Paolo Di Simplicio

The actin cytoskeleton response to oxidants: from small heat shock protein phosphorylation to changes in the redox state of actin itself

Actin is the major constituent of the cytoskeleton of almost all the eukaryotic cells. In vitro experiments have indicated that oxidant-stressed nonmuscle mammalian cells undergo remarkable changes in their morphology and in the structure of the actin cytoskeleton, often resulting in plasma membrane blebbing. Although the microfilament network is one of the earliest targets of oxidative stress, the mechanism by which oxidants change both the structure and the spatial organization of actin filaments is still a matter of debate and far from being fully elucidated. Starting from the 2-fold role of oxidants as injurious by-products of cellular metabolism and essential participants in cell signaling and regulation, this review attempts to gather the most relevant information related to (i) the activation of mitogen-activated protein (MAP) kinase stress-activated protein kinase-2/p38 (SAPK2/p38) which, via MAP kinase-activated protein (MAPKAP) kinase 2/3, leads to the phosphorylation of the actin polymerization (F-actin) modulator 25/27 kDa heat shock protein (HSP25/27), whose phosphorylation is causally related to the regulation of microfilament dynamics following oxidative stress; (ii) the alteration of the redox state of actin or some actin regulatory proteins. The actin cytoskeleton response to oxidants is discussed on the basis of the growing body of evidence indicating the actin system as the most sensitive constituent of the cytoskeleton to the oxidant attack.

Preferential transport of glutathione versus glutathione disulfide in rat liver microsomal vesicles.

A bi-directional, saturable transport of glutathione (GSH) was found in rat liver microsomal vesicles. GSH transport could be inhibited by the anion transport blockers flufenamic acid and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid. A part of GSH taken up by the vesicles was metabolized to glutathione disulfide (GSSG) in the lumen. Microsomal membrane was virtually nonpermeable toward GSSG; accordingly, GSSG generated in the microsomal lumen could hardly exit. Therefore, GSH transport, contrary to previous assumptions, is preferred in the endoplasmic reticulum, and GSSG entrapped and accumulated in the lumen creates the oxidized state of its redox buffer.

S-NO-actin: S-nitrosylation kinetics and the effect on isolated vascular smooth muscle

We describe the modification of reactive actin sulfhydryls by S-nitrosoglutathione. Kinetics of S-nitrosylation and denitrosylation suggest that only one cysteine of actin is involved in the reactions. By using the bifunctional sulfhydryl cross-linking reagent N,N′-1,4-phenylenebismaleimide and the monofunctional reagent N-iodoacetyl-N′-(5-sulpho-1-naphthyl)ethylenediamine, we identified this residue as Cys374. The time course of filament formation followed by high-shear viscosity changes revealed that S-nitrosylated G-actin polymerizes less efficiently than native monomers. The observed decrease in specific viscosity at steady state is due mainly to a marked inhibition of filament end-to-end annealing and, partially, to a reduction in F-actin concentration. Finally, S-nitrosylated actin acts as nitric oxide donor showing a fast, potent vasodilating activity at unusually low concentrations, being comparable with that of low molecular weight nitrosothiols.

Thiol groups in proteins as endogenous reductants to determine glutathione-protein mixed disulphides in biological systems

A novel method for glutathione-protein mixed disulphide (GSSP) determination, based on the use of protein sulphydryl groups as endogenous reductant and on the spectrophotometric determination of reduced glutathione, is described. The procedure is based on the observation that acid-precipitated proteins from different rat tissues rapidly release GSH from GSSP when brought to neutral pH. The basal GSSP content determined in rat liver, heart, lung, testis, spleen and brain corresponded to that reported in the literature and determined by more complex sample preparation or labor-intensive analytical procedures.

Different Metabolizing Ability of Thiol Reactants in Human and Rat Blood BIOCHEMICAL AND PHARMACOLOGICAL IMPLICATIONS

The effect of oxidants, electrophiles, and NO donors in rat or human erythrocytes was analyzed to investigate the influence of protein sulfhydryl groups on the metabolism of these thiol reactants. Oxidant-evoked alterations in thiolic homeostasis were significantly different in the two models; large amounts of glutathione protein mixed disulfides were produced in rat but not in human erythrocytes by treatment with hydroperoxides or diamide. The disappearance of all forms of glutathione (reduced, disulfide, protein mixed disulfide) was induced by menadione only in human erythrocytes. The treatment of rat red blood cells with electrophiles produced glutathione S-conjugates to a much lower extent than in human red blood cells; GSH was only minimally depleted in rat red blood cells. The NO donor S-nitrosocysteine induced a rapid transnitrosation reaction with hemoglobin in rat erythrocytes producing high levels of S-nitrosohemoglobin; this reaction in human red blood cells was negligible. All drugs were cleared more rapidly in rat than in human erythrocytes. Unlike human Hb, rat hemoglobin contains three families of protein SH groups; one of these located at position β125 is directly implicated in the metabolism of thiol reactants. This is thought to influence significantly the biochemical, pharmacological, and toxicological effects of some drugs.

