A Mouse Model of Argininosuccinic Aciduria: Biochemical Characterization

Molecular Genetics and Metabolism 78 (2003) 11–16 www.elsevier.com/locate/ymgme

A mouse model of argininosuccinic aciduria: biochemical characterization

V. Reid Sutton,a,1 Yanzhen Pan,a,1 Erica C. Davis,a and William J. Craigena,b,*

a Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA b Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

Received 11 October 2002; received in revised form 6 November 2002; accepted 6 November 2002

Abstract

Argininosuccinate lyase (AL) has several roles in intermediary metabolism. It is an essential component of the urea cycle, providing a pathway for the disposal of excess nitrogen in mammals. AL links the urea cycle to the tricarboxylic acid (TCA) cycle by generating fumarate. Finally, AL is required for the endogenous production of arginine. In this latter role it may function outside ureagenic organs to provide arginine as a substrate for nitric oxide synthases (NOS). Increasing evidence suggests that argininosuccinate synthetase (AS) and AL are more globally expressed, and the coordinate regulation of AS and AL gene expression with that of the inducible form of NOS (iNOS) provides evidence that this may facilitate the regulation of NOS activity. Deficiency of AL leads to the human urea cycle disorder argininosuccinic aciduria. We produced an AL deficient mouse by gene targeting in order to investigate the role of AL in endogenous arginine production. This mouse also provides a model of the human disorder to explore the pathogenesis of the disorder and possible new treatments. Metabolic studies of these mice demonstrated that they have the same biochemical phenotype as humans, with hyperammonemia, elevated plasma argininosuccinic acid and low plasma arginine. Plasma nitrites, derived from NO, were not reduced in AL deficient mice and there was no significant difference is the level of cyclic GMP, the second messenger induced by NO. Ó 2002 Elsevier Science (USA). All rights reserved.

Keywords: Argininosuccinate lyase; Nitric oxide; Mouse; Urea cycle

Introduction This channeling of substrates in the urea cycle has been demonstrated by in situ studies of permeabilized rat Argininosuccinate lyase (AL; EC 4.3.2.1) is respon- hepatocytes [2,3]. AL may also play a critical role in the sible for cleaving argininosuccinate into fumarate and production of nitric oxide (NO). AL participates in the arginine. AL is a 52-kDa cytosolic enzyme that func- arginine–citrulline cycle to generate arginine from which tions as a tetramer and participates in two important NOS generates NO and regenerates citrulline. AL and metabolic pathways: the urea cycle, where it participates argininosuccinate synthetase (AS) both participate in in the disposal of waste nitrogen, and the arginine–cit- this cycle and are expressed in all tissues that have been rulline cycle, in which arginine is metabolized by nitric examined [4,5]. Evidence to suggest a link between oxide synthases (NOS) to generate nitric oxide and cit- de novo arginine synthesis and NO production is pro- rulline. Because of its role in the urea cycle, AL is highly vided by co-induction studies. Rats injected with lipo- expressed in the liver and kidney, where nitrogen in the polysaccharide (LPS), a known inducer of NO synthesis, form of ammonium is processed into urea [1]. The urea have been used for kinetic studies of NO production. In cycle enzymes preferentially use substrates produced these studies, inducible NOS (iNOS), AS, and AL were within the cell rather than from extracellular sources. all co-induced, inferring that citrulline–arginine recy- cling appears to be important in NO synthesis [6]. This suggests that AL provides iNOS with a preferred cellu- * Corresponding author. Fax: +713-798-5386. E-mail address: [email protected] (W.J. Craigen). lar source of arginine, reminiscent of the channeling of 1 These authors contributed equally. substrates in the urea cycle [2]. Further evidence in hu-

