Teresa Iantomasi

Gluthatione level is altered in lymphoblasts from patients with familial Alzheimer's disease

Intracellular levels of glutathione (GSH), glutathione disulphide (GSSG), glutamic acid and gamma-glutamyl cysteine synthetase (gamma-GCS) were measured in lymphoblast lines from patients with familial and sporadic Alzheimers disease (AD) and from age-matched controls. Lymphoblasts carrying presenilins (PS) and amyloid precursor protein (APP) genes mutations showed significantly decreased GSH content with respect to controls. Levels of GSSG and glutamic acid, as well as the activity of gamma-GCS were not significantly different in lymphoblasts carrying genes mutations as compared with control cells. These results indicate that even peripheral cells not involved in the neurodegenerative process of AD show altered GSH content when carrying PS and APP genes mutations. The provided data appear to be in accordance with the known alteration of GSH levels in central nervous system and strengthen the hypothesis of oxidative stress as an important, possibly crucial mechanism in the pathogenesis of AD.

Effect of orally administered glutathione on glutathione levels in some organs of rats: role of specific transporters

The present study reports data on absorption of orally administered glutathione (GSH) in rat jejunum and in other organs, and the possible role of specific transport systems of GSH and gamma-glutamyltranspeptidase (EC 2.3.2.1; gamma-GT) activity. GSH levels were measured simultaneously in various organs after oral GSH administration to untreated rats and rats treated with L-buthionine sulfoximine (BSO) or acivicin (AT125). BSO selectively inhibits GSH intracellular synthesis and AT125 is a specific inhibitor of gamma-GT activity. GSH levels were also measured after oral administration of an equivalent amount of the constituent amino acids of GSH to untreated and BSO-treated rats. Significant increases in GSH levels were found in jejunum, lung, heart, liver and brain after oral GSH administration to untreated rats. GSH increases were also obtained in all organs, except liver, when GSH was administered to rats previously GHS-depleted by treatment with BSO. The analysis of all results allowed us to distinguish between the increase in GSH intracellular levels due to intact GSH uptake by specific transporters, and that due to GSH degradation by gamma-GT activity and subsequent absorption of degradation products with intracellular resynthesis of GSH; both these mechanisms seemed to be involved in increasing GSH content in heart after oral GSH administration. Jejunum, lung and brain took up GSH mostly intact, by specific transport systems, while in liver GSH uptake occurred only by its breakdown by gamma-GT activity followed by intracellular resynthesis.

Age and GSH metabolism in rat cerebral cortex, as related to oxidative and energy parameters.

A comprehensive study on GSH metabolism in relation to some markers of oxidative and energy status in rat cerebral cortex as a function of age was performed. Reduced GSH, total GSH and the GSH Redox Index decreased both during growth (defined as the period between 1 and 5 months) and during aging (defined as the period between 5 and 27 months) while GSSG levels increased during the two periods, but most significantly during aging. Also GSH-associated enzymes and adenine-pyridine nucleotide levels show age characteristic changes. The obtained results suggest that decreases in oxidative and energy metabolism occur during aging. They probably contribute to decreases in the activity of the biosynthetic processes (i.e., NADP+(H) and GSH synthesis) and in the antioxidant capacity of the GSH system. However, the oxidative stress does not seem to be a typical characteristic of the aging period; as an oxidative status is present during the growth period too. Typical parameters of aging process are mainly the low levels of reduced GSH, total GSH and GSH Redox Index and the high levels of GSSG as well as the high levels of GSH peroxidase and GSH transferase and the low levels of gamma-glutamylcysteine synthetase.

Intestinal glutathione transport system: a possible detoxication role

The epithelium of the small intestine act by the formation of GSH-S-conjugation, as a first line of defence against various ingested toxic chemicals. GSH and GSH-dependent enzymes are present in the gastrointestinal wall. We and others have characterized the GSH-specific transport systems in intestinal brush-border and in basolateral membrane vesicles, in which gamma-glutamyltranspeptidase (gamma-GT) activity was inactivated by AT-125. In the present study we use inhibition experiments, kinetic studies, trans-stimulation of GSH uptake and HPLC determination to demonstrate (for the first time) that GSH and two GSH-S-conjugates (chosen as model compounds) share a common transport system. Plasma GSH-S-conjugates that may enter the intestinal cells via basolateral membrane, and GSH-S-conjugates that form in intestinal cells, may be eliminated directly by this GSH transporter across brush-border membranes or transported into lumen to the active site of gamma-GT; they are then further metabolized and excreted by various routes. This transport system may thus contribute to the intestinal detoxication role.

