Lithium Desensitizes Brain Mitochondria to Calcium, Antagonizes Permeability Transition, and Diminishes Cytochrome c Release*

Among the numerous effects of lithium on intracellular targets, its possible action on mitochondria remains poorly explored. In the experiments with suspension of isolated brain mitochondria, replacement of KCl by LiCl suppressed mitochondrial swelling, depolarization, and a release of cytochrome c induced by a single Ca2+ bolus. Li+ robustly protected individual brain mitochondria loaded with rhodamine 123 against Ca2+-induced depolarization. In the experiments with slow calcium infusion, replacement of KCl by LiCl in the incubation medium increased resilience of synaptic and nonsynaptic brain mitochondria as well as resilience of liver and heart mitochondria to the deleterious effect of Ca2+. In LiCl medium, mitochondria accumulated larger amounts of Ca2+ before they lost the ability to sequester Ca2+. However, lithium appeared to be ineffective if mitochondria were challenged by Sr2+ instead of Ca2+. Cyclosporin A, sanglifehrin A, and Mg2+, inhibitors of the mitochondrial permeability transition (mPT), increased mitochondrial Ca2+ capacity in KCl medium but failed to do so in LiCl medium. This suggests that the mPT might be a common target for Li+ and mPT inhibitors. In addition, lithium protected mitochondria against high Ca2+ in the presence of ATP, where cyclosporin A was reported to be ineffective. SB216763 and SB415286, inhibitors of glycogen synthase kinase-3β, which is implicated in regulating reactive oxygen species-induced mPT in cardiac mitochondria, did not increase Ca2+ capacity of brain mitochondria. Altogether, these findings suggest that Li+ desensitizes mitochondria to elevated Ca2+ and diminishes cytochrome c release from brain mitochondria by antagonizing the Ca2+-induced mPT.

Mitochondrial injury plays a crucial role in excitotoxic neuronal death induced by prolonged activation of glutamate receptors (1). Exposure of neurons to excitotoxic glutamate causes massive influx of Ca 2ϩ into the cytosol through the glu-tamate receptors and possibly through the reverse mode of the Na ϩ /Ca 2ϩ exchanger (2)(3)(4)(5). Mitochondria contribute to clearance of elevated cytosolic Ca 2ϩ by accumulating Ca 2ϩ in the mitochondrial matrix (6 -11). However, excessive accumulation of Ca 2ϩ in the matrix might induce the mitochondrial permeability transition (mPT) 2 that leads to depolarization and swelling of the organelles (12). Both manifestations of the mPT are dangerous for the cell. The former leads to inhibition of oxidative phosphorylation and suppression of Ca 2ϩ uptake, whereas the latter might cause the rupture of the outer mitochondrial membrane (OMM) and a release of mitochondrial apoptogenic proteins (12). Therefore, it is of utmost importance to understand the mechanisms of induction and regulation of the mPT.
Numerous factors and pharmacological agents influence the probability of the mPT (13,14). Cyclosporin A (CsA) is considered to be the most prominent and widely used inhibitor (or "desensitizer" as recently suggested by Dr. Paolo Bernardi (12)) of the mPT, but it also leads to inhibition of calcineurin, a cytosolic phosphatase (15). NIM811 and UNIL025, more selective CsA derivatives that do not suppress calcineurin, are probably the most potent and specific inhibitors of the mPT (16). CsA as well as its congeners bind to mitochondrial cyclophilin D, desensitizing the mPT pore to Ca 2ϩ (17)(18)(19)(20). Sanglifehrin, another potent inhibitor of the mPT, also binds to cyclophilin D and suppresses the mPT (21). In addition, ADP and ATP also inhibit the mPT (22)(23)(24). There is evidence that adenine nucleotides can inhibit the mPT due to binding to adenine nucleotide translocase, a putative conducting pathway of the mPT pore in the inner mitochondrial membrane (23). Additionally, mitochondria possess at least two separate mPT-related binding sites for divalent cations (Me 2ϩ ) (25). One type of binding site was found inside of mitochondria (internal site), whereas another type (external) was located on the outer side of the IMM. Binding of Ca 2ϩ to the internal site increased probability of the mPT, whereas binding of other Me 2ϩ (Sr 2ϩ , Mn 2ϩ , and Mg 2ϩ ) proved to be inhibitory. Binding of any Me 2ϩ to the external site decreased the probability of the mPT (25). Whether monovalent cations can bind to these sites and affect mPT induction is not known.
