Franco Zoccarato

Dopamine-derived Dopaminochrome Promotes H2O2 Release at Mitochondrial Complex I STIMULATION BY ROTENONE, CONTROL BY Ca2+, AND RELEVANCE TO PARKINSON DISEASE

Inhibitors of Complex I of the mitochondrial respiratory chain, such as rotenone, promote Parkinson disease-like symptoms and signs of oxidative stress. Dopamine (DA) oxidation products may be implicated in such a process. We show here that the o-quinone dopaminochrome (DACHR), a relatively stable DA oxidation product, promotes concentration (0.1–0.2 μm)- and respiration-dependent generation of H2O2 at Complex I in brain mitochondria, with further stimulation by low concentrations of rotenone (5–30 nm). The rotenone effect required that contaminating Ca2+ (8–10 μm) was not removed. DACHR apparently extracts an electron from the constitutively autoxidizable site in Complex I, producing a semiquinone, which then transfers an electron to O2, generating \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} and then H2O2. Mitochondrial removal of H2O2 monoamine, formed by either oxidase activity or DACHR, was performed largely by glutathione peroxidase and glutathione reductase, which were negatively regulated by low intramitochondrial Ca2+ levels. Thus, the H2O2 formed accumulated in the medium if contaminating Ca2+ was present; in the absence of Ca2+, H2O2 was completely removed if it originated from monoamine oxidase, but was less completely removed if it originated from DACHR. We propose that the primary action of rotenone is to promote extracellular \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} release via activation of NADPH oxidase in the microglia. In turn, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} oxidizes DA to DACHR extracellularly. (The reaction is favored by the lack of GSH, which would otherwise preferably produce GSH adducts of dopaminoquinone.) Once formed, DACHR (which is resistant to GSH) enters neurons to activate the rotenone-stimulated redox cycle described.

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.

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.

Hydrogen Peroxide Induces a Long-Lasting Inhibition of the Ca2+-Dependent Glutamate Release in Cerebrocortical Synaptosomes Without Interfering with Cytosolic Ca2+

Abstract: We studied the action of H2O2 on the exocytosis of glutamate by cerebrocortical synaptosomes. The treatment of synaptosomes with H2O2 (50–150 µM) for a few minutes results in a long‐lasting depression of the Ca2+‐dependent exocytosis of glutamate, induced by KCl or by the K+‐channel inhibitor 4‐aminopyridine. The energy state of synaptosomes, as judged by the level of phosphocreatine and the ATP/ADP ratio, was not affected by H2O2, although a transient decrease was observed after the treatment. H2O2 did not promote peroxidation, as judged by the formation of malondialdehyde. In indo‐1‐loaded synaptosomes, the treatment with H2O2 did not modify significantly the KCl‐induced increase of [Ca2+]i. H2O2 inhibited exocytosis also when the latter was induced by increasing [Ca2+]i with the Ca2+ ionophore ionomycin. The effects of H2O2 were unchanged in the presence of superoxide dismutase and the presence of the Fe3+ chelator deferoxamine. These results appear to indicate that H2O2, apparently without damaging the synaptosomes, induces a long‐lasting inhibition of the exocytosis of glutamate by acting directly on the exocytotic process.

Involvement of endogenous phospholipase A2 in Ca2+ and Mg2+ movements induced by inorganic phosphate and diamide in rat liver mitochondria

Abstract Addition to rat liver mitochondria of 2 mM inorganic phosphate or 0.15 mM diamide, a thiol oxidizing agent, induced a respiration dependent efflux of Mg2+ which was prevented by both antimycin A and tetracaine. Tetracaine also inhibited the release of respiration induced by phosphate or diamide. Endogenous Ca2+ were retained by mitochondria until 50–60 per cent of endogenous Mg2+ has been lost. Tetracaine retarded Ca2+ release. The involvement of mitochondrial phospholipase A2 is demonstrated both by its inhibition by tetracaine and its activation by diamide or phosphate. The failure of these compounds to activate phospholipase A2 in the presence of added Ca2+ makes reasonable the assumption that the activation of phospholipase A2 is secondary to respiration dependent Ca2+ movements, which would favour the interaction Ca2+-phospholipase A2.

