MMM1 encodes a mitochondrial outer membrane protein essential for establishing and maintaining the structure of yeast mitochondria

In the yeast Saccharomyces cerevisiae, mitochondria are elongated organelles which form a reticulum around the cell periphery. To determine the mechanism by which mitochondrial shape is established and maintained, we screened yeast mutants for those defective in mitochondrial morphology. One of these mutants, mmm1, is temperature- sensitive for the external shape of its mitochondria. At the restrictive temperature, elongated mitochondria appear to quickly collapse into large, spherical organelles. Upon return to the permissive temperature, wild-type mitochondrial structure is restored. The morphology of other cellular organelles is not affected in mmm1 mutants, and mmm1 does not disrupt normal actin or tubulin organization. Cells disrupted in the MMM1 gene are inviable when grown on nonfermentable carbon sources and show abnormal mitochondrial morphology at all temperatures. The lethality of mmm1 mutants appears to result from the inability to segregate the aberrant-shaped mitochondria into daughter cells. Mitochondrial structure is therefore important for normal cell function. Mmm1p is located in the mitochondrial outer membrane, with a large carboxyl-terminal domain facing the cytosol. We propose that Mmm1p maintains mitochondria in an elongated shape by attaching the mitochondrion to an external framework, such as the cytoskeleton.     

Factors affecting the reactivation of the mitochondrial adenosine 5'-triphosphatase and the release of ATPase inhibitor protein during and following the reenergization of mitochondria from ischemic cardiac muscle

In the present study we examined three factors affecting the reversal of the ischemia-induced inhibition of the mitochondrial ATPase described by us earlier (W. Rouslin (1983) J. Biol. Chem. 258, 9657-9661). These factors were the pH, the MgATP concentration, and the pCa of the medium in which mitochondria were sonicated following their reenergization in vitro. It was found that the extent of ATPase reactivation, on the one hand, and the extent of inhibitor protein release, on the other, following the reenergization in vitro and subsequent sonication of intact mitochondria isolated from 20-min-ischemic canine cardiac muscle were affected differently by each of the three factors studied. While raising the pH of the medium in which the mitochondria were sonicated subsequent to reenergization from approximately 7.0 to approximately 8.2 resulted in marked parallel increases in both ATPase reactivation and inhibitor protein release, lowering the pH of the medium to approximately 6.4 resulted in a marked decrease in ATPase reactivation but also in the apparent irreversible binding and/or denaturation of a portion of the ATPase inhibitor. Increasing the MgATP concentration of the sonication medium from zero to 2.0 mM resulted in approximately a one-third decrease in ATPase reactivation. The effect upon inhibitor release was more dramatic. MgATP at 2 mM decreased inhibitor release by approximately two-thirds. The pCa of the sonication medium was varied between 9.0 and 3.5 using Ca-ethylenebis(oxyethylenenitrilo)-tetraacetic acid (EGTA) buffers. Decreasing the pCa of the medium from 9.0 to 3.5 had a paradoxical effect. It resulted in increases both in ATPase reactivation and in the amount of inhibitor bound to the particles. Such a paradoxical effect may be explained if one assumes the existence of two kinds of inhibitor-enzyme interaction sites, namely, regulatory and nonregulatory binding sites. Thus, decreasing the pCa may decrease interaction at regulatory sites while enhancing interaction at nonregulatory inhibitor binding sites.     

Factors affecting the reactivation of the oligomycin-sensitive adenosine 5'-triphosphatase and the release of ATPase inhibitor protein during the re-energization of intact mitochondria from ischemic cardiac muscle

In the present study we examined factors affecting the reversal of the ischemia-induced protonic inhibition of the mitochondrial ATPase described earlier (Rouslin, W. (1983) J. Biol. Chem. 258, 9657-9661). It was found that ATPase reactivation and accompanying inhibitor protein release during the re-energization of intact mitochondria isolated from 20-min ischemic canine heart muscle could be blocked completely by either carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or nigericin but was unaffected by valinomycin at 35 mM K+. At higher K+ concentrations, valinomycin also blocked ATPase reactivation but not quite as completely as did nigericin. These observations suggest that ATPase reactivation and inhibitor protein release are particularly dependent upon either the trans-inner membrane pH gradient (delta pH) or possibly upon matrix pH per se and slightly less dependent upon membrane potential (delta psi) in intact cardiac muscle mitochondria. The addition of FCCP at the end of the re-energization incubations limited partially the extent of both ATPase reactivation and inhibitor protein release. This latter effect appears to have been mediated by a partial reassociation of the inhibitor protein with the enzyme, and it was accentuated (when FCCP was added at the end of the incubations) or mimicked (when FCCP was absent) by lowering the pH of the re-energization medium. A close examination of the first 10 min of the time course of enzyme activation and of inhibitor protein release revealed that while the former process was essentially finished in 1 min or less, the latter required approximately 10 min for completion. This observation led to the proposal of a two-site model of enzyme-inhibitor interaction which is discussed.     

