Oxidized Lipoproteins Suppress Nitric Oxide Synthase in Macrophages: Study of Glucocorticoid Receptor Involvement

Activated cholesterol-laden macrophages in atherosclerotic lesions are believed to influence the progression of this disease. The induction of nitric oxide synthase (iNOS) activity was investigated in control and cholesterol-laden J774 macrophages, obtained by pre-incubation with oxidized or acetylated low density lipoproteins (oxLDL, acLDL). Loading with oxLDL caused a small induction of NOS activity in unstimulated cells, as indicated by nitrite and citrulline accumulation in the supernatant. However, it suppressed the iNOS activity resulting from stimulation of the cells with lipopolysaccharide with or without interferon-γ. AcLDL had no inhibitory effect, indicating that cholesterol accumulation as such was not responsible. Since the induction of NOS in macrophages is inhibited by glucocorticoids, the possibility that a glucocorticoid-like factor, formed during oxidation of LDL, may cause the inhibition, was investigated. However, addition of the glucocorticoid receptor antagonist mifepristone did not prevent the oxLDL-dependent NOS inhibition, indicating that the glucocorticoid receptor is not involved in the suppressive effect of oxLDL

Liver Transplantation Prevents Progressive Neurological Impairment in Argininemia

Argininemia is a rare hereditary disease due to a deficiency of hepatic arginase, which is the last enzyme of the urea cycle and hydrolyzes arginine to ornithine and urea. The onset of the disease is usually in childhood, and clinical manifestations include progressive spastic paraparesis and mental retardation. Liver involvement is less frequent and usually not as severe as observed in other UCDs. For this reason, and because usually there is a major neurological disease at diagnosis, patients with argininemia are rarely considered as candidates for OLT despite its capacity to replace the deficient enzyme by an active one. We report on long-term follow-up of two patients with argininemia. Patient 1 was diagnosed by the age of 20 months and despite appropriate conventional treatment progressed to spastic paraparesis with marked limp. OLT was performed at 10 years of age with normalization of plasmatic arginine levels and guanidino compounds. Ten years post-OLT, under free diet, there is no progression of neurological lesions. The second patient (previously reported by our group) was diagnosed at 2 months of age, during a neonatal cholestasis workup study. OLT was performed at the age of 7 years, due to liver cirrhosis with portal hypertension, in the absence of neurological lesions and an almost-normal brain MRI. After OLT, under free diet, there was normalization of plasmatic arginine levels and guanidino compounds. Twelve years post-OLT, she presents a normal neurological examination. We conclude that OLT prevents progressive neurological impairment in argininemia and should be considered when appropriate conventional treatment fails

γ-Aminobutyric Acid Transporter 2 Mediates the Hepatic Uptake of Guanidinoacetate, the Creatine Biosynthetic Precursor, in Rats

Guanidinoacetic acid (GAA) is the biosynthetic precursor of creatine which is involved in storage and transmission of phosphate-bound energy. Hepatocytes readily convert GAA to creatine, raising the possibility that the active uptake of GAA by hepatocytes is a regulatory factor. The purpose of this study is to investigate and identify the transporter responsible for GAA uptake by hepatocytes. The characteristics of [14C]GAA uptake by hepatocytes were elucidated using the in vivo liver uptake method, freshly isolated rat hepatocytes, an expression system of Xenopus laevis oocytes, gene knockdown, and an immunohistochemical technique. In vivo injection of [14C]GAA into the rat femoral vein and portal vein results in the rapid uptake of [14C]GAA by the liver. The uptake was markedly inhibited by γ-aminobutyric acid (GABA) and nipecotinic acid, an inhibitor of GABA transporters (GATs). The characteristics of Na+- and Cl−-dependent [14C]GAA uptake by freshly isolated rat hepatocytes were consistent with those of GAT2. The Km value of the GAA uptake (134 µM) was close to that of GAT2-mediated GAA transport (78.9 µM). GABA caused a marked inhibition with an IC50 value of 8.81 µM. The [14C]GAA uptake exhibited a significant reduction corresponding to the reduction in GAT2 protein expression. GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. This distribution pattern was consistent with that of the creatine biosynthetic enzyme, S-adenosylmethionine∶guanidinoacetate N-methyltransferase. GAT2 makes a major contribution to the sinusoidal GAA uptake by periportal hepatocytes, thus regulating creatine biosynthesis in the liver

Transport characteristics of guanidino compounds at the blood-brain barrier and blood-cerebrospinal fluid barrier: relevance to neural disorders

Guanidino compounds (GCs), such as creatine, phosphocreatine, guanidinoacetic acid, creatinine, methylguanidine, guanidinosuccinic acid, γ-guanidinobutyric acid, β-guanidinopropionic acid, guanidinoethane sulfonic acid and α-guanidinoglutaric acid, are present in the mammalian brain. Although creatine and phosphocreatine play important roles in energy homeostasis in the brain, accumulation of GCs may induce epileptic discharges and convulsions. This review focuses on how physiologically important and/or neurotoxic GCs are distributed in the brain under physiological and pathological conditions. Transporters for GCs at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier (BCSFB) have emerged as substantial contributors to GCs distribution in the brain. Creatine transporter (CRT/solute carrier (SLC) 6A8) expressed at the BBB regulates creatine concentration in the brain, and represents a major pathway for supply of creatine from the circulating blood to the brain. CRT may be a key factor facilitating blood-to-brain guanidinoacetate transport in patients deficient in S-adenosylmethionine:guanidinoacetate N-methyltransferase, the creatine biosynthetic enzyme, resulting in cerebral accumulation of guanidinoacetate. CRT, taurine transporter (TauT/SLC6A6) and organic cation transporter (OCT3/SLC22A3) expressed at the BCSFB are involved in guanidinoacetic acid or creatinine efflux transport from CSF. Interestingly, BBB efflux transport of GCs, including guanidinoacetate and creatinine, is negligible, though the BBB has a variety of efflux transport systems for synthetic precursors of GCs, such as amino acids and neurotransmitters. Instead, the BCSFB functions as a major cerebral clearance system for GCs. In conclusion, transport of GCs at the BBB and BCSFB appears to be the key determinant of the cerebral levels of GCs, and changes in the transport characteristics may cause the abnormal distribution of GCs in the brain seen in patients with certain neurological disorders

