Induction Of Permeability Transition Pore Research Articles

The Mitochondrial Effects of Small Organic Ligands of BCL-2: SENSITIZATION OF BCL-2-OVEREXPRESSING CELLS TO APOPTOSIS BY A PYRIMIDINE-2,4,6-TRIONE DERIVATIVE

We have investigated the mitochondrial effects of BH3I-2', Chelerythrine, and HA14-1, small organic molecules that share the ability to bind the BH3 domain of BCL-2. All compounds displayed a biphasic effect on mitochondrial respiration with uncoupling at low concentrations and respiratory inhibition at higher concentrations, the relative uncoupling potency being BH3I-2' (half-maximal uncoupling at about 80 nm) > Chelerythrine (half-maximal uncoupling at about 2 microm) > HA14-1 (half-maximal uncoupling at about 20 microm). At concentrations lower than required for uncoupling all compounds sensitized the permeability transition pore (PTP) to opening both in isolated mitochondria and intact cells. To assess whether the effects on BCL-2 binding, PTP induction and respiration could be due to different structural determinants we have tested a set of HA14-1 analogs from the Hoffmann-La Roche chemical library. We have identified 5-(6-chloro-2,4-dioxo-1,3,4,10-tetrahydro-2H-9-oxa-1,3-diaza-anthracen-10-yl)-pyrimidine-2,4,6-trione (EM20-25) as a molecule devoid of effects on respiration that is able to induce PTP opening, to disrupt the BCL-2/BAX interactions in situ and to activate caspase-9 in BCL-2-overexpressing cells. EM20-25 neutralized the antiapoptotic activity of overexpressed BCL-2 toward staurosporine and sensitized BCL-2-expressing cells from leukemic patients to the killing effects of staurosporine, chlorambucil, and fludarabine. These results provide a proof of principle that the potentially toxic effects of BCL-2 ligands on mitochondrial respiration are not essential for their antiapoptotic activity and represent an important step forward in the development of tumor-selective drugs acting on BCL-2.

Exercise training induces respiratory substrate-specific decrease in Ca2+-induced permeability transition pore opening in heart mitochondria

The purpose of this study was to determine whether regular exercise (treadmill running, 10 wk) alters the susceptibility of rat isolated heart mitochondria to Ca(2+)-induced permeability transition pore (PTP) opening and whether this could be associated with changes in the modulation of PTP opening by selected physiological effectors. Basal leak-driven and ADP-stimulated respiration in the presence of substrates for complex I, II, and IV were not affected by training. Fluorimetric studies revealed that in the control and exercise-trained groups, the amount of Ca(2+) required to trigger PTP opening was greater in the presence of complex II vs. I substrates (230 +/- 12 vs. 134 +/- 7 nmol Ca(2+)/mg protein, P < 0.01; pooled average of control and trained groups). In addition, with a substrate feeding the complex II, training increased by 45% (P < 0.01) the amount of Ca(2+) required to trigger PTP opening both in the presence and absence of the PTP inhibitor cyclosporin A. However, membrane potential, reactive oxygen species production, NAD(P)H ratio, and Ca(2+) uptake kinetics were not different in mitochondria from both groups. Together, these results suggest the existence of a substrate-specific regulation of the PTP in heart mitochondria and suggest that regular exercise results in a reduced sensitivity to Ca(2+)-induced PTP opening in presence of complex II substrates.

