$1033 Anxiety-Related Behaviors in Cholecystokinin-A, B, and Ab Receptor Gene Knockout Mice in the Plus-Maze Kyoko Miyasaka, Minoru Ohta, Setsuko Kanai, Yuki Yoshida, Hiroko Hosoya, Saeko Takano, Soichi Takignchi, Yutaka Takata, Takako Kawauami, Akihiro Fuuakoshi Background: CCK is one of the most abundant neurotransmitter peptides expressed in the brain. CCK-A receptor (R) is expressed in specific brain regions such as the amygdala, nucleus tractns solitarius, posterior nucleus accumbens, ventral tegmental area, hypothala- mus, substantia nigra, hippocampus, area postrema, and raphe nucleus, whereas CCK- BR is widely distributed throughout the central nervous system. Several reports including phamaacological studies have shown that the CCK-BR is involved in anxiety, while CCK- AR has been implicated in satiety and behavior. However, the cross-reactivity of each antagonist could not be excluded in those studies, moreover, the expression patterns of these receptors o~,erlap in the brain/Aims: We recently generated the CCK-AR(-/-), BR(-/- ) and CCK-AR(-/-)BR(-/.) mice. Here we have examined the anxiety-related behavior of these mutant mice during the elevated plus-maze test. Methods: The animals were handled for 3 m/n/day for 3 days prior to the test. In each test, the following parameters were measured over a period of 5 rain (300 s); l) the frequency of entry into the open and enclosed arms of the maze (with an arm entry defined as the placing of all four paws into an arrd); 2) the proportion of time spent in the open and enclosed arms; and 3) the number of partial entries (the placing of only two paws) into the open arms. Results: CCK-AR(-/-) mice showed a significantly higher frequency/of open-arm entries than wild-type and CCK-BR(-/-) mice, whereas % open-arm entry values in CCK-AR(-/-) mice did not differ from those in wild- type mice. By contrast, CCK-BR(-/-) mice showed significantly lower % open-arm entry values and spent significantly less time in the open- arms than wild-type and CCK-AR(-/-) mice. Although the frequency of open-arm entries for CCK-BR(-/-) mice was not significantly different from that for wild-type mice, it was significantly lower than that for CCK,AR(-/-) mice. The % open-arm entry values for CCK-BR(-/-) was significantly lower than those fdr wild-type and CCK-AR(-/-) mice. The time,/spent in the enclosed-arfns differed significantly among the four genotypes, and was significantly higher for both CCK-BR(-/-) arid CCK- AR(-/-)BR(-/-) mice than for CCK-AR(-/-) and wild-type mice. Conclusions: A lack of CCK- BR increases the anxiety-related behavior of the mouse in the elevated plus- maze. The discrepancy between previous pharmacological studies tflaf administration of a CCK-BR ligand induces anxiety and our resuh should be further investigated. 51034 Overexpression of Glycine-Extended Gastrin Maintains Add Secretion and Prevents the Development of Preneoplasia in the Stomach Via Reducing Parietal Cell Lost in Transgenic Mice Guangfin Cui, Theodore J. Koh, Duan Chen, Chun-Mei Zhao, Graham J. Dockray, Andrea Varro, James G. Fox, Timothy C Wang Background and Aims: Recent studies from our group have shown that giycine-extended gastrin (G-gly) when infused into gastrin-deficient mice can synergize with G-I7 to stimulate acid secretion and thus modify its biologic properties. We thus examined the phenotypic changes induced by overexpression of G-gly, G-17 or both peptides in the stomach of transgenic mice, as well as the effects of Helicobacter fehs (H. fells) infection in these mice. Methods: Wild-type (FVB/N) mice, INS-GAS (which have increased G-17), INS-GAS/G-gly at ages of 2-3, 5-6, 10-12, and over 18 months, or MT/G-gly transgenic mice (increased G- gly) at 2 months old, were included in this study. Some of these mice were also infected with H. fells for 6 months. Gastric acid secretion and peptic ulcer susceptibility were assessed using the pyloric ligation model. Analysis for histological changes (endocrine cells, inflammation, gastric atrophy and dysplasia etc.) were done by H&E staining and immunohis- tochemistry. Results: Uninfected two-month old INS-GAS/G-gly showed a two-fold increase in acid secretion and a high rate (50% of mice) of peptic ulcer compared to INS-GAS mice by pylotic ligation technique, but no in MT/G-gly