Nuclear Magnetic Resonance metabolomics reveals an excretory metabolic signature of renal cell carcinoma

RCC usually develops and progresses asymptomatically and, when detected, it is frequently at advanced stages and metastatic, entailing a dismal prognosis. Therefore, there is an obvious demand for new strategies enabling an earlier diagnosis. The importance of metabolic rearrangements for carcinogenesis unlocked a new approach for cancer research, catalyzing the increased use of metabolomics. The present study aimed the NMR metabolic profiling of RCC in urine samples from a cohort of RCC patients (n = 42) and controls (n = 49). The methodology entailed variable selection of the spectra in tandem with multivariate analysis and validation procedures. The retrieval of a disease signature was preceded by a systematic evaluation of the impacts of subject age, gender, BMI, and smoking habits. The impact of confounders on the urine metabolomics profile of this population is residual compared to that of RCC. A 32-metabolite/resonance signature descriptive of RCC was unveiled, successfully distinguishing RCC patients from controls in principal component analysis. This work demonstrates the value of a systematic metabolomics workflow for the identification of robust urinary metabolic biomarkers of RCC. Future studies should entail the validation of the 32-metabolite/resonance signature found for RCC in independent cohorts, as well as biological validation of the putative hypotheses advanced

Guanidino compounds in guanidinoacetate methyltransferase deficiency, a new inborn error of creatine synthesis

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Renal handling of guanidino compounds in rat and rabbit.

1. Guanidino compounds (GCs) have been quantified in different mammalian tissues such as brain, liver, muscle and kidney. The high anatomical heterogeneity of the kidney suggests that GCs could be unevenly distributed along the corticopapillary axis of the kidney in different species. 2. This study was designed to quantify twelve GCs in the different zones of rat and rabbit kidney. The kidneys were sliced and pieces of seven definite zones were weighed and homogenized for further GC extraction. GCs were determined by liquid chromatography. 3. The results indicate that: (1) GCs were unevenly distributed along rat and rabbit kidney; (2) qualitative and quantitative studies proved that each GC shows a particular distribution pattern along the corticopapillary axis for a given species; (3) in rats, alpha-keto-delta-guanidinovaleric acid, guanidinosuccinic acid, creatinine (CTN), methylguanidine and to a lesser extent gamma-guanidinobutyric acid increased steeply along the inner medulla in parallel to urea, whereas in rabbits, most of the GCs reached a plateau in the inner medulla and remained constant at this level; (4) gamma-guanidinobutyric acid was specifically found in the rat kidney; (5) argininic acid was higher in rabbit compared with rat kidney; (6) significantly higher levels of homoarginine were found in all zones of the rat kidney compared with the rabbit kidney. 4. The results suggest that: (1) GCs are mostly localized within the nephron segments; (2) an accumulation of GCs in the inner medulla might be explained either by a recycling process or by an intracellular storage as has been reported for urea, amino acids and organic osmolytes; (3) some GCs might be synthesized in nephron segments as reported for arginine (Arg) and guanidinoacetic acid (GAA); (4) several metabolic pathways of the GCs seemed to differ between rat and rabbit; (5) except for creatine, CTN, Arg and GAA, it seems unlikely that GCs might significantly increase the intracellular osmolality

Chemical models of epilepsy with some reference to their applicability in the development of anticonvulsants

This paper reviews chemical models of epilepsy and their relevance in the identification and characterization of anticonvulsants. For each convulsant we discuss possible modes of administration, clinical type(s) of seizures induced, proposed mechanism(s) of epileptogenesis and, where available, responsiveness of the induced seizures to anticonvulsants. The following compounds are reviewed: pentylenetetrazol, bicuculline, penicillin, picrotoxin, ,β-carbolines, 3-mercaptopropionic acid, hydrazides, allylglycine; the glycine antagonist strychnine; y-hydroxybutyrate; excitatory amino acids (glutamate, aspartate, N-methyl-D-aspartate, quisqualate, kainate, quinolinic acid); monosubstituted guanidino compounds, metals (alumina, cobalt, zinc, iron); neuropeptides (opioid peptides, corticotropin releasing factor, somatostatin, vasopressin); cholinergic agents (acetylcholine, acetylcholinesterase inhibitors, pilocarpine); tetanus toxin; flurothyl; folates; homocysteine and colchicine. Although there are a multitude of chemical models of epilepsy, only a limited number are applied in the routine screening of potential anticonvulsants. Some chemical models have a predictive value with regard to the clinical profile of efficacy of the tested anticonvulsants. Some chemical models may contribute to a better understanding of possible mechanisms of epileptogenesisstatus: publishe