April 2000

1410FORMATION OF ARTERIOVENOUS ANASTOMOSIS DURINGHEALING OF ACETIC ACID·INDUCED ULCER: INlllBITORYEFFECT OF BFGF AND REGULATION BY NNOS·POSITIVENERVE FIBERS,Masahiko Nakamura, Kanji Tsuchimoto, Hiromasa Ishii, The KitasatoInstitute, Tokyo , Japan; Kitasato Institute Hosp, Tokyo, Japan.

Considerable number of arteriovenous anastomoses (AVA) have beenshown to exist in the ears and skin and to play an important role in bloodflow regulation, while most studies concerning the gastric microcirculationhave denied the existence of AVA. Aim: The present study was undertakento clarify the alteration of the microcirculatory architecture during thehealing of acetic acid-induced gastric ulcer, effect of basic fibroblastgrowth factor (bFGF) and changes of the autonomic nerve distribution.Methods: Wistar strain male rats, weighing 200 to 250 g, were used. Aceticacid-induced ulcers were induced by the direct application of 100% aceticacid on the serosal surface of the glandular stomach for 30 sec. Three,seven, and fourteen days after the treatment, a polyethylene catheter wasinserted into either abdominal aorta or inferior vena cava and 4% aqueoussolution of agar mixed with FITC-dextran was infused orthodromically orretrogradely, and fixed with Zamboni' s solution. 20J.L cryostat sectionswere made and observed by confocal laser microscopy (Zeiss LSM41O),which made it possible to reconstruct the microcirculatory system. Inaddition, some rats were treated with oral administration of CS23, acid­stable human recombinant bFGF (IJ.LgllOOg b.w.) every twelve hours afterthe ulcer formation. To clarify the localization of cholinergic, adrenergicand neuronal nitric oxide synthase (nNOS)-positive nerve fibers, indirectfluorescent immunohistochemical studies were performed using anti-cho­line acetyltransferase, dopamine f3-hydroxylase and nNOS monoclonalantibodies. Results: In the control rat stomach, the gastric mucosal micro­vascular network consisted of arterioles, venules in the basal portion andtrue capillaries but no AVA were found. Fourteen days after ulcer forma­tion, direct communication of arterioles and venules, i.e. AVA, was ob­served in the basal portion of the fundic mucosa in three out of five rats,while no AVA was found in the bFGF-treated rats. nNOS immunoreactivenerve fibers were densely distributed surrounding the AVA. The additionof NO donor, nitroprusside (40J.Lgl l OOg b.w.) brought about the markedopening of this AVA. Conclusions: From these observations, it was con­cluded that AVA was often recognized in the healing process of aceticacid-induced ulcer, while bFGF administration inhibited the AVA forma­tion. This new blood channel was innervated by nNOS-positive nervefibers and could be regulated by NO.

1411CELL VOLUME REGULATION DURING HYPEROSMOLARSHRINKAGE IS MEDIATED BY NA.../K ...·ATPASE IN NECTRU·RUS GASTRIC SURFACE EPITHELIAL CELLS,Outi Nylander-Koski, Harri Mustonen, Tuula Kiviluoto, Eero Kivilaakso,Univ of Helsinki, Dept of Surg, Helsinki, Finland.

The gastric epithelium is often exposed to hypertonic solutions, which areknown to cause cell injury and death. This study investigates the effects ofhypertonic luminal NaCI on gastric epithelial cell volume and the regula­tory mechanisms underlying volume changes. METHODS: Isolated Nee­turus antral mucosa was chambered and perfused on serosal side (S) withRinger solution buffered to pH 7.25 with HC03 and gassed with 95%0 21

5%C02 on both sides. The mucosal side (M) was exposed successively for5 min to 150,200,300,400 and 500 mM NaC!. Amiloride (1mM) andOuabain (ImM) were added to block Na+lH+ antiport and Na+/K+-ATPase, respectively. Intracellular Na" activity (Na.) in surface cells wasmeasured with liquid sensor Na+-PD-microelectrodes. Cell volumechanges (%) compared Ringer solution were measured with microelec­trodes using intracellular tetramethylammonium as volume indicator. RE­SULTS:Most of the increase in (Na ) (see table) is attributable to thedecrease in cell volume and only ouabain treatment leads to uncontrollablecell shrinkage and increase in (Na.), Treatment (20 min) with serosalouabain alone (no NaClload) caused only minor changes in Na, from (5.5:!:0.5 to 8.9 :!:1.1 mM *) or in cell volume (2:!:3'1'0). CONCLUSIONS: Thecell volume regulation in gastric epithelial surface cells during mucosalhyperosmolar exposure is mainly maintained by the basolateral Na+IK+­ATPase, and the Na+IH+ antiport seems to have a less significant role.

Na,(mMI Control Ouabain(5) Amiloride(5) Amiloride(M)

Ringer 5.7±O.6 62±1.6 6.9±1.1 6.5±1.9NaCll50 mM 6.2±0.5 10.0±1.8't 8.4±1 .7 8.0±1.5NaCI300 mM 8.0±0.8t 23.0±5.2't 9.0±1 .S 11.5±2.3NaCI500 mM 17.7±41t 63.7±187't 189±5.6 23.3±4.2

Volume Change (±%) Control Ouabain(S) Amiloride(S) Amiloride(M)

NaCI150 mM -8.1±3.2t -24.9±7.S' t -6.1±3.4 -9,8±4.4NaCI300 mM -34.7±6.3t -50.9±9.2t -31 .6±10.4t -27.8±7.4:j:NaCI500 mM -398±7.4t -85.2±18S' t -45.0+6.7:j: -39.6±14.7t

Mean±SEM, N=5-8, 'p<O.05 compared tocontrol, t p<O.05 comparedtoRinger

AGAA239

1412A POSSmLE ROLE OF ZINC L·CARNOSINE AS A POTENTCHAPERON INDUCER IN CULTURED RAT GASTRIC MUCO·SAL CELLS AND RAT GASTRIC MUCOSA,Masaru Odashima, Michiro Otaka, Noriaki Konishi, Isao Wada, Ivan IvanPacheco, Toshihiro Sato, Sayuri Kato, Sumio Watanabe, Akita Univ Sch ofMedicine, Akita, Japan.

