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Elimination of trypsin enzyme activity by hemodiafiltration
A488 AGA ABSTRACTS • G1986
THE RAS-RELATED SMALL GTP-BINDING PROTEIN RAB4 IS INVOLVED IN REGULATED EXOCYTOSIS OF RAT PANCREATIC ACINI. Hirohide Ohnishi, Tetsuya Mine, Tomohiro Tsuchida, and Toshiro Fujita. Department of Intemal Medicine IV, University of Tokyo School of Medicine, Tokyo, Japan. Rab4, a member of the Rab family of Ras-related small GTP-binding proteins, has recently been suggested to participate in exocytosis in addition to its well known role in recycling of GLUT4 and transferrin receptors. The present study was conducted to elucidate the function of Rab4 in regulated exocytosis. Western blotting showed Rab4 to be localized to zymogen granule membranes of pancreatic acini as well as to endosome rich fraction. When the synthetic peptide corresponding to the Rab4 hypervariable carboxyl-terminai domain was introduced into streptolysin-O permeabilized acini, the amylase release induced by 10 laM free calcium was potentiated by 80% at maximum in a concentration-dependent manner. By contrast, another synthetic peptide corresponding to the Rab3C hypervariable carboxyl-terminal domain showed no effect on calcium-induced amylase release from permeabilized acini. Furthermore, anti-Rab4 antibody enhanced calcium-triggered amylase release from permeabilized acini. In cntrast, non-immune IgG revealed no effect on calcium-induced amylase release from permeabilized acini. To further elucidate the involvement of Rab4 in the stimulus-secretion coupling, we examined the effect of CCK on GTP-binding rate to Rab4. GTP-binding rate was measured by introducing radiolabelled GTP into streptolysin-O permeabilized acini and immunoprecipitating GTP-bound Rab4. Stimulation of pancreatic acini with 100 pM CCK increased the incorporation of radiolabelled GTP into Rab4 by 75% compared to control. TPA also enhanced the GTP-binding rate to Rab4 in pancreatic acini. In addition, pretreatment of pancreatic acini with 1 ~tM calphostin C, a protein kinase C inhibitor, attenuated the stimulatory effect of CCK on GTP-binding to Rab4. These results suggest that Rab4 protein on zymogen granules plays a crucial role in regulated exocytosis and that Rab4 is involved in the stimulus-secretion coupling of pancreatic acini via protein kinase C dependent signaling pathway. G1987 REGIONAL STOP-FLOW CItEMOTHERAPY FOR NON-RESECTABLE PANCREATIC CARCINOMA. Ohshima S, Sakon M, Ohsato H*, Nakamori S, Aoki T, Higaki N, Yamada T, Hasuike Y**, Yoshikawa T**, Gotoh M, and Monden M. Department of Surgery II, Osaka University Medical School, **Osaka National Hospital, Osaka, *Kansai Rosai Hospital, Hyogo, Japan. B A C K G R O U N D : Prognosis of non-resectable pancreatic cancer is still poor in spite of the use of various therapeutic modalities. Stop-flow chemotherapy with aortic clamping is thought to be effective, but various serious complications, such gastroduodenal ulceration, may develop. In this study, we evaluated the effect of regional stop-flow chemotherapy which allows a direct infusion of the anti-cancer dmg into the pancreas. M E T H O D S : Nine consecutive patients with non-resectable pancreatic carcinomas were treated by regional stop-flow chemotherapy. After laparotomy, the proper hepatic artery, right and left gastric arteries, splenic artery, and celiac and superior mesenteric arteries were clamped. A cocktail of CDDP (20-40 mg), MMC (10-30 mg) and 5-FU (250-500 mg) in 250-300 ml of saline was infused through a catheter introduced into the common hepatic artery, followed by vascular clamping for 20-30 min. The tumor tissue blood flow was measured by laser Doppler flowmetry to evaluate the degree of ischemia. The catheter was again used for arterial infusion chemotherapy (CDDP 10-20 mg and 5-FU 250-500 mg) every two weeks. CT scan or MRI was performed every month after surgery to evaluate tumor progression. RESULTS: Pancreatic tissue blood flow markedly decreased after regional vascular clamping. Although chemotherapy did not reduce the size of the tumor in every patient, it arrested the growth of the main tumor for 2 tO 6 months in all patients except Case 9. The level of CA 19-9 diminished or did not Change for 2 to 8 months except in two patients who also had liver metastasis. In patients without liver