Fast-reacting Thiols in Rat Hemoglobins Can Intercept Damaging Species in Erythrocytes More Efficiently Than Glutathione

The S-conjugation rates of the free-reacting thiols present on each component of rat hemoglobin with 5,5-dithio-bis(2,2-nitrobenzoic acid) (DTNB) have been studied under a variety of conditions. On the basis of their reactivity with DTNB (0.5 mm), three classes of thiols have been defined as follows: fast reacting (fHbSH), with t½ <100 ms; slow reacting (sHbSH), with t½ 30–50 s; and very slow reacting (vsHbSH), with t½ 180–270 s. Under paraphysiological conditions, fHbSH (identified with Cys-125β(H3)) conjugates with DTNB 100 times faster than glutathione and ∼4000 times more rapidly than (v)sHbSH (Cys-13α(A11) and Cys-93β(F9)). Such characteristics of fHbSH reactivity that are independent of the quaternary state of hemoglobin are mainly due to the following: (i) its low pK (∼6.9, the cysteinyl anion being stabilized by a hydrogen bond with Ser-123β(H1)) and (ii) the large exposure to the solvent (as measured by analysis of a model of the molecular surface) and make these thiols the kinetically preferred groups in rat erythrocytes for S-conjugation. In addition, because of the high cellular concentration (8 mm, i.e. four times that of glutathione), fHbSHs are expected to intercept damaging species in erythrocytes more efficiently than glutathione, thus adding a new physiopathological role (direct involvement in cellular strategies of antioxidant defense) to cysteinyl residues in proteins.

Glutathione recycling and antioxidant enzyme activities in erythrocytes of term and preterm newborns at birth.

We previously demonstrated a high susceptibility of neonatal red blood cells (RBC) to oxidative stress at birth. The aim of this study was to compare the RBC antioxidant capacity and redox cycle enzyme activities as well as glutathione (GSH) recycling in full-term and preterm infants at birth and in normal adults. GSH and GSH disulfide (GSSG) concentrations, GSH/GSSG ratio, and the activities of glucose-6-phosphate dehydrogenase (G-6-PDH), GSH peroxidase, GSH reductase (GR), catalase (CAT), superoxide dismutase (SOD), and hexokinase (HK) were measured in RBC of 25 healthy adults and 56 newborns (23 term, 33 preterm) at birth. The GSH recycling was measured in adult and newborn RBC exposed to oxidative stress (1 mM tert-butylhydroperoxide). The RBC of term and preterm babies showed higher GSH, GSSG, G-6-PDH, GR, and HK levels/activities and lower GSH/GSSG ratios and higher GSH-recycling rates than those of adults. In preterm babies significant correlations were found between G-6-PDH and CAT, GSH, GSH/GSSG ratio, and GSSG (r = –0.67, r = 0.71, r = –0.66, p < 0.01; r = 0.71, p < 0.05, respectively). In term newborns, statistically significant correlations were observed between G-6-PDH and CAT, SOD, and GSH (r = –0.65, r = –0.65, r = –0.69, p < 0.01, respectively). The results indicate the central role of the G-6-PDH activity in antioxidant defenses. We speculate that preterm babies have prompter involvement of antioxidant defenses than term babies.

Alteration in the redox state of plasma in heart-transplant patients with moderate hyperhomocysteinemia ☆