mice. However, while male INS-GAS mice showed marked (50%) reductions in parietal cell and ECL cell densities at 6 and 12 months of age, the male INS-GAS/G-gly mice showed no such reductions. Furthermore, in response to H. fells infection, INS-GAS/G-gly mice showed lesser degrees of [undic atrophy and hyperplasia of mucous neck ceils than INS-GAS mice did. However, overexpression of G- gly did not prevent the development of gastric cancer that was the same in both single and double transgenic mice. Expression of TFF 1 in the stomach was initially increased in younger INS GAS mice, but decreased in older mice, particular in area of deep glands. Expression of TFF2 was found in fundic metaplasia and cancer cells in both INS GAS and INS GAS/ Gly mice. Conclusions: Overexpression of G-gly and G-17 results in an increased acid secretion and a high susceptibility of peptic ulcer. However, it attenuates and even prevents the development of preneoplasia in the stomach seen either in uninfected INS-GAS mice or in H. fells infected INS-GAS mice, but does not prevents progression of cancer. S1035 Bombesin-lnduced Inhibition of Gastric Acid Secretion Is Mediated by Somatostatin: Evidence in SSTR2 Knockout Mice Laura Piqueras, Yvette Tache, Vicente Martinez In vitro studies in isolated mouse stomach indicate that bombesin (BBS) releases somatostatin (SS'i'). However, the functional significance of this observation is not clear and both SST- dependent and independent mechanisms have been suggested for BBS actions on gastric acid secretion (GAS). AIMS: To characterize the effects of BBS on GAS in mice and to determine the involvement of SST, by using SSTR2 knockout mice, the selective SSTR2 antagonist, PRL-2903, and in vivo immunoneutralization of the peptide. METHODS: GAS was monitored in urethane-anesthetized wild-type and SSTR2 knockout mice (SSTR2 -/-, Merck Res. Labs.) by continuous intragastric perfusion and backntration of perfusates to pH 7.0. The effects of BBS 00-40 tsg/kg, IV) on basal and pentagastrin-, histamine- or bethanecol-stimulated GAS were investigated. PRL-2903 (bolus of 1.5 mg/kg + IV infusion of 1.5 mg/kg/h) was given 10 min before IV BBS or $5T-14 (20 Izg/kg/h). The monoclonal SST antibody CURE S.6 (150 /ig/mouse) was given IV 30 rain before the infusion of BBS or SST-14 (20 v,g/kg/h). Data are mean_+SE RESULTS: In wild-type mice, BBS inhibited dose-dependently secretagognes-stimuhted GAS, with an antisecretory effect against penta- gastnn > bethanecol > histamine. Urethane-anesthetized SSTR2 -/- mice had a basal GAS (1.43_-+0.10 ~mol/10 rain) 10-12 fold higher than wild-type mice (0.12_+0.01 ~mol/10 min; P<.05). In SSTR2 4-, BBS (40 ~g/kg/h) did not modify the high basal GAS (vehicle: 9.21 _+ 1.77 p,mol/h; BBS: 9.67-+ 0.33 ~moi/h; n = 5-6). In wild-type mice, PRL-2903 increased GAS to 1.04_+0.05 ~mol/10 toni (P<.05 vs basal: 0.09-+0.01 ~mol/10 min, n = 5). Infusion of either SST-14 or BBS failed to affect PRL-2903-stimulated GAS (SST-14: 5.45--.0.48 v, mol/h; BBS: 6.17-+0.8 p, mol/h; both P>.05 vs vehicle: 6.17_+ 1.2 g,mol/h; n=4-8). In wild-type mice, immunoneutralization of %1 increased GAS by 4-5 fold over basal and blocked SST-14 or BBS antisecretory action (SST-14: 2.29-+ 0.10 ~molA; BBS: 1.90-+0.10 }xmol/h; both P>.05 vs vehicle: 2.01-+0.10 Ixmol/h; n=3-5). In SSTR2 -/- mice, neither PRL-2903 (13.59_+1.57 p.mol/h, n=4) nor the SST antibody (8.82_+2.97 Ixmol/h, n= 4) affected the high basal GAS observed under urethane anesthesia (vehicle for PRL-2903:12.0_+5.67 IxmoI/h, n=4; control antibody: 9.75_+1.18 panoI/h, n=4). CONCLUSIONS: These results demonstrate that in mice, BBS inhibits GAS through the release of SST and the activation of SSTR2 receptors. These data funher support that SST inhibitory action on GAS is mediated by SSTR2 receptors. S1036 The M1 Muscarinic Receptor is Required for Full Expression of Cholinergic Agonist-Induced Pepsinogen Secretion Guofeng Xie, MasahLsa Yamada, Jurgen Wess, Jean-Pierre Raufman Cholinergic agonLsts are potent stimulants of pepsinogen secretion. Studies with antagonists suggest that, of the 5 mnscarinic receptor subtypes, M3 receptors mediate pepsinogen secretion. However, in situ hybridization indicates that rat chief cells express M1 receptors. Objectives: (1) To determine the roles of M1, M2 