Mariagrazia Coassin

Microsomal lipid peroxidation: effect of vitamin E and its functional interaction with phospholipid hydroperoxide glutathione peroxidase.

The role of vitamin E in the protection against iron dependent lipid peroxidation was studied in rat liver microsomes and Triton-dispersed microsomal lipid micelles. In these systems, an antioxidant effect of vitamin E at a physiological ratio to phospholipids could be observed only in the presence of phospholipid hydroperoxide glutathione peroxidase (PHGPX) and glutathione. The rationale of this cooperation is discussed on the basis of the hydroperoxyl radical scavenging capacity of vitamin E and the reduction of membrane hydroperoxides by PHGPX. The scavenging of lipid hydroperoxyl radicals by vitamin E, although inhibiting propagation of the peroxidative chain, produces lipid hydroperoxides from which ferrous iron generates alkoxyl radicals that react with vitamin E almost as fast as with fatty acids. Therefore, only if membrane hydroperoxides are continuously reduced by this specific peroxidase does the scavenging of hydroperoxyl radicals by vitamin E lead to an effective inhibition of lipid peroxidation.

Kinetic mechanism and substrate specificity of glutathione peroxidase activity of ebselen (PZ51)

The glutathione peroxidase activity of ebselen (PZ51) was studied using different hydroperoxidic substrates. The single progression curves obtained in the spectrophotometric test were processed by a computer to fit the integrated rate equation that describes the ping pong reaction of the Se glutathione peroxidase. Ebselen catalyzes the GSH peroxidase reaction with a mechanism that appears kinetically identical to the mechanism of the enzymes. The inactivation of the catalytic properties of ebselen by iodoacetate suggests that a selenol moiety is involved. Among the substrates tested, the best hydroperoxidic substrates are the hydroperoxy derivatives of phosphatidyl choline. Ebselen is active also on membrane hydroperoxides as does phospholipid hydroperoxide glutathione peroxidase but not glutathione peroxidase.

Prostacyclin and sodium nitroprusside inhibit the activity of the platelet inositol 1, 4, 5-trisphosphate receptor and promote its phosphorylation

Prostaglandin I (PGI) and sodium nitroprusside (SNP) induce a rapid decay of the thrombin-promoted increase of [Ca] in aspirin-treated platelets incubated in the absence of external Ca. The mechanism of their effect was studied with a new method which utilizes ionomycin to increase [Ca], followed by bovine serum albumin (BSA) to remove the Ca ionophore. The rapid decay of [Ca] after BSA is mostly due to the reuptake into the stores, since it is strongly inhibited by the endomembrane Ca-ATPase inhibitor thapsigargin. PGI and SNP are without effect on the BSA-promoted decay both with and without thapsigargin, showing that they do not affect the activity of the Ca-ATPases. The fast decay of [Ca] after BSA is decreased by thrombin which produces the Ca releaser inositol 1,4,5-trisphosphate (InsP), thus counteracting the activity of the endomembrane Ca pump. When added after thrombin, PGI and SNP accelerate the BSA-activated decay of [Ca]. However, under the same conditions, they do not decrease the concentration of InsP. In saponin-permeabilized platelets, cAMP and cGMP counteract the Ca release induced by exogenous InsP. Their inhibitory effect disappears at high InsP concentrations. This demonstrates that PGI and SNP potentiate Ca reuptake by inhibiting the InsP receptor. Two bands of approximately 260 kDa are recognized by a monoclonal antibody recognizing the C-terminal region of the InsP receptor. Both are phosphorylated rapidly, the heavier more intensely, in the presence of PGI and SNP. The phosphorylation of the InsP receptor is fast enough to be compatible with its involvement in the inhibition of the receptor by cyclic nucleotides.

Antioxidant effect of manganese

The antioxidant effects of manganese and other transition metals were studied as the inhibition of microsomal lipid peroxidation and crocin bleaching by peroxyl radicals. The peroxyl radical scavenging capacity was measured by competition kinetics analysis. While Zn(II), Ni(II), and Fe(II) were almost completely ineffective, Mn(II) and Co(II) showed a free radical scavenging capacity, exhibiting relative rate constant ratios respectively of 0.513 and 0.287. This indicates that Mn(II) is by far the most active. Therefore, the chain-breaking antioxidant capacity of Mn(II) seems to be related to the rapid quenching of peroxyl radicals according to the reaction R-OO. + Mn(II) + H(+)-->ROOH+Mn(III). The antioxidant mechanism is discussed considering the different reduction potentials of the examined cations.