Kinetics of Pi-Pi exchange in rat liver mitochondria. Rapid filtration experiments in the millisecond time range

Phosphate-phosphate exchange through the inorganic phosphate (Pi) carrier of rat liver mitochondria was investigated by a new rapid filtration technique, which does not require the use of transport inhibitors to stop the reaction and offers high time resolution (starting from 10 ms), thus allowing kinetic measurements on a fine time scale even at room temperature. At approximately 22 degrees C, isotopic equilibrium of [32P]Pi is achieved within 0.8-2.5 s--depending on the Pi concentration--and an initial linear phase, lasting for 400-500 ms, is observed. Complete inhibition of Pi exchange by an excess (33 nmol/mg) of mersalyl, a well-known organomercurial inhibitor, required 200 ms, pointing to the insufficiency of this reagent for effective inhibitor stop. On the other hand, investigation of the effect of mersalyl (allowed to react with mitochondria for at least 20 s) on the initial rate of Pi exchange supports earlier observations on the protective effect of this inhibitor; i.e., up to 3 nmol of mersalyl/mg of protein does not decrease the transport rate whereas these low concentrations protect approximately 50% of the transport capacity from irreversible inactivation by N-ethylmaleimide. In nonrespiring mitochondria, at pH 7.3, Pi exchange exhibited a Km of 1.6 mM and a Vmax of 3.0 mumol min-1 (mg of mitochondrial protein)-1. The increase of the membrane potential without any concomitant change of delta pH had no significant influence on the kinetic parameters. The maximal velocity of Pi transport is significantly higher than the maximal velocity of all the other components of oxidative phosphorylation at comparable temperatures. The possible physiological significance of this excess capacity is discussed.     

BH-3-only BIK functions at the endoplasmic reticulum to stimulate cytochrome c release from mitochondria

Stimulation of apoptosis by p53 is accompanied by induction of the BH-3-only proapoptotic member of the BCL-2 family, BIK, and ectopic expression of BIK in p53-null cells caused the release of cytochrome c from mitochondria and activation of caspases, dependent on a functional BH-3 domain. A significant fraction of BIK, which contains a predicted transmembrane segment at its COOH terminus, was found inserted in the endoplasmic reticulum (ER) membrane, with the bulk of the protein facing the cytosol. Restriction of BIK to this membrane by replacing its transmembrane segment with the ER-selective membrane anchor of cytochrome b(5) also retained the cytochrome c release and cell death-inducing activity of BIK. Whereas induction of cell death by BIK was strongly inhibited by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, the inhibitor was without effect on the ability of BIK to stimulate egress of cytochrome c from mitochondria. This benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone-insensitive pathway for stimulating cytochrome c release from mitochondria by ER BIK was successfully reconstituted in vitro and identified the requirement for components present in the light membrane (ER) and cytosol as necessary for this activity. Collectively, the results identify BIK as an initiator of cytochrome c release from mitochondria operating from a location at the ER.     

Bcl-x(L) blocks a mitochondrial inner membrane channel and prevents Ca2+ overload-mediated cell death

Apoptosis is an active process that plays a key role in many physiological and pathological conditions. One of the most important organelles involved in apoptosis regulation is the mitochondrion. An increase in intracellular Ca(2+) is a general mechanism of toxicity in neurons which occurs in response to different noxious stimuli like excitotoxicity and ischemia producing apoptotic and necrotic cell death through mitochondria-dependent mechanisms. The Bcl-2 family of proteins modulate the release of pro-apoptotic factors from the mitochondrial intermembrane space during cell death induction by different stimuli. In this work, we have studied, using single-cell imaging and patch-clamp single channel recording, the mitochondrial mechanisms involved in the neuroprotective effect of Bcl-x(L) on Ca(2+) overload-mediated cell death in human neuroblastoma SH-SY5Y cells. We have found that Bcl-x(L) neuroprotective actions take place at mitochondria where this antiapoptotic protein delays both mitochondrial potential collapse and opening of the permeability transition pore by preventing Ca(2+)-mediated mitochondrial multiple conductance channel opening. Bcl-x(L) neuroprotective actions were antagonized by the Bcl-x(L) inhibitor ABT-737 and potentiated by the Ca(2+) chelator BAPTA-AM. As a consequence, this would prevent free radical production, mitochondrial membrane permeabilization, release from mitochondria of pro-apoptotic molecules, caspase activation and cellular death.     