and M3 receptors in mediating carbachol- induced pepsinogen secretion. (2) To develop an assay for murine pepsinogen secretion. (3) To determine whether loss of muscarinic receptor subtypes alters chief cell morphology. Methods: M1, M2 and M3 receptor knockout (KO) mice were generated by gene targeting and 6- to 10-wk wild type (WT) and KO mice were used. Cross-sections of gastric mucosa were stained using H + E and toluidine blue (to visualize zymogen granules). Pepsinogen secretion from mouse gastric glands was determined by adapting methods used for rabbit and rat stomach; collagenase digestion and mechanical shearing of gastric mucosa. Peptic activity was determined using ~251-albuminas substrate and expressed as a percentage of total cellular pepsinogen released during incubation. Results: Gastric glands obtained from WT and KO mice were similar in shape and yield [6 • 1.5 x 105 glands (mean -+ SE)/ stomach]. Histology indicated that chief cells from KO mice have normal morphology (preserved polarity and zymogen granule content). Pepsinogen secretion from WT, M1, M2 and M3 KO mice was detected with 1 laM and maximal with 100 btM carbachol. Cholecystoki- nin (CCK)-induced secretion from WT and KO mice was detected at 0.01 and maximal at 1 nM. There was no difference in the potency and efficacy of carbachol or CCK when companng M2 and M3 KO to WT mouse glands, or when comparing mouse glands to rat or rabbit glands and to dispersed chief ceils from guinea pig. In these experiments, maximum concentrations of carbachol and CCK caused a 3- and 2.5-fold increase in secretion, respec- tively. In contrast, in M1-KO mice, the maximal efhcacy of carbachol, but not CCK, was reduced by 25%. Conclusions: Mammal efficacy of carbachol-induced pepsinogen secretion is dependent on expression of M1 receptors. Nevertheless, in the absence of M1 receptors, mammal secretion remains 75% of that observed in WT mice. These findings suggest that other muscarinic receptors, like the M3 subtype, compensate to maintain secretion. This murine secretory model will facilitate use of transgenic mice to investigate the role of mnscarinic receptors and other signaling components in regulating pepsinogen secretion. $1037 Dextran Sulfate Sodium-Induced Enterocolitis is Attenuated in Vanilloid Receptor-1 Knockout Mice Kazunori Fujino, Sebastian G. De La Fuente, Theodore N. Pappas, Christopher R. Mantyh Background: Stimulation of the vanifioid receptor subtype one (VR-1), results in the release of proinflammatory peptides, which in turn mediate inflammation. We have previously shown that pretreatment with capsazepine, a competitive antagonist of VR-1, significantly reduces dextran-sulfate sodium (DSS)-induced colitis. In this study, we examined whether enterocolitLs is attenuated in mice genetically deficient of VR-1. Methods: Colitis was induced in VR-1 gene-deficient (VR-1 (-/-) mice, as confirmed by PCR, by oral administration of 5% DSS in drinking water for 7 days (n=6). C57BU6 wild-type mice were used as controls (n = 6). Animals were checked on a daily basis from day 0 to 7 of the DSS regimen for weight loss, presence of occult blood in feces or gross bleeding, and stool consistency. Based on these parameters, a well-established disease activity index (DAI) was used to clinically evaluate severity of inflammation in both study groups. The DAI score ranges from 0 to 12 total score and represents the sum of scores for weight loss, stool consistency and rectal bleeding. Comparison between groups was performed with Student-t test; significance was assumed to occur at p<0.05. Results: DA1 was increased daily in w/ld-type animals adminis- tered with DSS compared to VR-1 (-/-) mice; VR-1 (-/-) mice exhibited lower levels of DAI throughout the DSS regimen. The differences in DAI between groups reached statistical significance at day 2 of the DSS administrating regimen and continued marked throughout the study period (day 7, 1.111 _+ 0.141 VR-1 (-/-) vs. 2.278 -+ 0.181 wild-type, p<0.05). Conclusion: Animals lacking the VR-1 receptor are protected against the damaging effects of DSS, a well-estabfisbed model of intestinal inflammation. These findings confirm previous observations in that VR-1 in sensory neurons play a crucial role in induction of colonic inflam- mation. A-141 AGA Abstracts