I. INTRODUCTION

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The first great wave of mitochondrial research stretched from the 1940s, with the pioneering work of Lehninger, Chance, Slater, Ernster and others through the "chemiosmotic revolution" initiated by Peter Mitchell, to the early 1980s. During this period, the basic mitochondrial functions were identified and characterized with increasing precision, and the organelle emerged as a beautifully self-regulating machine for generating ATP (see Ref. 416 for review). In view of the central role played by mitochondria in cellular metabolism, it was assumed that the organelle was highly reliable and associated with few disease states. Indeed, almost the only mitochondrial dysfunction known was the extremely rare Luft's disease (344) in which "loose coupling" was detected in mitochondria from a hypermetabolic patient. Mitochondrial research therefore became increasingly focused on the elucidation of the molecular machinery of the proton pumps. However, in recent years, there has been a resurgence of interest in the functional aspects of mitochondria with the realization that, far from being perfect machines, mitochondria are fragile structures operating near their physicochemical limits, whose dysfunction appears to underlie a host of degenerative disease states in the brain. Furthermore, it has recently been discovered that mitochondria have an additional role, participating directly in the signaling pathways that culminate in apoptosis.

A complex and disparate variety of factors can influence cell survival, but in this review we focus on bioenergetic aspects, and in particular how mitochondrial function controls cell survival. Initial sections review the bioenergetics of mitochondria both in isolation and within the healthy cell. This is followed by discussion of the application of these studies to pathophysiological and degenerative conditions. To keep this review within manageable bounds, the emphasis is on neurons and neuronal mitochondria, but general principles obtained with other cell types are also discussed. Related topics including mitochondrial mutation (105, 187, 199, 334, 505, 506, 510, 626) and mitochondria and aging (126, 187) have been recently reviewed and so are not covered here.

	II. MITOCHONDRIAL PROTON CIRCUIT

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Mitochondria exert a multifactorial influence on cell function. In addition to ATP synthesis they can accumulate Ca²⁺ whenever the local cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_c) rises above a critical "set point" (11, 265, 410); under some conditions this can result in a nonspecific permeabilization of the inner membrane, the "mitochondrial permeability transition" (MPT) (for review, see Refs. 42, 634). In addition, changes in mitochondrial Ca²⁺ can regulate tricarboxylic acid cycle enzymes (367). The mitochondrial respiratory chain is also the major site for the generation of superoxide radicals (O₂·) (for review, see Refs. 534, 575). Each of these parameters is influenced by, and can in turn influence, the mitochondrial membrane potential (_m). Through the ATP/ADP pool, mitochondria can influence glycolysis, the activity of Ca²⁺- and Na⁺-K⁺-ATPases at the plasma membrane and consequently the activity of Na⁺-coupled plasma membrane transporters.

Because the manipulation of a single mitochondrial parameter may affect many cellular functions, experiments must be designed with care. It follows that an understanding of the basic bioenergetics of isolated mitochondria is essential to be able to monitor and manipulate mitochondrial function in the much more complex environment of the intact cell.

Electrons from NADH enter complex I of the respiratory chain (Fig. 1) at a redox potential of 300 mV and emerge to reduce a membrane-associated ubiquinol/ubiquinone (UQH₂/UQ) pool close to 0 mV. Electrons from flavin-linked dehydrogenases (such as succinate dehydrogenase) have an insufficiently negative redox potential to enter complex I and instead reduce the UQH₂/UQ pool via complex II. Ubiquinol transfers electrons to complex III, which in turn reduces cytochrome c (at a redox potential of approximately +250 mV). Cytochrome c then reduces the terminal acceptor complex IV (also called cytochrome-c oxidase) which then transfers four electrons to molecular oxygen. Complexes I, III, and IV function as proton pumps, acting in series with respect to the electron flux and in parallel with respect to the proton circuit (Fig. 1). The fall in redox potential of the electrons passing through these complexes is used to generate a proton electrochemical potential gradient, µ_H⁺, usually expressed in electrical potential units as the proton-motive force (p)

(1)

where _m is the mitochondrial membrane potential (a positive value corresponding to a negative matrix), pH is the pH gradient across the inner membrane (a positive value indicating an acidic matrix), and R, T and F refer to the gas constant, the absolute temperature, and the Faraday constant, respectively. Under most conditions, _m is the dominant component of p, accounting for 150-180 mV of the total proton-motive force of 200-220 mV (387, 408). A fourth component of the inner mitochondrial membrane, the energy-linked transhydrogenase (237), utilizes p to maintain a high level of reduction of the matrix NADP(H) pool by driving the following reaction to the right

(2)

In a typical mitochondrion, the NAD pool is ~10% reduced while the NADP pool is >90% reduced (493).

View larger version (25K): [in this window] [in a new window]   Fig. 1. Chemisomotic proton circuit. A: electrons from NADH and FADH2 enter respiratory chain at, respectively, complex I and II. Complexes I, III, and IV function as proton pumps, in series with respect to protons and in parallel with respect to proton circuit. Proton reentry can occur through ATP synthase to generate ATP and also through transhydrogenase (TrH) to reduce NADP+. B: proton circuit is analogous to an equivalent electrical circuit and can be quantified by Ohm's law, where proton-motive force (pmf) is equivalent to voltage term and magnitude of proton current is defined by membrane resistence to protons, which is in turn controlled by rate of ATP synthesis.

The ATP synthase is the dominant pathway for the reentry of protons into the mitochondrial matrix. The proton-motive force forces this ATP-hydrolyzing proton pump to run in reverse and synthesize ATP. ATP is not a "high-energy compound"; rather, the mitochondrion holds the ATP synthesis reaction (ADP + P_i = ATP) up to 10 orders of magnitude away from equilibrium (408, 482). The Gibbs free energy (G) for a reaction in a cell is simply a measure of this displacement from equilibrium; at 37°C, this means that the G for ATP synthesis in the cytoplasm is close to +60 kJ/mol.