Absence of heat shock protein synthesis in isolated mitochondria and plastids from maize

Examination of the proteins synthesized by isolated mitochondria, chloroplasts, or proplastids from maize tissues showed that a heat treatment at 40 degrees C does not induce or enhance the synthesis of any protein when compared to preparations treated at the control temperature of 28 degrees C. These observations are consistent with the results obtained by labeling proteins in vivo under sterile conditions. In vivo labeling in the presence of cycloheximide during heat shock showed no heat shock protein synthesis. Labeling in the presence of chloramphenicol during heat shock showed a similar heat shock protein pattern as in the absence of the inhibitor. It is concluded that maize organelles do not synthesize heat shock proteins and that, if present, they may be due to bacterial contamination.     

Exit of proteins and fragments thereof from mitochondria is accelerated by the import of cytosolic synthesized proteins

Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria. Incubation of ³⁵S-methionine labeled mitochondria from rat hepatocytes with proteins synthesized in a cell-free system, using messenger RNA from rat liver, dramatically increased the release of mitochondrial proteins and fragments thereof into the medium. Since the synthesized proteins include cytosolic precursors of mitochondrial proteins, our results strongly suggest that import of proteins from the cytosol into mitochondria influences the half-life of proteins in these organelles. The use of this simple approach — i.e. combining the study of protein import and exit with mitochondria — to further clarify intracellular protein turnover and its regulation is suggested.     

The cytosol as site of phosphorylation of the cyclic AMP-dependent protein kinase in adrenal steroidogenesis

The mitochondria, the microsomes and the cystosol have been described as possible sites of cAMP-dependent phosphorylation. However, there has been no direct demonstration of a cAMP-dependent kinase associated with the activation of the side-chain cleavage of cholesterol. We have investigated the site of action of the cAMP-dependent kinase using a sensitive cell-free assay. Cytosol derived from cells stimulated with ACTH or cAMP was capable of increasing progesterone synthesis in isolated mitochondria when combined with the microsomal fraction. Cytosol derived from cyclase or kinase of negative mutant cells did not. Cyclic AMP and cAMP-dependent protein kinase stimulated in vitro a cytosol derived from unstimulated adrenal cells. This cytosol was capable of stimulating progesterone synthesis in isolated mitochondria. Inhibitor of cAMP-dependent protein kinase abolished the effect of the cAMP. ACTH stimulation of cytosol factors is a rapid process observable with a half maximal stimulation at about 3 pM ACTH. The effect was also abolished by inhibitor of arachidonic acid release. The function of cytosolic phosphorylation is still unclear. The effect of inhibitors of arachidonic acid release, and the necessity for the microsomal compartment in order to stimulate mitochondrial steroidogenesis, suggest that the factor in the cytosol may play a role in arachidonic acid release.     

Effect of Temperature on Respiration of Mitochondria and Shoot Segments from Cold hardened and Nonhardened Wheat and Rye Seedlings

The effect of temperature on respiration of mitochondria and tissue segments from three wheat (Triticum aestivum L.) and one rye (Secale cereale L.) cultivar grown at 2 and 24 C has been examined. Discontinuities in Arrhenius plots of respiratory activity against temperature were observed for mitochondria and tissue segments from seedlings grown at both temperatures. The rates of respiration decreased abruptly below the transition temperatures, resulting in increased energy of activation values for respiration. Transition temperatures were observed from 6 to 10 C during tissue segment respiration, and from 10 to 14 C during respiration by isolated mitochondria. Respiratory control and efficiency of phosphorylation were not affected markedly by either reaction temperature or growth temperature of the seedlings. No correlation was observed between the cold hardiness of the cultivars and the temperature at which structural transitions occurred in the mitochondria. Dry matter content of the seedlings increased markedly during growth at 2 C, but no appreciable changes in the levels of mitochondrial protein were observed. The results support the view that changes other than fatty acid unsaturation are involved in the abrupt change in mitochondrial membrane properties at low temperature.     