Among other monovalent cations, Li ϩ has several remarkable properties. Its ionic radius is very close to Mg 2ϩ , and therefore in some cases, Li ϩ exert actions similar to Mg 2ϩ (26). It was shown in early studies that Li ϩ binds to mitochondria with higher affinity than other monovalent cations (27). Later, it was demonstrated that Li ϩ binds to phospholipids (28,29). Lithium has numerous intracellular targets and pharmacological actions (30). For more than 50 years, lithium has been used in the psychiatric treatment of bipolar disorder, although the mechanism of the lithium action remains unknown (30,31). Recently, Li ϩ was found to be protective against the ROS-induced mPT in experiments with cardiomyocytes and isolated cardiac mitochondria (32). Since Li ϩ is an inhibitor of glycogen synthase kinase-3␤ (GSK-3␤) (33) and since other inhibitors of GSK-3␤ also provided protection against the mPT in cardiac mitochondria, the authors concluded that the GSK-3␤ is a convergence point for the mPT inhibition by a variety of agents, including Li ϩ (32).
In long term experiments, low concentrations of lithium were found to interfere with phosphoinositol metabolism in neuronal cells (34,35). The long term treatment of cerebellar granule cells led to robust inhibition of Ca 2ϩ influx through N-methyl-D-aspartate-type glutamate receptors and also led to protection against excitotoxicity (36). An acute exposure to low concentrations of lithium did not affect Ca 2ϩ influx into neurons induced by glutamate/N-methyl-D-aspartate (36). Upon glutamate stimulation, Na ϩ and Li ϩ can enter neurons through the nonselective ion channel of the glutamate/N-methyl-D-aspartate receptor (37)(38)(39). It remains unknown whether Na ϩ or Li ϩ can change the susceptibility of brain mitochondria to Ca 2ϩ -induced mPT and thus potentially contribute to sensitization or desensitization of neurons to glutamate exposure. In the present study, we addressed this question. Using various experimental approaches to evaluate induction of the mPT in isolated synaptic and nonsynaptic brain mitochondria, we found that Li ϩ remarkably desensitizes brain mitochondria to Ca 2ϩ . We hypothesize that this effect might be due to Li ϩ binding to the external mPT-related cation binding site proposed earlier by Dr. Paolo Bernardi for divalent cations (25). We further speculate that Li ϩ -evoked desensitization of neuronal mitochondria to Ca 2ϩ might contribute to protection against delayed Ca 2ϩ deregulation in cultured neurons exposed to excitotoxic glutamate in LiCl-based medium.

EXPERIMENTAL PROCEDURES
Isolation of Brain, Liver, and Heart Mitochondria-To isolate mitochondria, male Sprague-Dawley rats (225-250 g) (Harlan, Indianapolis, IN) were sacrificed by decapitation according to an Institutional Animal Care and Use Committee-approved protocol. Nonsynaptic mitochondria were isolated in mannitol-sucrose medium and purified on a discontinuous Percoll gradient (40,41). Synaptic mitochondria were isolated from synaptosomes by the nitrogen cavitation method using a nitrogen cell disruption bomb, model 4639 (Parr Instrument Company, Moline, IL), cooled on ice (42) with some modifications. Briefly, the synaptosomes obtained during preparation of nonsynaptic mitochondria were transferred in a cooled plastic beaker and placed into the nitrogen bomb on ice under 1,000 p.s.i. for 13 min. Then the synaptosomes were layered on top of the discontinuous Percoll gradient (26%/40%) and centrifuged at 31,000 ϫ g for 28 min in a Beckman SW-41 rotor. The mitochondrial fraction in the interface between Percoll layers was transferred into a fresh tube, diluted 1:5 with medium containing 394 mM sucrose, 0.1 mM EGTA, 10 mM HEPES, pH 7.4, and centrifuged at 31,000 ϫ g for 20 min. The pellet was resuspended in 0.5 ml of the latter medium and kept on ice. Liver and heart mitochondria were isolated and purified in the same way as nonsynaptic brain mitochondria, except the lower level in the discontinuous density gradient contained 60% Percoll (43). Mitochondrial protein concentration was measured by the Bradford method (44) with bovine serum albumin as a standard.
Measurements of Light Scattering, Mitochondrial Membrane Potential (⌬), Respiration, and Ca 2ϩ Uptake-Mitochondrial swelling was evaluated by following changes in the scattering of transmitted light by mitochondrial suspension at 525 nm in a 0.3-ml chamber at 37°C and continuous stirring. In the experiments with slow Ca 2ϩ infusion, mitochondrial swelling was followed by monitoring the scattering of light beam directed on mitochondrial suspension under 90°to the axis of the photodetector at 525 nm in a 0.4-ml cuvette at 37°C and continuous stirring using a PerkinElmer Life Sciences LS-55 luminescence spectrometer. ⌬ was monitored by following the distribution of TPP ϩ between the external medium (initially 1.8 M TPP ϩ -Cl) and the mitochondrial matrix with a TPP ϩ -sensitive electrode (45) in the standard incubation medium containing 125 mM KCl, 10 mM HEPES, pH 7.4, 0.5 mM MgCl 2 , 3 mM KH 2 PO 4 , 10 M EGTA, 0.1% bovine serum albumin (free from fatty acids), 0.1 mM ADP, and 1 M oligomycin supplemented with 3 mM glutamate and 1 mM malate unless stated otherwise. A decline in the external TPP ϩ concentration corresponded to mitochondrial polarization, whereas a rise in the TPP ϩ concentration in the medium corresponded to depolarization. Mitochondrial Ca 2ϩ uptake was measured by following the disappearance of Ca 2ϩ from the incubation medium with a miniature Ca 2ϩ -selective electrode under the same conditions as the light scattering and ⌬. Mitochondrial respiration was recorded as described previously (46). Respiratory activities and respiratory control ratios of mitochondrial preparations used in this study were similar to those published earlier (46,47). Where stated, experiments have been performed in hypotonic 75 mM KCl-or LiCl-based medium or medium where 125 mM KCl was substituted for equimolar concentrations of LiCl, NaCl, CsCl, RbCl, or N-methyl-D-glucamine. In all experiments with isolated mitochondria, the concentration of mitochondrial protein in the chamber was 0.1 mg/ml. All data traces shown are representative of at least three separate experiments.
