Biomedical subjects
Publications and source records attributed to A Alexandre.
The authors report several cases of inferior alveolar nerve injuries caused by endodontic cements. The surgical treatment which is suggested is the decompression of the nerve by microsurgical neurolysis.
We studied the action of the glutathione transferase substrate, 1-chloro-2,4-dinitrobenzene (CDNB) on the synaptosomal production of H2O2. We found that CDNB (30-40 microM) readily depletes the cytosolic glutathione but is almost without effect on the mitochondrial fraction. The depletion of the cytosolic glutathione induced by CDNB affords the detection in the extracellular space of H2O2 produced intrasynaptosomally upon increasing the cytosolic Ca2+ concentration that is otherwise destroyed by glutathione peroxidase. Higher concentrations of CDNB induce a H2O2 production which is not related to the glutathione content. This H2O2 is of mitochondrial origin and requires that NAD be reduced. The primary product of the mitochondrial CD-NB-dependent oxygen reduction is at least in part the superoxide anion.
We previously reported on the release of hydrogen peroxide from guinea pig cerebral cortex synaptosomes (13). An important finding was that in glutathione depleted synaptosomes a linear release of hydrogen peroxide is rapidly induced on addition of the Ca++ -ionophore ionomycin (in the presence of Ca++) or upon depolarization of the plasma membrane. We report here that the ionomycin induced hydrogen peroxide is reversed following the addition of bovine serum albumin which strongly binds the ionophore, to be reactivated by further addition of excess ionomycin, or of the depolarizing agent KC1. Similarly, the effect of ionomycin is removed on decreasing the concentration of free Ca++. Bovine serum albumin, which counteracts the effect of ionomycin on the release of H2O2, also counteracts the effect of the ionophore on the movements of Ca++ and the release of gamma-aminobutyrate. These findings support the idea that the synaptosomal production of H2O2 is a carefully controlled important physiological event.
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.
Guinea-pig cerebral cortex synaptosomes steadily release H2O2 into the suspending medium, at the rate of 20-30 pmol min-1 mg protein-1. A transient increase of the H2O2 release is induced by the addition of 1 mM Ca2+, which declines within 60-90 s to a rate identical or slightly higher than that before Ca2+. The extra H2O2 following Ca2+ addition varies between 40-100 pmol/mg protein and is insensitive to verapamil. The H2O2 release increases strongly (up to 250 pmol min-1 mg-1) upon depletion of the synaptosomal glutathione by treatment with 1-chloro-2,4-dinitrobenzene, a substrate for glutathione transferase. This treatment however has no effect on the Ca2+-induced H2O2 transient. In these treated synaptosomes a further increase of the output of H2O2 is rapidly induced upon addition of the Ca2+ ionophore ionomycin. This increase (about 100 pmol min-1 mg-1) lasts several minutes and requires the presence of Ca2+. A similar, though less pronounced increased H2O2 release is obtained (also in the absence of Ca2+) upon depolarization of the synaptosomal plasma membrane with KCl or with veratridine.
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.
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.
We present observations on the action of the phenolic antioxidants 2,3-tert-butyl-4-methoxyphenol (BHT); 2,6-di-tert-butyl-4-methylphenol (BHA); n-propyl-gallate and nordihydroguaiaretic acid (NDGA) on the function of guinea pig cerebral cortex synaptosomes. While the Ca2+ uptake observed under non depolarizing conditions is not affected by these agents, the depolarization induced Ca2+ uptake is strongly inhibited. Similarly the phenolic antioxidants studied inhibit the Ca2+ plus depolarization induced exocytosis of GABA and glutamate. These results which are similar to those previously obtained in blood platelets (Alexandre, A. et. al. Biochem. Biophys. Res. Comm. 139, 509-514, 1986), may indicate that free radical intermediates are involved in stimulus-secretion coupling.
The synthetic antioxidants butylated hydroxytoluene (BHT), nordihydroguaiaretic acid and the one-electron donor 1,1'-dimethylferrocene, inhibit cytosolic Ca++ increase, shape change, aggregation and ATP secretion in aspirinated washed human platelets stimulated by thrombin, vasopressin and platelet-activating factor. The antioxidants also inhibit cytosolic Ca++ increase originating from intracellular stores in the presence of EGTA. The effect of phorbol ester (TPA), which promotes platelet aggregation and secretion without raising the cytosolic Ca++, is also antioxidant-sensitive. Since agonist activation of aspirinated platelets does not involve cyclooxygenase or lipoxygenase metabolites, it is suggested that other yet unknown free radical-dependent pathways are involved in the mechanism of platelet activation, both in the protein kinase C-independent events leading to the cytosolic Ca++ increase, and in those, largely protein kinase C-dependent, leading to aggregation and ATP secretion.