Characterization of tBid-induced cytochrome c release from mitochondria and liposomes

tBid, the cleaved form of Bid, can induce cytochrome c (Cyt. c) release from rat heart mitochondria more efficiently and reproducibly than that from liver or brain mitochondria. Unlike Bax, such release was not prevented by cyclosphorin A, an inhibitor of the opening of permeability transition pore. Carbonyl-cyanide m-chlorophenyl-hydrazone or oligomycin also have no obvious effect on the release of Cyt. c. In contrast to ceramide, tBid-mediated Cyt. c release from mitochondria is independent of the redox state of Cyt. c. Furthermore, Bid or tBid can directly trigger the efflux of encapsulated Cyt. c or trypsin within liposomes without involvement of other protein factors.     

Membranotropic effects of ω-hydroxypalmitic acid and Ca<Superscript>2+</Superscript> on rat liver mitochondria and lecithin liposomes. Aggregation and membrane permeabilization

The paper examines membranotropic Ca²⁺-dependent effects of ω-hydroxypalmitic acid (HPA), a product of ω-oxidation of fatty acids, on the isolated rat liver mitochondria and artificial membrane systems (liposomes). It was established that in the presence of Ca²⁺, HPA induced aggregation of liver mitochondria, which was accompanied by the release of cytochrome c from the organelles. It was further demonstrated that the addition of Ca²⁺ to HPA-containing liposomes induced their aggregation and/or fusion. Ca²⁺ also caused the release of the fluorescent dye sulforhodamine B from liposomes, indicating their permeabilization. HPA was shown to induce a high-amplitude swelling of Ca²⁺-loaded mitochondria, to decrease their membrane potential, to induce the release of Ca²⁺ from the organelles and to result in the oxidation of the mitochondrial NAD(P)H pool. Those effects of HPA were not blocked by the MPT pore inhibitor CsA, but were suppressed by the mitochondrial calcium uniporter inhibitor ruthenium red. The effects of HPA were also observed when Ca²⁺ was replaced with Sr²⁺ (but not with Ba²⁺ or Mg²⁺). A supposition is made that HPA can induce a Ca²⁺-dependent aggregation of mitochondria, as well as Ca²⁺dependent CsA-insensitive permeabilization of the inner mitochondrial membrane – with the subsequent lysis of the organelles.     

Lithocholic acid induces two different calcium-dependent inner membrane permeability systems in liver mitochondria

The effect of the most hydrophobic bile acid–lithocholic–as an inducer of two different Ca²⁺-dependent inner membrane permeability systems was studied on isolated rat liver mitochondria. It is shown that the addition of lithocholic acid at a concentration of 20 μM to the Ca²⁺-loaded mitochondria leads to swelling of the organelles, rapid release of Ca²⁺ from the matrix and almost complete collapse of Δψ. Mitochondrial pore blocker cyclosporin A (CsA) eliminates mitochondrial swelling but has no effect on the process of Ca²⁺ release and Δψ collapse. In the absence of Ca²⁺ lithocholic acid causes only a transient decrease of Δψ with subsequent complete recovery. Ruthenium red, inhibitor of mitochondrial Ca²⁺ uniporter, which blocks Ca²⁺ influx into the matrix, prevents mitochondrial swelling induced by lithocholic acid. At the same time, ruthenium red, which is added before lithocholic acid to the Ca²⁺-preloaded mitochondria, does not affect the swelling of the organelles but reduces the CsA-insensitive drop in Δψ. It is concluded that lithocholic acid is able to induce two Ca²⁺-dependent energy dissipation systems in the inner membrane of liver mitochondria: CsA-sensitive mitochondrial pore and CsA-insensitive permeability, which exhibits sensitivity to ruthenium red. It is found that the effect of this bile acid as an inductor of CsA-sensitive mitochondrial pore is not associated with the modulation of Pi effects. It is assumed that CsA-insensitive action of lithocholic acid is associated with the induction of Ca²⁺ efflux from the matrix in exchange for protons. In this case, the energy-dependent Ca²⁺ transport in the opposite direction with the participation of mitochondrial calcium uniporter sensitive to ruthenium red leads to the formation of calcium cycle and thereby to energy dissipation.     

