Further studies of the effects of aging on arginine metabolites in the rat vestibular nucleus and cerebellum.

Some studies have demonstrated that aging is associated with impaired vestibular reflexes, especially otolithic reflexes, resulting in postural instability. However, the neurochemical basis of these age-related changes is still poorly understood. The l-arginine metabolic system has been implicated in changes in the brain associated with aging. In the current study, we examined the levels of l-arginine and its metabolizing enzymes and downstream metabolites in the vestibular nucleus complex (VNC) and cerebellum (CE) of rats with and without behavioral testing which were young (4months old), middle-aged (12months old) or aged (24months old). We found that aging was associated with lower nitric oxide synthase activity in the CE of animals with testing and increased arginase in the VNC and CE of animals with testing. l-citrulline and l-ornithine were lower in the VNC of aged animals irrespective of testing, while l-arginine and l-citrulline were lower in the CE with and without testing, respectively. In the VNC and CE, aging was associated with lower levels of glutamate in the VNC, irrespective of testing. In the VNC it was associated with higher levels of agmatine and putrescine, irrespective of testing. In the CE, aging was associated with higher levels of putrescine in animals without testing and with higher levels of spermine in animals with testing, and spermidine, irrespective of testing. Multivariate analyses indicated significant predictive relationships between the different variables, and there were correlations between some of the neurochemical variables and behavioral measurements. Cluster analyses revealed that aging altered the relationships between l-arginine and its metabolites. The results of this study demonstrate that there are major changes occurring in l-arginine metabolism in the VNC and CE as a result of age, as well as behavioral activity.

Altered brain arginine metabolism in schizophrenia.

Previous research implicates altered metabolism of l-arginine, a versatile amino acid with a number of bioactive metabolites, in the pathogenesis of schizophrenia. The present study, for we believe the first time, systematically compared the metabolic profile of l-arginine in the frontal cortex (Brodmann's area 8) obtained post-mortem from schizophrenic individuals and age- and gender-matched non-psychiatric controls (n=20 per group). The enzyme assays revealed no change in total nitric oxide synthase (NOS) activity, but significantly increased arginase activity in the schizophrenia group. Western blot showed reduced endothelial NOS protein expression and increased arginase II protein level in the disease group. High-performance liquid chromatography and liquid chromatography/mass spectrometric assays confirmed significantly reduced levels of γ-aminobutyric acid (GABA), but increased agmatine concentration and glutamate/GABA ratio in the schizophrenia cases. Regression analysis indicated positive correlations between arginase activity and the age of disease onset and between l-ornithine level and the duration of illness. Moreover, cluster analyses revealed that l-arginine and its main metabolites l-citrulline, l-ornithine and agmatine formed distinct groups, which were altered in the schizophrenia group. The present study provides further evidence of altered brain arginine metabolism in schizophrenia, which enhances our understanding of the pathogenesis of schizophrenia and may lead to the future development of novel preventions and/or therapeutics for the disease.

Regional variations and age-related changes in arginine metabolism in the rat brain stem and spinal cord.

Accumulating evidence suggests that the metabolism of l-arginine, a metabolically versatile amino acid, is critically involved in the aging process. The present study compared the activity and protein expression of nitric oxide synthase (NOS) and arginase, and the levels of l-arginine and its eight down-stream metabolites in the brain stem (pons and medulla) and the cervical spinal cord in 3- (young) and 22- (aged) month-old male Sprague-Dawley rats. Total NOS activity was significantly reduced with age in the spinal cord (but not brain stem), and there were no age-related changes in arginase activity in both regions. Western blot revealed decreased protein expression of endothelial NOS, but not neuronal NOS, with age in both regions. Furthermore, there were significantly decreased l-arginine, glutamate, GABA and spermine levels and increased putrescine and spermidine levels with age in both regions. Although the absolute concentrations of l-arginine and six metabolites were significantly different between the brain stem and spinal cord in both age groups, there were similar clusters between l-arginine and its three main metabolites (l-citrulline, l-ornithine and agmatine) in both regions, which changed as a function of age. These findings, for the first time, demonstrate the regional variations and age-related changes in arginine metabolism in the rat brain stem and spinal cord. Future research is required to understand the functional significance of these changes and the underlying mechanisms.

Ageing alters behavioural function and brain arginine metabolism in male Sprague-Dawley rats.

A growing body of evidence suggests the involvement of L-arginine and its metabolites in the ageing and neurodegenerative processes. The present study assessed behavioural performance in 4- (young), 12- (middle-aged) and 24- (aged) month-old male Sprague-Dawley rats, and investigated age-related changes in the activity of two key arginine metabolic enzymes, nitric oxide synthase (NOS) and arginase, and the levels of L-arginine and its downstream metabolites in a number of memory-related brain structures. Aged rats were less anxious and performed poorly in the water maze task relative to the young and middle-aged rats, and both middle-aged and aged rats displayed reduced exploratory activity relative to the young ones. There were significant age-related changes in NOS and arginase activities, and the levels of L-arginine, L-citrulline, L-ornithine, agmatine, putrescine, spermidine, spermine and glutamate, but not γ-aminobutyric acid, in the CA1, CA2/3 and dentate gyrus sub-regions of the hippocampus and the prefrontal, entorhinal, perirhinal, postrhinal and temporal (an auditory cortex) cortices in a region-specific manner. Cluster analyses revealed that the nine related neurochemical variables formed distinct groups, which changed as a function of ageing. Multiple regression analyses revealed a number of significant correlations between the neurochemical and behavioural variables. The present study further supports the involvement of arginine metabolism in the ageing process, and provides further evidence of the effects of animals' behavioural experience on arginine metabolism.

