5

Arginine, Citrulline, and Ornithine

H. Wiesinger

1 2 2.1 2.2 3 3.1 3.1.1 3.1.2 3.1.3 3.2 3.2.1 3.2.2 3.3 3.3.1 3.3.2 4 4.1 4.2 5

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 Tissue and CSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 Cellular Localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Metabolism and Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Arginine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Citrulline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 Ornithine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Arginine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Citrulline and Ornithine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Arginine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Citrulline and Ornithine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 Physiology and Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 Arginine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 Citrulline and Ornithine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

Springer-Verlag Berlin Heidelberg 2007

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Abstract: Metabolism as well as function of the three amino acids, L-arginine, L-citrulline, and Lornithine, are closely intertwined and related to the homeostasis of nitric oxide at the tissue and cellular levels. For the three compounds data on neural tissue and CSF levels are summarized and localization in brain mainly derived from immunohistochemical experiments is reported. A complex pattern of differential cellular localization, subcellular compartmentation and expression of isoforms holds for the enzymes involved in the synthesis and degradation of all three amino acids in the nervous system. Trafcking of arginine or citrulline between particular neural cell populations is likely, and transport mechanisms and proteins involved are discussed. Up-regulation of arginine synthesis and transport by proinammatory agents satises the need of induced nitric oxide synthase for its substrate arginine especially in glial cells. Nitric oxide-related pathologies in the nervous system cannot be separated from the metabolic network established for the three amino acids; however, pharmacological intervention has to take into account the manyfold functions of arginine, citrulline, and ornithine in undisturbed neural cell physiology. List of abbreviations: AD, Alzheimers disease; ADC, Arginine decarboxylase; ADMA, Asymmetric NG, NG-dimethylarginine; AGAT, Arginine: glycine amidinotransferase; ASL, Argininosuccinate lyase; ASS, Argininosuccinate synthetase; CAT, Cationic amino acid transporter; CPS, Carbamoylphosphate synthetase; DDAH, NG, NG-dimethyl-L-arginine dimethylaminohydrolase; IFN-, Interferon-; LPS, Lipopolysaccharide; NMMA, NG-monomethylarginine; NOS, Nitric oxide synthase; OAT, Ornithine aminotransferase; ODC, Ornithine decarboxylase; OTC, Ornithine carbamoyltransferase; PAD, Peptidylarginine deiminase

Introduction

Since the last edition of this handbook, knowledge about the metabolically interrelated amino acids, Larginine, Lcitrulline, and Lornithine, has tremendously increased (stereochemical denotation will be abandoned from hereon). This is mainly due to the discovery in the late 1980s that the small radical molecule nitric oxide (NO) is ubiquitously distributed in the mammalian body and has profound physiological functions and that NO is generated in vivo exclusively from arginine with citrulline as coproduct. This perception shed new light on the role of the rudimentary urea cycle in nonhepatic tissues and in particular the various cell types of the nervous system, and recent ndings on arginase added to a rethinking on established facts of arginine and ornithine in physiology and disease. Finally, it was recognized that agmatine is an important metabolite of arginine also in higher eukaryotes with functions of its own and that Nmethylated arginine analogs as well as a number of guanidino compounds derived ultimately from arginine are worth being considered as (patho)physiological mediators. Since metabolism as well as function of the three amino acids and the arginine metabolites mentioned are closely interrelated, these compounds will be treated together under the aspects of occurrence, synthesis and degradation (including transport), and physiological role with pathophysiological implications. Emphasis will be put on ndings in brain tissue and in neuronal and glial cell cultures with reference to the peripheral nervous system (PNS) whenever appropriate. It should be mentioned that all chapters of this handbook dealing with generation and functions of NO should also be consulted when information on the roles of arginine and its metabolites in normal and pathophysiology of the nervous system is desired.

Occurrence

2.1 Tissue and CSF

Established analytical methods have been used for quantifying amino acids in nervous tissue and CSF. Data on arginine, citrulline, and ornithine in the brain can be found in the article by Perry (1982) and on arginine in the CSF in the one by Wood (1982) in the second edition of this handbook. A compilation of data from several literature sources is given by Wiesinger (2001). A mean value of arginine concentration in human CSF can be given as about 22 mM (Wood, 1982) and does not change in several disease states such as

