(NAB), in relation to CYP2CI9 genotype status, Methods: In a crossover pharmacod}mamic study, 10 healthy male subjects without Helicobac ter pylorf inti:ction, whose CYP2C 19 geno- type status was precisely determined, were given rabeprazole lOmB twice daily" or 20mg tw*ce daily or water only (base'line data) tbr 7 days each, in a randomized manner. On day 7 of each reglmen, 2 4 h iutragasrnc pH-metry was performed, The intragastric mean pH valne, the pH>3 holding time (pH>3HT), and tbe occurrence of NAB, defined as intragastric p H < 4 continuously tot 60 min, were compared. Results are expressed as means +/-S.E,M Resuhs: The 24-fi mean pH value and pH>3HT during rabeprazole 20mg twice daily (58 + / - 0 1 and 972 +/-1,2% in the homozygous extensive metaboiizers: homo- EMs (n = 6), 5 4 + / - 0 3 and 93,6 + / 4 0 % in the heteruzygons extensive metaboheers: hetero- EMs (n = 5), 6.3 +/-0.2 and 1000 +/-0,0% in the pool" metabolizers: PMs (n = 5), respec- tively) were not signdicantly different from rabeprazole 10rag twice daily (5.5+/-0.2 and 93,3+A1,9% in the homo-.EMs, 54+ / -0 .3 and 87.0+/-7.6% in the hetero-EMs, 6 ,0+/- 0.1 and 9 9 8 + / - 0 2 % in the PMs, respectively) in each of the three different CYP2C19 genotype gioups, No significant differences were also observed in these parameters among the thr'ee CYP2C19 genotype groups in each dosage. NAB occurred in 3 of the homo-EMs (50.0%), 3 of the hetero-EMs (60 0%), and none of the PMs (0.0%) with rabeprazole lOmg twice daily and it occurred in 1 of the homo-EMs (16.7%), 1 of the hetero-EMs (20.0%), and none ot the PMs (00%) with rabeprazole 20rag twice daily, Conclusions: No significant differences were observed in gastric acid suppression with rabeprazole lOmB twice daily and 20mg twke daily in any CTP2C19 genotype group, However, to prevent NAB, 20mg twice daily was superior in its efficacy.

T903

Does Chronic Hypercalcemia Influence Serum Pepsinogens Levels? Anna Ingegnoli, Giovanni Ferreri, Paolo Del Rio, Elena cerat i Ginha M Cavestro, kucas G. Cavallam, Gioacchino Lean&o, Daniela Basso, Mario Plehain, Mario Sianesi, Francesco Di Mano

Background: hicreased levels of serum pepsinogen I (PGI) have been reported in components ot t:amifial muhiple endocrine neoplasm type I Syndrome (MEN 1) with hypergastrinemia. Aim: to evaluate (he role of chronic hypercalcemia in stinmlate serum pepsinogens secretion in patients wuh hFperpamthyroidism without gastmenterologic symptoms, and to determine if parathyroi&ctomy lead to a decrease ot serum pepsinogen levels Patients and methods: levels of serum pepsinogens, gastrin were measured nsing a commercial kit (Biohit- Helsinki- Finland) in 13 consecutive patients (M: 1; F: 12) sutfering tram hyperparathyroidlsm and in 9 pauents (M:6; F:3) wnh hyperthyroidism (control group). Serum levels of parathyroid hormones (PTH) and plasma calcium were also evaluated. Furthermore serum levels of PGI and PGII were measured in 7 patients that underwent parathyruidectomy 4 days after sm'gery Data were presented as mean _+standard deviation Statistical analysis was made by meat~s of Mann-Whitney rank-sum test and Wilcoxon signed-rank test, as appropriate. Results: compared to the control group, patients with hyperparathyroidism showed a statisti- call)," significant increa~se in PG1 (178_+88 vs. 96_+21; p = 0.0148). PGII showed similar results in the two groups (19_+ 16 vs. 15_+8). After parathyroidectomy was observed a decrease in PGI (before 172_+89 vs 130_+96 after surgery; p=0.22), calcium (before 10,7 _+ 0 4 vs 9 1 _+ ] ,2 atter surgery; p = 006) and PTH (before 290 _+ 280 vs 91 _+ 24,9 atier surgery; p = 0 0 3 ) Conclusions: These results indicate that serum levels of PGI are higher in patients with hyperparathF~'oidism and decreased 4 days after pamthyroidectomy such as calcium levels.

