44 MAPK in human intestinal microvascular endothelial cells (HIMEC)

GASTROENTEROLOGY Vol. 118, No.4

A828 AGA ABSTRACTS

4365 GASTRIC VASODILATATION IN THE RAT BY CANNABINOID (CD) RECEPTOR ACTIVATION: INVOLVEMENT OF SENSORY NEURONS AND NITRIC OXIDE (NO). Inngard Th Lippe, Birgit Brodacz, Peter Holzer, Dept of Exp & Clin Pharmacology, Univ of Graz, Graz, Austria. Background : CB receptors have been implicated in inflammation and pain. CB receptor agonists are potent vasodilators in different vascular preparations, but the mechanism of action is unclear. We investigated the effect of methanandamide, a CB receptor agonist, on the gastric vasculature of the rat stomach in vitro. In particular, we addressed the possibility that methanandamide activated capsaicin-sensitive afferent neurons which are known to release vasoactive substances upon stimulation. Methods: The rat isolated stomach was perfused via the left gastric artery with oxygenated Krebs solution by means of a peristaltic pump. Vasoconstrictor responses were recorded by an increase in intravascular pressure. The lumen of the stomach was filled with 5 ml of saline, gastric contractions being recorded by an increase in intraluminal pressure. The vasculature was precontracted by continuous infusion of methoxamine (10 ILM). Results: Vascular perfusion ofrnethanandarnide (2 or 20 ILM) and capsaicin (0.2 or 2 ILM) caused significant concentration-dependent relaxation of the precontracted va~culature\{lut ha\i no effect on gastric muscle activity. The vasorelaxant activity of n1I:thanandamide was significantly counteracted by the cannabinoid CB t receptor antagonist SR-141716A (l ILM, 30 min, about 50% inhibition) and by L-NAME, an inhibitor of nitric oxide synthase (100 ILM, 30 min, about 30% reduction). The vasorelaxant effect of capsaicin was likewise reduced in the presence of L-NAME (37% reduction) and SR141716A (33% reduction). Neither SR-141716A nor L-NAME exerted any effects per se. Conclusions: Endogenous cannabinoids as well as their receptors are present in the vasculature of the gastrointestinal tract where they are thought to playa role in inflammation and pain. From our data it is conceivable that endogenous cannabinoids activate primary sensory nerve endings in the gastric vasculature and cause vasodilatation via a NO-mediated pathway. Endogenous cannabinoids may thus be pathophysiological stimulants of capsaicin-sensitive afferent neurons that regulate local blood flow and other homeostatic mechanisms of the stomach. Supported by the Austrian Science Foundation (grant P1l834-Med) and the Jubilee Foundation of the Austrian National Bank (grant 7843)

4366 HEAT SHOCK RESPONSE DURING ISCHEMIA AND REPERFU· SION IN PIG SMALL INTESTINE IN VIVO. Niku Kj Oksala, Kai Kaarniranta, Jyrki J. Tenhunen, Raine Tiihonen, Antero Heino, Lea Sistonen, Hannu Paimela, Esko Alhava, Dept of Surg, Kuopio Univ Hosp, Kuopio, Finland; Dept of Anatomy, Kuopio Univ, Kuopio, Finland; Dept of Anesthesiol IIntens care, Kuopio Univ Hosp, Kuopio, Finland; Turku Ctr for Biotech, Univ of TurkulAbo Akademi, Turku, Finland; Dept of Surg, Kanta-Hame Cent Hosp, Hameenlinna, Finland. Background: Intestinal ischemia and reperfusion may disturb mucosal barrier function and lead to multiple organ failure. In response to diverse chemical or physical stresses, heat shock genes are induced to express heat shock proteins (Hsps). Hsps function as molecular chaperones ensuring the survival of many organisms during stress. Aim: To investigate heat shock response in the jejunum during intestinal ischemia and reperfusion. Methods: Five female pigs were anesthesized and connected to intensive care monitoring equipment . After a stabilization period (90 min) control samples for ex vivo incubation were taken and the pigs were subjected to ischemia (60 min) by occlusion of the superior mesenteric artery followed by reperfusion (360 min). Systemic, pulmonary and mesenteric hemodynamics, intestinal luminal microdialysate lactate (MDL) and jejunal mucosal-arterial pCOz gradient (dpCOz) were monitored. Antimesenterial open tissue biopsies from the jejunum were obtained for histological analysis and the analysis of heat shock transcription factor I (HSF1) by electrophoretic gel mobility shift assay, the stress inducible heat shock protein 70 (Hsp70) by Western blotting and its mRNA by Northern blotting in the mucosal and muscular layers. Results: One animal was excluded because of low total hemoglobin concentration . Ischemia resulted in increased MDL(p= .OOOI , Friedman) (n = 4) and jejunal dpCO z (p = .0013, Friedman) (n= 4) and histological mucosal injury characterized by detachment of epithelial cells from the villous stroma and necrosis. In reperfusion MDL and dpCO z returned to baseline values and no apparent increase in the degree of histological mucosal injury was observed. Neither surgical procedures, as analyzed from the control samples incubated ex vivo nor 60 min ischemia in vivo induced heat shock response at any timepoint analyzed, whereas during subsequent reperfusion HSFI was activated, levels

