Regulation of cyclosporin A sensitive mitochondrial permeability transition by the redox state of pyridine nucleotides

The mechanisms involved in the induction of cyclosporine A sensitive mitochondrial swelling by oxidative stress were investigated in isolated guinea pig liver mitochondria. The aim of our study was to investigate, if swelling is inevitably associated with the oxidation of pyridine nucleotides, and if the oxidized pyridine nucleotides have to be hydrolysed for the induction of mitochondrial swelling. Quantitative measurement of oxidized pyridine nucleotides was performed with HPLC. Mitochondrial swelling was recorded by monitoring the decrease in light scattering of the mitochondrial suspension. Reduction and oxidation of pyridine nucleotides were followed by monitoring the changes of the autofluorescence signal of reduced pyridine nucleotides. Qualitative measurement of mitochondrial membrane potential was performed with the fluorescence indicator rhodamine 123. Neither t-butyl hydroperoxide nor the dissipation of the mitochondrial inner membrane potential with FCCP (carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone) induced the opening of the membrane permeability transition pore, unless an extensive oxidation of mitochondrial pyridine nucleotides took place. Mitochondrial swelling induced by our experimental conditions was always sensitive to cyclosporine A and accompanied by a cyclosporine A sensitive release of inner mitochondrial pyridine nucleotides without pyridine nucleotide hydrolysis. Not the cycling of calcium across the mitochondrial inner membrane but the accumulation of calcium inside the mitochondria was a prerequisite for mitochondrial swelling. The mitochondrial membrane permeability transition is neither caused nor accompanied by the hydrolysis of mitochondrial pyridine nucleotides.     

Carbon monoxide, oxidative stress, and mitochondrial permeability pore transition

The cellular effects of carbon monoxide (CO) are produced primarily by CO binding to iron or other transition metals, which may also promote prooxidant activities of the more reactive gases, oxygen and nitric oxide. We tested the hypothesis that prooxidant effects of CO deregulate the calcium-dependent mitochondrial pore transition (MPT), which disrupts membrane potential and releases apoptogenic proteins. Rats were exposed to either CO (50 ppm) or hypobaric hypoxia (HH) for 1, 3, or 7 days, and liver mitochondria harvested to study protein expression and sensitivity to MPT by calcium and oxidants. Both exposures induced hypoxia-sensitive protein expression: hypoxia-inducible factor 1alpha (HIF-1alpha), heme oxygenase-1 (HO-1), and manganese SOD (SOD2), but SOD2 induction was greater by CO than by HH, especially at 7 days. Relative to HH, CO also caused significant early mitochondrial oxidative and nitrosative stress shown by decreases in GSH/GSSG and increases in protein 3-nitrotyrosine (3-NT) and protein mixed disulfide formation. This altered MPT sensitivity to calcium through an effect on the "S-site," causing loss of pore protection by adenine nucleotides. By 7 days, despite continued CO, nitrosative stress decreased and adenine nucleotide protection was restored to preexposure levels. This is the first evidence of functional mitochondrial pore stress caused by CO independently of its hypoxic effect, as well as a compensatory response exemplifying a mitochondrial phenotype shift. The implications are that cellular CO can activate or deactivate mitochondria for initiation of apoptosis in vivo.     

The participation of pyridine nucleotides redox state and reactive oxygen in the fatty acid-induced permeability transition in rat liver mitochondria