Protein denaturation in intact hepatocytes and isolated cellular organelles during heat shock

There is circumstantial evidence that protein denaturation occurs in cells during heat shock at hyperthermic temperatures and that denatured or damaged protein is the primary inducer of the heat shock response. However, there is no direct evidence regarding the extent of denaturation of normal cellular proteins during heat shock. Differential scanning calorimetry (DSC) is the most direct method of monitoring protein denaturation or unfolding. Due to the fundamental parameter measured, heat flow, DSC can be used to detect and quantitate endothermic transitions in complex structures such as isolated organelles and even intact cells. DSC profiles with common features are obtained for isolated rat hepatocytes, liver homogenate, and Chinese hamster lung V79 fibroblasts. Five main transitions (A-E), several of which are resolvable into subcomponents, are observed with transition temperatures (Tm) of 45-98 degrees C. The onset temperature is approximately 40 degrees C, but some transitions may extend as low as 37-38 degrees C. In addition to acting as the primary signal for heat shock protein synthesis, the inactivation of critical proteins may lead to cell death. Critical target analysis implies that the rate limiting step of cell killing for V79 cells is the inactivation of a protein with Tm = 46 degrees C within the A transition. Isolated microsomal membranes, mitochondria, nuclei, and a cytosolic fraction from rat liver have distinct DSC profiles that contribute to different peaks in the profile for intact hepatocytes. Thus, the DSC profiles for intact cells appears to be the sum of the profiles of all subcellular organelles and components. The presence of endothermic transitions in the isolated organelles is strong evidence that they are due to protein denaturation. Each isolated organelle has an onset for denaturation near 40 degrees C and contains thermolabile proteins denaturing at the predicted Tm (46 degrees C) for the critical target. The extent of denaturation at any temperature can be approximately by the fractional calorimetric enthalpy. After scanning to 45 degrees C at 1 degree C/min and immediately cooling, a relatively mild heat shock, an estimated fraction denaturation of 4-7% is found in hepatocytes, V79 cells, and the isolated organelles other than nuclei, which undergo only 1% denaturation because of the high thermostability of chromatin. Thus, thermolabile proteins appear to be present in all cellular organelles and components, and protein denaturation is widespread and extensive after even mild heat shock.     

Detailing the role of Bax translocation, cytochrome c release, and perinuclear clustering of the mitochondria in the killing of HeLa cells by TNF

Induction of cell death in HeLa cells with TNF and cycloheximide (CHX) required an adequate ATP supply and was accompanied by decrease in intracellular pH, translocation of Bax, perinuclear clustering of the mitochondria, and cytochrome c release. The chloride channel inhibitor furosemide prevented the intracellular acidification, the translocation of Bax and the cell death. Cyclosporin A (CyA) or bongkrekic acid (BK) inhibited the induction of the MPT, the release of cytochrome c and the cell death without affecting the perinuclear clustering of the mitochondria or the translocation of Bax. Energy depletion with the ATP synthase inhibitor oligomycin or the uncoupler FCCP in the presence of 2-deoxy-glucose prevented the perinuclear clustering of the mitochondria and the cell killing. However, mitochondrial translocation of Bax was still observed. By contrast, cytochrome c was released in the oligomycin-treated cells but not in the same cells treated with FCCP. The data demonstrate that apoptosis in HeLa cells is ATP dependent and requires the translocation of Bax. The movement of Bax to the mitochondria occurs before and during the perinuclear clustering of these organelles and does not require the presence of ATP. The release of cytochrome c depends on the induction of the mitochondrial permeability transition but not ATP content.     

The course of etoposide-induced apoptosis from damage to DNA and p53 activation to mitochondrial release of cytochrome c

Treatment of L929 fibroblasts by the topoisomerase II inhibitor etoposide killed 50% of the cells within 72 h. The cell killing was preceded by the release of cytochrome c from the mitochondria. Simultaneous treatment of the cells with wortmannin, cycloheximide, furosemide, cyclosporin A, or decylubiquinone prevented the release of cytochrome c and significantly reduced the loss of viability. Etoposide caused the phosphorylation of p53 within 6 h, an effect prevented by wortmannin, an inhibitor of DNA-dependent protein kinase (DNA-PK). The activation of p53 by etoposide resulted in the up-regulation of the pro-apoptotic protein Bax, a result that was prevented by the protein synthesis inhibitor cycloheximide. The increase in the content of Bax was followed by the translocation of this protein from the cytosol to the mitochondria, an event that was inhibited by furosemide, a chloride channel inhibitor. Stably transfected L929 fibroblasts that overexpress Akt were resistant to etoposide and did not translocate Bax to the mitochondria or release cytochrome c. Bax levels in these transfected cells were comparable with the wild-type cells. The release of cytochrome c upon translocation of Bax has been attributed to induction of the mitochondrial permeability transition (MPT). Cyclosporin A and decylubiquinone, inhibitors of MPT, prevented the release of cytochrome c without affecting Bax translocation. These data define a sequence of biochemical events that mediates the apoptosis induced by etoposide. This cascade proceeds by coupling DNA damage to p53 phosphorylation through the action of DNA-PK. The activation of p53 increases Bax synthesis. The translocation of Bax to the mitochondria induces the MPT, the event that releases cytochrome c and culminates in the death of the cells.     