Physiological Ca 2+ level and Ca 2+-induced Permeability Transition Pore control protein phosphorylation in rat brain mitochondria

Phosphorylation of several low molecular mass proteins (3.5, 17, 23 and 29 kDa) was observed in rat brain mitochondria (RBM) at ATP concentration close to that in the mitochondrial matrix. Furthermore, regulatory effects of Ca 2+ on phosphorylation of these proteins were investigated. Protein phosphorylation was found to be modulated by Ca 2+ in the physiological concentration range (10 −8 to 10 −6 M free Ca 2+). Incorporation of 32 P from [ γ- 32 P ]ATP into the 17 kDa protein was dramatically increased within the 10 −7 to 10 −6 M free Ca 2+ range, whereas an opposite effect was observed for the 3.5 kDa polypeptide. Strong de-phosphorylation of the 3.5 kDa polypeptide and enhanced 32 P -incorporation into the 17 and 23 kDa proteins were found with supra-threshold Ca 2+ loads and these effects were eliminated or reduced in the presence of cyclosporin A, an inhibitor of Permeability Transition Pore (PTP) opening. In the presence of calmidazolium (Cmz), a calmodulin antagonist, enhanced levels of phosphorylation of the 17 and 3.5 kDa polypeptides were observed and the 17 kDa protein phosphorylation was suppressed by H-8, a protein kinase A inhibitor. It is concluded that Ca 2+ in physiological concentrations, as a second messenger, can control phosphorylation of the low molecular mass phospoproteins in RBM, in addition to well known regulation of some Krebs cycle dehydrogenases by Ca 2+. The protein phosphorylation was strongly dependent on the Ca 2+-induced PTP opening.

Improved Efficiency of Hepatic Mitochondrial Function in Rats with Cholestasis Induced by an Acute Dose of α-Naphthylisothiocyanate

Cholestasis is a feature of many chronic human liver diseases. Previous studies pointed out an impairment of mitochondrial function as a cause of hepatocyte dysfunction leading to cholestatic liver injury. This study was aimed to evaluate liver mitochondrial bioenergetics of α-naphthylisothiocyanate-treated Wistar rats, an experimental model of cholestasis. Serum markers of liver injury and endogenous adenine nucleotides were measured. Changes in membrane potential, mitochondrial respiration, and alterations in mitochondrial permeability transition pore induction were monitored. In rats injected with α-naphthylisothiocyanate, liver injury with cholestasis developed within 48 h, as indicated by both serum enzyme activities and total bilirubin concentration. Liver mitochondria isolated from α-naphthylisothiocyanate-treated rats had a higher state 3 respiration, respiratory control ratio, ADP/O, and endogenous ATP/ADP ratio compared to controls. No change in state 4 respiration was observed. Associated with these parameters, cholestatic mitochondria exhibited an increased resistance to disruption of mitochondrial calcium homeostasis due to permeability transition pore induction. Hepatic mitochondria isolated from α-naphthylisothiocyanate-treated rats exhibited an improved efficiency. These data indicate that an adaptive response to resist cell death occurs during α-naphthylisothiocyanate-induced acute cholestasis.

Dependence of permeability transition pore opening and cytochrome C release from mitochondria on mitochondria energetic status.

The dependence of Ca2+-induced permeability transition pore (PTP) opening and cytochrome c (Cyt. c) release from mitochondria on mitochondria energetic status was investigated. Results of test the PTP opening and Cyt. c release of isolated rat liver mitochondria with different mitochondrial respiratory substrates, electron transport inhibitors and uncoupler by spectrophotometry and western blotting showed that, Cyt. c release from mitochondria by PTP opening. PTP opening and Cyt. c release showed more sensitive and responsive with FADH-linked succinate than with NADH-linked glutamate plus malate as substrate. Partial or full inhibition of electron flow with electron flow inhibitors resulted in partial or full inhibition of PTP opening and Cyt. c release, respectively. Partial recovery of electron flow with electron sponsors resulted in partial recovery of PTP opening and Cyt. c release. PTP opening and Cyt. c release were completely interrupted by uncoupler. These results indicated PTP opening and Cyt. c release are characterized by respiratory substrate selectivity, electron flow dependence and energy coupling reliance. Hence, PTP opening and Cyt. c release tightly depend on mitochondria energetic status. These findings suggested also that it is possible to regulate apoptosis by altering mitochondrial energetic status to alter PTP opening and Cyt. c release.

