Transport properties of the calcium ionophore ETH-129.

The transport mechanism and specificities of ionophore ETH-29 have been investigated in a highly defined phospholipid vesicle system, with the goal of facilitating the application of this compound to biological problems. ETH-129 transports Ca(2+) via an electrogenic mechanism, in contrast to A23187 and ionomycin, which function in a charge neutral manner. The rate of transport is a function of membrane potential, increasing by 3.9-fold per 59 mV over a broad range of that parameter. Rate is independent of the transmembrane pH gradient and strongly stimulated by the uncoupler carbonyl cyanide m-chlorophenylhydrazone when no external potential has been applied. The effect of uncoupler reflects the collapse of an opposing potential arising during Ca(2+) transport, but also reflects the formation of a mixed complex between the uncoupler, ETH-129, and Ca(2+) that readily permeates the vesicle membrane. Oleate does not substitute for the uncoupler in either regard. ETH-129 transports polyvalent cations according to the selectivity sequence La(3+) > Ca(2+) > Zn(2+) approximately equal to Sr(2+) > Co(2+) approximately equal to Ni(2+) approximately equal to Mn(2+), with the magnitude of the selectivity coefficients reflecting the cation concentration range considered. There is little or no activity for the transport of Na(+), K(+), and Mg(2+). These properties suggest that ETH-129 will be useful for investigating the consequences of a mitochondrial Ca(2+) overload in mammalian cells, which is difficult to pursue through the application of electroneutral ionophores.

Free fatty acids activate a vigorous Ca(2+):2H(+) antiport activity in yeast mitochondria.

The accumulation and retention of Ca(2+) by yeast mitochondria (Saccharomyces cerevisiae) mediated by ionophore ETH 129 occurs with a variable efficiency in different preparations. Ineffective Ca(2+) transport and a depressed membrane potential occur in parallel, are exacerbated in parallel by exogenous free fatty acids, and are corrected in parallel by the addition of bovine serum albumin. Bovine serum albumin is not required to develop a high membrane potential when either Ca(2+) or ETH 129 are absent, and when both are present membrane potential is restored by the addition of EGTA in a concentration-dependent manner. Respiration and swelling data indicate that the permeability transition pore does not open in yeast mitochondria that are treated with Ca(2+) and ETH 129, whereas fatty acid concentration studies and the inaction of carboxyatractyloside indicate that fatty acid-derived uncoupling does not underlie the other observations. It is concluded that yeast mitochondria contain a previously unrecognized Ca(2+):2H(+) antiporter that is highly active in the presence of free fatty acids and leads to a futile cycle of Ca(2+) accumulation and release when exogenous Ca(2+) and ETH 129 are available. It is also shown that isolated yeast mitochondria degrade their phospholipids at a relatively rapid rate. The activity responsible is also previously unrecognized. It is Ca(2+)-independent, little affected by the presence or absence of a respiratory substrate, and leads to the hydrolysis of ester linkages at both the sn-1 and sn-2 positions of the glycerophospholipids. The products of this activity, through their actions on the antiporter, explain the variable behavior of yeast mitochondria treated with Ca(2+) plus ETH 129.

Release of Ca2+ from mitochondria via the saturable mechanisms and the permeability transition.

The literature, reviewed in the previous article, supports three physiological roles for sequestration of calcium by mitochondria: 1) control of the rate of ATP production, 2) activation of the Ca2+-induced mitochondrial permeability transition (PT), and 3) modulation of cytosolic Ca2+ transients. Removal of Ca2+ from mitochondria permits rapid and efficient changes in the rate of ATP production to adapt to changing demands and can reverse the process of PT induction. Two separate, saturable mechanisms for facilitating Ca2+ efflux from mitochondria exist. In addition, the permeability transition or PT, which may also remove Ca2+ from the mitochondrial matrix, is intimately involved in other important functions such as apoptosis. Here we briefly review what is known about these important mitochondrial mechanisms and from their behavior speculate on their possible and probable functions.

Topology of the mitochondrial inner membrane: dynamics and bioenergetic implications.