Experiments with Individual Mitochondria Loaded with Rhodamine 123-Membrane potential of individual brain nonsynaptic mitochondria was monitored following fluorescence of rhodamine 123 (Rh123) loaded into mitochondria (48,49). Briefly, isolated mitochondria were placed into a glass-bottomed 35-mm Petri dish and continuously perfused with a standard incubation medium supplemented with 0.2 M Rh123. In some experiments, KCl was substituted with equimolar LiCl. A Nikon Eclipse TE2000-S inverted microscope (100% output at the right optical port) equipped with a Nikon CFI Plan Apo VC ϫ100 1.4 numerical aperture objective and SimplePCI 6.1 software (Compix, Sewickley, PA) was used to visualize individual mitochondria loaded with Rh123 in nonquenching mode. In all experiments, mitochondria were perfused using a ValveBank 8 perfusion system (AutoMate Scientific, San Francisco, CA). Mitochondria were illuminated at 480 Ϯ 20 nm using a Lambda LS lighting system (Sutter Instrument, Novato, CA) with a 175-watt xenon lamp. The light beam was attenuated by neutral density filters to 1%. Fluorescence was collected through a 505-nm dichroic mirror and a 535 Ϯ 25-nm emission filter by a back-illuminated EM-CCD Hamamatsu C9100-12 camera (Hamamatsu, Bridgewater, NJ). Images were taken at room temperature every 15 s throughout the experiment.
Slow Ca 2ϩ Infusion-The experiments with slow Ca 2ϩ infusion were performed as described previously (50) with some modifications. Mitochondria were incubated in a 0.3-ml chamber at 37°C and continuous stirring. Mitochondrial Ca 2ϩ accumulation was followed by monitoring the disappearance of Ca 2ϩ from the incubation medium with a miniature Ca 2ϩ -selective electrode. Where stated, experiments have been performed in the standard incubation medium or in the media where KCl was substituted for equimolar LiCl, NaCl, CsCl, RbCl, or N-methyl-D-glucamine. All media were supplemented with 0.1 mM ADP and 1 M oligomycin unless stated otherwise. A solution of CaCl 2 was delivered into the chamber at a constant rate 333 nmol/mg protein ϫ min using a KDS 100 syringe pump (KD Scientific, Holliston, MA) equipped with a Hamilton microsyringe. The Ca 2ϩ capacity was estimated as the amount of accumulated Ca 2ϩ (mol/mg mitochondrial protein) before the failure to further accumulate Ca 2ϩ . This was determined by linear fitting of the initial fragment of the experimental trace and the final linear fragment of the trace and then finding the intersection point of these linear graph lines (Fig. 3).
Measurements of ROS Production with Amplex Red Assay-Production of ROS by nonsynaptic brain mitochondria incubated in media of various cationic compositions was assessed using the Amplex Red assay for H 2 O 2 (Invitrogen), as described previously (51).
Immunoblotting-Release of cytochrome c from isolated brain mitochondria was assessed using Western blotting in supernatants obtained through incubation of mitochondria either in 75 mM KCl-or 75 mM LiCl-based medium for 15 min at 37°C with or without Ca 2ϩ . Western blotting was performed as previously described (51). The release of cytochrome c from mitochondria treated with alamethicin (30 g/ml) was used as a control for maximal cytochrome c release.
Statistics-Statistical analyses of experimental data consisted of one-way analysis of variance followed by Bonferroni's posttest (GraphPad Prism, version 4.0; GraphPad Software, San Diego, CA). The data represent mean Ϯ S.E. of at least three separate, independent experiments.

Ca 2ϩ Pulse Experiments with Purified Mitochondrial
Suspension-In these experiments, we simultaneously evaluated two main manifestations of the mPT, mitochondrial depolarization and swelling (12). Ca 2ϩ pulses of various magnitudes were applied to mitochondria, and distribution of TPP ϩ between external medium and mitochondrial matrix (indica-tive of ⌬) and light scattering of mitochondrial suspension (indicative of matrix volume) were followed simultaneously in either KCl-or LiCl-based medium (Fig. 1a).
