Mild stress induces brain region-specific alterations of selective ER stress markers' mRNA expression in Wfs1-deficient mice.

In this work, the effect of mild stress (elevated plus maze test, EPM) on the expression of endoplasmic reticulum (ER) stress markers in different brain areas of wild type (WT) and Wfs1-deficient (Wfs1KO) mice was investigated. The following ER stress markers were studied: activating transcription factor 6α (Atf6α), protein kinase-like ER kinase (Perk), X-box binding protein 1 (Xbp1) and its spliced form (Xbp1s), 78-kilodalton glucose regulated protein (Grp78), 94-kilodalton glucose regulated protein (Grp94), C/EBP homologous protein (Chop). Wfs1KO and WT mice, not exposed to EPM, had similar patterns of ER stress markers in the studied brain areas. The exploratory activity of Wfs1KO mice in the EPM was inhibited compared to WT mice, probably reflecting increased anxiety in genetically modified mice. In response to the EPM, activation of inositol-requiring transmembrane kinase and endonuclease 1α (Ire1α) ER stress pathway was seen in both genotypes, but in different brain areas. Such a brain region-specific Ire1α activation was linked with dominant behavioural trends in these mice as more anxious, neophobic Wfs1KO mice had increased ER stress markers expression in the temporal lobe, the brain region related to anxiety, and more curious WT mice had ER stress markers increased in the ventral striatum which is related to the exploratory drive. The molecular mechanism triggering respective changes in ER stress markers in these brain regions is likely related to altered levels of monoamine neurotransmitters (serotonin, dopamine) in Wfs1KO mice.

Inflammatory, cardio-metabolic and diabetic profiling of chronic schizophrenia.

BACKGROUND: There is a growing interest in low-grade inflammatory and metabolic alterations in patients with chronic schizophrenia (SCH). METHODS: Inflammatory (tumor-necrosis factor-α [TNF-α], interferon-γ [IFN-γ], interleukins [IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10], monocyte chemo-attractant protein-1 [MCP-1]) and growth factors (vascular endothelial growth factor [VEGF], epidermal growth factor [EGF]) were measured in blood serum samples of 105 SCH patients and 148 control subjects (CS). Simultaneously the clinical biomarkers (C-reactive protein [CRP], triglycerides [TG], low-density lipoprotein [LDL-c] and high-density lipoprotein [HDL-c] cholesterol, glycated hemoglobin [HbA1c]) were measured, and body mass index (BMI) was calculated for patients. RESULTS: Several cyto-/chemokines (IFN-γ, MCP-1, IL-2, IL-6, IL-8 and IL-10) were significantly (P<0.0000001) elevated in SCH patients compared to CS. Odds ratios, obtained from logistic regression analyses, were significantly elevated for IL-2, IL-6, IL-10, INF-γ, and decreased for TNF-α in SCH group. Among the patients, higher IL-2, IL-6, INF-γ and lower MCP-1 levels as well as male gender were together significant (P<0.000001) predictors of higher HbA1c levels, and TG/HDL-c parameter was associated with ratios of INF-γ/IL-10 (P=0.004), and INF-γ/IL-4 (P=0.049), HbA1c (P=0.005), INF-γ (P=0.009), as well as LDL-c (P=0.02) levels. CONCLUSIONS: IL-2, IL-6, IL-10 and IFN-γ were the most significant SCH-related markers among the measured cytokines in our patient group. Furthermore, significant associations between pro-/anti-inflammatory imbalance and HbA1c as well as cardio-metabolic risk marker (TG/HDL-c) were observed, indicating higher risks of diabetes and cardiovascular diseases among SCH patients.

Cortical thickness and surface area correlates with cognitive dysfunction among first-episode psychosis patients.

BACKGROUND: In studies using magnetic resonance imaging (MRI), some have reported specific brain structure-function relationships among first-episode psychosis (FEP) patients, but findings are inconsistent. We aimed to localize the brain regions where cortical thickness (CTh) and surface area (cortical area; CA) relate to neurocognition, by performing an MRI on participants and measuring their neurocognitive performance using the Cambridge Neuropsychological Test Automated Battery (CANTAB), in order to investigate any significant differences between FEP patients and control subjects (CS). METHOD: Exploration of potential correlations between specific cognitive functions and brain structure was performed using CANTAB computer-based neurocognitive testing and a vertex-by-vertex whole-brain MRI analysis of 63 FEP patients and 30 CS. RESULTS: Significant correlations were found between cortical parameters in the frontal, temporal, cingular and occipital brain regions and performance in set-shifting, working memory manipulation, strategy usage and sustained attention tests. These correlations were significantly dissimilar between FEP patients and CS. CONCLUSIONS: Significant correlations between CTh and CA with neurocognitive performance were localized in brain areas known to be involved in cognition. The results also suggested a disrupted structure-function relationship in FEP patients compared with CS.

