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

Guanidino compounds that are increased in hyperargininemia inhibit GABA and glycine responses on mouse neurons in cell culture

The effects of arginine, homoarginine, [alpha]-keto-[delta]-guanidinovaleric acid and argininic acid (guanidino compounds that were found to be increased in hyperargininemia) were evaluated on responses to [gamma]-aminoburtyric acid (GABA) and glycine (Gly) on mouse neurons in primary dissociated cell culture. GABA and Gly were applied iontophoretically and intracellular microelectrode recording techniques were used.The guanidino compounds rapidly and reversibly inhibited both GABA and Gly responses. The guanidino compounds inhibited GABA responses in a concentration-dependent manner and inhibited Gly responses at a concentration of 10 mM. Argininic acid was the most potent in reducing inhibitory amino acid responses, followed in decreasing potency by [alpha]-keto-[delta]-guanidinovaleric acid, homoarginine and arginine. The guanidino compounds were equally potent in decreasing Gly and GABA responses. Co-application of CGS 9896, a benzodiazepine receptor antagonist, did not antagonize the guanidino compound-induced inhibition of GABA responses. These findings suggest that the guanidino compounds inhibited responses to the inhibitory neurotransmitters GABA and Gly by blocking the chloride channel. This effect might underlie the in vivo epileptogenicity of some of the guanidino compounds and might contribute to the pathogenesis of seizures in hyperargininemia.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29427/1/0000508.pd

Homeostatic Plasticity of Striatal Neurons Intrinsic Excitability following Dopamine Depletion

The striatum is the major input structure of basal ganglia and is involved in adaptive control of behaviour through the selection of relevant informations. Dopaminergic neurons that innervate striatum die in Parkinson disease, leading to inefficient adaptive behaviour. Neuronal activity of striatal medium spiny neurons (MSN) is modulated by dopamine receptors. Although dopamine signalling had received substantial attention, consequences of dopamine depletion on MSN intrinsic excitability remain unclear. Here we show, by performing perforated patch clamp recordings on brain slices, that dopamine depletion leads to an increase in MSN intrinsic excitability through the decrease of an inactivating A-type potassium current, IA. Despite the large decrease in their excitatory synaptic inputs determined by the decreased dendritic spines density and the increase in minimal current to evoke the first EPSP, this increase in intrinsic excitability resulted in an enhanced responsiveness to their remaining synapses, allowing them to fire similarly or more efficiently following input stimulation than in control condition. Therefore, this increase in intrinsic excitability through the regulation of IA represents a form of homeostatic plasticity allowing neurons to compensate for perturbations in synaptic transmission and to promote stability in firing. The present observations show that this homeostatic ability to maintain firing rates within functional range also occurs in pathological conditions, allowing stabilizing neural computation within affected neuronal networks

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

Extinction and renewal of conditioned behaviour : the effect of context and discrete cue

Classical conditioning concerns the paired presentation of a conditioned stimulus (CS) and an unconditioned stimulus (US, biologically significant event that elicits a certain behaviour). Because of the paired presentation of the CS and the US, an association between the CS and the US is formed that results in conditioned behaviour to the CS. A subsequent extinction procedure wherein the CS is presented in the absence of the US will gradually lead to the disappearance of the conditioned behaviour. However, research has shown that a context change after extinction can cause a return of conditioned behaviour. This post extinction phenomenon is referred to as renewal. Together with several other post extinction phenomena such as spontaneous recovery, reinstatement and rapid reacquisition, renewal demonstrates that extinction does not entail the unlearning of the CS-US association. Instead, extinction is now described as the learning of a new, inhibitory association between the CS and the US (CSnoUS). Extinction is viewed as an essential element in most treatments of anxiety and specific phobias. In an exposure treatment for specific phobias, the object of fear is presented repeatedly to the patient until the fear disappears. Clinical studies have demonstrated that, when patients leave the treatment context, fear often returns. These observations demonstrate that the renewal effect may be an important model for the return of fear after exposure treatment. The renewal effect has mainly been demonstrated in animal conditioning studies. More recently, the renewal effect has also been demonstrated in experimental research with humans. From the field of animal conditioning, several mechanisms have been proposed for the role of context in the learning of the inhibitory association during extinction. Research has provided evidence against the view that the context enters into a direct association with the US. Instead, evidence has been gathered suggesting that the context modulates the CS-noUS association. To date, research that addresses the mechanisms underlying extinction and renewal in humans is scarce. The research that is described in the present dissertation aimed at addressing this need. In the first part of the dissertation, a fear conditioning preparation was used to investigate whether the different forms of renewal that have been observed in animals can also be observed in humans. In addition, because findings from the animal laboratory have suggested that a change in the temporal context can also elicit renewal, the effect of a change in a temporal context after extinction was also investigated. More specifically, the effect of a retention interval (spontaneous recovery) and of a change in intertrial interval after extinction was investigated. In a second part of the dissertation, a predictive learning task was used to investigate two possible methods to reduce renewal: (1) by presenting an extinction reminder cue, and (2) by conducting extinction in multiple contexts. Within this second part of the dissertation, the mechanism underlying the effect of such a reminder cue was also directly tested because our results differed from the findings that have been obtained with animals. The present findings show that multiple processes may underlie the role of context in renewal. In the present studies, it was shown that the efficiency of methods to reduce renewal is dependent on the degree to which the acquisition and extinction context enter into a direct association with the US, or on the degree to which the reminder cue enters into a direct association with the US.status: publishe