April 1995 Growth, Development, and Nutrition A741

• DIFFERENCES IN EPITHELIAL APOPTOSIS AND APOPTOSIS- RELATED GENE EXPRESSION BETWEEN THE RAT SMALL AND L A R G E INTESTINE SF Moss. N Arber, R Guan, AR I-Ieydari, S Krajewski, JC Reed, PR Holt. Dept of Medicine, St.Luke's-Roosevelt Hospital Center/Columbia Univ, NY; Univ of Texas, San Antonio, TX & La Jolla Cancer Research Foundation, CA.

Apoptosis (programmed cell death) is important for physiological control of growth in rapidly-renewing tissues such as the intestine; abnormalities of apoptosis may contribute to carcinogenesis. The Bcl-2 family of genes are known as regulators of this process in vitro. To determine whether they are involved in the control of intestinal apoptosis in vivo, we compared enteroeyte apoptosis in rat colon and ileum and the expression of the bcl-2 gene family. Apoptotie cells were identified by in-situ end labelling using TdT & digoxigenin-labeled dUTP; the expression of bel-2 and bax, (mel-1 and bclx) were cOmpared by Northern and Western blotting of mueosal serapings. Results: Apoptotie cells in the ileum were rare and mainly at the villus tips. In the colon they were much more frequent(38__+29% of epithelial cells versus 1 .6+1.5 in the ileum, P < 0.01) and found in both surface and throughout the crypt epithelium. There were also differences in the expression of bel-2 related genes:

I leum Colon Bcl-2 -- + + Bax + + + BcI~L + + + ÷ Bcl~S - - - - Mcl-1 + +

Conclusion: Enterocyte apoptosis is more frequent in the colon than the ileum and may be due to different levels of expression of bel-2 related genes. Differences in physiologic apoptosis between these tissues may explain the propensity to neoplasia of the colon compared with the ileum.

EVIDENCE FOR ZNVOL%'IDIENT OF OENITMINE DECARBOX~rLASE IN TRANSFORMING GROWTIIFACTORRLFA-STII~/Id%TEDGASTRIC EPI~IIELIAL CELL PROLIFERATION. N. Naka~ime, Hajime Kuwayama, Y. ~akawa. Department of Medicine, Nihon University School of Medicine, Tokyo, Japan.

II~ZRODUCTION| Gastrointestinal mucoea is one of the most rapidly proliferating tissues in the body. This constant renewal is important for mucosal protection and repair, but its regulatory mechanisms are imprecisely defined. Recent studies have suggested that a number of growth factors, including transforming growth factor alfa (TGFa), may play an important role in regulating epithelial proliferation. Ornithine decarboxylase has also been shown to play an essential role in cell proliferation. The purpose of the present study was to determine if ornithine decarboxylaee is involved in TGFa's action.

METHODSI Monolayer gaetricepithelial cells, prepared from rabbit fetus stomach, were incubated with TGFa for 20 hrs with or without difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase. Cells were further incubated with tritiated thymidine (3H-TdRI for 1 hr in order to label proliferating cells. The specific ~H-TdR incorporation into DNA was evaluated by scintillation counting using calf thymus DNA as a standard,

RESI/LTSI TGFa increased ~H-TdR incorporation into fetal rabbit gastric epithelial cell at DNA in a dose dependent fashion, with maximal stimulation at 10~M.

Thvmidine incorooration into DNA ( % of control) TGFalI04M) TGFatI0~M}+DFMOtlO-SM) 180.77±49.50, 128.13±28.72 *Significant difference from control, i><0.05.

Our results indicate that DFMO, a specific inhibitor of ornithine decarboxylaee, inhibited TGFa-stimulated gastric epithelial cell DNA synthesis.

CONCLUSION| Ornithine decarboxylase may be involved in TGFa's stimulatory action on gastric epithelial proliferation.

• MEASUREMENT OF DUODENAL MUCOSAL PROTEIN SYNTHESIS AFTER DELIVERY OF [13C] LEUCINE AND [13C] VALINE BY INTRAVENOUS AND INTRAGASTRIC ROUTES. I M Nakshabendi, W Obiedat, S Downie*, M J Rennie*, R I Russell. Department of Gastroenterology, Royal Infirmary, Glasgow and Dept. of Anatomy & Physiology , University of Dundee,Scotland.