of hsp70 mRNA increased and Hsp70 protein accumulated in both the mucosal and muscular layers. The response could be observed within 30 min in the mucosa and 60 min in the muscularis during reperfusion. Conclusion: Heat shock response in pig jejunum subjected to both ischemia and reperfusion is characterized by transcriptional activation of the heat shock genes and accumulation of Hsp70 protein during reperfusion. These findings may provide novel diagnostic strategies for intestinal damage caused by ischemia and reperfusion. 4367 CGRP IN THE PREVENTION OF ISCHEMIAIREPERFUSION IN· DUCED MYOELECTRIC INJURY OF THE SMALL INTESTINE, Wieslaw W. Pawlik, Michal Pawlik, Ryszard Sendur, Jaroslaw Biernat, Jolanta Jaworek, Tomasz Pawelec, Piotr Thor, Dept of Physiology GielIonian Univ Med Sch, Krakow, Poland. Calcitonin gene related peptide (CGRP) is widely distributed in the myenteric neurons along GI tract. Biological effects of CGRP on gastrointestinal tract include increase in the intestinal blood flow, relaxation of the small muscle and slight increase in the slow waves amplitude (SWA) with no effect on frequency (SWF) of intestinal myoelectric activity (lMA) . The aim of this study was to evaluate the possible protective effects of endoand exogenous CORP against ischemia/reperfusion induced bowel injury in rats. Experiments were performed on 30 fasted anesthetized Wistar rats. Systemic arterial blood pressure (AP), mesenteric blood flow (MBF) and microcirculatory intestinal blood flow (LDBF) were measured. Oxygen consumption (V02) was estimated from MBF and mesenteric AV02 difference. IMA was recorded by 4 monopolar electrodes in proximal jejunum and analyzed using computer program with Fourrier analysis of SWF. Control ischemia induced by 30 min. total occlusion of anterior mesenteric artery (AMA) reduced SWA by 25:!:5% and SWF by 24:!:4%. At the end of 60 minute reperfusion period SWA and SWF values were restored to control values but SWF showed instability. At the 15th minute of reperfusion period MBF, LDBF and V02 incresed to I09:!:6, 1l9:!:8 and 120:!:7% of control values respectively. Infusion of exogenous CGRP (0,16 ILglkglmin i.a.) increased MBF, LDBF and V02 by 26:!:6, 31:!:9 and 20:!:4% respectively in comparison to control values. In the same experimental group SWA increase of 14% was observed with not significant changes in SWF. In the group where CGRP was administrated before and during 30 min. period of intestinal ischemia MBF, LDBF and V02 values at 15th minute ofreperfusion were significantly higher by 23:!:5, 32:!:6 and 17:!:5% respectively in comparison to the values observed in the control reperfusion. In that group SWA values were restored to preocclusion values at 15th minute of reperfusion yet and SWF showed much more stability. Infusion of CGRP receptor antagonist (CGRP 8-37) reduced MBF, LDBF and V02 by 12:!:7, 14:!:8 and II :!:6% respectively (differences not significant versus control). In IIR group when CGRP 8-37 was given hemodynamic parameters (during reperfusion) were significantly lower and IMA parameters were not restored to preocclusion values, We conclude, that endogenous and exogenous CGRP attenuate circulatory parameters of the small intestine during ischemia and reperfusion. A direct correlation exists between hemodynamic, metabolic and myoelectric effects of CORP.

