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The effect of urethane anesthetic on basal pancreatic secretion in the rat after acute and chronic pancreatic duct cannulation
April 1995
• THE EFFECT OF URETHANE ANESTHETIC ON BASAL PANCREATIC SECRETION IN THE RAT AFTER ACUTE AND CHRONIC PANCREATIC DUCT CANNULATION. DC Whitcomb. ET Curtis, YB Huh, A Spanangel, A Sved. Depts. Medicine and Neuroscience, Univ. Pittsburgh, VAMC Pittsburgh, PA and Dept. Physiology, Univ. Texas Health Science Center, San Antonio TX. Basal and stimulated pancreatic exocrine secretion in urethane anesthetized rats, after acute placement of pancreatic, biliary and intestinal cannulas, is about 10% of the secretion seen in conscious rats that have recovered from surgery. This profound effect has important implications for the study of the mechanism controlling pancreatic secretion. However some experiments require that the rat is anesthetized (e.g. microinjection of regulatory peptides into the brainstem). AIM The aim of this study is to determine whether the diminished fluid output in anesthetized rat with acutely cannulated pancreatic duct is due primarily to the surgery or the anesthetic. M E T H O D S Male rats were divided into two groups and prepared with pancreatic, biliary, duodenal and intravenous cannulas. Pancreatic juice & bile was collected and continuously returned to the intestine. Basal pancreatic exocrine secretion was measured immediately after surgery under urethane anesthesia (1.2 mg/kg IV), and after recovery from surgery (3 days) + urethane. Cloralose (55 mg/kg) was also tested. RESULTS: Basal pancreatic fluid secretion in conscious recovered rats was 7.17+0.55 ~tl/min. (n=7). Basal pancreatic fluid secretion immediately after surgery under urethane anesthesia averaged 0.47+0.06 i.tl/min. (n=l 1) and represented a 93% reduction in basal pancreatic secretion. Infusion of urethane in recovered rats resulted in a 30 to 40% reduction in basal fluid and protein secretion. On the other hand, chloralose anesthesia (55 mg/kg) resulted in only a 9% reduction in basal pancreatic fluid secretion (n=4). Urethane also blunted CCK-8 and 2~ deoxyglucose stimulated pancreatic secretion. CONCLUSION: Basal pancreatic secretion is altered by immediate surgery and urethane anesthesia, although immediate surgery appears to have the greatest impact of basal pancreatic secretion. Since normal pancreatic secretion requires normal neural input, anesthetics with minimal effects on the autonomic nervous system should be evaluated in order to study neurohormonal control mechanisms regulating pancreatic secretion. (NIH R29 DK45781, Dr. Whitcomb)
• G R A N U L O C Y T E A C T I V A T I O N OCCURS EARLY IN ACUTE P A N C R E A T I T I S (AP). A.L. Widdison, R.E. May. Dept of Surgery, F r e n c h a y Hospital, Bristol, England. M u l t i p l e o r g a n failure (MOF) frequently complicates severe AP. A c t i v a t e d granulocytes are important in the p a t h o g e n e s i s of MOF. We investigated g r a n u l o c y t e function early in AP. M e t h o d s : V e n o u s b l o o d was taken from patients with AP w i t h i n 48 hrs. Granulocytes were separated and zymosan stimulated luminol enhanced c h e m i l u m i n e s c e n c e (a m e a s u r e of oxidative metabolism), c o m p l e m e n t m e d i a t e d antibody independent opsinisation, chemotactic activity toward endotoxin, and random motility, measured. Data was e x p r e s s e d as percent of controls, m e d i a n ( i n t e r - q u a r t i l e range) and compared using Mann W h i t n e y test. Results: 14 p a t i e n t s with mild (Imrie score <3) and 6 w i t h severe AP were of similar age, sex and aetiology. In p a t i e n t s w i t h severe AP APACHE scores w e r e h i g h e r (15(5) vs 6(5) in mild AP, p
Pancreatic Disorders
A401
