Evidence for increased anionic arginase activity in pig enterocytes during development.

We have recently reported that the flux of L-arginine through arginase in enterocytes is increased in weaned pigs when compared with suckling animals (Blachier et al. 1993, Eur. J. Biochem. 216, 109-117). The aim of the present study was to characterize arginase activities at both stages of development. Enterocytes isolated from suckling animals were found to possess an anionic (50%) and a non-anionic (50%) form of arginase as judged from activities recovered from DEAE-cellulose ion exchange chromatography. In enterocytes isolated from weaned animals, anionic arginase was the major form representing 89% of arginase activity. This isoform is characterized by increased affinity for L-arginine (2 fold) and increased maximal velocity (39 fold) when compared with the anionic form originating from suckling piglet enterocytes. In conclusion, our data demonstrate that pig enterocytes are equipped with at least 2 isoforms of arginase and that anionic form of arginase activity appeared to be mainly responsible for the capacity of weaned pig enterocytes to catabolize L-arginine.

Metabolism of L-arginine through polyamine and nitric oxide synthase pathways in proliferative or differentiated human colon carcinoma cells.

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.

Transglutaminase activity in enterocytes isolated from pig jejunum.

Polyamines appear to be involved in the turnover, growth and maintenance of intestinal mucosa integrity. Since polyamines could act -in part at least- through their incorporation into cellular proteins as catalyzed by transglutaminase, we have measured this enzyme activity in villus enterocytes isolated from pig jejunum and in homogenate derived from isolated cells. A part of putrescine, spermidine and spermine taken up by enterocytes is incorporated in TCA precipitable material derived from cells and this corresponds to the presence of transglutaminase activity in cellular homogenates. This activity which is time and substrate concentration dependent is strongly inhibited by the transglutaminase inhibitor glycine methyl ester. The capacity for de novo production of polyamines from L-arginine or L-glutamine is very limited in isolated enterocytes, and this coincided with a very low ornithine decarboxylase activity when compared with polyamine cell content. It is concluded that the main source of polyamines for pig enterocytes is extracellular and that exogenous polyamines are substrates for enterocyte transglutaminase.

Characterization and ontogenesis of nitric oxide synthase activity in pig enterocytes.

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-L-biopterin. Conversely exogenous FAD and FMN did not appear to be necessary for enzyme activity. The enzyme activity was not affected by added Ca2+ or EGTA and was inhibited by the arginine analogs NG-monomethyl-L-arginine and N omega-nitro-L-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.

Intestinal arginine metabolism during development. Evidence for de novo synthesis of L-arginine in newborn pig enterocytes.

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 L-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 +/- 0.45 nmol x 10(6) cells-1 x 30 min-1), or from L-ornithine (0.29 +/- 0.06 nmol x 10(6) cells-1 x 30 min-1) 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 +/- 0.15 nmol x 10(6) cells-1 x 30 min-1). 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 L-citrulline was very low in post-weaned animals, it was significantly enhanced in the presence of L-glutamine, 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.

Polyamine metabolism in enterocytes isolated from newborn pigs.

In the pig, the growth of intestinal mucosa is very intense after birth. Since the polyamines are key elements affecting cell proliferation and differentiation, the present work was undertaken in order to know whether this hypertrophy is associated with an adaptation of polyamine metabolism. Villus enterocytes isolated from pig immediately after birth or 2 days later were found to contain similar amounts of putrescine, spermidine and spermine, i.e., 0.23; 0.41 and 1.24 nmol/10(6) cells, respectively. At birth, despite a relatively high ODC activity, putrescine synthesis from 1 mM L-arginine or 2 mM L-glutamine was very low in isolated enterocytes (6.4 +/- 3.8 pmol/10(6) cells per 30 min), while spermidine and spermine production were not detectable. This could be explained by a very low L-ornithine generation from both amino acids and to an inhibitory effect of polyamines on ODC activity. Two days later, polyamine synthesis from L-arginine remained undetectable despite a higher L-ornithine generation. This was concomitant with a dramatic fall in ODC activity. At both stages, enterocytes were able to take up polyamines from the extracellular medium in a temperature-dependent manner. It is concluded that de-novo synthesis of polyamines from L-arginine or L-glutamine does not play a significant role in the control of polyamine content of pig enterocytes during the postnatal period. In contrast, polyamine uptake by enterocytes would contribute to maintain a steady-state polyamine content during this period.

Stimulation by D-glucose of the direct conversion of arginine to citrulline in enterocytes isolated from pig jejunum.

In enterocytes isolated from pig jejunum, L-arginine is metabolized to L-citrulline either directly or indirectly through the sequence of reactions catalysed by arginase and ornithine transcarbamylase. In the presence of 5 mM D-glucose, the direct conversion of 1mM L-[guanido-14C] arginine to L-citrulline was increased more than 4 times. Isolated enterocytes exhibit a high glycolytic capacity. Furthermore, the decarboxylation of 5mM D-[1-14C] glucose was 3.6 fold higher than the decarboxylation of 5 mM D-[6-14C] glucose which suggests the presence of a pentose phosphate pathway in enterocytes. Since the production of labelled L-citrulline from L-[guanido-14C] arginine in pig enterocyte homogenates was markedly increased in the presence of NADPH, it is proposed that the direct conversion of L-arginine to L-citrulline could be stimulated by the production of NADPH from D-glucose in the pentose phosphate pathway.