A242 AGA ABSTRACTS

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PEROXISOME PROLIFERATOR·ACTIVATED RECEPTOR rREGULATES THE EXPRESSION OF TREFOIL FACTORS INGASTRIC EPITHELIAL CELLS.Tadahito Shimada, Kazuo Kojima, Yasuo Mitobe, Hideyuki Hiraishi,Akira Terano, Dept of Gastroenterology, Dokkyo Univ, Tochigi, Japan.

BACKGROUND:Trefoil factors (TFF) are a family of peptides which bearthe three-loop trefoil domains. At least three trefoil peptides are known atpresent, TFFI (pS2), TFF2 (spasmolytic peptide), and TFF3 (intestinaltrefoil factor). Gastric epithelial cells express TFFI and TFF2, and thesetrefoil factors are believed to play important roles in mucosal defense andrepair. Peroxisome proliferator-activated receptor -y(PPAR-y) is a memberof the nuclear receptor family and has been shown to be closely associatedwith adipocyte differentiation. It has recently been shown that gastrointes­tinal epithelial cells also express PPARy, and that PPARy is involved incell differentiation and growth suppression. Since the regulation mecha­nisms of TFF expression is not well understood, we examined the effectsof the activators of PPARy on the expression of TFF in gastric epithelialcells. METHODS:MKN45 cells, derived from human gastric carcinoma,were used as a model of gastric epithelial cells. Expression of PPARy wasexamined by RT-PCR and Western blot analysis. Troglitazone (TG) and15-deoxy-PGJiPGJ2) were used to activate PPARy. Growth response ofthe cells (DNA synthesis) was assessed by ELISA which quantifies BrdUincorporation. Quantitative RT-PCR analysis using an ABI-PRISM 7700Sequence Detector (Perkin-Elmer) was performed to determine the level ofTFF mRNA expression in response to the activators of PPARy. l3-actinmRNA expression was also measured for standardization. RESULTS:RT­PCR and Western blot analysis showed the expression of PPARy mRNAand protein in MKN45 cells. Treatment of the cells with PGJz or TG for24 h resulted in the suppression of DNA synthesis in a dose-dependentmanner. In the control condition, average TFFI/I3-actin, TFF2/I3-actin, andTFF3/I3-actin mRNA ratios were 0.16, 0.003, and 0.0001, respectively.Incubation of the cells with PGJ2(10 1J.M) for 24 h caused 1.3-fold increasein TFFI mRNA expression and 4.7-fold increase in TFF2 mRNA expres­sion, while TFF3 expression was not affected. Similarly, 24 h-treatmentwith TG (30 1J.M) caused 1.7-fold increase in TFFI expression and 13.7­fold increase in TFF2 expression but had no effect on TFF3 expression.CONCLUSIONS:TFFI and TFF2 expression in MKN45 cells appears tobe modulated by PPARy. PPARy may playa role in gastric mucosaldefense and repair through regulation of TFF expression.

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THE DIFFERENCE IN THE RATE OF PROSTAGLANDIN E2 SE·CRETION WITHIN THE LOWER AND THE UPPER ESOPHA·GEAL MUCOSA: ITS POTENTIAL PATHOGENETIC IMPLICA·TlONS.Cezary Poplawski, Mazen Asadi, Tomasz Skoczylas, Michele Loftis, Rich­ard W. McCallum, Jerzy Sarosiek, Kumc, Kansas City, KS.

We have recently demonstrated that the lower segment of the humanesophagus exhibits a significant secretory function in terms of mucus andprostaglandin E, (PGEz), related to its anatomical presence of submucosalmucous glands (Gastro, 106:973-81, 1994; AJG, 89:588-94, 1994). Itremains unknown, however, if the rate of PGEz secretion within the loweresophageal mucosa, the major location of the gastroesophageal reflux­related mucosal injury, is different from the upper esophagus. Aim: Toinvestigate the esophageal PGEz release in asymptomatic volunteers beforeand during exposure to HClIPepsin, mimicking the natural gastroesopha­geal reflux scenario, in the lower and the upper esophagus. Subjects &Methods: The study approved by lRB was conducted in 10 asymptomaticvolunteers (4F & 6M, mean age of 33). Esophageal secretions werecollected during mucosal exposure to NaCI, HClIPepsin (pH 2.1), andNaCI using esophageal perfusion catheter (Wilson-Cook Med. Inc. NC),with the distal balloon placed 35 em and 25 cm from incisors. EsophagealPGEz was measured by RIA (Amersham, IL). Statistical analysis wasperformed using ~-Stat software (Jandel, CAY. Results: The volumes andthe pH of the esophageal perfusates were similar in both secretory tests.The basal rate of the lower esophageal PGEz release, during perfusion withsaline, was only 13% higher than within the upper esophagus (4401 2::2329vs 3895 2:: 1822 pg/min, P>0.20). The rate, however, of the esophagealPGEz release during the mucosal exposure to HCI/pepsin, mimicking thenatural gastroesophageal reflux episode, was significantly greater (by103%) within the lower esophageal mucosa (3009 2::704 vs 1479 2::268pg/min, P<0.05). The rate of the esophageal PGEz release within the loweresophageal mucosa still remained significantly greater (136%) after dis­continuation of HClIPepsin and infusion of the final saline (3572 2:: 1071 vs1513 ±3l5 pglmin, P<0.05). Conclusions: 1. The significantly higher rateof the esophageal PGEz release within the lower esophageal mucosa mayindicate that the lower segment of the human esophagus is better equippedin terms of its protective power to combat injury by the gastroesophagealrefluxate. 2. This higher content of the esophageal PGEz within the loweresophageal segment may play pivotal role during episodes of nocturnalreflux when mucosal defense cannot rely on swallow-induced flow ofsalivary protective factors.

