Salvatore Passarella

Increase of proton electrochemical potential and ATP synthesis in rat liver mitochondria irradiated in vitro by helium-neon laser

Mitochondria Laser Transmembrane proton gradient Membrane potential ATP synthesis Oxygen uptake

Production of reactive oxygen species, alteration of cytosolic ascorbate peroxidase, and impairment of mitochondrial metabolism are early events in heat shock-induced programmed cell death in tobacco Bright-Yellow 2 cells

To gain some insight into the mechanisms by which plant cells die as a result of abiotic stress, we exposed tobacco (Nicotiana tabacum) Bright-Yellow 2 cells to heat shock and investigated cell survival as a function of time after heat shock induction. Heat treatment at 55°C triggered processes leading to programmed cell death (PCD) that was complete after 72 h. In the early phase, cells undergoing PCD showed an immediate burst in hydrogen peroxide (H2O2) and superoxide (O2·-) anion production. Consistently, death was prevented by the antioxidants ascorbate (ASC) and superoxide dismutase (SOD). Actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively, also prevented cell death, but with a lower efficiency. Induction of PCD resulted in gradual oxidation of endogenous ASC; this was accompanied by a decrease in both the amount and the specific activity of the cytosolic ASC peroxidase (cAPX). A reduction in cAPX gene expression was also found in the late PCD phase. Moreover, changes of cAPX kinetic properties were found in PCD cells. Production of ROS in PCD cells was accompanied by early inhibition of glucose (Glc) oxidation, with a strong impairment of mitochondrial function as shown by an increase in cellular NAD(P)H fluorescence, and by failure of mitochondria isolated from cells undergoing PCD to generate membrane potential and to oxidize succinate in a manner controlled by ADP. Thus, we propose that in the early phase of tobacco Bright-Yellow 2 cell PCD, ROS production occurs, perhaps because of damage of the cell antioxidant system, with impairment of the mitochondrial oxidative phosphorylation.

Glutamate neurotoxicity, oxidative stress and mitochondria

The excitatory neurotransmitter glutamate plays a major role in determining certain neurological disorders. This situation, referred to as ‘glutamate neurotoxicity’ (GNT), is characterized by an increasing damage of cell components, including mitochondria, leading to cell death. In the death process, reactive oxygen species (ROS) are generated. The present study describes the state of art in the field of GNT with a special emphasis on the oxidative stress and mitochondria. In particular, we report how ROS are generated and how they affect mitochondrial function in GNT. The relationship between ROS generation and cytochrome c release is described in detail, with the released cytochrome c playing a role in the cell defense mechanism against neurotoxicity.

Cytochrome c Is Released from Mitochondria in a Reactive Oxygen Species (ROS)-dependent Fashion and Can Operate as a ROS Scavenger and as a Respiratory Substrate in Cerebellar Neurons Undergoing Excitotoxic Death

In rat cerebellar granule cells both reactive oxygen species production and release of cytochrome c take place during glutamate toxicity. This investigation was aimed (i) to ascertain whether and how these two processes are related and (ii) to gain insight into the role played by the released cytochrome c in the onset of neurotoxicity. Cytochrome c release takes place owing to the generation of reactive oxygen species both in glutamate-treated cerebellar granule cells and in sister control cultures incubated in the presence of the reactive oxygen species-generating system consisting of xanthine plus xanthine oxidase. In the early phase of neurotoxicity (30-min glutamate exposure) about 40% of the maximum (as measured at 3 h of glutamate exposure) cytochrome c release was found to occur in cerebellar granule cells from mitochondria that were essentially coupled and intact and that had a negligible production of oxygen free radicals. Contrarily, mitochondria from cells treated with glutamate for 3 h were mostly uncoupled and produced reactive oxygen species at a high rate. The cytosolic fraction containing the released cytochrome c was able to transfer electrons from superoxide anion to molecular oxygen via the respiratory chain and was found to partially prevent glutamate toxicity when added externally to cerebellar neurons undergoing necrosis. In the light of these findings, we propose that in the early phase of neurotoxicity, cytochromec release can be part of a cellular and mitochondrial defense mechanism against oxidative stress.