The ability of low concentrations (5-15 microM) of long-chain fatty acids to open the permeability transition pore (PTP) in Ca(2+)-loaded mitochondria has been ascribed to their protonophoric effect mediated by mitochondrial anion carriers, as well as to a direct interaction with the pore assembly [M.R. Wieckowski and L. Wojtczak, FEBS Lett. 423 (1998) 339-342]. Here, we have compared the PTP opening ability of arachidonic acid (AA) with that of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) at concentrations that cause similar quantitative dissipation of the membrane potential (DeltaPsi) in Ca(2+)-loaded rat liver mitochondria respiring on succinate. The initial protonophoric effects of AA and FCCP were only slightly modified by carboxyatractyloside and were followed by PTP opening, as indicated by a second phase of DeltaPsi disruption sensitive to EGTA, ADP, dithiothreitol and cyclosporin A. This second phase of DeltaPsi dissipation could also be prevented by rotenone or NAD(P)H-linked substrates which decrease the pyridine nucleotide (PN) oxidation that follows the stimulation of oxygen consumption induced by AA or FCCP. These results suggest that, under the experimental conditions used here, the PTP opening induced by AA or FCCP was a consequence of PN oxidation. Exogenous catalase also inhibited both AA- and FCCP-induced PTP opening. These results indicate that a condition of oxidative stress associated with the oxidized state of PN underlies membrane protein thiol oxidation and PTP opening.     

Sulforaphane inhibits mitochondrial permeability transition and oxidative stress

Exposure of mitochondria to oxidative stress and elevated Ca²⁺ promotes opening of the mitochondrial permeability transition pore (PTP), resulting in membrane depolarization, uncoupling of oxidative phosphorylation, and potentially cell death. This study tested the hypothesis that treatment of rats with sulforaphane (SFP), an activator of the Nrf2 pathway of antioxidant gene expression, increases the resistance of liver mitochondria to redox-regulated PTP opening and elevates mitochondrial levels of antioxidants. Rats were injected with SFP or drug vehicle and liver mitochondria were isolated 40 h later. Respiring mitochondria actively accumulated added Ca²⁺, which was then released through PTP opening induced by agents that either cause an oxidized shift in the mitochondrial redox state or directly oxidize protein thiol groups. SFP treatment of rats inhibited the rate of pro-oxidant-induced mitochondrial Ca²⁺ release and increased expression of the glutathione peroxidase/reductase system, thioredoxin, and malic enzyme. These results are the first to demonstrate that SFP treatment of animals increases liver mitochondrial antioxidant defenses and inhibits redox-sensitive PTP opening. This novel form of preconditioning could protect against a variety of pathologies that include oxidative stress and mitochondrial dysfunction in their etiologies.     

The mitochondrial megachannel is the permeability transition pore

Single-channel electrophysiological recordings from rat liver mitoplast membranes showed that the 1.3-nS mitochondrial megachannel was activated by Ca⁺⁺ and inhibited by Mg⁺⁺, Cyclosporin A, and ADP, probably acting at matrix-side sites. These agents are known to modulate the so-called mitochondrial permeability transition pore (Gunter, T. E., and Pfeiffer, D. R. (1990)Am. J. Physiol. 258, C755–C786) in the same manner. Furthermore, the megachannel is unselective, and the minimum pore size calculated from its conductance is in agreement with independent estimates of the minimum size of the permeabilization pore. The results support the tentative identification of the megachannel with the pore believed to be involved in the permeabilization process.Abbreviations used: PT: permeability transition; PTP: permeability transition pore; MMC: mitochondrial megachannel; IMAC: inner membrane anion channel. P_A: permeability of ion A. CSP: Cyclosporin A.     

Developmental changes of the sensitivity of cardiac and liver mitochondrial permeability transition pore to calcium load and oxidative stress

Opening of the mitochondrial membrane permeability transition pore (MPTP) is an important factor in the activation of apoptotic and necrotic processes in mammalian cells. In a previous paper we have shown that cardiac mitochondria from neonatal rats are more resistant to calcium load than mitochondria from adult animals. In this study we have analyzed the ontogenetic development of this parameter both in heart and in liver mitochondria. We found that the high resistance of heart mitochondria decreases from day 14 to adulthood. On the other hand, we did not observe a similar age-dependent sensitivity in liver mitochondria, particularly in the neonatal period. Some significant but relatively smaller increase could be observed only after day 30. When compared with liver mitochondria cardiac mitochondria were more resistant also to the peroxide activating effect on calcium-induced mitochondrial swelling. These data thus indicate that the MPTP of heart mitochondria is better protected against damaging effects of the calcium load and oxidative stress. We can only speculate that the lower sensitivity to calcium-induced swelling may be related to the higher ischemic tolerance of the neonatal heart.     