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Alzheimers disease (AD) or Parkinsons disease (Kuiper et al., 2000). Citrulline levels appear to be an order of magnitude lower (Kuiper et al., 2000), although a wide range of data can be taken from early reports (Wiesinger, 2001). Data on brain concentrations were gathered from common laboratory animals and the biopsied human brain; 0.1 to 0.3mmol/g wet weight is a reliable value for arginine in whole brain, but also for frontal or cerebellar cortex (Perry, 1982; de Jonge et al., 2001; Wiesinger, 2001). Citrulline again appears to be present in somewhat lower concentrations of 0.02 to 0.1mmol/g wet weight (Perry, 1982). With a full urea cycle lacking in the brain, the nevertheless substantial amounts of citrulline in this organ can be explained by the presence of a neural citrullineNO cycle operating not in a stoichiometric fashion (see > Sect. 3.1). Concentrations reported for ornithine range from 0.01 to 0.05mmol/g wet weight (Perry, 1982; Seiler and DauneAnglard, 1993). An even distribution of arginine and ornithine in the brain may be assumed since data do not differ when reported for whole brain, frontal and cerebellar cortex (Perry, 1982) or brain stem, cerebellum, and medulla (Seiler and DauneAnglard, 1993). A slightly elevated value of citrulline in cerebellar cortex (Perry, 1982) may reect the substantial constitutive generation of NO in this region (Garthwaite and Boulton, 1995). Cerebellar concentrations of arginine were the same when measured in 3 to 5monthold (young) and 18 to 22monthold (aged) rats (4.4 mmol/g of soluble protein; Mistry et al., 2002).

2.2 Cellular Localization

Direct localization of amino acids by immunohistochemistry was made possible by developing protocols for the generation of antibodies directed against these small molecules. Arginine appears to be distributed unevenly among CNS cell populations. In the rat brain and spinal cord, arginine immunoreactivity was localized mainly in astrocytes whereas oligodendrocytes were not immunoreactive (Aoki et al., 1991b). Bergmann glial cells in the cerebellum stained positive for arginine, neighboring basket and Purkinje cells did not. However, some neurons in the cerebellum showed weak arginine immunoreactivity, several bers in the brain stem and the spinal cord were prominently stained (Aoki et al., 1991b). Similarly, in the PNS arginine immunoreactivity was predominant in glial components, i.e., satellite and supporting cells surrounding neuronal structures (Aoki et al., 1991a). Arginine immunoreactivity was seen in astroglial endfeet wrapping endothelial cells of the vasculature (Aoki et al., 1991b) and a glial localization of immunoreactive arginine was reported for the ventroposterior thalamic nucleus of the rat (Kharazia et al., 1997) and the rat neurohypophysis (Pow, 1994). These immunohistochemical data correlate well with the nding of high concentrations of arginine in cultured astrocytes (Yudkoff et al., 1989). By immunohistochemical methods, citrulline was detected in the rat brain exclusively in neurons; these neurons also contained NADPH diaphorase, an enzyme activity taken as being identical with neuronal nitric oxide synthase NOS1 (Pasqualotto et al., 1991; for critical discussion, see Wolf, 1997). A neuronal localization of citrulline immunoreactivity was also found in the rat neurohypophysis (Pow, 1994). In addition to NOScontaining neurons, activated microglial cells in the brain stained positively for citrulline. Citrulline immunoreactivity was lacking in neurons of inducible NOS2 knockout mice and was increased when recycling of citrulline to arginine was blocked (Keilhoff et al., 2000). With confocal laser scanning microscopy, costaining of citrulline and NOS2 was found predominantly at postsynaptic densities (Martinelli et al., 2002). From these results it was concluded that citrulline immunohistochemistry is a reliable means for studying NOS activity in the brain at the single cell and subcellular level. However, it should be kept in mind that citrulline can be generated posttranslationally within proteins from arginine by the action of any isoform of calciumdependent peptidylarginine deiminase (PAD; EC 3.5.3.15; Vossenaar et al., 2003). Therefore, in addition to free citrulline generated in the brain mainly by NOS activity, antisera may also detect proteinbound citrulline. Indeed, antisera stained citrulline containing proteins in Western blot analysis, and staining increased with increasing concentration of intracellular calcium (Keilhoff and Wolf, 2003). Citrullinated epitopes in proteins were investigated systematically in the rat and human brains after generation of a monoclonal antibody, which was selected for reacting with human citrullinated myelin basic protein (Nicholas and Whitaker, 2002). Immunohistochemical localization of citrullinecontaining

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proteins in the brain revealed staining of white matter myelin, but also of a subset of astrocytes associated with blood vessels and ventricular surfaces; in these cells glial brillary acidic protein (GFAP) may be present in multiple citrullinated isoforms (Nicholas and Whitaker, 2002; Nicholas et al., 2003). Deimination of arginine in myelin basic protein was increased in protein isolated from white matter of multiple sclerosis patients and may be related to the pathogenesis of this demyelinating disease (Kim et al., 2003). Membranebound PAD was elevated in a demyelinating mouse model prior to loss of myelin (Moscarello et al., 2002). Cellular localization of PAD type II revealed expression of the enzyme in immature oligodendrocytes and in astrocytes and microglial cells after kainateinduced neurodegeneration (Akiyama et al., 1999; Asaga et al., 2002). Direct localization of ornithine has not been reported. However, localization of arginine, citrulline, and ornithine can be inferred from localization of their biosynthetic machineries. With this in mind, the reader may consult the appropriate paragraphs of this chapter (see > Sect. 3.1).