Electron tomography indicates that the mitochondrial inner membrane is not normally comprised of baffle-like folds as depicted in textbooks. In actuality, this membrane is pleomorphic, with narrow tubular regions connecting the internal compartments (cristae) to each other and to the membrane periphery. The membrane topologies observed in condensed (matrix contracted) and orthodox (matrix expanded) mitochondria cannot be interconverted by passive folding and unfolding. Instead, transitions between these morphological states likely involve membrane fusion and fission. Formation of tubular junctions in the inner membrane appears to be energetically favored, because they form spontaneously in yeast mitochondria following large-amplitude swelling and recontraction. However, aberrant, unattached, vesicular cristae are also observed in these mitochondria, suggesting that formation of cristae junctions depends on factors (such as the distribution of key proteins and/or lipids) that are disrupted during extreme swelling. Computer modeling studies using the "Virtual Cell" program suggest that the shape of the inner membrane can influence mitochondrial function. Simulations indicate that narrow cristae junctions restrict diffusion between intracristal and external compartments, causing depletion of ADP and decreased ATP output inside the cristae.

Ionomycin, a carboxylic acid ionophore, transports Pb(2+) with high selectivity.

Studies utilizing phospholipid vesicle loaded with chelator/indicators for polyvalent cations show that ionomycin transports divalent cations with the selectivity sequence Pb(2+) > Cd(2+) > Zn(2+) > Mn(2+) > Ca(2+) > Cu(2+) > Co(2+) > Ni(2+) > Sr(2+). The selectivity of this ionophore for Pb(2+) is in contrast to that observed for A23178 and 4-BrA23187, which transport Pb(2+) at efficiencies that are intermediate between those of other cations. When the selectivity difference of ionomycin for Pb(2+) versus Ca(2+) was calculated from relative rates of transport, with either cation present individually and all other conditions held constant, a value of approximately 450 was obtained. This rose to approximately 3200 when both cations were present and transported simultaneously. 1 microM Pb(2+) inhibited the transport of 1 mM Ca(2+) by approximately 50%, whereas the rate of Pb(2+) transport approached a maximum at a concentration of 10 microM Pb(2+) when 1 mM Ca(2+) was also present. Plots of log rate versus log ionomycin or log Pb(2+) concentration indicated that the transporting species is of 1:1 stoichiometry, ionophore to Pb(2+), but that complexes containing an additional Pb(2+) may occur. The species transporting Pb(2+) may include H.IPb.OH, wherein ionomycin is ionized once and the presence of OH(-) maintains charge neutrality. Ionomycin retained a high efficiency for Pb(2+) transport in A20 B lymphoma cells loaded with Indo-1. Both Pb(2+) entry and efflux were observed. Ionomycin should be considered primarily as an ionophore for Pb(2+), rather than Ca(2+), of possible value for the investigation and treatment of Pb(2+) intoxication.

Proton selective substate of the mitochondrial permeability transition pore: regulation by the redox state of the electron transport chain.

The permeability transition pore of rat liver mitochondria can be closed by chelating free Ca2+, with respect to the passage of large molecules such as mannitol and sucrose. However, an apparent H+-conducting substate remains open under these conditions, as indicated by the persistence of maximal O2 consumption rates and by the failure to recover a membrane potential. Agents which favor a closed pore, such as cyclosporin A, ADP, Mg2+, or bovine serum albumin, do not close the H+-conducting substate, but it closes spontaneously when respiration becomes limited by the availability of O2. Closure provoked by an O2 limitation requires free Mg2+ in the sub-micromolar concentration range and becomes less efficient with increasing time spent in the presence of free Ca2+. The H+-conducting substate is apparently regulated by the redox status of the electron transport chain, with a reduced form favoring closure. A physical association (or equivalence) between the pore and one of the respiratory chain complexes is supported. These characteristics suggest that the transition is irreversible in vivo, if it involves a small fraction of total mitochondria, and would lead to their elimination and/or replacement by the cell. The implications of this proposal are considered, as they relate to a possible role for the transition in cellular apoptosis and the elimination of mitochondria containing mutated DNA.