An instructive analogy is to compare the proton circuit to an equivalent electrical circuit (Fig. 1), where p is the potential term, the effective proton conductance of the inner membrane corresponds to the conductance (reciprocal resistance) of a component, and the consequent proton current flowing round the circuit is governed by Ohm's law (for review, see Ref. 416). Because the proton circuit is completed by the reentry of protons through the ATP synthase, which in turn is tightly coupled to ATP synthesis, it follows that the proton current responds automatically to ATP demand. Tight coupling between electron flux and proton pumping in each respiratory chain complex means that electron flux (and hence respiration) is in turn controlled by ATP demand, the phenomenon of respiratory control. The fixed H⁺/2e stoichiometry of the individual respiratory chain complexes means that the proton current can be calculated from the respiratory rate multiplied by the H⁺/2e (406). Because the potential term p can be calculated (see sect. IIIA), application of Ohm's law allows the effective proton conductance of the membrane to be derived under a wide range of experimental conditions (406, 409).

Isolated mitochondria respire in state 4 (the state when there is no extramitochondrial ATP hydrolysis and hence no proton reentry via the ATP synthase) due to a constitutive proton leak across the inner membrane. This leak is highly potential dependent ("nonohmic"), being maximal in state 4 and dropping to almost zero in state 3 (the state when extramitochondrial ATP turnover and hence proton reentry via the ATP synthase is maximal) (409, 425, 480). The proton leak limits the respiratory control ratio (state 3 divided by state 4 respiration) to between 5 and 10 depending on the substrate (408, 409, 480). Substrates such as succinate that feed electrons into complex II generate a significantly higher p than complex I substrates and in consequence display a higher nonohmic proton leak (408). The nonohmic leak plays a major role in controlling basal metabolic rate (480) and in addition may limit the production of potentially dangerous reactive oxygen species (ROS) (534). The respiratory chain complexes are efficient transducers of redox potential into p; indeed, complexes I and III (but not IV) operate close to thermodynamic equilibrium. This means that changes in p affect the redox state of components within these complexes, and this in turn has important consequences for the cell's susceptibility to oxidative stress (see sect. VIID).

As with an electrical circuit, p drops slightly when the proton current is increased (409). The only communication between the respiratory chain and the ATP synthase is via the proton circuit, and it is the difference between the "static head" p, at which there would be no proton pumping, and the actual p that is responsible for controlling respiration. With isolated mitochondria, p in state 3 is 10-15% lower than in state 4 (387, 406). Although this difference is small, it can be further reduced in many cells under conditions of high metabolic activity by a Ca²⁺-mediated activation of key dehydrogenases (for review, see Refs. 211, 367). This generates an enhanced reduction of NADH, increasing the static head p to compensate for the greater disequilibrium required to drive the more rapid ATP generation (479).

Protonophores can be used to titrate the proton conductance of the membrane, and in excess can virtually collapse p, leading to maximal, uncontrolled respiration (387, 409). The ATP synthase will reverse in the presence of a protonophore, hydrolyzing extramitochondrial ATP; as will be discussed in the cellular context, protonophores are a highly effective means of depleting extramitochondrial ATP.

Mitochondria possess an inherent monovalent cation/H⁺ exchanger that prevents Na⁺ or K⁺ accumulation in the matrix in response to the high _m (386, 407). The ionophore nigericin (465) is a K⁺/H⁺-selective exchanger that in high K⁺ media (equivalent to a physiological cytoplasm) can decrease pH and cause a compensatory increase in _m. The K⁺ uniport ionophore valinomycin collapses _m in high K⁺ media and causes massive swelling.

Mitochondrial and cellular bioenergetics are intimately interlinked (Fig. 2). In neurons, the dominant mitochondrial substrate is pyruvate derived from glycolysis (230). Mitochondrial respiration is controlled by ATP turnover ("respiratory control"), which in neuronal cells is primarily a consequence of the ATP demand for plasma membrane ion pumping. The rate of glycolysis is controlled by mitochondrially generated ATP via the Pasteur effect (445); in neuronal preparations, inhibition of oxidative phosphorylation can lead to a 10-fold enhancement in the rate of glucose utilization (159, 160, 283). Importantly, a collapse in p will lead not only to a cessation of mitochondrial ATP synthesis, but to a rapid hydrolysis of cytoplasmic ATP as the ATP synthase reverses in an attempt to restore p. This can lead to profound depletion of ATP. Finally, to compound the complexity, the design of experiments to investigate mitochondrial function in intact cells must overcome the relative inaccessibility of the in situ mitochondria and the presence of other compartments and membrane systems.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Simplified ion circuitry across mitochondrial and plasma membranes of a typical neuron. Respiratory chain drives each of these circuits either directly or indirectly via ATP.

The isolated nerve terminal (synaptosome) preparation retains an intact plasma membrane, cytoplasm, mitochondria, associated metabolic pathways, and all the machinery for the uptake, storage, and release of neurotransmitter (for reviews, see Refs. 160, 372, 411, 606). Although the synaptosomal preparation is enriched in presynaptic nerve terminals and not representative of an intact neuron, it represents one of the simplest preparations in which to model neuronal mitochondrial/cellular interactions. However, it must be kept in mind that synaptic and nonsynaptic mitochondria differ somewhat in their respiratory capacities; notably, synaptic mitochondria have a lower threshold before inhibition of complex I restricts state 3 respiration (115).

Respiration, and thus the proton current flowing across the inner membrane of the in situ mitochondria, is one of the few bioenergetic parameters that is not more difficult to determine in synaptosomes than in isolated mitochondria. The respiratory stimulation produced by protonophores indicates that the in situ mitochondria within rat cortical synaptosomes have 500% spare respiratory capacity (160, 517). The Na⁺-K⁺-ATPase is the major ATP-requiring process, thus its inhibition with ouabain substantially inhibits basal respiration (160, 272, 517), while addition of veratridine to prevent voltage-activated Na⁺ channels from inactivating results in a 300% increase in respiration driving the rapid cycling of Na⁺ and K⁺ across the plasma membrane (156, 160, 272, 517, 585, 588). The residual respiration in the presence of oligomycin (which inhibits the ATP synthase) can be used to quantify the proton leakage across the mitochondrial membrane and can detect classic "uncoupling" of the in situ mitochondria.

Accurate estimation of the G for the cytoplasmic adenine nucleotide pool is difficult. The G is a function of free [ATP]/([ADP][P_i]) in the cytoplasm rather than the ATP level per se. Whole cell ATP/ADP greater than ~5 generally reflect a healthy synaptosomal preparation (73, 160), whereas ratios approaching 15:1 are characteristic of primary cultures of neurons and glia (526). However, it must be kept in mind that these values are a combination of the mitochondrial and cytoplasmic pools.