T904

Significance of an Exaggerated Meal-Stimulated Gastrin Response in The Pathogenesis of Helicobacter Pylori-Negative Duodenal Ulcer Tomoan Koamada, Kuniaki Sugiu, Qian Dong Mei, Hiroaki Kusunoki, Ken Haruma, Masanon ho, Sbinji Tanaka

Background: It is expected that H. pylori-negatree ulcer may increase fi-om now on by the decline of 1-tehcobacter pylon (H. D4ori) infection, and the spread of eradication treatment of H, pylon. "t'ne aim of this study was to darit}" [he pathogenesis of H. pylori-negative duodenal ulcer (DU) by investigating the meal-stimulated serum gastrin (SG) response. Patients and Methods: Subiects were nine patients w'ith H. pylori-negative DU, 28 H. pylori- positive DU, l 1 H. pylori positive volunteers, and 30 H. pylori-negative volunteers. Blood samples were taken beiore and after consumption of a test meal. The integrated 1-h gastrin response (IGR) was taken to be the area under the SG time curve, calculated by the trapezoid method H, pyIori iniectlon statns was determined by histology, serology, and the 13C-urea breath test Results: The mean basal SG concentration was lower in patients with H. pylori- uegauve DU than in those w~ffi H. pylori-positive DU (61,0 vs 85.0 pg/ml). The mean IGR in patmnts with H. pylorimegative DU was closer to that ot the H, pylori-posinve volunteers (8,842.0 vs 8,1629 pg/ml ram) than to that of H. pylorimegative volunteers. In addition, an ex.aggerated IGR was observed in three patients (33.3%) with H. pylori-negative DU. Conclusion: Our findings indicate that an exaggerated meal-stimulated gastrin response may contribute to the pathogenesls of H. pylori-negative DU,

T905

Increased TRPC1 Expression and Capacitative Ca ~+ Entry in Differentiated Intestinal Epithelial Cells during Restitution Jaladanki N, Rao, Xin Guo, I.an Lm, Tong-Tong Zou, Vera A, Golovina, Jason X-J. Yuan, Jian-Ying Wang

Early mucosal restitution is a primary repair modafi U in the GI tract and occurs b)" epithelial cell migration to reseal superfnial wounds. Diftel~ntiated intestinal epithelial cells (IEC- C&;2LI cells) induced by torced expression of the Cdx2 gene migrate over the wounded edge much faster than undifierentiated parental cells (IEC-6 line) in an in vitro model. We recently demonstrate that this increased migration ot differentiated IEC-Cdx2L1 cells is dependent on cytoplasmic Ca '+ Depletion of intracellu[ar Ca 2 § stores triggers capacitative

Ca 2~ en W (CCE), which is involved in maintaining Ca > intlnx and refilling Ca > stores Transient receptor potential channels (TRPC) are store-operated Ca 2+ channels that are activated by Ca 2+ store depletion. This study determined if TRPC1 and CCE play a role in Ca z+ influx and migration after wounding in IEC-Cdx2L1 ceils. Methods: TRPC1 expression was measured by RT-PCR and Western blot analysis CCE was elicited by passive store depletion using cycloptazonic acid. An antisense ofigonucleotide (AS) specifically designed to cleave TRPC1 mRNA was transfected into the cells. Results: Differentiated IEC-Cdx2LI cells highly expressed TRPC1 mRNA and protein but did not express L-type voltage-gated Ca ~+ channels Basal level of TRPC1 protei n in difterenttated IEC-Cdx2LI ceils was ~5 times that of parental IEC-6 cells. Resting cytosolic free Ca 2 + concentration ([Ca 2 +]~y,) and amplitude of CCE were significantly greater in differentiated IEC-Cdx2L1 cell than in parental IEC-6 ceils. Migration of differentiated IEOCdx2L1 ceils was ~4-fold that of parental IEC-6 cdls, Inhibition of TRPC1 expression by transfection of TRPCl-AS not only reduced CCE but also inhibited cell migration after wounding. CCE was decreased by ~60% in AS-treated calls and migration was inhibited by ~30% during restitution. Conclusions: These results suggest that TRPC1 is a store-operated Ca ~+ channel in intestinal epithelial cells and plays a critical role in cell migration during restitution by regnlating CCE and [Ca2+l~,

T906

The Novel Delta (8) Isoform of PKC Causes iNOS & NO Upregulation: A Unique Mechanism for Oxidant-induced Carbonylation & Disassembly of the Cytoskeleton and Disruption of Barrier of Intestinal Epithelia All Banan, Jeremy Fields, Ashkan Farhadi, Maliha Shaikh, Lijuan Zhang, All Keshavarzian

