April 1995 Pancreatic Disorders A401

• 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 cannu- lated pancreatic duct is due primarily to the surgery or the anes- thetic. METHODS Male rats were divided into two groups and prepared with pancreatic, biliary, duodenal and intravenous cannu- las. 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 repre- sented 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 pan- creatic 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 evalu- ated in order to study neurohormonal control mechanisms regulating pancreatic secretion. (NIH R29 DK45781, Dr. Whitcomb)

• CIRCULATING LYMPHOCYTES EARLY IN ACUTE PANCREATITIS (AP). A.L. Widdison, R.E. May. Dept of Surgery, Frenchay Hospital, Bristol, England.

Depressed immune function may 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 activation early in AP.

Methods: Venous blood was taken from AP patients within 48 hrs. Lymphocytes were isolated and surface antigen expression measured using indirect immunofluorescence. Data given as mean number of cells x 109/L ± standard error of mean and compared using Mann Whitney test.

Results: 20 controls, 14 patients with mild AP (Imrie <3) and 6 with severe AP were studied. Age, sex and aetiologies of AP were similar. Two patients with severe AP died. Interleukin 2 receptor expression 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<O.02 vs mild AP. CD4/CD8 ratio was the same in mild AP (1.7±0.3), severe AP (1.7±0.5) and controls (1.5±0.1).

Conclusions: T lymphocytes are activated early in AP. This and the unchanged CD4/CD8 ratio suggest reduced circulating lymphocyte and T cell counts are due to sequestration not immune depression.

• GRANULOCYTE ACTIVATION OCCURS EARLY IN ACUTE PANCREATITIS (AP). A.L. Widdison, R.E. May. Dept of Surgery, Frenchay Hospital, Bristol, England.

Multiple organ failure (MOF) frequently complicates severe AP. Activated granulocytes are important in the pathogenesis of MOF. We investigated granulocyte function early in AP.

Methods: Venous blood was taken from patients with AP within 48 hrs. Granulocytes were separated and zymosan stimulated luminol enhanced chemiluminescence (a measure of oxidative metabolism), complement mediated antibody independent opsinisation, chemotactic activity toward endotoxin, and random motility, measured. Data was expressed as percent of controls, median(inter-quartile range) and compared using Mann Whitney test.

Results: 14 patients with mild (Imrie score <3) and 6 with severe AP were of similar age, sex and aetiology. In patients with severe AP APACHE scores were higher (15(5) vs 6(5) in mild AP, p<O.01), and organ failure (2(3) vs O(0) in mild AP, P<0.OI) more frequent. Two patients with severe AP died. In AP chemiluminescence was increased (189(322)% of controls, P=O.05), particularly in severe AP 257(301)% (P<0.05 vs controls). Complement mediated antibody independent opsinisation was normal (NS vs controls). Chemotactic activity was normal (109(20)% of controlsin mild, and 95(25)% in severe AP, NS). Random motility was 92(14)% of controls (P<0.01) and was inversely proportional to Imrie scores (r=-0.8, P=O.02).

Conclusions: Granulocytes are metabolically hyperactive early in AP. Reduced random motility reflected this. Chemotaxis and complement mediated opsinization were normal. In AP activated granulocytes may be important in causing MOF.

• EVIDENCE THAT CCK SHORT PEPTIDES EVOKE Ca 2+ 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 Ca z+ 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-Phe33- NI-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 Ca 2+ 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, o10 gM) 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 Ca 2+ since the intracellular Ca 2+ 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 JMV- 180, 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.