Agmatine prevents the Ca2+-dependent induction of permeability transition in rat brain mitochondria

The arginine metabolite agmatine is able to protect brain mitochondria against the drop in energy capacity by the Ca²⁺-dependent induction of permeability transition (MPT) in rat brain mitochondria. At normal levels, the amine maintains the respiratory control index and ADP/O ratio and prevents mitochondrial colloid-osmotic swelling and any electrical potential (ΔΨ) drop. MPT is due to oxidative stress induced by the interaction of Ca²⁺ with the mitochondrial membrane, leading to the production of hydrogen peroxide and, subsequently, other reactive oxygen species (ROS) such as hydroxyl radicals. This production of ROS induces oxidation of sulfhydryl groups, in particular those of two critical cysteines, most probably located on adenine nucleotide translocase, and also oxidation of pyridine nucleotides, resulting in transition pore opening. The protective effect of agmatine is attributable to a scavenging effect on the most toxic ROS, i.e., the hydroxyl radical, thus preventing oxidative stress and consequent bioenergetic collapse.     

Studies elucidating the mechanisms of calcium induced mitochondrial depolarization in individual isolated brain mitochondria

Among other mitochondrial functions, energy production and Ca²⁺ uptake are crucial for maintaining neuronal viability. Both of these functions are critically dependent on mitochondrial membrane potential (ΔΨ_m). Mitochondrial Ca²⁺ overload causing a dissipation of ΔΨ_m is a key component of several neuronal pathologies. However, the mechanism of Ca²⁺-induced depolarization in neuronal mitochondria remains unclear. Typically, ΔΨ_m has been evaluated as a single overall estimate from all mitochondria present in a given cell or tissue. However, recent data showed that the population of mitochondria isolated from tissues is not homogeneous, and averaged parameters from the whole population do not necessarily reflect the processes taking place in a single organelle. This review summarizes our recent studies of Ca²⁺-induced depolarization in individual mitochondria isolated from rat forebrain and immobilized to coverslips. Fluorescence imaging techniques and potentiometric fluorescent dyes were effectively used to study ΔΨ_m changes. The data have shown that Ca²⁺ triggers ΔΨ_m oscillations in brain mitochondria followed by a complete depolarization. Further investigation of this phenomenon led us to suggest that Ca²⁺-induced ΔΨ_m oscillations can represent an intermediate unstable state that may lead to irreversible mitochondrial dysfunction. Therefore, further study of this phenomenon would help to understand what causes the irreversible damage of mitochondria during cytosolic/mitochondrial Ca²⁺ overload. Here we discuss the effects of different modulators of the mitochondrial permeability transition pore on Ca²⁺-induced depolarization in brain mitochondria and in liver mitochondria, where the mechanism of Ca²⁺-depolarization is better understood. A comparison of these effects in brain and liver mitochondria led us to conclude that Ca²⁺ can induce reversible “low conductance” permeability transition in brain mitochondria, the phenomenon which requires a transient conformational change of the adenine nucleotide translocator from a specific transporter to a non-specific pore. The article is published in the original.     

Mechanism of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> death induced by <Emphasis Type="Italic">Cratylia mollis</Emphasis> seed lectin

Incubation of T. cruzi epimastigotes with the lectin Cramoll 1,4 in Ca²⁺ containing medium led to agglutination and inhibition of cell proliferation. The lectin (50 μg/ml) induced plasma membrane permeabilization followed by Ca²⁺ influx and mitochondrial Ca²⁺ accumulation, a result that resembles the classical effect of digitonin. Cramoll 1,4 stimulated (five-fold) mitochondrial reactive oxygen species (ROS) production, significantly decreased the electrical mitochondrial membrane potential (ΔΨ_m) and impaired ADP phosphorylation. The rate of uncoupled respiration in epimastigotes was not affected by Cramoll 1,4 plus Ca²⁺ treatment, but oligomycin-induced resting respiration was 65% higher in treated cells than in controls. Experiments using T. cruzi mitochondrial fractions showed that, in contrast to digitonin, the lectin significantly decreased ΔΨ_m by a mechanism sensitive to EGTA. In agreement with the results showing plasma membrane permeabilization and impairment of oxidative phosphorylation by the lectin, fluorescence microscopy experiments using propidium iodide revealed that Cramoll 1,4 induced epimastigotes death by necrosis.     