Wfs1-deficient animals have brain-region-specific changes of Na+, K+-ATPase activity and mRNA expression of α1 and ß1 subunits.

Mutations in the WFS1 gene, which encodes the endoplasmic reticulum (ER) glycoprotein, cause Wolfram syndrome, a disease characterized by juvenile-onset diabetes mellitus, optic atrophy, deafness, and different psychiatric abnormalities. Loss of neuronal cells and pancreatic ß-cells in Wolfram syndrome patients is probably related to the dysfunction of ER stress regulation, which leads to cell apoptosis. The present study shows that Wfs1-deficient mice have brain-region-specific changes in Na(+),K(+)-ATPase activity and in the expression of the α1 and ß1 subunits. We found a significant (1.6-fold) increase of Na-pump activity and ß1 subunit mRNA expression in mice lacking the Wfs1 gene in the temporal lobe compared with their wild-type littermates. By contrast, exposure of mice to the elevated plus maze (EPM) model of anxiety decreased Na-pump activity 1.3-fold in the midbrain and dorsal striatum and 2.0-fold in the ventral striatum of homozygous animals compared with the nonexposed group. Na-pump α1 -subunit mRNA was significantly decreased in the dorsal striatum and midbrain of Wfs1-deficient homozygous animals compared with wild-type littermates. In the temporal lobe, an increase in the activity of the Na-pump is probably related to increased anxiety established in Wfs1-deficient mice, whereas the blunted dopamine function in the forebrain of Wfs1-deficient mice may be associated with a decrease of Na-pump activity in the dorsal and ventral striatum and in the midbrain after exposure to the EPM.

Energy metabolism and thyroid function of mice with deleted wolframin (Wfs1) gene.

There is no data about the energy metabolism of patients with Wolfram syndrome caused by mutations in the wolframin (Wfs1) gene. The aim of this study was to investigate the role of Wfs1 in energy metabolism and thyroid function in Wfs1 deficient mice (Wfs1KO). 16 male (8 Wfs1KO, 8 wild type (wt)) and 16 female (8 Wfs1KO, 8wt) mice aged 11-13 weeks were studied alone in a specific metabolic cage for 48 h. Body weight, food, water and O2 consumption, motor activity, CO2 and heat production of mice were recorded. At the age of 14-20 weeks, plasma levels of thyroxine (T4), TSH and leptin were measured and histology of thyroid tissues examined. Mean CO2 and heat production was not different between the groups. Mean O2 consumption was higher in the Wfs1KO females compared to the Wfs1KO males (3 410.0±127.0 vs. 2 806.0±82.4 ml/kg/h; p<0.05), but not compared to the wt mice. The mean movement activity was not different between the groups except that the Wfs1KO females reared up more often than the wt females (199.8±63.46 vs. 39.26±24.71 cnts/48 h; p<0.05). Both male and female Wfs1KO mice had significantly lower body mass and food intake than wt mice. Male Wfs1KO mice also lost more weight in metabolic cage than wt males (20.43±0.41 vs. 16.07±0.86%; p<0.05) indicating more pronounced response to isolation. Male Wfs1KO mice had significantly lower levels of plasma leptin than wt male mice (3.37±0.40 vs. 5.82±0.71 ng/ml; p<0.01). Thyroid function measured by serum TSH and T4 levels was not different between Wfs1KO and wt groups, but both Wfs1KO and wt male mice had significantly higher mean T4 levels than female mice. The histology of thyroid tissue of Wfs1KO males showed a trend to a smaller mean number of epithelial cells per follicle than the wt male mice.Although Wfs1KO mice were smaller and lost more weight during the experiment, their energy metabolism was not different from wt mice except that the female Wfs1KO mice consumed more O2. As mice in this study were relatively young, longitudinal studies in older mice are necessary to clarify whether Wfs1 has a role in energy metabolism when the disease progresses further.

Whole Transcriptome Analysis (RNA Sequencing) of Peripheral Blood Mononuclear Cells of Vitiligo Patients.