Mechanism and specificity of lanthanide series cation transport by ionophores A23187, 4-BrA23187, and ionomycin.

A23187, 4-BrA23187, and ionomycin transport several lanthanide series trivalent cations at efficiencies similar to Ca2+, when compared at cation concentrations of approximately 10(-5) M, ionophore concentrations of approximately 10(-6) M, and a pH of 7.00. Selectivity sequences and the range of relative rates are as follows: A23187, Nd3+ > La3+ > Eu3+ > Gd3+ > Er3+ > Yb3+ > Lu3+ (approximately 34-fold); 4-BrA23187, Nd3+ > Eu3+ > Gd3+ > La3+ > Er3+ > Yb3+ > Lu3+ (approximately 34-fold); ionomycin, La3+ > Yb3+ > Nd3+ > Lu3+ > Er3+ > Eu3+ > Gd3+ (approximately 4-fold). At concentrations between 9 and 250 microM, La3+ is transported by an electroneutral mechanism, predominately through mixed complexes of the type (ionophore)2La-OH (A23187 and 4-BrA23187) or (ionophore)La-OH (ionomycin), when no membrane potential is present. For all three ionophores, an induced potential of approximately 160 mV accelerates transport by approximately 50-100%. However, measured values of H+/La3+ exchange indicate that only 4-BrA23187 displays a significant electrogenic activity under these conditions. At a La3+ concentration of 17 mM, transport by all three ionophores is electroneutral and apparently occurs through complexes of type (ionophore)3La (A23187 and 4-BrA23187) or (ionophore)La-OH (ionomycin). Analysis of these patterns in a context of comproportionation equilibria involving the transporting species and free La3+ indicates that the species containing three ionophore molecules are formed on the membrane when aqueous phase solution conditions would strongly favor a 1:1 complex, based upon previous studies in solution. The implications of this and other findings are discussed.

Properties of a cyclosporin-insensitive permeability transition pore in yeast mitochondria.

Yeast mitochondria (Saccharomyces cerevisiae) contain a permeability transition pore which is regulated differently than the pore in mammalian mitochondria. In a mannitol medium containing 10 mM Pi and ethanol (oxidizable substrate), yeast mitochondria accumulate large amounts of Ca2+ (>400 nmol/mg of protein) upon the addition of an electrophoretic Ca2+ ionophore (ETH129). Pore opening does not occur following Ca2+ uptake, even though ruthenium red-inhibited rat liver mitochondria undergo rapid pore opening under analogous conditions. However, a pore does arise in yeast mitochondria when Ca2+ and Pi are not present, as monitored by swelling, ultrastructure, and matrix solute release. Pore opening is slow unless a respiratory substrate is provided (ethanol or NADH) but also occurs rapidly in response to ATP (2 mM) when oligomycin is present. Pi and ADP inhibit pore opening (EC50 approximately 1 and 4 mM, respectively), however, cyclosporin A (7 microg/ml), oligomycin (20 microg/ml), or carboxyatractyloside (25 microM) have no effect. The pore arising during respiration is also inhibited by nigericin or uncoupler, indicating that an acidic matrix pH antagonizes the process. Pi also inhibits pore opening by lowering the matrix pH (Pi/OH- antiport). However, inhibition of the ATP-induced pore by Pi is seen in the presence of mersalyl, suggesting a second mechanism of action. Since pore induction by ATP is not sensitive to carboxyatractyloside, ATP appears to act at an external site and Pi may antagonize the interaction. Isoosmotic polyethylene glycol-induced contraction of yeast mitochondria swollen during respiration, or in the presence of ATP, is 50% effective at a solute size of 1.0-1.1 kDa. This suggests that the same pore is induced in both cases and is comparable in size with the permeability transition pore of heart and liver mitochondria.

Regulation of the mitochondrial Ca2+ uniporter by external adenine nucleotides: the uniporter behaves like a gated channel which is regulated by nucleotides and divalent cations.