In KCl medium, low and moderate Ca 2ϩ pulses caused reversible swelling of brain mitochondria similar to those reported recently (46). In LiCl medium, the same Ca 2ϩ pulses FIGURE 1. Li ؉ desensitizes brain mitochondria to Ca 2؉ administered by a single bolus: decreased swelling, depolarization, and cytochrome c release induced by moderate Ca 2؉ pulses in LiCl medium. The LiCl medium favors Ca 2ϩ uptake. In a and b, isolated nonsynaptic brain mitochondria were incubated at 37°C either in 125 mM KCl medium or in 125 mM LiCl medium supplemented with 3 mM pyruvate plus 1 mM malate. Concentration of mitochondrial protein in the chamber was 0.1 mg/ml. a, mitochondrial swelling (thick, upper traces) followed by the light scattering assay was monitored simultaneously with ⌬ followed by a TPP ϩ assay (thin, lower traces). b, mitochondrial Ca 2ϩ uptake was measured with a Ca 2ϩ -selective electrode. The additions of CaCl 2 are indicated by arrowheads. The numbers near traces indicate the amount of added CaCl 2 (mol/mg protein). a and b, experimental traces are overlapped for comparison. c, Ca 2ϩ -induced cytochrome c release was detected by Western blotting in the KCl but not in LiCl medium following application of Ca 2ϩ pulses. As shown earlier, a decrease in the medium tonicity (75 instead of 125 mM KCl) favored the Ca 2ϩ -induced release of cytochrome c, presumably due to better osmotic balance between incubation medium and mitochondrial matrix (41). A decrease of medium osmolarity did not influence response to Ca 2ϩ , as judged by the light scattering assay (not shown). Therefore, in the cytochrome c release experiments, we used the 75 mM KCl-or LiCl-based media. Alamethicin (30 g/ml) produced maximal release of cytochrome c in both media. a.u., arbitrary units. produced a smaller amplitude of swelling. The effect of Li ϩ was concentration-dependent; smaller concentrations of Li ϩ produced less protection against swelling (supplemental Fig. 1). Larger Ca 2ϩ pulses equalized the amplitude of swelling in both KCl and LiCl medium. In addition, Ca 2ϩ pulses caused mitochondrial depolarization. In LiCl medium, a low Ca 2ϩ pulse caused less depolarization than the same pulse applied in KCl medium (Fig. 1a). Replacement of K ϩ by Na ϩ failed to increase resilience of mitochondria to Ca 2ϩ (not shown). The Ca 2ϩ -dependent depolarization and reversible swelling in brain mitochondria can be prevented by CsA and therefore can be attributed to the mPT (46). Hence, it is possible that Li ϩ also protected brain mitochondria due to desensitization of the mPT pore to Ca 2ϩ .
Li ϩ decreases apparent V max for Ca 2ϩ uptake by liver mitochondria (52). Since the mPT depends on Ca 2ϩ influx into mitochondria (12), we used parallel experiments to test whether replacement of K ϩ for Li ϩ affects mitochondrial Ca 2ϩ uptake. It appeared that mitochondria more effectively accumulated Ca 2ϩ in LiCl medium than in KCl medium (Fig. 1b). Thus, desensitization of mitochondria to Ca 2ϩ in LiCl medium cannot be explained by deficient Ca 2ϩ uptake.
The decreased mitochondrial swelling in LiCl medium correlated with a suppressed release of cytochrome c (Fig. 1c). The maximal cytochrome c release was observed after treatment of mitochondria with alamethicin, which eliminated the barrier properties of the OMM (41). In this case, the release was similar in both KCl and LiCl medium. Thus, the substitution of K ϩ by Li ϩ did not affect the maximal cytochrome c release, but it prevented the release induced by Ca 2ϩ pulses.