Cyclic GMP and nitroprusside inhibit the activation of human platelets by fluoroaluminate.

Sodium nitroprusside, an activator of the soluble guanylate cyclase, inhibits the intracellular Ca2+ mobilization, ATP secretion and aggregation of human platelets evoked by fluoroaluminate. Similar results are obtained with 8-bromo-cyclic GMP (8-Br-cGMP). Both nitroprusside and 8-Br-cGMP inhibit the protein kinase C-dependent phosphorylation of the 47 and 20 kDa proteins induced by fluoroaluminate, but not by the protein kinase C activators phorbol ester and diacylglycerol. Since fluoroaluminate interacts directly with a G protein, the present results suggest that the cGMP interferes with platelet activation at the level of G protein-phospholipase C.

Forskolin and prostacyclin inhibit fluoride induced platelet activation and protein kinase C dependent responses

Platelet activation (cytosolic [Ca2+] increase, aggregation and ATP secretion) was induced with A1F-4. This agent presumably interacts with a G protein which appears to mediate the coupling of the receptors for Ca mobilizing hormones and phospholipase C. All the A1F-4 evoked responses were inhibited by treatment with forskolin or prostacyclin, agents known to increase cellular cAMP. Thus the G protein-phospholipase C system appears to be the site of cAMP inhibition. Unexpectedly forskolin and prostacyclin also inhibited secretion and aggregation induced by the activators of protein kinase C, diglyceride and phorbol ester, suggesting that cAMP can also inhibit directly the protein kinase C dependent responses.

Involvement of nuclear factor-kappa B (NF-κB) activation in mitogen-induced lymphocyte proliferation: inhibitory effects of lymphoproliferation by salicylates acting as NF-κB inhibitors

The transcription factor nuclear factor-kappa B (NF-kappaB) is involved in the production of inflammatory cytokines and in the control of the inflammatory response. Some nonsteroidal anti-inflammatory drugs such as acetylsalicylic acid (ASA) or salicylate are known to exert some of their anti-inflammatory pharmacological properties independently of cyclooxygenase inhibition. For ASA and salicylate, an NF-kappaB inhibitory effect at mM concentrations (pharmacological plasma concentrations reached in vivo) has been shown. We studied the action of ASA, salicylate, and several NF-kappaB inhibitors on the mitogen-induced activation of peripheral blood lymphocytes (PBL) and purified T cells. We showed that ASA and salicylate (1-3 mM) (but not indomethacin, a specific cyclooxygenase inhibitor) as well as a group of chemically unrelated inhibitors of NF-kappaB (including the sesquiterpene lactone parthenolide, Bay 11-7082, sulfasalazine, the proteasome inhibitor MG-132 and the peptide SN-50, an inhibitor of the nuclear transfer of the p50 subunit of NF-kappaB), were potent inhibitors of phytohemoagglutinin-activated PBL and T cell proliferation. At the same concentrations, they inhibited NF-kappaB binding to DNA in nuclear extracts. The inhibition of proliferation was not relieved by exogenous interleukin (IL)-2. We concluded that NF-kappaB activation has a fundamental role in T cell proliferation independently of IL-2 production. Some pharmacological actions of ASA may be ascribed to the inhibition of immune cell proliferation via the inhibition of the transcription factor NF-kappaB.

Synergic action of calcium ions and diamide on mitochondrial swelling.

Abstract The characteristics of rat liver mitochondria swelling induced by diamide, an oxidizing agent for thiol groups, and by Ca ions are very similar. In both cases the swelling, which is initiated by addition of 0.5–1 mM phosphate or acetate, is prevented by FCCP, antimycin A, EGTA, Mg ++ and ruthenium red. Diamide potentiates the swelling action of Ca ++ , while DTE potentiates that of Mg ++ . The additive effects of calcium and diamide on rat liver mitochondria have been correlated with their synergic action in promoting the release of mitochondrial Mg ++ . The results strongly indicate that some of the effects of diamide are mediated by a mobilization of endogenous divalent ions and that the antagonism between Ca ++ and Mg ++ is closely correlated with the redox state of membrane bound thiol groups.