controls. The levels of protection ranged between a factor of 1.6 to 18. Administration of AbK-0600 (0.2 mg/kg/day b . t d ) tor 6 days prior and 4 days followmg irradiation (11 to 16 Gy) also significantly increased stem cell survival, with a protection factor ranging between 1.2 and 1.6 In contrast, administration of ALX-0600 (02 mg/kg/day b.i.d.) tbr 4 days tollowing irradiation (11 to 16 Gy) did not protect crypt stem cell survival. CONCLUSIONS: ALX-0600 reproducibly and signibcantly increased small intestinal crypt survival approxi- mately 2-f01d when admniistered prior to radiation exposure. The protective effect was not observed with post-irradiation only administration. These results indicate that ALX-0600 has clonogeinc stem cell modulatory effects, and may have useful clinicaI application in the prevention of cancer therapy induced mucosins.

W999

Elevated Plasma Concentrations Of Amidated Gastrin Cause Increased Crypt Survival In Murine Intestinal Epithelia After F-Radiation Penelope D Ottewell, Alastair Watson, Timothy Wang, Andrea Varro, Graham Dockray, Mark Pritchard

Background and aims: Amidated gastrin has well characterised mitogenic and morphogenic properties in the stomach, but its role in the distal intestine retrains unclear. We have previously demonstrated no differences in the levels of apoptosis and mitosis in small intestnial and colonic epithelia of mice that overexpress amidated gastrin (INS-GAS) com- pared to their wild-type counterparts, either in the untreated state or 4.5h following 8Gy ~/-radiatinn. In order to complete our analysis of the eftects of hypergastrinemia upon the distal intestine in vivo, we have now investigated the eflects of elevated plasma concentrations of amidated gastrin upon small intestinal and colonic clonogenic crypt survival following ',/-radiation. Methods: Mice analysed were adult (10-12 week) INS-GAS and their mild-type counterparts (FVB/N). Clonogenic c ~ t survival was assessed by light microscopy of the small intestinal and colonic crypts 4 days afier I0, 12 or 14Gy y-radiation. We coiRim~ed that the differences observed were specifically induced by hypergasmnemia by analysing INS-GAS mice treated with the specific gastrin/CCKu receptor antagrarist YF476 and FVB/ N mice treated wnb the proton pump inhibitor omeprazole. Results: 4 days following 12 and 14Gy ~/-radiation, INS-GAS mice exhibited significantly higher (-3 fold) small intestinal and colonic crypt survival compared with their FVBRq wild-type counterparts. INS-GAS mice dosed daily with 101*M/kg YF476 showed significantly lower (--4 fold) small intestinal and colonic crypt survival after 14Gy "/-radiation compared with mice receiving vehicle alone. FVB/N mice treated daily with 75mg/kg omeprazole &veloped hypergasmnemia resuhing in significantly increased (-4 fold) survival of small intestinal and colonic crypts alter 14Gy y-radiation. Conclusion.s: (1) Increased small intestinal and colonic crypt survival is observed in mice with elevated plasma concentrations of amidated gastrin following y- radiation. (2) This protective effect of hypergastrinemia occurs as a resah of gastrin signalling via the gastrirgCCK~ receptor.

W l 0 0 0

Glucagondike Peptide-2 Improves Intestinal Permeability and Crypt Cell Production In ParenteraUy Fed Rats With Short Bowel Syndrome Gary R. Martin, Laurie E. Wallace Jon B. Meddnigs, David L. Sigalet