1096-7192/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S1096-7192(02)00206-8 12 V. Reid Sutton et al. / Molecular Genetics and Metabolism 78 (2003) 11–16 mans comes from stable isotope studies in adult males vere episodes of hyperammonemia than patients with fed a regular diet followed by an arginine-free diet. other urea cycle disorders, it has been suggested that Restriction of dietary arginine leads to a decrease in the liver pathology is due to the toxicity of ar- serum arginine but does not alter the rate of whole body gininosuccinic acid or its anhydride metabolites. Al- NO synthesis [7]. This implies that endogenous arginine ternatively, liver disease in argininosuccinic aciduria synthesized by AL is the preferred source (over dietary may result at least in part from impaired NO synthesis arginine) for NOS. as a consequence of the inability to synthesize arginine. Deficiency of AL in humans was first described by It is possible that reduced NO production may lead to Coryell et al. [8], and is commonly referred to as ar- impaired blood pressure regulation in the liver. Per- gininosuccinic aciduria. Individuals with this autosomal turbed hepatic blood flow may result in hepatic fibrosis. recessive disease typically come to medical attention Alternatively, accumulation of argininosuccinic acid, early in life with symptoms of hyperammonemia such which is normally undetectable in the plasma, may be as seizures, lethargy, or coma. The disease is charac- fibrogenic and lead to cirrhosis. terized biochemically by hyperammonemia, detectable Since NO has been shown to regulate basal blood argininosuccinic acid in serum, urine and CSF, low flow and is produced from arginine [11–15], investigat- serum arginine concentrations and elevated serum ing the effect of reduced endogenous arginine synthesis citrulline levels. Treatment currently consists of a low- on NO production may provide insights into the role of protein diet, decreasing nitrogen flux through the urea arginine synthesis on the regulation and pathophysio- cycle, and arginine supplementation [1]. Despite this logic effects of NO. NO achieves its effect by acting on treatment, patients often exhibit intellectual impairment intracellular guanylyl cyclase, resulting in conversion of and delayed motor skills. These sequellae are thought GTP to cGMP. cGMP then acts on phosphodiesterases, to result from chronic hyperammonemia, although it cGMP-gated ion channels, and cGMP-dependent pro- has also been suggested that a deficit in TCA cycle tein kinases which produce the cardiovascular, neuro- intermediates may contribute to the phenotype [9]. nal, pancreatic and other effects associated with NO Patients with AL deficiency may also suffer from pro- [21,22]. cGMP is a potentially more stable indirect gressive hepatic disease [10], and may require ortho- measure of NO production. Here we describe the gen- topic liver transplantation due to cirrhosis. Because eration and initial biochemical characterization of a individuals with AL deficiency have fewer and less se- mouse strain deficient in AL activity.

Fig. 1. The targeting strategy for the mouse AL gene. (A) Restriction sites used for generating the targeting plasmid are indicated (B, BamH1; Ev, EcoRV; Xb, Xba1; E, EcoR1). (B) The probe depicted as a black bar detects a recombinant band of 3.9 kbp following homologous recombination and replacement of exons 8 and 9 with the neomycin gene. A representative Southern blot is shown with the 5 kbp wild type band and the 3.9 kbp recombinant band. V. Reid Sutton et al. / Molecular Genetics and Metabolism 78 (2003) 11–16 13

