April 2000

ing function of peritoneal Mfs against Candida parapsilosis with or with­out anti-Mac-I, and Mac-l-deficient mice, which lack this receptor, for toelucidate that phagocytic killing by Mfs required the signal via Mac-I.METHODS Peritoneal Mfs were isolated from normal mice and Mac-Iknockout mice after glycogen injection into the peritoneal space. Phago­cytic Killing by Mfs was evaluated using acridine orange-crystal violetstaining assay under the fluorescence microscope (AmJ.Physiol. 1996).RESULTS Anti-Mac-I down inhibited Phagocytic killing by Mfs. And,phagocytic killing of peritoneal Mfs, which were isolated from Mac-Iknockout mice, was suppressed compared with that of control mice.DISCUSSION Previous study showed that Mac-I was required for phago­cytosis of PML. Furthermore we elucidated the signal via Mac-I effectedphagocytic killing function of Mfs as a matter of fact. It was reported thatMac-I antibody reduced the incremental production of O2in polymorpho­nuclear leukocytes (PML). Our results showed that there was possibilityO2,which worked on phagocytic killing in Mfs against Cparapsilosis, wasincreased by the signal via Mac-I.

Phagocytic kiliing ofperitoneai macrophages(Mfs) against C.parapsilosis

%killing %killing (%control) pvalure

control Mis 15± 19 100 ± 0control Mis+controllGG 16.2 ± 32 107 ± 95

control MIs+anti·Mac·1 10.5±12 n.o± 19.60.17 (vs control Mis +

controllGG)Mac·1 kockout mice MIs 10.5 ± 0.5 71.1 ±8.9 0.03 (vs control MIs)

%kiliing =no. dead intracllular Candida organisms I total no. intracellular Candida organisms. Allgroup used 3mice.

5121

MEMBRANE TUMOR NECROSIS FACTOR RECEPTORS INLUNG AND KIDNEY OF MALNOURISHED RATS.Nilima Raina, Khursheed N. Jeejeebhoy, Univ OF TORONTO, Toronto,ON, Canada.

Malnutrition is a common cause of Pneumonia and tumor necrosis factor­alpha (TNF-ll') plays a pivotal role in the pathogenesis of lung disease.Previously we had shown that the nutritional status altered the metabolicresponse to TNF infusion, with severe metabolic abnormalities in TNF­treated weight-gaining rats. This effect was as a result of increased mem­brane and soluble receptors in weight-gaining rats suggesting stabilizationof TNF in circulation thereby augmenting its activity and prolonging itsaction. In light of the above observations, this study was designed to testthe hypothesis that malnutrition will alter the expression of membrane TNFreceptor (mTNFR) in lungs which is very susceptible to infection inundernourished subjects. In addition for comparison nutritional effects onthe expression of mTNFR in kidneys will also be measured. Twenty-twomale rats weighing 200-220 g were fed a liquid formula diet for 10 days,and divided equally into control rats meeting all nutritional requirements(C) and malnourished rats with protein energy restrictions (M). Lung(n= 10) and kidney (n= 12) plasma membranes (PM) were isolated byseveral steps of centrifugation using sucrose gradient. Purity of PM wasconfirmed by measuring the 5'-nucleotidase activity by a kinetic methodusing stabilized glutamate dehydrogenase. Recombinant rat TNF-a waslabeled with Nal25 using the Chloramine-T procedure. PM was cross­linked with 125I-ratTNF-ll' and electrophoresed on 4-15% SDS-PAGE(sodium dodecyl polyacrylamide gel electrophoresis). 125I-ratTNF-ll'binding was demonstrated in lung and kidney to proteins of molecularmasses of 33 to 45 kD and 66 to 92 kD regions, respectively. Excessunlabeled TNF-ll' displaced the binding showing its specificity. Thesefindings may reflect that different organs have cell-specific receptors withdifferent functions as a result of expression of both high- and low-affinityreceptors which are distinct and differ in size, glycosylation, peptide mapsand their degree of internalization and degradation. The degree of bindingto kidney PM did not show any significant changes between the C and Mgroups. This suggests low binding in kidneys which is not affected bynutritional status. On the contrary increased binding in lung PM of M ratsas compared with C rats, however, possibly, suggests that malnutrition inassociation with increased mTNFR may make lung susceptible to one ofthe most common infections namely, Pneumonia. Disclosure: This researchwas funded by Medical Research Council of Canada.

5122

EFFECTS OF SENSORY NEUROPEPTIDE RECEPTOR BLOCK­ADE ON TRANSFORMING GROWTH FACTOR ALPHA (TGFA)MEDIATED GASTROPROTECTION.1. Ren, L. A. Saymeh, Y. Xia, R. F. Harty, Oklahoma Univ Health Sci Ctr,Oklahoma City, OK.

