Coinduction of Nitric Oxide Synthase, Argininosuccinate Synthetase, and Argininosuccinate Lyase in Lipopolysaccharide-treated Rats

Abstract

Nitric oxide (NO) is synthesized from arginine by nitric oxide synthase (NOS), and citrulline which is generated can be recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). Rats were injected with bacterial lipopolysaccharide (LPS), and expression of the inducible isoform of NOS (iNOS), AS, and AL was analyzed. In RNA blot analysis, iNOS mRNA was undetectable before the LPS treatment but was induced by LPS in the lung, heart, liver, and spleen, and less strongly in the skeletal muscle and testis. AS mRNA was induced in the lung and spleen, and AL mRNA was weakly induced in these tissues. AS and AL mRNAs were abundant in the control liver and remained unchanged after the treatment. Kinetic studies showed that iNOS mRNA increased rapidly in both spleen and lung, reached a maximum 2-5 h after the treatment, and decreased thereafter. On the other hand, AS mRNA increased more slowly and reached a maximum in 6 -12 h (by about 10-fold in the spleen and 2-fold in the lung). AL mRNA in the spleen and lung increased slowly and remained high up to 24 h. In immunoblot analysis, increase of iNOS protein was evident in the lung, liver, and spleen, and there was an increase of AS protein in the lung and spleen. In immunohistochemical analysis, macrophages in the spleen that were negative for iNOS and AS before LPS treatment were strongly positive for both iNOS and AS after this treatment. As iNOS, AS, and AL were coinduced in rat tissues and cells, citrulline-arginine recycling seems to be important in NO synthesis under the conditions of stimulation.Nitric oxide (NO) is a major messenger molecule regulating blood vessel dilatation and immune function and functions as a neurotransmitter in the brain and peripheral nervous system (see Refs. 1-3 for reviews). NO is synthesized from arginine by nitric oxide synthase (NOS), 1 generating citrulline as another product. Cellular NO production is absolutely dependent on availability of arginine. This amino acid can be obtained from exogenous sources via the blood circulation, from intracellular protein degradation, or by endogenous synthesis of arginine.Major sites of arginine synthesis in ureotelic animals are the liver, where arginine generated in the urea cycle (ornithine cycle) is rapidly converted to urea and ornithine by arginase, and the kidney, where arginine is synthesized from citrulline and released into the blood circulation (see Ref. 4 for a review). However, other tissues and cell types also contain low levels of argininosuccinate synthetase (AS) and argininosuccinate lyase (AL), which together synthesize arginine from citrulline (5-8). Therefore, arginine can be generated from citrulline which is produced as a co-product of the NOS reaction, forming a cycle which could be termed the "citrulline-NO cycle" (9) or "arginine-citrulline cycle" (10). Vascular endothelial cells can convert citrulline to arginine (11), and this conversion is increased when cells are stimulated to produce NO (12). Cytokine-activated macrophages, which produ...

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“…Lipopolysaccharide-activated macrophages have also been shown to transform citrulline into arginine (16), which is further degraded into urea and ornithine by the lipopolysaccharide-activated arginase (17). Animal studies have demonstrated that metformin increases arginase I expression in various innate immune cells, such as microglia and activated macrophages, thus exhibiting anti-inflammatory effects while fueling the urea and ornithine cycle (18), as suggested by Adam et al (8).…”

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“…Although competition for arginine between the urea cycle and hepatic inducible NO synthase (iNOS) was claimed also by a third group [135], their study only showed that exogenously supplied arginine could be utilized by both arginase and iNOS ; it did not determine whether arginine synthesized within the urea cycle could be used for hepatic NO synthesis. Finally, the hepatic capacity for arginine synthesis within the urea cycle, as indicated by mRNA levels for ASS and ASL, is not affected by inflammatory stimuli such as lipopolysaccharide (LPS) [136,137].…”

“…In rat tissues, ASL is also co-induced with iNOS [136,137]. Because basal expression of ASS differs greatly among different cell types, the magnitude of ASS induction by inflammatory stimuli is highly variable.…”

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“…The eNOS, nNOS, and iNOS proteins were detected with selective monoclonal antibodies (1:500 for each antibody) with lysates of endothelial cells, pituitary cells, and cytokine-activated macrophages as positive controls, respectively. AS and AL proteins (1:1,000) were detected with polyclonal antibodies that were raised in rabbits against recombinant proteins and were used previously to detect these proteins in the diaphragm and other rat tissues (19). The formation of 3-nitrotyrosine was detected with a monoclonal antibody as previously indicated (5).…”