Arginine metabolism in porcine enterocytes

Abstract

Enterocytes contain a functional urea cycle and may play a pivotal role in arginine metabolism and homeostasis in growing pigs. The activities of carbamoyl phosphate synthase-1 (CPS-1), ornithine carbamoyltransferase (OCT), argininosuccinate synthase (ASS), argininosuccinate lyase (ASL), and arginase were determined in the cytosol and mitochondria of enterocytes. CPS-1 and OCT were mitochondrial, whereas ASS and ASL were cytosolic enzymes. Arginase was present in both the cytosol and mitochondria. Mitochondrial arginase activity was decreased by preheating at 550C. Vmax.. values of CPS-1 (6.12 ︢0.25 nmol citrulline-min-l-mg protein-'), ASS (1.91 ︢0.13 nmol argininosuccinate-min-l-mg protein-'), ASL (3.57 ︢0.24 nmol arginine-min-'-mg protein-1 ), and arginase (68.1 + 4.5 nmol urea-min-l-mg protein-' in pre-heated cytosol) were lower than those in mammalian liver. Vmax, of OCT (690.5 ︢36.3 nmol citrulline-min-l-mg protein-') was similar to hepatic OCT. The substrate Km values (mmol/L) for arginase (91.3 ︢ 5.5 for cytosol), OCT (5.13 ︢0.24 and 17.1 ︢1.2 for ornithine and carbamoyl phosphate, respectively), CPS-1 (1.34 ︢0.11, 58.6 ︢2.4, 15.2 ︢1.4 for NH4, HCO3-, and ATP, respectively), ASS (2.86 ︢0.16, 0.054 0.003, 0.15 ︢ 0.01 for ATP, aspartate, and citrulline, respectively), and ASL (0.63 0.04) were higher than the hepatic enzymes with the exception of CPS-1 for NH4+. The kinetic properties of enterocyte urea cycle enzymes are consistent with the role of the small intestine in the net production of citrulline for endogenous synthesis of arginine in pigs. Pyrroline-5-carboxylate (P5C) synthase and ornithine aminotransferase (OAT) activities were measured in various tissues. P5C synthase was detected only in jejunal enterocytes. OAT was ubiquitous in tissues. Gabaculine (Ki 19 ︢2 4mol/L) was a potent inhibitor of OAT and prevented net synthesis of ornithine and citrulline from glutamine in isolated enterocytes. The administration of gabaculine to 60-day-old fasted pigs increased plasma concentrations of ornithine and proline but decreased plasma concentrations of arginine and citruiline. In 60-day-old fed pigs, gabaculine administration increased plasma concentrations of proline and had no effect on plasma concentrations of ornithine, citrulline or arginine. The results suggest that endogenous arginine synthesis plays an important role in arginine homeostasis in food-deprived 60-day-old pigs but not in fed pigs.