Metabolism and Transport

The three amino acids arginine, citrulline, and ornithine are metabolically intertwined. Synthesis of one amino acid is brought about by degradation of another one, and vice versa (> Figure 5-1). Differential cellular localization of the enzymes involved, subcellular compartmentalization, and expression of various isoenzymes have to be considered when drawing a complete picture of the metabolic pathways involved. For proper quantitative consideration of neural homeostasis of all three amino acids, uptake from the periphery has to be taken into consideration (see > Sect. 3.3). For current knowledge about the metabolism of the amino acids in peripheral tissues, about arginine being asemiessential amino acid, and about interorgan trafcking of citrulline and arginine, the reader is referred to reviews (Levillain, 2003; Cederbaum et al., 2004; Morris, 2004; Wu and Morris, 2004).

. Figure 5-1 Overview of interrelated metabolism of arginine, citrulline, and ornithine in neural cells. For details of cosubstrates or coproducts, see > Figures 5-2 and > 5-3. Enzymes are written in italics, abbreviations as in the text. Argininosucc., argininosuccinate; GSA, glutamic semialdehyde; Glu, glutamate; PM, plasma membrane; Pro, proline. Differential cellular expression and mitochondrial localization of some of the enzymes are described in the text. Exact biochemistry of the reactions of GSA to Glu and Pro, respectively, is not shown

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3.1 Synthesis
3.1.1 Arginine
The central pivot in the metabolism of arginine is the urea cycle as the mammalian bodys means to dispose of surplus nitrogen and detoxify ammonia (Withers, 1998). The existence of urea cycle enzymes in the brain has been investigated early and is summarized in the rst edition of this handbook (Buniatian, 1971; see Wiesinger, 2001). Activities of the mitochondrial enzymes carbamoylphosphate synthetase I (CPS I; EC 6.3.4.16) and ornithine carbamoyltransferase (OTC; EC 2.1.3.3) were not detected in rodent CNS, which implies that neural cells are not capable of de novo synthesis of arginine and that urea is not the molecule used for disposal of nitrogen in the brain. In contrast it was clear from the early studies that activities of the extramitochondrial enzymes of the urea cycle can be measured in brain (Buniatian, 1971; Sadasivudu and Rao, 1976). Nevertheless, a functional role of argininosuccinate synthetase (ASS; EC 6.3.4.5) and argininosuccinate lyase (ASL; EC 4.3.2.1) in neural tissue was not evident until it was conceived that a citrullineNO cycle might explain the considerable amounts of citrulline present in the brain (see > Sect. 2.1). A renaissance of investigations on arginine-synthesizing enzymes in the nervous system followed with emphasis on ASS, the ratelimiting enzyme of the pathway (> Figures 5-1 and > 5-2). A localization of ASS and ASL in neural cells was attempted with rened immunological and molecular biology methods. From the rst reports on brain tissue, it appeared as if ASS was exclusively expressed in neurons (Nakamura et al., 1991a; ArntRamos et al., 1992; summarized by Wiesinger, 2001). ASS immunoreactivity was not detected in glial cells of the brain (ArntRamos et al., 1992), which also did not exhibit any signal for ASS transcript in an extensive in situ hybridization study (Braissant et al., 1999a). However, an exclusive neuronal expression of ASS in CNS tissue is in contrast to ndings in cell culture. 15 Nlabeled aspartate was incorporated into cellular arginine in astroglial culturesa reaction, which necessitates the presence of ASS (Yudkoff et al., 1987), and ASS enzyme activity was measured in homogenates of cultured astrocytes (Jackson et al., 1996). Therefore, the presence of ASS protein in glial
. Figure 5-2 CitrullineNO cycle. Enzymes are written in italics. Pi, inorganic phosphate; other abbreviations, see text