Vitiligo is an idiopathic disorder characterized by depigmented patches on the skin due to a loss of melanocytes. The cause of melanocyte destruction is not fully understood. The aim of this study was to detect the potential pathways involved in the vitiligo pathogenesis to further understand the causes and entity of vitiligo. For that the transcriptome of peripheral blood mononuclear cells of 4 vitiligo patients and 4 control subjects was analyzed using the SOLiD System platform and whole transcriptome RNA sequencing application. Altogether 2,470 genes were expressed differently and GRID2IP showed the highest deviation in patients compared to controls. Using functional analysis, altogether 993 associations between the gene groups and diseases were found. The analysis revealed associations between vitiligo and diseases such as lichen planus, limb-girdle muscular dystrophy type 2B, and facioscapulohumeral muscular dystrophy. Additionally, the gene groups with an altered expression pattern are participating in processes such as cell death, survival and signaling, inflammation, and oxidative stress. In conclusion, vitiligo is rather a systemic than a local skin disease; the findings from an enormous amount of RNA sequencing data support the previous findings about vitiligo and should be further analyzed.

Associations between LSAMP gene polymorphisms and major depressive disorder and panic disorder.

The purpose of this case-control genetic association study was to explore potential relationships between polymorphisms in the limbic system-associated membrane protein (LSAMP) gene and mood and anxiety disorders. A total of 21 single-nucleotide polymorphisms (SNPs) from the LSAMP gene were analyzed in 591 unrelated patients with the diagnoses of major depressive disorder (MDD) or panic disorder (PD) and in 384 healthy control subjects. The results showed a strong association between LSAMP SNPs and MDD, and a suggestive association between LSAMP SNPs and PD. This is the first evidence of a possible role of LSAMP gene in mood and anxiety disorders in humans.

The antidepressant action of imipramine and venlafaxine involves suppression of nitric oxide synthesis.

Depressive disorders represent a major public health problem worldwide. The limitations of current antidepressant drugs have warranted on-going research to identify pharmacological agents and strategies that offer a greater therapeutic efficacy. The NMDA/L-arginine nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) cascade is an important signaling pathway that is also implicated in the regulation of depression. In animal models detecting antidepressant activity, distinct NO synthase inhibitors display antidepressant-like action. Therefore, the aim of current study was to evaluate whether pretreatment with L-arginine (precursor of NO) could counteract antidepressant-like effects of distinct antidepressant classes in the mouse forced swimming test (FST), and whether these drugs are able to modulate the nitric oxide synthesis in the brain. We found in the FST that pretreatment with L-arginine (500 mg/kg) counteracted the antidepressant-like effect of imipramine (IMI, 15 mg/kg) and venlafaxine (VENL, 6 mg/kg), but not the effects of bupropion (BUPR, 20mg/kg) or fluoxetine (FLX, 20mg/kg). Increasing the dose of L-Arg to 1000 mg/kg attenuated the antidepressant-like effects of BUPR, but did not modify the action of FLX. L-Arginine was devoid of any locomotor effects on the animals. The effect of antidepressants on brain NO metabolism paralleled their behavioral action in case of IMI and VENL which decreased the nitrite+nitrate concentration in the brain. BUPR and FLX did not have any effect on brain nitrite+nitrate concentration. These results support the idea that some antidepressants are able to inhibit nitric oxide synthesis in the brain, an effect which could be mechanistically related to the ability of L-arginine to counteract their antidepressant-like effects.

Sex differences in the development of diabetes in mice with deleted wolframin (Wfs1) gene.