Using monolayers of intestinal (Caco-2) cells, we reported that oxidant induced disruption of barrier integrity requires the disassembly of microtubule (MT) cytoskeleton and that expression of the 75 kDa PKC-deha (8) isoform was required tbr barrier disruption. The underlying mechanism for this disruption is unknown. Since iNOS upregnlation is key to oxidant stress, we hypothesized that PKC-8 activation is essential in oxidant-induced iNOS upregnlation and the consequent cytoskeletal oxidation & disarray and monolayer bamer dysfunction. METHODS: Cells were stably transtected with varymg levels of an inducible plasmid to over-express PKC-8 or with a dominam negative to inhibit the activity of native PKC-& These clones were then incubated with oxidant (H202) -+ modulators. Parental ceils were treated similarly. RESULTS: ~A. Exposure to oxidant disrupted monolayers by increasing native PKC-8 activity, increasing six iNOS pathway related variables (iNOS activity & protein, NO, oxidative stress, tubulin oxidation & nitration), decreasing polymerized tubulin while increasing nsonomeric tubulin, disrupting the cytoarchitecture of MT, and causing monolayer barrier dysfunction. ~Be Induction of PKC-8 over-expression by itself ( + 3 5 fold) led to oxidantdike disruptive effects, including activation of iNOS driven pathways. Over-expres- sion induced upregulation of iNOS driven reactions was potentiated by oxidant. <C~ Domi- nant inhibition of nat*re PKC-g activity (-985%) prevented all the various measures of oxidant-induced iNOS upregn"lation while protecting the monolayer cytoskeleton and barrier integrity. CONCLUSIONS: 1) oxidams induce loss of intestinal epithelial barrier integrity by oxidizing & disassembling the cytoskeleton, in part, through the activation of PKC-8 isoform and up-regulation of iNOS; 2) Over-expression and activation of PKC-5 is by itself required for cellular injury induced by oxidative stress of iNOS up-regulation. 3) We thus repov a unique pathophysiological mechanism, activation of iNOS driven pathway and its injurious consequences to the c}aoskeletal subunits including oxidation and nitration, among the novel snblamily of PKC isoforms in ceils, Thus, suppressing PKC-g signaling is a potentially unique therapeutic strategy in intestinal disorders such as intlammatory bowd disease (IBD) that are associated with oxidative stress of iNOS.

T907

Cellular Protective Role of a Novel Vanilloid Receptor Subtype Expressed Peripherally in Rat Gastric Mucosal Epithelial Cells Shinichi Kato, Eitaro Aihara, Koji Takeuchi

VainUoid receptors of type 1 [VP.1) are nonselective cation channels, that are expressed mainly in sensor)," C and A/xfibers and gated by nociceptive stimulation, such as acid and heat, in addition to capsaicm, Several ;q/1 homologues have so far been cloned, and all of them are believed to exist only m either nem'ons or ganglions. We recently found a \:N1 homologne in the rat stomach and further demonstrated the expression of the 'vqR1 homoIogue peripherally in gastnc epithelial cells. In the present study, we investigated the role of the newly identified VR1 homologue on cellular protection against injury in rat gastric mucosal epithelial cells in vRro. Methods The rat gastric mucosal epithelial cell line, RGM-1, was used m this study. Cell injury was induced by treatment with either the medium containing 10% ethanol or acidified phosphate buffer saline (pH 4) for 20 or 30 min, respectively, and the cell viability was determined by lvlTT assay, Capsaicin or resiniferatoxin (RTX) was added 30 rain before challenge with ethanol or acid, while capsazepine or mtheninm red (a VR1 antagonist) was added simnhaneonsly with capsaicin. The expression of VR1 was examined by RT-PCR as well as Western blot using two different anti-VR1 antibodies; C- terminal and N-terminal epitopes on VR1 protein. Results: ]'he apparent expression of'vN1 mRNA was observed in RGM-1 ceils by RT-PCR. Using both C- and N-terminal anti-VR1 antibodies, the VR1 protein expression was found in membrane fraction of RGM-1 cells, However, the amount of band detected by the C-terminal antibody was much greater than that by the N-terminal antibody. On the other hand, the cell viability of RGMq decreased to 60-70% after the treatment with ethanol or acid. Pretreatment with capsaidn dose- dependently prevented the cell injury against either ethanol or acid, The protective elfect of capsaiein was mimicked by RTX and almost totally abolished by co-addition with capsazep- ine or ruthenium red. Conclusion: These resuhs suggest that certain VR1 homologne is also expressed m RGM-I cells and plays a protective role against cellular injury caused by ethanol or acid, The VR1 homologne expressed in RGM-1 cells may include some unknown VR1, probably differed from the authentic VR1, regarding the site of N-terminal.

AGA Abstracts A-446