Polycations induce the release of soluble intermembrane mitochondrial proteins

The release of proapoptotic proteins from the intermembrane space of mitochondria is an early critical step in many pathways to apoptosis. Induction of the mitochondrial permeability transition pore (PTP) was suggested to be the mechanism of the release of soluble mitochondrial intermembrane proteins (SIMP) in apoptosis. However, several studies suggested that proapoptotic proteins (e.g. Bax and Bid) can induce the release of SIMP (e.g. cytochrome c (cyt c) and adenylate kinase 2 (AK2)) in vivo and in vitro independent of PTP. We have found that a number of structurally diverse polycations, such as aliphatic polyamines (e.g. spermine and to a lesser extent spermidine), aminoglycosides (e.g. streptomycin, gentamicin and neomycin), and cytotoxic peptides (e.g. melittin), induce the release of SIMP from liver mitochondria, in vitro. All the polycations released AK2 together with cyt c, suggesting that rupture of the outer membrane is a common mechanism of cyt c release by these polycations. Several polycations (e.g. spermine, spermidine and neomycin) induced SIMP release without inducing significant swelling, and this release was not inhibited significantly by the PTP inhibitor cyclosporin. In contrast, under the same conditions, streptomycin and melittin induced swelling and SIMP release that was inhibited strongly by cyclosporin. Gentamicin-induced swelling and release of SIMP were partially inhibited by cyclosporin. The affinity of polyamines to the anionic phospholipids of the mitochondrial membranes (spermine=neomycin>gentamicin>streptomycin=spermidine) correlated roughly with their ability to induce PTP-independent release of SIMP, which suggests that the binding of polycations to the anionic phospholipids of the outer mitochondrial membrane facilitates the rupture of this membrane. However, some polycations facilitated the induction of PTP, possibly by binding to cardiolipin on the inner membrane. This dual mechanism may be relevant to the induction of SIMP release in apoptosis.

Bcl-2 does not inhibit the permeability transition pore in mouse liver mitochondria

The mechanism by which the mitochondrially-localized Bcl-2 protein inhibits apoptosis is still unclear. Some authors have proposed that apoptosis is dependent on induction of the mitochondrial permeability transition pore (PTP), and that activators of apoptosis such as Bax work through activation of PTP, whereas inhibitors of apoptosis such as Bcl-2 work through inhibition of PTP, and the consequent activation or inhibition of PTP-dependent release of mitochondrial apoptotic factors, including cytochrome c. PTP opening is classically measured by a light-scattering assay of large-amplitude swelling of rodent liver mitochondria in sucrose media. Thus to test the hypothesis that Bcl-2 inhibits either the PTP or the PTP-dependent release of cytochrome c, the rate and extent of PTP, and PTP-dependent release of cytochrome c were compared in liver mitochondria from control and Bcl-2 transgenic mice. We demonstrated that Bcl-2 protein was expressed to high levels in mitochondria of transgenics versus controls. We confirmed that while control mice undergo massive hepatic cell death upon exposure to anti-Fas antibody, the Bcl-2 transgenic livers were resistant, by the criteria of gross morphology, serum enzyme release, and caspase 3 activity. We purified mitochondria from livers of the Bcl-2 transgenics and measured PTP directly by the mitochondrial swelling assay. Purified mitochondria from both transgenics and controls were induced to undergo large-amplitude swelling that was dependent on the classical PTP inducers calcium ion (Ca 2+), t-butyl hydroperoxide (tBOOH) and atractyloside (Atr); and as expected, pretreatment of mitochondria with cyclosporin A (CsA) completely abolished mitochondrial swelling. However, there was no difference in the rate or final extent of PTP induction in Bcl-2 overexpressors versus control mitochondria. Furthermore, there was no difference in the PTP dependent release of cytochrome c from Bcl-2 overexpressors versus control mitochondria. Therefore, while we observe a strong inhibition of Fas-dependent apoptosis by Bcl-2 overexpression in mouse liver, we observe no effect of Bcl-2 overexpression on either the rate or extent of mitochondrial PTP, or upon the release of cytochrome c from mitochondria in which the PTP has been induced. The simplest explanation of these results is that Bcl-2 inhibits neither PTP nor PTP-dependent release of cytochrome c, however, other possibilities are discussed.