• CIRCULATING L Y M P H O C Y T E S EARLY IN ACUTE PANCREATITIS (AP). A.L. Widdison, R.E. May. Dept of Surgery, F r e n c h a y Hospital, Bristol, England. Depressed immune function m a y increase the risk of infection in AP. Lymphocytes are key components of the immune system. We measured circulating lymphocyte counts, T cell subsets and T lymphocyte a c t i v a t i o n early in AP. Methods: V e n o u s blood was taken from AP patients w i t h i n 48 hrs. Lymphocytes were isolated and surface a n t i g e n expression measured using indirect immunofluorescence. Data given as mean number of cells x 109/L ± standard error of mean and c o m p a r e d u s i n g M a n n W h i t n e y test. Results: 20 controls, 14 patients with mild AP (Imrie <3) and 6 w i t h severe AP were studied. Age, sex and aetiologies of AP were similar. Two patients w i t h severe AP died. Interleukin 2 receptor e x p r e s s i o n was increased in mild AP (5±1%) and severe AP (14±6%) (P<0.05 vs controls) suggesting lymphocyte activation. Imrie Total count CD3 CD4 CD8 Controls 2.1±0.i 1.5±0.1 0.9±0.1 0.6±0.0 Mild 1.5±0.2 A 1.0±0.2 A 0.6±0.I A 0.4±0.1 A Severe 0.8±0.i AB 0.4±0.i AB 0.2±0.0 AB 0.2±0.0 AB A P<0.05 VS controls, B P
• EVIDENCE THAT CCK SHORT PEPTIDES EVOKE Ca2+ SIGNALING AND PANCREATIC AMYLASE SECRETION BY MECHANISMS INDEPENDENT OF PHOSPHOLIPASE C AND A2 PATHWAYS. H Yoshida, Y Tsunoda, C Owyang. Department of Internal Medicine, University of Michigan, Ann Arbor, MI. CCK-8 and its analogue JMV-180 utilize different 2nd messenger systems to evoke Caz+ signaling and pancreatic amylase secretion. Key amino acids to activate phospholipase (PLC) pathways are Met28 (or Thr28) whilst Nle28 is critical to evoke phospholipase A2 (PLA2) cascade. This study further determined the structural requirements critical for CCK peptides to stimulate amylase secretion. Using dispersed rat pancreatic acini, we showed that CCK-6 (Met28-Gly29-Trp30-Met31-Asp32-Phe33NI-12), CCK-5 (29-33) and CCK-4 (30-33) stimulated monophasic amylase secretion with EC50s of 3, 20 and 30 riM, respectively. In comparison, CCK-7 was much more potent with an EC50 of 0.7 pM whereas CCK-3 (31-33) failed to evoked amylase secretion. This suggests that Tyr [SO3H]27 and Trp30 are key amino acids for biological activity of the CCK peptides. Furthermore, neither ely-extended CCK-8 (or 5)-OH nor CCK-5-OH caused amylase secretion indicating the importance of the Phe33 amidation. Similar to other CCK peptides, CCK-6, -5 and -4 all caused Ca2+ oscillations. We next investigated the 2rid messenger systems utilized by these short peptides. In contrast to CCK-8 or JMV-180, neither the PLC inhibitor (U73122, 5 IxM) nor the PLA2 inhibitor (ONO-RS-082, o10gM) inhibited amylase secretion induced by CCK-6, -5 or -4 (10-12-10 °M). Similarly, the protein tyrosine kinase (PTK) inhibitor (genistein 100 p-M) also failed to affect amylase secretion stimulated by these short peptides. In separate studies, we showed that in contrast to CCK-8 which stimulated IP3 and PTK activities or JMV-180, which increased intracellular arachidonic acid levels, CCK-6, -5 and -4 (10-12-10-9M) failed to stimulate IP3 and arachidonic acid production as well as PTK activity. On the other hand, amylase secretion stimulated by these peptides was dependent on intracellular Ca2+ since the intracellular Ca2+ chelator BAPTA totally abolished their actions. In conclusion, our studies demonstrated that Tyr[SO3H]27 and Trp30 of the CCK peptides are key amino acids in evoking amylase secretion. In contrast to CCK-8 or JMV180, short CCK peptides like CCK-6, -5 and -4 stimulate pancreatic secretion by novel intracellular mechanism(s) which are independent of the PLC, PLA2 and PTK pathways. This provides the framework for understanding the structure-function relationship for CCK-like peptides and the signal transduction pathways.
