Lucia Cavallini

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.

Inhibitory action of quercetin on xanthine oxidase and xanthine dehydrogenase activity

Quercetin is an equally good inhibitor of xanthine oxidase (type O, oxygen-reducing enzyme) and xanthine dehydrogenase (type D, NAD+-reducing enzyme) activity of a preparation of the xanthine-oxidizing enzyme partially purified from rat liver. The inhibition seems competitive with the oxidase form and non-competitive (mixed-type) with the dehydrogenase form of the enzyme. These inhibitory properties should be referred to the flavonoid structure of quercetin rather than to its antioxidant power. The antioxidant properties of quercetin and its inhibitory effect on the xanthine-oxidizing enzyme are discussed with reference to hyperuricemic and ischemic states.

Inhibitory action of silymarin of lipid peroxide formation in rat liver mitochondria and microsomes

Abstract Silymarin, a 3-oxyflavone present in Silybum Marianum, protected both liver mitochondria and microsomes from lipid peroxide formation induced by various agents. The antiperoxidative action exhibited by Silymarin was 10-fold higher than that of α-tocopherol, and was present when the drug was added as well as after the peroxidant agents. Data obtained rule out the possibility that the antiperoxidative action of Silymarin was due to an interaction of this drug with Fe 2+ . The reported results are compatible with an interaction of Silymarin with free radical species responsible for lipid peroxidation.

Comparative evaluation of antiperoxidative action of silymarin and other flavonoids.

Summary The antiperoxidative action of silymarin has been compared to that of quercetin, dihydroquercetin and quercitrin in microsomes and mitochondria from rat liver exposed in vitro to two peroxidizing systems: dihydroxyfumaric acid plus FeSo 4 and potassium peroxychromate. It has been found that silymarin is about as active as quercetin and dihydroquercetin, and more active than quercitrin as antiperoxidative agent, irrespective to the system used for inducing peroxidation. The results obtained are also consistent with the view that all flavonoids tested act as “scavengers” of free radicals and not simply as metal complexing agents.

The pH-Sensitive Dye Acridine Orange as a Tool to MonitorExocytosis/Endocytosis in Synaptosomes

Abstract : We introduce the use of the pH‐sensitive dye acridine orange (AO) to monitor exo/endocytosis of acidic neurotransmitter‐containing vesicles in synaptosomes. AO is accumulated exclusively in acidic v‐ATPase‐dependent bafilomycin (Baf)‐sensitive compartments. A fraction of the accumulated AO is rapidly released (fluorescence increase) upon depolarization with KCl in the presence of Ca2+. The release (completed in 5‐6 s) is followed by reuptake to values below the predepolarization baseline. The reuptake, but not the release, is inhibited by Baf added 5 s prior to KCl. In a similar protocol, Baf does not affect the initial fast phase of glutamate release measured enzymatically, but it abolishes the subsequent slow phase. Thus, the fast AO release corresponds to the rapid phase of glutamate release and the slow phase depends on vesicle cycling. AO reuptake depends in part on the progressive accumulation of acid‐loaded vesicles during cycling. Stopping exocytosis at selected times after KCl by Ca2+ removal with EGTA evidences endocytosis : Its T1/2 was 12 ± 0.6 s. The KA+, channel inhibitors 4‐aminopyridine (100 μM) and α‐dendrotoxin (10‐100 nM) are known to induce glutamate release by inducing the firing of Na+ channels ; their action is potentiated by the activation of protein kinase C. Also these agents promote a Ca2+‐dependent AO release, which is prevented by the Na+ channel inhibitor tetrodotoxin and potentiated by 4β‐phorbol 12‐myristate 13‐acetate (PMA). With α‐dendrotoxin, endocytosis was monitored by stopping exocytosis at selected times with EGTA or alternatively with Cd2+ or tetrodotoxin. The T1/2 of endocytosis, which was unaffected by PMA, was 12 ± 0.4 s with EGTA and Cd2+ and 9.5 ± 0.5 s with tetrodotoxin. Protein kinase C activation appeared to facilitate vesicle turnover.

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.