Materials and methods noassay (EIA) (Amersham-Pharmacia Biotech) that combines the use of a peroxidase-labeled cGMP conju- Gene targeting of the AL gene gate, a specific antiserum which can be immobilized to pre-coated microtiter plates and a one-pot stabilized A previously isolated cDNA fragment [16] was used substrate solution. Measurement of cGMP in concen- to screen a 129SvEv mouse genomic library by plaque trations of femtomoles/well was carried out by spectro- hybridization to isolate a 12 kbp portion of the mouse photometric measurement at 450 nm. AL gene. Using standard molecular biology cloning strategies, exons 8 and 9 were replaced with a 1.4 kbp neomycin cassette which serves to delete 2 of the 13 Results exons and generate an out of frame mRNA beginning at exon 10 (Fig. 1). This AL gene construct was electro- Generation of AL deficient mice porated into the embryonic stem cell line AB2.2, the cells subject to positive and negative selection with G418 Following microinjection of ES cells into blastocysts, and gancyclovir, and numerous ES cell clones were heterozygous mice were generated from chimeric foun- identified by Southern blotting using the mini-Southern der mice (Fig. 1). Twenty-two litters (179 pups) from procedure [17]. We generated mice heterozygous for the heterozygous intercrosses were genotyped by PCR mutant allele and bred them to produce homozygous shortly after birth. Genotypes of offspring were consis- deficient mice. Genotyping was carried out using the tent with the expected Mendelian ratio (1:2:1), indicat- PCR primers: (50 AAG ATG GGT TCC GCA GAT ing that there is no increased in utero lethality in TAG GAT C 30;50 TGT AGG CAT CTG AGA GTT homozygous null (ALðÀ=ÀÞ) or heterozygous mice. GCA CAA A 30;50 TCT TGT CGA TCA GGA TGA ALðÀ=ÀÞ pups are indistinguishable in weight (ALðÀ=ÀÞ TCT GGA C 30). PCR was used to identify a wild type average 1.37 g vs. wild type average 1.45 g; v2 ¼ :002) or 600 bp sized fragment containing exons 8 and 9 and a appearance at birth from wild type littermates and all smaller 400 bp fragment primed from the neomycin initially feed well. Within 48 h ALðÀ=ÀÞ mice became less cassette. Animal care conformed to institutional stan- active, stopped feeding and rapidly expired. dards following an approved animal protocol. Characterization of urea cycle enzymes and biochemical Biochemical assays parameters

Argininosuccinate lyase activity in homogenized liver In ALðÀ=ÀÞ mice there was a complete deﬁciency of samples was measured by colorimetric assay of urea AL activity (Fig. 2). Enzyme activity in ALðÀ=ÀÞ liver production from argininosuccinate, and the activities of ranged from 14–77 lmol/h/g tissue, while wild type liver ornithine transcarbamylase, carbamyl phosphate syn- had AL activity of 673–1540 lmol/h/g tissue, consistent thetase, argininosuccinate synthetase, and argninase with a null mutation generated by deleting a large por- were measured as previously described [18]. Serum tion of the carboxy terminal end of the protein. To de- amino-acid analysis was performed by high pressure li- termine whether the absence of AL leads to a down- quid chromotography utilizing the ninhydrin reaction and detection at 570 nm (440 nm for proline). Plasma ammonia levels were assayed based on reductive ami- nation of 2-oxoglutarate, using glutamate dehydroge- nase and reduced NADPH. The decrease in absorbance at 340 nm due to the oxidation of NADPH is propor- tional to the plasma ammonia concentration. Guani- dinoacetate and creatine concentrations were measured in aqueous extracts of homogenized heart, skeletal muscle, and liver using tandem mass spectrometry [19].

Measurement of nitric oxide degradation products and related metabolites Fig. 2. Selected serum amino-acid levels. Values are indicated with the Measurement of the degradation products of NO was standard error for each group (n ¼ 4). The animals deficient for argi- performed on urine samples using the Greiss reaction, a ninosuccinate lyase have elevated levels of glutamine (gln), citrulline (cit), and argininosuccinic acid (ASA) and low levels of arginine (arg) colorimetric detection of nitrite [20]. cGMP levels in relative to wild type littermates. ASA is undetectable in wild type mice. urine and homogenized liver were measured using the All differences are highly significant (P > 0:001). These biochemical commercially available Biotrak cGMP enzyme immu- abnormalities recapitulate the human disease argininosuccinic aciduria. 14 V. Reid Sutton et al. / Molecular Genetics and Metabolism 78 (2003) 11–16