The redox state of pyridine nucleotides controls permeability of uncoupled mitochondria to K+

Heart mitochondria respiring in the presence of P_i release endogenous K⁺ to a sucrose medium when an uncoupler is added. The uncoupled mitochondria retain K⁺, however, if the oxidation of NAD(P)H is prevented by the addition of rotenone or antimycin. Addition of rotenone, once the uncoupler-dependent K⁺-efflux has been initiated, results in a rapid reduction of NAD(P) and a simultaneous decrease in permeability to K⁺. These changes are independent of respiration. The results suggest that a latent pathway for K⁺-permeability is present in the membrane, that it can be opened and closed reversibly, and that it reflects, either directly or indirectly, the redox status of mitochondrial pyridine nucleotides. The possible relationship of this putative pathway to those available for Ca²⁺ uptake and release is considered.     

Prooxidants open both the mitochondrial permeability transition pore and a low-conductance channel in the inner mitochondrial membrane

Mitochondria can be induced by a variety of agents/conditions to undergo a permeability transition (MPT), which nonselectively increases the permeability of the inner membrane (i.m.) to small (<1500 Da) solutes. Prooxidants are generally considered to trigger the MPT, but some investigators suggest instead that prooxidants open a Ca(2+)-selective channel in the inner mitochondrial membrane and that the opening of this channel, when coupled with Ca(2+) cycling mediated by the Ca(2+) uniporter, leads ultimately to the observed increase in mitochondrial permeability [see, e.g., Schlegel et al. (1992) Biochem. J. 285, 65]. S. A. Novgorodov and T. I. Gudz [J. Bioenerg. Biomembr. (1996) 28, 139] propose that the i.m. contains a pore that, upon exposure to prooxidants, can open to two states, one of which conducts only H(+) and one of which is the classic MPT pore. Given the current interest in increased mitochondrial permeability as a factor in apoptotic cell death, it is important to determine whether i.m. permeability is regulated in one or multiple ways and, in the latter event, to characterize each regulatory mechanism in detail. This study examined the effects of the prooxidants diamide and t-butylhydroperoxide (t-BuOOH) on the permeability of isolated rat liver mitochondria. Under the experimental conditions used, t-BuOOH induced mitochondrial swelling only in the presence of exogenous Ca(2+) (>2 microM), whereas diamide was effective in its absence. In the absence of exogenous inorganic phosphate (P(i)), (1) both prooxidants caused a collapse of the membrane potential (DeltaPsi) that preceded the onset of mitochondrial swelling; (2) cyclosporin A eliminated the swelling induced by diamide and dramatically slowed that elicited by t-BuOOH, without altering prooxidant-induced depolarization; (3) collapse of DeltaPsi was associated with Ca(2+) efflux but not with efflux of glutathione; (4) neither Ca(2+) efflux nor DeltaPsi collapse was sensitive to ruthenium red; (5) collapse of DeltaPsi was accompanied by an increase in matrix pH; no stimulation of respiration was observed; (6) Sr(2+) was able to substitute for Ca(2+) in supporting t-BuOOH-induced i.m. depolarization, but not swelling; (7) in addition to being insensitive to CsA, the collapse of DeltaPsi was also resistant to trifluoperazine, spermine, and Mg(2+), all of which block the MPT; and (8) DeltaPsi was restored (and its collapse was inhibited) upon addition of dithiothreitol, ADP, ATP or EGTA. We suggest that these results indicate that prooxidants open two channels in the i.m.: the classic MPT and a low-conductance channel with clearly distinct properties. Opening of the low-conductance channel requires sulfhydryl group oxidation and the presence of a divalent cation; both Ca(2+) and Sr(2+) are effective. The channel permits the passage of cations, including Ca(2+), but not of protons. It is insensitive to inhibitors of the classic MPT.     

