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

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

Bcl-2 inhibits apoptosis induced by mitochondrial uncoupling but does not prevent mitochondrial transmembrane depolarization

Bcl-2 overexpression protects cells from apoptosis induced by many cytotoxic agents. In this study, we investigated the effects of uncoupling mitochondrial electron transport in both HL60 wild-type and Bcl-2-overexpressing cells using the protonophore carbonyl cyanide m-chlorophenylhydrazone. We found that uncoupling mitochondrial electron transport induced apoptosis in wild-type, but not in Bcl-2-overexpressing cells. To investigate the mechanism of action of Bcl-2-mediated inhibition of cyanide m-chlorophenylhydrazone-induced apoptosis, we measured the mitochondrial transmembrane potential (DeltaPsi(m)) after uncoupling mitochondrial electron transport and found that both HL-60 wild-type and Bcl-2-overexpressing cells similarly depolarize following cyanide m-chlorophenylhydrazone exposure. Western blot analysis demonstrated that Bcl-2 overexpression did not completely block cytochrome c release from mitochondria after uncoupling mitochondrial electron transport. Since Bcl-2 may act as an antioxidant, we studied the effect of altering the cellular redox state prior to uncoupling mitochondrial electron transport in Bcl-2-overexpressing cells. Depletion of mitochondrial (but not cytosolic) glutathione induced apoptosis in Bcl-2-overexpressing cells and negated the protective effect of Bcl-2. Furthermore, following glutathione depletion, Bcl-2-overexpressing cells were sensitized to undergo cyanide m-chlorophenylhydrazone-induced apoptosis. These data suggest that the action of Bcl-2 is dependent, in part, on the cellular and mitochondrial redox state.     

On the state of calcium ions in isolated rat liver mitochondria. I. Ion fluxes and volume changes upon Ca2+ uptake under various ionic conditions

As to functional consequences of Ca2+ uptake in isolated rat liver mitochondria, we simultaneously measured 3H2O and [14C]sucrose spaces, monovalent cation distribution, membrane potential and delta pH across the inner membrane, and [32P]phosphate and 45Ca2+ content in parallel incubations of different ionic composition. Without added Ca2+ and phosphate, mitochondrial matrix volume, membrane potential, and delta pH depended on the concentration and permeability of monovalent cations. Despite large differences in membrane potential, maximal Ca2+ uptake was identical under all conditions. Ca2+ uptake never provoked a volume change from which an osmotic active state of mitochondrial Ca2+ could be concluded. If matrix volume shrunk this could be totally accounted for by the loss of alkali ions exchanging for calcium ions. Even phosphate taken up in conjunction with Ca2+ was osmotically silent. Volume increases here occurring if K+ was permeabilized, solely resulted from K+ uptake, though this condition may give rise to irreversible mitochondrial damage with Ca2+ and phosphate release. As mitochondrial Ca2+ is bound, an electro-chemical equilibrium across the membrane is impossible for this ion. This has to be considered in any model describing equilibria of Ca2+ with mitochondria, though present models neglect this state of mitochondrial Ca2+.     

Bioenergetic consequences of opening the ATP-sensitive K(+) channel of heart mitochondria

There is an emerging consensus that pharmacological opening of the mitochondrial ATP-sensitive K(+) (K(ATP)) channel protects the heart against ischemia-reperfusion damage; however, there are widely divergent views on the effects of openers on isolated heart mitochondria. We have examined the effects of diazoxide and pinacidil on the bioenergetic properties of rat heart mitochondria. As expected of hydrophobic compounds, these drugs have toxic, as well as pharmacological, effects on mitochondria. Both drugs inhibit respiration and increase membrane proton permeability as a function of concentration, causing a decrease in mitochondrial membrane potential and a consequent decrease in Ca(2+) uptake, but these effects are not caused by opening mitochondrial K(ATP) channels. In pharmacological doses (<50 microM), both drugs open mitochondrial K(ATP) channels, and resulting changes in membrane potential and respiration are minimal. The increased K(+) influx associated with mitochondrial K(ATP) channel opening is approximately 30 nmol. min(-1). mg(-1), a very low rate that will depolarize by only 1-2 mV. However, this increase in K(+) influx causes a significant increase in matrix volume. The volume increase is sufficient to reverse matrix contraction caused by oxidative phosphorylation and can be observed even when respiration is inhibited and the membrane potential is supported by ATP hydrolysis, conditions expected during ischemia. Thus opening mitochondrial K(ATP) channels has little direct effect on respiration, membrane potential, or Ca(2+) uptake but has important effects on matrix and intermembrane space volumes.     

