Increased tenascin mRNA levels, timing of production, and location during gastric ulcer healing in rats

Increased tenascin mRNA levels, timing of production, and location during gastric ulcer healing in rats


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• INCREASED TENASCIN mRNA LEVELS, TIMING OF PRODUCTION, AND LOCATION DURING GASTRIC ULCER HEALING IN RATS..M. Tanaka, T. Arakawa, K. Tominaga, S. Kim,* T. Fukuda, K. Higuchi, H. Nakamura, H, lwao, * K. Kobayashi. Third Dept. of Internal Medicine and *Dept. of Pharmacology, Osaka City University Medical School, Japan.

Epithelial cell migration, for which a basement membrane is needed, is important in gastric ulcer healing. Tenascin, an extracellular matrix protein, is abundant in the mesenchyme that surrounds budding and neoplastic epithelial cells. Tenascin may be involved in the migration or proliferation of epithelial cells in vitro, participating in wound healing via these functions. However, the role of tenascin in gastric ulcer healing is unknown. This study was done to investigate the mRNA levels, timing of production, and location of tenascin compared with collagen IV and laminin, main components of the basement membrane, during such healing in rats. Gastric ulcers were produced in male Wistar rats by direct exposure of the serosa to 100% acetic acid. The rats were killed at 12 h oron day 1, 3, 5, 7, 11, 18, 32, oi" 60 after this treatment. There were two groups at each time, one for northern blotting and the other for an immunohistochemical study. Tissues of the ulcerated area and intact tissues in sham-operated rats (controls) were excised and RNA was extracted and assayed by northern blotting with specific eDNA probes. Immunn0histochemical staining was done by the streptoavidin-biotin peroxidase method with mouse monoclonai antibody to human tenascin. Other sections were stained for collagen IV, laminin, or proliferating cell nuclear antigen'(PCNA). The level of tenascin mRNA in ulcerous tissues peaked twice, at 12 h (sixfold that of the controls) and on day 5 (sevenfold), de.easing later to the control level. However, the mRNA levels of collagen IV and !aminin in ulcerous tissues were higher on clays 3 to 32 than in the controls, with peaks on days 5 and 18. The little tenascin. found in control gastric tissues was in the interstitium of gastric glands. At 12 h and on day 5, tenascin was abundant in the interstifium around new epithelial cells on the margin of the ulcer. Collagen IV and laminin were not detected there. PCNA was not found in the new epithelial':cells. The increases in tenascin mRNA and in the gene product in uleeroiJs tissues preceded such increases in collagen IV and laminin during healing. Tenascin was in the interstitium around cells that were'Orobablynot proliferating but migrating. The role of tenascin in such healing may differ from the roles of collagen IV and laminin; tenascin may act as a kind of a transient 'basement membrane' for contact guidance of epithelial cells at ulcerous marginal areas before collagen IV and laminin are produced.

• INDOMETHACIN DOES N O T A B O U S H PENTAGASTRINASSOCIATED ENHANCEMENTS OF RAT GASTRIC MUCOSAL DEFENSE MECHANISMS IN VIVO. S. Tanak~ P.H. Guth andJ.D. Kaun~z. I~s. Svc.and Dept oTMed., West LA VAMC & UCLA Sch.of Med., Los Angeles. Pentagastdn(Gas)enhancesgastric mucosalbarrier function in vivo by increasing mucusgel thickness and the hyperemic responseto add. Sincethese effectsare also produced by exogenousprostaglandim(F~), we hypothesizedtha¢Gas enhancesdefensivemechanismsby inducJng~ s of endogenousPGs. Methods: IntracelullarpH (pH~ and mucusgel thid
AO (IJmollminlcm') 0 min 130 rain

MGT ~)/obaseline) 30 min t 50 rain

control 0.21_+0.03 0.22_+0.08 control 99.1_+0.9102.0_+1.3 Gas 0.16_+0.030.57-+0.05* Gas* 116.6-+2.4 116.6-+2.4 Indo 0.27_+0.030.29_+0.07 Indo 99.0_+1.0 101.7-+1.8 Indo+Gas 0.19+0.04 0.74_+0.08* Indo+Gas*t 109.6_+2.8 112.7_+3.7 GMBF ( ~ b ~ U , e ) ~i 30 min140 mini 50 rain 30 min 140 min 150 mln control 90_+1 88_+1 79_+1 control 7.05_+0.03633-+0.02 6.49_+0.03 Gas* 91-+2 120_+6 140_+5 Gas* 7.05_+0.04 6.69_+0.03 6.72_+0.03 Indo 84-+3 85+-4 79+-8 Indo 7.05_+0.04 658-+0.03 639-+0.04 Indo+Gas*t 101-+1i 117-+13133-+11|ndo+Ges* 7.09-+0.05 6.67--.0.02 6.72-+0.05 * p < 0.05 vs. corr¢ol by single or repeatedANOVA; tp < 0.05vs. Gas by repeatedANOV,A. Conclusions: Indodid not affectGas-inducedenhancementof add output, but partial~impaired Gas-~sodamdaugmentationof mucusgel thickness and the hyperemicresponseto add. Indo did not alterthe benefidaleffectof Gason pH~ Thesedataindicatethat Gas-associatedenhancementsof gastricmucosalbarrier funclJonresultonly partiallyfrom synthesisof endogenousprostaglandins.The major effectsof Gason gastricdefensemechanismsoccureither directlyor throughthe releaseof'other mediators.

