Yuri V. Evtodienko

Mechanisms regulating cytochrome c release in pancreatic mitochondria

Background: Mechanisms of acinar cell death in pancreatitis are poorly understood. Cytochrome c release is a central event in apoptosis in pancreatitis. Here, we assessed the regulation of pancreatic cytochrome c release by Ca2+, mitochondrial membrane potential (ΔΨm), and reactive oxygen species (ROS), the signals involved in acute pancreatitis. We used both isolated rat pancreatic mitochondria and intact acinar cells hyperstimulated with cholecystokinin-8 (CCK-8; in vitro model of acute pancreatitis). Results: Micromolar amounts of Ca2+ depolarised isolated pancreatic mitochondria through a mechanism different from the “classical” (ie, liver) mitochondrial permeability transition pore (mPTP). In contrast with liver, Ca2+-induced mPTP opening caused a dramatic decrease in ROS and was not associated with pancreatic mitochondria swelling. Importantly, we found that Ca2+-induced depolarisation inhibited cytochrome c release from pancreatic mitochondria, due to blockade of ROS production. As a result, Ca2+ exerted two opposite effects on cytochrome c release: Ca2+ per se stimulated the release, whereas Ca2+-induced depolarisation inhibited it. This dual effect caused a non-monotonous dose-dependence of cytochrome c release on Ca2+. In intact acinar cells, cytochrome c release, caspase activation and apoptosis were all stimulated by ROS and Ca2+, and inhibited by depolarisation, corroborating the findings on isolated pancreatic mitochondria. Conclusions: These data implicate ROS as a key mediator of CCK-induced apoptotic responses. The results indicate a major role for mitochondria in the effects of Ca2+ and ROS on acinar cell death. They suggest that the extent of apoptosis in pancreatitis is regulated by the interplay between ROS, ΔΨm and Ca2+. Stabilising mitochondria against loss of ΔΨm may represent a strategy to mitigate the severity of pancreatitis.

Phosphorylation of a Peptide Related to Subunit c of the F0F1-ATPase/ATP Synthase and Relationship to Permeability Transition Pore Opening in Mitochondria

A phosphorylated polypeptide (ScIRP) from the inner membrane of rat liver mitochondria with an apparent molecular mass of 3.5 kDa was found to be immunoreactive with specific antibodies against subunit c of F0F1-ATPase/ATP synthase (Azarashvily, T. S., Tyynelä, J., Baumann, M., Evtodienko, Yu. V., and Saris, N.-E. L. (2000). Biochem. Biophys. Res. Commun. 270, 741–744. In the present paper we show that the dephosphorylation of ScIRP was promoted by the Ca2+-induced mitochondrial permeability transition (MPT) and prevented by cyclosporin A. Preincubation of ScIRP isolated in its dephosphorylated form with the mitochondrial suspension decreased the membrane potential (ΔΨM) and the Ca2+-uptake capacity by promoting MPT. Incorporation of ScIRP into black-lipid membranes increased the membrane conductivity by inducing channel formation that was also suppressed by antibodies to subunit c. These data indicate that the phosphorylation level of ScIRP is influenced by the MPT pore state, presumably by stimulation of calcineurin phosphatase by the Ca2+ used to induce MPT. The possibility of ScIRP being part of the MPT pore assembly is discussed in view of its capability to induced channel activity.

Calcium-induced permeability transition in rat brain mitochondria is promoted by carbenoxolone through targeting connexin43

Carbenoxolone (Cbx), a substance from medicinal licorice, is used for antiinflammatory treatments. We investigated the mechanism of action of Cbx on Ca(2+)-induced permeability transition pore (PTP) opening in synaptic and nonsynaptic rat brain mitochondria (RBM), as well as in rat liver mitochondria (RLM), in an attempt to identify the molecular target of Cbx in mitochondria. Exposure to threshold Ca(2+) load induced PTP opening, as seen by sudden Ca(2+) efflux from the mitochondrial matrix and membrane potential collapse. In synaptic RBM, Cbx (1 μM) facilitated the Ca(2+)-induced, cyclosporine A-sensitive PTP opening, while in nonsynaptic mitochondria the Cbx threshold concentration was higher. A well-known molecular target of Cbx is the connexin (Cx) family, gap junction proteins. Moreover, Cx43 was previously found in heart mitochondria and attributed to the preconditioning mechanism of protection. Thus, we hypothesized that Cx43 might be a target for Cbx in brain mitochondria. For the first time, we detected Cx43 by Western blot in RBM, but Cx43 was absent in RLM. Interestingly, two anti-Cx43 antibodies, directed against amino acids 252 to 270 of rat Cx43, abolished the Cbx-induced enhancement of PTP opening in total RBM and in synaptic mitochondria, but not in RLM. In total RBM and in synaptic mitochondria, PTP caused dephosphorylation of Cx43 at serine 368. The phosphorylation level of serine 368 was decreased at threshold calcium concentration and additionally in the combined presence of Cbx in synaptic mitochondria. In conclusion, active mitochondrial Cx43 appears to counteract the Ca(2+)-induced PTP opening and thus might inhibit the PTP-ensuing mitochondrial demise and cell death. Consequently, we suggest that activity of Cx43 in brain mitochondria represents a novel molecular target for protection.

