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The presence of nitric oxide synthase (NOS), argininosuccinate synthetase (ASS), and argininosuccinate lyase (ASL) and their coexistence with NADPH-diaphorase (NADPHd), a marker for NOS, in the porcine sphenopalatine ganglia (SPG), pial veins, and the anterior cerebral arteries was examined using immunohistochemical and histochemical staining techniques. NOS-immunoreactive (I), ASS-I, and ASL-I fibers were found in pial veins and the anterior cerebral arteries. NOS, ASS, and ASL immunoreactivities were also found in neuronal cell bodies in the SPG. Almost all neuronal cell bodies in the SPG and nerve fibers in pial veins and the anterior cerebral arteries that were reactive to ASS, ASL, and NOS were also stained positively with NADPHd, suggesting that ASS, ASL, and NOS were colocalized in the same neurons in the SPG and perivascular nerves. With the use of in vitro tissue bath techniques, L-citrulline but not D-citrulline reversed inhibition of neurogenic vasodilation in isolated porcine pial veins produced by NOS inhibitors such as NG-nitro-L-arginine methyl ester. In the presence of L-aspartate, L-arginine was synthesized from L-citrulline in homogenates of SPG and endothelium-denuded cerebral arteries and pial veins. These results provide evidence indicating that perivascular nerves in pial veins like cerebral arteries can convert L-citrulline to L-arginine for synthesizing nitric oxide. The conversion is most likely via an argininosuccinate pathway.

This study was designed to determine whether cortisol plays a role in arginine and glutamine metabolism in enterocytes and, more specifically, whether cortisol regulates metabolic changes in these cells during weaning. Twenty-eight 21-day-old suckling pigs were randomly assigned to one of four groups (7 animals in each) and received intramuscular injections of vehicle solution (sesame oil) (control group), hydrocortisone 21-acetate (HYD) (25 mg/kg body wt), RU-486 (10 mg/kg body wt) (a potent blocker of glucocorticoid receptors), or HYD plus RU-486. At 29 days of age, pigs were killed for preparation ofjejunal enterocytes. During the entire experimental period, pigs were nursed by sows. Activities of argininosuccinate synthase, argininosuccinate lyase (ASL), arginase, and pyrroline-5-carboxylate (P5C) synthase were measured. For metabolic studies, enterocytes were incubated for 30 min at 37 degrees C in 2 ml of Krebs-bicarbonate buffer (pH 7.4) containing 0, 0.5, or 2 mM [U-(14)C]arginine or [U-(14)C]glutamine. Compared with control, cortisol administration increased 1) the activities of ASL and arginase and the production of CO(2), ornithine, and proline from arginine, and 2) P5C synthase activity and the formation of glutamate, alanine, aspartate, ornithine, citrulline, proline, and CO(2) from glutamine in enterocytes. The stimulating effects of cortisol on the enzyme activities and the metabolism of arginine and glutamine were abolished by coadministration of RU-486. Our data suggest that cortisol plays an important role in regulating arginine and glutamine metabolism in enterocytes via a glucocorticoid receptor-mediated mechanism.

Both non-arginine-depleted and arginine-depleted pulmonary artery endothelial cells (PAEC) actively convert citrulline into arginine. Exposure to hypoxia for 4-24 h inhibited arginine synthesis from citrulline in intact cells and in cell homogenates. The conversion of L-citrulline to L-argininosuccinate by argininosuccinate synthetase (AS) was inhibited by exposure to hypoxia for 4, 12, or 24 h. The conversion of argininosuccinate to arginine by argininosuccinate lyase was inhibited by exposure to hypoxia for 24 h but not for 4-12 h. The decrease of L-arginine biosynthesis during hypoxia coincided with the increase of intracellular glutamine content and was abrogated by preventing an increase in intracellular glutamine. In addition, AS activity was inversely related to glutamine content in the medium. These results indicate that hypoxia inhibited the L-arginine biosynthetic pathway via decreased activity of AS. The latter is related to increased glutamine content. Hypoxic inhibition of arginine synthesis from citrulline did not result in a decrease of arginine content, suggesting that PAEC are able to maintain intracellular arginine for up to 24 h despite reduction in the L-arginine biosynthetic pathway.

Arginine is deficient in porcine colostrum and milk, and yet the piglet has a particularly high requirement for this essential amino acid for rapid postnatal growth. To explain this paradox, arginine synthesis was quantified in enterocytes from newborn (0-day-old) and 2- to 7-day-old suckling pigs. Arginine was found to be synthesized from glutamine in 0- to 7-day-old pig enterocytes, but the rates of arginine synthesis were three- to fourfold greater in 0- to 2-day-old pigs than in 7-day-old pigs. To elucidate the developmental change of the intestinal arginine synthesis, the metabolism of glutamine to citrulline, the conversion of citrulline to arginine, and the activities of the enzymes involved were measured. The rates of metabolism of glutamine to citrulline were 2.5- to 3.5-fold greater in enterocytes from 0- to 2-day-old pigs than in cells from 7-day-old pigs, as were the rates of conversion of citrulline to arginine. The activities of all enzymes that synthesize arginine from glutamine, except pyrroline-5-carboxylate synthase and argininosuccinate lyase (ASL), increased in enterocytes from 2-day-old pigs compared with 0-day-old pigs. The activities of all these enzymes decreased by approximately 75% in 7-day-old pigs compared with 2-day-old pigs. Arginase activity was negligible in enterocytes from 0- to 7-day-old pigs, thus minimizing intestinal hydrolysis of newly synthesized arginine and maximizing the endogenous provision of arginine. The results of this study demonstrate the presence of arginine-synthesizing enzymes and their developmental changes in postnatal pig enterocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

The urea biosynthetic pathway functions in mammalian liver to convert excess ammonia to urea and to maintain the concentration of ammonia in blood at nontoxic levels. This action is accomplished by enzymatic adaptation to quantitative changes in dietary protein. The first two enzymes of the pathway are found in the intestine of the adult mouse, but they do not adapt to dietary change. The enzymes in the intestine produce citrulline, which is carried by the bloodstream to the kidney, where it is converted by the next two enzymes of the pathway to arginine. This mechanism serves as the major source of circulating arginine. We have demonstrated that, at birth, the arginine-synthesizing enzymes in the kidney of the C57Bl/6 mouse are minimally developed, whereas in the intestine activity of carbamoyl-phosphate synthase is elevated and argininosuccinate synthase and lyase, usually present only in trace quantities in the adult intestine, are markedly increased in the newborn. The arginine formed cannot be converted to urea, since arginase does not appear in intestinal cells of the mouse until the age of 15 days. Except for liver, intestine has the most rapid protein turnover of any normal tissue. Our study indicates that, at a time when no other endogenous source of arginine for protein synthesis is available, the intestine of the newborn C57Bl mouse is capable of synthesizing arginine from either citrulline or NH3 and CO2.