Reconstitution and partial purification of the glibenclamide-sensitive, ATP-dependent K+ channel from rat liver and beef heart mitochondria.

The transport properties of mitochondria are such that net potassium flux across the inner membrane determines mitochondrial volume. It has been known that K+ uptake is mediated by diffusive leak driven by the high electrical membrane potential maintained by redox-driven, electrogenic proton ejection and that regulated K+ efflux is mediated by an 82-kDa inner membrane K+/H+ antiporter. There is also long-standing suggestive evidence for the existence of an inner membrane protein designed to catalyze electrophoretic K+ uptake into mitochondria. We report reconstitution of a highly purified inner membrane protein fraction from rat liver and beef heart mitochondria that catalyzes electrophoretic K+ flux in liposomes and channel activity in planar lipid bilayers. The unit conductance of the channel at saturating [K+] is about 30 pS. Reconstituted K+ flux is inhibited with high affinity by ATP and ADP in the presence of divalent cations and by glibenclamide in the absence of divalent cations. The mitochondrial ATP-dependent K+ channel is selective for K+, with a Km of 32 mM, and does not transport Na+. K+ transport depends on voltage in a manner consistent with a channel activity that is not voltage-regulated. Thus, the mitochondrial ATP-dependent K+ channel exhibits properties that are remarkably similar to those of the ATP-dependent K+ channels of plasma membranes.     

Regulation of the mitochondrial matrix volume in vivo and in vitro. The role of calcium.

The ability of alpha-adrenergic agonists and vasopressin to increase the mitochondrial volume in hepatocytes is dependent on the presence of extracellular Ca2+. Addition of Ca2+ to hormone-treated cells incubated in the absence of Ca2+ initiates mitochondrial swelling. In the presence of extracellular Ca2+, A23187 (7.5 microM) induces mitochondrial swelling and stimulates gluconeogenesis from L-lactate. Isolated liver mitochondria incubated in KCl medium in the presence of 2.5 mM-phosphate undergo energy-dependent swelling, which is associated with electrogenic K+ uptake and reaches an equilibrium when the volume has increased to about 1.3-1.5 microliter/mg of protein. This K+-dependent swelling is stimulated by the presence of 0.3-1.0 microM-Ca2+, leading to an increase in matrix volume at equilibrium that is dependent on [Ca2+]. Ca2+-activated K+-dependent swelling requires phosphate and shows a strong preference for K+ over Na+, Li+ or choline. It is not associated with either uncoupling of mitochondria or any non-specific permeability changes and cannot be produced by Ba2+, Mn2+ or Sr2+. Ca2+-activated K+-dependent swelling is not prevented by any known inhibitors of plasma-membrane ion-transport systems, nor by inhibitors of mitochondrial phospholipase A2. Swelling is inhibited by 65% and 35% by 1 mM-ATP and 100 microM-quinine respectively. The effect of Ca2+ is blocked by Ruthenium Red (5 micrograms/ml) at low [Ca2+]. Spermine (0.25 mM) enhanced the swelling seen on addition of Ca2+, correlating with its ability to increase Ca2+ uptake into the mitochondria as measured by using Arsenazo-III. Mitochondria derived from rats treated with glucagon showed less swelling than did control mitochondria. In the presence of Ruthenium Red and higher [Ca2+], the mitochondria from hormone-treated animals showed greater swelling than did control mitochondria. These data imply that an increase in intramitochondrial [Ca2+] can increase the electrogenic flux of K+ into mitochondria by an unknown mechanism and thereby cause swelling. It is proposed that this is the mechanism by which alpha-agonists and vasopressin cause an increase in mitochondrial volume in situ.     