The protective action of pyruvate on recovery of ischemic rat heart: Comparison with other oxidizable substrates

The recovery of both contractile performance and metabolic response of rat heart following 1 h of ischemia after equilibration with glucose + insulin (glucose-ischemia) or with pyruvate (pyruvate-ischemia), was tested in normoxic reperfusion in the presence of glucose + insulin, pyruvate, lactate or acetate. In glucose-ischemia only the reperfusion with pyruvate results in a complete recovery of the contractile force (left ventricular pressure, LVP) (170%) and good recovery of high energy phosphate compounds. Lower LVP and tissue energy charge were found in glucose reperfusion and even less in lactate and acetate reperfusion. Disappearance of the IMP accumulated during ischemia is evident only in the pyruvate reperfusion indicating a higher metabolic recovery. On the contrary in pyruvate-ischemia all types of reperfusion tested were effective in reactivating the contractile force (although acetate to a lesser extent); the contractile activity was accompanied by a good recovery of phosphocreatine, ATP, energy charge and by the decrease of IMP. Large decreases of adenine nucleotides and NADP and lower decreases of NAD are observed during ischemia/reperfusion in both systems. Pyruvate-ischemia is quite similar to, if not worse than glucose-ischemia, for all the metabolic parameters considered, but not worse for the possibility of recovery. Some specific effect of pyruvate should be exerted during the ischemic phase. The mechanism of pyruvate protection is discussed in relationship to: (i) the possible activation of pyruvate dehydrogenase, (ii) the activation of NADPH-dependent peroxide scavenging systems, (iii) the direct scavenging action of pyruvate on H2O2.

Mitochondrial lipid peroxidation by cumene hydroperoxide and its prevention by succinate

Rat liver mitochondria form lipid hydroperoxides when they are incubated aerobically with cumene hydroperoxide. The rate of reaction is dependent on the initial concentration of the latter and involves the consumption of oxygen. Gradient-separated and cytochrome c-depleted mitochondria, mitoplasts and submitochondrial fractions also undergo this peroxidation. Mitochondrial lipid peroxidation by cumene hydroperoxide is strongly inhibited by SKF52A (an inhibitor of cytochrome P-450), by antioxidants and to a lesser extent by the enzymes superoxide dismutase and catalase. Conversely, rotenone and N-ethylmaleimide stimulate the reaction. Succinate protects against the lipid peroxidation and in some mitochondrial fractions the associated oxygen uptake is also inhibited. This protection by succinate is prevented by malonate but not by N-ethylmaleimide or antimycin. Lipid hydroperoxides present in previously peroxidised mitochondria are partly lost on reincubation with succinate and this reaction is also unaffected by N-ethylmaleimide but inhibited by both malonate and antimycin. The results suggest that reduction of mitochondrial ubiquinone may prevent the generation of lipid hydroperoxides but that their subsequent removal may require reduction at or beyond cytochrome b.

Succinate modulation of H2O2 release at NADH:ubiquinone oxidoreductase (Complex I) in brain mitochondria

Complex I (NADH:ubiquinone oxidoreductase) is responsible for most of the mitochondrial H2O2 release, both during the oxidation of NAD-linked substrates and during succinate oxidation. The much faster succinate-dependent H2O2 production is ascribed to Complex I, being rotenone-sensitive. In the present paper, we report high-affinity succinate-supported H2O2 generation in the absence as well as in the presence of GM (glutamate/malate) (1 or 2 mM of each). In brain mitochondria, their only effect was to increase from 0.35 to 0.5 or to 0.65 mM the succinate concentration evoking the semi-maximal H2O2 release. GM are still oxidized in the presence of succinate, as indicated by the oxygen-consumption rates, which are intermediate between those of GM and of succinate alone when all substrates are present together. This effect is removed by rotenone, showing that it is not due to inhibition of succinate influx. Moreover, alpha-oxoglutarate production from GM, a measure of the activity of Complex I, is decreased, but not stopped, by succinate. It is concluded that succinate-induced H2O2 production occurs under conditions of regular downward electron flow in Complex I. Succinate concentration appears to modulate the rate of H2O2 release, probably by controlling the hydroquinone/quinone ratio.

Pathways of hydrogen peroxide generation in guinea pig cerebral cortex mitochondria

The production of H2O2 by brain mitochondria was monitored employing a new technique based on the horseradish peroxidase dependent oxidation of acetylated ferrocytochrome c. It was shown that brain mitochondria release H2O2 by an intermediate autooxidation at the QH2-cytochrome c oxidoreductase level (induced by antimycin A and inhibited by myxothiazol). With both succinate and pyruvate plus malate this H2O2 release is inhibited at high substrate concentrations. With pyruvate plus malate a second source of H2O2 could be detected, apparently from autoxidation at the NADH dehydrogenase level. With alpha-glycerophosphate some H2O2 derives from autooxidation at the alpha-glycerophosphate dehydrogenase. The NADH dehydrogenase dependent, but not the QH2-cytochrome c oxidoreductase dependent H2O2 was significantly stimulated upon depletion of the mitochondrial glutathione.