Because ionophores induce ion permeabilities in lipid bilayers, they display virtually no membrane selectivity (465). Ionophores may induce relatively nonselective cation channels in membranes (e.g., gramicidin) or may be selective mobile carriers catalyzing the electrogenic uniport of a single ion [e.g., protonophores (H⁺) or valinomycin (K⁺ or Rb⁺)] or the electroneutral exchange of two species [e.g., K⁺/H⁺ (nigericin) or Ca²⁺/2H⁺ (ionomycin or A-23187)].

Protonophores have been widely employed in the cellular context to depolarize mitochondria (e.g., Refs. 118, 173, 292, 293, 392, 448, 449, 539, 549, 550, 559, 566, 598). However, protonophore addition to an intact cell has multiple consequences; the ionophore will acidify the cytoplasm by equilibrating protons across the plasma membrane (569, 597) and deplete synaptic vesicles of their contents, particularly amino acids, by collapsing the transvesicular p (80, 419, 599). At the mitochondrion, the protonophore will collapse _m (inhibiting mitochondrial Ca²⁺ accumulation and releasing any accumulated Ca²⁺), reverse the ATP synthase (leading to a rapid hydrolysis of cytoplasmic ATP, Ref. 73), inhibit the generation of O₂·, and perhaps induce the MPT. Finally, the decreased cytoplasmic ATP may prevent Na⁺ and Ca²⁺ extrusion from the cell and lead to a subsequent failure of glycolysis if ATP levels are below those needed by hexokinase. For a cell to survive in the presence of protonophore, the capacity of glycolytic ATP generation must therefore exceed that required by the cell plus that consumed by the reversed ATP synthase; this only seems to be the case for cells with a limited population of mitochondria.

Great care must be taken in the use of other ionophores in the cellular context. The K⁺ uniport ionophore valinomycin cannot be used with intact cells unless it is the intention to swell and depolarize the in situ mitochondria (184, 489). Although nigericin (465) collapses intracellular pH gradients between compartments containing an equal concentration of K⁺ and consequently allows mitochondria to hyperpolarize (604), the K⁺ > Na⁺ selectivity for nigericin is only 25-45 (461), allowing high concentrations of the ionophore (>1 µM) to depolarize the plasma membrane, dissipate the Na⁺ and K⁺ gradients across the plasma membrane, and short-circuit the Na⁺-K⁺-ATPase (158, 160).

A variety of inhibitors exist selective for each respiratory chain complex (Fig. 1). Because respiratory chain inhibitors target specific mitochondrial components, they are free of the inherent membrane nonselectivity characteristic of ionophores, although some complex IV inhibitors have additional, nonmitochondrial, sites of action. Inhibition of any one complex will block electron transfer through the entire respiratory chain. Complex III inhibition (by, for example, antimycin A or myxathiazol) or complex IV inhibition (by, for example, azide, cyanide, carbon monoxide, or NO·) will inhibit electron flow to the terminal oxidase. Even though two separate complexes (I and II) reduce complex III, inhibition of either will affect respiration in an intact cell, since the supply of succinate for complex II is dependent on preceding NAD⁺-linked dehydrogenases in the tricarboxylic acid cycle, whereas inhibition of succinate dehydrogenase or complex II will also block the cycle, limiting the generation of NADH.

Because redox components upstream of the site of inhibition will become reduced, whereas those downstream will be oxidized, the inhibitors can be used to control the redox state of specific regions of the respiratory chain. This is of particular interest in complex III, where myxathiazol inhibits upstream of the ubisemiquinone site, which is the major source of mitochondrial O₂·, whereas antimycin A inhibits downstream, and, by increasing the occupancy of this site, increases O₂· generation (576). The immediate bioenergetic consequences of respiratory chain inhibition are largely independent of the site of inhibition. Because respiratory chain-linked proton extrusion ceases, p decays, stopping ATP synthesis and allowing the ATP synthase to reverse. The resultant hydrolysis of cytoplasmic ATP occurs more slowly than in the presence of protonophore (see sect. IIC) since the inner membrane retains its low permeability to protons. This slow hydrolysis of ATP is sufficient to maintain _m at levels only slightly lower than in the absence of inhibitor (517).

In synaptosomes, rotenone inhibition of complex I (517) or 3-nitropropionic acid (3-NPA) inhibition of complex II (157) leads to a fall in ATP and phosphocreatine levels. The ability of a cell to maintain function in the presence of respiratory chain inhibitors appears in the short term to be governed by the ability of glycolysis to supply sufficient ATP for cell metabolism as well as for this ATP synthase reversal. As any spare ATP-generating capacity of the cell is depleted, the susceptibility of cells to factors that increase ATP demand is enhanced proportionately.

Oligomycin is a selective inhibitor of the F_o proton well of the mitochondrial ATP synthase (270), although high concentrations may also inhibit the plasma membrane Na⁺-K⁺-ATPase (169). Because both synthesis and hydrolysis of ATP by the mitochondrion are prevented, the organelle will not consume ATP regardless of subsequent bioenergetic manipulations. Oligomycin thus allows the key mitochondrial function of ATP synthesis to be selectively inhibited, and the cell will survive as long as the glycolytic capacity of the cell is sufficient to maintain ATP in the absence of oxidative phosphorylation. Mitochondrial membrane potential is not only maintained in the presence of oligomycin but is increased by a few millivolts (46, 143, 489, 517) due to the inhibition of proton reentry via the ATP synthase. As a consequence, _m-dependent functions including mitochondrial Ca²⁺ transport and the generation of O₂· will be maintained, or even increased as a consequence of the slight mitochondrial hyperpolarization. Most importantly, the further addition of a protonophore or respiratory chain inhibitor will not a priori cause a depletion of ATP, allowing _m to be manipulated without the complications of bioenergetic failure (73, 74, 141, 188, 325).

The ability of cells to maintain bioenergetic homeostasis in the presence of oligomycin will depend on their glycolytic capacity. Cerebellar granule cells in culture have a high glycolytic capacity and can be maintained for several hours in the presence of the inhibitor (73, 74). However, cultured hippocampal neurons in some studies (63, 139, 508, 539) but not others (62) show signs of energetic limitation in the presence of the inhibitor.

