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Localization of cholecystokinin (CCK)-A receptor in the human duodenum
A460 AGA ABSTRACTS G1870 EXPERIMENTAL INVESTIGATION INTO THE ONSET MECHANISM OF CHRONIC ALCOHOLIC PANCREATITIS: CANINE MODEL OF PANCREAS DIVISUM. T Fujii, T Tanaka, Y Kodo, Y Matsugu, K Koide, Y Miura, and K Dohi. Second Department of Surgery, Hiroshima University School of Medicine, Japan. Aims: Many aspects of the onset mechanism of chronic alcoholic pancreatitis (CAP) are still unclear. As reported previously, we succeeded in preparing an experimental model which is very similar to human CAP, by slightly narrowing the pancreatic duct prior to alcohol administration. It is known that in human pancreas divisum, obstructive pancreatitis only occurs on the dorsal side of the pancreas. Under these circumstances, we prepared a canine model of pancreas divisum and evaluated the effect of alcohol on this model. Materials and Methods: Adult mongrel dogs, weighing 8-15 kg, were used. A segment of pancreas was cut out from between the greater and the lesser pancreatic ducts. The cut ends of the remaining pancreatic regions, including the cut end of the pancreatic duct, were then sutured together to form two separate pancreatic segments. As a result of this procedure, the dorsal region of the pancreas was drained by the lesser pancreatic duct, the ventral region was drained by the greater pancreatic duct, and thus, the dorsal pancreatic region was relatively less drained. This was the model of pancreas divisum (Group PD). In a second group, administration of ethanol at 2.0 g/kg/day in addition to the same treatment as in Group PD was performed (Group PD+A). After three months, re-laparotomy was performed in both groups, and the pancreas was isolated and examined histopathologically. Results: < Macroscopic findings > Hardness of the organ was increased only in the dorsal pancreatic region in Group PD+A, whereas changes were little noted in the ventral region in Group PD+A or in either region in Group PD. < Histopathological findings > In Group PD, the dorsal pancreatic region presented mild fibrosis around the pancreatic duct, while no change was found for the ventral region. No change was noted in the ventral region in Group PD+A. In the dorsal pancreatic region in Group PD+A, however, moderate fibrosis with accompanying infiltration of inflammatory cells was noted mainly in the interlobular areas, indicating chronic pancreatitis. Conclusion: The histopathological findings noted in the dorsal region of pancreas in Group PD+A were very similar to those found for human CAP. We conclude that narrowing of the pancreatic duct intensifies alcohol-induced damage tO parenchymal cells of the pancreas. • G1871 MOLECULAR MECHANISM OF GROWTH INHIBITION BY A RAR a-SELECTIVE RETINOIC ACID IN PANCREATIC CANCER CELL LINES. K. Fujimoto. R. Hosotani, R. Doi, M. Wada, J.-Uk. Lee, T. Koshiba, Y. Miyamoto, S. Tsuji, S. Nakajima, M. Imamura. Department of Surgery and Surgical Basic Science, Kyoto University, Kyoto, 606-01, Japan. Recent publications have reported that all-trans retinoic acid, which almost equally binds to retinoic acid receptor (RAR) a, 13, and T, inhibits pancreatic cancer cell growth (Rosewicz-S et al. Gastroenterology, 109, 1995). We show here that TAC-101 (Am555S), which selectively binds to RAR a, has the potent growth-inhibitory effect on pancreatic cancer cell lines. Furthermore, to obtain information regarding the growth-inhibitory effect of TAC-101 on pancreatic cancer cell lines, differences in the induction of apoptosis, the inhibition of cell cycle progression and the expression of Gl-phase cell cycleregulating factors (the retinoblastoma gene product (pRB) and CDK inhibitor p21 ) were studied in retinoic acid responsive and non-responsive cell lines. Methods: The cell growth inhibition mediated by TAC-101 in four pancreatic cancer cell lines (BxPC-3, MIAPaCa-2, CFPAC-I, and AsPC-1) was determined by measuring the number of cells using Coulter counter. Induction of apoptosis was confirmed by DNA gel electrophoresis and Hoechst staining in TAC-101 