Bcl-2 prevents loss of mitochondria in CCCP-induced apoptosis

Bcl-2 family proteins regulate apoptosis at the level of mitochondria. To examine the mechanism of Bcl-2 function, we investigated the effects of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on two hematopoietic cell lines and Bcl-2 overexpressing transfectants. CCCP directly interferes with mitochondrial function and induces apoptosis. We show that Bcl-2 inhibits apoptosis and that the antiapoptotic effect of Bcl-2 takes place upstream of caspase activation and nuclear changes associated with apoptosis, since these were markedly inhibited in cells overexpressing Bcl-2. Bcl-2 does not prevent the decrease in mitochondrial membrane potential nor the alterations in cellular ATP content induced by CCCP in FL5.12 and Jurkat cells. A higher number of mitochondria was observed in untreated Bcl-2 transfected cells compared to parental cells, as shown by electron microscopy. Exposure to CCCP induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, with apparent swelling and loss of cristae in parental cells. Bcl-2 clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. These data suggest that CCCP induces apoptosis by structural disruption of mitochondria and that Bcl-2 prevents apoptosis and mitochondrial degeneration by preserving mitochondrial integrity.     

Aranorosin and a novel derivative inhibit the anti-apoptotic functions regulated by Bcl-2

Bcl-2 is an intracellular membrane protein that prevents cells from undergoing apoptosis in response to various cell-death signals. It negatively regulates mitochondrial outer membrane permeabilization, which is responsible for the release of apoptogenic factors and the subsequent activation of caspases. A microbial metabolite, aranorosin, was identified as an inhibitor of the anti-apoptotic function of Bcl-2. Based on its structure, a more potent derivative, K050, was synthesized. Apoptosis could be induced in a cell line that overexpressed Bcl-2 when cells were treated with an anti-Fas antibody in addition to K050, at sub-micromolar concentrations. Furthermore, K050 inhibited anti-apoptotic functions regulated by Bcl-2, resulting in a Fas-triggered mitochondrial transmembrane potential loss, the activation of caspase-9, and a morphological change to apoptosis. Inhibition of cell-based function of Bcl-2 and its anti-apoptotic effects could serve as useful pharmacological effects. Thus, a novel aranorosin derivative, K050, could be a potent therapeutic agent against Bcl-2-overexpressing human malignancies.     

Characterization of functionally distinct mitochondrial subpopulations

Mitochondrial stress results in changes in mitochondrial function, morphology and homeostasis (biogenesis, fission/fusion, mitophagy) and may lead to changes in mitochondrial subpopulations. While flow cytometric techniques have been developed to quantify features of individual mitochondria related to volume, Ca²⁺ concentration, mtDNA content, respiratory capacity and oxidative damage, less information is available concerning the identification and characterization of mitochondrial subpopulations, particularly in epithelial cells. Mitochondria from rabbit kidneys were stained with molecular probes for cardiolipin content (nonyl acridine orange, NAO) and membrane potential (tetramethylrhodamine, TMRM) and analyzed using flow cytometry. We validated that side scatter was a better indicator of volume and that as side scatter (SSC) decreased mitochondrial volume increased. Furthermore, those mitochondria with the highest NAO content had greater side scattering and were most highly charged. Mitochondria with average NAO content were of average side scattering and maintained an intermediate charge. Those mitochondria with low NAO content had minimal side scattering and exhibited minimal charge. Upon titration with the uncoupler carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), it was found that the high NAO content subpopulations were more resistant to uncoupling than lower NAO content populations. Ca²⁺-induced swelling of mitochondria was evaluated using probability binning (PB) analyses of SSC. Interestingly, only 30 % of the mitochondria showed changes in response to Ca²⁺, which was blocked by cyclosporine A. In addition, the small, high NAO content mitochondria swelled differentially in response to Ca²⁺ over time. Our results demonstrate that flow cytometry can be used to identify mitochondrial subpopulations based on high, mid and low NAO content and/or volume/complexity. These subpopulations showed differences in membrane potential, volume, and responses to uncoupling and Ca²⁺-induced swelling.     

