This study was designed to determine whether pyrroline-5-carboxylate (P-5-C) synthase is deficient in chick enterocytes therefore resulting in the lack of synthesis of ornithine and citrulline from glutamine. Post-weaning pig enterocytes, which are known to contain P-5-C synthase and to synthesize both ornithine and citrulline from glutamine, were used as positive controls. Enterocytes were incubated at 37 degrees C for 0-30 min in the presence of 2 mM [U-14C]glutamine or 2 mM ornithine plus 2 mM NH4Cl. In chick enterocytes, glutamine was metabolized to NH3, CO2, glutamate, alanine and aspartate, but not to ornithine, citrulline, arginine or proline. Likewise, there was no formation of citrulline, arginine, alanine or aspartate from ornithine in chick enterocytes. Furthermore, the rate of conversion of ornithine into proline in chick enterocytes was only about 4% of that in cells from pigs. To elucidate the reason for the inability of chick enterocytes to synthesize ornithine and citrulline from glutamine, the activities of the enzymes involved were measured. No activity of P-5-C synthase or ornithine carbamoyltransferase was found in chick enterocytes, in contrast with cells from post-weaning pigs. It was also demonstrated that the activity of ornithine aminotransferase in chick enterocytes was only 3% of that in cells from pigs. Thus the present findings elucidate the biochemical reason for the lack of endogenous synthesis of ornithine and citrulline in chicks. Our results also explain previous observations that ornithine cannot replace arginine or proline in the diet of chicks. We suggest that the absence of P-5-C synthase and ornithine carbamoyltransferase in enterocytes is the metabolic basis for the nutritional requirement of arginine in the chick.
The synthesis of citrulline from glutamine was quantified in enterocytes from pre-weaning (14-21 days old) and post-weaning (29-58 days old) pigs. The cells were incubated at 37 degrees C for 30 min in Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 0, 0.5, 2 and 5 mM glutamine. Oxygen consumption was linear during the 30 min incubation period. The rates of citrulline synthesis were low or negligible in enterocytes from 14-21-day-old pigs, but increased 10-20-fold in the cells from 29-58-day-old pigs. This marked elevation of citrulline synthesis coincided with an increase in the activity of pyrroline-5-carboxylate synthase with the animal's post-weaning growth. In contrast, decreases in the activities of phosphate-dependent glutaminase, ornithine aminotransferase, ornithine carbamoyltransferase and carbamoyl-phosphate synthase were observed as the age of the pigs increased. The concentrations of carbamoyl phosphate in enterocytes from pre-weaning pigs were higher than, or similar to, those in the cells from post-weaning pigs. It is possible that the low rate of citrulline synthesis from glutamine in enterocytes from pre-weaning pigs was due to a limited availability of ornithine, rather than that of carbamoyl phosphate. We suggest that this limited availability of ornithine in pre-weaning-pig enterocytes results from (i) the low rate of pyrroline-5-carboxylate synthesis from glutamate, due to the low activity of pyrroline-5-carboxylate synthase, and (ii) the competitive conversion of pyrroline-5-carboxylate into proline. Our present findings on the developmental aspect of citrulline synthesis in pig enterocytes may offer a biochemical mechanism for the previous observations that arginine is a nutritionally essential amino acid for suckling piglets, but not for adult pigs.
The activity of the arginine-citrulline cycle was investigated in macrophages from the spontaneous immunologically mediated diabetic BB rat. Peritoneal macrophages were prepared from male diabetes-prone (BBdp), diabetic (BBd) and age-matched non-diabetes-prone (BBn) rats. Cells were incubated at 37 degrees C for 2 h in Krebs-Henseleit bicarbonate buffer containing 0.5 mM L-arginine, 0.1 mM L-[ureido-14C]citrulline and 5 mM D-glucose to measure the activity of the arginine-citrulline cycle. The uptakes of citrulline and arginine by macrophages were measured during a 5 min incubation period with L-[ureido-14C]citrulline and L-[2,3-3H] arginine respectively. The production of NO3- (the major stable oxidation product of NO) increased (P < 0.01) by 112% and 151% in 75-day-old BBdp and 115-day-old BBd macrophages respectively, compared with age-matched BBn cells. The conversion of [14C]citrulline into [14C]arginine increased (P < 0.01) by 704%, 892% and 904% in 50- and 75-day-old BBdp and 115-day-old BBd macrophages respectively, compared with age-matched BBn cells. The enhanced NO synthesis in BBdp and BBd macrophages was associated with a 25-35% increase in the uptake of L-arginine. However, there were no differences in the uptake of citrulline between BBdp or BBd macrophages and age-matched BBn cells. Our results demonstrate for the first time the activation of the arginine-citrulline cycle in macrophages in an autoimmune condition. The inherent increase in the recycling of L-citrulline to L-arginine in BBdp and BBd macrophages may reflect an innate metabolic disorder in these cells. This increased L-arginine synthesis from L-citrulline may play a role in sustaining a sufficient intracellular L-arginine concentration for prolonged generation of NO in BBdp and BBd macrophages. A role for NO in the autoimmune destruction of pancreatic beta-cells in insulin-dependent diabetes mellitus warrants further investigation.