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• DOES A WHEAT AMYLASE INHIBITOR (WAI) AFFECT INTRADUODENAL AMYLASE ACTIVITY IN HUMANS? A.Choudhury and E.P. DiMagno. GI Unit, Mayo Clinic, Rochester, MN

Previously we showed that a partially purified white bean amylase inhibitor decreased human amylase activity within the intestinal tract. In normal and type II diabetics, >90% amylase inhibition, was accompanied by slowing of carbohydrate absorption and gastric emptying, and decreased levels of postprandial plasma glucose and insulin (Gastroenterology 1986;91:41). WAI is a by product of gluten and wheat starch production. Therefore, WAI is a potentially inexpensive and easily available commercial source. In rats and dogs, we showed that WAI ingested for 3 to 9 wks reduces carbohydrate digestion and absorption and plasma insulin levels without producing pancreatic hypertrophy or hyperplasia. In addition WAI reduces food intake and weight gain in growing rats. Our aims were 1) to determine ifinfasion of WAI into the duodenum affects intraluminal amylase activity and plasma levels of glucose and hormones; and 2) to determine the amount of intraduodenal WAI needed to inhibit >90% of secreted amylase activity. Methods: After an overnight fast, gastric and duodenal tubes were placed under fluoroseopie control in 4 healthy persons (3 men and 1 woman, ages 25-45). The gastric tube, which had infusion and aspiration ports, was placed along the greater curve of the antrum. The duodenal infusion port was positioned at the junction of the descending and third parts of the duodenum and the sampling port was placed at the duodenojejunal junction. To achieve a 50% maximal stimulation of pancreatic enzyme secretion, a mixture of 118 mM essential amino acids was infused into the duodenum at a rate of 4 ml/min for the entire 270 min of the study. During the middle 90 min,WAl was added to the infusate to achieve intraduodenal concentrations of 3, 4.5 or 6 mg per ml. Throughout the study, 5-ml duodenal samples were obtained every 15 min for measurement of pancreatic amylase and markers. Blood samples were obtained every 15 min. for measurement of plasma glucose and hormones (insulin, neurotensin,and PYY). Results: lntraduodenal amylase activity was inhibited (p<O.03) by 57, 62 and 94% by infusion of 3, 4.5 and 6 mg/ml concentrations of WAI, respectively: Plasma glucose and concentrations of the hormones were not affected (p>0.19) by infusion of the essential amino acid infusion or WAI. Summary: Intradaodenal amylase activity is inhibited by WAI. The intraduodenal concentrations of WAl that will inhibit > 90% amylase activity should be between 4.5 and 6.0 mg/ml. Conclusion: WAI in doses 0f4.5 to 6.0 g with meals should inhibit postprandial intraduodenal amylase activity by >90%. In the absence of ingesting carbohydrate, WAI has no affect on plasma glucose, insulin or other hormones.

PANCREATIC FLUID HYPERSECRETION IN ACUTE EXPERIMENTAL PANCREATITIS. Laszlo Czako. and Makoto Otsuki. Third Dept. Int. Med., University of Occupational and Environmental Health, Japan, School of Medicine, Kitakyushu, Japan.

B a c k g r o u n d / A i m : There are only few reports concerning pancreatic secretory function in the early phase of acute pancreatitis, although drugs and procedures inhibiting pancreatic secretion have been used. Our aim was, therefore, to evaluate pancreatic exocrine function at specified time points after acute pancreatitis in two different experimental models. M e t h o d s : Acute pancreatitis was induced in rats by a total of four s.c. injections of 20/ . tg/kg body wt cerulein (Cn) at hourly intervals or by retrograde intraductal infusion of 40 ktl/100 g body wt o f 3% sodium taurocholate (NaTc). Basal, Cn- and secret in-st imulated pancreat ic exocrine secretion was determined under urethaue anesthesia. Results: Basal pancreatic juice (PJ) flow at 6 h after the first Cn injection was significantly elevated (27.6 + 3.7 vs 16.8 + 2.0 111/30 rain in control, P<0.01) and further increased with time reaching its peak at 24 h (105.1 -+ 4.6 111/30 min). Thereafter it gradually decreased and normalized on day 7. Similar results were obtained in NaTc-iuduced postpancreatitic rats (58.7 + 6.4 vs 15.2 + 2.6 111/30 min in sham-operated control, P<0.001). Intravenous infusion of loxiglumide, atropine, or anti-secretin serum was unable to modify the elevated basal PJ. Loxiglumide, even when given 8 and 16 h after the ftrst Cn injection, could not inhibit the PJ hypersecretion seen at 24 h. However, loxiglumide when given 30 min before the first Cn injection markedly reduced the PJ hypersecretion and ameliorated acute pancreat i t is . The vascular and ducta l permeabi l i ty evaluated by extravasation of Evans blue dye was significantly increased in the NaTc- pancreatitis but it was not seen in Cn model at 24 h after pancreatitis. Cn (0.038-9.84 nmol/kg body wt/h) when given early after acute pancreatitis could not stimulate fur ther increase in PJ but supramaximal doses decreased it. Secretin (0.20-25.76 nmol/kg body wt/h) stimulated dose- dependent increases in PJ and bicarbonate concentrations in both models, a l though the secretory responsiveness o f bicarbonate was markedly decreased compared with the control rats. Conelusion: These results indicate that in the postpancreatitic state basal juice f low increases not by an increased permeabil i ty but by some unknown mechanism other than CCK, secretin, or cholinergic mechanisms and that the juice flow from the damaged pancreas was resistant to Cn but not to secretin stimulation.