Bile acids affect liver mitochondrial bioenergetics: possible relevance for cholestasis therapy.

It has been pointed out that intracellular accumulation of bile acids cause hepatocyte injury in cholestatic disease process. This study was aimed to test if cytotoxicity of these compounds is mediated through mitochondria dysfunction. Bile acids effects on isolated rat liver mitochondrial were analyzed by monitoring changes in membrane potential and mitochondrial respiration, as well as alterations in H(+) membrane permeability and mitochondrial permeability transition pore induction. Increasing concentrations of the bile acids litocholic (LCA), deoxycholic (DCA), ursodeoxycholic (UDCA), chenodeoxycholic (CDCA), glycochenodeoxycholic (GCDC), or taurochenodeoxycholic (TCDC) decrease transmembrane potential (delta psi) developed upon succinate energization. These compounds also decreased state 3 respiration and enhanced state 4. We have also demonstrated that the observed concentration-dependent stimulation of state 4 by LCA, DCA, CDCA, TCDC, and GCDC, is associated with an enhanced permeability of mitochondria to H(+). Addition of LCA, DCA, CDCA, TCDC, GCDC, and UDCA to mitochondria energized with succinate resulted in a dose-dependent membrane depolarization and stimulation of mitochondrial permeability transition. Tauroursodeoxycholate (TUDC) elicited no significant effect on succinate-supported mitochondrial bioenergetics. In contrast, in the presence of glycoursodeoxycholic (GUDC), delta psi increases as a function of bile salt concentration. The results of this investigation demonstrate that at toxicologically relevant concentrations, most but not all bile acids alter mitochondrial bioenergetics, so impairment of mitochondrial function can be clinically relevant for patients with cholestasis.

The Ca 2+-induced permeability transition pore is involved in Ca 2+-induced mitochondrial oscillations : A study on permeabilised Ehrlich ascites tumour cells

The Ca 2+-induced permeability transition of the mitochondrial inner membrane was studied in digitonin-permeabilized Ehrlich ascites tumour cells respiring on succinate in an isotonic medium. Addition of a sufficient amount of Ca 2+ to induce an efflux of accumulated Ca 2+ from mitochondria produced an oscillatory state with periodically changing rates of respiration, transmembrane potential, ΔpH and direction of Ca 2+ fluxes. This contrasts with liver mitochondria in which only a Ca 2+ efflux is induced under these conditions. Addition of traces of cyclosporin A (approximately 0.1 nM) damped the oscillations by inhibiting the phase in which Ca 2+ efflux occurs and promoting the reestablishment of a higher transmembrane potential. Efflux was also prevented by addition of ATP or ADP, ATP being more potent. Efflux was also inhibited by low concentrations of spermine. It is concluded that Ca 2+-induced oscillations involve the cyclosporin A-sensitive pore and that the Ehrlich ascites tumour cell mitochondria differ from liver mitochondria in being far more sensitive to cyclosporin A and ATP. The possible physiological role of the oscillatory state is discussed.

Modulation of the mitochondrial cyclosporin A-sensitive permeability transition pore by the proton electrochemical gradient. Evidence that the pore can be opened by membrane depolarization.