GASTROENTEROLOGY Vol. US, No.4

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GASTRIC ULCERATION TRIGGERS ACTIVATION OF ANGlO·POIETIN-l,-2 AND TIE2 GENES IMPORTANT FOR ANGIOGEN·ESIS.A. S. Tarnawski, R. Pai, M. K. Jones, I. L. Szabo, H. Kawanaka, I. Sarfeh,VA Medical Ctr, Long Beach, CA; Univ of CA, Irvine, CA.

Angiogenesis - the formation of new capillary blood vessels - is essentialfor ulcer healing because of nutrient and oxygen delivery to the healingsite. Recently discovered endothelial specific growth factors, angiopoietins-1 and -2 (Angl and Ang2) and their common receptor, Tie2 play impor­tant roles in angiogenesis during embryonic development and are essentialfor microvessel maturation and integrity. Whether they participate inangiogenesis during gastric ulcer healing is unknown, forming the basis forthis study. METHODS: Gastric ulcers were developed in 52 SpragueDawley rats by local (4 mm i.d.) serosal application of acetic acid. Normalgastric mucosa, ulcer or dissected granulation tissue were obtained 7 daysafter ulcer induction. STUDIES: 1) Quantitative histology, 2) TransmissionEM, 3) Vascular cast study/scanning EM to assess angioarchitecture, 4)Angl, Ang2 and Tie2 mRNAs by RTIPCR, and proteins by immunohis­tochemistry and Western blotting. RESULTS: At 7 days after gastric ulcerinduction, granulation tissue was well developed at the ulcer base. Itconsisted of fibroblasts and proliferating endothelial cells forming mi­crovessels. Ang2 and Tie2 mRNAs were significantly upregulated(3202::20% and 3902::30% increases, respectively; both p<O.OOI; andAngl was increased by 362::4% (p<0.05). The increased expression ofAng2 and Tie2 closely correlated with the number of branching microves­sels reflecting angiogenesis. CONCLUSIONS: 1) Ang2 and Tie2 mRNAand proteins are strongly upregulated in granulation tissue at the gastriculcer base. 2) Their expression level strongly correlates with angiogenesis.3) These data demonstrate for the first time that Angl, Ang2 and Tie2 areinvolved in gastric (granulation tissue) angiogenesis, essential for the ulcerhealing.

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EVIDENCE FOR TWO DISCRETE MUCUS SECRETIONS IN THENORMAL STOMACH.Catherine Taylor, Adrian Allen, Peter W. Dettmar, Jeffrey P. Pearson,Univ of Newcastle upon Tyne, Newcastle, United Kingdom; Reckitt &Colman Products Ltd, Hull, United Kingdom.

Introduction The functions of the gastric mucus are twofold. I) Mucusprovides a protective layer over the mucosal surface which is a barrier topepsin. This layer also allows the maintenance of a pH gradient whichprotects the mucosa from luminal acid. 2) Mucus acts as a lubricantattenuating shear stress at the mucosa resulting from the mechanical forcesof digestion. The pig is an excellent model to investigate the properties ofgastric mucus. Pig gastric mucin gene products are very similar to theirhuman counterparts, for example human MUC5AC and isolated pig gastricmucin have >80% homology in the cysteine rich non-tandem repeatregion. Isolated mucins from pig and human stomach have also been shownto have comparable functional properties. Methods Mucus gels are ob­tained by gently scraping the mucosa of freshly killed pigs stomachsRheological measurements were carried out on a Bohlin rheometer withparallel plates operating in oscillatory mode. ELISA s were carried outusing the primary antibody was NCL-HGM-45MI which is raised to aportion of the human MUC5AC gene product outside the tandem repeatand has been shown to cross-react with pig gastric mucin. Results Twodistinct mucus gels can be isolated from the stomach of fasted animals.These gels were designated firm and sloppy. The firm gel is found adherentto the mucosa over the whole. The sloppy gel is found lying over the firmgel and is found in the corpus, antrum and pylorus. In the stomach of fedanimals only the firm gel remains adherent to the mucosa. Rheologicalmeasurements have shown that both these secretions are true gels (G' >G"), however they show completely different behaviour profiles. The firm gelis a stronger gel than the sloppy gel (lower phase angle) and the sloppy gelshows a characteristic loss of gel structure when shear stress is applied,liquid like behaviour becoming dominant at around 2Pa. This breakdownis not seen in the firm gel up to a stress of lOPa. Treatment of the firm gelwith pepsin at pH2 (48 hours) does not alter the rheological propertiesdemonstrating that the sloppy gel is not a breakdown product of the firmgel. The sloppy gel has a five fold greater MUC5AC like reactivity than thefirm gel giving rise to the possibility that the two gels are composed ofdifferent MUC gene products. Conclusions The stomach is protected bytwo types of mucus gel: a firm gel that is resistant to pepsin degradationcovering the whole stomach and a sloppy gel covering the firm gel in thedistal part of the stomach which flows under low shear (presumably havinga lubricative function).