Oxidative state and IL‐6 production in intestinal myofibroblasts of Crohn's disease patients

Background: Intestinal subepithelial myofibroblasts (ISEMFs) produce inflammatory cytokines in response to certain stimuli. In the intestine of patients with Crohns disease (CD), cytokine synthesis is modified and an increased number of myofibroblasts has been observed. The intracellular redox state influences cytokine production and oxidative stress is present in the intestinal mucosa of CD patients. Methods: This study was performed in ISEMFs isolated from the colon of patients with active CD and in a myofibroblast cell line derived from human colonic mucosa: 18Co cells. Cellular glutathione (GSH) levels were modulated by treatment with buthionine sulfoximine, an inhibitor of GSH synthesis, or N‐acetylcysteine, a GSH precursor. GSH and oxidized glutathione (GSSG) levels were measured by high‐performance liquid chromatography (HPLC) methods. Interleukin (IL)‐6 production was detected by enzyme‐linked immunosorbent assay (ELISA). Results: ISEMFs of CD patients exhibited an increased oxidative state due to a decrease in the GSH/GSSG ratio, which is related to an increase in basal IL‐6 production or is stimulated by tumor necrosis factor alpha (TNF&agr;) or bacterial products. This relationship was also confirmed in 18Co cells. Phosphorylation and activation of ERK1/2 and p38 MAPK, which are signaling factors involved in the IL‐6 synthesis, were also increased when there is oxidative stress in ISEMFs. Conclusions: This study shows for the first time in ISEMFs of CD patients an increased production of IL‐6 synthesis related to the decrease in the GSH/GSSH ratio, suggesting redox regulation with the involvement of specific kinase activation. The present data shed light on the pathogenesis of inflammatory chronic processes and relapses that occur in this pathology. (Inflamm Bowel Dis 2010;)

Glutathione-mediated transport across intestinal brush-border membranes.

Glutathione transport was studied in brush-border membrane vesicles of rabbit small intestine in which gamma-glutamyl transpeptidase (EC 2.3.2.2) had been inactivated by a specific affinity-labeling reagent, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT125). Transport of intact [glycine-2-3H]GSH occurred into an osmotically active intravesicular space of AT125-treated membranes. The 0.1 M NaSCN gradient (Na+ inside greater than Na+ outside) in the transport medium could be replaced with KSCN or NaCl without affecting transport activity. The initial rate of GSH transport followed Michaelis-Menten saturation kinetics (Km = 17 microM). The results suggest that, in these membranes, there was an Na+-independent mediated transport for intact GSH with marked specificity and affinity. In fact glycine, glutamic acid and cysteine did not decrease GSH uptake, as was also true for glycylglycine and glycylglycylglycine; only gamma-glutamylcysteinylglycyl ester, a derivative of GSH, partially inhibited GSH transport.

Redox State and O2•- Production in Neutrophils of Crohn's Disease Patients:

The aim of this in vitro study was to evaluate the intracellular redox state and respiratory burst (RB) in neutrophils of patients with Crohns disease (CD). The intracellular redox state and RB in neutrophils was assessed by the superoxide anion (O2•-) production induced in these cells after stimulation by various factors related to the molecular mechanisms that, if altered, may be responsible for an abnormal immune response. This can, in part, cause the onset of inflammation and tissue damage seen in CD. This study demonstrated a decreased glutathione/glutathione disulfide (GSH/GSSG) ratio index of an increased oxidative state in CD patient neutrophils. Moreover, our findings showed a decrease in tumor necrosis factor (TNF-α)- or phorbol 12-myristate 13-acetate (PMA)–induced O2•- production in CD patient neutrophils adherent to fibronectin as compared with controls. A decreased adhesion was also demonstrated. For this reason, the involvement of altered mechanisms of protein kinase C (PKC) and β-integrin activation in CD patient neutrophils is suggested. These data also showed that the harmful effects of TNF-α cannot be caused by excessive reactive oxygen species (ROS) production Induced by neutrophils. Decreased cell viability after a prolonged time of adhesion (20 hrs) was also measured in CD patient neutrophils. The findings of this study demonstrate, for the first time, that granulocyte-macrophage colony–stimulating factor (GM-CSF), a compound recently used in CD therapy, is able to activate the RB for a prolonged time both in control and CD patient neutrophils. Increased viability of CD patient neutrophils caused by GM-CSF stimulation was also observed. In conclusion, our results indicate that decreased O2•- production and adhesion, caused, in part, by an anomalous response to TNF-α, together with low GSH level and low cell viability, may be responsible for the defective neutrophil function found in CD patients. This can contribute to the chronic inflammation and relapses that characterize this pathology. A possible role of GM-CSF in inducing O2•- production and in restoring the defensive role of neutrophils in CD patients is suggested.