Experiments with Individual Rh123-loaded Brain Mitochondria-Experiments with mitochondrial suspension treated with Ca 2ϩ pulses provided valuable insight into the ability of mitochondria to withstand Ca 2ϩ insult. However, this type of experiment requires a relatively high amount of purified mitochondria (in our hands, 30 g of protein/assay) and correspondingly high nonphysiological concentrations of Ca 2ϩ (50 -300 M in a 0.3-ml volume). In addition, submillimolar ADP or ATP prevented Ca 2ϩ -induced depolarization as well as swelling in this type of experiment (not shown). The use of individual brain mitochondria loaded with Rh123 to monitor ⌬ helped to overcome these problems. Fig. 2a shows a representative fluorescence image of isolated brain mitochondria loaded with Rh123 in nonquenching mode. A similar approach was used previously with liver and brain mitochondria (48,49). Prior to Ca 2ϩ application, mitochondria maintained a stable ⌬ (Fig.  2b). Perfusion with a solution containing 1 mM ATP and 0.5 M of free Ca 2ϩ (calculated using MaxChelator software Webmaxc Standard by C. Patton) caused depolarization in most of the mitochondria. In many mitochondria, perfusion with Ca 2ϩ led to rapid fluctuations of ⌬, as previously observed (53). At the end of the experiment, 1 M carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone was applied to collapse the ⌬ completely. The Ca 2ϩ -induced depolarization of mitochondria might be a consequence of the mPT (12). Indeed, CsA antagonized Ca 2ϩ -induced depolarizations, linking them to the mPT (Fig. 2c). Li ϩ robustly protected mitochondria against Ca 2ϩinduced depolarization (Fig. 2d). Fig. 2, e and f, summarizes these results, showing averaged traces for each representative experiment. Thus, the experiments with individual mitochondria substantiated the observations made in Ca 2ϩ -pulse exper- . Li ؉ , in contrast to Na ؉ , increases Ca 2؉ capacity of synaptic and nonsynaptic brain mitochondria in the experiments with slow Ca 2؉ infusion. a and b, isolated synaptic and nonsynaptic brain mitochondria were incubated at 37°C in either 125 mM KCl medium, 125 mM NaCl medium, or 125 mM LiCl medium supplemented with 3 mM glutamate plus 1 mM malate. Ca 2ϩ infusion was initiated as indicated. c, a summary plot demonstrating the Ca 2ϩ capacity of brain mitochondria incubated in different media calculated as described under "Experimental Procedures." *, p Ͻ 0.01 between Ca 2ϩ capacities in KCl versus LiCl medium. Data are mean Ϯ S.E., n ϭ 3.
iments with mitochondrial suspension. In both cases, Li ϩ desensitized mitochondria to Ca 2ϩ and antagonized the mPT.
Slow Ca 2ϩ Infusion-Although collapse of the ⌬ appears to be the main outcome of the mPT (12), inhibition of oxidative phosphorylation and the failure to accumulate Ca 2ϩ represent the major consequences of mitochondrial depolarization. In neurons exposed to glutamate, Ca 2ϩ influx continues until glutamate is present (54). A slow, continuous infusion of Ca 2ϩ into a suspension of isolated mitochondria mimics this situation (50,55,56). Obviously, mitochondria could contribute to clearance of cytosolic Ca 2ϩ in situ and in vitro only until they become unable to accumulate Ca 2ϩ (6,8). Indeed, in our exper-iments, both synaptic and nonsynaptic mitochondria maintained low Ca 2ϩ until they lose the ability to sequester Ca 2ϩ (Fig. 3). Since Ca 2ϩ infusion continued, external Ca 2ϩ raised dramatically. In addition to Ca 2ϩ coming into the chamber with infusion, the release of accumulated Ca 2ϩ from mitochondria might contribute to the rise of external Ca 2ϩ . Replacement of K ϩ by Na ϩ did not increase Ca 2ϩ buffering capacity in either synaptic or nonsynaptic mitochondria (Fig. 3). In contrast, substitution of K ϩ with Li ϩ augmented Ca 2ϩ capacity in both types of brain mitochondria. The similar increase in Ca 2ϩ capacity was found with rat liver and heart mitochondria incubated in LiCl medium(supplementalFig.2).TheeffectofLi ϩ wasconcentrationdependent and appeared to be more pronounced at higher con-  . Li ؉ defers mitochondrial depolarization and antagonizes mitochondrial swelling caused by a slow Ca 2؉ infusion. Isolated nonsynaptic brain mitochondria were incubated at 37°C either in 125 mM KCl medium or in 125 mM LiCl medium supplemented with 3 mM glutamate plus 1 mM malate. a, ⌬ was followed by monitoring the distribution of TPP ϩ between the mitochondrial matrix and the external medium as described under "Experimental Procedures." b, mitochondrial swelling was followed by monitoring light scattering at 525 nm in a 0.4-ml cuvette at 37°C and continuous stirring using a PerkinElmer LS-55 luminescence spectrometer. Alamethicin (30 g/ml) was used to produce maximal swelling. Ca 2ϩ infusion was initiated as indicated.
centrations of Li ϩ (Fig. 4). The replacement of KCl by RbCl, CsCl, or N-methyl-D-glucamine did not increase the Ca 2ϩ capacity of brain mitochondria (supplemental Fig. 3). The increase of Ca 2ϩ capacity in LiCl medium occurred in parallel to improved ability of mitochondria to maintain ⌬ (Fig. 5a). In addition, Li ϩ antagonized mitochondrial swelling caused by an infusion of excessive Ca 2ϩ (Fig. 5b). Thus, Li ϩ protected mitochondria against Ca 2ϩ and increased the ability of mitochondria to sequester Ca 2ϩ .