responsive and non-responsive cell lines. Cell cycle progression and levels of expression of pRB and p21 proteins in TAC-101 responsive and non-responsive cell lines were analysed by flow cytometry and Western blotting, respectively. Results: (1) Growth inhibition by TAC-101 was observed in a time-and dosedependent manner in three cell lines, BxPC-3, MIAPaCa-2, and CFPAC-1, whereas AsPC-1 cells showed no inhibitory effect. Table. The inhibitory potency of TAC-101 on cell growth after a 6 day incubation period. BxPC-3 MIAPaCa-2 CFPAC-1 AsPC-1 ICs0 (/1 M) 13 4- 4 24 4- 6 46 4- 7 > 100 (2) In TAC-101 responsive BxPC-3 cells, DNA laddering and fragmentation were observed at 24, 48 h after treatment with 40 p M, whereas these phenomena were not observed in non-resp0nsive AsPC-1 cells. (3) In BxPC-3 cells, the population of G1 phase ceils markedly increased at 48 h after treatment (control 35.3% and TAC-101 81.6%), whereas AsPC-1 cells showed no change (control 38.9% and TAC-101 36.6%). (4) The retinoblastoma protein showed hyperphosphorylated form at 24 h after serum was added to starved cells, but in the presence of TAC-101 pRB remained hypophosphory lated in BxPC-3 cells. (5) In MIAPaCa-2 cells, an increase in p21 protein was observed at 12 and 24 h after TAC-101 treatment, whereas p21 remained unchanged in AsPC-1 cells.
GASTROENTEROLOGYVol. 114, No. 4 Conclusions: The present study showed that RAR a-selective retinoic acid induces an apoptotic cell death and produces a G1 cell-cycle arrest by upregulating p21 protein and preventing pRB phosphorylation as an early event, and leads to growth inhibition in responsive pancreatic cancer cell lines. RAR a specific agonist might therefore provide a powerful tool in the treatment of pancreatic carcinoma. G1872
LOCALIZATION OF CHOLECYSTOKININ (CCK)-A RECEPTOR IN THE HUMAN DUODENUM. A. Funakoshi. Fukuoka: Y. Fukamizu, Kyoto; A. Jimi, Kurume, Fukuoka; H. Shinozaki, Inatsuki, Fukuoka; Y. Yasunami, S. Ikeda, Fukuoka; K. Miyasaka, Tokyo, Japan. CCK is a major physiological mediator of gallbladder contraction and pancreatic enzyme secretion through binding to CCK-A receptors. Infusion of CCK-A receptor antagonists dosedependently inhibited CCK-8-stimulated pancreatic enzyme responses in humans. However, by in vitro autoradiography human pancreas predominantly expresses CCK-B receptors, whereas only CCK-A receptors were localized in the human gallbladder. Thus, these data suggested that the effects of CCK on the human pancreas are mediated by CCK-A receptors, although it probably resides on neurons that may even be extrapancreatic. We tried to confirm the localization of CCK-A receptor in the human duodenum which may be another candidate of receptor site of intestinal phase of pancreatic secretion. Methods: Expressions of the CCK-A and B receptor genes in human duodenum, pancreas and gallbladder were examined by the Northern blot analysis and RT-PCR followed by Southern blot hybridization. The autoradiographic study of CCK-A & -B receptor in the duodenum was examined in vitro and results were compared with those of the pancreas. To determine the subtypes of CCK receptors in the pancreas or duodenum, we studied the abilities of CCK-A and CCK-B receptor agonists (CCK-8 and gastrin) and antagonists (loxiglumide and L365,260) to inhibit binding of 125I-Bolton-Hunter sulfated CCK-8 under identical conditions. Results: CCK-A receptor mRNA was not expressed in the pancreas, but was expressed in the gallbladder and duodenum by Northem blot analysis, although it was expressed in these tissues by RT-PCR followed by Southern blot analysis. CCK-B receptor mRNA was expressed in the pancreas, but not in the duodenum. High concentrations of CCK-A receptors were detected in the mucosa and muscularis propria of duodenum, although in the pancreas only CCK-B receptors were detected. Conclusion: Localization of CCK-A receptor in human duodenum provides a biochemical and morphological basis for the some physiological functions of CCK. G1873 SNARE PROTEINS (Mune 18c/SYNTAXIN-4/SNAP-23/VAMP-2) MED1ATE BASOLATERAL PLASMA MEMBRANE EXOCYTOSIS DURING EXPERIMENTAL