Cytochrome c Is Released in a Reactive Oxygen Species-Dependent Manner and Is Degraded via Caspase-Like Proteases in Tobacco Bright-Yellow 2 Cells en Route to Heat Shock-Induced Cell Death

To gain some insight into the mechanism of plant programmed cell death, certain features of cytochrome c (cyt c) release were investigated in heat-shocked tobacco (Nicotiana tabacum) Bright-Yellow 2 cells in the 2- to 6-h time range. We found that 2 h after heat shock, cyt c is released from intact mitochondria into the cytoplasm as a functionally active protein. Such a release did not occur in the presence of superoxide anion dismutase and catalase, thus showing that it depends on reactive oxygen species (ROS). Interestingly, ROS production due to xanthine plus xanthine oxidase results in cyt c release in sister control cultures. Maximal cyt c release was found 2 h after heat shock; later, activation of caspase-3-like protease was found to increase with time. Activation of this protease did not occur in the presence of ROS scavenger enzymes. The released cyt c was found to be progressively degraded in a manner prevented by either the broad-range caspase inhibitor (zVAD-fmk) or the specific inhibitor of caspase-3 (AC-DEVD-CHO), which have no effect on cyt c release. In the presence of these inhibitors, a significant increase in survival of the cells undergoing programmed cell death was found. We conclude that ROS can trigger release of cyt c, but do not cause cell death, which requires caspase-like activation.

Specific helium-neon laser sensitivity of the purified cytochrome c oxidase.

Purpose : In order to gain some insight into the mechanism of interaction between Helium-Neon (He-Ne) laser light and mitochondrial cytochromes, the sensitivity of cytochrome electron transfer activity to He-Ne laser was tested. Materials and methods : Irradiation of solutions containing either purified cytochromes or dissolved rat liver mitochondria was carried out (wavelength 632.8nm, fluence rate 10 m W cm -2, fluence 2 J cm -2) ; the irradiation conditions were the ones able to affect cytochrome c oxidase (COX) activity in mitochondria (Pastore et al., 1994). Results : Cytochrome c oxidation catalysed by COX was affected by He-Ne laser irradiation of the purified enzyme. This result was obtained from measurements of the pseudo-first-order kinetic constant and from determinations of the turnover number of the enzyme, performed at different cytochrome c/COX ratios. Consistently, the kinetic parameters of COX changed. On the contrary, no alteration in the rate of electron transfer catalysed by either cytochrome c or bc 1 complex was found. Conclusions : This study shows that purified COX is a specific target of He-Ne laser light; therefore, COX may be considered to be a mitochondrial photo-acceptor.PURPOSE In order to gain some insight into the mechanism of interaction between Helium-Neon (He-Ne) laser light and mitochondrial cytochromes, the sensitivity of cytochrome electron transfer activity to He-Ne laser was tested. MATERIALS AND METHODS Irradiation of solutions containing either purified cytochromes or dissolved rat liver mitochondria was carried out (wavelength 632.8 nm, fluence rate 10 mW cm(-2), fluence 2 J cm(-2)); the irradiation conditions were the ones able to affect cytochrome c oxidase (COX) activity in mitochondria (Pastore et al., 1994). RESULTS Cytochrome c oxidation catalysed by COX was affected by He-Ne laser irradiation of the purified enzyme. This result was obtained from measurements of the pseudo-first-order kinetic constant and from determinations of the turnover number of the enzyme, performed at different cytochrome c/COX ratios. Consistently, the kinetic parameters of COX changed. On the contrary, no alteration in the rate of electron transfer catalysed by either cytochrome c or bc1 complex was found. CONCLUSIONS This study shows that purified COX is a specific target of He-Ne laser light; therefore, COX may be considered to be a mitochondrial photo-acceptor.

Increase in the ADP/ATP exchange in rat liver mitochondria irradiated in vitro by helium-neon laser.