High‐affinity peripheral benzodiazepine receptor ligand, PK11195, regulates protein phosphorylation in rat brain mitochondria under control of Ca2+

The effects of PK11195, a high‐affinity peripheral benzodiazepine receptor (PBR) ligand, on protein phosphorylation in isolated purified rat brain mitochondria were investigated. The isoquinoline carboxamide ligand of PBR, PK11195, but not the benzodiazepine ligand Ro5–4864, in the nanomolar concentration range strongly increased the phosphorylation of 3.5 and 17 kDa polypeptides. The effect of PK11195 was seen in the presence of elevated Ca2+ levels (3 × 10−7 to 10−6 m), but not at very low Ca2+ levels (10‐8 to 3 × 10−8 m). This indicates that PBR involves Ca2+ as a second messenger in the regulation of protein phosphorylation. Staurosporine, an inhibitor of protein kinase activity was able to suppress the PK11195‐promoted protein phosphorylation. When the permeability transition pore (PTP) was opened by threshold Ca2+ load, phosphorylation of the 3.5‐kDa polypeptide was diminished, but strong phosphorylation of the 43‐kDa protein was revealed. The 43‐kDa protein appears to be a PTP‐specific phosphoprotein. If PTP was opened, PK11195 did not increase the phosphorylation of the 3.5 and 17‐kDa proteins but suppressed the phosphorylation of the PTP‐specific 43‐kDa phosphoprotein. The ability of PK11195 to increase the protein phosphorylation, which was lost under Ca2+‐induced PTP opening, was restored again in the presence of calmidazolium, an antagonist of calmodulin and inhibitor of protein phosphatase PP2B. These results show a tight interaction of PBR with the PTP complex in rat brain mitochondria. In conclusion, a novel function of PBR in brain mitochondria has been revealed, and the PBR‐mediated protein phosphorylation has to be considered an important element of the PBR‐associated signal transducing cascades in mitochondria and cells.

INHIBITION BY CA2+ OF THE HYDROLYSIS AND THE SYNTHESIS OF ATP IN EHRLICH ASCITES TUMOUR MITOCHONDRIA: RELATION TO THE CRABTREE EFFECT

Phosphorylation of ADP and hydrolysis of ATP by isolated mitochondria from Ehrlich ascites tumour cells is greatly reduced when the mitochondria have been preloaded with Ca2+ (50 nmol/mg protein or more). Translocation of ADP is diminished in Ca(2+)-loaded mitochondria. However, ATPase in toluene-permeabilized mitochondria and in inside-out submitochondrial particles is also strongly inhibited by micromolar concentrations of Ca2+, indicating that, independently of adenine nucleotide transport, F1Fo-ATPase is also affected. ATP hydrolysis by submitochondrial particles depleted of the inhibitory subunit of F1Fo-ATPase (the Pullman-Monroy protein inhibitor) is insensitive to Ca2+; however, this sensitivity is restored when the particles are supplemented with the inhibitory subunit isolated from beef heart mitochondria. In view of the previous observations that glucose elicits in Ehrlich ascites tumour cells an increase of cytoplasmic free Ca2+ (Teplova, V.V., Bogucka, K., Czyz, A., Evtodienko, Yu.V., Duszyński, J. and Wojtczak, L. (1993) Biochem. Biophys. Res. Commun. 196, 1148-1154) and that this calcium is then taken up by mitochondria, resulting in a strong inhibition of coupled respiration (Evtodienko, Yu.V., Teplova, V.V., Duszyński, J., Bogucka, K. and Wojtczak, L. (1994) Cell Calcium 15, 439-446), the present results are discussed in terms of the mechanism of the Crabtree effect in tumour cells.