We have previously used measurements of uncoupler-enforced reverse activity to demonstrate that the mitochondrial Ca2+ uniporter is strongly inhibited by external EGTA plus free Mg2+, following a brief period of rapid activity. Using the same approach, we now show that in addition to divalent cations, the uniporter is regulated by external adenine nucleotides and by other components of the cytosol. Inhibition produced by EGTA plus free Mg2+ is reversed by spermine (EC0.5 approximately 40 microM) and reduced when mitochondria are purified by an isoosmotic density-gradient method. Under either condition, inhibition is restored by external adenine nucleotides in a concentration-dependent manner. The order of effectiveness is ATP > ADP > AMP, with the nucleoside adenosine being ineffective. Among nucleotide triphosphates, the order is ATP > CTP approximately UTP > GTP. The effectiveness of ATP (EC50 approximately 0.6 mM) is the same in mitochondria and mitoplasts, the same as that of AMPPNP, and is not altered by the presence of oligomycin, carboxyatractyloside, or AP5A, used alone or in combinations. These findings indicate that ATP acts at a site located on the outer surface of the inner membrane through a mechanism which does not require its hydrolysis. Phosphate also inhibits reverse uniport under some conditions (EC50 approximately 20 microM). The sites at which free ATP and free Mg2+ inhibit the uniporter can be distinguished by chymotrypsin treatment of mitoplasts, which eliminates the action of Mg2+ but does not affect the action of ATP. Data are interpreted within the context of a model in which the uniporter is considered to be a gated channel that is controlled, in part, by specific external effector sites that accept divalent cations or nucleotides. The possible consequences of the model for cell Ca2+ regulation by mitochondria and regulation of TCA cycle activity by the matrix free Ca2+ concentration are considered.

Evidence against formation of A23187 dimers and oligomers in solution: photo-induced degradation of Ionophore A23187.

Ionophore A23187 has been proposed to form Ca(2+)- conducting channels that arise from dimers and oligomers of the compound (e.g., Balasubramanian, S. V., and Easwaran, K. R. K. (1989) Biochem. Biophys. Res. Commun. 158, 891-897). To investigate this possibility, the solution behavior of A23187 in chloroform, n-hexane, ethanol, 80% methanol-water, and palmitoyloleoylphosphatidyl choline (POPC) vesicles was investigated using UV-VIS, circular dichroism (CD), and 1H NMR techniques. The concentration dependence of the UV-VIS and CD spectra obtained in freshly prepared chloroform solutions indicates that neutral A23187 (HA) exists as a monomer for ionophore concentrations in the range of 50-1000 microM. The cause of time- and concentration-dependent spectral alterations which gave rise to the dimer/channel hypothesis was also investigated. For solutions of 50-1000 microM A23187 in chloroform, n-hexane, and ethanol stored in the dark, no spectral changes were observed for periods of 2 months. However, solutions in these solvents did show time-dependent spectral changes when exposed to light. In 80% methanol-water or phospholipid vesicles, similar spectral changes were observed, even when the solutions were protected from light. Application of TLC and MS methods indicate that the time-dependent spectral changes reflect degradation of A23187, not dimer or oligomer formation. The degradative processes proceed with half-lives ranging from approximately 75 to > 400 h, and are influenced by several factors, including solvent, exposure to light, ionophore concentration, pH, and the presence of metal ions, EDTA, dissolved oxygen, and a radical inhibitor. The kinetics of Ca2+ transport into Quin-2-loaded POPC vesicles by authentic A23187 give no evidence of a channel mechanism, even following a previous and lengthy coincubation of the ionophore with the vesicles.

Ionophore 4-BrA23187 transports Zn2+ and Mn2+ with high selectivity over Ca2+.