	III. MITOCHONDRIAL MEMBRANE POTENTIAL, pH GRADIENT, AND PROTON-MOTIVE FORCE

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The membrane potential _m is the mitochondrial parameter most frequently estimated in intact cells. We first review the methodologies that have been employed with isolated mitochondria, while bearing in mind that their extrapolation to intact cells may not always be valid. The two components of p are determined separately. The _m value is estimated from the gradient of a permeable cation across the inner membrane. Initially, K⁺ or Rb⁺ was employed in a low K⁺ medium in the presence of valinomycin (387, 408). Subsequently, membrane-permeant phosphonium cations, triphenylmethylphosphonium (TPMP⁺), or tetraphenylphosphonium (TPP⁺), were used, either as isotopes (543) or in combination with a selective phosphonium electrode (273).

To calculate _m from the phosphonium ion accumulation, it is necessary to determine the matrix volume (as sucrose-impermeable, H₂O-permeable space, Ref. 417) and the activity coefficient of the cation within the matrix by establishing a calibration from diffusion potentials generated by valinomycin in the presence of defined extramitochondrial K⁺ concentrations (609). The pH is determined from the equilibrium distribution of weak acids such as acetic acid that are membrane permeant in the protonated but not anionic forms (387, 408). Finally, p is calculated from these experimentally determined values (Eq. 1).

Cationic membrane-permeant fluorescent probes have been much employed to monitor _m. Their use with isolated mitochondria is dependent on the quenching of their fluorescence due to stacking of the probe molecules at the high concentrations achieved within the polarized mitochondrial matrix (339). Under these conditions, the total fluorescence of a suspension of mitochondria will decrease if the organelles hyperpolarize and more probe is sequestered within the matrix. Because this technique depends largely on an empirical aggregation or stacking of the probe, results are highly dependent on dye concentration, and results are generally calibrated by reference to K⁺ diffusion potentials obtained in the presence of valinomycin. Furthermore, great care must be taken to ensure that mitochondrial function is not compromised by the accumulated probe; for example, the cationic cyanine dye 3,3'-dihexyloxacarbocyanine iodide [DiOC₆(3)] has been reported to be a particularly potent inhibitor of mitochondrial complex I (50% inhibition by <10 nM dye equilibrated with cells; Refs. 124, 488, 489).

In the presence of physiological concentrations of phosphate and Mg²⁺, isolated mitochondria from liver, heart, brown fat, or brain (387, 408, 543) maintain a total p of some 220 mV in state 4, of which the membrane potential _m comprises 150-180 mV with a pH of 0.5 to 1 pH unit contributing the remaining 30-60 mV (Eq. 1). In the presence of excess phosphate, pH remains small and _m generally shadows p. This may justify the general failure to consider the pH component in discussions of in situ mitochondrial energization, although as discussed in section IVA there are conditions associated with Ca²⁺ accumulation where this relationship falls down.

The determination of both components of p for mitochondria in situ is complex. In addition to the need to determine matrix volume and probe binding, the presence of the intervening plasma membrane, with its own membrane potential and pH gradient has to be considered (128, 517). Hoek et al. (236) first quantified p for in situ hepatocyte mitochondria; _m was determined from the Nernstian distribution of [³H]TPMP⁺ between the cytoplasm and matrix, after correcting for the plasma membrane potential (_p), while pH was measured by weak acid distribution, again controlling for the gradient across the plasma membrane. A value for p of >200 mV was consistent with values obtained with isolated mitochondria. Subsequently, the phosphonium cations have been used as isotopes or with a TPP⁺-selective electrode (273) to quantify the _m component (typically some 150 mV) for mitochondria within isolated nerve terminals (6, 517) and a variety of cells, including sea urchin sperm (504), basophilic leukemia cells (494), lymphocytes (59), thymocytes (60), hepatocytes (206, 425, 426), and perfused rat hearts (596).

The distribution of phosphonium cations such as TPMP⁺ and TPP⁺ is responsive to both _m and _p

(3)

(4)

(5)

where the potentials have their normal sign conventions (resting _p is negative, _m is positive). The logarithmic nature of the above equations requires comment, particularly in the context of the fluorescent probes discussed below. A signal that is proportional to the concentration of the probe in the mitochondrion does not translate into a linear function of membrane potential. Thus, for example, a report of a 30% decrease in signal (e.g., Ref. 78) does not translate into a 30% decrease in _m (e.g., from 180 to 125 mV) but rather a much more modest decrease from 180 to 170 mV. Attempts to quantify _m from the extent of phosphonium cation binding after washing to remove free cation (82) are of course invalid.

The extension of the use of cationic fluorescent membrane potential indicators from isolated mitochondria to cells requires considerable care to avoid misleading artifacts. The probes are accumulated into intracellular mitochondria in the same way as the phosphonium cations: first across the plasma membrane in response to _p and then from cytoplasm to matrix in response to _m. Although confocal microscopy can resolve the fluorescence of single intracellular mitochondria loaded with a membrane potential probe (340), direct determination of the Nernstian gradient between matrix and cytoplasm by confocal microscopy is extremely difficult because of the small size of the mitochondria, the enormous dynamic range required to quantify the 300-fold gradient corresponding to a 150-mV potential, and the need to work at low concentrations of probe that do not produce quenching (or inhibition) within the matrix. Although it is virtually impossible to exclude extramitochondrial volume from the optical slice, Loew and co-workers (167) have devised compensating deconvolution algorithms for tetramethylrhodamine ester fluorescence from single in situ mitochondria. In addition, Ubl et al. (583) have evaluated the possibility of comparing the fluorescence intensity of mitochondria-poor regions (such as the nucleus in a thin optical section) with that of the mitochondria-rich cytoplasm and have concluded that with appropriate calibration (valinomycin-induced K⁺ diffusion potentials in permeabilized cells) that an estimate of _m up to 140 mV may be obtained with an accuracy of ±20 mV.

Plasma membrane and mitochondrial membrane potentials are determined from the free rather than bound concentrations of probe; thus the fixable cationic probe chloromethyl-tetramethyl-rosamine methyl ester (CMTMR; Mitotracker orange) (54, 593) poses serious limitations. The principle is that the cationic probe accumulates in the matrix and then reacts via its chloromethyl group with thiol groups, allowing the cells to be washed, fixed, and monitored. Unfortunately, it is not clear that the amount of chemically reacted CMTR is directly proportional to the free matrix concentration and whether the thiol groups remain in excess or become saturated.