• THE EFFECT OF URETHANE ANESTHETIC ON BASAL PANCREATIC SECRETION IN THE RAT AFTER ACUTE AND CHRONIC PANCREATIC DUCT CANNULATION. DC Whitcomb. ET Curtis, YB Huh, A Spanangel, A Sved. Depts. Medicine and Neuroscience, Univ. Pittsburgh, VAMC Pittsburgh, PA and Dept. Physiology, Univ. Texas Health Science Center, San Antonio TX. Basal and stimulated pancreatic exocrine secretion in urethane anesthetized rats, after acute placement of pancreatic, biliary and intestinal cannulas, is about 10% of the secretion seen in conscious rats that have recovered from surgery. This profound effect has important implications for the study of the mechanism controlling pancreatic secretion. However some experiments require that the rat is anesthetized (e.g. microinjection of regulatory peptides into the brainstem). AIM The aim of this study is to determine whether the diminished fluid output in anesthetized rat with acutely cannulated pancreatic duct is due primarily to the surgery or the anesthetic. M E T H O D S Male rats were divided into two groups and prepared with pancreatic, biliary, duodenal and intravenous cannulas. Pancreatic juice & bile was collected and continuously returned to the intestine. Basal pancreatic exocrine secretion was measured immediately after surgery under urethane anesthesia (1.2 mg/kg IV), and after recovery from surgery (3 days) + urethane. Cloralose (55 mg/kg) was also tested. RESULTS: Basal pancreatic fluid secretion in conscious recovered rats was 7.17+0.55 ~tl/min. (n=7). Basal pancreatic fluid secretion immediately after surgery under urethane anesthesia averaged 0.47+0.06 i.tl/min. (n=l 1) and represented a 93% reduction in basal pancreatic secretion. Infusion of urethane in recovered rats resulted in a 30 to 40% reduction in basal fluid and protein secretion. On the other hand, chloralose anesthesia (55 mg/kg) resulted in only a 9% reduction in basal pancreatic fluid secretion (n=4). Urethane also blunted CCK-8 and 2~ deoxyglucose stimulated pancreatic secretion. CONCLUSION: Basal pancreatic secretion is altered by immediate surgery and urethane anesthesia, although immediate surgery appears to have the greatest impact of basal pancreatic secretion. Since normal pancreatic secretion requires normal neural input, anesthetics with minimal effects on the autonomic nervous system should be evaluated in order to study neurohormonal control mechanisms regulating pancreatic secretion. (NIH R29 DK45781, Dr. Whitcomb)
• G R A N U L O C Y T E A C T I V A T I O N OCCURS EARLY IN ACUTE P A N C R E A T I T I S (AP). A.L. Widdison, R.E. May. Dept of Surgery, F r e n c h a y Hospital, Bristol, England. M u l t i p l e o r g a n failure (MOF) frequently complicates severe AP. A c t i v a t e d granulocytes are important in the p a t h o g e n e s i s of MOF. We investigated g r a n u l o c y t e function early in AP. M e t h o d s : V e n o u s b l o o d was taken from patients with AP w i t h i n 48 hrs. Granulocytes were separated and zymosan stimulated luminol enhanced c h e m i l u m i n e s c e n c e (a m e a s u r e of oxidative metabolism), c o m p l e m e n t m e d i a t e d antibody independent opsinisation, chemotactic activity toward endotoxin, and random motility, measured. Data was e x p r e s s e d as percent of controls, m e d i a n ( i n t e r - q u a r t i l e range) and compared using Mann W h i t n e y test. Results: 14 p a t i e n t s with mild (Imrie score <3) and 6 w i t h severe AP were of similar age, sex and aetiology. In p a t i e n t s w i t h severe AP APACHE scores w e r e h i g h e r (15(5) vs 6(5) in mild AP, p
Pancreatic Disorders
A401