Table 1 Urea cycle enzyme activity levels in ALðÀ=ÀÞ mice and wild type littermates Enzyme ALðÀ=ÀÞ mice Wild type littermates Means ( SE) in lmol/h/g liver Means ( SE) in lmol/h/g liver Carbamyl phosphate synthetase 17.2 ð2:5Þ 12.2 ð1:5Þ Ornithine transcarbamylase 597 ð86Þ 713 ð164Þ Arginase 12.8 ð1:6Þ 21.3 ð2:7Þ Argininosuccinate lyase 40 ð19Þ 968 ð286Þ Arginase activity is lower in ALðÀ=ÀÞ mice relative to control mice. * P < 0:05. regulation of the overall pathway, other urea cycle en- (mean ¼ 34:1 10:2) and ALðÀ=ÀÞ animals (mean ¼ zymes were assayed in liver tissue. ALðÀ=ÀÞ liver has 26:7 13:7). reduced levels of arginase activity relative to control To examine the signal transduction pathway acti- liver (Table 1). vated by NO, additional experiments were performed to Serum amino-acid analysis corroborates the enzyme determine guanosine 30,50-cyclic monophosphate (cGMP) findings. ALðÀ=ÀÞ mice have statistically significant ele- levels in the urine of the affected and control animals. vations of serum argininosuccinic acid, citrulline, and Pooled urine from eight litters was assayed. The mean glutamine and low serum arginine levels when compared level in ALðÀ=ÀÞ mice was 11,080 (SE ¼ 1081) femto- to wild type littermates (Fig. 2; P < 0:001). Plasma moles/well while mean urine cGMP in wild type animals ammonia levels were assayed in samples from ALðÀ=ÀÞ was 11,410 (SE ¼ 589) femtomoles/well. There was no and wild type mice. ALðÀ=ÀÞ mice had a statistically significant difference in the concentration of cGMP in significant increase in plasma ammonia levels compared the urine of ALðÀ=ÀÞ mice when compared to wild type to wild type mice (Fig. 3; P < 0:05). These results are all mice. consistent with the loss of AL activity in this mutant mouse strain, resulting in hyperammonemia. Since arginine is a precursor in the synthesis of cre- Discussion atine via guanidinoacetate, we measured the concentration of each compound in pooled homogenized The urea cycle of mammals serves two functions; the skeletal muscle, heart, and liver samples. There was no de novo biosynthesis of arginine and the disposal of difference detected between ALðÀ=ÀÞ and wild type mice excess nitrogen beyond that needed for net protein in the pooled tissues (wild type 0:73 0:135 lM(N ¼ 6) biosynthesis. Arginine is also used as a substrate in vsALðÀ=ÀÞ 0:658 0:094 lM(N ¼ 5)). several other pathways including polyamine, creatine Because NO has a short half-life (t1=2 ¼ 10 s), direct and NO biosynthesis. Argininosuccinic aciduria is a measurement is difficult. Therefore, measurement of the rare, autosomal recessive disorder of nitrogen metabo- NO degradation product nitrite was measured by the lism that results from a deficiency of the enzyme argi- Greiss reaction. There was no observable difference of ninosuccinate lyase. Individuals with argininosuccinic plasma nitrite concentration among between wild type aciduria typically come to medical attention in the newborn period or during infancy as a result of vomit- ing, lethargy, developmental delay and hyperammonemia. The biochemical hallmarks of this disease are elevations of the amino acids argininosuccinic acid and citrulline and low levels of plasma arginine. Despite treatment to normalize plasma arginine levels and decrease episodes of hyperammonemia, individuals often have impaired motor and intellectual development, although this is not a universal finding [9]. In