At single-cell resolution, two general protocols have been adopted. In the first, the putative change in _m is established before addition of the probe. Total cell fluorescence, i.e., the sum of nonmitochondrial and mitochondrial contributions, is monitored, a decrease relative to controls being ascribed to a mitochondrial depolarization. Unfortunately, cells are frequently washed in the absence of probe before or during measurement, thus disturbing the Nernst distribution across the plasma membrane. The fluorescence will then depend on the rate at which the membrane-permeant probe effluxes from the cell. In addition, most of these studies fail to take account of the extent of fluorescence quenching of the highly concentrated probe within the mitochondrial matrix (155).

Matrix quenching is deliberately exploited in the second protocol, which is suitable in principle for the continuous monitoring of _m. If trans-plasma membrane equilibration of the probe is slow in relation to that across the mitochondrial inner membrane, a decrease in _m will be reported as an increased whole cell fluorescence as more probe moves from the relatively quenched environment of the matrix to the cytoplasm (339). Conversely, a mitochondrial hyperpolarization will cause a decreased whole cell fluorescence. Commonly employed dyes include rhodamine-123 (47, 73, 74, 141, 155, 257, 267, 286, 290, 463), DiOC₆(3) (84, 348, 349, 488, 489, 495, 623), and the tetramethylrhodamine methyl or ethyl esters [TMRM or TMRE (148, 340, 423, 508)]. The cationic 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyanine iodide (JC-1) (477) has been used in a different mode (107, 240, 476, 604). Above a critical concentration in the matrix, the probe forms red-emitting (590 nm) J-aggregates and below this concentration green-emitting (527 nm) monomers.

Redistribution of probe between the matrix and cytoplasm in response to a decrease in _m will of course increase the concentration in the latter compartment. However, because the probe is permeant across the plasma membrane (the pathway for its initial loading), it follows that the resultant increase in cytoplasmic concentration should lead to a secondary efflux of probe from the cytoplasm to restore the plasma membrane Nernst equilibrium with a consequent decrease in signal. The actual shape of this biphasic fluorescent response to mitochondrial depolarization will depend on the relative permeability of the mitochondrial and plasma membranes to the probe and the geometry of the cell. The high surface-to-volume ratio possessed by small cells and cell processes such as neurites means that the secondary decrease in fluorescence follows rapidly upon the initial increase (415), whereas in large cell bodies the secondary efflux can be slower.

Because the cytoplasmic concentration of these probes at equilibrium is sensitive to _p, the probes are also responsive to plasma membrane depolarization, as shown for the phosphonium cations (Eq. 2). This consideration is of particular relevance under conditions where _p changes, for example, KCl depolarization of neurons or N-methyl-D-aspartate (NMDA) receptor activation. The actual behavior of individual probes is largely empirical; thus the slow decrease in TMRM fluorescence seen when cultured neurons are challenged with glutamate (508) primarily reflects the decrease in _p accompanying receptor activation (415).

The fluorescent signal from some probes [e.g., rhodamine-123 (141) and JC-1 (495)] has been reported to be insensitive to changes in plasma membrane potential, although Davis et al. (119) observe a strong _p dependency of rhodamine-123 fluorescence in MCF-7 cells. Furthermore, changes induced by high KCl (141) or glutamate in the absence of Ca²⁺ (604) fail to affect the red-to-green emission ratio of JC-1. This could either imply that redistribution across the plasma membrane of these probes is so slow that loss of probe from the cells is unimportant or that the signal results from predominantly bound rather than free probe, which would imply that the probes are not responding in the Nernstian mode required for interpretation of the fluorescence changes. A recent paper by Rottenberg and Wu (489) analyzed in great detail the use of DiOC₃(6) for the determination of _m in lymphocytes by flow cytometry, a technique which has been widely employed to estimate changes in _m (84, 348, 349, 439, 623). Rottenberg and Wu (489) conclude that the concentrations at which the dye is normally employed (40-100 nM) result in a 90% inhibition of cell respiration (hence lowering _m), quenching of the signal within the matrix, and a response selective for _p rather than _m (489).

It is important to remain cautious in the detailed interpretation of the fluorescence changes of _m monitoring probes and to seek some independent confirmation of the bioenergetic status of the in situ mitochondria. In particular, reliable application of a particular probe to the measurement of _m in one cell type should not be taken to mean that it can be used in the same way with other cells. Calibration with defined effectors of _m and _p should be performed with each new cell type.

Very recently, a promising technique for monitoring the pH of intracellular compartments has been described, based on the pH-sensitive fluorescence of targeted Aequoria victoria green-fluorescent protein mutants (296, 336). Mitochondria in situ within HeLa cells displayed a pH of 0.5 units (336) consistent with values obtained with isolated mitochondria.

	IV. MITOCHONDRIAL CALCIUM TRANSPORT

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The inner mitochondrial membrane possesses a uniporter, which remains to be identified at a molecular level, able to transport Ca²⁺, but not Mg²⁺, into the matrix (for review, see Refs. 203, 211, 412). The high mitochondrial membrane potential dictates that thermodynamic equilibrium would be attained if the free matrix Ca²⁺ rose to a value 10⁵-10⁶ higher than in the extramitochondrial medium. This potentially lethal accumulation does not however occur, due to the presence in the inner membrane of a separate efflux pathway which exchanges Ca²⁺ for protons (e.g., liver mitochondria) or for Na⁺ (e.g., heart, brain, and brown fat mitochondria) (114). In the latter tissues, a mitochondrial Na⁺/H⁺ transporter is present, and the overall ion flux under conditions of constant mitochondrial Ca²⁺ loading consists of sequential Ca²⁺, Na⁺, and H⁺ cycling (113, 114), the last driven by the respiratory chain (Fig. 2).

In the presence of physiological concentrations of phosphate, an osmotically inactive Ca²⁺-phosphate complex forms in the matrix at a critical Ca²⁺ loading (318), and the activity of the efflux pathway is found to be invariant above this value (632). The complex is not a hydroxyapetite-like precipitate, since depolarization of the inner membrane leads to a rapid independent efflux of Ca²⁺ via the reversed uniporter and phosphate on its transporter. The factors that prevent precipitation of calcium phosphate in the matrix are unclear, although phosphocitrate has been proposed as an antinucleation agent preventing crystallization (564). When there is sufficient Ca²⁺ and phosphate to form this complex, mitochondria behave as perfect buffers of extramitochondrial Ca²⁺ ([Ca²⁺]_o), accumulating the cation whenever its concentration rises above the set point at which uptake and efflux balance and releasing Ca²⁺ below this value (40, 410). The set point can vary between 0.3 and 1 µM (58, 323, 410, 418), with the actual value depending on the kinetics of the two pathways. Thus the set point can be shifted to a higher [Ca²⁺]_o by activating the efflux pathway or inhibiting the uniporter. This can be observed with isolated brain mitochondria where increasing the Na⁺ concentration in the medium stimulates the efflux pathway (418). It is important that the set point is above the resting [Ca²⁺]_c of typical cells, since otherwise there would be an inexorable increase in mitochondrial Ca²⁺. Ordinarily, this mechanism is adapted to cope with peak elevations in [Ca²⁺]_c (for review, see Ref. 412).