We have previously shown that the gastroprotective effects of TGFll'against ethanol injury in rats were prevented by chemical (capsaicin)sensory denervation. Aim: The present studies examined involvement ofthe sensory neuropeptides calcitonin gene-related peptide (CGRP) andsubstance P (SP) in TGFa-mediated gastric mucosal protection againstethanol injury. Methods: Fasted adult male SD rats received intragastricallyeither saline (l m!) or ethanol (I ml; 75% v!v). Prior (30 min.) to orogastricdosing animals were pretreated with either TGFll' (50 mg/kg; i.p.) orvehicle. Additional pre-treatment strategies included intravenous adminis-

AGAAll07

tration of either CGRP antagonist, CGRP 8-37 (0.1 mg/kg; iv), or SPantagonist, [D-Pro 2, D-Trp 7, 9] (0.5 mgt kg, iv). Peptide receptorantagonists were given immediately prior to TGFll' administration.Results:Severe gastric ulceration was observed in the corpus 30 minutes afterethanol gavage and the area of injury measured 48.6 :t 7.5 mnr'. TGFll'pretreatment inhibited significantly ethanol induced mucosal injury: 5.1 :t0.7 rnm";p <0.001 vs ethanol alone. CGRP receptor antagonist, CGRPs_37 ,

prevented completely the protective effects ofTGFll' and the area of injury(44.3 :t 5.1 mm/) was not different from corresponding results withethanol alone (p>O.1). In contrast, SP antagonist did not alter TGFll'­mediated gastroprotection: area of injury 4.8 :t 0.6 mrrr': p <0.001.Conclusion: I) TGFll'-mediated gastroprotection involves sensory neuronactivation and neuropeptide release and 2) CGRP receptor blockade pre­vented TGFll' protective effect. Summary: These results suggest that thegastroprotective actions of TGFll' are, at least in part, indirect and involvesensory neuron activation and CGRP release.

5123

ALGINATE AND EPIDERMAL GROWTH FACTOR UP·REGU­LATE MICROPINOCYTOSISIN OESOPHAGEAL SQUAMOUSEPITHELIAL CELLS.Peter E. Ross, Paul M. McPherson, Carol Gallacher, Ian G. Jolliffe, PeterW. Dettmar, Univ of Dundee, Dundee, United Kingdom; Reckitt & Col­man Products Ltd, Hull, United Kingdom.

Introduction Little is known of the cellular patho-physiology of oesopha­geal mucosa. This laboratory established a method to measure fluid phaseendocytosis, a well conserved costitutive function involving integratedcellular activity and energy production. In oesophageal mucosa fluid phaseendocytosis is up-regulated in inflammation (I) but little is known aboutcontrol of this endocytic process. This report describes the effect ofepidermal growth factor, a protective factor secreted in saliva, and alginate,a component of some medications to treat symptoms of acid reflux into theoesophagus. Methods Pig oesophageal mucosa was incubated in an Ussingchamber for I hour with fluorescent microspheres (0.02/Lm). Tissue wasdisagregated and the resultant single cell suspension measured by flowcytometry using trypan blue to demonstrate cell viability and the excludefluorescence from non-internalised microspheres. An oesophageal cell line,KYSE30 which over-expresses EGF receptor was investigated in culture.Results EGF up-regulates fluid phase endocytosis Alginate up-regulatesfluid phase endocytosis Phorbal ester up-regulates fluid phase endocytosisAlginate does not up-regulate receptor mediated endocytosis of EGFConclusions EGF up-regulates fluid phase endocytosis. The increase issimilar to that seen with phorbol ester, suggesting the EGF effect ismediated by activation of protein kinase C. Alginate also increases fluidphase endocytosis but this was not associated with increased internalisationof EGF (I) Hopwood et al Gut (1995)37 pp598-602

5124

SMAD4 INDEPENDENT INDUCTION OF COX-2 BY TRANS­FORMING GROWTH FACTOR-HI (TGF-H1) IN HUMAN PAN·CREATIC CARCINOMA (BXPC-3) CELLS.Debabrata Saha, Michihiko Wada, Jason D. Morrow, Robert DanielBeauchamp, Vanderbilt Univ Med Ctr, Nashville, TN.

Purpose: TGF-f31 treatment induces COX-2 expression and prostaglandinsynthesis in rat intestinal epithelial (RIE-I) cells. RIE-I cells chronicallyexposed to TGF-f3I, attain a malignant phenotype and overexpress COX-2.The purpose of this study is to determine whether this induction of COX-2by TGF-f31 is mediated by classical Smad signaling pathway. For thisstudy, we have selected three different Smad4 deficient human carcinomacell lines. These are SW480.7 (colon carcinoma), MDA-MB-468 (breastcarcinoma) and BxPC-3 (pancreatic carcinoma) cells. Methods: BxPC-3cells are maintained in RPMI with 10% PBS whereas, SW480.7 andMDA-MB-468 are maintained in DMEM with 10% FBS. Cells were serumstarved for 24 hrs and then treated with TGF-f31 (5ng/ml) in serum freecondition. Lysates were collected at different times and COX-2 and Smad4level was determined by western blot analysis. PGE2 release was deter­mined by gas chromatography and mass spectrometry. In some experi­ments cells were infected (MOl of 10) with recombinant adenoviruscontaining cDNA of either wild type Smad4 or f3-galactosidase (negativecontrol). After 36 hrs of infection cells were serum starved and treated withTGF-f31 as mentioned earlier. Efficiency of infection was analyzed bystandard J3-galactosidasestaining method. Results: Only BxPC-3 cells havedetectable level of COX-2 protein. TGF-f31 induces the COX-2 proteinlevel up to 3-fold whereas, PGE2 release was increased up to 17-fold in 8hrs. COX-2 protein was not detected and PGE2 levels were not changed inresponse to TGF-f31 in either SW480.7 or MDA-MB-468 cells. TheCOX-2 induction pattern by TGF-f31 was not changed after reconstitutingthe Smad4 signaling pathway in BxPC-3 cells by infection with Smad4­adenovirus (wild type). Conclusions: Our results showed that TGF-f31mediated induction ofCOX-2 expression in BxPC-3 cells was independentof the conventional Smad signaling pathway. We predict that this inductionof COX-2 expression and PGE2 release is due to the TGF-f31 mediatedalternative signaling cascades. This TGF-f31 dependent alternative signal­ing may be responsible for tumorigenesis in selected conditions.