What are the mechanisms that incapacitated mitochondria in the experiments with slow, continuous Ca 2ϩ infusion? Previously, it was shown that CsA augmented Ca 2ϩ capacity of isolated nonsynaptic brain mitochondria incubated in NaClbased medium (50). In our experiments, CsA increased the Ca 2ϩ capacity of synaptic as well as nonsynaptic mitochondria in both KCl and NaCl media (Fig. 6). It seemed reasonable to assume that excessive accumulation of Ca 2ϩ in mitochondria induced the mPT, leading to depolarization and thus preventing further Ca 2ϩ uptake. However, in LiCl medium, where Ca 2ϩ capacity was already augmented, CsA failed to increase it further. A similar result was obtained with sanglifehrin, another potent inhibitor of the mPT (21) (supplemental Fig. 4). Thus, these observations suggest that the mPT is a critical factor determining mitochondrial Ca 2ϩ capacity and a common target for CsA, sanglifehrin, and Li ϩ .
Mg 2ϩ antagonizes the Ca 2ϩ -induced mPT (22,57). In the following experiments, we tested whether Mg 2ϩ protection can be observed in LiCl medium. In KCl medium, increasing Mg 2ϩ concentrations augmented the Ca 2ϩ capacity of brain mitochondria, presumably due to suppression of the mPT (Fig. 7). In contrast, increased Mg 2ϩ concentration in LiCl medium did not further enhance the already augmented Ca 2ϩ capacity (Fig.  7). Since it is most likely that Mg 2ϩ influences Ca 2ϩ capacity due to the inhibition of the mPT, these results support the idea that Li ϩ and Mg 2ϩ might act at a common target, the mPT. . CsA augments mitochondrial Ca 2؉ capacity in KCl or NaCl medium but failed to increase Ca 2؉ capacity in LiCl medium. a and b, traces from a representative experiment are shown. Isolated synaptic and nonsynaptic brain mitochondria were incubated at 37°C in either 125 mM KCl medium, 125 mM NaCl medium, or 125 mM LiCl medium supplemented with 3 mM glutamate plus 1 mM malate. 1 M CsA was present in the incubation medium as indicated. c, summary plots show Ca 2ϩ capacity of brain mitochondria incubated in different media with and without CsA. *, p Ͻ 0.05; **, p Ͻ 0.01 between Ca 2ϩ capacities with and without CsA. Data are mean Ϯ S.E., n ϭ 3. ATP also augmented Ca 2ϩ capacity of brain mitochondria (Fig. 8). ATP as well as ADP are inhibitors of the mPT (22,23,58). In contrast to ATP, GTP, which does not inhibit the mPT, failed to increase Ca 2ϩ capacity (Fig. 8). The substitution of K ϩ with Li ϩ increased Ca 2ϩ capacity of mitochondria incubated in the presence of 0.1 or 0.5 mM ATP. With 1 mM ATP, Ca 2ϩ capacity in KCl or LiCl medium became closer, but in LiCl medium, mitochondria still accumulated more Ca 2ϩ than in KCl medium.
Both Ca 2ϩ and Sr 2ϩ can be accumulated by mitochondria (59,60), but in contrast to Ca 2ϩ , Sr 2ϩ does not induce the classical CsA-sensitive mPT (22,46). Accordingly, Li ϩ failed to increase the ability of mitochondria to sequester Sr 2ϩ (Fig. 9). In addition, neither CsA nor ATP affected the Sr 2ϩ capacity of brain mitochondria (not shown). Thus, loss of the ability to sequester Sr 2ϩ appeared to be mPT-independent. The failure of Li ϩ to increase Sr 2ϩ accumulation suggests that Li ϩ action is specifically directed at the mPT.
Taken together, all of these results indicate that Li ϩ augments the Ca 2ϩ capacity of mitochondria by desensitizing them FIGURE 8. Li ؉ increases Ca 2؉ capacity of brain mitochondria in the presence of ATP. A and B, a, no ATP in the medium; b, 0.5 mM GTP; c, 0.1 mM ATP; d, 0.5 mM ATP; e, 1 mM ATP. Isolated nonsynaptic brain mitochondria were incubated at 37°C either in 125 mM KCl medium or in 125 mM LiCl medium with 3 mM succinate plus 3 mM glutamate without ADP and oligomycin. Similar results were obtained with 3 mM glutamate plus 1 mM malate. Slow infusion of Ca 2ϩ was initiated as indicated. FIGURE 9. Li ؉ fails to increase Sr 2؉ capacity of brain mitochondria. a and b, traces from representative experiments are shown. Isolated nonsynaptic brain mitochondria were incubated at 37°C either in 125 mM KCl medium or in 125 mM LiCl medium supplemented with 3 mM glutamate plus 1 mM malate. Slow infusion of Ca 2ϩ (a) or Sr 2ϩ (b) was initiated as indicated. c, the summary plot shows Ca 2ϩ and Sr 2ϩ capacity of brain mitochondria incubated in KCl medium or in LiCl media. *, p Ͻ 0.01 between divalent cation (Me 2ϩ ) capacities in KCl versus LiCl medium. Data are mean Ϯ S.E., n ϭ 3.
to Ca 2ϩ and thus deferring the mPT. However, the mechanisms of Li ϩ -evoked desensitization of mitochondria to Ca 2ϩ remain unclear.