metastasis, the prognosis was better compared with historical control subjects, but in those with liver metastasis, it was still poor and 4 of 5 patients died within 8 months. Regional stop-flow chemotherapy was complicated by liver abscess in one patient but no other serious complications were noted. CONCLUSIONS: Regional stop-flow chemotherapy is effective in nonresectable pancreatic carcinomas, however, for a better prognosis it is important to control liver metastasis. G1988 ELIMINATION OF TRYPSIN ENZYME ACTIVITY BY HEMODIAFILTRATION. T. Okahisa, M. Sogabe, S. Ichikawa, T Bando, S. Taoka, Y. Ohkita, A. Tsutsui, T. Fukuda, S. Hayashi, N. Muguruma, S. Okamura, H. Shibata, S. Ito, Second Department of Internal Medicine, School of Medicine, The University of Tokushima, Tokushima, Japan. Hemodiafiltration (HDF) has been carded out on patients with severe acute pancreatitis with renal failure. We performed experimental studies on the effect of eliminating blood Trypsin like enzyme activity (TLA) by HDF, and on its mechanism of action, by using bovine blood tank models. HDF was
GASTROENTEROLOGY Vol. 114, No. 4
performed for 5 hrs (blood flow; 100 ml/min, dialisate flow; 10 ml/min, filtrate flow; 10 ml/min) using a polysulfone filter (PS filter CF, surface area; 0.7m 2, Kuraray Co. Ltd., Japan). Blood TLA was 4 5 . 4 - 0.9 nmol/ml/min immediately after the addition of porcine pancreatic trypsin (Wako Pure Chemical Industries, Ltd., Japan) to the 5L of blood in the tank. Although the elimination rate of TLA after 5 hours was 5.5 ± 1.4% in the HDF group where heparin sodium (HS, Shimizu Pharmaceuticals, Japan) was used at I000 unit/hr as the anticoagulant, the elimination rate was 92.6 _+2.5% in the HDF group where Nafamostat mesilate (NM, Torii Co. Ltd., Japan) was used at 30 mg/hr as the anticoagulant, and was 91.8 ± 3.0% in the circulating flow group where NM was used at 30 mg/hr as the anticoagulant. The decrease rate of TLA after passing through the circuit in the HDF group where HS was used at 1000 unit/hr as the anticoagulant was 0.90 ± 0.54%. From the above results, it is concluded that the decrease of blood TLA during HDF is caused neither by removal of TLA into the waste fluid nor by removal due to adsorption on the filter, but mostly by an inhibiton by NM used as the anticoagulant. For severe acute pancreatitis, HDF using NM was considered to be an effective treatment in respect of the inhibition of TLA. G1989 A. CLINICAL STUDY OF AUTOIMMUE-RELATED CHRONIC PANCREATITIS. K. Okazaki K. Uchida, T. Chiba, Department of Gastroenterology and Hepatology, and Endoscopy, Kyoto University, Kyoto, Japan. M. Ohana, H. Takakuwa, K. Hajiro, Tenri Hospital, Nara, Japan. Aim: An autoimmune mechanism may be involved in some patients with idiopathic chronic pancreatitis. However, the clinical features and pathophysiology are still unknown. To clarify it, we analyzed 38 patients with autoimmune-related chronic pancreatitis. Materials and Methods: Thirty-eight patients with autoimmune-ralated pancreatitis were diagnosed as follows: (1) increased pancreatic enzymes, pancreatic exocrine dysfunction, and abnormal pancreatogram, (2) hypergammaglobulinemia, autoantibodies, or complication with other autoimmune diseases, (3) histopathologically, infiltration of lymphocytes, (4) effective steroid therapy. In 38 patients (26 males and 12 females, mean age: 63 yr.) with autoimmune pancreatitis, we analyzed clinical features, ERCP images, biochemistry, and immunological findings. We studied autoantibodies (against DNA, smooth muscle, mitochondria, lactoferrin, carbonic anhydrase II, or rheumatoid factor), immuno-histochemistry, or flow-cytometriy. Results: Fourteen of 38 patients had jaundice. Seven patients were complicated with sicca syndrome, 5 with primary sclerosing cholangitis, and 14 with diabetes mellitus. All patients showed segmental or diffuse stenosis of the main pancreatic duct. Fifteen patients showed stenosis of the common bile duct. BT-PABA excretion test showed abnormal pancreatic exocrine function in 16 patients. Twenty-four patients had hyper-gammaglobulinemia. Rheumatoid factor was detected in 4 of 11, antinuclear antibody in 4 of 12, anti-smooth muscle antibody in 2 of 7, anti-carbonic anhydrase II antibody in 3 of 9, and anti-lactoferrin antibody in 3 of 6. None had anti-mitochondrial antibody. Microscopic