Critical Review. Release of Ca 2+ from Mitochondria via the Saturable Mechanisms and the Permeability Transition

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1 IUBMB Life, 52: , 2001 Copyright c 2001 IUBMB /01 $ Critical Review Release of Ca 2+ from Mitochondria via the Saturable Mechanisms and the Permeability Transition Douglas R. Pfeiffer, 2 Thomas E. Gunter, 1 Roman Eliseev, 1 Kimberly M. Broekemeier, 3 and Karlene K. Gunter 1 1 Department of Biochemistry and Biophysics, University of Rochester, School of Medicine and Dentistry, Rochester, New York Department of Molecular and Cellular Biochemistry, The Ohio State University, School of Medicine, Columbus, Ohio Department of Chemistry, Ohio Northern University, Ada, Ohio Summary 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 Ca 2+ - induced mitochondrial permeability transition (PT), and 3) modulation of cytosolic Ca 2+ transients. Removal of Ca 2+ from mitochondria permits rapid and ef cient 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 Ca 2+ ef ux from mitochondria exist. In addition, the permeability transition or PT, which may also remove Ca 2+ from the mitochondrial matrix, is intimately involved in other important functions such as apoptosis. Here we brie y review what is known about these important mitochondrial mechanisms and from their behavior speculate on their possible and probable functions. IUBMB Life, 52: , 2001 Keywords Ca 2C ef ux; Na C -dependent ef ux; Na C -independent ef ux; mitochondrial permeability transition. THE SATURABLE EFFLUX MECHANISMS Two separate, saturable Ca 2C ef ux mechanisms have been identi ed in mitochondria, Na C -dependent and Na C - independent ef ux. Both transport Ca 2C or related ions from the mitochondrial matrix to the intermembrane or the external space. This requires these mechanisms to supply approximately 33 kj of energy to transport each mole of Ca 2C outward against its Received 31 August 2001; accepted 19 September Address correspondence to Thomas Gunter, Dept. of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, 575 Elmwood Avenue, Rochester, NY Fax: thomas gunter@urmc.rochester.edu electrochemical gradient (1). This energy could come from the electrochemical gradients of exchanged or cotransported ions, or from chemical sources (e.g., from the electron transport chain). These mechanisms show very different kinetics, different sensitivities to inhibitors, have different selectivity series, and are clearly distinct mechanisms, which probably mediate somewhat different functions. The characteristics and functions of these mechanisms have been more completely described in earlier reviews and are discussed only brie y here (1 3). The Na C -Dependent Ef ux Mechanism. The kinetics of the Na C -dependent ef ux mechanism have been found to be rstorder in Ca 2C and either second- or third-order in Na C (1, 4). This ef ux mechanism is the primary mechanism in brain, heart, skeletal muscle, parotid gland, brown fat, and most tumor mitochondria (1, 4). V max for this mechanism varies by about a factor of 30 between brain and liver mitochondria (1). On the other hand, the K m is about 8 to 10 nmoles/mg for intramitochondrial Ca 2C and 9 to 10 mm for external Na C in both heart and liver mitochondria (1, 4). The velocity of this mechanism in liver mitochondria has then been given as: v D V max f[ca 2C ]=(K Ca C [Ca 2C ])gf[na C ] 2 =(K 2 Na C [NaC ] 2 )g; where V max D 2:6 0:5 nmoles/(mg min), K Ca D 8:1 1:4 nmole/mg protein, and K Na D 9:4 0:6 mm (1). This Na C - dependent mechanism can pump Ca 2C out of mitochondria against a Ca 2C gradient whose energy is well over twice that of the Na C gradient. This requires that either more than two Na C ions be used to supply the energy for Ca 2C ef ux or that some other energy source be used in addition to the energy of transport of Na C ions. Reexamination of earlier data, led to the conclusion that this transport was electrogenic and that the transport stoichiometry was probably 3:1 instead of 2:1. See (2) for a review. 205