• CENTRAL VAGAL STIMLATION ENHANCES RAT GASTRIC MUCOSAL BARRIER F U N C T I O N IN V/VO S. Tanaka, K. Kato, H. Kaneko, Y. Tachd, and J.D. Kaunitz. CURE, Research Service and Department of Medicine, VAMC & UCLA, Los Angeles, CA. Pentagastrin enhancesgastric defense mechanisms. Since vagal stimulation increases acid output independently of gastrin, we tested the hypothesisthat vagal stimulation enhancesgastric defense mechanisms in a similar fashion. Methods: Under urethane anesthesia, a thyrotropin-releasing hormone analogue, RX77368 (RAY),in a 10pl volume, was injected i.c at time=0 for vagal stimulation. Injury model: FIX at dose 0-300 ng; 60% EtOH (4 ml/kg) at time=30 rain; gross lesions measured at time=90 min. In v/vo model: IntracellularpH (pH~and mucusgel thickness(MGT) were measuredby in v/v0 microscopy. Gastricmucosalbloodflow (GMBF)was measuredby laser-Dopper flowmetTyand acidoutput (AO) by back-dtTadon.Gastric mucosaewere superEused with pH 7,4 buffer during time=0-30 min and pH Ii0 during time=30-50 min. (n = 6-12/dose);*p<0.0S vs. saline (t-test or ANOVA):

Gross Lesions (% mucosal area)

GMBF(% b~eline)

(doseof RXin ng) Saline 15.6-+2.5 RXI0 RX0'5 4. _+3.3 RX30 RXI 12.2_+1.7 RXI00 111(3 12.6_+2.4 RX300

[ 20 rain 130 min J 50 min 8.7_+1.8 Saline 92_+5 86_+3 72_+5 3.6_+0.6~ RX30~ 41-+17 24_+7 111_+14 8.3_+2.5 9.8_+4.0

MOT(%) Slinea 30 min I 50 min

pHt IdO (/./mol/min/cn{) 30 min I 50 min 30 min

98.7-+1.3 98.7+13 Saline 7.05-+0.03 6.49_+0.03Saline 0.12_+0.04 RX30* 1125.1-+4.2 125.5_+4.1RX30* 7. 9-+0.03 6.74-+0.02 IIX30* 0.6 ±0. 3 Results: Rx protected against gross injury only at the 30 ng dose. RX increased AO to rates comparable to maximal pentagastrin stimulation. RX increased MGT and GMBF; no further increase occurred during luminal acidification. FIX increased pH~ during acid superfusion. Conclusions: Central vagai stimulation enhanced gastric mucosal defense mechanisms, although, in contrast to pentagastrin stimulation, a hyperemic response to acid was not observed. Diverse secretory stimuli enhance gastric defense mechanisms at several levels, suggesting that secondary mediators common to each stimulus may be involved.

• INDOMETHACIN INCREASES MUCUS BISMUTH CONCENTRATION IN RANITIDINE BISMUTH C I T R A T E T R E A T E D R A T S S. Tanaka and J.D. Kaunitz. Research Service and Dept. of Medicine, VAMC & UCLA, Los Angeles, CA Bismuth compounds have been used in regimens to eradicate Helicobacter pylori (HP) in clinical studios. A drawback of conventional bismuth preparations is that their antibacterial concentrations in gastric mucus are generally of inadequate duration to eradicate HP without frequent dosing. Using in wvo microscopy, we found that the gastric mucus of indomethacin (Indo)-treated rats dosed orally with ranitidine bismuth citrate, GR12231 IX (GR), but not rats treated with GR alone, had a grayish color suggestiveof high Bi3+ concentrations. We thus hypothesized that indomethacin increased Bi3+ concentrations in the gastric mucus. Methods: Ratswere treated with GR, 100 mg/kg p.o. and Indo (60 mg/kg) s.c. Ratswere killed 3, 6, and 12 hrs after drug treatment, and gastric and duodenal mucus were Collected by scraping (after washing) and were assayedfor Bi3+ using plasma-coupled atomic emissiGn .' W" Results: (Bi3+ concentrati o n ,/Jg/g w e t W (ight) Treatment Organ 3 hr I 6 hr 12 hr GR


97.7+_ 16.6 32.4+_5.3




1430 -+ 551

878 +_466

207 + 20. I




37.1_+ 15.7


GR+lndo Duodenum 227+_117 51.4+_13.7 29.7+_ 12.8 "p < 0.05 vs. GR, unpaired t-test. Conclusions: BiJ+ was preferentially localized in gastric mucus. Although this dose of Indo causesgastric erosions, these were not present at 3 hr when Bi3+ concentrations were highest, consistent with Bi3+ localization in mucus and not in erosions. Indo increased Bi3+ concentration in gastric mucus over 1000%, and increased Bi3 + concentrations in duodenal mucus to a lesser extent, possibly as an effect of inhibition of pepsin activity (by Bi3+) and mucus synthesis (by Indo). Bi3+ concentrations in GR+lndo rats were well above the HP inhibitory concentration for Bi3+ of 32/lgJml. These results suggest that GR, even in a lesser dose, plus an NSAID, might be effective in eradicating HP without an additional antibiotic. (Supported by Glaxo Pharmaceuticals).