Mitochondrial Calcium Transport and Mitochondrial Dysfunction After Global Brain Ischemia in Rat Hippocampus

Here we report effect of ischemia-reperfusion on mitochondrial Ca²⁺ uptake and activity of complexes I and IV in rat hippocampus. By performing 4-vessel occlusion model of global brain ischemia, we observed that 15 min ischemia led to significant decrease of mitochondrial capacity to accumulate Ca²⁺ to 80.8% of control whereas rate of Ca²⁺ uptake was not significantly changed. Reperfusion did not significantly change mitochondrial Ca²⁺ transport. Ischemia induced progressive inhibition of complex I, affecting final electron transfer to decylubiquinone. Minimal activity of complex I was observed 24 h after ischemia (63% of control). Inhibition of complex IV activity to 80.6% of control was observed 1 h after ischemia. To explain the discrepancy between impact of ischemia on rate of Ca²⁺ uptake and activities of both complexes, we performed titration experiments to study relationship between inhibition of particular complex and generation of mitochondrial transmembrane potential (ΔΨ_m). Generation of a threshold curves showed that complex I and IV activities must be decreased by approximately 40, and 60%, respectively, before significant decline in ΔΨ_m was documented. Thus, mitochondrial Ca²⁺ uptake was not significantly affected by ischemia-reperfusion, apparently due to excess capacity of the complexes I and IV. Inhibition of complex I is favourable of reactive oxygen species (ROS) generation. Maximal oxidative modification of membrane proteins was documented 1 h after ischemia. Although enhanced formation of ROS might contribute to neuronal injury, depressed activities of complex I and IV together with unaltered rate of Ca²⁺ uptake are conditions favourable of initiation of other cell degenerative pathways like opening of mitochondrial permeability transition pore or apoptosis initiation, and might represent important mechanism of ischemic damage to neurones.     

Role of calcium in nitric oxide-induced programmed cell death in tobacco protoplasts

We tried to determine the mechanisms by which Ca²⁺ mediated NO-induced programmed cell death (PCD) in tobacco protoplasts. Treatment of tobacco protoplasts with the NO donor sodium nitroprusside (SNP) resulted in a rapid [Ca²⁺]_cyt accumulation and decrease in mitochondrial membrane potential (ΔΨ_m) before the appearance of PCD. NO-induced PCD could be largely prevented not only by NO scavenger c-PTIO, but also by EGTA (Ca²⁺ chelator), LaCl₃ (Ca²⁺-channel blocker) or CsA (a specific mitochondrial permeability transition pore inhibitor, which also inhibit Ca²⁺ cycling by mitochondria). All results suggested that NO-induced PCD is mediated through mitochondrial pathway and regulated by Ca²⁺.     

Resveratrol-induced mitochondrial dysfunction and apoptosis are associated with Ca2+ and mCICR-mediated MPT activation in HepG2 cells

Resveratrol, a natural polyphenolic antioxidant, has been reported to possess the cancer chemopreventive potential in wide range by means of triggering tumor cells apoptosis through various pathways. It induced apoptosis through the activation of the mitochondrial pathway in some kinds of cells. In the present reports, we showed that resveratrol-induced HepG2 cell apoptosis and mitochondrial dysfunction was dependent on the induction of the mitochondrial permeability transition (MPT), because resveratrol caused the collapse of the mitochondrial membrane potential (ΔΨ_m) with the concomitant release of cytochrome c (Cyt.c). In addition, resveratrol induced a rapid and sustained elevation of intracellular [Ca²⁺], which compromised the mitochondrial ΔΨ_m and triggered the process of HepG2 cell apoptosis. In permeabilized HepG2 cells, we further demonstrated that the effect of the resveratrol was indeed synergistic with that of Ca²⁺ and Ca²⁺ is necessary for resveratrol-induced MPT opening. Calcium-induced calcium release from mitochondria (mCICR) played a key role in mitochondrial dysfunction and cell apoptosis: (1) mCICR inhibitor, ruthenium red (RR), prevent MPT opening and Cyt.c release; and (2) RR attenuated resveratrol-induced HepG2 cell apoptotic death. Furthermore, resveratrol promotes MPT opening by lowering Ca²⁺-threshold. These data suggest modifying mCICR and Ca²⁺ threshold to modulate MPT opening may be a potential target to control cell apoptosis induced by resveratrol. Xuemei Tian—Foundation item: Chinese National Natural Science Foundation (No.30300455).     