In mitochondria operating below the set point, matrix [Ca²⁺] ([Ca²⁺]_m) is low and responsive to changes in [Ca²⁺]_c. Three key metabolic enzymes, pyruvate dehydrogenase, isocitrate dehydrogenase, and 2-oxoglutarate dehydrogenase, are activated by increases in [Ca²⁺]_m, and the increased flux of the citric acid cycle is reflected in an increased reduction state of NADH and a slight increase in p when [Ca²⁺]_m responds to an increased [Ca²⁺]_c (211, 367). This is a means of activating the respiratory chain in response to hormonal stimuli that lead to a physiological increase in [Ca²⁺]_c, allowing an increased "static head" p to offset the increased drop in potential as the proton current is increased in response to increased cellular ATP demand, or net Ca²⁺ accumulation itself (reviewed in Ref. 211). Thus an increase in [Ca²⁺]_c in synaptosomes resulted in an increased activity of pyruvate dehydrogenase (212), while NAD(P)H autofluorescence (which largely reflects the mitochondrial pool) was enhanced by increase in [Ca²⁺]_c in dissociated mouse sensory neurons (141).

Calcium accumulation by isolated mitochondria can lower _m in one of three ways (Fig. 3). First, in the absence of phosphate, the capacity of isolated mitochondria to accumulate Ca²⁺ is restricted, since the protons extruded by the respiratory chain during Ca²⁺ accumulation are no longer neutralized by phosphate uptake (632). Under these conditions, a progressive alkalinization of the matrix occurs, which can ultimately result in almost 2 pH units across the inner membrane and a _m reduced to ~100 mV (410).

View larger version (14K): [in this window] [in a new window]   Fig. 3. Relationships between proton-motive force (p), mitochondrial membrane potential (m), and pH gradient across inner membrane (pH) for isolated mitochondria accumulating Ca2+. Schematic data are shown for time course of changes in p, m, and 60pH during loading of Ca2+ (bar). A: in presence of Pi, there is a reversible depolarization of p shadowed by m. B: in strict absence of Pi, protons extruded to drive Ca2+ uptake are no longer neutralized, and pH builds up at expense of m. C: permeability transition is signaled by a total collapse of ion gradients. Horizontal dashed line represents approximate value of p which is in thermodynamic equilibrium with ATP/ADP pool. Above this value ATP synthesis is possible; below this value cytoplasmic ATP will be hydrolyzed.

The second means by which Ca²⁺ can lower _m occurs in the presence of excess phosphate (Fig. 3). Coaccumulation of phosphate with Ca²⁺ neutralizes pH and forms the osmotically inactive but rapidly dissociable calcium phosphate complex. Under these conditions, there are transient depressions in both the _m and pH components of p during the uptake of the cation, due to the utilization of the proton gradient by the Ca²⁺ plus phosphate transport, and these can be sufficient to interrupt ATP synthesis or even to cause reversal of the ATP synthase (484). Acetate can also be used as a permeant anion to neutralize pH (since acetate enters together with a proton), but this combination causes matrix swelling since calcium acetate is osmotically active.

The accumulation of Ca²⁺ in the presence of excess phosphate should be capable of continuing indefinitely, since phosphate neutralizes the alkalinization of the matrix (Fig. 3), as long as there is space in the matrix for the osmotically inactive calcium-phosphate complex. In practice, mitochondria incubated in media approximating to the physiological cytoplasm (including adenine nucleotides and Mg²⁺) can accumulate and retain in excess of 100 mM total Ca²⁺ (e.g., >200 nmol Ca²⁺/mg mitochondrial protein in a matrix volume of ~1 µl/mg protein; Ref. 418) with no apparent deterioration in their capacity to maintain a high p or to generate ATP (418, 484).

The third mechanism by which Ca²⁺ accumulation may lower _m (Fig. 3) is the condition of Ca²⁺ overload leading to mitochondrial swelling (as evidenced by a decreased 90° light scattering at 540 nm), loss of respiratory control, collapse of _m, and release of matrix Ca²⁺ caused by a permeabilization of the mitochondrial inner membrane to sucrose and other molecules up to ~1.5 kDa (11a). This MPT is most readily observed with isolated mitochondria incubated in the presence of phosphate and Ca²⁺, but in the absence of adenine nucleotides and Mg²⁺ (see Refs. 42, 44, 634 for reviews). It is unclear what relation free as opposed to total matrix Ca²⁺ has to mitochondrial dysfunction. Thus the deleterious effects of Ca²⁺ increase with phosphate concentration, whereas at the same time [Ca²⁺]_m decreases due to the formation of the Ca²⁺-phosphate complex (632).

That the MPT is mediated by a pore in the inner membrane, rather than by membrane damage during the isolation of mitochondria, was demonstrated by Hunter and Haworth and co-workers (246, 247). They showed, by using hyperosmotic solutions of polyethylene glycol of different molecular weights, that the MPT possessed a permeability cut off for solutes at ~1,500 Da (247). Direct evidence for the existence of a pore was later obtained with electrophysiological measurements of a "mitochondrial megachannel" with properties qualitatively similar to the MPT (633). The maximum conductance of the megachannel is reported to be between 1 and 1.5 nS (633), with long-lasting closed states. The molecular identity of the pore remains elusive, but a number of components including the adenine nucleotide translocator, mitochondrial porin, and the peripheral benzodiazepine receptor are implicated in either its regulation or formation at membrane contact sites (208, 209).

