x a m i n e d by RT-PCR method, because the distribution of CCK receptor binding sites has been reported in the vagal afferent nerve terminal. R e s u l t s : Pancreatic responses to graded d o s e s of intravenous injection of secretin arid acetylcholine, and to intracerebroventricular a d m i n i s t r a t i o n ' o f T R H were comparable for OLETF and LETO rats. H o w e v e r , responses to intravenous injection of neuromedin C to intraduodenal injection of capsaicin and to intragastric' injection of liquid meal were impaired in OLETF rats. T h e C C K - A recelJtor m R N A w a s not expressed in the small intestine of OLETF rats but was in LETO rats. Conclusions: T h e p a n c r e a t i c r e s p o n s e s to v a r i o u s stimulations i n OLETF rats were well conserved except for the case of involvement of CCK-A receptor functions.
AGA ABSTRACTS
CHANGES OF S E R U M A M Y L A S E AND SYMPTOMS IN ERCP INDUCED ACUTE PANCREATITIS. A.Mirmiran-Yazdy, S. Bank, CQ Pan, B. Stark, D i v i s i o n of Gastroenterology, Long Island Jewish Medical Center, New Hyde Park, NY, the L o n g Island Campus for Albert E i n s t e i n College of Medicine. Backqround: E n d o s c o p i c Retrograde C h o l a n g i o p a n c r e a t o g r a p h y (ERCP) induced acute p a n c r e a t i t i s has been reported with an incidence of 1% to 17% of patients having the procedure. P u b l i s h e d s t u d i e s have s h o w n that h y p e r a m y l a s e m i a occurs as early as two hours after ERCP. The duration, peak t i m e and level of amylase that are a s s o c i a t e d with symptoms are uncertain. Methods: Our study reviewed the r e c o r d s o f 20 patients who had ERCP induced acute pancreatitis. The data include serum amylase levels at 6, 12~ 24 hours and at the following 24 hour intervals until the levels returned to normal; symptoms of fever~ new onset or new p a t t e r n of a b d o m i n a l pain, nausea, vomiting, and white cell count after ERCP. Results: All patients had h y p e r a m y l a s e m i a (>300 u/L) 6 hours after ERCP. In 80% of patients amylase levels p e a k e d at 12 hours and 20% at 6 hours. H y p e r a m y l a s e m ~ a ( > 3 0 0 u/L) remained e l e v a t e d a t 48 hours then returned to normal at 72 to 96 hours. The clinical p r e s e n t a t i o n v a r i e d in different cases except the abdominal pain. Our study showed that during the first 6 hours all patients (i00%) had abdominal pain, 80% d e v e l o p e d fever, 50% had an e l e v a t e d white b l o o d cell count, and 35% d e v e l o p e d nausea and vomiting. Most symptoms were con- c u r r e n t with hyperamylasemia. In 6 0 % o f patients symptoms p e r s i s t e d after serum amylase returned to normal. Conclusion: In our study~ the symptoms of E R C P - i n d u c e d acute p a n c r e a t i t i s a s s o c i a t e d with hyperamylasemia (>300 u/L) d e v e l o p e d w i t h i n 6 hours after the procedure. Implication: Patients having ERCP should be m o n i t o r e d for at l e a s t 6 hours after the procedure. New onset or n e w p a t t e r n of abdominal pain and h y p e r a m y l a s e m i a (>300 u/L) strongly suggest ERCP ~ induced acute pancreatitis. A s y m p t o m a t i c p a t i e n t s with normal amylase are u n l i k e l y to develop p a n c r e a t i t i s after 6 hours.
• SUBCELLULAR REDISTRIBUTION OF LYSOSOMAL ENZYMES IN EXPERIMENTAL ACUTE PANCREATITIS IS ASSOCIATED WITH INCREASED PROTEASE ACTIVATION IN THE INTRAACINAR ZYMOGEN COMPARTMENT. K. Mithffer, C. Femandez-del Casfillo, D.W. Rattnerl A.L. Warshaw. Department of Surgery, Massachusetts General Hospital, Boston, MA.
Ectopic activation of pancreatic zymogens is believed to be a key event in the pathogenesis of acute pancreatitis. Intracellular redistribution of lysosomal enzymes into the zymogen compartment has been suggested as a potential mechanism for activation of inactive protease precursors and possible initiating event in acute pancreatitis. To investigate the pathophysiologic relevance of lysosomal enzyme redistribution f o r premature protease activation, rats were randomly infused with 0.9% NaC1 or caerulein at 0.25#g/kg/h or 5#g/kg/h for periods from 15min to 3 hrs (n > 4 at each time point). Early changes of serum amylase activity, pancreatic wet/dry weight ratio, concentration of trypsinogen activation peptide (TAP) in pancreatic tissue homogenates, and distribution of cathepsin B activity and TAP in the subcellular compartments after subcellular fractionation were investigated, RESULTS: No significant changes were noted in animals infused with saline or 0.25#g/kg/h caerulein. 5#g/kg/h caerulein increased pancreatic TAP content within 15 min (P < 0.05) while significant hyperamylasemia and pancreatic edema developed within 30 rain. All parameters increased continuously for 3 hrs. Activity of Cathepsin B in the zymogen compartment increased continuously over the 3h period (P<0.01). Concomitantly, increasing TAP generation was noted in the zymogen compartment (P<0.05) which correlated with the observed changes of Cathepsin B activity in the same compartment (r=0.87, P<0.001). CONCLUSIONS: Trypsinogen activation represents an early event in caerulein-induced acute pancreatitis. Redistribution of lysosomal enzymes into the subcellular zymogen compartment is associated with increased intracompartmental trypsinogen activation. This finding supports the relevance of the redistribution phenomenon in the early intraacinar changes in acute experimental pancreatitis.