Astaxanthin protects mitochondrial redox state and functional integrity against oxidative stress

Mitochondria combine the production of energy with an efficient chain of reduction–oxidation (redox) reactions but also with the unavoidable production of reactive oxygen species. Oxidative stress leading to mitochondrial dysfunction is a critical factor in many diseases, such as cancer and neurodegenerative and lifestyle-related diseases. Effective antioxidants thus offer great therapeutic and preventive promise. Investigating the efficacy of antioxidants, we found that a carotenoid, astaxanthin (AX), decreased physiologically occurring oxidative stress and protected cultured cells against strong oxidative stress induced with a respiratory inhibitor. Moreover, AX improved maintenance of a high mitochondrial membrane potential and stimulated respiration. Investigating how AX stimulates and interacts with mitochondria, a redox-sensitive fluorescent protein (roGFP1) was stably expressed in the cytosol and mitochondrial matrix to measure the redox state in the respective compartments. AX at nanomolar concentrations was effective in maintaining mitochondria in a reduced state. Additionally, AX improved the ability of mitochondria to remain in a reduced state under oxidative challenge. Taken together, these results suggest that AX is effective in improving mitochondrial function through retaining mitochondria in the reduced state.     

Physiological roles of the mitochondrial permeability transition pore

Neurons experience high metabolic demand during such processes as synaptic vesicle recycling, membrane potential maintenance and Ca²⁺ exchange/extrusion. The energy needs of these events are met in large part by mitochondrial production of ATP through the process of oxidative phosphorylation. The job of ATP production by the mitochondria is performed by the F₁F_O ATP synthase, a multi-protein enzyme that contains a membrane-inserted portion, an extra-membranous enzymatic portion and an extensive regulatory complex. Although required for ATP production by mitochondria, recent findings have confirmed that the membrane-confined portion of the c-subunit of the ATP synthase also houses a large conductance uncoupling channel, the mitochondrial permeability transition pore (mPTP), the persistent opening of which produces osmotic dysregulation of the inner mitochondrial membrane, uncoupling of oxidative phosphorylation and cell death. Recent advances in understanding the molecular components of mPTP and its regulatory mechanisms have determined that decreased uncoupling occurs in states of enhanced mitochondrial efficiency; relative closure of mPTP therefore contributes to cellular functions as diverse as cardiac development and synaptic efficacy.     

Bcl-2 prevents mitochondrial permeability transition and cytochrome c release via maintenance of reduced pyridine nucleotides

Digitonin-permeabilized PC12 and GT1-7 neural cells exhibited a cyclosporin A-sensitive decrease in mitochondrial membrane potential, increased volume, and release of the pro-apoptotic factor cytochrome c in the presence of Ca2+ and the mitochondrial permeability transition (MPT) inducers t-butyl hydroperoxide (t-bOOH) or phenylarsine oxide (PhAsO). Although the concentration of PhAsO required to induce the MPT was similar for Bcl-2 negative and Bcl-2 overexpressing transfected cells (Bcl-2(+)), the level of t-bOOH necessary for triggering the MPT was much higher for Bcl-2(+) cells. A higher concentration of t-bOOH was also necessary for promoting the oxidation of mitochondrial pyridine nucleotides in Bcl-2(+) cells. The sensitivity of Bcl-2(- ) cell mitochondria to t-bOOH but not PhAsO could be overcome by the use of conditions that protect the pyridine nucleotides against oxidation. We conclude that the increased ability of Bcl-2(+) cells to maintain mitochondrial pyridine nucleotides in a reduced redox state is a sufficient explanation for their resistance to MPT under conditions of oxidative stress induced by Ca2+ plus t-bOOH.     

