Abstract
L-citrulline (Cit) is a co-product of NO synthesis and a direct L-arginine (Arg) precursor for de novo NO synthesis. Acute liver failure (ALF) is associated with increased nitric oxide (NO) and cyclic GMP (cGMP) synthesis in the brain, indirectly implicating a role for active transport of Cit. In the present study we characterized [3H]Cit uptake to the cortical brain slices obtained from control rats and rats with thioacetamide (TAA)-induced ALF (“TAA slices”). In both control and TAA slices the uptake was partially Na+-dependent and markedly inhibited by substrates of systems L and N, including L-glutamine (Gln), which accumulates in excess in brain during ALF. Cit uptake was not affected by Arg, the y+/y+L transport system substrate, nor by amino acids taken up by systems A, xc −or XAG. The Vmax of the uptake in TAA slices was ~60 % higher than in control slices. Chromatographic (HPLC) analysis revealed a ~30 % increase of Cit concentration in the cerebral cortical homogenates of TAA rats. The activity of argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL), the two enzymes of Cit-NO cycle catalyzing synthesis of Arg, showed an increase in TAA rats, consistent with increased ASS and ASL protein expression, by ~30 and ~20 %, respectively. The increased Cit-NO cycle activity was paralleled by increased expression of mRNA coding for inducible nitric oxide synthase (iNOS). Taken together, the results suggest a role for Cit in the activation of cerebral NO synthesis during ALF.
Highlights
Hepatic encephalopathy (HE), a consequence of acute or chronic liver failure (ALF or CLF), is a complex neuropsychiatric disorder that results from impaired clearance from blood of ammonia and other toxins, and is compounded by peripheral or local inflammatory processes (Prakash and Mullen 2010)
Astrocytes are the main site of ammonia detoxification, through the amidation of glutamate (Glu) forming Gln (Cooper and Plum 1987), in the reaction catalyzed by glutamine synthetase (GS) (Martinez-Hernandez et al 1977)
Accumulation of Gln in the brain in HE patients or animal models of Acute liver failure (ALF) is one of the main factors that contribute to ammonia-induced neurotoxicity (Albrecht and Norenberg 2006)
Summary
Hepatic encephalopathy (HE), a consequence of acute or chronic liver failure (ALF or CLF), is a complex neuropsychiatric disorder that results from impaired clearance from blood of ammonia and other toxins, and is compounded by peripheral or local inflammatory processes (Prakash and Mullen 2010). At the molecular level the activation of ionotropic (mainly NMDA) glutamate receptors leads to increased intracellular free calcium which, after binding to calmodulin, activates nitric oxide synthase (NOS), leading to increased production of nitric oxide (NO) (Garthwaite et al 1988). In ALF, ammoniainduced increase of NO and subsequently extracellular cGMP is a good indicator of the over-stimulation of NMDA receptors in rat brain, a process that contributes to increased reactive oxygen and nitrogen species (ROS/RNS) production (Kosenko et al 2003; Hermenegildo et al 2000; Hilgier et al 2004). Increased NO synthesis under HE conditions in the brain, requires L-arginine (Arg) as a substrate for NOS, which generates NO and L-citrulline (Cit). Metab Brain Dis (2014) 29:1053–1060 considered Cit transport as a critical step within the Cit-NO cycle. The present study is, to the best of our knowledge, the first which attempted to characterize in more detail Cit transport in ex vivo brain tissue
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