The cation transport selectivities of the Ca2+ ionophores A23187, Ionomycin, and 4-BrA23187 have been determined using a model system comprised of phospholipid vesicles loaded with the chelator/indicator Quin-2. At pH 7.00 and a 100 microM concentration of the cations, A23187 displays the transport selectivity sequence Zn2+ > Mn2+ > Ca2+ > Co2+ > Ni2+ > Sr2+, with the absolute rates of transport spanning approximately 3 orders of magnitude. Similar data are obtained with Ionomycin, although the relative transport rates of Zn2+ and Mn2+ are equivalent, and the range of absolute rates is decreased by a factor of approximately 3. When values are normalized to those of Ca2+, transport selectivity is seen to be only weakly related to complexation or extraction selectivity. It is also seen that, when used to manipulate Ca2+ (or Mg2+), both ionophores can be expected to alter the distribution of additional divalent cations which have known biological activities. 4-BrA23187 is a low-activity ionophore for Ca2+, compared to A23187 and Ionomycin, while retaining comparable activities as an ionophore for the other cations. As a consequence, 4-BrA23187 is highly selective for the transport of Zn2+ and Mn2+, compared to Ca2+, with selectivity ratios approaching that of valinomycin for K+ over Na+ when conditions are optimal. Plots of the log of the rate of cation transport vs the log of the ionophore concentration indicate that Ca2+ is transported primarily as a 2:1 complex by A23187 and 4-BrA23187, but Zn2+ and Mn2+ are transported, in part, as 1:1 complexes. These findings, together with a postulated low stability of 2:1, compared to 1:1 complexes between 4-BrA23187 and divalent cations, partially explain the novel transport selectivity of this compound. Unlike A23187 or Ionomycin, 4-BrA23187 may be useful for investigating cell regulation by Zn2+ and Mn2+, without interference by regulatory mechanisms which respond to Ca2+.

On the use of fluorescent probes to estimate free Mg2+ in the matrix of heart mitochondria.

The fluorescent probe furaptra shows increases and decreases in the concentration of free magnesium ion, [Mg2+], in the mitochondrial matrix with changes in total Mg2+ and ligand availability. The factors involved in the calibration of these fluorescence changes in terms of absolute [Mg2+] have been investigated. The affinity of furaptra for Mg2+ is highly dependent on both temperature and ionic strength. The Kd for Mg-furaptra in solution in 100 mM KCl was found to be 2.1 +/- 0.1 mM at 25 degrees C. The use of this Kd to calculate matrix [Mg2+] is more reliable than in situ Kd measurements because ionophores, such as BrA23187 and ionomycin, do not equilibrate external Mg2+ with the matrix in an acceptable way. Furaptra is present at high concentrations (up to 500 microM) in the matrix when introduced by hydrolysis of the acetoxymethyl ester. However, absorbance spectra of aqueous solutions show no evidence of dimerization of the probe or other changes in properties at these concentrations. Fluorescence intensity at 340 nmex is strongly attenuated for matrix-sequestered furaptra, mag-fura-5, and mag-indo-1. This appears to result in part from preferential binding of the Mg-probe to mitochondrial proteins. The fluorescence of uncomplexed furaptra at 375-380 nmex seems unaffected by protein binding, however, and changes in intensity in this region of the spectrum can be used in conjunction with the Kd found in aqueous solution to estimate matrix [Mg2+]. The presence of secondary equilibria, such as protein binding, and possible changes in ionic strength may undermine exact quantitation by this method. However, values for matrix [Mg2+] obtained in this way (0.5 to 0.7 mM) correspond well to estimates by other available methods and each of these methods suffers from comparable uncertainties.

Inhibition of the mitochondrial permeability transition by cyclosporin A during long time frame experiments: relationship between pore opening and the activity of mitochondrial phospholipases.