Increased ROS generation favors an induction of the mPT (61,62). If mitochondrial production of ROS in LiCl medium were diminished, it might explain the Li ϩ -induced protection against Ca 2ϩ . To test this hypothesis, we measured H 2 O 2 release from isolated brain mitochondria in media of various cationic compositions. Surprisingly, the highest rate of basal H 2 O 2 release was found in CsCl medium, whereas in LiCl medium, the rate of ROS generation was lower than in KCl medium but very close to NaCl medium (Fig. 10). Ca 2ϩ infusion diminished the rate of H 2 O 2 release in KCl medium and to a lesser extent in LiCl medium, making them practically equal. Thus, despite some difference in basal H 2 O 2 release, the ROS production during Ca 2ϩ infusion was similar in KCl and LiCl media. Additionally, if the basal H 2 O 2 release determined the sensitivity of mitochondria to Ca 2ϩ , then mitochondria would be more sensitive to Ca 2ϩ in CsCl medium. However, this was not the case. Altogether, these results argue against the hypothesis that diminished ROS production in LiCl medium might be a cause for desensitization of brain mitochondria to Ca 2ϩ .
Previously, it was reported that 3 mM LiCl significantly protected cardiac mitochondria against ROS-induced mPT (32). Mitochondria contain GSK-3␤ (63,64), which is inhibited by Li ϩ (33,65). Since Li ϩ and other inhibitors of GSK-3␤ demonstrated similar protection, it was concluded that GSK-3␤ plays an essential role in regulation of mPT induction in heart mitochondria (32). In our hands, 3 mM LiCl did not protect brain mitochondria against Ca 2ϩ -induced mPT (not shown). We also tested SB216763 and SB415286, inhibitors of GSK-3␤ (66), in the experiments with a single Ca 2ϩ pulse and slow Ca 2ϩ infusion. These inhibitors neither protected against mitochondrial depolarization and swelling caused by Ca 2ϩ pulse (not shown) nor increased Ca 2ϩ capacity of brain mitochondria (Fig. 11). Thus, in contrast to heart mitochondria, in brain mitochondria, GSK-3␤ apparently does not play a role in the regulation of the mPT.

DISCUSSION
The main finding of this study is that Li ϩ desensitizes mitochondria to elevated Ca 2ϩ and diminishes cytochrome c release by antagonizing the Ca 2ϩ -induced mPT. Since CsA, SgfA, and high Mg 2ϩ had no protective effect in the LiCl medium, they all, including Li ϩ , apparently targeted the same process, the mPT. This conclusion is also supported by the lack of Li ϩ protection in the experiments with Sr 2ϩ , which damages mitochondria in an mPT-independent manner (46). In the experiments with individual mitochondria and slow Ca 2ϩ infusion, the protective effect of Li ϩ proved to be stronger than the effect of CsA. In addition, Li ϩ exerted desensitization of brain mitochondria to Ca 2ϩ even in the presence of ATP when CsA appeared to be ineffective (67). Li ϩ has numerous targets in the cell (30) and can possibly diminish Ca 2ϩ influx into neurons (4) (but see Refs. 68 and 69). At the same time, it is conceivable that the ability of Li ϩ to desensitize mitochondria to Ca 2ϩ and antagonize the mPT might contribute to the Li ϩ -induced protection of cultured neurons against delayed calcium deregulation caused by excitotoxic glutamate. 3 In the present study, we used three different experimental approaches to demonstrate Li ϩ -evoked desensitization of brain mitochondria to Ca 2ϩ : (i) simultaneous monitoring of light scattering and ⌬ in the purified, enriched suspension of isolated brain mitochondria; (ii) monitoring of ⌬ in individual isolated brain mitochondria loaded with Rh123 and attached to the glass-bottomed Petri dish; and (iii) monitoring of the Ca 2ϩ sequestration under conditions of slow, continuous Ca 2ϩ infusion that simulates Ca 2ϩ influx into neurons treated with glutamate. In all of these experiments, Li ϩ remarkably antagonized the Ca 2ϩ -inducible mPT. It is particularly important that the protective effect of Li ϩ was not due to mitochondrial depolarization and/or inhibition of Ca 2ϩ uptake. Rather, in LiCl medium, mitochondria better maintained ⌬ and more efficiently accumulated Ca 2ϩ than in any other tested media. Thus, 3 V. Li, T. Brustovetsky, A. Bolshakov, and N. Brustovetsky, unpublished results. it is likely that Li ϩ protects mitochondria by directly antagonizing an induction of the mPT pore.