addition, many patients develop progressive liver dysfunction and fibrosis. The pathophysiology of the liver disease is un- clear. It seems unlikely to be related to the hyperammonemia, since individuals with other urea cycle disorders typically have more episodes of hyperammonemia and do not develop cirrhosis with the frequency Fig. 3. Plasma ammonia levels from mutant mice and control littermates. Plasma ammonia levels in ALðÀ=ÀÞ (n ¼ 5) average four-times seen in individuals with argininosuccinic aciduria. This the levels seen in wild type littermates (n ¼ 3; P < 0:05). The standard has led to the hypothesis that argininosuccinic acid is errors are shown for the two groups. toxic to hepatocytes and results in cirrhosis, although a V. Reid Sutton et al. / Molecular Genetics and Metabolism 78 (2003) 11–16 15 correlation of plasma argininosuccinate levels with liver difference in the tissue concentrations of creatine or its disease has not been established. Two alternate mecha- precursor guanidinoacetate was detected in the mutant nisms of liver and brain injury related to arginine defi- mice, however, this may reflect the young age of the ciency seem plausible. Low tissue arginine levels may mutant mice and residual levels from maternal produc- reduce NO production and impair liver blood flow tion. Future experiments using a regulatable genetic regulation, and may lead to fibrosis [21,22]. Support for rescue of the AL neonatal lethality should allow a more this theory comes from two observations in humans. A definitive determination of the role of endogenous ar- study of normal individuals infused with the endothelial ginine synthesis in NO formation and the mechanism of NO inhibitor L-NMMA into the brachial artery liver fibrosis in argininosuccinic aciduria. demonstrated a 50% fall in basal blood flow and an attenuated response to the vasodilator acetylcholine [23]. Similarly, blood pressure perturbations have been Acknowledgments reported in a hypoargininemic newborn with argininosuccinic aciduria [24]. The infant exhibited hy- These studies were supported in part by the core pertension at the time of presentation, which resolved laboratories of the Baylor College of Medicine Mental with intravenous arginine infusion. In the same report a Retardation Research Center (HD24064) and Child second infant administered an intravenous ‘‘arginine Health Research Center. We thank Arthur Beaudet challenge’’ to evaluate pituitary function exhibited re- M.D. for his assistance. duced blood pressure with each infusion of arginine. In addition, rats administered the arginine analog/NO in- x hibitor N -nitro-L-arginine have sustained hypertension in comparison to control animals. This effect can be References attenuated by pretreatment with L-arginine [25]. [1] S.W. Brusilow, A.L. Horwich, Urea cycle enzymes, in: C.R. NO is an important signaling molecule used in a vast Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), The Metabolic number of biologic settings including the regulation of and Molecular Bases of Inherited Disease, McGraw-Hill, Inc, vascular tone, neurotransmitter release and learning, New York, 2001, pp. 1909–1963. immune function and skeletal muscle and cardiac energy [2] C.W. Cheung, N.S. Cohen, L. Raijman, Channeling of urea cycle regulation. NO functions by activating guanylyl cyclase intermediates in situ in permeabilized hepatocytes, J. Biol. Chem. 264 (1989) 4038–4044. to produce cGMP from GTP, which leads to a series of [3] N.S. Cohen, A. Kuda, Argininosuccinate synthetase and argini- aforementioned effects [26,27]. Recent studies indicate nosuccinate lyase are localized around mitochondria: an immu- that endogenously synthesized (as opposed to dietary) nocytochemical study, J. Cell Biochem. 