Inhibition of the mitochondrial permeability transition pore by cyclosporin A or trifluoperazine is transient on the time scale of cell injury studies (hours). However, these agents act synergistically and produce long-lasting inhibition when used in combination. The cause of this synergism has been investigated from the perspective of the known action of trifluoperazine as an inhibitor of mitochondrial phospholipase A2. Free fatty acids, which are phospholipase reaction products, facilitate pore opening in a concentration-dependent manner (I50 approximately 2 nmol/mg of mitochondrial protein). Endogenous and exogenous fatty acids are similarly effective. Fatty acids of differing structure are also similarly effective, but long-chain alcohols and alkanes are ineffective. Free fatty acids accumulate in cyclosporin A-treated mitochondria when Ca2+ plus tert-butyl hydroperoxide or Ca2+ plus N-ethylmaleimide is present, but do not accumulate when Ca2+ plus inorganic phosphate is present. In the presence of cyclosporin A, bovine serum albumin markedly delays pore opening induced by tert-butyl hydroperoxide or N-ethylmaleimide, but has little effect on pore opening induced by inorganic phosphate, which is subject to long-lasting inhibition by cyclosporin A without trifluoperazine. Free fatty acid accumulation is thus a factor which limits pore inhibition by cyclosporin A. However, trifluoperazine has no effect on free fatty acid accumulation in intact, cyclosporin-inhibited mitochondria and thus does not act by inhibiting phospholipases. Comparing the actions of free fatty acids, trifluoperazine, long-chain acyl cations, and other effectors on the pore suggests that a more negative membrane surface potential favors pore opening and a more positive potential favors a closed pore. Expected surface potential effects of trifluoperazine can explain the synergism between this compound and cyclosporin A as pore inhibitors. Surface potential may influence the pore through the voltage-sensing element which responds to transmembrane potential. The present data also suggest that long-lived, solute-selective forms of the pore exist when it is opened in the presence of inhibitors. The implications of these findings for pore regulation and for the use of cyclosporin A to identify pore opening as a component of cell injury mechanisms are discussed.

Effects of pH conditions on Ca2+ transport catalyzed by ionophores A23187, 4-BrA23187, and ionomycin suggest problems with common applications of these compounds in biological systems.

Phospholipid vesicles loaded with Quin-2 and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) have been used to investigate the effects of pH conditions on Ca2+ transport catalyzed by ionophores A23187, 4-BrA23187, and ionomycin. At an external pH of 7.0, a delta pH (inside basic) of 0.4-0.6 U decreases the rate of Ca2+ transport into the vesicles by severalfold under some conditions. The apparent extent of transport is also decreased. In contrast, raising the pH by 0.4-0.6 U in the absence of a delta pH increases both of these parameters, although by smaller factors. The relatively large effects of a delta pH on the transport properties of Ca2+ ionophores seem to reflect a partial equilibration of the transmembrane ionophore distribution with the H+ concentration gradient across the vesicle membrane. This unequal distribution of ionophore can cause a very slow or incomplete ionophore-dependent equilibration of delta pCa with delta pH. A second factor of less certain origin retards full equilibration of delta pCa when delta pH = 0. These findings call into question several ionophore-based methods that are used to investigate the regulatory activities of Ca2+ and other divalent cations in biological systems. Notable among these are the null-point titration method for determining the concentration of free cations within cells and the use of ionophores plus external cation buffers to calibrate intracellular cation indicators. The present findings also indicate that the transport mode of Ca2+ ionophores is more strictly electroneutral than was thought, based upon previous studies.

The peptide mastoparan is a potent facilitator of the mitochondrial permeability transition.

Mastoparan facilitates opening of the mitochondrial permeability transition pore through an apparent bimodal mechanism of action. In the submicromolar concentration range, the action of mastoparan is dependent upon the medium Ca2+ and phosphate concentration and is subject to inhibition by cyclosporin A. At concentrations above 1 microM, pore induction by mastoparan occurs without an apparent Ca2+ requirement and in a cyclosporin A insensitive manner. Studies utilizing phospholipid vesicles show that mastoparan perturbs bilayer membranes across both concentration ranges, through a mechanism which is strongly dependent upon transmembrane potential. However, solute size exclusion studies suggest that the pores formed in mitochondria in response to both low and high concentrations of mastoparan are the permeability transition pore. It is proposed that low concentrations of mastoparan influence the pore per se, with higher concentrations having the additional effect of depolarizing the mitochondrial inner membrane through an action exerted upon the lipid phase. It may be the combination of these effects which allow pore opening in the absence of Ca2+ and in the presence of cyclosporin A, although other interpretations remain viable. A comparison of the activities of mastoparan and its analog, MP14, on mitochondria and phospholipid vesicles provides an initial indication that a G-protein may participate in regulation of the permeability transition pore. These studies draw attention to peptides, in a broad sense, as potential pore regulators in cells, under both physiological and pathological conditions.