To gain some insight into the mechanism of cell photostimulation by laser light, measurements were made of the rate of ADP/ATP exchange in mitochondria irradiated with the low power continuous wave Helium Neon laser (energy dose 5 Joules/cm2). To do this a method has been developed to continuously monitor ATP efflux from phosphorylating mitochondria caused by externally added ADP, by photometrically following the NADP+ reduction which occurs in the presence of glucose, hexokinase, glucose-6-phosphate dehydrogenase and effluxed ATP. The NADP+ reduction rate shows hyperbolic dependence on ADP concentration (Km and Vmax values 8.5 +/- 0.87 microM and 20.7 +/- 0.49 nmoles NADP+ reduced/min x mg mitochondrial protein, respectively), and proves to measure the activity of the ADP/ATP translocator as shown by inhibition experiments using atracyloside, powerful inhibitor of this carrier. Irradiation was found to enhance the rate of ADP/ATP antiport, with externally added ADP ranging between 5 and 100 microM. As a result of experiments carried out with mitochondria loaded with either ATP or ADP, the increase in the activity of the ADP/ATP translocator is here proposed to depend on the increase in the electrochemical proton gradient which occurs owing to irradiation of mitochondria.

The Existence of the K+ Channel in Plant Mitochondria

In this study, evidence is given that a number of isolated coupled plant mitochondria (from durum wheat, bread wheat, spelt, rye, barley, potato, and spinach) can take up externally added K+ ions. This was observed by following mitochondrial swelling in isotonic KCl solutions and was confirmed by a novel method in which the membrane potential decrease due to externally added K+ is measured fluorimetrically by using safranine. A detailed investigation of K+ uptake by durum wheat mitochondria shows hyperbolic dependence on the ion concentration and specificity. K+ uptake electrogenicity and the non-competitive inhibition due to either ATP or NADH are also shown. In the whole, the experimental findings reported in this paper demonstrate the existence of the mitochondrial K+ ATPchannel in plants (PmitoKATP). Interestingly, Mg2+ and glyburide, which can inhibit mammalian K+ channel, have no effect on PmitoKATP. In the presence of the superoxide anion producing system (xanthine plus xanthine oxidase), PmitoKATP activation was found. Moreover, an inverse relationship was found between channel activity and mitochondrial superoxide anion formation, as measured via epinephrine photometric assay. These findings strongly suggest that mitochondrial K+ uptake could be involved in plant defense mechanism against oxidative stress due to reactive oxygen species generation.

Mitochondria and l-lactate metabolism

We review here the novel insights arisen from investigations on l‐lactate metabolism in mammalian, plant and yeast mitochondria. The presence of l‐lactate dehydrogenases inside mitochondria, where l‐lactate enters in a carrier‐mediated fashion, suggests that mitochondria play an important role in l‐lactate metabolism. Functional studies have demonstrated the occurrence of several l‐lactate carriers. Moreover, immunological investigations have proven the existence of monocarboxylate translocator isoforms in mitochondria.

Riboflavin Uptake and FAD Synthesis in Saccharomyces cerevisiae Mitochondria INVOLVEMENT OF THE Flx1p CARRIER IN FAD EXPORT

We have studied the functional steps by which Saccharomyces cerevisiae mitochondria can synthesize FAD from cytosolic riboflavin (Rf). Riboflavin uptake into mitochondria took place via a mechanism that is consistent with the existence of (at least two) carrier systems. FAD was synthesized inside mitochondria by a mitochondrial FAD synthetase (EC 2.7.7.2), and it was exported into the cytosol via an export system that was inhibited by lumiflavin, and which was different from the riboflavin uptake system. To understand the role of the putative mitochondrial FAD carrier, Flx1p, in this pathway, an flx1Δ mutant strain was constructed. Coupled mitochondria isolated from flx1Δ mutant cells were compared with wild-type mitochondria with respect to the capability to take up Rf, to synthesize FAD from it, and to export FAD into the extramitochondrial phase. Mitochondria isolated from flx1Δ mutant cells specifically lost the ability to export FAD, but did not lose the ability to take up Rf, FAD, or FMN and to synthesize FAD from Rf. Hence, Flx1p is proposed to be the mitochondrial FAD export carrier. Moreover, deletion of the FLX1 gene resulted in a specific reduction of the activities of mitochondrial lipoamide dehydrogenase and succinate dehydrogenase, which are FAD-binding enzymes. For the flavoprotein subunit of succinate dehydrogenase we could demonstrate that this was not due to a changed level of mitochondrial FAD or to a change in the degree of flavinylation of the protein. Instead, the amount of the flavoprotein subunit of succinate dehydrogenase was strongly reduced, indicating an additional regulatory role for Flx1p in protein synthesis or degradation.