findings showed infiltration of CD4(+) lymphocytes around the pancreatic duct, and HLA-DR was expressed on CD4(+) cells and pancreatic duct ceils. Flowcytometric analysis showed that the major infiltrating lymphocytes in the pancreas were activated T cells (CD4+, HLA-DR+, more than 80%). Conclusion: An autoimmune mechanism against carbonic anhydrase II or lactoferrin via CD4 positive cells may be involved in patients with immunerelated chronic pancreatitis. G1990 EFFECTS OF BILE ACIDS ON SECRETION BY DOG PANCREATIC DUCT EPITHELIAL CELLS. C. N. Okolo, M.W. Moody, T.D. Nguyen, Seattle V.A. Medical Center & University of Washington, Seattle, WA. Pancreatic duct epithelial cells (PDEC) mediate the exocrine pancreatic secretion of fluids and electrolytes, mainly bicarbonate. Because PDEC may be exposed to refluxed bile (e.g. in gallstone pancreatitis), we examined the effects of bile acids on the ion transport pathways of these cells. Non-transformed dog PDEC, isolated and cultured according to Oda & Lee (A.J.Path. 148: 977, 1996), were used. These cells express many of the ion transport pathways of PDEC, including CFFR and Ca2÷-activated C1- and K÷ channels (A.J.Phys. 272: G172, 1997). The effect of bile acids on C1° channels was determined by monitoring the efflux of 125I-, a radioactive marker for CI-, from pre-loaded cells. Taurodeoxycholic acid (TDCA) stimulated 12sI-efflux from PDEC at concentrations > 1 mM. The efflux stimulated by 1 mM TDCA was markedly inhibited by 500 laM NPPB and partially inhibited by 500 laM DIDS, similar to the inhibitory profile previously characterized for the Ca2+-activated C1- channels in these ceils. TDCA-stimulated efflux was also fully inhibited after preincubation with the cell-permeant Ca2÷ chelator, BAPTA/AM (50 laM), verifying that TDCA action is dependent on increased cytosolic Ca2÷. When cellular toxicity was assessed through LDH release from PDEC exposed to TDCA for 5 min, LDH activities in the supematant were unchanged from baseline for 0.5 and 1 mM TDCA, the concentration used in secretory studies. LDH activity increased with 2 mM and reached a maximal value at 5 mM TDCA. When different conjugated groups were evaluated, the peak 125I- efflux stimulated by 1 mM glycodeoxycholic acid
THE RAS-RELATED SMALL GTP-BINDING PROTEIN RAB4 IS INVOLVED IN REGULATED EXOCYTOSIS OF RAT PANCREATIC ACINI. Hirohide Ohnishi, Tetsuya Mine, Tomohiro Tsuchida, and Toshiro Fujita. Department of Intemal Medicine IV, University of Tokyo School of Medicine, Tokyo, Japan. Rab4, a member of the Rab family of Ras-related small GTP-binding proteins, has recently been suggested to participate in exocytosis in addition to its well known role in recycling of GLUT4 and transferrin receptors. The present study was conducted to elucidate the function of Rab4 in regulated exocytosis. Western blotting showed Rab4 to be localized to zymogen granule membranes of pancreatic acini as well as to endosome rich fraction. When the synthetic peptide corresponding to the Rab4 hypervariable carboxyl-terminai domain was introduced into streptolysin-O permeabilized acini, the amylase release induced by 10 laM free calcium was potentiated by 80% at maximum in a concentration-dependent manner. By contrast, another synthetic peptide corresponding to the Rab3C hypervariable carboxyl-terminal domain showed no effect on calcium-induced amylase release from permeabilized acini. Furthermore, anti-Rab4 antibody enhanced calcium-triggered amylase release from permeabilized acini. In cntrast, non-immune IgG revealed no effect on calcium-induced amylase release from permeabilized acini. To further elucidate the involvement of Rab4 in the stimulus-secretion coupling, we examined the effect of CCK on GTP-binding rate to Rab4. GTP-binding rate was measured by introducing radiolabelled GTP into streptolysin-O permeabilized acini and immunoprecipitating GTP-bound Rab4. Stimulation of pancreatic acini with 100 pM CCK increased the incorporation of radiolabelled GTP into Rab4 by 75% compared to control. TPA also enhanced the GTP-binding rate to Rab4 in pancreatic acini. In addition, pretreatment of pancreatic acini with 1 ~tM calphostin C, a protein kinase C inhibitor, attenuated the stimulatory effect of CCK on GTP-binding to Rab4. These results suggest that Rab4 protein on zymogen granules plays a crucial role in regulated