4368 VEGF INDUCES NUCLEAR TRANSLOCATION OF NUCLEAR FACTOR ACTIVATED T CELL (NFAT) AND ACTIVATION OF P42J44 MAPK IN HUMAN INTESTINAL MICROVASCULAR EN· DOTHELIAL CELLS (HIMEC). Parvaneh Rafiee, Mona S. Li, Pamela J. Fisher, Thomas H. Larnirand, Vinod B. Shidham, Christopher P. Johnson, David G. Binion, Med Coll of Wisconsin, Milwaukee, WI. Background and Aims: The endothelial growth factor VEGF, plays a key role in angiogenesis in wound healing, chronic inflammation, and tumor neovascularizaton, including colon cancer. VEGF binds two high affinity tyrosine kinase receptors on endothelial cells, resulting in intracellular signalling, gene expression and cell cycle re-entry. The intracellular transduction pathways, as well as the transcription factors that lead to gene expression and proliferation in tissue specific microvascular endothelial populations, are not well understood. We investigated the effect of VEGF on signalling pathways in primary cultures of colonic HlMEC, focusing on MAPK cascades and NFAT. Methods: Primary cultures of confluent and subconfluent HIMEC (GE 112:1895-1907, 1997) were used in all experiments. HIMEC growth and proliferation was quantified using cell enumeration. Intracellular signalling pathways were characterized using serumstarved HIMEC monolayers stimulated with 20 ng/ml VEGF . The nonselective NOS inhibitor L-NMMA , the antioxidant curcumin, and the MEK inhibitor PD98059, as well as the calcineurin inhibitor Cyclosporin A (CsA),which also inhibits NFAT nuclear translocation, were used to pretreat HIMEC prior to activation and Western analysis. Specific antibodies targeting p42/44 MAPK were used to detect phosphorylated signalling

AGAA829

April 2000

proteins. Nuclear extracts of HIMEC were used to detect NFAT translocation. Results: HIMEC proliferated in response to VEGF, doubling in approximately 48 hours, but their growth was completely inhibited by TNFaILPS. VEGF-induced phosphorylation of p42/44 MAPK was inhibited by pretreatmentwith PD98059, curcumin and NOS inhibitors. VEGF induced translocation of NFAT from cytosol to the nucleus in HIMEC. TNFalLPS also led to activation of p42/44 MAPK, but did not induce nuclear translocation of NFAT. CsA inhibited VEGF-induced NFAT nuclear translocation as well as HIMEC proliferation. Conclusions: VEGF and TNFalLPS both induce p42/44 MAPK activation in HIMEC. Inhibition of p42/44 MAPK by NOS inhibitors, suggest that NO acts as an upstreamsignal in VEGF inducedactivation.NFAT is activatedin HIMEC in responseto VEGF, and may be associatedwith endothelialproliferation. This data suggests that anti-angiogenic therapy may be tailored to block specific MAPK and NFAT signalling pathways involved in HIMEC proliferation.