2 206 PFEIFFER ET AL. The divalent cation selectivity series of the Na C -dependent mechanism seems more restricted than that of the Na C - independent mechanism. The Na C -dependent Ca 2C ef ux mechanism has been found to exchange Ca 2C for Ca 2C, Sr 2C, or Na C (1 4); furthermore, it has also been reported that Sr 2C strongly inhibits Ca 2C transport via this mechanism and that Sr 2C is only transported slowly (2). Mn 2C is not transported via this mechanism (2). Because the velocity of Na C -dependent ef ux is far greater than Na C -independent ef ux in brain mitochondria, this inability of the Na C -dependent mechanism to transport Mn 2C may be one reason why Mn 2C accumulates in brain mitochondria, perhaps leading to symptoms of Mn 2C toxicity, yet Mn 2C seems to be cleared easily from the liver (2). Na C -dependent mitochondrial Ca 2C ef ux is inhibited by a wide range of inhibitors. These include tri uoperazine, diltiazem, verapamil, clonazepam, tetraphenyl phosphonium, amiloride and its derivatives and very high levels of rutnenium red (1). The Na C -Independent Mechanism. The Na C -independent ef ux mechanism can transport either Ca 2C, Sr 2C, or Mn 2C outward, across the inner membrane, and against the Ca 2C electrochemical gradient (1 3). Although the maximum transport velocities of both Na C -dependent and Na C -independent mechanisms are similar in liver mitochondria studied through in vitro experiments in Mg 2C free medium, Mg 2C inhibits Na C - dependent ef ux signi cantly more than Na C -independent ef- ux (1). Inhibition of Na C -dependent ef ux by endogenous Mg 2C causes the Na C -independent mechanism to be the dominant mechanism in liver, kidney, lung, and smooth muscle mitochondria (1, 2). The Na C -independent mechanism is second order in Ca 2C and in liver mitochondria has been found to follow Adair-Pauling or nonessential activation kinetics as: v D V max f[ca 2C ] 2 C a[ca 2C ]g=fk 2 m C [Ca2C ] 2 C 2a[Ca 2C ]g; where V max D 1:2 0:1 nmoles/(mg min), K m D 8:4 0:6 nmole/ mg, and a D 0:9 0.2 nmole/mg (1). This transport is electroneutral and has been characterized as a 1 Ca 2C for 2 H C exchanger (1, 2). However, it has also been shown to transport Ca 2C outward against a Ca 2C gradient that is much greater than twice the electrochemical proton gradient (2). This demonstrates that some other energy input must be involved with transport via this mechanism. Furthermore, it has also been observed that the rate of transport via this mechanism can decrease with an increasing ph gradient, again suggesting that it is not a simple Ca 2C for 2 H C exchanger (2). Transport via this mechanism has been shown to be inhibited by CN suggesting that electron transport might be the source of this additional energy (1). The simplest explanation for its observed behavior is that it is an active Ca 2C for 2H C exchanger. In addition to being inhibited by CN, this mechanism can be inhibited by low levels of uncouplers such as CCCP and FCCP, and by very high levels of ruthenium red (1, 2). What appears to be an analogous mechanism has recently been identi ed in mitochondria of the yeast Saccharomyces cerevisiae. This yeast mechanism displays the novel characteristic of requiring low levels of free fatty acids for activity (5). Why are There Two Saturable Mitochondrial Ca 2+ Ef ux Mechanisms and How May Their Physiological Roles Differ? The Na C -dependent mechanism is dominant in heart and brain mitochondria where rapid release of Ca 2C may be important to mediate rapid changes in intromitochondrial [Ca 2C ], whereas the Na C -independent mechanism is dominant in liver and kidney, which are tissues involved in the clearance of metal ions from the body. Furthermore, the Na C -dependent mechanism has been found to transport Sr 2C only slowly and not to transport Mn 2C at all, yet the Na C -independent mechanism readily transports these ions. This suggests that an important physiological role of the Na C -independent mechanism may be to remove ions other than Ca 2C from the mitochondria in order to clear them from the cell (1, 2). The Ca 2+ -Induced Mitochondrial Permeability Transition Discovery and General Properties. Both the saturable ef ux mechanisms and the mitochondrial permeability transition (PT) can release Ca 2C from the mitochondrial matrix. They are considered together in part for historical reasons, and because this remains as a possible physiological function of the PT. Research on the PT dates from the mid-1970s when it was suggested that the damaging effects of Ca 2C on mitochondria might be of physiological signi cance. In adrenal cortex mitochondria, it was shown that Ca 2C induces a transformation that allows extramitochondrial pyridine nucleotides to gain access to the intramitochondrial compartment, and that the NADPH entering in this way supports the 11 ß hydroxylation of deoxycorticosterone (6 8). This transformation was recognised early on to have the following characteristics: it required Ca 2C accumulation speci cally, it was accompanied by swelling and marked ultrastructural changes, it was antagonized by Mg 2C, and it was reversed upon the chelation of free Ca 2C (6 8). An expanded set of similar phenomena occurring in heart mitochondria was referred to as a con gurational transition and it was recognised that energy coupling is among the activities of mitochondria that can be reversibly lost upon induction of this Ca 2C -induced transition (9). The transition phenomena were associated with Ca 2C release in 1978 (10), and thereafter Hunter and Haworth extended and assembled these ndings into a set of mitochondrial behaviors that has since been referred to as the permeability transition (11 14). These investigators attributed the PT to a reversible opening of a proteinaceous pore in the inner mitochondrial membrane, which has proven to be a key insight. Three subsequent important results in early PT research included the recognition that the many agents that promote mitochondrial Ca 2C release act by a common mechanism, the PT (15, 16), the discovery of cyclosporin A (Cys) as a universal inhibitor of the PT (17, 18), and the identi cation of membrane

3 MITOCHONDRIAL Ca 2C EFFLLUX AND THE PERMEABILITY TRANSITION 207 potential as a central determinant of the PT pore open/closed probability (19, 20). For an individual mitochondrion, the PT represents a catastrophic change in the permeability of the inner mitochondrial membrane from its usual rather impermeable condition to a condition in which it is readily permeable to all small ions and molecules. Following the PT, mitochondria cannot generate an electrochemical proton gradient and consequently cannot mediate oxidative phosphorylation. Furthermore, this wholesale change in permeability of the inner membrane is associated with prolonged exposure of the mitochondrial matrix space to high Ca 2C concentration. Numerous endogenous and external agents, known as inducing agents, accelerate induction of the PT and decrease the concentration of Ca 2C necessary for its induction. Usually, there is a lag between the time at which Ca 2C and the inducing agents are introduced and the onset of increased permeability. Endogenous inducing agents include ubiquitous metabolites such as inorganic phosphate (Pi), oxaloacetate, and acetoacetate, yet ADP, ATP, and Mg 2C are endogenous inhibitors. Recent studies show that low Pi concentrations increase the lag period in some cases (21), and that the anti-apoptotic protein bcl-2 has a similar effect (22). Although creatine and creatine phosphate were thought to inhibit the PT with a neuroprotective action, more recent evidence suggests that their effects are due to enhancement of cytosolic high energy phosphate levels and not to direct actions on the PT (23). Commonly used external inducing agents include N- ethylmaleimide, tert-butyl hydroperoxide, atractyloside, or carboxyatractyloside, and phenylarsine oxide (1). The most commonly used exogenous PT inhibitors are CsA, bongkrekate, and Ca 2C chelators (1). More inducing agents are identi ed with every passing year. Recently identi ed inducing agents include ibuprofen (24), parabens (25), arachidonic and palmitic acids (26), aluminum (27), Zn 2C (28), and chronic ethanol treatment (29). Recently described inhibitors are cinnarizine and unarizine (30) and certain quinones (3). The abundance of reagents that in uence the pore creates a challenge to those interested in its structure and regulation. Molecular Properties and Regulation of the PT Pore. The size of the pore opening was rst addressed by Hunter and Haworth who used a colloid osmotic pressure-based shrinkage assay, employing extramitochondrial polyethylene glycols (PEG) of various size, to show that the pore in heart mitochondria excludes solutes with a molecular weight larger than 1,500 Da (11). For a PEG of this size, the hydrodynamic radius is estimated to be 1.2 to 1.5 nm (31), an estimate of pore radius that is similar to that displayed by the mitochondrial megachannel. The latter structure is observed in the inner membrane by patch clamp methods. It displays a conductivity of about 1 ns, is sensitive to CsA, and is thought to represent the PT pore (PTP) (32 34). Pore heterogeniety is often related to pore size. Re nement and further application of PEG-based methods have shown that the average pore in liver mitochondria (Ca 2C plus Pi induction) excludes molecules of molecular weight greater than 650 Da (35). However much larger