Agmatine selectively improves behavioural function in aged male Sprague-Dawley rats.

L-arginine, a semi-essential amino acid, can be metabolized to form a number of bioactive molecules. Nitric oxide (NO), generated by NO synthase (NOS) from L-arginine, has been strongly implicated in the aging process. Agmatine, decarboxylated arginine, regulates the production of NO and other metabolites of L-arginine, modulates behavioural function, and has anti-inflammatory and neuroprotective effects. The present study investigated whether agmatine supplementation could improve behavioural function in aged male Sprague-Dawley rats, and could attenuate age-related changes in NOS activity and protein expression in memory-related structures. Aged rats treated with saline displayed significantly reduced exploratory activity and impaired spatial reference and working memory and object recognition memory. Agmatine (40 mg/kg) administered intraperitoneally significantly improved spatial working memory and object recognition memory in aged rats, suppressed age-related elevation in total NOS activity, and restored endothelial NOS protein to the normal level. However, agmatine supplementation was unable to improve exploratory activity and spatial reference learning and memory in aged rats. These findings suggest that exogenous agmatine selectively improves behavioural function in aged rats under the present experimental condition, and merit future investigation of its therapeutic potential in cognitive decline during aging.

Aging affects L-arginine and its metabolites in memory-associated brain structures at the tissue and synaptoneurosome levels.

L-arginine, one of the most metabolically versatile amino acids, can be metabolized to form a number of bioactive molecules. The present study systematically investigated age-related changes in L-arginine and its metabolites in the hippocampus, parahippocampal region, and prefrontal cortex at the tissue (crude homogenates) and synaptoneurosome (a subcellular preparation enriched for synaptic material) levels. As aging leads to reduced water content in the brain, age-related changes in neurochemical levels in tissue homogenates normalized by wet tissue weight and protein level were compared. There were significant differences in L-arginine, L-citrulline, L-ornithine, agmatine, putrescine, spermidine, spermine, and glutamate, but not GABA, in the CA1, CA2/3, and dentate gyrus sub-regions of the hippocampus and the prefrontal, entorhinal, perirhinal, and postrhinal cortices in 24 (aged) and 4 (young) months old rats in a region-specific manner. The overall pattern of age-related changes in amino acids (L-arginine, L-citrulline, L-ornithine, glutamate, and GABA) was largely similar between homogenates and synaptoneurosomes, whereas the pattern for the amines (agmatine, putrescine, spermidine, and spermine) was quite different. Furthermore, the pattern of age-related changes in neurochemical levels in tissue homogenates normalized by wet tissue weight and protein level was very similar for all 9 neurochemicals measured. These findings suggest that there are differential effects of aging on L-arginine metabolism at the tissue and synaptoneurosome levels and that the way of data normalization (tissue weight vs. protein level) has no or very minor effects on 9 neurochemicals measured.

Behavioral effects of agmatine in naive rats are task- and delay-dependent.

The present study systematically investigated the effects of agmatine administered i.p. in several commonly used behavioral tasks. In Experiment 1, pre-test treatment of agmatine (1 and 40 mg/kg) appeared to improve animals' performance in the water maze probe test conducted 24 h, but not 120 s, after training, when the effect was evaluated within subjects. In Experiment 2, pre-test agmatine treatment (40 mg/kg) did not affect animals' performance in the open field, and the place navigation, probe tests (1-4 and 6), reversal test and cued navigation in the water maze, but significantly facilitated performance in probe 5 which was conducted 96 h after training. In Experiment 3, rats with pre-test agmatine treatment (40 mg/kg) were less anxious relative to the controls, with no performance changes in the open field. In the water maze task, post-training agmatine treatment (40 mg/kg) did not affect place and cued navigation, but significantly improved animals' performance in the probe test conducted 24 h after training and the reversal test. In the working memory version of the task, agmatine treated rats took significantly less time and generated markedly shorter path length to reach the platform at the 180 s, but not 30 s, delay relative to the controls. In the object recognition task, rats with pre-test agmatine treatment (40 mg/kg) spent significantly more time exploring displaced objects, but not novel object, as compared to the controls. In Experiment 4, pre-test agmatine treatment (40 mg/kg) had no effect on the task acquisition in the delayed non-match to position task in the T-maze, but significantly facilitated performance at the 600 s delay. These results suggest that the behavioral effects of agmatine are task- and delay-dependent, and agmatine facilitates memory particularly when the task difficulty is increased due to memory trace decay and/or greater interference.