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cells was investigated with a monospecic antiserum generated against a peptide representing a partial sequence of mouse liver ASS in homogenates of the rat glioma cell line C6BU1 (Schmidlin et al., 1997) or pure mouse astroglial cultures (Schmidlin and Wiesinger, 1998). In both glial paradigms, weak basal ASS immunoreactivity increased considerably when the cells had been incubated with bacterial lipopolysaccharide (LPS) and/or interferong (IFNg), immunostimulants that also induce NO synthesis in these cells. ASS protein was reported in the C6 glioma cell line after stimulation with a mixture of immunostimulants and cytokines (Zhang et al., 1999), and ASS transcript was found in the glioma cells and in astrocytes in aggregating cell cultures from fetal rat telencephalon (Zhang et al., 1999; Braissant et al., 1999b). Coinduction of ASS and NOS2 mRNA and protein upon incubation with LPS and IFNg was reported for a murine microglial cell line and rat primary microglial cells (Kawahara et al., 2001). From the experiments described above, it became clear that ASS is expressed in unstimulated cultured glial cells at levels that are at the limits of detection with the antisera used. Glial ASS protein strongly increased after the cells had been incubated with molecules representing proinammatory signals. Pitfalls of cell culture systems were avoided when proinammatory conditions were generated in rat striatum by injecting a mixture of LPS and IFNg (Heneka et al., 1999). A detailed quantitative analysis revealed that ASS was predominantly localized in activated microglial cells and only occasionally in GFAPpositive astrocytes. Interneurons in cortex and striatum also stained positively for ASS; however, the same level of staining intensity was seen in control and injected animals (Heneka et al., 1999). Expression of ASS and NOS2 was investigated by immunohistochemistry in hippocampus, frontal, and entorhinal cortex of brains of AD patients and nondemented agematched controls (Heneka et al., 2001). ASS expression was detected in neurons of control brains; the number of ASSpositive neurons as well as the expression level increased in AD brains. Basal ASS expression and an increase in expression level were also observed in astrocytes without an increase in the number of ASSpositive cells. Only occasionally ASSpositive activated microglial cells were present in the surroundings of senile plaques. Colocalization of ASS and NOS2 was evident in neurons and, although to a lesser extent, in glial cells (Heneka et al., 2001). mRNA levels of ASS and NOS2 were increased in postmortem cortical tissue obtained from AD patients as compared with control brains (Haas et al., 2002). Thus, it is suggestive that a concomitant up-regulation and/or induction of ASS and NOS2 both in neurons and glial cells may be responsible for prolonged generation of potentially deleterious NO in AD brains. Indeed, bamyloid peptides induced transcription of NOS2 and ASS in mixed rat neuronalglial cultures (Haas et al., 2002). A complex picture emerged from studies on the localization of ASL (summarized in Wiesinger, 2001). ASLimmunoreactivity was detected in the spinal cord and throughout the brain in neurons which, in most cases, were lacking ASS immunoreactivity (Nakamura et al., 1990, 1999; Nakamura, 1997). Differential neuronal distribution of ASS and ASL implies transmembrane transport of argininosuccinate which, however, is hard to conceive and is not corroborated by any experiments (see discussion in Wiesinger, 2001). ASL transcript was found in neurons as well as in astrocytes in the rat brain (Braissant et al., 1999a). ASL activity, ASL transcript, and ASL immunoreactivity were detected in a variety of glial cell cultures (Yudkoff et al., 1987; Jackson et al., 1996; Bolla et al., 1999; Braissant et al., 1999b; Kawahara et al., 2001). In contrast to ASS, however, glial ASL expression levels appear not to be inuenced by proinammatory stimuli (Bolla et al., 1999; Zhang et al., 1999; Kawahara et al., 2001). In conclusion, it is clear that neurons as well as glial cells are able to synthesize arginine in a shortcut of the urea cycle, which was designated the citrullineNO cycle (Hecker et al., 1990; Husson et al., 2003; > Figure 5-2). Nevertheless, it remains to be established if neuronal generation of arginine depends on an interneuronal trafcking of the intermediate argininosuccinate and if an intercellular citrullineNO cycle may operate in the brain.

3.1.2 Citrulline
Substantial synthesis of citrulline in the brain occurs only through the action of any isoform of NOS (> Figure 5-2). The mitochondrial enzymes of the urea cycle are lacking (Buniatian, 1971; see > Sect. 3.1.1) and an indirect supply of citrulline from degradation of proteins in which arginine was posttranslationally

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deiminated by PAD (see > Sect. 2.2) does not appear to be of quantitative importance. Citrulline can also be generated by the activity of NG, NGdimethylLarginine dimethylaminohydrolase (DDAH; EC 3.5.3.18; G G > Figure 5-1) from N methylated arginine analogs, N monomethylarginine (NMMA), and asymmetric G G N ,N dimethylarginine (ADMA), both being inhibitors of NOS (Tran et al., 2003). However, DDAH, which is expressed in the brain (Leiper et al., 1999) and the spinal cord, is not colocalized with NOS1 (Mishima et al., 2004). NMMA and ADMA are derived from hydrolysis of proteins in which arginine has been posttranslationally methylated. Again, generation of citrulline by DDAH after protein breakdown does not appear to be of quantitative signicance given the fact that methylated proteins in the brain do not turnover rapidly under normal conditions. However, an up-regulation of protein degradation under pathological circumstances may necessitate increased hydrolysis of methylarginines as was found after motor nerve injury (Nakagomi et al., 1999), and net synthesis of citrulline from NMMA has been proposed for the striatum (Ohta et al., 1994). It may be summarized that citrulline in neural cells is almost exclusively generated as an intermediate in the citrullineNO cycle (> Figure 5-2), which is hardly replenished with citrulline from other pathways. Since no metabolic cycle operates in a stoichiometric fashion, considerable amounts of citrulline are detected in nervous tissue (see > Sect. 2.1).

3.1.3 Ornithine
Ornithine can be synthesized by the hydrolytic cleavage of arginine catalyzed by both isoforms of arginase and the action of arginine:glycine amidinotransferase (Seiler and DauneAnglard, 1993). Since both enzymes are pivotal in the degradation of arginine, ndings will be detailed in the next chapter (see > Sect. 3.2.1). Ornithine synthesis from glutamate or proline is not signicant in nervous tissue (Wu et al., 1997).