Glucagon-like peptide-2 (GLP-2) has shown to be trophic for the intestinal mucosa and to be correlated with intestinal adaptation following resection. Aims: We investigated the effects of GLP-2 in a total parenteral nutrition (TPN) supported model of experimental short bowel syndrome. Methods: The study was done using juvenile SD rats (250-275 g). Animals were housed in metabolic cages and fed a liquid meal tot 3 days prior to surgery, then underwent a 90 % small intestinal resection and jugular catheter insertion. Nutritional maintenance was isocaloric and contained equal amounts of protein, fat, and carbohydrate: Group 1, liquid enteral diet and Lv. saline infnsion; Group 2, parenteral nutrition only; Group 3, parenteral nutrition + 10 ug/kg/hr GLP-2 mixed with TPN and administered via continuous drip. All groups recewed 50 mg/kg/day i.v. of the antibiotic Cefazolin. Body weight was monitored. After 7 days, intestinal permeability was measured in vivo using orally gavaged/ urinary recover)' over 20 hours of 3-0 methylglucose, lacmlose, and manintol The absorption of probes was quantified by high performance hquid chromatography. Animals were pulsed for 1 hour with BrdU (50 mg/kg, Lp.) prior to euthanasia; intestinal tissue was harvested and processed tor morphological analysis and crypt cell proliferation rate (CCPR). Results: GLP-2 co-intb.sinn prevented bowd and body weight loss, maintained CCPR and epithelial bamer hraction, and sustained normal morphological adaptation (table 1). The TPN alone group had a signfl'icant decrease in CCPR, villus height, bowel and body weight, and also had a significant nicrease in intestinal permeability compared with GLP.2 infused and enterally ted groups There were no significant differences in post-resection adaptation between enterally maintanied and TPN + GLP-2 rats. Condusion: These results show that GLP-2 infusion alone stimulates intestinal adaptation in TPN maintained SBS animals. Further studies are warranted to detemnne the mechanisms involved and to explore the role of GLP-2 in preserving mucosal adaptation.

Table 1

CCPR Villus M C/V ratio lad man ratio bodywt Bowelwt

Enteral 9.6~1.5 655~26 0.24~0.01 018 0.02 -2.8 0.9 Z47 0.1

TPN+GLP. 7,t 0.8 558 16 0.22 0,02 0.23 ~001 -4.2 0.8 Z1 0.09 2

TPN 2s 1.8" 460 27' 0.30 0,32 ~ 0.03* -112 ~ 1.2 0.08* 0.02* 2,0'

Mean SEM. *P<0.05 vs. enteral and TPN + GLP.2 groups, CCPR, brdu immunoreactJvity/crypt reg~n; V~llus hi, urn; body wI, % change; bowel wt, g,

W1001

Up-Regulation of Hepatocyte Growth Factor Release from Human Intestinal Myofibroblasts by Polyunsaturated Fatty Acids, and Its Relationship to Prostaglandin Synthesis Natalya Cockcroft, Cathy Wells, Andy Bennett, Cj Hawkey

INTRODUCTION: Recent studies demonstrate sub-epithelial myofibroblasts orchestrate man), aspects of intestinal bamer function and repair, which are also affected by polyunsatu- rated fatty acids. Since hepatocyte growth factor (HGF) and prostaglandin (PGE2) are both important factors in wound restitution we investigated their release from human primary colonic myofibroblasts, and the effect of the n3 fatty add, eicosapentaenoic acid (EPA), and the n6 fatty acid, gamma-linoleic acid (GLA). METHODS: Nine human colon primary myoflbroblast cell lines at different passages (from 3 to 11) were grown to confluence in 75 cm 2 flasks, before culture for 4 further days with 30~M EPA or GLA or no addition. Similar experiments were conducted with selective and non-selective cyclooxygenase inhibitors. HGF and PGE (measured as PGE2) were assessed by ELISA. Calculation of cell number and the MTS test were used to monitor cell ~bili ty. Fluorescence microscopy was used to monitor changes in re-localization of COX-1 and COX-2. Analysis of variance (ANOVA) and paired t-test were used for statistical analysis. RESULTS: ANOVA showed that individual cell line, passage number and treatment were significant influences on HGF and PGE2 release In the absence of tatty acids, myofibroblasts released 3.3 (mean) +A 0.48 (SD) n g / ml of HGF (n = 17). This increased to 4.3 +/- 0.57 ng/ml with EPA (p<0.001) and 4.9 +/- 0.58 ng/ml with GLA (p<0.0001 vs control, p = 0.003 vs GLA). Unstimulated myofibroblasts released 91.5 +A 63.9 pg/ml of PGE2, compared to 194.4 +/- 115.0 (NS, p = 0.403) with EPA and 604.0 +/- 199.5 pg/ml with GLA (p = 0.032 vs control, 0.053 vs EPA) HGF and PGE2 release correlated for GLA (correlation co-efficient 0.54, p = 0.038), but not control or EPA. Treatment with EPA and GLA resulted in the apparent transloeation of COX-1 and COX-2 to the nucleus as monitored for two cell lines. The non-selective cydooxygenase inhibitor naproxen and the selective COX-1 inhibitor tended to reduce HGF release (p = 0.068). CONCLUSIONS: EPA and GLA increase HGF and PGE2 release by human colonic myoflbrobfasts, by mechanisms that are only partially PGE2-dependant Altered cyclooxygenase distribution may influence this spectrum of activity.