4369 ACTIVATION OF NFKB FOLLOWS INFLAMMATORY AND ANGIOGENIC STIMULI IN HUMAN INTESTINAL MICROV ASCULAR ENDOTHELIAL CELLS (HIMEC). Parvaneh Rafiee, Cara Olds, Pamela 1. Fisher, Mona S. Li, Thomas H. Lamirand, Vinod B. Shidham, Christopher P. Johnson, Galen M. Pieper, David G. Binion, Med Coli of Wisconsin, Milwaukee, WI. Background and Aims: Tissue specific microvascular endothelium is a stable, differentiatedcell population,that is capable of dramatic alteration in physiology and function when challenged by inflammatory and angiogenic stimuli. Nuclear factor kappa B (NFKB) is a major transcription factor for coordinated gene expression, present in multiple cell types, including vascular endothelial cells. We sought to characterize the signalling pathways that follow inflammatory (TNFalLPS) and angiogenic (VEGF) activation in colonic microvascular endothelial cells by using HIMEClines in vitro. We hypothesizedthat these stimuli, relevant to IBD and colon cancer, would result in NFKB activation in HIMEC. Methods: HIMEC isolated from colonic mucosa were used in all experiments. Endothelial monolayers were activated with TNFa/ LPS or VEGF, which had been previously demonstrated to induce inflammatory activation and growth (GE 112: 1895-1907, 1997). HIMEC were pretreated with specific inhibitors of NFK15~ncluding Bay II and NS-50, a p38 MAPK inhibitor SB203580, a specific\¥EK inhibitor PD98059, the antioxidant curcumin and the immunosuppressive agent Cyclosporin A (CsA). Intracellular signalling pathways were assessed using Western analysis of cell lysate. Nuclear fractions were analyzed with electromobilityshift assays (EMSA) using an NFKB consensus oligonucleotide sequence. Results: Following activation with TNFalLPS, HIMEC demonstrated cytosolic phosphorylation of MAPKsand IKB by Western, and nuclear translocation of NFKB by EMSA. The activationofNFKB was inhibitedin a concentrationdependent manner by curcumin, Bay 11, NS-50, and SB203580 but was not affected by PD98059and CsA. VEGF stimulationof HIMEC resulted in phosphorylation of p42/44 MAPK and activation of NFKB detected by nuclear translocation (EMSA), that was inhibited by all agents except SB203580. Conclusions: This data demonstrates that at least 2 signallingpathways are involved in inflammatory and angiogenic activation in HIMEC. NFKB activation following inflammatory (TNFILPS) and angiogenic (VEGF) activation were inhibitableby distinct pharmacologic agents. These results suggest that endothelial signalling pathways can be targeted for vascular therapy in inflammation as well as angiogenesis. 4370 INHIBITION OF ISCHEMIA REPERFUSION-INDUCED INTESTI· NAL INJURY AND SYSTEMIC INFLAMMATION BY A NOVEL COMPLEMENT REGULATORY PROTEIN. Scott T. Rehrig, Sherry D. Fleming, Joel Guthridge,Daniel Mulloy, David Ward, Steve Lawson, Michael Holers, Terez Shea-Donohue, George Tsokos, Usuhs, Bethesda,MD; UCHSC,Denver, CO; Walter Reed Army Med Ctr, Washington, DC. Complementactivationis a fundamental part of the inflammatory response to tissue injury. Complementreceptor l-related gene/protein y (Crry) is a potent murine membrane regulator resembling human complement receptor I. A recombinant soluble form of the mouse Crry was fused to IgGI hinge, CH2, and CH3 domains (Crry-Ig). It is active in both the classical