Calcium uptake by bovine epididymal spermatozoa is regulated by the redox state of the mitochondrial pyridine nucleotides

Immature caput epididymal sperm accumulate calcium from exogenous sources at a rate 2- to 4-fold greater than mature caudal sperm. Calcium accumulation by these cells, however, is maximal in the presence of lactate as external substrate. This stimulation of calcium uptake by optimum levels of lactate (0.8-1.0 mM) is about 5-fold in caput and 2-fold in caudal sperm compared to values observed with glucose as substrate. Calcium accumulation by intact sperm is almost entirely mitochondrial as evidenced by the inhibition of uptake by rotenone, antimycin, and ruthenium red. The differences in the ability of the various substrates in sustaining calcium uptake appeared to be related to their ability to generate NADH (nicotinamide adenine dinucleotide). Previous reports have documented that mitochondrial calcium accumulation in several somatic cells is regulated by the oxidation state of mitochondrial NADH. A similar situation obtains for bovine epididymal sperm since calcium uptake sustained by site III oxidation of ascorbate in the presence of tetramethyl phenylenediamine and rotenone was also stimulated by NADH-producing substrates, including lactate, and inhibited by substrates generating NAD+ (nicotinamide adenine dinucleotide, oxidized form). Further, calcium uptake by digitonin-permeabilized sperm in the presence of succinate was stimulated when NADH oxidation was inhibited by rotenone. The compounds alpha-keto butyric, valeric, and caproic acids, which generate NAD+, inhibited the maximal calcium uptake observed in the presence of succinate and rotenone, and the hydroxy acids lactate and beta-hydroxybutyrate reversed this inhibition. These results document the regulation of sperm calcium accumulation by the physiological substrate lactate, emphasize the importance of mitochondria in the accumulation of calcium by bovine epididymal sperm, and suggest that the mitochondrial location of the isozyme LDH-X in mammalian sperm may be involved in the regulation of calcium accumulation.     

The mitochondrial permeability transition pore and cyclophilin D in cardioprotection

Mitochondria play a central role in heart energy metabolism and Ca²⁺ homeostasis and are involved in the pathogenesis of many forms of heart disease. The body of knowledge on mitochondrial pathophysiology in living cells and organs is increasing, and so is the interest in mitochondria as potential targets for cardioprotection. This critical review will focus on the permeability transition pore (PTP) and its regulation by cyclophilin (CyP) D as effectors of endogenous protective mechanisms and as potential drug targets. The complexity of the regulatory interactions underlying control of mitochondrial function in vivo is beginning to emerge, and although apparently contradictory findings still exist we believe that the network of regulatory protein interactions involving the PTP and CyPs in physiology and pathology will increase our repertoire for therapeutic interventions in heart disease. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

The role of the mitochondrial permeability transition pore in heart disease

Like Dr. Jeckyll and Mr. Hyde, mitochondria possess two distinct persona. Under normal physiological conditions they synthesise ATP to meet the energy needs of the beating heart. Here calcium acts as a signal to balance the rate of ATP production with ATP demand. However, when the heart is overloaded with calcium, especially when this is accompanied by oxidative stress, mitochondria embrace their darker side, and induce necrotic cell death of the myocytes. This happens acutely in reperfusion injury and chronically in congestive heart failure. Here calcium overload, adenine nucleotide depletion and oxidative stress combine forces to induce the opening of a non-specific pore in the mitochondrial membrane, known as the mitochondrial permeability transition pore (mPTP). The molecular nature of the mPTP remains controversial but current evidence implicates a matrix protein, cyclophilin-D (CyP-D) and two inner membrane proteins, the adenine nucleotide translocase (ANT) and the phosphate carrier (PiC). Inhibition of mPTP opening can be achieved with inhibitors of each component, but targeting CyP-D with cyclosporin A (CsA) and its non-immunosuppressive analogues is the best described. In animal models, inhibition of mPTP opening by either CsA or genetic ablation of CyP-D provides strong protection from both reperfusion injury and congestive heart failure. This confirms the mPTP as a promising drug target in human cardiovascular disease. Indeed, the first clinical trials have shown CsA treatment improves recovery after treatment of a coronary thrombosis with angioplasty.     