60 (1996) 334–340. arginine as a substrate for NOS is important for NO [4] Y. Hattori, E.B. Campbell, S.S. Gross, Argininosuccinate synthe- production. That NOS preferentially uses endogenously tase mRNA and activity are induced by immunostimulants in vascular smooth muscle. Role in the regeneration or arginine for produced arginine is supported by evidence of chan- nitric oxide synthesis, J. Biol. Chem. 269 (1994) 9405–9408. neling of substrate via microcompartmentalization [5] Y. Yu, K. Terada, A. Nagasaki, M. Takiguchi, M. Mori, within the cell [28]. Thus, low tissue arginine levels Preparation of recombinant argininosuccinate synthetase and would be expected to impede NO production by de- argininosuccinate lyase: expression of the enzymes in rat tissues, J. creasing the availability of the substrate arginine and Biochem. 117 (1995) 952–957. [6] A. Nagasaki, T. Gotoh, M. Takeya, Y. Yu, M. Takiguchi, H. through impaired induction of NOS. To test the hy- Matsuzaki, K. Takatsuki, M. Mori, Coinduction of nitric oxide pothesis that low arginine levels restrict NO production, synthetase, argininosuccinate synthetase and argininosuccinate we measured degradation products (nitrites) and the lyase in lipopolysaccharide-treated rats, J. Biol. Chem. 271 (1996) second messenger (cGMP). There was no significant 2658–2662. difference in either plasma nitrite levels of mutant ani- [7] L. Castillo, M. Sanchez, J. Vogt, T.E. Chapman, T.C. DeRojas- Walker, S.R. Tannenbaum, A.M. Ahami, V.R. Young, Plasma mals or urine cGMP levels. A better method to test this arginine, citrulline and ornithine kinetics in adults, with observa- hypothesis would be to directly measure NO produc- tions on nitric oxide synthesis, Am. J. Physiol. 268 (1995) E360– tion. Unfortunately, the small size and short life span of E367. AL deficient mice combined with the short half-life of [8] M.E. Coryell, W.K. Hall, T.G. Thevaos, D.A. Welter, A.J. Gatz, NO did not permit direct measurement. Measurement of B.F. Horton, B.D. Sisson, J.W. Looper Jr., R.T. Farrow, A familial study of a human enzyme defect, argininosuccinic NO formation in cultured astrocytes following induc- aciduria, Biochem. Biophys. Res. Commun. 14 (1964) 307–312. tion with cytokines is in progress. [9] K. Widhalm, S. Koch, S. Scheibenreiter, E. Knoll, J.P. Colombo, Finally, low arginine levels may reduce creatine pro- C. Bachmann, O. Thalhammer, Long-term follow-up of 12 duction and thus reduce the energy capacity of the patients with the late-onset variant of argininosuccinic acid lyase brain, and/or the synthesis of NO in the brain may be deficiency: no impairment of intellectual and psychomotor development during therapy, Pediatrics 89 (1992) 1182–1184. impaired [29,30]. AL is known to be expressed in several [10] A. Zimmerman, C. Bachmann, R. Baumgartner, Severe liver cell types within the brain and its expression is induced fibrosis in argininosuccinic aciduria, Arch. Pathol. Lab. Med. 110 by elevated ammonia in a cell culture system [31,32]. No (1986) 136–140. 16 V. Reid Sutton et al. / Molecular Genetics and Metabolism 78 (2003) 11–16