Duality of effect of La3+ on mitochondrial permeability transition pore depending on the concentration

In order to explore the role of mitochondria in proliferation promotion and/or apoptosis induction of lanthanum, the mutual influences between La³⁺ and Ca²⁺ on mitochondrial permeability transition pore (PTP) opening were investigated with isolated mitochondria from rat liver. The experimental results revealed that La³⁺ influence the state of mitochondria in a concentration-dependent biphasic manner. La³⁺ in nanomolar concentrations, acting as a Ca²⁺ analog, entered mitochondrial matrix via the RuR sensitive Ca²⁺ channel and elevated ROS level, leading to opening of PTP indicated by mitochondrial swelling, reduction of ΔΨ_m and cytochrome c release. Inhibition of PTP with 10 μM CsA attenuated the effects of La³⁺. However, micromolar concentrations La³⁺ acted mainly as a Ca²⁺ antagonist, inhibiting PTP opening induced by Ca²⁺. We postulated that this action of La³⁺ on mitochondria through interaction with Ca²⁺ might be involved in the proliferation-promoting and apoptosis induction by La³⁺.     

Neuroprotective Effect of KB-R7943 Against Glutamate Excitotoxicity is Related to Mild Mitochondrial Depolarization

KB-R7943, an inhibitor of a reversed Na⁺/Ca²⁺ exchanger, exhibits neuroprotection against glutamate excitotoxicity. Taking into consideration that prolonged exposure of neurons to glutamate induces delayed calcium deregulation (DCD) and irreversible decrease of mitochondrial membrane potential (Δψ_mit), we examined the effect of KB-R7943 on glutamate and kainate-induced [Ca²⁺]_i and on Δψ_mit changes in rat cultured cerebellar granule neurons. 15 μmol/l KB-R7943 significantly delayed the onset of DCD in response to kainate but not in response to glutamate. In spite of [Ca²⁺]_i overload, KB-R7943 considerably improved the [Ca²⁺]_i recovery and restoration of Δψ_mit after glutamate and kainate washout and increased cell viability after glutamate exposure. In resting neurons, KB-R7943 induced a statistically significant decrease in Δψ_mit. KB-R7943 also depolarized isolated brain mitochondria and slightly inhibited mitochondrial Ca²⁺ uptake. These findings suggest that mild mitochondrial depolarization and diminution of Ca²⁺ accumulation in the organelles might contribute to neuroprotective effect of KB-R7943.     

Protective Effects of the Synthetic Cannabinoids CP55,940 and JWH-015 on Rat Brain Mitochondria upon Paraquat Exposure

The effects of cannabinoids in mitochondria after acute oxidative stress insult are not fully established. We investigated the ability of CP55,940 and JWH-015 to scavenge reactive oxygen species and their effect on mitochondria permeability transition (MPT) in either a mitochondria-free superoxide anion generation system, intact rat brain mitochondria or in sub-mitochondrial particles (SMP) treated with paraquat (PQ). Oxygen consumption, mitochondrial membrane potential (Δψ_m) and MPT were determined as parameters of mitochondrial function. It is found that both cannabinoids effectively attenuate mitochondrial damage against PQ-induced oxidative stress by scavenging anion superoxide radical (O₂ ^∙−) and hydrogen peroxide (H₂O₂), maintaining Δψ_m and by avoiding Ca²⁺-induced mitochondrial swelling. Understanding the mechanistic action of cannabinoids on mitochondria might provide new insights into more effective therapeutic approaches for oxidative stress related disorders.     