Wolfram syndrome, caused by mutations in the wolframin (Wfs1) gene, is characterised by juvenile-onset diabetes mellitus, progressive optic atrophy, diabetes insipidus and deafness. Diabetes tend to start earlier in boys. This study investigated sex differences in longitudinal changes in blood glucose concentration (BGC) in wolframin-deficient mice (Wfs1KO) and compared their plasma proinsulin and insulin levels with those of wild-type (wt) mice. Non-fasting BGC was measured weekly in 42 (21 males) mice from both groups at nine weeks of age. An intraperitoneal glucose tolerance test (IPGTT) was conducted at the 30 (th) week and plasma insulin, c-peptide and proinsulin levels were measured at the 32 (nd) week. At the 32 (nd) week, Wfs1KO males had increased BGC compared to wt males (9.40±0.60 mmol/l vs. 7.91±0.20 mmol/l; p<0.05). The opposite tendency was seen in females. Both male and female Wfs1KO mice had impaired glucose tolerance on IPGTT. Wfs1KO males had significantly lower mean plasma insulin levels than wt males (57.78±1.80 ng/ml vs. 69.42±3.06 ng/ml; p<0.01) and Wfs1KO females (70.30±4.42 ng/ml; p<0.05). Wfs1KO males had a higher proinsulin/insulin ratio than wt males (0.09±0.02 vs. 0.05±0.01; p=0.05) and Wfs1KO females (0.04±0.01; p<0.05). Plasma c-peptide levels in males were lower in Wfs1KO males (mean 55.3±14.0 pg/ml vs. 112.7±21.9 pg/ml; p<0.05). Male Wfs1KO mice had a greater risk of developing diabetes than female Wfs1KO mice. Low plasma insulin concentration with an increased proinsulin/insulin ratio in Wfs1KO males indicates possible disturbances in converting proinsulin to insulin which in long-term may lead to insulin deficiency. Further investigation is needed to clarify the mechanism for the sex differences in the development of diabetes in Wolfram syndrome.

Temperature dependence of the sodium pump is altered in the cerebral cortex of CCK2 receptor-deficient mice.

Previously we have shown that the temperature dependence of the sodium pump (Na(+),K(+)-ATPase) is altered under different neuropathological conditions. In this study we compared temperature dependence of the Na(+),K(+)-ATPase in the fronto-parietal cortex of CCK(2) receptor-deficient (homo- and heterozygous) and normal (wild-type) mice. The Arrhenius plot for Na(+),K(+)-ATPase from wild-type brain is non-linear with a breakpoint at 20.3 +/- 0.4 degrees C. In case of the brain cell membrane of CCK(2) receptor-deficient mice (homo- and heterozygous) the breakpoint on Arrhenius plot was detected at 26.0 +/- 1.1 degrees C and 25.4 +/- 0.4 degrees C, respectively. The shift of the breakpoint on the Arrhenius plot established in CCK(2) receptor-deficiency as well as in case of some other pathological conditions confirms that such kind of alteration in the Na(+),K(+)-ATPase temperature dependence is likely related to the homeostatic adjustment of altered function of the sodium pump.

Relation of exploratory behaviour to plasma corticosterone and Wfs1 gene expression in Wistar rats.

Male Wistar rats exhibit significant variations in exploratory behaviour in the elevated plus-maze (EPM) model of anxiety. We have now investigated the relation between exploratory behaviour and levels of corticosterone and systemic oxidative stress. Also, the expression levels of endocannabinoid-related and wolframin (Wfs1) genes were measured in the forebrain structures. The rats were divided into high, intermediate and low exploratory activity groups. Exposure to EPM significantly elevated the serum levels of corticosterone in all rats, but especially in the high exploratory group. Oxidative stress indices and expression of endocannabinoid-related genes were not significantly affected by exposure to EPM. Wfs1 mRNA level was highly dependent on exploratory behaviour of animals. In low exploratory activity rats, Wfs1 gene expression was reduced in the temporal lobe, whereas in high exploratory activity group it was reduced in the mesolimbic area and hippocampus. Altogether, present study indicates that in high exploratory activity rats, the activation of brain areas related to novelty seeking is apparent, whereas in low exploratory activity group the brain structures linked to anxiety are activated.

Wfs1 gene deletion causes growth retardation in mice and interferes with the growth hormone pathway.

The aim of present study was to describe changes in gene expression in the temporal lobe of mice induced by deletion of the Wfs1 gene. Temporal lobes samples were analyzed using Affymetrix Mouse Genome 420 2 GeneChips and expression profiles were functionally annotated with GSEA and Ingenuity Pathway Analysis. We found that Wfs1 mutant mice are significantly smaller (20.9 +/- 1.6 g) than their wild-type counterparts (31.0 +/- 0.6 g, P < 0.0001). This difference existed in 129S6 and C57B6 backgrounds. Interestingly, microarray analysis identified upregulation of growth hormone (GH) transcripts and functional analysis revealed activation of GH pathways. In line with microarray data, the level of IGF-1 in the plasma of Wfs1 mutant mice was significantly increased (P < 0.05). Thus, Wfs1 deletion induces growth retardation, whereas the GH pathway is activated. To test the interaction between the Wfs1 deletion and genomic background, mutant mice were backcrossed to two different genetic backgrounds. In line with previous studies, an interaction between a gene knockout and genetic background was found in gene expression profiles in the congenic region. However, genetic background did not alter the effect of the Wfs1 mutation on either body weight or GH pathway activation. Further studies are needed to describe biochemical and molecular changes of the growth hormone axis as well as in other hormones to clarify their role in growth retardation in the Wfs1 mutant mice.

