Alfonso Pompella

Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism

Many studies have supported a genetic etiology for autism. Here we report mutations in two X-linked genes encoding neuroligins NLGN3 and NLGN4 in siblings with autism-spectrum disorders. These mutations affect cell-adhesion molecules localized at the synapse and suggest that a defect of synaptogenesis may predispose to autism.

Human Atherosclerotic Plaques Contain Gamma-Glutamyl Transpeptidase Enzyme Activity

During the last decade, growing evidence has shown that serum gamma-glutamyl transpeptidase (GGT) is an independent prognostic marker for cardiac death and reinfarction, both in unselected populations and in patients with coronary artery disease. Clinical and epidemiological evidence indicates that the prognostic value of GGT is largely independent of other risk factors for cardiovascular disease and alcohol consumption. The catalytic activity of GGT, which is present on the surface of cell membranes and in serum, is …

Glutathione catabolism as a signaling mechanism

Glutathione (GSH) is the main intracellular thiol antioxidant, and as such participates in a number of cellular antitoxic and defensive functions. Nevertheless, non-antioxidant functions of GSH have also been described, e.g. in modulation of cell proliferation and immune response. Recent studies from our and other laboratories have provided evidence for a third functional aspect of GSH, i.e. the prooxidant roles played by molecular species originating during its catabolism by the membrane ectoenzyme gamma-glutamyl transpeptidase (GGT). The reduction of metal ions effected by GSH catabolites is capable to induce redox cycling processes leading to the production of reactive oxygen species (superoxide, hydrogen peroxide), as well as of other free radicals. Through the action of these reactive compounds, GSH catabolism can ultimately lead to oxidative modifications on a variety of molecular targets, involving oxidation and/or S-thiolation of protein thiol groups in the first place. Modulating effects of this kind have been observed on several important, redox-sensitive components of the signal transduction chains, such as cell surface receptors, protein phosphatase activities and transcription factors. Against this background, the prooxidant reactions induced by GSH catabolism appear to represent a novel, as yet unrecognized mechanism for modulation of cellular signal transduction.

Hydrogen peroxide produced during gamma-glutamyl transpeptidase activity is involved in prevention of apoptosis and maintainance of proliferation in U937 cells.

It has been reported in several cell lines that exposure to low levels of reactive oxygen species can exert a stimulatory effect on their proliferation. We have previously shown that mild oxidative conditions can also counteract apoptotic stimuli. A constitutive cellular production of low levels of superoxide and hydrogen peroxide originates from various sources; among these, γ‐glutamyl transpeptidase (GGT), the plasma membrane‐bound activity in charge of metabolizing extracellular reduced glutathione, has recently been included. Since the inhibition of GGT is a sufficient stimulus for the induction of apoptosis in selected cell lines, we investigated whether this effect might result from the suppression of the mentioned GGT‐dependent prooxidant reactions, on the theory that the latter may represent a basal antiapoptotic and proliferative signal for the cell. Experiments showed that: 1) GGT activity in U937 monoblastoid cells is associated with the production of low levels of hydrogen peroxide, and two independent GGT inhibitors cause a dose‐dependent decrease of such GGT‐dependent production of H2O2; 2) GGT inhibition with acivicin results in cell growth arrest, and induces cell death and DNA fragmentation with the ladder appearance of apoptosis; 3) treatment of cells with catalase—and even more with Trolox C—is able to decrease their proliferative rate; 4) GGT inhibition (with suppression of H2O2 production) results in a down‐regulation of poly(ADP‐ribose) polimerase (PARP) activity, which precedes the proteolytic cleavage of PARP molecule, such as that typically induced by caspases. The reported data suggest that the low H2O2 levels originating as a by‐product during GGT activity are able to act as sort of a ‘life signal’ in U937 cells, insofar as they can maintain cell proliferation and protect against apoptosis, possibly through an up‐regulation of PARP activity. —Del Bello, B., Paolicchi, A., Comporti, M., Pompella, A., Maellaro, E. Hydrogen peroxide produced during γ‐glutamyl transpeptidase activity is involved in prevention of apoptosis andmaintainance of proliferation in U937 cells. FASEB J. 13, 69–79 (1999)

Redox modulation of cell surface protein thiols in U937 lymphoma cells: the role of γ-glutamyl transpeptidase-dependent H2O2 production and S-thiolation