Activation of latent K+ uniport in mitochondria treated with the ionophore A23187

Addition of A23187 plus EDTA to energized mitochondria in KCl medium determines a rapid osmotic swelling due to K+ uptake. The swelling is fully reversed by uncoupler, is stimulated by quinine, and is accompanied by membrane depolarization and increased rate of respiration. A23187-treated mitochondria passively swell in K+ thiocyanate at neutral pH, under conditions where the H+-K+ antiporter appears to be silent. These data indicate that A23187 activates electrophoretic K+ flux, supporting the notion that Mg2+ depletion unmasks several ionic conductance pathways whose concerted interplay could provide a sensitive regulation of mitochondrial volume homeostasis.     

Evidence for an electrogenic 3-deoxy-2-oxo-D-gluconate--proton co-transport driven by the protonmotive force in Escherichia coli K12.

Evidence is presented indicating that the carrier-mediated uptake of 3-deoxy-2-oxo-D-gluconate and D-glucuronate in Escherichia coli K12 is driven by the deltapH and deltapsi components of the protonmotive force. 1. Approximately two protons enter the cells with each sugar molecule, independent of the sugar and the strain used. 2. In respiring cells, the magnitude of the pH gradient alone, as measured by distribution of [3H]acetate, appears to be insufficient to account for the chemical gradient of 3-deoxy-2-oxo-D-gluconate that is developed between pH 6.0 and 8.0. 3. If the external pH is varied between 5.5 and 8.0, 3-deoxy-2-oxo-D-gluconate uptake is gradually inhibited by valinomycin plus K+ ions, whereas the inhibition caused by nigericin is concomitantly relieved, thus reflecting the relative contribution of deltapH and deltapsi to the total protonmotive force at each external pH. 4. 3-Deoxy-2-oxo-D-gluconate can be transiently accumulated into isolated membrane vesicles in response to an artificially induced pH gradient. The process is stimulated when the membrane potential is collapsed by valinomycin in the presence of K+ ions.     

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established¹, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca²⁺ homeostasis², bioenergetics and redox signaling ³.mtPTP opening is triggered by matrix Ca²⁺ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids⁴. In vitro, mtPTP opening can be achieved by increasing Ca²⁺ concentration inside the mitochondrial matrix through exogenous additions of Ca²⁺ (calcium retention capacity). When Ca²⁺ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca²⁺ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca²⁺ handling (uptake and release, as measured by Ca²⁺ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca²⁺ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca²⁺, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.     

An estimation of the light-induced electrochemical potential difference of protons across the membrane of Halobacterium halobium.

The light-dependent uptake of triphenylmethylphosphonium (TPMP+) and of 5,5-dimethyloxazolidine-2,4-dione (DMO) by starved purple cells of Halobacterium halobium was investigated. DMO uptake was used to calculate the pH difference (deltapH) across the membrane, and TPMP+ was used as an index of the electrical potential difference, deltapsi. Under most conditions, both in the light and in the dark, the cells are more alkaline than the medium. In the light at pH 6.6, deltapH amounts to 0.6-0.8 pH unit. Its value can be increased to 1.5-2.0 by either incubating the cells with TPMP+ (10(-3) M) or at low external pH (5.5). --deltapH can be lowered by uncoupler or by nigericin. The TPMP+ uptake by the cells indicates a large deltapsi across the membrane, negative inside. It was estimated that in the light, at pH 6.6, deltapsi might reach a value of about 100 mV and that consequently the electrical equivalent of the proton electrochemical potential difference, deltamuH+/F, amounts under these conditions to about 140 mV. The effects of different ionophores on the light-drive proton extrusion by the cells were in agreement with the effects of these compounds on --deltapH.     

