The capacity for L-arginine metabolism was studied in villus enterocytes isolated from pigs at birth, after 2-8 days suckling and after weaning. Immediately after birth, enterocytes were able to convert 1 mM L-citrulline, 2 mM ~-glutamine or 1 mM L-ornithine to L-arginine. In 2-8-day-old animals, the net production of L-arginine from L-citrulline (2.00 f 0.45 nmol . lo6 cells-' . 30 min-'), or from L-ornithine (0.29 -+ 0.06 nmol . 10' cells-' . 30 min-') was similar to the values obtained at birth. Furthermore, 40% of L-arginine synthetized de novo from L-citrulline were released into the incubation medium. In 2 -8-day-old animals, the production of L-arginine from L-glutamine represented only 5% of the production at birth (the latter being 0.73 f 0.15 nmol . lo6 cells-' . 30 min-').In enterocytes isolated from post-weaned pigs, no significant production of L-arginine from either L-glutamine or L-ornithine was detected. In contrast, although the L-arginine production from Lcitrulline was very low in post-weaned animals, it was significantly enhanced in the presence of Lglutamine, representing 23% of the production measured in suckling animals. The capacity of enterocytes to cleave L-arginine to L-ornithine and urea was very limited at birth, but was increased more than threefold in 2-day-old animals. This was concomitant with a marked increase in arginase activity. In post-weaned animals, the flux through arginase in intact enterocytes, and the arginase activity were both threefold higher than in 2 -8-day-old animals. It is concluded that enterocytes isolated from neonatal pigs exhibit the capacity for a net production of L-arginine since the metabolism of this amino acid is oriented to anabolism rather than catabolism. The results are discussed in relation to L-arginine metabolism in the neonatal liver.Apart from their well-identified function of nutrient translocation, including that of amino acids, from the intestinal lumen to the bloodstream, enterocytes are able to partially metabolize these amino acids during their transcellular journey. L-Glutamine is highly utilized by intestinal absorptive cells where it plays the role of a respiratory fuel [l]. In contrast, the oxidative catabolism of L-arginine in enterocytes is negligible relative to its conversion to L-ornithine and L-citrulline [2]. The adult intestine intensively degrades absorbed L-arginine resulting in a high L-ornithine, urea and L-citrulline release into blood [3]. Intestinal arginase cleaves L-arginine into L-ornithine and urea, and L-ornithine is considered as an important contributor to urea synthesis from excess ammonia in liver [4-61. Further metabolism of L-ornithine in adult intestine is limited to L-citrulline generation since a complete urea cycle does not operate in adult intestinal mucosa (71, the two enzymes responsible for the conversion of L-citrulline to L-arginine [argininosuccinate

HT-29 Glc-/+ cells originate from a human colon adenocarcinoma. These cells have been selected in a glucose-free culture medium and switched back in a glucose-containing medium. In this condition, they can spontaneously differentiate after confluency in enterocyte-like cells according to the activity of the brush-border associated hydrolase dipeptidyl peptidase IV. Since L-arginine can generate polyamines which are necessary for cellular proliferation and also differentiation, and nitric oxide with reported anti-proliferative property, the metabolism of this amino acid was examined in proliferative and differentiated isolated HT-29 cells. Proliferative HT-29 cells were characterized by micromolar intracellular concentration of putrescine and millimolar concentration of spermidine and spermine. In these cells, L-arginine is converted to L-ornithine and putrescine and to a minor part to nitric oxide and L-citrulline. Putrescine was taken up by HT-29 cells, leading to the production of a modest amount of spermidine. The diamine was slightly incorporated into cellular proteins and largely released in the incubation medium. The proliferative HT-29 cells take up spermidine and spermine but do not catabolize these polyamines and slightly released spermidine. Differentiation of HT-29 cells is not associated with change in intracellular polyamine content but is paralleled by an almost complete extinction of de novo synthesis of putrescine (due to a dramatic decrease of ornithine decarboxylase activity) and by a reduced release capacity of putrescine. In contrast, putrescine net uptake and incorporation into cellular proteins remained unchanged after differentiation. Furthermore, spermidine and spermine metabolism as well as the circulation of L-arginine in the nitric oxide synthase pathway were also not modified after differentiation. In conclusion, putrescine is the L-arginine-derived molecule, the metabolism of which is specifically and markedly modified when HT-29 cells move from proliferative to differentiated state.

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Nitric oxide has been implicated as a local modulator of several gastrointestinal functions. In this study, we have measured nitric oxide synthase activity in homogenates of enterocytes isolated from post-weaned pigs. The enzyme required the presence of NADPH and 6-(R,S)-5,6,7,8-tetrahydro+biopterin.Conversely exogenous FAD and FMN did not appear to be necessary for enzyme activity. The enzyme activity was not affected by added Ca'+ or EGTA and was inhibited by the arginine analogs NO-monomethyl+arginine and N"-nitro+arginine. NO synthase activity was not detectable in enterocytes isolated at birth and increased slightly in suckling animals. NO synthase activity was found to be present mostly in the cytosolic fraction isolated from post-weaned pigs enterocytes.