exocytosis and that Rab4 is involved in the stimulus-secretion coupling of pancreatic acini via protein kinase C dependent signaling pathway. G1987 REGIONAL STOP-FLOW CItEMOTHERAPY FOR NON-RESECTABLE PANCREATIC CARCINOMA. Ohshima S, Sakon M, Ohsato H*, Nakamori S, Aoki T, Higaki N, Yamada T, Hasuike Y**, Yoshikawa T**, Gotoh M, and Monden M. Department of Surgery II, Osaka University Medical School, **Osaka National Hospital, Osaka, *Kansai Rosai Hospital, Hyogo, Japan. B A C K G R O U N D : Prognosis of non-resectable pancreatic cancer is still poor in spite of the use of various therapeutic modalities. Stop-flow chemotherapy with aortic clamping is thought to be effective, but various serious complications, such gastroduodenal ulceration, may develop. In this study, we evaluated the effect of regional stop-flow chemotherapy which allows a direct infusion of the anti-cancer dmg into the pancreas. M E T H O D S : Nine consecutive patients with non-resectable pancreatic carcinomas were treated by regional stop-flow chemotherapy. After laparotomy, the proper hepatic artery, right and left gastric arteries, splenic artery, and celiac and superior mesenteric arteries were clamped. A cocktail of CDDP (20-40 mg), MMC (10-30 mg) and 5-FU (250-500 mg) in 250-300 ml of saline was infused through a catheter introduced into the common hepatic artery, followed by vascular clamping for 20-30 min. The tumor tissue blood flow was measured by laser Doppler flowmetry to evaluate the degree of ischemia. The catheter was again used for arterial infusion chemotherapy (CDDP 10-20 mg and 5-FU 250-500 mg) every two weeks. CT scan or MRI was performed every month after surgery to evaluate tumor progression. RESULTS: Pancreatic tissue blood flow markedly decreased after regional vascular clamping. Although chemotherapy did not reduce the size of the tumor in every patient, it arrested the growth of the main tumor for 2 tO 6 months in all patients except Case 9. The level of CA 19-9 diminished or did not Change for 2 to 8 months except in two patients who also had liver metastasis. In patients without liver metastasis, the prognosis was better compared with historical control subjects, but in those with liver metastasis, it was still poor and 4 of 5 patients died within 8 months. Regional stop-flow chemotherapy was complicated by liver abscess in one patient but no other serious complications were noted. CONCLUSIONS: Regional stop-flow chemotherapy is effective in nonresectable pancreatic carcinomas, however, for a better prognosis it is important to control liver metastasis. G1988 ELIMINATION OF TRYPSIN ENZYME ACTIVITY BY HEMODIAFILTRATION. T. Okahisa, M. Sogabe, S. Ichikawa, T Bando, S. Taoka, Y. Ohkita, A. Tsutsui, T. Fukuda, S. Hayashi, N. Muguruma, S. Okamura, H. Shibata, S. Ito, Second Department of Internal Medicine, School of Medicine, The University of Tokushima, Tokushima, Japan. Hemodiafiltration (HDF) has been carded out on patients with severe acute pancreatitis with renal failure. We performed experimental studies on the effect of eliminating blood Trypsin like enzyme activity (TLA) by HDF, and on its mechanism of action, by using bovine blood tank models. HDF was
GASTROENTEROLOGY Vol. 114, No. 4
performed for 5 hrs (blood flow; 100 ml/min, dialisate flow; 10 ml/min, filtrate flow; 10 ml/min) using a polysulfone filter (PS filter CF, surface area; 0.7m 2, Kuraray Co. Ltd., Japan). Blood TLA was 4 5 . 4 - 0.9 nmol/ml/min immediately after the addition of porcine pancreatic trypsin (Wako Pure Chemical Industries, Ltd., Japan) to the 5L of blood in the tank. Although the elimination rate of TLA after 5 hours was 5.5 ± 1.4% in the HDF group where heparin sodium (HS, Shimizu Pharmaceuticals, Japan) was used at I000 unit/hr as the anticoagulant, the elimination rate was 92.6 _+2.5% in the HDF group where Nafamostat mesilate (NM, Torii Co. Ltd., Japan) was used at 30 mg/hr as the anticoagulant, and was 91.8 ± 3.0% in the circulating flow group where NM was used at 30 mg/hr as the anticoagulant. The decrease rate of TLA after passing through the circuit in the HDF group where HS was used at 1000 unit/hr as the anticoagulant was 0.90 ± 0.54%. From the above results, it is concluded that the decrease of blood TLA during HDF is caused neither by removal of TLA into the waste fluid nor by removal due to adsorption on the filter, but