The C. elegans B-cell lymphoma 2 (Bcl-2) homolog cell death abnormal 9 (CED-9) associates with and remodels LIPID membranes

Bcl-2 proteins associate with and remodel mitochondria to regulate apoptosis. While the C. elegans Bcl-2 homolog CED-9 constitutively associates with mitochondria, it is unclear whether or not this association reflects an innate ability of CED-9 to directly remodel mitochondrial membranes. To address this question, we have characterized the effects of recombinantly expressed and purified CED-9 on synthetic lipid vesicles. We found that CED-9 associates with anionic lipid vesicles at neutral pH, and that association can occur independently of the C-terminal transmembrane domain. Membrane association changes the environment of CED-9 tryptophans and results in an apparent increase in α-helical structure. Upon association, CED-9 alters the permeability of membranes resulting in leakage of encapsulated dyes. Furthermore, this membrane remodeling promotes membrane fusion upon protonation of CED-9. Bypass of this protonation trigger can be achieved by mutating two conserved glutamates (E187K/E190K) or removing the N-terminal 67 residues. Together, these in vitro results suggest that CED-9 retains the amphitropic ability of mammalian Bcl-2 proteins to associate with cellular membranes. We therefore discuss the possibility that CED-9 and other Bcl-2 homologs localize at mitochondria to regulate mitochondrial homeostasis by either modulating mitochondrial membrane permeability or fusion.     

Selective localization of Bcl-2 to the inner mitochondrial and smooth endoplasmic reticulum membranes in mammalian cells

Bcl-2, an anti-apoptotic protein, is believed to be localized in the outer mitochondrial membrane, endoplasmic reticulum, and nuclear envelope. However, Bcl-2 has also been suggested as playing a role in the maintenance of mitochondrial membrane potential, indicating its possible association with the inner mitochondrial membrane. We therefore further examined the exact localization of Bcl-2 in mitochondria purified from wild-type and bcl-2-transfected PC12 cells and pre- and postnatal rat brains. Double immunostaining demonstrated that Bcl-2 was co-localized with subunit beta of F1F0ATPase in the inner mitochondrial membrane. Biochemical analysis of isolated mitochondria using digitonin and trypsin suggests an association of Bcl-2 with the inner mitochondrial membrane. More interestingly, the majority of Bcl-2 disappeared from the inner membrane of mitochondria when cultured under serum deprivation. These results suggest that Bcl-2 acts as an anti-apoptotic regulator by localizing mainly to the inner mitochondrial and smooth ER membranes.     

Role of Bcl-2 family of proteins in mediating apoptotic death of PC12 cells exposed to oxygen and glucose deprivation

Apoptotic cell death has been observed in many in vivo and in vitro models of ischemia. However, the molecular pathways involved in ischemia-induced apoptosis remain unclear. We have examined the role of Bcl-2 family of proteins in mediating apoptosis of PC12 cells exposed to the conditions of oxygen and glucose deprivation (OGD) or OGD followed by restoration of oxygen and glucose (OGD-restoration, OGD-R). OGD decreased mitochondrial membrane potential and induced necrosis of PC12 cells, which were both prevented by the overexpression of Bcl-2 proteins. OGD-R caused apoptotic cell death, induced cytochrome C release from mitochondria and caspase-3 activation, decreased mitochondrial membrane potential, and increased levels of pro-apoptotic Bax translocated to the mitochondrial membrane, all of which were reversed by overexpression of Bcl-2. These results demonstrate that the cell death induced by OGD and OGD-R in PC12 cells is potentially mediated through the regulation of mitochondrial membrane potential by the Bcl-2 family of proteins. It also reveals the importance of developing therapeutic strategies for maintaining the mitochondrial membrane potential as a possible way of reducing necrotic and apoptotic cell death that occurs following an ischemic insult.     

Differential regulation of doxorubicin-induced mitochondrial dysfunction and apoptosis by Bcl-2 in mammary adenocarcinoma (MTLn3) cells