pores may also arise under certain conditions, as shown by the observation that matrix space proteins are sometimes released in a CsA-sensitive fashion (36). Furthermore, PEG actions on PT-dependant swelling suggest that individual mitochondria form pores of a relatively distinct size, but that the size varies throughout a given population (35, 37). Pore size may also vary when different inducing agents are employed (38), and when mitochondria from different organs are compared. Heterogeneity can also be caused by differing properties of substrates. Because NAD C /NADH diffuses out of mitochondria upon PTP opening more easily than other metabolites, substrate differences also cause signi cant differences in characteristics of the PT in mitochondria, in myocytes (39), and in neurons (40). Thus, the picture emerging is one of pore heterogeneity, rather than a single molecular entity that is responsible for the PT. There seem to be several modalities through which modulators of the pore are acting. One of the strongest relates to the conformation of the adenine nucleotide translocase (ANT). When bound to ADP, the ANT faces inward, toward the mitochondrial matrix, in what is called the m conformation. Similarly, it is in the m conformation when bound to the PT antagonist bongkrekate; however, it faces the cytosol and is in the c conformation when bound to the inducing agents atractyloside or carboxyatractyloside (41). Bongkrekate and the atractylosides were rst known as inhibitors of adenine nucleotide transport via the ANT. The relationships between these compounds and regulation of the PT were noticed by Hunter and Haworth, who suggested that the ANT plays a role in induction of the PT (12). Recognition that agents that stabilize the ANT in the m conformation act as inhibitors of the PT, and those that stabilize the c conformation act as inducing agents arose later, subsequent to work conducted in several labs (42 46). These observations lead to a current model of pore structure, in which the ANT interacts with VDAC from the outer membrane to form a pore that reaches from outside the mitochondrion to the matrix space. Within the model these entities are located at contact sites and are further interacting with other mitochondrial proteins that participate in pore regulation [see (41) and (47) for review]. Inhibition of the PT by CsA occurs by an interaction with the matrix protein cyclophilin D, which is one of the proteins that are thought to interact with the ANT/VDAC multimer comprising the pore (41). This action of CsA is distinct from its activity as an immunosuppressant, which occurs through binding to cytosolic cyclophilin A, and the further interaction of that complex with a protein phosphatase (calcineurin), which is thereby inhibited. Cyclophilin D is a peptidyl-prolyl cis-trans isomerase that participates in the folding of imported proteins. As a pore-related protein it is thought to interact with the ANT in a Ca 2C -dependent manner, and to comprise a structural component. Several inducing agents, including t-butyl hydroperoxide, diamide, and phenylarsine oxide increase cyclophilin D binding to the inner mitochondrial membrane (48), and inhibit the

4 208 PFEIFFER ET AL. binding of ADP and ATP, which are PT inhibitors as already noted (44, 47, 49, 50). As additional support for this particular model of pore structure, several studies show that the isolated reconstituted ANT can in fact form pores [reviewed in (47)]. On the other hand, chemical cross-linking experiments have so far failed to demonstrate that cyclophilin D-membrane binding in fact represents an interaction with the ANT (41). In addition, the model does not easily account for pore regulation in the presence of carboxyatractyloside, as has been observed (51), for the apparent pore size heterogeneity described previously, and for the transient action of CsA as an inhibitor of the PT (62). The latter factor in particular suggests that it is the established catalytic activity of cyclophilin D, rather than a structural involvement, that is important in pore formation (52). Thus, many believe that the ANT is located topologically near to the PT pore, plays a central role in pore activity, and may be a crucial component the pore complex; however, there are still many questions about its precise role that remain to be answered (3, 53). Other proteins thought to interact with the PT and contribute to its regulation include creatine kinase, hexokinase, and the mitochondrial porin, VDAC (45, 46, 54 56). Creatine kinase and hexokinase were proposed earlier to mediate a preferential use of ATP recently synthesized by oxidative phosphorylation, and VDAC could mediate a rapid transport of ATP into the cytosol. The alignment of the very large VDAC pore with the large PT pore could create a rapid diffusion pathway for small ions and molecules out of the matrix space. Cyclophilin D was found to bind to the ANT in this VDAC-ANT complex and when the entire complex was incorporated into liposomes, addition of Ca 2C and Pi (which activate the PT) opened a CsA-sensitive pore large enough to allow permeability of uorescein sulphonate [(54), see also (41)]. The apparent association of creatine kinase and hexokinase with the ANT/VDAC multimer could not only modify ATP distribution but also, if the ANT is an inherent part of the PT pore, the regulatory characteristics of the PT. Several additional factors contribute to the regulation of pore opening. The oxidation state of pyridine nucleotides is an important factor regulating the pore, with the oxidized condition promoting opening. Recent work has shown that aging may also be a factor (57). Bernardi and coworkers have shown that in addition to being modulated by endogenous inducing agents and inhibitors, pore opening is also strongly affected by ph and by mitochondrial membrane potential. Acid ph (or increasing [H C ]) provides a strong protection through the protonation of histidyl residues and this action can be blocked by diethylpyrocarbonate (3, 58). The protection afforded by a high membrane potential is also marked (19) and may be one reason why mitochondria hydrolyze ATP to maintain their polarization during anoxia and other periods of stress (3). For more complete reviews of PT inducers, inhibitors and modes of action see (1, 3, 41, 47, 53, 59). Pore Substates and Flickering. Electrophysiological measurements have shown that many channels icker (i.e., open transiently and asynchronousl y for periods that are characteristic of the channel in question). This ickering has been reported in electrophysiological studies of the mitochondrial megachannel, which some believe to be the PT (60). Transient, asynchronous ickering of mitochondrial membrane potential has been observed in isolated heart mitochondria loaded with the uorescent dye tetramethyl rhodamine ethyl ester (TMRE) (61). Initially, the mitochondrial membrane was impermeable to calcein (molecular weight D 623) during these short periods of depolarization; however, with time the periods of depolarization became longer lasting and the inner membrane became permeable to calcein. These longer periods of depolarization could be inhibited by CsA suggesting that PT induction was underlying this behavior. Shorter periods of pore opening could also be followed using calcein uorescence in the presence and absence of uorescent (tetramethylrhodamine methyl ester) and non uorescent (Co 2C ) quenchers (62). Pore ickering may correspond to the properties of a lower conductance state of the PT, permeable to H C, K C, and Ca 2C, but not to larger molecules such as sucrose (63, 64). Flickering and eventual longer-term opening of the PTP induced by light-induced generation of free radicals has also been described in confocal microscopy studies using TMRE. It was suggested that this ickering might lead to release of Ca 2C but not larger molecules (61). Recent work has suggested that pro-oxidants open both the PTP and a separate low conductance channel in the inner membrane that permits passage of cations (but not protons). This low conductance channel is insensitive to the usual inhibitors of the PT (65). [See (64) or (3) for a more complete review of work on the low-conductance state of the PT.] Function of the Mitochondrial PT. Loss of oxidative phosphorylation would deprive the cell of approximately 95% of its supply of ATP. Why then are the properties of the PT conserved and found in almost all types of mitochondria? The answer to this question may be emerging as we learn more about the role of mitochondria in apoptosis. Because of space constraints, we discuss this area selectively, and emphasize recent references. The reader is referred to longer reviews for more detailed information (66 69). A number of agents that induce apoptosis incorporate mitochondria into the mechanism that they initiate (53, 70, 71). Mitochondrial participation involves signals that trigger the release of several factors from the intermembrane space, including