The ER-mitochondria interface: the social network of cell death

When cellular organelles communicate bad things can happen. Recent findings uncovered that the junction between the endoplasmic reticulum (ER) and the mitochondria holds a crucial role for cell death regulation. Not only does this locale connect the two best-known organelles in apoptosis, numerous regulators of cell death are concentrated at this spot, providing a terrain for intense signal transfers. Ca2+ is the most prominent signalling factor that is released from the ER and, at high concentration, mediates the transfer of an apoptosis signal to mitochondria as the executioner organelle for cell death. An elaborate array of checks and balances is fine-tuning this process including Bcl-2 family members. Moreover, MAMs, "mitochondria-associated membranes", are distinct membrane sections at the ER that are in close contact with mitochondria and have been found to exchange lipids and lipid-derived molecules such as ceramide for apoptosis induction. Recent work has also described a reverse transfer of apoptosis signals, from mitochondria to the ER, via cytochrome c release and prolonged IP3R opening or through the mitochondrial fission factor Fis1 and Bap31 at the ER, which form the ARCosome, a novel caspase-activation complex.     

31P NMR saturation-transfer study of the in situ kinetics of the mitochondrial adenine nucleotide translocase

The exchange of intramitochondrial ATP (ATP(in)) for extramitochondrial ATP (ATP(out)) was measured by using 31P NMR spectroscopy over a range of temperatures in isolated rat liver mitochondria oxidizing glutamate and succinate in the presence of external ATP but no added ADP (state 4). The rate of this exchange is more than an order of magnitude faster than rates reported previously that were determined by using isotopic techniques in the presence of oligomycin, the potent ATPase inhibitor. Differences are ascribed in part to the low levels of matrix ATP present in oligomycin-treated mitochondria. The addition of oligomycin to mitochondrial suspensions decreases intramitochondrial ATP levels from 17 +/- 3 (SEM) nmol/mg of protein in state 4 to 1.51 +/- 0.1 nmol/mg of protein in the presence of inhibitor at 8 degrees C. Simultaneously, transporter flux falls from 960 +/- 55 nmol/min.mg to undetectable levels (less than 300 nmol/min.mg). Although transport rates are much faster when measured by saturation-transfer than by conventional isotopic methods, the enthalpy values obtained by determining the effect of temperature on flux are very similar to those reported in the past that were determined by using isotopic techniques. Intramitochondrial ATP content regulates the rate of the ATP(in)/ATP(out) exchange. At 18 degrees C, the concentration of internal ATP that produces half-maximal transport rate is 6.6 +/- 0.12 nmol/mg of mitochondrial protein. The relationship between substrate concentration and flux is sigmoidal and is 90% saturated at 11.3 +/- 0.18 nmol/mg of mitochondrial protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estradiol-induced protection against ischemia-induced heart mitochondrial damage and caspase activation is mediated by protein kinase G

We have previously reported that estradiol can protect heart mitochondria from the ischemia-induced mitochondrial permeability transition pore-related release of cytochrome c and subsequent apoptosis. In this study we investigated whether the effect of 17-beta-estradiol on ischemia-induced mitochondrial dysfunctions and apoptosis is mediated by activation of signaling protein kinases in a Langendorff-perfused rat heart model of stop-flow ischemia. We found that pre-perfusion of non-ischemic hearts with 100 nM estradiol increased the resistance of subsequently isolated mitochondria to the calcium-induced opening of mitochondrial permeability transition pore and this was mediated by protein kinase G. Loading of the hearts with estradiol prevented ischemia-induced loss of cytochrome c from mitochondria and respiratory inhibition and these effects were reversed in the presence of the inhibitor of Akt kinase, NO synthase inhibitor L-NAME, guanylyl cyclase inhibitor ODQ and protein kinase G inhibitor KT5823. Estradiol prevented ischemia-induced activation of caspases and this was also reversed by KT5823. These findings suggest that estradiol may protect the heart against ischemia-induced injury activating the signaling cascade which involves Akt kinase, NO synthase, guanylyl cyclase and protein kinase G, and results in blockage of mitochondrial permeability transition pore-induced release of cytochrome c from mitochondria, respiratory inhibition and activation of caspases.