The MPT is facilitated by factors that enhance oxidative stress or deplete the matrix adenine nucleotide pool such as pyrophosphate and atractylate (which locks the adenine nucleotide translocator in the C-conformation in which the binding site faces the cytoplasm). Conversely, the MPT is largely prevented by including Mg²⁺ and adenine nucleotides in the medium, by bongkrekate (which locks the adenine nucleotide in the matrix or M-conformation), and by cyclosporin derivatives that interact with a mitochondrial cyclophilin which associates with the adenine nucleotide translocator. Chelation of extramitochondrial Ca²⁺ with EGTA can also reverse the MPT (11a). However, a recent confocal study of immobilized mitochondria (248) has shown that individual mitochondria can undergo stochastic, cyclosporin-sensitive, large-amplitude fluctuations in _m even in the absence of Ca²⁺.

The Ca²⁺ ionophores that have been commonly used in mitochondrial studies are ionomycin and A-23187, which function as electroneutral Ca²⁺/2H⁺ exchangers. Their effect in mitochondria is to create an additional Ca²⁺ efflux pathway in the inner membrane, depleting the matrix of Ca²⁺ (456, 475). In contrast to the slow Ca²⁺ cycling that occurs between the uniporter and the relatively low activity endogenous efflux pathway, the rate of Ca²⁺ cycling in the presence of excess ionophore is only limited by the activity of the Ca²⁺ uniporter and is hence controlled by [Ca²⁺]_o. In elevated [Ca²⁺]_o (>1-2 µM), this dissipative cycling can have a similar effect to a protonophore, collapsing _m and inducing uncontrolled respiration (219).

Calcium homeostasis, metabolism, and bioenergetics are intimately interconnected in the intact cell and must be considered as part of an integrated system (Fig. 2). Mitochondrial respiration drives both ATP synthesis and Ca²⁺ accumulation. Because these two processes compete for the proton circuit, they should be considered together in an analysis of the effects of cellular Ca²⁺ loading; thus, although Ca²⁺ accumulation by the mitochondria may affect ATP synthesis, alterations in ATP synthesis will in turn affect the activity of ion pumps responsible for removing Ca²⁺ from the cytoplasm.

The early studies with isolated mitochondria discussed above predicted that mitochondria would accumulate Ca²⁺ whenever [Ca²⁺]_o rose above the set point at which uptake and efflux balanced (410). A major goal of recent studies with intact neurons has been to determine how mitochondrial Ca²⁺ accumulation might affect cellular Ca²⁺ homeostasis, particularly in response to the Ca²⁺ loading via voltage-activated Ca²⁺ channels or NMDA receptor activation. The cytoplasmic Ca²⁺ transients measured with fluorescent Ca²⁺ probes during depolarization of neurons with KCl (103, 141, 144), glutamate (102, 104, 473, 594), or trains of action potentials (109, 602) rise well above the predicted mitochondrial set point and should therefore be influenced by mitochondrial Ca²⁺ sequestration. Qualitative confirmation that the mitochondria have accumulated Ca²⁺ has been obtained by protonophore addition to a Ca²⁺-loaded cell. This will release any accumulated mitochondrial Ca²⁺ to the cytoplasm, giving a transient elevation in [Ca²⁺]_c preceding any subsequent effects due to ATP depletion. When such experiments are performed with resting, polarized neurons, no cytoplasmic transients are observed, consistent with mitochondria being largely depleted of Ca²⁺ below their set point; however, protonophore addition to cells after Ca²⁺ loading produces the predicted spike in [Ca²⁺]_c (73, 565, 566, 598, 602, 603).

Although the Ca²⁺-2H⁺ antiport ionophores ionomycin or A-23187 are frequently added to cells in an attempt to increase [Ca²⁺]_c (e.g., Refs. 89, 303, 312, 434, 447, 525), the incorporation of the ionophore into the inner mitochondrial membrane and resulting dissipative Ca²⁺ cycling can depolarize _m with deleterious effects on ATP levels comparable to a protonophore (7).

The kinetics of mitochondrial Ca²⁺ transport suggested a model in which the organelles would act as temporary stores of Ca²⁺ during cytoplasmic Ca²⁺ peaks, blunting the cytoplasmic response and releasing the Ca²⁺ back to the cytoplasm when [Ca²⁺]_c recovered to below the set point (8, 412). In 1990, Thayer and Miller (565) obtained direct evidence for this by showing that brief KCl-mediated depolarization of dorsal root ganglion cells was followed by a recovery in [Ca²⁺]_c to a plateau of 200-600 nM (549, 565). This elevated plateau was interpreted to be a consequence of the slow release from the mitochondrion of Ca²⁺ accumulated at the peak [Ca²⁺]_c. A similar plateau was observed in these cells following trains of action potentials (602). Comparable plateaus have subsequently been reported for bullfrog sympathetic neurons (173) or chromaffin cells (29, 229).

The cationic fluorescent Ca²⁺ indicator rhod 2 can be localized within the mitochondrial matrix to monitor [Ca²⁺]_m (268, 385, 447, 448, 570). Rhod 2 has been used to detect histamine-induced oscillations in [Ca²⁺]_m (268) and mitochondrial Ca²⁺ accumulation in both glutamate-exposed striatal neurons (447, 448) and field-stimulated lizard nerve terminals (118). In the last example, a mitochondrial signal was only observed after a train of 15-20 pulses, consistent with the need to exceed the threshold imposed by the set point. A limitation, however, with rhod 2 is its relatively high affinity (dissociation constant = 0.6 µM), which means that it is saturated by >5 µM free Ca²⁺.

The problem inherent in studies aimed at assessing the role of mitochondrial Ca²⁺ transport lies in the ability to perform appropriate control experiments in which mitochondrial Ca²⁺ uptake is selectively inhibited. There are currently no specific, cell-permeant inhibitors of the mitochondrial Ca²⁺ uniporter, although the ruthenium amine complex Ru-360 is a possible candidate (363). The hexavalent glycoprotein stain ruthenium red (343, 560) is effective with isolated mitochondria at low concentrations; however, there is little evidence that it can permeate across the plasma membrane of neurons. Experiments in which ruthenium red or La³⁺ have been shown to antagonize the neurotoxic effects of NMDA receptor activation (e.g., Refs. 131, 150, 257) may be primarily due to inhibition of plasma membrane voltage-activated Ca²⁺ channels or NMDA receptors (352, 560). Penetration of the stain into the somata of cultured neurons accompanying excitotoxicity has been reported (560), but it is not clear whether the appearance of ruthenium red in the cytoplasm preceded plasma membrane permeabilization. This criticism of course does not apply to experiments in which ruthenium red is loaded via the electrode in whole cell patch-clamp experiments (e.g., Refs. 221, 438, 565).