Role of oxidative stress in the ammonia-induced mitochondrial permeability transition in cultured astrocytes

Ammonia is a neurotoxin that has been strongly implicated in the pathogenesis of hepatic encephalopathy (HE) and other neurological disorders, and astrocytes are thought to be the principal target of ammonia toxicity. While the precise mechanisms of ammonia neurotoxicity remain to be more clearly defined, altered bioenergetics and oxidative stress appear to be critical factors in its pathogenesis. It has recently been demonstrated that pathophysiological concentrations of ammonia induce the mitochondrial permeability transition (MPT) in cultured astrocytes, a process associated with mitochondrial dysfunction, and frequently caused by oxidative stress. This study investigated the potential role of oxidative stress in the induction of the MPT by ammonia. Accordingly, the effect of various antioxidants on the induction of the MPT by ammonia in cultured astrocytes was examined. Astrocytes were subjected to NH4Cl (5 mM) treatment for 2 days with or without various antioxidants. The MPT was assessed by quantitative fluorescence imaging for the mitochondrial membrane potential (DeltaPsim), employing the potentiometric dye TMRE; by changes in mitochondrial calcein fluorescence and by 2-deoxyglucose-6-phosphate (2-DG-6-P) changes in mitochondrial permeability. Astrocytes treated with ammonia significantly dissipated the DeltaPsim, which was blocked by the MPT inhibitor, cyclosporin A, caused a decrease in mitochondrial calcein fluorescence and increased 2-DG-6-P permeability into mitochondria. All of these findings are consistent with induction of the MPT. Pretreatment with SOD, catalase, desferroxamine, Vitamin E, PBN and the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), completely blocked the ammonia-induced MPT. These data provide strong evidence that oxidative stress is involved in the induction of the MPT by ammonia, and suggest that oxidative stress and the subsequent induction of the MPT contribute to the pathogenesis of HE and other hyperammonemic disorders.     

The permeability transition pore controls cardiac mitochondrial maturation and myocyte differentiation

Although mature myocytes rely on mitochondria as the primary source of energy, the role of mitochondria in the developing heart is not well known. Here, we find that closure of the mitochondrial permeability transition pore (mPTP) drives maturation of mitochondrial structure and function and myocyte differentiation. Cardiomyocytes at embryonic day (E) 9.5, when compared to E13.5, displayed fragmented mitochondria with few cristae, a less-polarized mitochondrial membrane potential, higher reactive oxygen species (ROS) levels, and an open mPTP. Pharmacologic and genetic closing of the mPTP yielded maturation of mitochondrial structure and function, lowered ROS, and increased myocyte differentiation (measured by counting Z bands). Furthermore, myocyte differentiation was inhibited and enhanced with oxidant and antioxidant treatment, respectively, suggesting that redox-signaling pathways lie downstream of mitochondria to regulate cardiac myocyte differentiation.     

Electrophysiology clarifies the megariddles of the mitochondrial permeability transition pore

After a brief review of the early history of mitochondrial electrophysiology, the contribution of this approach to the study of the mitochondrial permeability transition (MPT) is recapitulated. It has for example provided evidence for a dimeric nature of the MPT pore, allowed the distinction between two levels of control of its activity, and underscored the relevance of redox events for the phenomenon. Single-channel recording provides a means to finally solve the riddle of the biochemical entity underlying it by comparing the characteristics of the pore with those of channels formed by candidate molecules or complexes. The possibility that this entity may be the protein import machinery of the inner mitochondrial membrane is emphasized.