The mechanisms of Li ϩ antagonism are not yet clear. The Li ϩ -evoked desensitization of brain mitochondria could be due to competition with Ca 2ϩ for the binding sites associated with the induction of the mPT pore. It was determined in early studies that Ca 2ϩ induces the mPT due to interaction with the binding site located inside of mitochondria (25,70). The IMM is obviously impermeable to Li ϩ , because mitochondria do not swell in LiCl medium unless Ca 2ϩ is added, and mitochondria remain tightly coupled in LiCl medium (47). Therefore, it seems more likely that Li ϩ antagonizes the Ca 2ϩ -induced mPT by acting on the external binding site. In their study on the mPT in liver mitochondria, Bernardi et al. (25) found internal and external Me 2ϩ binding sites involved in the regulation of the mPT. Interaction of Ca 2ϩ with the internal site was shown to lead to the mPT, whereas interaction of Ca 2ϩ and other divalent cations, including Mg 2ϩ , with the external site resulted in inhibition of the mPT (25). Interestingly, Li ϩ , with an ionic radius of 0.60 Å, is the closest to Mg 2ϩ , with an ionic radius of 0.66 Å (26). Both Li ϩ and Mg 2ϩ depressed apparent V max for Ca 2ϩ uptake, although Li ϩ appeared to be a much weaker inhibitor of Ca 2ϩ uniporter than Mg 2ϩ (26,52). Li ϩ was shown to bind to mitochondria in a ⌬-independent manner (27). The site of Li ϩ binding was not determined in the early studies. However, it was later found that Li ϩ can bind to different membrane phospholipids (28,29). All of these observations taken together suggest that Li ϩ might interact with the external binding site antagonizing the mPT in brain mitochondria.
The effect of Li ϩ could be mediated by modulating the surface potential of the IMM. In early studies, the membrane surface potential was proposed to be one of the modulators of the mPT (71). Agents that make the surface potential more negative (e.g. free fatty acids) were found to be activators of the mPT, whereas agents that shifted the surface potential toward more positive values were found to be inhibitors of the mPT. Hence, it seems possible that Li ϩ bound to the membrane makes the surface potential of the IMM more positive and thus antagonizes the mPT.
The Li ϩ -evoked desensitization proved not to be unique to brain mitochondria. In the experiments with slow Ca 2ϩ infusion, isolated heart and liver mitochondria also responded to Li ϩ by increasing their Ca 2ϩ capacity. Interestingly, isolated brain mitochondria, both synaptic and nonsynaptic, have the largest Ca 2ϩ capacity, followed by heart and liver mitochondria. In all cases, Li ϩ protection appeared to be qualitatively similar, with presumably a common mechanism in different types of mitochondria.
Provided that Li ϩ can enter neurons (37)(38)(39)72), Li ϩ -induced desensitization of brain mitochondria translated into a delay of the mPT could increase resilience of neurons to glutamate insult. Previously, the mPT was implicated in disturbance of neuronal Ca 2ϩ homeostasis, mitochondrial depolarization, and eventual neuronal death caused by exposure to excitotoxic glutamate (73)(74)(75)(76)(77)(78). Later, the role of the mPT was questioned, mainly due to the lack of a reliable pharmacological tool to identify the mPT-related phenomena (79 -81). In addition, it was reported that CsA did not increase Ca 2ϩ capacity of mito- FIGURE 11. SB216763 and SB415286, inhibitors of GSK-3␤, fail to increase Ca 2؉ capacity of brain mitochondria. a and b, traces from representative experiments are shown. Isolated nonsynaptic brain mitochondria were incubated at 37°C either in 125 mM KCl medium or in 125 mM LiCl medium supplemented with 3 mM glutamate plus 1 mM malate. a, 3 M SB216763 was added to the KCl medium prior to the addition of mitochondria. For comparison, Ca 2ϩ sequestration in LiCl medium is shown. b, 30 M SB415286 was present in the KCl medium. c, a summary plot shows Ca 2ϩ capacities of brain mitochondria incubated in LiCl medium and in KCl medium in the presence of SB216763 or SB415286. *, p Ͻ 0.01 between Ca 2ϩ capacities in LiCl versus KCl medium and between Ca 2ϩ capacities in KCl medium with and without SB216763. Data are mean Ϯ S.E., n ϭ 3. chondria in digitonin-permeabilized cerebellar granule neurons (82) and that CsA applied to isolated nonsynaptic brain mitochondria became ineffective in the presence of ATP (67). At the same time, the elusive nature of the CsA protection, especially in brain mitochondria or in long lasting experiments, is well documented (83,84). Thus, the lack of CsA protection does not necessarily indicate a lack of the mPT. In addition, in the neuron treated with glutamate, the response of individual mitochondria might differ, as it occurs in the experiments with isolated mitochondria (85). Therefore, it seems possible that in the same cell, some mitochondria might undergo the mPT, whereas other mitochondria maintain integrity and the ability to sequester Ca 2ϩ and/or produce ATP. It is conceivable that Li ϩ , which might enter neurons following stimulation of glutamate receptors, could change the proportion of these two mitochondrial subpopulations and thus influence the neuronal fate.