The transport of Mg2+ into and out of guinea pig cerebral cortex synaptosomes was studied. Experiments are reported showing that the influx of Mg2+ (at low synaptosomal ATP) specifically requires a physiologically oriented Na+ gradient across the plasma membrane. It is also shown that an outwardly directed energy dependent Mg2+ pump is present in the synaptosomal plasma membrane; this pump, which requires ATP, is inhibited at high synaptosomal (Na+).
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Much evidence indicates that superoxide is generated from O2 in a cyanide-sensitive reaction involving a reduced component of complex III of the mitochondrial respiratory chain, particularly when antimycin A is present. Although it is generally believed that ubisemiquinone is the electron donor to O2, little experimental evidence supporting this view has been reported. Experiments with succinate as electron donor in the presence of antimycin A in intact rat heart mitochondria, which contain much superoxide dismutase but little catalase, showed that myxothiazol, which inhibits reduction of the Rieske iron-sulfur center, prevented formation of hydrogen peroxide, determined spectrophotometrically as the H2O2-peroxidase complex. Similarly, depletion of the mitochondria of their cytochrome c also inhibited formation of H2O2, which was restored by addition of cytochrome c. These observations indicate that factors preventing the formation of ubisemiquinone also prevent H2O2 formation. They also exclude ubiquinol, which remains reduced under these conditions, as the reductant of O2. Since cytochrome b also remains fully reduced when myxothiazol is added to succinate- and antimycin A-supplemented mitochondria, reduced cytochrome b may also be excluded as the reductant of O2. These observations, which are consistent with the Q-cycle reactions, by exclusion of other possibilities leave ubisemiquinone as the only reduced electron carrier in complex III capable of reducing O2 to O2-.
Two different bypasses around the antimycin block of electron transport from succinate to cytochrome c via the ubiquinol-cytochrome c oxidoreductase of intact rat liver mitochondria were analyzed, one promoted by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and the other by 2,6-dichlorophenolindophenol (DCIP). Both bypasses are inhibited by myxothiazol, which blocks electron flow from ubiquinol to the Rieske iron-sulfur center, and by 2-hydroxy-3-undecyl-1,4-naphthoquinone, which inhibits electron flow from the iron-sulfur center to cytochrome c1. In the bypass promoted by TMPD its oxidized form (Wurster's blue) acts as an electron acceptor from some reduced component prior to the antimycin block, which by exclusion of other possibilities is ubisemiquinone. In the DCIP bypass its reduced form acts as an electron donor, by reducing ubisemiquinone to ubiquinol; reduced DCIP is regenerated again at the expense of either succinate or ascorbate. The observations described are consistent with and support current models of the Q cycle. Bypasses promoted by artificial electron carriers provide an independent approach to analysis of electron flow through ubiquinol-cytochrome c oxidoreductase.
The mechanistic stoichiometry for vectorial H+ ejection coupled to electron transport through energy-conserving segments 1 + 2 was determined on cyanide-inhibited mitochondria from rat liver, rat heart, and Ehrlich ascites tumor cells, and on rat liver mitoplasts with ferricyanide or ferricytochrome c as electron acceptors. K+ (+ valinomycin) and Ca2+ were employed as permeant cations. Three different methods were employed. In the first, known pulses of ferricyanide were added, and the total H+ ejected was determined with a glass electrode. Such measurements gave H+/2e-values exceeding 7.0 for both normal and tumor mitochondria with beta-hydroxybutyrate and other NAD-linked substrates; uptake of Ca2+ was also measured and gave the expected q+/2e-ratios. The second type of measurement was initiated by addition of ferricytochrome c to rat liver mitoplasts, with H+ ejection monitored with the glass electrode and ferricytochrome c reduction by dual-wavelength spectrophotometry; the H+/2e-ratios generally exceeded 7.0. In the third type of measurement, mixing and dilution artifacts were eliminated by oxidizing ferrocytochrome c in situ with a small amount of ferricyanide. H+/2e-ratios for rat liver mitoplasts oxidizing beta-hydroxybutyrate consistently approached or exceeded 7.5. Over 150 measurements made under a variety of conditions gave observed H+/2e-ejection ratios significantly exceeding 7.0, which correlated closely with H+/2e-measurements on sites 1 + 2 + 3, sites 2 + 3, and site 2. Factors leading to the deficit of the observed ratios from the integral value 8 for sites 1 + 2 were discussed.