[11] M. Hecker, W.C. Sessa, H.J. Harris, E.E. Anggard, J.R. Vane, substrate arginine for nitric oxide synthesis, Biochem. Biophys. The metabolism of L-arginine and its significance for the Res. Commun. 205 (1994) 1391–1398. biosynthesis of endothelium-derived relaxing factor: cultured [23] P. Vallance, J. Collier, S. Moncada, Effects of endothelium- endothelial cells recycle L-citrulline to L-arginine, Proc. Natl. derived nitric oxide on peripheral arteriolar tone in man, Lancet 2 Acad. Sci. USA 87 (1990) 8612–8616. (8670) (1989) 997–1000. [12] P.L. Huang, M.C. Fishman, Genetic analysis of nitric oxide [24] C.R. Fakler, H.A. Kaftan, L.D. Nelin, Two cases suggesting a synthase isoforms: targeted mutation in mice, J. Mol. Med. 74 role for the L-arginine nitric oxide pathway in neonatal blood (1996) 415–421. pressure regulation, Acta Paediatr. 84 (1995) 460–462. [13] R.G. Knowles, S. Moncada, Nitric oxide synthases in mammals, [25] Y. Ohashi, S. Kawashima, K. Hirata, T. Yamashita, T. Ishida, N. Biochem. J. 298 (1994) 249–258. Inoue, T. Sakoda, H. Kurihara, Y. Yazaki, M. Yokoyama, [14] J.A. Mitchell, M. Hecker, J.R. Vane, The generation of L-arginine Hypotension and reduced nitric oxide-elicited vasorelaxation in in endothelial cells is linked to the release of endothelium-derived transgenic mice overexpressing endothelial nitric oxide synthase, J. relaxing factor, Eur. J. Pharmacol. 176 (1990) 253–254. Clin. Invest. 102 (1998) 2061–2071. [15] S. Moncada, A. Higgs, The L-arginine–nitric oxide pathway, N. [26] F. Murad, S. Waldman, C. Molina, B. Bennett, D. Leitman, Engl. J. Med. 329 (1993) 2002–2012. Regulation and role of guanylate cyclase–cyclic GMP in vascular [16] V.R. Sutton, Y. Pan, W.J. Craigen, Regional localization of the relaxation, Prog. Clin. Biol. Res. 249 (1987) 65–76. mouse argininosuccinate lyase gene to chromosome 5, Mamm. [27] A.B. Vaangrager, H.R. deJonge, Signalling by cGMP-dependent Genome 8 (1997) 871. protein kinases, Mol. Cell. Biochem. 157 (1996) 23–30. [17] R. Ramirez-Solis, J. Rivera-Perez, J.D. Wallace, M. Wims, H. [28] L. Xie, Y. Hattori, N. Tume, S.S. Gross, The preferred source of Zheng, A. Bradley, Genomic DNA microextraction: a method to arginine for high-output nitric oxide synthesis in blood vessels, screen numerous samples, Anal. Biochem. 201 (1992) 331–335. Semin. Perinatol. 24 (2000) 42–45. [18] C.T. Nuzum, P.J. Snodgrass, in: S. Grisola, R. Baguena, F. [29] O. Braissant, H. Henry, M. Loup, B. Eilers, C. Bachmann, Mayor (Eds.), The Urea Cycle, Wiley, New York, 1976, pp. 325– Endogenous synthesis and transport of creatine in the rat brain: 349. an in situ hybridization study, Brain Res. Mol. Brain Res. 86 [19] O.A. Bodamer, S.M. Bloesch, A.R. Gregg, S. Stockler-Ipsiroglu, (2001) 193–201. W.E. OÕBrien, Analysis of guanidinoacetate and creatine by [30] T.J. Lee, J.G. Yu, L-Citrulline recycle for synthesis of NO in isotope dilution electrospray tandem mass spectrometry, Clin. cerebral perivascular nerves and endothelial cells, Ann. NY Acad. Chem. Acta 308 (2001) 173–176. Sci. 962 (2002) 73–80. [20] L.C. Green, D.A. Wagner, J. Glogowski, P.L. Skipper, J.S. [31] O. Braissant, T. Gotoh, M. Loup, M. Mori, C. Bachmann, L- Wishnok, S.R. Tannenbaum, Analysis of nitrate, nitrite and Arginine uptake, the citrulline–NO cycle and arginase II in the rat [15N]nitrate in biological fluids, Anal. Biochem. 126 (1982) 131– brain: an in situ hybridization study, Brain Res. Mol. Brain Res. 138. 70 (1999) 231–241. [21] H. Suzuki, M. Menegazzi, A. Carcereri de Prati, S. Mariotto, U. [32] O. Braissant, P. Honegger, M. Loup, K. Iwase, M. Takiguchi, Armato, Nitric oxide in the liver: pathophysiological roles, Adv. C. Bachmann, Hyperammonemia: regulation of argininosuccin- Neuroimmunol. 5 (1995) 379–410. ate synthetase and argininosuccinate lyase genes in aggregating [22] Y. Wakabayashi, E. Yamada, T. Yoshida, H. Takahashi, Defi- cell cultures of fetal rat brain, Neurosci. Lett. 266 (1999) 89– ciency of endogenous arginine evokes hypertension by exhausting 92.