Mitochondria in Ca2+ Signaling and Apoptosis

Cellular Ca²⁺ signals are crucial in the control of most physiological processes, cell injuryand programmed cell death; mitochondria play a pivotal role in the regulation of such cytosolicCa²⁺ ([Ca²⁺]_c) signals. Mitochondria are endowed with multiple Ca²⁺ transport mechanismsby which they take up and release Ca²⁺ across their inner membrane. These transport processesfunction to regulate local and global [Ca²⁺]_c, thereby regulating a number of Ca²⁺-sensitivecellular mechanisms. The permeability transition pore (PTP) forms the major Ca²⁺ effluxpathway from mitochondria. In addition, Ca²⁺ efflux from the mitochondrial matrix occursby the reversal of the uniporter and through the inner membrane Na⁺/Ca²⁺ exchanger. Duringcellular Ca²⁺ overload, mitochondria take up [Ca²⁺]_c, which, in turn, induces opening of PTP,disruption of mitochondrial membrane potential (_m) and cell death. In apoptosis signaling,collapse of ;_m and cytochrome c release from mitochondria occur followed by activationof caspases, DNA fragmentation, and cell death. Translocation of Bax, an apoptotic signalingprotein from the cytosol to the mitochondrial membrane, is another step during thisapoptosis-signaling pathway. The role of permeability transition in the context of cell death in relationto Bcl-2 family of proteins is discussed.     

Role of Oxidative Stress,Apoptosis, and Intracellular Homeostasis in Primary Cultures of Rat Proximal Tubular Cells Exposed to Cadmium

Cadmium (Cd) is a known nephrotoxic element. In this study, the primary cultures of rat proximal tubular (rPT) cells were treated with low doses of cadmium acetate (2.5 and 5 μM) to investigate its cytotoxic mechanism. A progressive loss in cell viability, together with a significant increase in the number of apoptotic and necrotic cells, were seen in the experiment. Simultaneously, elevation of intracellular [Ca²⁺]i and reactive oxygen species (ROS) levels, significant depletion of mitochondrial membrane potential(Δ Ψ) and cellular glutathione (GSH), intracellular acidification, and inhibition of Na⁺, K⁺-ATPase and Ca²⁺-ATPase activities were revealed in a dose-dependent manner during the exposure, while the cellular death and the apoptosis could be markedly reversed by N-acetyl-l-cysteine (NAC). Also, the calcium overload and GSH depletion were significantly affected by NAC. In conclusion, exposure of rPT cells to low-dose cadmium led to cellular death, mediated by an apoptotic and a necrotic mechanism. The apoptotic death might be the chief mechanism, which may be mediated by oxidative stress. Also, a disorder of intracellular homeostasis induced by oxidative stress and mitochondrial dysfunction is a trigger of apoptosis in rPT cells.     

Sildenafil citrate concentrations not affecting oxidative phosphorylation depress H2O2 generation by rat heart mitochondria