The expression of gamma-glutamyl transpeptidase (GGT), a plasma membrane ectoenzyme involved in the metabolism of extracellular reduced glutathione (GSH), is a marker of neoplastic progression in several experimental models, and occurs in a number of human malignant neoplasms and their metastases. Because it favors the supply of precursors for the synthesis of GSH, GGT expression has been interpreted as a member in cellular antioxidant defense systems. However, thiol metabolites generated at the cell surface during GGT activity can induce prooxidant reactions, leading to production of free radical oxidant species. The present study was designed to characterize the prooxidant reactions occurring during GGT ectoactivity, and their possible effects on the thiol redox status of proteins of the cell surface. Results indicate that: (i) in U937 cells, expressing significant amounts of membrane-bound GGT, GGT-mediated metabolism of GSH is coupled with the extracellular production of hydrogen peroxide; (ii) GGT activity also results in decreased levels of protein thiols at the cell surface; (iii) GGT-dependent decrease in protein thiols is due to sulfhydryl oxidation and protein S-thiolation reactions; and (iv) GGT irreversible inhibition by acivicin is sufficient to produce an increase of protein thiols at the cell surface. Membrane receptors and transcription factors have been shown to possess critical thiols involved in the transduction of proliferative signals. Furthermore, it was suggested that S-thiolation of cellular proteins may represent a mechanism for protection of vulnerable thiols against irreversible damage by prooxidant agents. Thus, the findings reported here provide additional explanations for the envisaged role played by membrane-bound GGT activity in the proliferative attitude of malignant cells and their resistance to prooxidant drugs and radiation therapy.

Restoration of nitric oxide availability after calcium antagonist treatment in essential hypertension.

Essential hypertension is associated with impaired endothelium-dependent vasodilation caused by oxygen free radical–induced nitric oxide (NO) breakdown. Because calcium antagonists can improve endothelial function in patients with essential hypertension, in this study we tested the hypothesis that this beneficial effect could be related to restoration of NO availability by antioxidant properties. In 15 healthy subjects and 15 hypertensive patients, we studied forearm blood flow (strain-gauge plethysmography) modifications induced by intrabrachial acetylcholine (ACh; 0.15, 0.45, 1.5, 4.5, and 15 &mgr;g/100 mL per minute), an endothelium-dependent vasodilator in basal conditions, during infusion of NG-monomethyl-l-arginine (L-NMMA, 100 &mgr;g/100 mL forearm tissue per minute), an NO-synthase inhibitor, vitamin C (8 mg/100 mL forearm tissue per minute), and finally, simultaneous infusion of L-NMMA and vitamin C. The response to sodium nitroprusside (SNP; 1, 2, and 4 &mgr;g/100 mL forearm tissue per minute) was also evaluated. In control subjects, vasodilation to ACh was inhibited by L-NMMA and not changed by vitamin C. In hypertensive patients, vasodilation to ACh was blunted as compared with control subjects and resistant to L-NMMA. Vitamin C, which decreased plasma isoprostanes and increased plasma antioxidant capacity, increased the response to ACh and restored the inhibiting effect of L-NMMA. In hypertensive patients, the study was repeated after 3-month treatment with nifedipine gastrointestinal therapeutic system (30 to 60 mg/daily). Nifedipine treatment decreased circulating plasma lipoperoxides and isoprostanes and increased plasma antioxidant capacity. Moreover, nifedipine increased the vasodilation to ACh but not to SNP and restored the inhibiting effect of L-NMMA on ACh-induced vasodilation, whereas vitamin C no longer exerted its facilitating activity. These results indicate that nifedipine increases endothelium-dependent vasodilation by restoring NO availability, an effect probably determined by antioxidant activity.

Cellular pathways for transport and efflux of ascorbate and dehydroascorbate

The mechanisms allowing the cellular transport of ascorbic acid represent a primary aspect for the understanding of the roles played by this vitamin in pathophysiology. Considerable research effort has been spent in the field, on several animal models and different cell types. Several mechanisms have been described to date, mediating the movements of different redox forms of ascorbic acid across cell membranes. Vitamin C can enter cells both in its reduced and oxidized form, ascorbic acid (AA) and dehydroascorbate (DHA), utilizing respectively sodium-dependent transporters (SVCT) or glucose transporters (GLUT). Modulation of SVCT expression and function has been described by cytokines, steroids and post-translational protein modification. Cellular uptake of DHA is followed by its intracellular reduction to AA by several enzymatic and non-enzymatic systems. Efflux of vitamin C has been also described in a number of cell types and different pathophysiological functions were proposed for this phenomenon, in dependence of the cell model studied. Cellular efflux of AA is mediated through volume-sensitive (VSOAC) and Ca(2+)-dependent anion channels, gap-junction hemichannels, exocytosis of secretory vesicles and possibly through homo- and hetero-exchange systems at the plasma membrane level. Altogether, available data suggest that cellular efflux of ascorbic acid - besides its uptake - should be taken into account when evaluating the cellular homeostasis and functions of this important vitamin.

Plasma total antioxidant capacity in hemodialyzed patients and its relationships to other biomarkers of oxidative stress and lipid peroxidation.