3.2 Degradation
3.2.1 Arginine
A major pathway of degradation of arginine in nervous tissue is the reaction catalyzed by the three isoforms of NOS (EC 1.14.13.39; > Figure 5-2). Constitutive neuronal isoform, NOS1, is widespread in the PNS and is established as furnishing NO as a neurotransmitter, e.g., the transmitter released from nonadrenergic, noncholinergic myenteric plexus neurons in the gastrointestinal tract (Bult et al., 1990; Grozdanovic et al., 1992). In the CNS constitutive isoform NOS3 is particularly expressed in the endothelial cells lining the capillaries (Seidel et al., 1997). NOS1 is found in many parts of the brain, most prominently in the cerebellum and hippocampus, but also in the cerebral cortex and the striatum. Finally, inducible isoform NOS2 is expressed under many pathological conditions, most notably in glial cells (see > Sect. 4). Arginine is the only physiological nitrogencontaining substrate of any NOS isoform, and neural supply of arginine by the citrullineNO cycle or by transmembrane transport has been discussed (Wiesinger, 2001). Arginine catabolism by the two isoforms of arginase (EC 3.5.3.1; > Figure 5-3) yields urea and ornithine. A distinction that cytosolic arginase I is predominantly expressed in liver whereas mitochondrial arginase II is more ubiquitously distributed may become obsolete (Cederbaum et al., 2004). In the nervous system, arginase activity was detected in the rat brain in the cerebellum, cerebral cortex, and brain stem (Sadasivudu and Rao, 1976); arginase activity and protein (as probed by Western blotting) were found in the rat hippocampus and were not dependent on age or functional activity (Liu et al., 2003a, b). Arginase I immunoreactivity was localized in neurons of the olfactory bulb, the cerebellar cortex, and the facial motor nucleus (Nakamura et al., 1990). By in situ hybridization and immunohistochemistry, arginase I as well as arginase II were detected exclusively in neurons of the mouse brain with arginase I being more strongly expressed than the mitochondrial isoform (Yu et al., 2001). On the other hand, cultured microglia as well as astrocytes exhibited arginase activity (Xu et al., 2003), and the transcript for mitochondrial arginase II was present in neurons and glial cells throughout the rat brain, most notably also in oligodendrocytes of cerebellar white matter (Braissant et al., 1999a). Arginase I was strongly expressed in sympathetic ganglia of

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. Figure 5-3 Metabolism of arginine. By hydrolysis (arginase), decarboxylation (ADC), or amidino transfer reaction (AGAT). Enzymes are written in italics. Abbreviations, see text

the PNS (Yu et al., 2002, 2003), which may explain the retardation symptoms in arginase I deciency (Yu et al., 2002). A role for arginase I in autoimmune inammation of the spinal cord was suggested since the gene for the enzyme was the most up-regulated gene in mice during experimental autoimmune encephalomyelitis and since inhibition of arginase activity ameliorated the symptoms (Xu et al., 2003). Production of polyamines downstream of arginase activity may be essential for axonal regeneration (Cai et al., 2002); however, there is no unequivocal data in the literature which isoform of arginase supplies ornithine as a precursor for polyamine synthesis in the brain. Arginine in the brain is also catabolized by arginine:glycine amidinotransferase (AGAT; EC 2.1.4.1; > Figure 5-3), the rst enzyme in the biosynthetic pathway leading to creatine. AGAT is ubiquitously distributed in the adult rat brain in neurons and glial cells, suggesting that every cell type in the CNS is able to synthesize creatine from arginine (Braissant et al., 2001). Indeed, NMR studies hinted at creatine synthesis in cultured astroglial cells of the rat brain (Dringen et al., 1998). Deciency in AGAT leads to retardation of speech and mental development (Item et al., 2001; Leuzzi, 2002), and the importance of creatine for proper brain functioning and in neuroprotection has been documented (Schulze, 2003; Klivenyi et al., 2004). Due to its low substrate specicity, AGAT may also be responsible for the formation of other guanidino compounds, i.e., neurotoxic guanidinopropionic acid or guanidinobutyric acid (de Deyn et al., 2001; see > Sect. 4). Finally, the irreversible decarboxylation of arginine yielding 4(aminobutyl)guanidine (agmatine) is catalyzed by mitochondrial arginine decarboxylase (ADC; EC 4.1.1.19; > Figure 5-3). ADC activity was detected in the brain and cultured rat glioma cells (Regunathan and Reis, 2000), and Northern blot analysis with probes for human ADC showed expression of the mRNA in the human brain (Zhu et al., 2004). Roles for agmatine at imidazolinebinding sites, a2receptors, and at the NMDAtype glutamate receptor, and in the regulation of cellular polyamine content and consequently cell proliferation have been described (Li et al., 1994, 2003; Olmos et al., 1999; Abe et al., 2003; Berkels et al., 2004). Agmatine (or its aldehyde metabolite) is a potent inhibitor of NOS (Satriano, 2003). Agmatine reduced NO production in cultured microglia (Abe et al., 2000); NOS2 activity as well as the amount of NOS2 protein were decreased when cultured astrocytes had been incubated with immunostimulants in the presence of agmatine, and synthesis of agmatine was up-regulated (Regunathan and Piletz, 2003). These latter ndings may explain the neuroprotecive effects of agmatine in models of brain injury (Gilad et al., 1996; Feng et al., 2002). However, whether agmatine is indeed a key molecule in regulating the generation of potentially harmful NO by NOS2 remains to be elucidated.