GASTROENTEROLOGY, Vol. 108, No. 4
• DOSE DEPENDENCE OF ECTOPIC PROTEASE ACTIVATION AND PANCREATIC INJURY IN HYPERCALCEMIA-INDUCED EXPERIMENTAL PANCREATITIS. K. Mithffer, T.W. Frick, C. Femandez-del Castillo, K.B. Lewandrowski, D.W. Rattner. A.L. Warshaw. Departments of Surgery and Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA.
Clinical reports have shown the association of high calcium doses with the development of pancreatic injury after cardiopulmoaary bypass. Recent experimental studies demonstrated development of acute pancreatitis with increased intrap~creatic trypsmogen activation after administration of high quantities of calcium. In order to investigate if this effect of calcium is limited to high doses, rats randomly received bolus injections of 2ml 0.9% NaCI. or CaCI2 at 50mg/kg, 100mg/kg, 150mg/kg or 200mg/kg (n=5 each). Changes of serum [Ca2+], serum amylase activity, pancreatic Trypsinogen Activation Peptide (TAP), and pancreatic wet/dry weight ratio were then evaluated at lh following CaCI2 administration. RESULTS: All investigated parameters remained unchanged after saline infusion. Serum [Ca2+] increased in proportion to the amount of infused CaCI2
o°O 0
" P
s, P
~t.5
~o
15o 200 Calcium Dose ( m g / k g )
0
+ P
100 200 Calcium Dose ( m g / k g )
(P< 0.001, r=0.96). Similarly, serum amylase activity and pancreatic wet/dry weight ratio both increased in a dose dependent manner (Figures). While tissue TAP was not significantly elevated after 50 mg/kg CaCI2 (1044-22 nM/L. g), a significant rise of TAP to 236 4-37, 247 4-60, and 293_+72 nM/L • g (P < 0.05) was observed lh after infusion of 100. 150. and 200 mg/kg, respectively. CONCLUSIONS: These results indicate a direct relationship between the degree of hypercalcemia and severity of biochemical and morphologic damage in the pancreas after acute hypercalcemic challenges.
• NEUROHORMONAL REGULATION OF PANCREATIC EXOCRINE F U N C T I O N IN A N E W R A T M O D E L W I T H O U T GENE E X P R E S S I O N O F C H O L E C Y S T O K I N I N - A RECEPTOR. K. ' M. M a s u d a , 1'. K a w a n a m i , A. Funakosh'iT. t o f Clinical P h y s i o l o g y , Tokyo Metropolitan nstitute of Gerontology, Tokyo-173, National Kyushu Cancer Center, F u k u o k a - 8 1 5 W e have reported that Otsuka L o n g - E v a n s T o k u s h i m a Fatty (OLETF) rats revealed none or less e x p r e s s i o n of cholecystokinin (CCK)-A receptor gene in the pancreas, and that the pancreas of this strain of rats did not respond C C K stimulation (BBRC, 1994). In the present study, we examined w h e t h e r the p a n c r e a t i c responses to hormonal and neural stimulations except for CCK in OLETF rats were maintained. Methods: The rats were prepared with cannula draining bile and pancreatic juice, separately and with a duodenal cannula and a jugular v e i n cannula. In Some animals, an additional cannula was inserted into the left lateral ventricle. T h e p a n c r e a t i c r e s p o n s e s t o i n t r a v e n o u s injection o f neuromedin C, secretin and acetylcholine, to intraduodenal injection of capsaicin (013 mg/rat) which stimulated vagal afferent nerve, to cerebroventricular injection of TRH which stimulates vagal efferent nerve and to intragastric injection of liquid meal, were examined in vivo without anesthesia, and compared with t h e control ( L o n g - E v a n s - T o k u s h i m a = LETO) rats. Moreover, the gene expres.stons of CCK receptors in the small intestine were e x a m i n e d by RT-PCR method, because the distribution of CCK receptor binding sites has been reported in the vagal afferent nerve terminal. R e s u l t s : Pancreatic responses to graded d o s e s of intravenous injection of secretin arid acetylcholine, and to intracerebroventricular a d m i n i s t r a t i o n ' o f T R H were comparable for OLETF and LETO rats. H o w e v e r , responses to intravenous injection of neuromedin C to intraduodenal injection of capsaicin and to intragastric' injection of liquid meal were impaired in OLETF rats. T h e C C K - A recelJtor m R N A w a s not expressed in the small intestine of OLETF rats but was in LETO rats. Conclusions: T h e p a n c r e a t i c r e s p o n s e s to v a r i o u s stimulations i n OLETF rats were well conserved except for the case of involvement of CCK-A receptor functions.