W1002

Expression of Grp and Its Receptor in Well Differentiated Human Colon Cancer Cells Correlates wi th The Presence of Focal Adhesion Kinase Phosphorylated at Tyrosines 397 and 407 Sarah Glover, Kristina A. Matkowskyj, Kristin Keller, Richard V. Benya

Background: Gastrin releasing peptide (GRP) and its receptor (GRP-R) are not normally expressed by epithelial cells lining the colon, but are aberrandy expressed by colorectal cancer cells where they act as morphogens and regulate tumor cell differentiation. Studies of colon cancer formation in mice genetically incapable of synthesizing GRP-R suggested that this receptor's morphogenic properties might be mediated via focal adhesion kinasr (FAK)(Cdl Growth Diff 2000; 11: 385-393). We therefore set out to determine the presence of both total and phosphorylated forms of FAK in human colon cancer specimens as a function of tumor cell differentiation and GRP/GRF-R co-expression. Methods: Ten colon cancers containing 25 regions of distinct differentiation were randomly selected from the UIG GI Cancer Tumor Bank. All specimens were immunohistochemically probed using antibodies directed against GRP, GRP-R, total FAK, as well as those recognizing FAK specifi- cally phosphorylated at tyrosine (Y) 397, 407, 576, 577, 861, and 925. Antibody-specific chromogen was determined by quantitative immunohistochemistry (J Histochem Cytoch~ 2003, in press) for each cellular region of defined differentiation. Results: We confirm tha~ GRWGRP-R co<xpression is a function of differentiation, with highest levels observed m well differentiated tumor cells, However, there was no link between tumor stage and GRP/ GRP-R expression. In contrast, we show that the amount of total FAK, as well as FAK phosphorylated at Y397 and Y407, tightly correlates with differentiation as well as with the amount of GRP/GRP-R co-expressinn. Importantly, FAK phosphorylated at other sites did not correlate with either tumor cell differentiation or with GRP/GRP-R quantity. Conclusions: These findings are consistent with the morphogenic properties of GRP being mediated by its cognate receptor acting to increase total FAK quantity, and by inducing this enzymes phosphorylatinn at tyrosines 397 and 407.

W1003

Substance P Stimulates Ca 2+ Release and Migration in Intestinal Epithelial Cells Douglas J. Turner, Bernard S. Marasa, Jaladanki N Rao, Xm Guo, Jian-Ying Wang, Barbara L. Bass

The enteric nervous system modulates a wide variety of intestinal functions, although a clear role m intestinal mucosal growth and repair remains elusive. The objective of our studies is to characterize the direct epithelial effects of ENS peptides on the process of mucosal restitution. Restitution is the mucosal repair process in which adjacent epithelial cells migrate across areas of superficial iNury to restore mucosal integrity. We hypothesize that t':,e neumpeptides of the ENS, which are densely distributed within the mucosa, will directly effect epithelial rmgration. We tested the ability of Substance P (SPY, a ubiquitous ENS neurotransmitter, to modulate epithelial migration in our in vitro enterocyte model of restitu- tion. Further, we examined the role Ca 2+ activation in this process. Methods: 1EC-6 ceils were grown on plastic dishes to confluence. As an in vitro model of restitution, cell mwatlon was measured by counting the number of cells that migrated onto the denuded surface 6 hours after scraping the cells from the dish, reported as calls/ram Ca 2+ levels were measured by fura-2 imaging. Studies were completed with control media and in the presence of varying concentrations of SP with or without an SP receptor antagonist (NK1 receptor subtype). Results: Exposure of cells to SP demonstrated dose-dependent elevation of cy~osolic Ca > levels at physiologic levels (1 nM to 1 pM). Co-incubation with the SP receptor (NKi receptor subtype) antagonist completely blocked this Ca 2+ response at a dose of 100 izM Exposure of cells to a dose of SP that caused elevation of cytosolic Ca ~ + (1 nM) also mmuhted

AGA Abstracts A-610

entemcyle migration (from 50 +_ 6 to 73 +_ 6 cells/nnn) after wounding Conclusions: These results indicate that SP directly stimulates IEC-6 enterocytes in vitro, increasing cytosolic Qa '+ levels and stimulating epithelial mlgmtinn. These studies support the hypothesis that enterocytes may be directly responsive to nenrntra~smitters of the ENS suggesting a local effeetor function tbr these peptides in the intestinal mucosa.