3.2.2 Citrulline and Ornithine

Recycling of citrulline to arginine in the citrullineNO cycle is the only metabolic pathway of the NOS product in CNS. The rst and ratelimiting enzyme of this transformation has been dealt with extensively in this chapter (see > Sect. 3.1.1; > Figure 5-2).

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Two major catabolic pathways of ornithine in neural tissue have to be considered, i.e., decarboxylation to putrescine by ornithine decarboxylase (ODC; EC 4.1.1.17) and transamination by mitochondrial ornithine aminotransferase (OAT; EC 2.6.1.13; > Figure 5-1). Citrulline formation from ornithine does not take place in the CNS (see > Sect. 3.1). ODC activity in the brain is highest during pre and early postnatal stages, whereas in the mature brain ODC activity is low not the least due to the presence of ODC antizyme. ODC as well as antizyme transcripts and proteins have been colocalized in neurons throughout the adult rat brain (Kilpelainen et al., 2000). ODC appears to be up-regulated in some brain regions in AD (Morrison et al., 1998) and in cerebral ischemia (Babu et al., 2003). ODC is induced in glial cells upon long ller, 1999), and in neurons and microglial cells lasting activation of neuronal functions (Bernstein and Mu after systemic administration of LPS (Soulet and Rivest, 2003). Early data on regulation of ODC in the brain ller, 1999). has been summarized (Seiler and DauneAnglard, 1993; Bernstein and Mu Transamination of ornithine yields glutamic semialdehyde which equilibrates nonenzymatically with 1 LD pyrroline5carboxylate, a precursor of glutamate or proline (> Figure 5-1). Different isoforms of OAT may exist in neurons and astrocytes (Drejer and Schousboe, 1984). A possible role of ODC in the formation of GABA has been discussed (Seiler and DauneAnglard, 1993).

3.3 Transport
3.3.1 Arginine
Several transport systems for the cationic amino acids arginine, lysine, and ornithine have been described at the physiological and molecular level (Deves and Boyd, 1998; Closs and Mann, 1999). Uptake of arginine in neural cells is mediated by systems y, Ly, B0,, and b0,, with y being by far the most important transport system. A compilation of data is given by Wiesinger (2001). System y is not sodium dependent, thus operating according to the laws of facilitated diffusion and consequently being unable to transport the amino acid against a concentration gradient. System y is present in cultured neurons and glial cells, but also in endothelial cells of the microvessels and in the choroid plexus epithelium, thus rendering peripheral arginine available to the brain parenchyma and the CSF (Segal et al., 1990; Stoll et al., 1993; Stuhlmiller and Boje, 1995). System y also seems to mediate uptake of arginine into mitochondria (Dolinska and Albrecht, 1998), where arginine catabolizing enzymes arginase II and ADC are located (see > Sect. 3.2.1). Upon incubation with LPS transport activity of system y in cultured astroglial cells was up-regulated in parallel with the induction of NO production (Schmidlin and Wiesinger, 1994, 1995). On the other hand, arginine transport in neuronal primary cultures was insensitive against proinammatory stimuli (Stevens and Vo, 1998). Arginine uptake into astrocytes and cells of the glioma cell line C6 was down-regulated by prolonged exposure to noradrenaline, isoproterenol, or dibutyryl cyclic AMP (Feinstein and Rozelman, 1997). Arginine transport in neural cells may be inuenced by pathological conditions such as hepatic encephalopathy (Rao et al., 1997; Hazell and Norenberg, 1998). Several transcripts related to system y have been detected in brain tissue as well as in neural cell cultures. Constitutive CAT1 has been detected in neurons of the brain, in cultured neurons and glial cells, and in microvessels and the choroid plexus, and mediates basic arginine supply to cells of the CNS (Stoll et al., 1993; Stevens et al., 1996; Stevens and Vo, 1998; Braissant et al., 1999a). CAT2 splice variants are responsible for up-regulation of arginine transport, and a particular splice variant of CAT2 was found in cultured astroglial cells upon incubation of the cells with immunostimulants (Stevens et al., 1996). Up-regulation of CAT2 by LPS and IFNg was also reported for a murine microglial cell line (Kawahara et al., 2001), and the importance of arginine uptake by CAT2 for astroglial NO production by NOS2 has been stressed (Manner et al., 2003). The signicance of up-regulated CAT1 mRNA in microglial cells of mice expressing the APOE4 gene allelea risk factor in ADis not clear (Czapiga and Colton, 2003). Transcript CAT3 appears to be brain specic and was mainly localized along the midbrainthalamus hypothalamus axis (Hosokawa et al., 1997; Ito and Groudine, 1997). CAT3 transcript was detected neither in capillary endothelial nor in glial cells, but prominently in neurons (Hosokawa et al., 1999; Braissant et al., 1999a). However, an antiserum prepared against a rat CAT3 peptide stained neurons in the ventromedial