mostly by an inhibiton by NM used as the anticoagulant. For severe acute pancreatitis, HDF using NM was considered to be an effective treatment in respect of the inhibition of TLA. G1989 A. CLINICAL STUDY OF AUTOIMMUE-RELATED CHRONIC PANCREATITIS. K. Okazaki K. Uchida, T. Chiba, Department of Gastroenterology and Hepatology, and Endoscopy, Kyoto University, Kyoto, Japan. M. Ohana, H. Takakuwa, K. Hajiro, Tenri Hospital, Nara, Japan. Aim: An autoimmune mechanism may be involved in some patients with idiopathic chronic pancreatitis. However, the clinical features and pathophysiology are still unknown. To clarify it, we analyzed 38 patients with autoimmune-related chronic pancreatitis. Materials and Methods: Thirty-eight patients with autoimmune-ralated pancreatitis were diagnosed as follows: (1) increased pancreatic enzymes, pancreatic exocrine dysfunction, and abnormal pancreatogram, (2) hypergammaglobulinemia, autoantibodies, or complication with other autoimmune diseases, (3) histopathologically, infiltration of lymphocytes, (4) effective steroid therapy. In 38 patients (26 males and 12 females, mean age: 63 yr.) with autoimmune pancreatitis, we analyzed clinical features, ERCP images, biochemistry, and immunological findings. We studied autoantibodies (against DNA, smooth muscle, mitochondria, lactoferrin, carbonic anhydrase II, or rheumatoid factor), immuno-histochemistry, or flow-cytometriy. Results: Fourteen of 38 patients had jaundice. Seven patients were complicated with sicca syndrome, 5 with primary sclerosing cholangitis, and 14 with diabetes mellitus. All patients showed segmental or diffuse stenosis of the main pancreatic duct. Fifteen patients showed stenosis of the common bile duct. BT-PABA excretion test showed abnormal pancreatic exocrine function in 16 patients. Twenty-four patients had hyper-gammaglobulinemia. Rheumatoid factor was detected in 4 of 11, antinuclear antibody in 4 of 12, anti-smooth muscle antibody in 2 of 7, anti-carbonic anhydrase II antibody in 3 of 9, and anti-lactoferrin antibody in 3 of 6. None had anti-mitochondrial antibody. Microscopic findings showed infiltration of CD4(+) lymphocytes around the pancreatic duct, and HLA-DR was expressed on CD4(+) cells and pancreatic duct ceils. Flowcytometric analysis showed that the major infiltrating lymphocytes in the pancreas were activated T cells (CD4+, HLA-DR+, more than 80%). Conclusion: An autoimmune mechanism against carbonic anhydrase II or lactoferrin via CD4 positive cells may be involved in patients with immunerelated chronic pancreatitis. G1990 EFFECTS OF BILE ACIDS ON SECRETION BY DOG PANCREATIC DUCT EPITHELIAL CELLS. C. N. Okolo, M.W. Moody, T.D. Nguyen, Seattle V.A. Medical Center & University of Washington, Seattle, WA. Pancreatic duct epithelial cells (PDEC) mediate the exocrine pancreatic secretion of fluids and electrolytes, mainly bicarbonate. Because PDEC may be exposed to refluxed bile (e.g. in gallstone pancreatitis), we examined the effects of bile acids on the ion transport pathways of these cells. Non-transformed dog PDEC, isolated and cultured according to Oda & Lee (A.J.Path. 148: 977, 1996), were used. These cells express many of the ion transport pathways of PDEC, including CFFR and Ca2÷-activated C1- and K÷ channels (A.J.Phys. 272: G172, 1997). The effect of bile acids on C1° channels was determined by monitoring the efflux of 125I-, a radioactive marker for CI-, from pre-loaded cells. Taurodeoxycholic acid (TDCA) stimulated 12sI-efflux from PDEC at concentrations > 1 mM. The efflux stimulated by 1 mM TDCA was markedly inhibited by 500 laM NPPB and partially inhibited by 500 laM DIDS, similar to the inhibitory profile previously characterized for the Ca2+-activated C1- channels in these ceils. TDCA-stimulated efflux was also fully inhibited after preincubation with the cell-permeant Ca2÷ chelator, BAPTA/AM (50 laM), verifying that TDCA action is dependent on increased cytosolic Ca2÷. When cellular toxicity was assessed through LDH release from PDEC exposed to TDCA for 5 min, LDH activities in the supematant were unchanged from baseline for 0.5 and 1 mM TDCA, the concentration used in secretory studies. LDH activity increased with 2 mM and reached a maximal value at 5 mM TDCA. When different conjugated groups were evaluated, the peak 125I- efflux stimulated by 1 mM glycodeoxycholic acid