• CIRCULATING L Y M P H O C Y T E S EARLY IN ACUTE PANCREATITIS (AP). A.L. Widdison, R.E. May. Dept of Surgery, F r e n c h a y Hospital, Bristol, England. Depressed immune function m a y increase the risk of infection in AP. Lymphocytes are key components of the immune system. We measured circulating lymphocyte counts, T cell subsets and T lymphocyte a c t i v a t i o n early in AP. Methods: V e n o u s blood was taken from AP patients w i t h i n 48 hrs. Lymphocytes were isolated and surface a n t i g e n expression measured using indirect immunofluorescence. Data given as mean number of cells x 109/L ± standard error of mean and c o m p a r e d u s i n g M a n n W h i t n e y test. Results: 20 controls, 14 patients with mild AP (Imrie <3) and 6 w i t h severe AP were studied. Age, sex and aetiologies of AP were similar. Two patients w i t h severe AP died. Interleukin 2 receptor e x p r e s s i o n was increased in mild AP (5±1%) and severe AP (14±6%) (P<0.05 vs controls) suggesting lymphocyte activation. Imrie Total count CD3 CD4 CD8 Controls 2.1±0.i 1.5±0.1 0.9±0.1 0.6±0.0 Mild 1.5±0.2 A 1.0±0.2 A 0.6±0.I A 0.4±0.1 A Severe 0.8±0.i AB 0.4±0.i AB 0.2±0.0 AB 0.2±0.0 AB A P<0.05 VS controls, B P
• EVIDENCE THAT CCK SHORT PEPTIDES EVOKE Ca2+ SIGNALING AND PANCREATIC AMYLASE SECRETION BY MECHANISMS INDEPENDENT OF PHOSPHOLIPASE C AND A2 PATHWAYS. H Yoshida, Y Tsunoda, C Owyang. Department of Internal Medicine, University of Michigan, Ann Arbor, MI. CCK-8 and its analogue JMV-180 utilize different 2nd messenger systems to evoke Caz+ signaling and pancreatic amylase secretion. Key amino acids to activate phospholipase (PLC) pathways are Met28 (or Thr28) whilst Nle28 is critical to evoke phospholipase A2 (PLA2) cascade. This study further determined the structural requirements critical for CCK peptides to stimulate amylase secretion. Using dispersed rat pancreatic acini, we showed that CCK-6 (Met28-Gly29-Trp30-Met31-Asp32-Phe33NI-12), CCK-5 (29-33) and CCK-4 (30-33) stimulated monophasic amylase secretion with EC50s of 3, 20 and 30 riM, respectively. In comparison, CCK-7 was much more potent with an EC50 of 0.7 pM whereas CCK-3 (31-33) failed to evoked amylase secretion. This suggests that Tyr [SO3H]27 and Trp30 are key amino acids for biological activity of the CCK peptides. Furthermore, neither ely-extended CCK-8 (or 5)-OH nor CCK-5-OH caused amylase secretion indicating the importance of the Phe33 amidation. Similar to other CCK peptides, CCK-6, -5 and -4 all caused Ca2+ oscillations. We next investigated the 2rid messenger systems utilized by these short peptides. In contrast to CCK-8 or JMV-180, neither the PLC inhibitor (U73122, 5 IxM) nor the PLA2 inhibitor (ONO-RS-082, o10gM) inhibited amylase secretion induced by CCK-6, -5 or -4 (10-12-10 °M). Similarly, the protein tyrosine kinase (PTK) inhibitor (genistein 100 p-M) also failed to affect amylase secretion stimulated by these short peptides. In separate studies, we showed that in contrast to CCK-8 which stimulated IP3 and PTK activities or JMV-180, which increased intracellular arachidonic acid levels, CCK-6, -5 and -4 (10-12-10-9M) failed to stimulate IP3 and arachidonic acid production as well as PTK activity. On the other hand, amylase secretion stimulated by these peptides was dependent on intracellular Ca2+ since the intracellular Ca2+ chelator BAPTA totally abolished their actions. In conclusion, our studies demonstrated that Tyr[SO3H]27 and Trp30 of the CCK peptides are key amino acids in evoking amylase secretion. In contrast to CCK-8 or JMV180, short CCK peptides like CCK-6, -5 and -4 stimulate pancreatic secretion by novel intracellular mechanism(s) which are independent of the PLC, PLA2 and PTK pathways. This provides the framework for understanding the structure-function relationship for CCK-like peptides and the signal transduction pathways.