A possible inhibitor of the mitochondrial Ca²⁺ efflux pathway has been investigated by a number of groups (33, 241, 529, 604, 605). 7-Chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-one (CGP-37157) inhibits the mitochondrial Na⁺/Ca²⁺ exchanger and would therefore be predicted to lower the mitochondrial set point and potentially to lead to massive, essentially irreversible, Ca²⁺ accumulation by the mitochondria. Addition of the inhibitor immediately subsequent to glutamate facilitates the restoration of basal [Ca²⁺]_c in cultured forebrain neurons (605). Unfortunately, the inhibitor also inhibits voltage-gated Ca²⁺ channels, preventing its application to such studies (33). The lipophilic cation TPP⁺, discussed in section IIIB has been reported to inhibit the Na⁺-dependent mitochondrial Ca²⁺ efflux pathway (278, 608), and the cation has been used in a recent study (559) in an attempt to establish the role of presynaptic mitochondrial Ca²⁺ transport in the posttetanic potentiation of synaptic transmission. However, the extracellular concentration employed, 10 µM, would give an estimated 100 mM within the mitochondrial matrix (see Eq. 5) which would result in extensive mitochondrial swelling and depolarization, with consequent compromised ATP synthesis and Ca²⁺ sequestration.

In the absence of selective, permeant mitochondrial Ca²⁺ uniport inhibitors, an indirect approach has to be taken to inhibit mitochondrial Ca²⁺ transport in intact neurons. That most frequently adopted has been to depolarize the mitochondria. However, addition of a protonophore can lead to profound ATP depletion as well as causing cytoplasmic acidification and releasing glutamate from synaptic vesicles. With this in mind, it has generally been reported that the addition of a protonophore leads to an increased cytoplasmic Ca²⁺ elevation in response to a Ca²⁺ load induced by elevated KCl (73, 549, 565) or glutamate activation of NMDA receptors (292, 566). However, in such experiments, it is difficult to distinguish between an increased [Ca²⁺]_c as a consequence of failed mitochondrial Ca²⁺ sequestration and one due to inhibited Ca²⁺ extrusion after ATP depletion. This was already recognized in 1990, when Duchen et al. (144) reported enhanced cytoplasmic Ca²⁺ transients in dorsal root ganglion cells exposed to a range of metabolic inhibitors, including protonophores, cyanide, and glucose removal, but observed that these effects could be a consequence of impaired Ca²⁺ extrusion from the cells as well as inhibited mitochondrial sequestration. Similar results have been obtained with cortical neurons (472), hippocampal neurons (598), avian cochlear neurons (392), and cerebellar granule cells (73).

In cells with active glycolysis, the ATP synthase inhibitor oligomycin can be employed to reduce the number of parameters affected by mitochondrial depolarization. Comparison of the [Ca²⁺]_c signal evoked, for example, by elevated KCl in cells in the presence of oligomycin with those incubated with oligomycin plus a respiratory chain inhibitor allows the effect of inhibited mitochondrial Ca²⁺ sequestration to be observed in the absence of ATP synthase reversal (73, 74, 85, 141, 517, 519). In designing such experiments, the reversibility of inhibitor binding needs to be taken into consideration. High-affinity inhibitors such as rotenone, antimycin A, and oligomycin (116) are effectively irreversible, whereas cyanide and ionophores including carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) can be readily removed by washing.

In view of the evidence that mitochondria sequester much of the Ca²⁺ load imposed by KCl depolarization, it would be predicted that abolition of the mitochondrial pool would enhance a subsequent cytoplasmic Ca²⁺ transient. However, the results are surprising; granule cells maintaining a high ATP/ADP in the presence of rotenone/oligomycin (73) and display reduced cytoplasmic Ca²⁺ responses to KCl depolarization or NMDA receptor activation (73, 74, 85), whereas increased signals are obtained in cells treated with protonophore before Ca²⁺ loading. Although a decreased bulk cytoplasmic Ca²⁺ elevation when the cell's main Ca²⁺ sink is inactivated is counterintuitive, it is supported by the decreased total accumulation of ⁴⁵Ca²⁺ by neurons under these conditions (74).

	V. REACTIVE OXYGEN SPECIES AND MITOCHONDRIA

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Oxidative stress is a common feature of many different forms of neurodegenerative disease (for review, see Ref. 38). To rationalize a large and complex field, it is helpful to first consider the mechanisms for the endogenous generation of ROS.

Although molecular oxygen is reduced to water in the terminal complex IV by a sequential four-electron transfer, a minor proportion can be reduced by a 1e addition that occurs predominantly in complex III (56, 362, 544) but also in complex I (228, 577). The cyclic electron transfer pathway within complex III (the Q cycle; see Ref. 416 for review) involves a site close to the cytoplasmic face of the membrane where UQH₂ transfers a single electron to cytochrome c₁ via the Rieske iron-sulfur protein, leaving a highly reactive ubisemiquinone UQ·. Loss of the second electron and generation of UQ is dependent on the transfer of the electron through two sequential cytochrome b prosthetic groups located on opposite sides of the inner membrane (628). This transfer is therefore opposed by _m such that a high _m enhances the occupancy of the ubisemiquinone binding site, where a chance exists that this second electron can be transferred to molecular oxygen, generating the superoxide anion O₂· (576). The inhibitor antimycin A increases O₂· production by inhibiting reduction of the second b cytochrome, while myxathiazol has the opposite effect by inhibiting the initial generation of the semiquinone (307, 576). Recently, a Q cycle-related pathway of electron transfer has been proposed for complex I (146).

Very high membrane potentials (e.g., during succinate respiration in state 4) are required to generate significant amounts of O₂· (534) or H₂O₂ (57). Succinate-supported O₂· production is strongly inhibited in state 3 (57), although complex I substrates are unaffected by the decrease in _m and may be the main contributors to O₂· production during state 3 respiration (228). The nonohmic proton leak (408, 425) may function to limit _m and hence potentially toxic O₂· generation (534). Proton-motive force can also be regulated by uncoupling proteins (UCP). In addition to the original UCP, UCP1 (220), other isoforms have recently been identified (168). The protein UCP2 is widely expressed and has been proposed to control ROS generation by limiting _m (403).

Calcium loading of isolated mitochondria in the presence of phosphate increases the production of O₂· (14