Gene expression study of IL10 family genes in vitiligo skin biopsies, peripheral blood mononuclear cells and sera.

BACKGROUND: Vitiligo is a pigmentation disorder, the cause of which is complex and not yet fully understood. There is a significant change of epidermal cytokines in involved skin of patients with vitiligo compared with uninvolved skin and skin of healthy controls, thus suggesting a possible involvement of cytokines in the pathogenesis of vitiligo. OBJECTIVES: To evaluate potential roles of IL10 family cytokines (IL10, IL19, IL20, IL22 and IL24) in vitiligo. Along with the selected cytokines, we investigated subunits of the receptors (IL10RA, IL10RB, IL20RA and IL22RA1) which are involved in the signalling pathway of the cytokines. METHODS: Quantitative real-time polymerase chain reaction was used to detect mRNA expression levels in samples extracted from skin biopsies and peripheral blood mononuclear cells and an enzyme-linked immunosorbent assay was used to measure protein concentrations in serum from patients with vitiligo and healthy controls. RESULTS: IL22 is significantly associated with vitiligo, especially with the active stage of vitiligo, as shown by results of mRNA expression and supported by results of protein level in sera. IL22 may provoke inflammation which leads to destruction of melanocytes. CONCLUSIONS: The actual role of IL22 during pathogenesis of vitiligo remains to be better characterized. Signal transductions of other investigated cytokines seem to be regulated on the expression level of their receptor complex subunits.

Alpha-synuclein A30P point-mutation generates age-dependent nigrostriatal deficiency in mice.

Lewy bodies are mainly composed of alpha-synuclein (SNCA) and specific mutations in SNCA gene are related to familial forms of Parkinson's disease (PD). The purpose of our study was to generate a mouse line with A30P knock-in point mutation in SNCA gene and to test if a single point-mutation is able to turn otherwise normal SNCA into a toxic form. The behavioral profile of SNCA A30P mice was followed for 16 months. Generally, these mice are healthy and viable without any obvious abnormalities. Starting from the age of 13 months mice developed a significant deficit in motor performance tests related to nigrostriatal function (ink-test and beam walk). In other tests (motility boxes, rotarod) mice continuously performed normally. Moreover, SNCA A30P mice expressed the altered sensitivity to VMAT2 inhibitor reserpine, possibly reflecting a functional deficiency of dopamine. Indeed, mice at 15 months of age had significantly reduced levels of dopamine and its major metabolite DOPAC in the striatum, and reduced levels of dopamine in the mesolimbic system. The present study confirms that SNCA plays an important role in the development of PD and an insertion of a single point mutation is sufficient to generate age-related decline in specific motor performance. The generated mouse line has a potential to become a model for PD with comparable time course and phenotype.

Association analysis of IL20RA and IL20RB genes in psoriasis.

The interleukin-20-receptor I complex (IL-20-RI) is composed of two chains, IL20RA and IL20RB. Its ligands are the three members of the IL19 subfamily of cytokines, IL-19, IL-20 and IL-24. These cytokines are important in the manifestation of psoriatic lesions and, recently, an association of polymorphisms of IL20 with psoriasis has been described. In the present study we tested the hypotheses that genetic variations of the IL-20-RI influence susceptibility to psoriasis and investigated single nucleotide polymorphisms (SNPs) in the IL20RA and IL20RB genes in psoriasis patients (n=254) and healthy controls (n=224). We found no association of any of the investigated SNPs with the disease. Analysis of pairwise linkage disequilibrium (LD) across studied markers revealed a strong level of LD between SNPs within the IL20RA gene and SNPs within the IL20RB gene, and, for both genes six common haplotypes were identified with an estimated frequency >or=1%. Haplotype analyses suggested that the IL20RA haplotype CCG (rs1184860, rs1167846, rs1167849) is significantly associated with psoriasis (OR 3.14, 95% CI 1.61-6.14), whereas the TTG haplotype had a protective effect (OR 0.20, 95% CI 0.07-0.55). The risk haplotype defining SNPs 1167846 and 1184860 were found to modify paired box 5 and homeobox A9 sites, respectively, two transcription factors related to the differentiation of immune cells. Further studies are needed to confirm the genetic association and to investigate the functional relevance of IL20RA haplotypes in psoriasis.