Redox regulation of ERK1/2 activation induced by sphingosine 1-phosphate in fibroblasts: Involvement of NADPH oxidase and platelet-derived growth factor receptor

BACKGROUND Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite synthesized after stimulation with growth factors or cytokines. S1P extracellular effects are mediated through specific Gi-protein coupled receptors (GPCRs). Recently, we demonstrated in NIH3T3 fibroblasts stimulated by platelet-derived growth factor (PDGF) or S1P the NADPH oxidase activation and the H(2)O(2) intracellular level increase trough the Gi protein involvement. METHODS NIH3T3 fibroblast cell cultures were used. Western blot and quantitative analyses by Chemidoc-Quantity-One software were performed. H(2)O(2) level was assayed by fluorescence spectrophotometric analysis, and cell proliferation by counted manually or ELISA kit. RESULTS This study demonstrates, in NIH 3T3 fibroblasts, a novel redox regulated mechanism of S1P-induced activation of ERK 1/2 related to NADPH oxidase activity and intracellular H(2)O(2) level increase with PDGF receptor tyrosine kinase involvement through a transactivation mechanism. This event is mediated by S1P(1) and S1P(3) receptors by Gi proteins and can contribute to S1P mitogenic signaling. CONCLUSION These results can be related to mechanisms of cross-talk previously identified between receptor tyrosine kinase, including PDGFreceptor, and several GPCR ligands. GENERAL SIGNIFICANCE The redox-sensitive ERK1/2 and PDGFr tyrosine kinase activity could be targets for therapies in diseases in which deregulation of intracellular oxidative status and the consequent alteration of S1P and/or PDGF signaling pathway are involved.

Relationship between age and GSH metabolism in synaptosomes of rat cerebral cortex

A comprehensive analysis on glutathione metabolism in rat cerebral cortex synaptosomes as a function of age was performed. All different glutathione system components (GSH, GSSG, total GSH, and GSH redox index) changed significantly only during aging. GSH, total GSH, and GSH redox index decreased by about 40%, 24%, and 52%, respectively, while GSSG showed a remarkable increase of about 60%. On the contrary, some GSH-related enzyme activities showed characteristic changes both during growth and aging. GSH peroxidase and GSH-S-transferase activities significantly increased both during growth and aging, GSH reductase and gamma-glutamylcysteine synthetase activities showed lower levels only during aging, while glucose-6-phosphate dehydrogenase activity did not change throughout the life of the rat. The results obtained suggest an increase of the oxidative status due to a reduced antioxidant capacity of the GSH system in the synaptosomal compartment during aging. The main cause of these metabolic modifications is a lowering of the rates of both GSSG reduction to GSH and GSH synthesis. Moreover, an irreversible loss of GSH as GSH-S-conjugates due to a high detoxification mechanism during aging is also possible. These alterations in glutathione metabolism, found mainly during aging in rat cerebral cortex synaptosomes may contribute to clarify some aspects of cerebral diseases.

Glutathione transport across intestinal brush-border membranes: effects of ions, pH, Δψ, and inhibitors

We characterized glutathione transport in brush-border membrane vesicles (BBMV) that were prepared from rabbit small intestine in which γ-glutamyl transpeptidases (γ-glutamyltransferases, EC 2.3.2.2) had been inactivated by a specific affinity-labeling reagent (AT 125 ). Intact GSH transport was strongly increased by the presence of Na + , K + , LI + , Ca 2+ and Mn 2+ and, of all these, the Ca 2+ activation effect was prevalent. This cation effect was selective and catalytic but not energetic; V max obtained in the presence of both Na + and Ca 2+ was about 6-times higher than it was in their absence, while K m did not change. Moreover, these cations almost completely eliminated GSH binding on the membrane surface. Na + activation cannot be explained as a stimulation effect on the Na + H + antiport system, since a GSH proton-driven transport was excluded. We determined a pH optimum (7.5), while low or high extravesicular pH values diminished the GSH uptake rate. The Ca 2+ effect on GSH transport, when an electrical potential difference was imposed across BBMV, was different from that of monovalent cations. Indeed, experiments performed by valinomycin-induced K + diffusion potential or by anion substitution showed that the GSH transport system was an electroneutral process in the presence of Na + or K + , but that it was electrogenic in the presence of Ca 2+ or in the absence of extravesicular cations. These results suggest that GSH is also contransported with these cations, without its accumulation inside vesicles. Moreover, since GSH is negatively charged, the effect of pH changes and of cation activation on GSH transport is arguably mediated by changes in the ionization state of certain groups as the carrier site and of GSH itself, indicating the electrostatic nature of GSH binding sites on the transporter. The high Ca 2+ activation effect is perhaps also partly due to fluidity changes in the lipoproteic microenvironment of the GSH transporter. Moreover, this transport system has high affinity with GSH, given the low K m value (17 μM) and the fact that it was only inhibited by GSH S-derivatives and by GSH monoethyl ester, which probably share the same transport system.