Sildenafil citrate (Viagra) is a potent and specific inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5), which exhibits cardioprotective action against ischemia/reperfusion injury in intact and isolated heart. The mechanism of its cardioprotective action is not completely understood, but some results suggested that sildenafil exerts cardioprotection through the opening of mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP). However, the impact of sildenafil citrate per se on isolated heart mitochondrial function is unknown. The goal of this study was to investigate the influence of the compound on mitochondrial function (bioenergetics, Ca²⁺-induced mitochondrial permeability transition, and hydrogen peroxide (H₂O₂) generation) in an attempt to correlate its known actions with effects on heart mitochondria. It was observed that sildenafil citrate concentrations of up to 50 μM did not significantly affect glutamate/malate-supported respiration in states 2, 3, 4, oligomycin-inhibited state 3, and uncoupled respiration. The respiratory control ratio (RCR), the ADP to oxygen ratio (ADP/O), the transmembrane potential (ΔΨ), the phosphorylation rate, and the membrane permeability to H⁺, K⁺ and Ca²⁺ were not affected either. However, sildenafil citrate decreased H₂O₂ generation by mitochondria respiring glutamate/malate, and also decreased the formation of superoxide radical (O₂^•−) generated in a hypoxantine/xantine oxidase system. It was concluded that sildenafil citrate concentrations of up to 50 μM do not affect either rat heart mitochondrial bioenergetics or Ca²⁺-induced mitochondrial permeability transition, but it depresses H₂O₂ generation by acting as a superoxide dismutase (SOD)-mimetic. By preventing reactive oxygen species (ROS) generation, sildenafil citrate may preserve heart mitochondrial function.     

Gliclazide may have an antiapoptotic effect related to its antioxidant properties in human normal and cancer cells

Experimental and clinical studies suggest that gliclazide may protect pancreatic β-cells from apoptosis induced by an oxidative stress. However, the precise mechanism(s) of this action are not fully understood and requires further clarification. Therefore, using human normal and cancer cells we examined whether the anti-apoptotic effects of this sulfonylurea is due to its free radical scavenger properties. Hydrogen peroxide (H₂O₂) as a model trigger of oxidative stress was used to induce cell death. Our experiments were performed on human normal cell line (human umbilical vein endothelial cell line, HUVEC-c) and human cancer cell lines (human mammary gland cell line, Hs578T; human pancreatic duct epithelioid carcinoma cell line, PANC-1). To assess the effect of gliclazide the cells were pre-treated with the drug. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay was employed to measure the impact of gliclazide on cell viability. Generation of reactive oxygen species, mitochondrial membrane potential (∆Ψ_m), and intracellular Ca²⁺ concentration [Ca²⁺] were monitored. Furthermore, the morphological changes associated with apoptosis were determined using double staining with Hoechst 33258-propidium iodide (PI). Gliclazide protects the tested cells from H₂O₂-induced cell death most likely throughout the inhibition of ROS production. Moreover, the drug restored loss of ΔΨ_m and diminished intracellular [Ca²⁺] evoked by H₂O₂. Double staining with Hoechst 33258-PI revealed that pre-treatment with gliclazide diminished the number of apoptotic cells. Our findings indicate that gliclazide may protect both normal and cancer human cells against apoptosis induced by H₂O₂. It appears that the anti-apoptotic effect of the drug is most likely associated with reduction of oxidative stress.     

Amyloid beta-peptide promotes permeability transition pore in brain mitochondria

In this work the effect of the neurotoxic amino acid sequence, A_25–35, on brain mitochondrial permeability transition pore (PTP) was studied. For the purpose, the mitochondrial transmembrane potential (m), mitochondrial respiration and the calcium fluxes were examined. It was observed that A_25–35, in the presence of Ca²⁺, decreased the m, the capacity of brain mitochondria to accumulate calcium and led to a complete uncoupling of the respiration. However, the reverse sequence of the peptide A_25–35 (A_35–25) did not promote the PTP. The alterations promoted by A_35–25 and/or Ca²⁺ could be reversed when Ca²⁺ was removed by EGTA or when ADP plus oligomycin were present. The pre-treatment with CsA or ADP plus oligomycin prevented the m drop and preserved the capacity of mitochondria to accumulate Ca²⁺. These results suggest that A_25–35 can promote the PTP induced by Ca²⁺.     