ACUTE PANCREATITIS. HY Gaisano 1, J Leser 2, L Sheu 1, Y Tamori 3, WS Trimble4, MP Lutz 2 from the Depts. of IMedicine and 1,4physiology, University of Toronto, 2University of Ulm, Germany, 3Kobe University School of Medicine, Japan; 4Division of Cell Biology, Hospital for Sick Children of Toronto, Canada. Whereas exocytotic fusion of the zymogen granule with the basolateral plasma membrane (PM) had been proposed as a mechanism of pancreatitis, the molecular mechanism mediating this pathologic membrane fusion event remains undefined. We had reported in pancreatic acinar cells, the presence and distinct cellular locations of vesicle (v-, zymogen granule) SNAREs (VAMP-2 and ceUubrevin) and target (t-, plasma membrane) SNAREs (syntaxins-2 thrn -4, and SNAP-23). Secl/Munc 18a proteins, identified in yeasts and the nervous system, had been proposed to negatively control vesicle docking/fusion by binding to Syntaxin. Using confocal microscopy and an antibody generated against the specific C-terminal 15 amino acid of the recently described non-neuronal Munc 18c protein, we now show that Munc 18c, like Syntaxin-4 and SNAP-23, to be present on the basolateral PM of dispersed rat pancreatic acinar cells, as well as in intact rat pancreatic tissues. Whereas submaximal (10-100pM) CCK stimulation of acini up to 1 hr had no effects on Munc 18c levels on the basolateral PM, supramaximal (10nM) CCK caused a time-dependent diminution of PM staining paralleled by an increase in cytosolic staining of Munc 18c. These changes, initially noted at 15 mins and nearly complete at 30 mins, are indicative of the displacement of Munc 18c from the PM to the cytosol. Translocation of Munc 18c from the PM to the cytosol was confirmed by Western blotting of these subcellular fractions from 10nM CCK (15 mins)-stimulated acini. To determine the role of Munc 18c and SNARE proteins in pancreatitis, rats were infused with supramaximal CCK up to 6 hr. In these pancreatitis tissues, we observed a more delayed but nonetheless, similarly progressive diminution of Munc 18c staining of the basolateral PM, which was noticeable at lh, and was nearly complete at 4h of CCK infusion. These changes correlated well to the progressive disruption and disintegration of phalloidin (used as a double label) staining of the PM, the latter of which is indicative of actin disassembly, a hallmark of pancreatitis. No significant changes were observed with the cellular location and intensity of Syntaxin-4 and SNAP-23 staining on the basolateral PM in the acini or pancreatitis tissues. Taken together, these results are consistent with the general thinking of SNARE Hypothesis of vesicular fusion, and may explain the pathologic fusion events in pancreatic
GASTROENTEROLOGYVol. 114, No. 4 Conclusions: The present study showed that RAR a-selective retinoic acid induces an apoptotic cell death and produces a G1 cell-cycle arrest by upregulating p21 protein and preventing pRB phosphorylation as an early event, and leads to growth inhibition in responsive pancreatic cancer cell lines. RAR a specific agonist might therefore provide a powerful tool in the treatment of pancreatic carcinoma. G1872
LOCALIZATION OF CHOLECYSTOKININ (CCK)-A RECEPTOR IN THE HUMAN DUODENUM. A. Funakoshi. Fukuoka: Y. Fukamizu, Kyoto; A. Jimi, Kurume, Fukuoka; H. Shinozaki, Inatsuki, Fukuoka; Y. Yasunami, S. Ikeda, Fukuoka; K. Miyasaka, Tokyo, Japan. CCK is a major physiological mediator of gallbladder contraction and pancreatic enzyme secretion through binding to CCK-A receptors. Infusion of CCK-A receptor antagonists dosedependently inhibited CCK-8-stimulated pancreatic enzyme responses in humans. However, by in vitro autoradiography human pancreas predominantly expresses CCK-B receptors, whereas only CCK-A receptors were localized in the human gallbladder. Thus, these data suggested that the effects of CCK on the human pancreas are mediated by CCK-A receptors, although it probably resides on neurons that may even be extrapancreatic. We tried to confirm the localization of CCK-A receptor in the human duodenum which may be another candidate of receptor site of