Inhibition of the mitochondrial Ca2+ uniporter by pure and impure ruthenium red.

Commercial ruthenium red is often purified by a single recrystallization as described by Luft, J.H. (1971) Anat Rec 171, 347-368, which yields small amounts of material having an apparent molar extinction coefficient of approximately 67,400 at 533 nm. A simple modification to the procedure dramatically improves the yield, allowing crystallization to be repeated. Three times recrystallized ruthenium red has an apparent extinction coefficient of approximately 85,900, the highest value reported to date. Both crude and highly purified ruthenium red can be shown to inhibit reverse activity of the mitochondrial Ca2+ uniporter (uncoupled mitochondria), provided that care is taken to minimize and account for Ca2+ release through the permeability transition pore. Crude ruthenium red is 7-10 fold more potent than the highly purified material in this regard, on an actual ruthenium red concentration basis. The same relative potency is seen against forward uniport (coupled mitochondria), however, the I50 values are 10 fold lower for both the crude and purified preparations. These data demonstrate unambiguously that the energy state of mitochondria affects the sensitivity of the Ca2+ uniporter to ruthenium red preparations, and that both the forward and reverse reactions are subject to complete inhibition. The data suggest, however, that the active inhibitor may not be ruthenium red per se, but one or more of the other ruthenium complexes which are present in ruthenium red preparations.

Recent progress on regulation of the mitochondrial permeability transition pore; a cyclosporin-sensitive pore in the inner mitochondrial membrane.

The mitochondrial permeability transition pore allows solutes with a m.w. approximately less than 1500 to equilibrate across the inner membrane. A closed pore is favored by cyclosporin A acting at a high-affinity site, which may be the matrix space cylophilin isozyme. Early results obtained with cyclosporin A analogs and metabolites support this hypothesis. Inhibition by cyclosporin does not appear to require inhibition of calcineurin activity; however, it may relate to inhibition of cyclophilin peptide bond isomerase activity. The permeability transition pore is strongly regulated by both the membrane potential (delta psi) and delta pH components of the mitochondrial protonmotive force. A voltage sensor which is influenced by the disulfide/sulhydryl state of vicinal sulfhydryls is proposed to render pore opening sensitive to delta psi. Early results indicate that this sensor is also responsive to membrane surface potential and/or to surface potential gradients. Histidine residues located on the matrix side of the inner membrane render the pore responsive to delta pH. The pore is also regulated by several ions and metabolites which act at sites that are interactive. There are many analogies between the systems which regulate the permeability transition pore and the NMDA receptor channel. These suggest structural similarities and that the permeability transition pore belongs to the family of ligand gated ion channels.

Magnesium ion modulates the sensitivity of the mitochondrial permeability transition pore to cyclosporin A and ADP.