A method for measurement of gut mucosal protein synthesis (GMPS) would be valuable in a numbQr of clinical situations. We have developed such a method using stable isotope tracer (SIT) technology and used it to investigate the effect of the route of delivery of tracer amino acids on GMPS. METHODS: Seven normal subjects ( 6 males, 1 female, 25.5-59 yr, 56-86 kg) were studied. All subjects received primed constant infusions of [13C] leucine and [13C] valine for 4 hrs. In four of the subjects leucine was given intravenously (IV) and valine intragastrically (IG) and in the three others the routes of delivery were reversed. Biopsies of distal duodenal mucosa (-50 mg total) were obtained at 4 hr using endoscopy. Analysis of free blood and tissue tracer was by gas chromatography mass spectrometry, analysis of protein tracer incorporation was by preparative gas chromatography and isotope ratio mass spectrometry. RESULTS: Plateau labelling of-5 atom % excess of tracers was obtained in the plasma in all subjects within 1 hr. AS expected there was a marked gradient of enrichment between the plasma amino acid pool and the intracellular free amino acid pool, the size and direction of which depended upon the route of administration of the tracer; for IV route the ratio was 1.73 ± 0.16, but for IG route the ratio was reversed, ie 0.65 ± 0.12. The absolute rates of incorporation of tracer into duodenal mucosal protein were higher for the IG route, but when the protein synthetic rates were calculated on the basis of the free tissue enrichment there was no difference in the rate of GMPS, ie IV tracer 2.58 ± 0.32 % /h -1, IG tracer 2.46 ± 0.67 % /h -1. CONCLUSION: We describe a reproducible method to measure GMPS using SIT which can be applied in various disease states.

Nitrogen (N) absorption from protein-based (PBD), peptide-based (PEPD), or amino acid diet (ASD) in the isolated perfused rat intestine. F.Neuwirth, A.Raible, D.Veillard-Baron, M.Gregor, M.Plauth. Medizinische Klinik, Eberhard- Karls-Universifiit, Otfried-Mfiller-Str. 10, 72076 Tiibingen

Following pancreatectomy 61% of N are absorbed from intact lactalbumin administered by nasogastric tube. It was thus speculated, that the small intestine was capable of relevant N-absorption from intact protein. To exclude digestive action of gastric and pancreatic secretions we determined mesenteric-vanous amino acid appearance rates in the isolated vascularly and luminally perfused rat small intestine as a measure of nitrogen absorption from enteral liquid diets.

After a control phase (C) small intestines of 5 rats each were luminally perfused with either PBD (Biosorb R Sonde), or PEPD (PeptisorbR), or ASD (Nutri2000R). Vascular exchange rates were calculated from av concentration-differences of amino acids (measured by HPLC) and perfusate flow. The vascular perfusate consisted of a fluorocarbon emulsion, Krebs-Henseleit buffer, 0.6mM glutamine and 10mM glucose. Exchange rates of Gin, Ala, and Leu are given in table 1. In table 2 exchange rates of Leu, lie, and Val are normalized on the basis of a luminal nitrogen content of lg N/100ml diet.

Tabl: Exchange rates (nmol/mirdg; x+SEM; *p < 0.05, **p < 0.01, paired t-test) C PBD C PEPD C ASD

Gin -85+8 -50+3** -83+11 -60+ 9 -76-+5 -49+12" Ala 54+4 52-+3 70-+51 23-1-15" 66+8 81-1-10 Leu 5+1 9+1"* 104- 2 95-+13"* 9+2 63+ 8**

Tab2: Exchange rates (nmol/min/g; x_+SEM) normalized to lg N/100ml diet C PBD C PEPD C ASD

Leu 8+1 14+2"* 16--+3 159+22"* 25-+5 168+--21"* Val 7+1 15+2"* 13-1-2 105-+14"* 21+5 137+13"* lie 5-+1 10-+1"* 9+1 89-+11"* 14-+3 113+14"*

When applied directly into the small intestine PBD was a poor substrate for nitrogen absorption. Obviously, without gastric or pancreatic secretions rat small intestine is not capable of relevant N-assimilation from intact casein. N-Absorption seems to occur more effieiantly from PEPD than from ASD when compared to the appropriate controls. Neither PBD nor PEPD provided glutamine in amounts suffieiant to achieve net glutamine appearance in mesenteric venous perfusate.