Abstract Patients undergoing long-term hemodialysis (HD) exhibit increased levels of oxidative stress, likely contributing to the increased rate of cardiovascular disease. The present study represents a critical evaluation of some of the most widely used oxidative indicators, as applied to the monitoring of hemodialysis-associated oxidative stress. Total plasma antioxidant capacity was determined by two independent procedures, the total antioxidant status (TAS) and the ferric reducing ability of plasma (FRAP) methods. Plasma lipid peroxidation was assessed by determining the peroxidation products malonaldehyde and 4-hydroxynonenal (MDA-4HNE) as well as lipid hydroperoxides (“Fox-2” and “d-ROMs” methods). Total plasma thiols and plasma b-tocopherol were also determined. MDA-4HNE levels were higher in HD patients and decreased following HD, possibly due to passive diffusion across dialysis filters. d-ROMs were also higher in HD patients but exhibited a further increase following the dialysis procedure. Serum α-tocopherol did not show any significant differences. Plasma thiols were lower in HD patients and were restored following HD. Plasma total antioxidant capacity determined with either method was unexpectedly higher in HD patients compared to controls, and decreased following HD. These data indicate that, of the biomarkers studied, d-ROMs level is the one more accurately reflecting the oxidative alterations taking place in HD patients, while determination of MDA-4HNE fails to detect oxidation occurring during the HD sessions. In addition, our findings point out that the determination of total antioxidant capacity in HD patients is severely affected by the concomitant fluctuations in plasma urate levels and therefore needs careful interpretation.

Oxidative stress and antioxidant therapy in cystic fibrosis.

Cystic fibrosis is a lethal autosomal recessive condition caused by a defect of the transmembrane conductance regulator gene that has a key role in cell homeostasis. A dysfunctional cystic fibrosis transmembrane conductance regulator impairs the efflux of cell anions such as chloride and bicarbonate, and also that of other solutes such as reduced glutathione. This defect produces an increased viscosity of secretions together with other metabolic defects of epithelia that ultimately promote the obstruction and fibrosis of organs. Recurrent pulmonary infections and respiratory dysfunction are main clinical consequences of these pathogenetic events, followed by pancreatic and liver insufficiency, diabetes, protein-energy malnutrition, etc. This complex comorbidity is associated with the extensive injury of different biomolecular targets by reactive oxygen species, which is the biochemical hallmark of oxidative stress. These biological lesions are particularly pronounced in the lung, in which the extent of oxidative markers parallels that of inflammatory markers between chronic events and acute exacerbations along the progression of the disease. Herein, an abnormal flux of reactive oxygen species is present by the sustained activation of neutrophils and other cystic fibrosis-derived defects in the homeostatic processes of pulmonary epithelia and lining fluids. A sub-optimal antioxidant protection is believed to represent a main contributor to oxidative stress and to the poor control of immuno-inflammatory pathways in these patients. Observed defects include an impaired reduced glutathione metabolism and lowered intake and absorption of fat-soluble antioxidants (vitamin E, carotenoids, coenzyme Q-10, some polyunsaturated fatty acids, etc.) and oligoelements (such as Se, Cu and Zn) that are involved in reactive oxygen species detoxification by means of enzymatic defenses. Oral supplements and aerosolized formulations of thiols have been used in the antioxidant therapy of this inherited disease with the main aim of reducing the extent of oxidative lesions and the rate of lung deterioration. Despite positive effects on laboratory end points, poor evidence was obtained on the side of clinical outcome so far. These aspects examined in this critical review of the literature clearly suggest that further and more rigorous trials are needed together with new generations of pharmacological tools to a more effective antioxidant and anti-inflammatory therapy of cystic fibrosis patients. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.

Gamma-Glutamyl Transpeptidase-Dependent Lipid Peroxidation in Isolated Hepatocytes and HepG2 Hepatoma Cells

Gamma-glutamyltranspeptidase (GGT), a plasma membrane-bound enzyme, provides the only activity capable to effect the hydrolysis of extracellular glutathione (GSH), thus favoring the cellular utilization of its constituent amino acids. Recent studies have shown however that in the presence of chelated iron prooxidant species can be originated during GGT-mediated metabolism of GSH, and that a process of lipid peroxidation can be started eventually in suitable lipid substrates. The present study was undertaken to verify if a GGT-dependent lipid peroxidation process can be induced in the lipids of biological membranes, including living cells, and if this effect can be sustained by the GGT highly expressed at the surface of HepG2 human hepatoma cells. In rat liver microsomes (chosen as model membrane lipid substrate) exposed to GSH and ADP-chelated iron, the addition of GGT caused a marked stimulation of lipid peroxidation, which was further enhanced by the addition of the GGT co-substrate glycyl-glycine. The same was observed in primary cultures of isolated rat hepatocytes, where the lipid peroxidation process did not induce acute toxic effects. GGT-stimulation of lipid peroxidation was dependent both on the concentration of GSH and of ADP-chelated iron. In GGT-rich HepG2 human hepatoma cells, the exposure to GSH, glycyl-glycine, and ADP-chelated iron resulted in a nontoxic lipid peroxidation process, which could be prevented by means of GGT inhibitors such as acivicin and the serine-boric acid complex. In addition, by co-incubation of HepG2 cells with rat liver microsomes, it was observed that the GGT owned by HepG2 cells can act extracellularly, as a stimulant on the GSH- and iron-dependent lipid peroxidation of microsomes. The data reported indicate that the lipid peroxidation of liver microsomes and of living cells can be stimulated by the GGT-mediated metabolism of GSH. Due to the well established interactions of lipid peroxidation products with cell proliferation, the phenomenon may bear particular significance in the carcinogenic process, where a relationship between the expression of GGT and tumor progression has been envisaged.