[Background and Aim] Many recent studies have shown the importance ofheat shock proteins (HSPs) for cells survival under stress conditions. In thegastric mucosa, a 72-kDa heat shock protein (stress-inducible HSP70,HSP72) has crucial cytoprotective function mediated by their functions as"molecular chaperon". On the other hand, it is well known that zinc caninduce HSPs in vitro and in vivo. Zinc L-eamosine (polaprezinc) is anantiulcer drug developed in Japan. Zinc L-camosine prevented manyexperimental gastric lesions, although little is known about the mecha­nisms. In this study, the influence of zinc L-eamosine on expression ofHSP72 in rat gastric mucosal cells (RGMI ) and rat gastric mucosa in vivowas investigated. In addition, we investigated the effect of pre-induction ofHSP72 on ethanol-induced mucosal injury. Materials and Methods: 15­25-week old male Sprague-Dawley rats and rat gastric mucosal cells(RGMI) were used in this experiment. RGMI cells exposed to zincLcarnosine, zinc sulfate or L-camos ine (1 to 3oomM) for 6 hours, and thesoluble protein was extracted. Six hours after intragastric administration ofzinc L-camosine, zinc sulfate (30 or lOOmglkg) and L-camosine (76mg/kg), rats were sacrificed and the gastric mucosa was removed. The expres­sion of HSP72 was determined by Western blotting and densitometricanalysis. The effect of pre-induction of HSP72 by polaprezinc on ethanolinduced gastric mucosal lesion were evaluated. The effect of pre-inductionof HSP72 in RGMI cells after ethanol exposure was assessed by LDHrelease. Results: Exposure of RGMI cells to zinc L-camosine and zincsulfate significantly increased the expression of HSP72. Intragastric ad­ministration of zinc L-camosine and zinc sulfate showed significant incre­ment in HSP72 in rat gastric mucosa also in vivo. Induction ability ofHSP72 is approximately 3 times higher in zinc L-carnosine compared withzinc sulfate based on molar concentration in vivo. However, L-eamosinedid not increase the expression of HSP72 in vitro and in vivo. Polaprezincprevented ethanol-induced injury in vitro and in vivo. Conclusion: Themucosal protection by zinc L-eamosine might be partially mediated byinduction of HSP72, and zinc could be a potent "chaperon inducer" ingastric mucosal cells.

1413IS STRAIN STRESS A POTENT "CHAPERON REDUCER" INGASTRIC MUCOSAL CELLS? THE EFFECT OF DECREASEDEXPRESSION OF HSP72 ON WOUND RESTORATION.Michiro Otaka, Sayuri Kato, Masaru Odashima, Noriaki Konishi, IsaoWada, Toshihiro Sato, Ivan Ivan Pacheco, Sumio Watanabe, Akita UnivSch of Medicine, Akita, Japan.

Background: Many recent studies have indicated the importance of heatshock proteins (HSPs) for survival of cells under stress conditions. SomeHSPs are known to be involved in cytoprotection against environmentalstresses mediated by their function as a "molecular chaperon". We havereported that a 72-kDa heat shock protein (HSP72, stress-inducible HSP70)plays essential role in the gastric and colonic mucosa in vivo (Dig Dis Sci44: 1401-7, 1999, Dig Dis Sci 42:833-46, 1997). On the other hand, it hasbeen reported that mechanical strain stress delayed wound restoration incultured gastric mucosal cells. In the presend study, the influence ofmechanical strain stress on the expression of heat shock proteins andwound restoration was investigated. Materials and Methods: Rat gastricmucosal cell line (RGM-I) was cultured to make a confluent cell sheet inCO2 incubator. Mechanical strain was applied to adherent RGM-I cells inorder to increase the length of the cells by an average of 18% at Icycles/sec for 12 h using FLEXERCELL cell-strain system. Using specificantibodies, expression of HSP60, HSP72 and HSP90 was investigated byimmunoblot and densitometric analysis before and after the stress. Rsto­ration of artificially created wound was monitored with or without strainstress. Also, the effect of a potent "chaperon inducer" (zinc Lcarnosine,1-300J.LM ) on the expression of HSPs was studied. Results: (1) Expressionof HSP60 (chaperonin homolog) was significantly increased by mechanicalstrain stress (P < 0.01 vs. control). (2) On the other hand, HSP72 expres­sion was significantly decreased by strain stress (P < 0.01 vs. control).Expression of HSP90 was not affected by the stress. (3) Strain stresssignificantly delayed artificial wound restoration. (4) Pretreatment the cellswith zinc L-eamosine dose-dependently prevented the reduction of HSP72level by strain stress. [Conclusions] Mechanical strain stress showed dif­ferent effect on the expression of HSP60, HSP72 and HSP90. Also, it issuggested that expression of HSP72 might play an important role in gastricwound restoration by modifying cellular migration or proliferation. Inaddition, zinc Lcamosine, a potent chaperon inducer, could be effectivefor chaperon-reducing events or stresses in which wound restoration isinhibited.