Interpretation of knockout experiments: the congenic footprint.

In gene targeting experiments, the importance of genetic background is now widely appreciated, and knockout alleles are routinely backcrossed onto a standard inbred background. This produces a congenic strain with a substantial segment of embryonic stem (ES)-cell-derived chromosome still flanking the knockout allele, a phenomenon often neglected in knockout studies. In cholecystokynin 2 (Cckbr) knockout mice backcrossed with C57BL/6, we have found a clear 'congenic footprint' of expression differences in at least 10 genes across 40 Mb sequence flanking the Cckbr locus, each of which is potentially responsible for aspects of the 'knockout' phenotype. The expression differences are overwhelmingly in the knockout-low direction, which may point to a general phenomenon of background dependence. This finding emphasizes the need for caution in using gene knockouts to attribute phenotypic effects to genes. This is especially the case when the gene is of unknown function or the phenotype is unexpected, and is a particular concern for large-scale knockout and phenotypic screening programmes. However, the impact of genetic background should not be simply viewed as a potential confound, but as a unique opportunity to study the broader responses of a system to a specific (genetic) perturbation.

Association analysis of IL19, IL20 and IL24 genes in palmoplantar pustulosis.

BACKGROUND: Interleukin (IL) 19, IL-20 and IL-24 belong to the IL-10 cytokine family and have been identified to play a role in the regulation of epidermal functions and in inflammation. The genes encoding IL-19, IL-20 and IL-24 are located within a gene cluster on chromosome 1q31-32 and carry frequent genetic variations. OBJECTIVES: This study investigated whether variations in the IL19, IL20 and IL24 genes that have previously been associated with plaque-type psoriasis may also play a role in palmoplantar pustulosis (PPP). PATIENTS: Fifteen polymorphisms were analysed in 43 patients with PPP and in 149 healthy control subjects. RESULTS: The rare allele of IL20 1380 A-->G (rs2981573) was less frequent in patients with PPP compared with healthy controls (OR 1 x 95, 95% CI 1 x 00-3 x 79). Haplotype analyses of IL19 and IL20 suggested an increased risk for PPP associated with IL20 haplotype GAA (OR 2 x 39, 95% CI 1 x 17-4 x 86) and a reduced risk for PPP associated both with IL19 haplotype GATGATA (OR 0 x 41, 95% CI 0 x 16-1 x 05) and IL20 haplotype GGG (OR 0 x 48, 95% CI 0 x 23-0 x 98). Extended haplotype analysis revealed an association of IL19/IL20 haplotype GACACCGGAA with a higher risk for PPP (OR 2 x 31, 95% CI 1 x 05-5 x 10) and of IL20/IL24 haplotype CAAAC with a reduced risk for PPP (OR 0 x 12, 95% CI 0 x 02-0 x 82). CONCLUSIONS: This exploratory study supports the hypothesis that variations of genes of the IL-19 subfamily of cytokines influence susceptibility to PPP. However, due to the limited size of the study samples, this current concept should be considered as preliminary and the results need to be confirmed in future independent studies.

High-performance liquid chromatography method with radiochemical detection for measurement of nitric oxide synthase, arginase, and arginine decarboxylase activities.

Nitric oxide has been shown to be involved in numerous biological processes, and many studies have aimed to measure nitric oxide synthase (NOS) activity. Recently, it has been demonstrated that arginase and arginine decarboxylase (ADC), two enzymes that also employ arginine as a substrate, may regulate NOS activity. We aimed to develop a HPLC-based method to measure simultaneously the products of these three enzymes. Traditionally, the separation of amino acids and related compounds with HPLC has been carried out with precolumn derivatization and reverse phase chromatography. We describe here a simple and fast HPLC method with radiochemical detection to separate radiolabeled L-arginine, L-citrulline, L-ornithine, and agmatine. 3H-labeled L-arginine, L-citrulline, agmatine, and 14C-labeled L-citrulline were used as standards. These compounds were separated in the normal phase column (Allure Acidix 250 x 4.6 mm i.d.) under isocratic conditions in less than 20 min with good sensitivity. Using the current method, we have shown the formation of L-citrulline and L-ornithine in vitro using brain tissue homogenate of rats and that of agmatine by Escherichia coli ADC.