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Arginine, citrulline, and ornithine

part of the rat brain only weakly, whereas good colocalization of immunocytochemical and RNA hybridization signals was found in the cerebral cortex, the hippocampus, and the cerebellum. Immunoreactivity was conned to neuronal cell bodies thus conrming CAT3 as a neuronspecic transporter. A role for human CAT4 being transcribed in the brain remains to be established (Sperandeo et al., 1998).

3.3.2 Citrulline and Ornithine

The necessity of intercellular trafcking of citrulline in neural cells may arise if recycling of citrulline to arginine in a citrullineNO cycle is spatially separated from the generation of NO (Wiesinger, 2001). All neural cell populations appear to have the capacity to take up citrulline, however, not by a specic transport system of its own, but with the help of the transporter classied physiologically as sodiumindependent system L for large neutral amino acids (Schmidlin et al., 2000). System L can operate in both directions across the plasma membrane; thus a prerequisite of an intercellular citrullineNO cycle is indeed given. However, effective utilization of extracellular citrulline depends on the concentration of all neutral amino acids present, which holds in particular for a possible neural uptake of citrulline from the blood. Mitochondrial transport of citrulline (Palmieri, 2004) is not relevant for brain cells that do not express the full urea cycle (see > Sect. 3.1.1). Data on transport of ornithine across the plasma membrane of neural cells are lacking. However, it is highly probable that ornithine is accepted by system y since it is also a cationic amino acid and indeed inhibits uptake of arginine into neural cells (Schmidlin and Wiesinger, 1994). Since y is present at the bloodbrain barrier and since plasma concentrations of arginine and ornithine differ by less than an order of magnitude supply of neural ornithine by uptake from the periphery has to be considered. Mitochondrial transport of ornithine may be restricted to the liver with its fully expressed urea cycle (Begum et al., 2002).

Physiology and Pathophysiology

4.1 Arginine
Arginine is an amino acid with widespread roles in metabolism and the regulation of physiological functions. Nutritional and therapeutic aspects of arginine are under active investigation, and endocrine activity (Jun and Wennmalm, 1994), effects on wound healing (Witte and Barbul, 2003), modulation of the ger and BodeBo ger, 2001; immune system (Bansal and Ochoa, 2003), and regulation of vascular tone (Bo Ignarro and Napoli, 2004) have been reviewed. Since arginine is the only nitrogencontaining substrate of any NOS isoform, effects of arginine in vivo or in cell and tissue culture may reect the effect of the amino acid on the generation of NO, and modulation of NO synthesis by the availability of arginine is well established (Hallemeesch et al., 2002). Indeed, NMDA receptorstimulated generation of NO in arginine deprived brain slices was dependent on refeeding of arginine (Garthwaite et al., 1989), and systemic as well as central administration of arginine enhanced NO production in the rat brain (Salter et al., 1996; Yamada et al., 1997). Neuroprotective effects of arginine have been reported for traumatic brain injury (Liu et al., 2002; Cherian et al., 2003) or for cerebral ischemia (Temiz et al., 2003); in contrast, arginine worsened ischemic damage when given with a delayed time frame (Zhao et al., 2003). In all cases the effect of arginine on the generation of NO has been implicated. For more information on the roles of NO in the physiology and pathophysiology of the nervous system, the reader is referred to reviews (Garthwaite and Boulton, 1995; Bredt, 1999; Prast and Philippu, 2001) and respective chapters in this handbook. Arginine as substrate for neural NO synthesis has been reviewed (Wiesinger, 2001). Some pathophysiological consequences of arginine metabolism have already been mentioned in the previous paragraphs. A particular role for astrocytes in neural arginine metabolism has been proposed (Wiesinger, 2001). Release of arginine from astrocytes upon stimulation by glutamate or peroxynitrite (Grima et al., 1997; VegaAgapito et al., 2002) ts well into the picture of astrocytes as storage compartments of arginine