intestinal phase of pancreatic secretion. Methods: Expressions of the CCK-A and B receptor genes in human duodenum, pancreas and gallbladder were examined by the Northern blot analysis and RT-PCR followed by Southern blot hybridization. The autoradiographic study of CCK-A & -B receptor in the duodenum was examined in vitro and results were compared with those of the pancreas. To determine the subtypes of CCK receptors in the pancreas or duodenum, we studied the abilities of CCK-A and CCK-B receptor agonists (CCK-8 and gastrin) and antagonists (loxiglumide and L365,260) to inhibit binding of 125I-Bolton-Hunter sulfated CCK-8 under identical conditions. Results: CCK-A receptor mRNA was not expressed in the pancreas, but was expressed in the gallbladder and duodenum by Northem blot analysis, although it was expressed in these tissues by RT-PCR followed by Southern blot analysis. CCK-B receptor mRNA was expressed in the pancreas, but not in the duodenum. High concentrations of CCK-A receptors were detected in the mucosa and muscularis propria of duodenum, although in the pancreas only CCK-B receptors were detected. Conclusion: Localization of CCK-A receptor in human duodenum provides a biochemical and morphological basis for the some physiological functions of CCK. G1873 SNARE PROTEINS (Mune 18c/SYNTAXIN-4/SNAP-23/VAMP-2) MED1ATE BASOLATERAL PLASMA MEMBRANE EXOCYTOSIS DURING EXPERIMENTAL ACUTE PANCREATITIS. HY Gaisano 1, J Leser 2, L Sheu 1, Y Tamori 3, WS Trimble4, MP Lutz 2 from the Depts. of IMedicine and 1,4physiology, University of Toronto, 2University of Ulm, Germany, 3Kobe University School of Medicine, Japan; 4Division of Cell Biology, Hospital for Sick Children of Toronto, Canada. Whereas exocytotic fusion of the zymogen granule with the basolateral plasma membrane (PM) had been proposed as a mechanism of pancreatitis, the molecular mechanism mediating this pathologic membrane fusion event remains undefined. We had reported in pancreatic acinar cells, the presence and distinct cellular locations of vesicle (v-, zymogen granule) SNAREs (VAMP-2 and ceUubrevin) and target (t-, plasma membrane) SNAREs (syntaxins-2 thrn -4, and SNAP-23). Secl/Munc 18a proteins, identified in yeasts and the nervous system, had been proposed to negatively control vesicle docking/fusion by binding to Syntaxin. Using confocal microscopy and an antibody generated against the specific C-terminal 15 amino acid of the recently described non-neuronal Munc 18c protein, we now show that Munc 18c, like Syntaxin-4 and SNAP-23, to be present on the basolateral PM of dispersed rat pancreatic acinar cells, as well as in intact rat pancreatic tissues. Whereas submaximal (10-100pM) CCK stimulation of acini up to 1 hr had no effects on Munc 18c levels on the basolateral PM, supramaximal (10nM) CCK caused a time-dependent diminution of PM staining paralleled by an increase in cytosolic staining of Munc 18c. These changes, initially noted at 15 mins and nearly complete at 30 mins, are indicative of the displacement of Munc 18c from the PM to the cytosol. Translocation of Munc 18c from the PM to the cytosol was confirmed by Western blotting of these subcellular fractions from 10nM CCK (15 mins)-stimulated acini. To determine the role of Munc 18c and SNARE proteins in pancreatitis, rats were infused with supramaximal CCK up to 6 hr. In these pancreatitis tissues, we observed a more delayed but nonetheless, similarly progressive diminution of Munc 18c staining of the basolateral PM, which was noticeable at lh, and was nearly complete at 4h of CCK infusion. These changes correlated well to the progressive disruption and disintegration of phalloidin (used as a double label) staining of the PM, the latter of which is indicative of actin disassembly, a hallmark of pancreatitis. No significant changes were observed with the cellular location and intensity of Syntaxin-4 and SNAP-23 staining on the basolateral PM in the acini or pancreatitis tissues. Taken together, these results are consistent with the general thinking of SNARE Hypothesis of vesicular fusion, and may explain the pathologic fusion events in pancreatic