• STIMULATION OF BOTH CCKA AND CCKB RECEPTORS ACTIVATES MAP KINASES IN AR42J AND RECEPTOR TRANSFECTED CHO CELLS. A. Dabrowski, K.M. Detjen, C.D. Logsdon & J.A. Williams. Depts of Physiology and Internal Medicine, Univ. of Michigan, Ann Arbor, M148109.

It was recently found that CCK activates MAP kinases in isolated rat pancreatic acini. CCK is known to interact with two types of receptors - CCKA and CCKB/gastrin. The aim of the present study was therefore to evaluate whether one or both types of CCK receptors are capable of MAP kinase activation. We used the pancreatic AR42J acinar cell line as well as CHO cells transfected with CCKA or CCKB receptors. In serum starved AR42J cells stimulation with 10 nM CCK~ caused a time-dependent increase in MAP kinase activity, as assessed by an in-gel kinase assay using myelin basic protein as substrate, which was greater than the response to 10% fetal bovine serum. CCK significantly increased p44 [MAPK1] and p42 [MAPK2] kinase activity within 1 min, with a maximal activation [16-fold and 7-fold respectively] observed after 5 rain. Although the response subsequently declined, it was still elevated at 30 min. AR42J cells were then incubated for 5 min with different doses of CCK or gasttinl7 I [(3]. Minimal, one-half maximal and maximal responses were observed at 30 pM, 500 pM and 10 nM respectively, after CCK stimulation, and at 100 pM, 1.5 nM and 30 nM respectively, after G stimulation. Stimulation with 30 nM G activated MAPK1 and MAPK2 activities similar to l0 nM CCK. To distinguish the effects of different CCK receptors on MAP kinases activation, the cells were pretrented for 5 min with 100 nM of the CCKA receptor inhibitor L364,718 or/and the CCKs receptor inhibitor L365,260 and then stimulated for 5 min with 1 nM CCK or 3 nM G. It was found that L365,260 almost totally blocked MAP kinases activation in AR42J cells after stimulation with G and substantially reduced roy 77%] the activation of both kinases by CCK, while the effect of L364,718 was much less. The combination of both inhibitors totally prevented MAP kinases activation by CCK. In an alternative approach, stably transfeoted CHO cells bearing either CCKA or CCKB receptors were stimulated with l0 nM CCK. Each receptor induced a time-dependent increase in activity of both MAP kinases. The maximal response was observed after 10 rain with MAPK1 and MAPK2 activity being increased by 8-fold and 6-fold respectively in CCKA and by 7-fuld and 4-fold in CCKB beating cells. This represented about 30% of the maximal stimulation observed after stimulation with 10% fetal bovine serum in this cell type. In conelnsion, stimulation of both CCKA and CCKB receptors activates MAP kinases in AR42J cells and in transfected CHO ceils.

ENHANCED EXPRESSION OF APOPTOTIC FACTORS IMPLICATE A ROLE IN HUMAN PANCREATIC CANCER

J. Deflorin, H. Friess, R. Schmid*, L. Zs. Horvath, M. Korc +, G. Adler*, M.W. Bfchler. Department of Visceral and Transplantation Surgery, University of Berne, Switzedand; *Department of Gastroenterology, University of UIm, Germany; +Departments of Medicine and Biological Chemistry, University of California Irvine, USA

Apoptosis also occurs spontaneously in malignant tumors, often markedly retarding their growth, and it is increased in tumors responding to physical and chemical treatments. Pancreatic cancer is known for its aggressiveness, its poor prognosis and its unresponsiveness to chemo- and radiotherapy. As drug resistance in cancer is thought to be a resistance to apoptosis, the investigation of apoptotic factors in pancreatic carcinomas is of great interest and could supply us some additional information about its rapid progression in particular, and a better understanding of drug resistance in cancer in general. Apoptosis is a complex process which is regulated by different genes, some of which are descnbed to date. BCI-2 and Bcl-xL belong to a proto-oncogene family that inhibits apoptosis. Contrary, overexpression of Bax accelerates apoptofic death as does Apo-1, when cross-linked with anti-Apo-l-antibodies. Patients: Cancerous tissue samples were obtained from 6 female and 8 male patients with a median age of 58 years (range: 36-73 years), undergoing surgery for pancreatic cancer. Normal human pancreatic tissue samples were obtained from healthy individuals through an organ donor program. Methods: Tissue samples were frozen in liquid nitrogen immediately after surgical removal and stored at -80°C until use. Additionally tissue samples were fixed in Bouin solution and paraffin embedded for histological analysis. Expression of Bcl- xL, Bax and Apo-1 was determined by Northern blot analysis using specific cRNA probes. Immunohistocbemical analysis was performed with specific monoclonal antibodies. Results: Northern blot analysis revealed a marked increase in Bax mRNA in the cancer samples compared with the normal control tissue. BCI-xL mRNA expression was less, but clearly increased in pancreas carcinomas compared with normal tissue samples. In contrast Apo-1 seems to be expressed in very little amounts in normal controls as well as in cancerous tissue and is hardly detectable on Northern blots containing 20p.g total RNA. For Bcl-2 the experiments are in progress and therefore, no final results are available. Conclusions: These preliminary results indicate that the apoptosis inducing factor Bax is overexpressed in pancreatic cancer in comparison with normal controls, in addition, Bci-xL which has been reported as an apoptosis inhibiting factor was also expressed at higher levels in cancerous tissues. These findings indicate that apoptosis may be inhibited in pancreatic cancer. This disturbance in apoptosis might play a role in the pathogenesis of pancreatic cancer and might contribute to its aggressiveness.