Neuropsychological outcome of 100 patients with severe head injuries is correlated with their clinical condition on admission. The aim of this paper is to estimate the reliability of several early indices of brain damage severity in formulating a prognosis. Both the Glasgow Coma Scale and the neurological syndrome involved appeared to be highly reliable in this respect. The duration of coma and of posttraumatic amnesia was not as significant. Since these two parameters were assessed when reviewing the cases, they have to be considered only as retrospective judgments. The findings emphasized the importance of reflex ocular motility and the value of recording electroencephalographic (EEG) activity, particularly EEG sleep patterns. The relationship between rapid eye movement sleep impairment and cognitive defects was highly significant. Analysis of surgical observations demonstrated that purely compressive expanding lesions were not as harmful as parenchymal damage. Of paramount importance was the time lapse between injury and surgery, since secondary lesions may develop and affect cerebral areas that are involved both in sleep organization and in memory functions.
The mechanistic stoichiometry of vectorial H+ ejection coupled to electron transport from added ferrocytochrome c to oxygen by the cytochrome oxidase (EC 1.9.3.1) of rat liver mitoplasts was determined from measurements of the initial rates of electron flow and H+ ejection in the presence of K+ (with valinomycin). Three different methods of measuring electron flow were used: (a) dual-wavelength spectrophotometry of ferrocytochrome c oxidation, (b) uptake of scalar H+ for the reduction of O2 in the presence of a protonophore, and (c) a fast-responding membraneless oxygen electrode. The reliability of the rate measurements was first established against the known stoichiometry of the scalar reaction of cytochrome oxidase (2ferrocytochrome c + 2H+ + 1/2O2 leads to 2ferricytochrome c + H2O) in the presence of excess protonophore. With all three methods the directly observed vectorial H+/O ejection ratios in the presence of K+ + valinomycin significantly exceeded 3.0. However, because the rate of backflow of the ejected H+ into the mitoplasts is very high and increases with the increasing delta pH generated across the membrane, there is a very rapid decline in the observed H+/O ratio from the beginning of the reaction. Kinetic analysis of ferrocytochrome c oxidation by the mitoplasts, carried out with a fast-responding membraneless oxygen electrode, showed the reaction to be first order in O2 and allowed accurate extrapolation of the rates of O2 uptake and H+ ejection to zero time. At this point, at which there is zero delta pH across the membrane, the H+/O ejection ratio of the cytochrome oxidase reaction, obtained from the rates at zero time, is close to 4.0.
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The location of the H+-translocating reactions within energy-conserving Site 2 of the mitochondrial electron transport chain was evaluated from two sets of data. In the first, the H+/2e- ejection ratios and Ca2+/2e- uptake ratios were compared for electron flow from succinate dehydrogenase, whose active site is on the matrix side of the inner membrane and from glycerol phosphate dehydrogenase, whose active site is on the cytosolic side. In intact rat liver mitochondria both substrates yielded H+/2e- ejection ratios close to 4.0 and Ca2+/2e- uptake ratios close to 1.0 during antimycin-sensitive reduction of ferricyanide. With rat liver mitoplasts and ferricytochrome c as electron acceptor, both substrates again gave the same stoichiometric ratios. The second approach involved determination of the sidedness of H+ formation during electron flow from succinate to ferricyanide via bypass of the antimycin block of the cytochrome b.c1 complex provided by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), under conditions in which the TMPD-TMPD+ couple does not act as a membrane-penetrating protonophore. Electron flow in this system was inhibited by 2-then-oyltrifluoroacetone, indicating that TMPD probably accepts electrons from ubiquinol. The 2 H+ formed in this system were not delivered into the matrix but appeared directly in the medium in the absence of a protonophore. To accommodate the available evidence on Site 2 substrates, it is concluded that the substrate hydrogens are first transferred to ubiquinone, 2 H+ per 2e then appear in the medium by protolytic dehydrogenation of a species of ubiquinol or ubiquinol-protein having the appropriate sidedness (designated Site 2A), and the other 2 H+ are translocated from the matrix to the medium on passage of 2e- through the cytochrome b x c1 complex (designated Site 2B).