Effects of Tl<Superscript>+</Superscript> on ion permeability,membrane potential and respiration of isolated rat liver mitochondria

It is known that permeability of the inner mitochondrial membrane is low to most univalent cations (K⁺, Na⁺, H⁺) but high to Tl⁺. Swelling, state 4, state 3, and 2,4-dinitrophenol (DNP)-stimulated respiration as well as the membrane potential (ΔΨ_mito) of rat liver mitochondria were studied in media containing 0–75 mM TlNO₃ either with 250 mM sucrose or with 125 mM nitrate salts of other monovalent cations (KNO₃, or NaNO₃, or NH₄NO₃). Tl⁺ increased permeability of the inner mitochondrial membrane to K⁺, Na⁺, and H⁺, that was manifested as stimulation of the swelling of nonenergized and energized mitochondria as well as via an increase of state 4 and dissipation of ΔΨ_mito. These effects of Tl⁺ increased in the order of sucrose <K⁺ <Na⁺ ≤ NH₄⁺. They were stimulated by inorganic phosphate and decreased by ADP, Mg²⁺, and cyclosporine A. Contraction of energized mitochondria, swollen in the nitrate media, was markedly inhibited by quinine. It suggests participation of the mitochondrial K⁺/H⁺ exchanger in extruding of Tl⁺-induced excess of univalent cations from the mitochondrial matrix. It is discussed that Tl⁺ (like Cd²⁺ and other heavy metals) increases the ion permeability of the inner membrane of mitochondria regardless of their energization and stimulates the mitochondrial permeability transition pore in low conductance state. The observed decrease of state 3 and DNP-stimulated respiration in the nitrate media resulted from the mitochondrial swelling rather than from an inhibition of respiratory enzymes as is the case with the bivalent heavy metals.     

The mechanism of lead-induced mitochondrial Ca2+ efflux

Addition of Pb²⁺ to rat kidney mitochondria is followed by induction of several reactions: inhibition of Ca²⁺ uptake, collapse of the transmembrane potential, oxidation of pyridine nucleotides, and a fast release of accumulated Ca²⁺. When the incubation media are supplemented with ruthenium red, the effect of Pb²⁺ on NAD(P)H oxidation, membrane , and Ca²⁺ release are not prevented if malate-glutamate are the oxidizing substrates; however, the latter two lead-induced reactions are prevented by ruthenium red if succinate is the electron donor. It is proposed that in mitochondria oxidizing NAD-dependent substrates, Pb²⁺ induces Ca²⁺ release by promoting NAD(P)H oxidation and a parallel drop in due to its binding to thiol groups, located in the cytosol side of the inner membrane. In addition, it is proposed that with succinate as substrate, the Ca²⁺-releasing effect of lead is due to the collapse of the transmembrane potential as a consequence of the uptake of Pb²⁺ through the calcium uniporter, since such effect is ruthenium red sensitive.     

High Bcl-2/Bax ratio in Walker tumor cells protects mitochondria but does not prevent H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced apoptosis via calcineurin pathways

It has been previously shown that Walker 256 tumor cells express a high content of the anti-apoptotic protein Bcl-2 which protects mitochondria against the damaging effects of Ca²⁺. In the present study, we analyze H₂O₂-induced apoptotic death in two different types of tumor cells: Walker 256 and SCC-25. Treatment with H₂O₂ (4mM) increased reactive oxygen species generation and the concentration of cytosolic free Ca²⁺. These alterations preceded apoptosis in both cell lines. In Walker cells, which show a high Bcl-2/Bax ratio, apoptosis was dependent on calcineurin activation and independent of changes in mitochondrial membrane potential (Δ < eqid1 > _m), as well as cytochrome c release. In contrast, in SCC-25 cells, which show a lower Bcl-2/Bax ratio, apoptosis was preceded by a decrease in Δ < eqid2 > _m, mitochondrial permeability transition, and cytochrome c release. Caspase-3 activation occurred in both cell lines. The data suggest that although the high Bcl-2/Bax ratio protected the mitochondria of Walker cells from oxidative stress, it was not sufficient to prevent apoptosis through calcineurin pathways.     