Arginine, citrulline, and ornithine

109

possibly for the benet of surrounding neurons. The fact that exogenous arginine enhances NOSmediated NO production, although the concentration of intracellular arginine is saturating for the enzyme was termed the arginine paradox. Proximity of endothelial NOS and arginine transport system (McDonald et al., 1997) and relief of inhibition of NOS by endogenous inhibitors (Tsikas et al., 2000) have been invoked to explain the phenomenon. In astrocytes, availability of extracellular arginine regulates the expression level of inducible NOS2 by inuencing phosphorylation of eukaryotic initiation factor eIF2a; this may explain the arginine paradox in the case of NOS2 (Lee et al., 2003). Coupling of arginine levels to synthesis of NOS2 ensures that the enzyme is not expressed under conditions that favor its activity being directed toward generation of harmful superoxide. Neurotoxic guanidino compounds are derived from arginine by several mechanisms and accumulate during uremia, but also hyperargininemia (De Deyn et al., 2001; De Jonge et al., 2001). AGAT may generate guanidinopropionic and guanidinobutyric acid (see > Sect. 3.2.1). Formation of guanidinosuccinic acid may be a side reaction of AGAT, but also of ASS. In addition, nonenzymatic oxidation of argininosuccinate leads to guanidinosuccinic acid (Aoyagi et al., 2001), similar to a nonenzymatic formation of methylguanidine from creatinine and reactive oxygen species (Nakamura et al., 1991b). The guanidino compounds are associated with cognitive malfunction, behavioral decits, seizures and convulsions, and coma (Hiramatsu, 2003), and molecular mechanism:s are sought in the inhibition of Na, KATPase or NMDA receptor activation (DHooge et al., 1996; da Silva et al., 1999). Not the least arginine is a proteinogenic amino acid which is of particular interest when taking into account the high turnover of neuroactive peptides and proteins in the nervous system (see chapter in this handbook). It should also be remembered that proteinbound arginine is subject to posttranslational modication, and that citrulline and methylated arginines liberated during proteolysis can exert physiological functions and enter their appropriate metabolic routes (see > Sect. 3.1.2). Arginine is involved in protein degradation since it tags unstable polypeptides that are degraded further by the ubiquitin proteasome pathway (Bohley et al., 1991); thus one may even draw a link to the hypothesis of neurodegeneration and cell death being caused by proteasomal dysfunction (Halliwell, 2002). As a precursor of urea, arginine has a central function in the hepatic detoxication of peripheral ammonia derived from amino acid and nucleotide catabolism. Therefore, acute or chronic liver failure as well as inborn errors of metabolism affecting the urea cycle enzymes lead to hyperammonemia with severe pathophysiological consequences, including mental retardation (Shih, 1978; Butterworth, 2003; see chapters in this handbook). Neurochemical consequences of hypoargininemia can be studied in an OTC decient mouse model (Qureshi and Rao, 1997). Since the OTC gene is located on the Xchromosome, affected male infants die soon after birth due to hyperammonemic coma. Chronic hyperammonemia caused by OTCdeciency results in behavioral abnormalities and encephalopathy already in the young. Citrullinemia may be caused by a deciency in ASS (Patejunas et al., 1994) and in this case is manifest right after birth (neonatal type I citrullinemia). That adultonset type II citrullinemia is resulting from a defect of the gene for the mitochondrial transport protein for citrulline (Kobayashi et al., 1999; Saheki and Kobayashi, 2002) has been questioned (Sinasac et al., 2004). Nevertheless, citrullinemia patients suffer from disturbed consciousness and coma and die with cerebral edema within a few years of onset. Hyperargininemia due to arginase deciency is associated with elevated levels of arginine in the CNS and leads to mental retardation (Shih, 1978).

4.2 Citrulline and Ornithine

Since citrulline is a precursor of arginine in the citrullineNO cycle, biochemistry and physiology of the nonproteinogenic amino acid: are related to the generation of NO. However, extracellular citrulline cannot maintain maximal rates of NO synthesis, e.g., in cultured astrocytes (Schmidlin and Wiesinger, 1998), because it competes with other amino acids for the uptake system (see > Sect. 3.3.2) or cannot enter easily the intracellular NOgenerating compartment (Wiesinger, 2001). Ornithinederived putrescine is the precursor of the polyamines spermidine and spermine, and, therefore, activity of ODC (see > Sect. 3.2.2) is pivotal for proper cell proliferation in the developing

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5
5

Arginine, citrulline, and ornithine

nervous system. In addition, ndings on polyamines as modulators of ion channels (Williams, 1997) shed ller, 1999). Other compounds genernew light on neural decarboxylation of ornithine (Bernstein and Mu ated in pathways starting from ornithine are the amino acids proline and glutamate. A physiological signicance of ornithinederived neural glutamate, which ultimately may be decarboxylated to the inhibitory transmitter GABA, is far from being clear (Seiler and DauneAnglard, 1993).

Concluding Remarks

It has become evident that also in the nervous system, arginine is one of the most versatile amino acids (Wu and Morris, 1998). Since arginine is metabolically tied to citrulline and ornithine, the dynamic interrelationship between the three amino acids determines the physiological roles of any of them. Interest in synthesis, degradation, and transport of the compounds has been stimulated by the discovery of arginine dependent NO synthesis in virtually every mammalian organ. Targeted pharmacological manipulation of a complex metabolic network for treatment of disease is a challenge for future studies, not the least in the nervous system.

Acknowledgments
bingen, for critical reading of the manuscript and for preparation of the The author thanks J. Hullmann, Tu gures.

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