This paper reports an investigation on the relationship between the proton electrochemical gradient (delta mu H+) and the cyclosporin A-sensitive permeability transition pore (PTP) in rat liver mitochondria. Using the SH group cross-linker phenylarsine oxide as the inducer, we show that both matrix pH and the membrane potential can modulate the process of PTP induction independently of Ca2+. We find that membrane depolarization induces the PTP per se when pHi is above 7.0, while at acidic matrix pH values PTP induction is effectively prevented. Since Ca2+ uptake leads to major modifications of the delta mu H+ (i.e. matrix alkalinization and membrane depolarization), we have explored the possibility that the Ca(2+)-induced changes of the delta mu H+ may contribute to PTP induction by Ca2+. Our data in mitochondria treated with Ca2+ plus N-ethylmaleimide and Ca2+ plus phosphate show that membrane depolarization is a powerful inducer of the PTP. Taken together, our observations indicate that the PTP can be controlled directly by the delta mu H+ both in the absence and presence of Ca2+, and suggest that a collapse of the membrane potential may be the cause rather than the consequence of PTP induction under many experimental conditions. Thus, many inducers may converge on dissipation of the membrane potential component of the delta mu H+ by a variety of mechanisms.

Modulation of the mitochondrial permeability transition pore. Effect of protons and divalent cations.

We have studied the induction of the mitochondrial cyclosporin A-sensitive permeability transition pore (PTP) by the bifunctional SH group reagent phenylarsine oxide (PhAsO). Addition of nanomolar concentrations of the electroneutral H(+)-K+ ionophore nigericin to nonrespiring mitochondria in sucrose medium determines a dramatic increase of the time required for PTP induction by PhAsO, while no effect of nigericin is apparent in KCl medium. Using mitochondria loaded with the internal pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, we show that the effect of nigericin is mediated by the ionophore-induced acidification of matrix pH. Indeed, experimental manipulation of pHi by a number of treatments indicates that PTP induction is directly related to matrix pH, in that the PTP induction process becomes slower as pHi decreases at constant pHo. PTP induction by PhAsO in respiration-inhibited mitochondria is stimulated by Ca2+ and inhibited by a series of divalent cations. Since PhAsO induces the PTP even in the presence of excess EGTA and in the absence of respiration (Lenartowicz, E., Bernardi, P., and Azzone, G.F. (1991) J. Bioenerg. Biomembr. 23, 679-688), we have been able to study the Ca2+ dependence of the induction process. We show that the apparent Km for Ca2+ activation is about 10(-5) M and that Ca2+, cyclosporin A, and inhibitory Me2+ ions behave as if they were competing for the same binding site(s) on the pore. Since similar results are obtained from patch-clamp experiments on the mitochondrial megachannel (Szabó, I., Bernardi, P., and Zoratti, M. (1992) J. Biol. Chem. 267, 2940-2946), we suggest that (i) the PTP and the mitochondrial megachannel are the same molecular structures and (ii) the same factors affect both the process of pore induction and its open-closed orientation.

Effect of physical conditioning on cardiac mitochondrial function.

ARTICLESEffect of physical conditioning on cardiac mitochondrial functionS. Penpargkul, A. Schwartz, and J. ScheuerS. Penpargkul, A. Schwartz, and J. ScheuerPublished Online:01 Dec 1978https://doi.org/10.1152/jappl.1978.45.6.978MoreSectionsPDF (2 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByExercise training induces respiratory substrate-specific decrease in Ca2+-induced permeability transition pore opening in heart mitochondriaMariannick Marcil, Karine Bourduas, Alexis Ascah, and Yan Burelle1 April 2006 | American Journal of Physiology-Heart and Circulatory Physiology, Vol. 290, No. 4Effects of β1- and β1 + β2-antagonists on training-induced myocardial hypertrophy and enzyme adaptationBiochemical Pharmacology, Vol. 36, No. 20Noninvasive assessment of changes in myocardial perfusion and ventricular performance following exercise trainingAmerican Heart Journal, Vol. 104, No. 2 More from this issue > Volume 45Issue 6December 1978Pages 978-986 Copyright & PermissionsCopyright © 1978 the American Physiological Societyhttps://doi.org/10.1152/jappl.1978.45.6.978PubMed730603History Published online 1 December 1978 Published in print 1 December 1978 Metrics