WlO04

Enhanced Plasma Ghrelin Levels in Streptozotocindnduced Diabetic Rats Tatsuhiro Masaoka, Hidekazu Suzuki, Hiroshi Hosoda, Takaynki Ota, Ynriko Minegishi, Hiroshi Nagata, Kenji Kangawa, Hirotnasa lshii

Background & Aim. Ghrelin, a novel growth hormone secretagogue mainly secreted from the stomach, stimulates growth hormone release, food intake and gastrointestinal motility. In humans and rodents, states of negative energy balance such as starvation or weight loss have been reported to increase plasma ghrdin levels. The streptozotocin (STZ)-induced diabetic rat which is a model of inaulm dependent diabetes mellitus, is known to present with reduced body' weight and hyperphagia. The present study was designed to investigate the dynamics of plasma and gastric ghrelin in STZ-indnced diabetic rats. Methods. 8week- old male Wistar rats were intraperitonaally injected with STZ (60 rag&g) and then examined four weeks later alter they had fasted for 16 hmtra. Ghrelin dynamics were measured by using radioimmunoassay, real-time reverse transcription polymerase chain reaction, and immunohistochemical techniques. Results. The total plasma ghrelin levels in STZ-induced diabetic rats were significantly increased (1651.7 • 256.9 fmol/ml) as compared with those in control rats (593.4+_44.6, p<0.01). The plasma active ghrelin levels in STZ-indnced diabetic rats were also significantly- increased (147.8_+26.8 fmol/ml) as compared with those in control rats (70.4 +_ 9.9, p<0.05). Gastric preprogh~elin mRNA expression levels normalized by GAPDH mRNA expression levels in STZ-induced diabetic rats were signifi- cantly increased (0,093 _+0.016) as compared with those in control rats (0,017+_ 0,004, p<0s C~a the other hand, the gastric total and active ghrelin levels in STZ-indnced diabetic rats were significantly decreased (total; 1053.6 +_ 124.6 fmol/mg, active; 2072 +- 38.5) as compared with those in control rats (total; 174L9 +- 43.9, p<0,001; active; 327.9 • 25.3, p<0,05). The density of ghrelin-immunoreactive cells in the gastric hindna in STZ-indnced diabetic rata was significantly decreased (119.6 • 7 .1/ram 2) as compared with those in control rats (1485 • p<0.05). Conclusion. Ghrelin secretion from the stomach into the bloodstream is significantly increased in STZqnduced diabetic rats. This suggests that the nagatwe energy balance in STZ-treated rats might trigger a signal to up- regulate preproghrelin mRNA expression levels and to stimulate ghrelin synthesis as well as secretion from ghrelin producing cells in the stomach.

W1005

COX-2 Inhibition Reverses Leptin-Stimulated Breast Cancer Growth In Vitro Ponnandai Somasundar, Linda C. Vona-Davis, Dale Riggs, Barbara jackson, David W. McFadden

INTRODUCTION: Breast cancer and the adipocyte hormone leptin have been associated with obesity and reproductive phenomena in women. Previous studies from our laboratory' show that physiological levels of leptin stimulate the growth of breast cancer cells in vitro. Rotecoxib is a non-steroidal anti-inflammatory agent that selectively inhibits cyclooxygenase- 2 (COX-2). The inducible isofoml of COX-2 is overexpressed in many tumors and appears to play an important role in cancer growth. We hyt~othesized that in vitro treatment with rotecoxib would inhibit the growth of breast cancer cells stimulated by leptin. METHODS: MCF-7 and ZR75-1 breast cancer cell lines, both estrogen-responsive and receptor-positive, were cultured using standard techniques. Cells were treated with either human recombinant leptin (0.4-8Ong/ml) or rntecoxib (625p~g/ml) for 24 and 48 hours under serum-enriched conditiona~ Cell viability, was measured by" MTT assay. Statistical analysis was performed by- ANOVA. RESULTS: Leptm significantly (P < 0.05) increased the growth of MCF-7 cells at the physiological dose 4.0ng/mI when compared with controls Proliferative effects were mo~ pronom~ced at 24 hours niter leptm administration. No change in cell viability was observed in the ZR75q cells treated with leptin. In the MCF-7 cells, rofecoxib alone caused significant (P < 0.O01) loss of cell viability and reversed the proliferative effects of leptin when treatments were combined. In contrast, rofecoxib was less eiIective in inhibiting the growth of ZR75-1 cells CONCLUSIONS: We have shown for the first time that rofecoxib, a selective COX-2 iohtbitor, reverses the proliferation of human breast cancer with Ieptin administration. Human studies are warranted to evaluate the potential eftlcacy in cancer therapy