Hyperhomocysteinemia has recently been suggested to contribute to the progression of the so-called chronic rejection or cardiac allograft vasculopathy (CAV) in heart-transplant patients in which the major determinant of the increase in homocysteine (Hcy) was the progressive decline of renal function. The exact mechanisms of tissue injury by Hcy is unknown, but some aspects of its toxicity have been related to its capacity for altering the redox state of plasma and forming protein adducts by intermediate lactone. To study the relationships between Hcy levels and variations in the redox state governed by thiols, plasma levels of Hcy, cysteine, glutathione, cysteinylglycine, and corresponding disulfides and protein-mixed disulfides were evaluated in subjects with moderate hyperhomocysteinemia represented by heart-transplant patients with (HTRF) and without (HT) renal failure, as well as patients with renal failure of different origin (RF), and compared with those of a control group (C) of normal subjects matched for age and sex. Plasma levels of Hcy and the corresponding protein mixed disulfides increased progressively in HTs, RFs, and HTRFs with respect to control. These changes were correlated with cysteine variations (as cystine and protein-mixed disulfides) but not with glutathione or cysteinylglycine that varied only as disulfides with a similar tendency. Moreover, an alteration in the plasma redox was evidenced by the decrease in thiol/disulfide ratios of cysteine, Hcy, and cysteinylglycine. In all groups, cysteine was directly correlated with Hcy but not with glutathione or cysteinylglycine, which in turn were correlated each other. Therefore levels of plasma cysteine were more linked to Hcy than to metabolism of glutathione. The clinical meaning of cysteine changes remains undefined and requires further study.

Protein-thiol substitution or protein dethiolation by thiol/disulfide exchange reactions: the albumin model.

Dethiolation experiments of thiolated albumin with thionitrobenzoic acid and thiols (glutathione, cysteine, homocysteine) were carried out to understand the role of albumin in plasma distribution of thiols and disulfide species by thiol/disulfide (SH/SS) exchange reactions. During these experiments we observed that thiolated albumin underwent thiol substitution (Alb‐SS‐X+RSH↔Alb‐SS‐R+XSH) or dethiolation (Alb‐SS‐X+XSH↔Alb‐SH+XSSX), depending on the different pKa values of thiols involved in protein–thiol mixed disulfides (Alb‐SS‐X). It appeared in these reactions that the compound with lower pKa in mixed disulfide was a good leaving group and that the pKa differences dictated the kind of reaction (substitution or dethiolation). Thionitrobenzoic acid, bound to albumin by mixed disulfide (Alb‐TNB), underwent rapid substitution after thiol addition, forming the corresponding Alb‐SS‐X (peaks at 0.25–1 min). In turn, Alb‐SS‐X were dethiolated by the excess nonprotein SH groups because of the lower pKa value in mixed disulfide with respect to that of other thiols. Dethiolation of Alb‐SS‐X was accompanied by formation of XSSX and Alb‐SH up to equilibrium levels at 35 min, which were different for each thiol. Structures by molecular simulation of thiolated albumin, carried out for understanding the role of sulfur exposure in mixed disulfides in dethiolation process, evidenced that the sulfur exposure is important for the rate but not for determining the kind of reaction (substitution or dethiolation). Our data underline the contribution of SH/SS exchanges to determine levels of various thiols as reduced and oxidized species in human plasma.

Effect of low doses of ethanol on platelet function in long-life abstainers and moderate-wine drinkers

In vitro, high concentrations of ethanol (EtOH) reduce platelet aggregation. Less is known about the effect of low EtOH doses on platelet function in a selected human population of long-life abstainers and low moderate-wine drinkers to avoid rebound effect of EtOH on platelet aggregation. Results of our experiments suggest that moderate-wine drinkers have higher levels of high density lipoprotein (HDL) than long-life abstainers while fibrinogen levels are unchanged. Furthermore, platelets obtained from these individuals do not differ in their response when stimulated by agonists such as AA and collagen. The effect of in vitro exposure of low doses of EtOH has been studied in PRP and in washed platelets. EtOH (0.1-10 mM) inhibits platelet aggregation induced by collagen at its ED50 while is ineffective when aggregation was triggered by U-46619 and by 1 microM adenosine diphosphate (ADP). 5-10 mM EtOH partially reduces the second wave of aggregation induced by 3 microM ADP. 0.1-10 mM EtOH dose-dependently lowers the aggregation induced by AA at its ED50 but it is less effective at ED75 of AA. The antiaggregating effect of EtOH on aggregation induced by AA is unchanged by inhibitor of nitric oxide synthase. In addition, 10 mM EtOH reduces thromboxane (Tx) formation. In washed platelets, 1-10 mM EtOH partially inhibits platelet aggregation induced by thrombin. In washed resting platelets, 10 mM EtOH does not change the resting [Ca++]i while significantly reduces the increase in [Ca++]i triggered by AA. The results of ex vivo experiments have demonstrated that wine increases the HDL. However, this observation may or may not influence the response of platelets to agonists. Results of our studies demonstrate that low doses of alcohol reduces platelet function.