Various anticancer drugs cause mitochondrial perturbations in association with apoptosis. Here we investigated the involvement of caspase- and Bcl-2-dependent pathways in doxorubicin-induced mitochondrial perturbations and apoptosis. For this purpose, we set up a novel three-color flow cytometric assay using rhodamine 123, annexin V-allophycocyanin, and propidium iodide to assess the involvement of the mitochondria in apoptosis caused by doxorubicin in the breast cancer cell line MTLn3. Doxorubicin-induced apoptosis was preceded by up-regulation of CD95 and CD95L and a collapse of mitochondrial membrane potential (Deltapsi) occurring prior to phosphatidylserine externalization. This drop in Deltapsi was independent of caspase activity, since benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone did not inhibit it. Benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone also blocked activation of caspase-8, thus excluding an involvement of the death receptor pathway in Deltapsi dissipation. Furthermore, although overexpression of Bcl-2 in MTLn3 cells inhibited apoptosis, dissipation of Deltapsi was still observed. No decrease in Deltapsi was observed in cells undergoing etoposide-induced apoptosis. Immunofluorescent analysis of Deltapsi and cytochrome c localization on a cell-to-cell basis indicates that the collapse of Deltapsi and cytochrome c release are mutually independent in both normal and Bcl-2-overexpressing cells. Together, these data indicate that doxorubicin-induced dissipation of the mitochondrial membrane potential precedes phosphatidylserine externalization and is independent of a caspase- or Bcl-2-controlled checkpoint.     

Fluorescent cationic probes of mitochondria. Metrics and mechanism of interaction.

Mitochondria strongly accumulate amphiphilic cations. We report here a study of the association of respiring rat liver mitochondria with several fluorescent cationic dyes from differing structural classes. Using gravimetric and fluorometric analysis of dye partition, we find that dyes and mitochondria interact in three ways: (a) uptake with fluorescence quenching, (b) uptake without change in fluorescence intensity, and (c) lack of uptake. For dyes that quench upon uptake, the extent of quenching correlates with the degree of aggregation of the dye to dimers, as predicted by theory (Tomov, T.C. 1986. J. Biochem. Biophys. Methods. 13:29-38). Also predicted is the relationship observed between quenching and the mitochondria concentration when constant dye is titrated with mitochondria. Not predicted is the relationship observed between quenching and dye concentration when constant mitochondria are titrated with dye. Because a limit to dye uptake exists, in this case, the degree of quenching decreases as dye is added. A Langmuir isotherm analysis gives phenomenological parameters that predict quenching when it is observed as a function of dye concentration. By allowing for a decrease in membrane potential, caused by incorporation of cationic dye into the lipid bilayer, a modification of the Tomov theory predicts the dye titration data. We present a model of cationic dye-mitochondria interaction and discuss the use of these as probes of mitochondrial membrane potential.     

Antagonistic Regulation of Parvalbumin Expression and Mitochondrial Calcium Handling Capacity in Renal Epithelial Cells

Parvalbumin (PV) is a cytosolic Ca²⁺-binding protein acting as a slow-onset Ca²⁺ buffer modulating the shape of Ca²⁺ transients in fast-twitch muscles and a subpopulation of neurons. PV is also expressed in non-excitable cells including distal convoluted tubule (DCT) cells of the kidney, where it might act as an intracellular Ca²⁺ shuttle facilitating transcellular Ca²⁺ resorption. In excitable cells, upregulation of mitochondria in “PV-ergic” cells in PV-/- mice appears to be a general hallmark, evidenced in fast-twitch muscles and cerebellar Purkinje cells. Using Gene Chip Arrays and qRT-PCR, we identified differentially expressed genes in the DCT of PV-/- mice. With a focus on genes implicated in mitochondrial Ca²⁺ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit β (Atp5b) were found to be up-upregulated. At the protein level, COX1 was increased by 31 ± 7%, while ATP-synthase subunit β was unchanged. This suggested that these mitochondria were better suited to uphold the electrochemical potential across the mitochondrial membrane, necessary for mitochondrial Ca²⁺ uptake. Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected. Suppression of PV by shRNA in PV-expressing MDCK cells led subsequently to an increase in COX1 expression. The collapsing of the mitochondrial membrane potential by the uncoupler CCCP occurred at lower concentrations in PV-expressing MDCK cells than in control cells. In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells. Deregulation of the cytoplasmic Ca²⁺ buffer PV in kidney cells was counterbalanced in vivo and in vitro by adjusting the relative mitochondrial volume and modifying the mitochondrial protein composition conceivably to increase their Ca²⁺-buffering/sequestration capacity.