Regulation of the mitochondrial permeability transition pore has been investigated following the release of matrix solutes which normally participate in pore regulation. Under these conditions, neither cyclosporin A nor ADP induces pore closure, as judged by restoration of delta psi, unless Mg2+ is also added. Mg2+ alone is ineffective. In liver mitochondria, the Mg2+ effect is expressed over a 0 to 0.5 mM concentration range with higher concentrations inhibiting repolarization. In heart mitochondria, the inhibitory action of high Mg2+ is not seen and it can be shown that the Mg2+ effect on repolarization increases progressively up to a concentration of 5 mM. In liver mitochondria, when the pore is closed by maximally effective concentrations of Mg2+ plus cyclosporin A or Mg2+ plus ADP, reopening occurs upon the addition of carboxyatractyloside. The latter compound, however, fails to reopen the pore when Mg2+, cyclosporin A, and ADP are present simultaneously. In heart mitochondria, where higher Mg2+ concentrations can be employed, Mg2+ plus cyclosporin A or Mg2+ plus ADP produces pore closure in a carboxyatractyloside insensitive manner. Titration experiments support the adenine nucleotide translocase as the site at which carboxyatractyloside acts to regulate the pore. However, the action of ADP appears to involve a translocase-independent site. In intact mitochondria the action of carboxyatractyloside on pore opening is counteracted by oligomycin, apparently through inhibition of the F1F0 ATP synthase, with a consequent increase in the matrix space ADP/ATP ratio. It is concluded that the permeability transition pore induced by Ca2+ plus P(i) is not formed from the adenine nucleotide translocase although the translocase conformation is one of several factors which regulate the pore. The matrix Mg2+ concentration is also one of these factors. Formation of the pore by a Ca2+ and ADP binding protein is one model which is consistent with the present data.

Ca2+ transport properties of ionophores A23187, ionomycin, and 4-BrA23187 in a well defined model system.

Models for the electroneutral transport of Ca2+ by ionophores A23187, ionomycin, and 4-BrA23187 have been tested in a defined system comprised of 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylcholine vesicles prepared by freeze-thaw extrusion. Quin-2-loaded and CaCl2-loaded vesicles were employed to allow the investigation of transport in both directions. Simultaneous or parallel measurements of H+ transport and membrane potential, respectively, indicate that for any of these ionophores, electrogenic transport events do not exceed 1 in 10,000 when there is no preexisting transmembrane potential. When a potential of approximately 150 mV is imposed across the membrane, transport catalyzed by A23187 remains electroneutral; however, for ionomycin and 4-BrA23187, approximately 10% of transport events may be electrogenic. The defined vesicle system has also been utilized to determine how the rate of Ca2+ transport varies as a function of ionophore and Ca2+ concentration and with the direction of transport. Some aspects of the results are unexpected and should be considered by investigators using ionophores in biological systems. These include the apparent failure of these compounds to fully equilibrate Ca2+ with a high affinity Ca2+ indicator when these species are separated by a membrane, rates of transport that vary markedly with the direction of transport, and extents of transport that are a function of ionophore concentration. At least some of these unexpected behaviors can be explained by a strong influence of delta pH on forward and reverse transport kinetics. In the case of A23187, the data also give some initial insights into the relationship between formation of the transporting species and the entry of this species into the membrane hydrophobic region.

Mitochondrial metabolism of 12- and 15-hydroxyeicosatetraenoic acids.

We have previously demonstrated that peroxisomal-deficient human skin fibroblasts and mutant Chinese hamster ovary cells do not convert 12- and 15-hydroxyeicosatetraenoic acids (HETEs) to chain-shortened, polar metabolites, suggesting that peroxisomes are the intracellular location for beta-oxidation of these compounds. This implies that mitochondria do not beta-oxidize HETEs. To test this hypothesis we incubated highly purified rat liver mitochondria with [3H]12-(S)- and [3H]15-(S)-HETE in the presence of carnitine and an acylcoenzyme A-generating system. Extracts obtained from these incubations were analyzed for radiolabeled polar metabolites. Both HETEs were converted to apparent products of beta-oxidation, although the 12-HETE compound was a markedly better substrate. The presence of 50 microM 2-tetradecyloxirane carboxylate, a potent inhibitor of carnitine palmitoyl transferase, completely blocked 12- and 15-HETE conversion to these metabolites as did omission of carnitine from the medium. These data demonstrate carnitine-dependent beta-oxidation of HETEs in isolated mitochondria and suggest that mitochondria are competent to carry out this metabolic process in eukaryotic cells. Prevailing metabolic conditions in subcellular compartments may have precluded observation of mitochondrial activity in our earlier work with cultured cells. Alternatively, transport mechanisms may exist in the cell types studied that distribute 12-(S)- and 15-(S)-HETEs specifically to peroxisomes.