cytochrome c (72, 73), AIF (74), and SMAC/DIABLO (75). These proteins acting alone, or in combination with other factors, initiate a cascade of caspase activation that culminates with activation of executioner caspases and cell death (76). It is possible that participation in apoptosis is a physiological role of the PT. The Bcl-2 family of proteins is closely involved in regulating apoptosis and also in uences the sensitivity of mitochondria to the PT. They consist of both pro- and antiapoptotic members (77) with the former group promoting the PT opening and the latter group being inhibitory. Interestingly, some of these proteins are constitutively associated with mitochondria,

5 MITOCHONDRIAL Ca 2C EFFLLUX AND THE PERMEABILITY TRANSITION 209 such as the anti-apoptotic Bcl-2 per se, and Bcl-xl (78). Others translocate to mitochondria upon the induction of apoptosis, including the pro-apoptotic proteins Bax, Bak, and Bid (79). In mitochondria, the proteins associate with each other and their regulatory properties are modulated through these interactions. It appears that they reside at the contact sites between the outer and inner mitochondrial membranes, and therefore colocalize with components of the PT pore (80). Recently, in patch clamp studies, Bcl-2 has been found to suppress Ca 2C activation of the mitochondrial megachannel (81), which may equate to the PT pore as noted before. That report follows an earlier demonstration that Bcl-2 enhances the loading capacity of mitochondria for Ca 2C, which was an early indication that the protein antagonizes the PT (82). The anti-apoptotic Bcl-2 proteins may function by maintaining pyridine nucleotides in the reduced state (83), which also inhibits opening of the PT pore. Their actions are in contrast to Bax, which sensitizes mitochondria to conditions that promote the PT when overexpressed (84). There is controversy regarding the mechanism(s) by which cytochrome c and the other regulators of apoptosis are released from mitochondria. One hypothesis holds that a selective transport mechanism associated with the pro-apoptotic members of the Bcl-2 family proteins are responsible (85 88). Another holds that PT-mediated swelling causes a nonspeci c release of intermembrane proteins by rupturing the outer membrane (71, 89, 90). In favor of the latter hypothesis are data showing extensive mitochondrial swelling (91, 92), depolarization (89, 93), and permeability of the inner membrane to large solutes (66); all preceding the release of cytochrome c and AIF. Sensitivity of these phenomena and of apoptosis to CsA is also supporting evidence. The treatment of cells with agents that activate the PT, in many instances results in triggering apoptosis (94). Elevated cytosolic [Ca 2C ], which is sometimes found in the early steps of apoptosis, is itself a prerequisite for the PT opening (95). Induction of the PT is generally held to be suf cient for induction of the apoptotic cascade, but many believe that it is not necessary. Thus, it is possible that mitochondria are subject to the PT as a consequence of their involvement in apoptosis; however, other reasons for its occurrence should also be considered. Ichas and other investigators emphasize that through the PT, mitochondria act collectively as excitable structures and that this may contribute to the regulation of cell Ca 2C by mitochondria (96). Crompton suggests that the PT pores of isolated mitochondria actually represent the structures that allow mitochondria to form extended networks in vivo (47). Others propose that the PT, as its most basic function, leads to the removal and replacement of poorly functioning mitochondria in cells (97 99). According to this hypothesis, mitochondria that are damaged by reactive oxygen species or exposure to chemical toxins, or those that contain mutated DNA, or are functioning poorly for other reasons undergo the PT more readily than normal mitochondria. Following permeabilization, the affected fraction is removed by autolysis (97), autophagy (98), because the import of new proteins/structural components no longer occurs (99), or by some combination of these processes and considerations. This concept is referred to as mitochondrial apoptosis or mitoptosis, and encompasses the known roles of the PT in cell death by specifying a different consequence of the phenomenon according to the fraction of the mitochondrial population that is involved. When the fraction is small, only those mitochondria undergoing the transition are affected