Modification of the xanthine-converting enzyme of perfused rat heart during ischemia and oxidative stress

The reversible and irreversible conversion of xanthine dehydrogenase to xanthine oxidase during ischemia/reperfusion and oxidative stress induced by hydrogen peroxide or diamide and its relationship with glutathione and protein SH groups were studied. The direct spectrophotometric measurement of the various forms of the xanthine-converting enzyme indicates that, in the fresh rat heart or after normoxic perfusion, there always is a basal level of 80% xanthine dehydrogenase and 20% of xanthine oxidase (15% irreversible and 5% reversible) that could contribute to the background production of free radicals. There is no significant increase of irreversible xanthine oxidase during ischemia nor during reperfusion. After global ischemia the reversible oxidase shows almost no increase while, when ischemia is followed by reperfusion, there is a limited increase (less then 9%) of the reversible xanthine oxidase. In the latter conditions there is a decrease of glutathione and of SH groups of about 70% and 25%, respectively. Perfusion for 1 h with oxidizing agents like hydrogen peroxide (60 microM) or diamide (100 microM) determines a marked conversion of xanthine dehydrogenase to reversible xanthine oxidase of about 40% and 60%, respectively; this oxidase activity partially reconverts to the dehydrogenase after withdrawing the oxidizing agents from the perfusion medium. The level of irreversible xanthine oxidase remains unchanged in all the conditions tested. Both hydrogen peroxide and diamide induce a strong decrease in SH groups and depletion of glutathione. The xanthine dehydrogenase----xanthine oxidase conversion thus appears to be sensitive to the redox state of thiol groups.

Increased ultra weak chemiluminescence emission from rat heart at postischemic reoxygenation: protective role of vitamin E

Aim of this study was to confirm an increased free radical generation rate during ischemia-reoxygenation, by ultra-weak chemiluminescence detection at the surface of perfused rat heart. We observed that reoxygenation following 30 min global ischemia, induces an increase of ultraweak chemiluminescence emission in isolated perfused heart only if partial depletion of vitamin E is induced by dietary manipulation. Moreover, in normal diet fed rats, vitamin E is partially consumed during global ischemia, but not during reoxygenation. Since chemiluminescence increases during post-ischemic reperfusion, when vitamin E myocardial content is lowered, the most probable free radicals involved are the hydroperoxyl radical derivatives of lipids. These radicals, indeed, are known both to produce photoemission by disproportion and to react with vitamin E. On the other hand, the nature of the reaction that consumes vitamin E during ischemia is still obscure. Accordingly, the basal level of vitamin E myocardial content seems to be a key factor for protecting the heart against reoxygenation injury and its consumption during ischemia could be a determinant of myocardial sensitivity to oxidative stress during reperfusion.

Conversion of Rat Xanthine Dehydrogenase to Xanthine Oxidase during Oxidative Stress

The enzyme “xanthine oxidase” is one of the best characterized sources of superoxide anion and hydrogen peroxide. “In vivo” it mainly acts as a dehydrogenase which, by reducing NAD+, appears to be the physiologic form. Nevertheless under a variety of conditions the enzyme can undergo a conversion to an oxidase which delivers the electrons to oxygen to form superoxide and hydrogen peroxide. The transformation xanthine dehydrogenase→xanthine oxidase is irreversible when it is induced by a proteolytic attack or reversible when the SH groups of the enzyme have been oxidized.

Diacylglycerol mediates the thrombin-induced, protein kinase C and Ca2+ independent activation of the Na+/H+ exchanger in platelets

Treatment of aspirinated platelets with the electroneutral K+/H+ exchanger nigericin induces a decrease in intraplatelet pH as measured with the intracellular fluorescent indicator BCECF. Under these conditions, the proton permeability of the plasma membrane is unaffected. The addition of thrombin induces a rapid partial recovery of pHi, which is completely abolished by the Na+/H+ exchanger inhibitor NHA. The effect is also evident in the presence of the PKC inhibitors GF 109203X or staurosporine and in the absence of both external (EGTA‐chelated) and internal (BAPTA‐chelated) Ca2+. This makes the thrombin‐induced activation of the exchanger independent of the involvement of the hitherto described activators, namely PKC and the increase in [Ca2+]i, as well of the recently reported activator arachidonic acid [Cavallini, L., Coassin, M., Borean, A., and Alexandre, A. (1996) Biochem. J. 319, 567–574], whose production requires a high [Ca2+]i. The thrombin‐dependent recovery of pHi is prevented by the phospholipase C inhibitor ET 18 O‐CH3 and is mimicked by the addition of the permeable diglyceride dioctanoyl glycerol (DiC8) exogenously supplied. The effect of thrombin and DiC8 is unaffected by inhibition of diacylglycerol lipase and diacylglycerol kinase. These experiments identify diglyceride as a novel activator of the Na+/H+ exchanger in platelets.