Molecular mechanisms of euplotin C-induced apoptosis: involvement of mitochondrial dysfunction,oxidative stress and proteases

The metabolite euplotin C (EC), isolated from the marine ciliate Euplotes crassus, is a powerful cytotoxic and pro-apoptotic agent in tumour cell lines. For instance, EC induces the rapid depletion of ryanodine Ca²⁺ stores, the release of cytochrome c from the mitochondria, and the activation of caspase-3, leading to apoptosis. The purpose of this study was to gain further insight into the mechanisms of EC-induced apoptosis in rat pheochromocytoma PC12 cells. We found that EC increases Bax/Bcl-2 ratio and that Bax is responsible of the EC-induced dissipation of the mitochondrial membrane potential (Δψ_m). In addition, EC induces the generation of reactive oxygene species (ROS) without involvement of p53. The inhibition of ROS generation prevents, at least in part, the pro-apoptotic effects of EC as well as the effects of EC on Bax, Δψ_m and intracellular free Ca²⁺, indicating a cross-talk between different pathways. However, definition of the effector cascade turns out to be more complex than expected and caspase-independent mechanisms, acting in parallel with caspases, should also be considered. Among them, EC increases the expression/activity of calpains downstream of ROS generation, although calpains seem to exert protective effects. D. Cervia and M. Garcia-Gil equally contributed to the work.     

Mitochondria from the salt-tolerant yeast <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis> (halophilic organelles?)

The yeast Debaryomyces hansenii is considered a marine organism. Sea water contains 0.6 M Na⁺ and 10 mM K⁺; these cations permeate into the cytoplasm of D. hansenii where proteins and organelles have to adapt to high salt concentrations. The effect of high concentrations of monovalent and divalent cations on isolated mitochondria from D. hansenii was explored. As in S. cerevisiae, these mitochondria underwent a phosphate-sensitive permeability transition (PT) which was inhibited by Ca²⁺ or Mg²⁺. However, D. hansenii mitochondria require higher phosphate concentrations to inhibit PT. In regard to K⁺ and Na⁺, and at variance with mitochondria from all other sources known, these monovalent cations promoted closure of the putative mitochondrial unspecific channel. This was evidenced by the K⁺/Na⁺-promoted increase in: respiratory control, transmembrane potential and synthesis of ATP. PT was equally sensitive to either Na⁺ or K⁺. In the presence of propyl-gallate PT was still observed while in the presence of cyanide the alternative pathway was not active enough to generate a ΔΨ due to a low AOX activity. In D. hansenii mitochondria K⁺ and Na⁺ optimize oxidative phosphorylation, providing an explanation for the higher growth efficiency in saline environments exhibited by this yeast.     

Relationship between endoplasmic reticulum- and Golgi-associated calcium homeostasis and 4-NQO-induced DNA repair in Saccharomyces cerevisiae

Calcium (Ca²⁺) is an important ion that is necessary for the activation of different DNA repair mechanisms. However, the mechanism by which DNA repair and Ca²⁺ homeostasis cooperate remains unclear. We undertook a systems biology approach to verify the relationship between proteins associated with Ca²⁺ homeostasis and DNA repair for Saccharomyces cerevisiae. Our data indicate that Pmr1p, a Ca²⁺ transporter of Golgi complex, interacts with Cod1p, which regulates Ca²⁺ levels in the endoplasmic reticulum (ER), and with Rad4p, which is a nucleotide excision repair (NER) protein. This information was used to construct single and double mutants defective for Pmr1p, Cod1p, and Rad4p followed by cytotoxic, cytostatic, and cell cycle arrest analyses after cell exposure to different concentrations of 4-nitroquinoline 1-oxide (4-NQO). The results indicated that cod1Δ, cod1Δrad4Δ, and cod1Δpmr1Δ strains have an elevated sensitivity to 4-NQO when compared to its wild-type (WT) strain. Moreover, both cod1Δpmr1Δ and cod1Δrad4Δ strains have a strong arrest at G₂/M phases of cell cycle after 4-NQO treatment, while pmr1Δrad4Δ have a similar sensitivity and cell cycle arrest profile when compared to rad4Δ after 4-NQO exposure. Taken together, our results indicate that deletion in Golgi- and ER-associated Ca²⁺ transporters affect the repair of 4-NQO-induced DNA damage.