and alternative pathways. Aim: to define the role of complement in mesenteric ischemia reperfusion (IR)-induced injury in mice. Methods: Mice were subjected to sham laparotomy or laparotomy with 30 minutes of ischemia followedby 120 minutes of reperfusion.Crry-Ig was given either 5 minutes prior (T-5)or 30 minutes (T+30) into the reperfusion period. Intestinal mucosal injury (SMI, 0 [normal)-5 [severe)), nitric oxide synthase (NOS)activity (NADPH diaphorase stain), neutrophil infiltration (PMN/hpf) and LTB4 generation were determined. The percent of PMN producing H202 was assessed using flow cytometry.Results: IR increased mucosal injury,PMN infiltration.L'TB, generation, and reduced NOS activity. Crry-Ig inhibited mucosal injury, but did not prevent PMN infiltration. IR-induced elevation of LTB4 production was inhibited by crry-Ig given at T+30, but not at T-5. The IR-induced reduction in NOS activity was prevented by T+30 crry-Ig. Systemically,47% of PMN produced H202 in IR compared to 7.7% in sham mice. SystemicPMN productionof H202 in T+30 crry-Ig was similar to sharn(7.5%). Conclusions: Crry-Ig protected against IR-induced intestinal mucosal injury despite elevated PMN infiltration. The improved protection after crry-Ig T+30 was coincident with a reduction in LTB4 generation and a preservation of NOS activity. In addition, the ability of crry-Ig to limit systemic PMN H202 production may suppress the development of the systemic inflammatory response syndrome after major surgery or trauma. Treatment Sham

IR120 IR120 Cny.lg T·5 IR120 Cny·lg T+30

MUCOSAL INJURY

PMN(#/hpfl

LTB, lpg/mgl

0.6 ± 03 2.6 ± 0.416± 0.5$ 08 ± O.4p

1±O 6± 19± 212±4-

35± 18 117±27158 ± 4836± 8.5

-Poo:0.05 vs. Sham; $Poo:0.05 vs. IR120

4371 CALCITONIN GENE-RELATED PEPTIDE CAN ATTENUATE THE ISCHEMIAIREPERFUSION INDUCED INJURY OF THE INTESTINAL MUCOSA. Ryszard Sendur, Jaroslaw Biernat, Wojciech Szczepanski, Rafal Koziol, Wieslaw W. Pawlik, Dept of Physiology Jagiellonian Univ Med Sch, Krakow, Poland. Sensory peptides plays an important role in the maintenance of intestinal circulation, myoelectric and metabolic activity. Calcitonin Gene related Peptide (CGRP), a potent mesenteric bed vasodilator can have a similar effect by affecting blood supply during reperfusionperiod. The aim of this study was to evaluate possible protectiveeffects of CGRP against intestinal ischemia/reperfusion injury (IIR). Experiments were carried out on 50 anesthetisedWistar rats. Systemicarterial pressure (AP), mesentericblood flow (MBF) and microcirculatory intestinal blood flow (LDBF) were measured continuously. Oxygen consumption (V02) was estimated from MBF and mesenteric AV02 difference.Intestinalischemiawas inducedby 30 min. total occlusion of anterior mesentericartery (AMA). At the end of experiments ischemic/reperfused segmentsof the rats small intestine were taken, which were ebedded in paraffineand stained with hematoxylineand eosine to obtain histological preparates. The degree of intestinal mucosal damage was established on semiquantative scale according to Chiu ( from to 5 grade). For groups comparison Kruskall-Wallis ANOVA significance test was. used. Rats were divided for five groups (10 animals each group). First group sham operated was taken as a control. In the second group of rats ischemia of 30 min. and reperfusion of 60 min. was performed. In the third group ischemia (alone) was made.. In the fourth IIR was performed on the background of CGRP (0.16 mg/kg/min i.a.), In the fifth group CGRP receptorantagonist (CGRP 8-37) was given before I1R. Results: The degree of mucosal damage in ischemia and ischemia/reperfusion group was 3,42 and 3,63 respectively(not significantdifference). In the fourth group infusion of CGRP elevated MBF,LDBF and V02 by 23::':5, 32::':6 and 17::':5% respectively during the reperfusion period. In this group we observed a significantreduction of the mucosal damage to 2,25 (p<0,05). Administration of CGRP receptor blocker before ischemia and reperfusionexacerbatedmucosaldamage to 3,75 but the result was not significantly different versus second group. It was accompanied by the marked reduction of MBF, LDBF and V02 by 15::':5,23::':7 and 12::':3% respectively. Conclusions: Above results of the present investgationsdemonstrate that exogenous CGRP has a marked protective effect against intestinal mucosal injury induced by I1R. It's action is probably related to increased intestinal tissue blood flow and increased oxygen delivery.

°