INHIBITION OF CATION EFFLUX BY ANTIOXIDANTS DURING OSCILLATORY ION TRANSPORT IN MITOCHONDRIA

It is well known that biological membranes are susceptible to lipid peroxidation [ 11. Effects of per- oxidative damage on energy-linked functions and membrane enzymes of mitochondria were reported [2]. In the presence of pro-oxidants isolated mito- chondria swell and lyse after incubation in salt media and concomitantly lipid peroxides were accumulated [3]. Inhibitory actions of antioxidants on these pro- cesses were noted [4]. Probably one of the earliest events following lipid peroxidation is an alteration in ionic permeability of the mitochondrial membrane. It was shown recently that during oscillations of ion fluxes in mitochondrial suspensions, periodic increases and decreases of membrane permeability and passive ion fluxes occur [5]. The obvious advan- tages of such a reversible system of mitochondrial ion transport prompted us to study the influence of different antioxidants on the process of mitochondrial oscillatory ion fluxes. The main effect of all anti- oxidants studied was the inhibition of ion efflux during the oscillatory cycle without significant influences on either active transport or oxidative phosphorylation 2.

The Ca2+ threshold for the mitochondrial permeability transition and the content of proteins related to Bcl-2 in rat liver and Zajdela hepatoma mitochondria.

Zajdela hepatoma mitochondria were able to accumulate two to five times more Ca2+ than rat liver mitochondria before the permeability transition was induced. Pulses of Ca2+ were given in series to determine the Ca2+ threshold by recording changes in [Ca2+] and membrane potential, the permeability transition causing the release of accumulated Ca2+ and collapse of the membrane potential. Hepatoma mitochondria had lower Ca2+ efflux rates, higher net Ca2+ uptake rates and lower phosphorylation rates than liver mitochondria. Since the differences in regard to induction of the permeability transition might be due to higher expression of the Bcl-2 protein in hepatoma cells than in hepatocytes, the transcription of Bcl-2 and the proteins reacting with a Bcl-2 polyclonal antiserum were estimated by Northern and Western blotting, respectively. Hepatoma cells had two Bcl-2 specific mRNA bands of 7 and 2.4 kb, and substantial amounts of the Bcl-2 protein, whereas in liver cells and mitochondria these were not detected. Both cell lines had a reactive band at 19-20 kDa, and hepatocytes a small band at 31-32 kDa. Bcl-2 antibodies stimulated the permeability transition potently in hepatoma mitochondria.

The stoichiometry of ion fluxes during Sr2+-induced oscillations in mitochondria

A quantitative study of H+, K+, Sr2+ and succinate fluxes in Sr2+-induced oscillatory state of rat liver mitochondria is presented. It was shown that oscillation of succinate content in mitochondria occurs synchronously with oscillations of the cation fluxes. Total charge transferred across the membrane by the registered cations and the succinate-anion is equal to zero. Passive H+-influx has been calculated at all stages of the oscillatory cycle. The conclusion is made that electroneutral 2 H+/Sr2+ exchange is periodically induced in mitochondria. A value of (2 +/- 0.2) . 10(-7) mol Sr2+/min per mg protein has been determined for Sr2+ by this type of exchange.

Involvement of periodic deacylation-acylation cycles of mitochondrial phospholipids during Sr2+-induced oscillatory ion transport in rat liver mitochondria

Lysophosphatidylcholine and lysophosphatidylethanolamine levels were determined during Sr2+-induced oscillating ion fluxes in mitochondria prelabelled in vivo with 32Pi. Periodic fluctuations of both lyso compounds were established with an increase at the stage of simultaneously monitored K+ influx and a decrease at K+ efflux. The periodic activations and inactivations of phospholipase were found to be associated with periodic changes in the incorporation rates of labelled polyunsaturated fatty acids with an apparent phase difference of 180 degrees. Periodic deacylation-acylation cycles of phospholipids accompanying the periodic cycles of reversible ion accumulation and release are suggested to be involved in the trigger mechanism generating the permeability changes during oscillatory ion transport.

Mechanisms of the resistance to the mitochondrial permeability transition in tumour cells

Abstract Tumour cell mitochondria often have a high Ca 2+ threshold for induction of the mitochondrial permeability transition (MPT). The mechanisms of inhibited MPT are briefly reviewed. MPT stimulates the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria. This is central in the signal pathways leading to apoptosis. Inhibited apoptosis may lead to excessive cell proliferation while induced apoptosis is the aim of cancer therapies. Also cell death by necrosis may result from impaired mitochondrial function due to MPT and the resulting decrease in cellular ATP levels. Many prooxidants trigger cell death by inducing MPT. Tumour cells may have greater resistance to prooxidants and free radicals formed also in radiation therapy. One mechanism for the inhibition of MPT could be increased expression of the Bcl-2 protein in tumour cells. We found this to be the case in Zajdela hepatoma mitochondria. Another mechanism could be the increased contents of Mg 2+ in tumour mitochondria, since Mg 2+ is an inhibitor of MPT. This was found to be the case in Ehrlich ascites tumour cell mitochondria. Other contributing factors could be inhibition of phospholipase A 2 by the Mg 2+ , changes in the amounts and properties of the adenine nucleotide translocator and/or mitochondrial ATP synthase.