MCF.7 ZR?5-1

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

0 0.4 4~0 40 gO Le~r~ ng,'rd Lepttn, ng/ml

- . - LepU.

Rofecoxib ~s2s ugJ.~)

W1006

Interaction of Bile Acids with Muscarinic Receptors: A Case of Molecular Mimicry Jean-Pierre Raufman, Ying Cben, Kunrong Cheng, Cesar Compadre, Lilia Compadre, Piotr Zimniak

Lithocholykaurine (LCT) interacts functionally with muscarinic receptors on gastric chief cells. We used CHO ceils expressing rat M3 receptors to characterize bile acid/muscarinic receptor interaction and molecular modeling to determine structural properties of LCT that might explain this interaction. Using N-[3H-methyl]scopolamine (NMS) binding, we observed that the potency of LCT and carbachol tbr inhibition of radioligand binding overlsppedi Inhibition of radioligand binding was detectable with 10 ~M LCT and concentrations in the 100 ~M range, achieved in human gallbladder, bile ducts and intestines, reduced binding by 30%. Further evidence of a f~.mctional interaction with muscarinic receptors was that LCT (0.1 and 1 raM) increased inositol phosphates (IP) in CHO-M3 ceils, but not in KI cells that do not express muscarinic receptors. Preincubation with cholinergic inverse agonists (atropine and NMS) inhibited LCTqnduced IP formation. Increasing concentrations of LCT progresswely inhibited acetylcholine (ACh)-induced MAP kinase (p44 and p42) phosphoryla- tion. Until now, ACh was the only known natural ligand of muscarinic receptors. To determine the structural basis for LCT imeraction with mnsearinic receptors, we considered a molecular comparison of LCT and ACh without perfect alignment of individual atoms, but with similar electrostatic concordance: the ester carbonyl group of ACh, that carries a partial negative charge, aligns with the sulfonie acid in LCT, and the amide of LCT aligals with the ammonium ion of ACh. We used molecular modeling to examine this alignment. The LCT conformation with the lowest ener~ ~ was generated and compared with this ACh structure. In the optimized alignment the geometries of the molecular surfaces show striking similarities. Moreover, the distribution of charges on the taurine amide of the bile acid side chain closely resembles the distribution of electrostatic charges of ACh. The similarity of surface charge distribution suggests a likely geometry of LCT imeraction with muscarinic receptors. Increasing evidence indicates that bile acids serve as signaling molecules. Bile acid imeraction with nuclear receptors alters regulation of bile acid production and metabolism. As shown here, bile acids interact at similar concentrations with mnacarinic receptors. The finding that bile acid signaling can affect more than bile acid metabolism has important implications regarding previously unanticipated actions of bile acids on mammalian physiol- ogy and pathophysiology.

W1007

Spinal cord st imulation (SCS) reduces perception of a visceral s t imulus induced by colorectal distention in rodents Beverley Greenwood-Van meerveld, Anthony C. Johnson, Sofia Siswanto

The mechanisms underlying the cause and treatment of visceral pain of gastrointestinal origin are poorly understood. Spinal cord stimulation (SCS) attenuates neuropathic and iscbemic pain, however the efficacy of SCS tot the treatment of abdominal pain are unknown. The overall goal of the current study' was to investigate in rodent models whether SCS has any' effect on nociceptive reflexes induced by colonic hypersensinvity. Methods: Under

A - 6 1 1 A G A A b s t r a c t s