because the release of proapoptotic factors is too limited to support cellular apoptosis, and because surviving mitochondria easily compensate for the loss of biochemical capacity at the cell level. However, as the permeable fraction grows, cellular apoptosis can be initiated, or in the extreme, necrotic cell death will occur if most of the mitochondria have been permeabilized (97 99). The extent to which mitochondria undergo autolysis following the PT has been little characterized, but the appearance of Ca 2C -dependent phospholipase activity during the PT has long been recognized [reviewed in (1)]. New data (Fig. 1) show that liver mitochondria in fact contain a much more substantial phospholipase activity, which is Ca 2C -independent and seen in media of high ionic strength. It is activated during the PT, when mitochondria are permeabilized by alamethicin, or when they are Figure 1. Ca 2C Independent Phospholipase A 2 Activity in Rat Liver Mitochondria. Incubations were conducted at 25 C in media containing 13 mm KCl, 10 mm succinate (Na C ), 3 mm Hepes (Na C ), ph 7.4, and mitochondria at 1.0 mg protein/ml., No further additions., The PT was induced by Ca 2C (0.1 mm) plus Pi (5 mm), which were present from 0 min., Pores were induced by the addition of alamethicin (1 g/ml) at 0 min., Mitochondria were uncoupled by the addition of CCCP (3 M) at min. Excess EGTA was present from the beginning in all cases, except for, were it was added after the PT had occurred. Free fatty acids arising from the C 2 position of mitochondrial phospholipids were determined by GLC.

6 210 PFEIFFER ET AL. simply uncoupled under conditions where pore formation does not occur. As will be demonstrated elsewhere, this newly discovered activity has a molecular weight near 80,000 (Broekemeier et al., manuscript submitted). It shows an inhibitor sensitivity pattern that is characteristic of Ca 2C -independent phospholipases A 2 from other sources, and is recognized by antibodies directed at a cytosolic phospholipase A 2 from CHO cells (Broekemeier et al., manuscript submitted). This phospholipase, which should become active in poorly energized mitochondria without the previous occurrence of cellular Ca 2C disregulation is of signi cance to the concept of mitochondrial apoptosis as a function of the PT. Even very low levels of free fatty acids make mitochondria labile to the PT, apparently through their effects on membrane surface charge (52, 59), and perhaps because they are uncouplers that further depress membrane potential. Thus this activity may be crucial in determining which mitochondria are subject to the PT in vivo. The extent of autolysis that can follow and the possible relationship of this to the onset of autophagy remain to be determined. FUTURE DIRECTIONS After almost 30 years of continuous investigation, it has become clear that mitochondria release Ca 2C through two distinct saturable mechanisms and are subject to the PT, which releases Ca 2C via pore formation. The kinetics of the saturable mechanisms have been clearly identi ed. Their molecular identities are of immediate interest for future research. Future research on the mitochondrial PT will likely be broader in context, re- ecting the range of physiological and pathophysiological processes that are in uenced by the phenomenon. The current models of pore structure and pore characteristics, while supported by data, require further testing under broader sets of conditions before consensus is reached. Perhaps the most signi cant set of questions regarding PT regulation concern the integration of the PT with other cellular processes such as apoptosis. Clearly, we must apply what we have learned about the PT in basic research to clinical research leading to the eventual management of this phenomenon during acute and chronic disease states. This need not await a further clari cation of basic issues, but will require the development of drugs that target the PT, including both activators and inhibitors that are suitable for use topically, or as systemic agents. ACKNOWLEDGMENTS Supported by NIH ES10041, CA71603, and AR40325, The Wallace Research Foundation, and a grant from the American Heart Association. We thank Dr. Claire Gavin, Dr. Gisela Beutner, and Ms. Kerstin Gunter for editing and critiquing the manuscript. REFERENCES 1. Gunter, T. E., and Pfeiffer, D. R. (1990) Mechanisms by which mitochondria transport calcium. Am. J. Physiol. 258, C755 C Gunter, T. 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