Rapid Ca2+-dependent increase in oxygen consumption by mitochondria in single mammalian central neurons

Oxygen consumption increases within a fraction of a second after the onset of neuronal activity, a phenomenon referred to as the "initial dip" in functional imaging studies of the living brain. The cellular mechanism that underlies this rapid increase in oxygen consumption has remained unclear, however. We have now used two-photon excitation imaging to characterize rapid activity-dependent mitochondrial responses in single neurons. This approach allowed simultaneous multicolor imaging of individual mitochondria in single mouse Purkinje neurons in culture. Mitochondrial depolarization was induced immediately when the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) exceeded 15 microM and was associated with oxidation of mitochondrial NAD(P)H, suggesting that Ca(2+)-induced mitochondrial depolarization mediated by the Ca(2+) uniporter directly facilitated oxidation of NAD(P)H. With the use of a miniature oxygen electrode, we detected a burst of oxygen consumption within 0.2s after the onset of cell depolarization in single Purkinje neurons, and this rapid increase in oxygen consumption was dependent on the increase in [Ca(2+)](i). We have thus demonstrated a rapid Ca(2+)-dependent consumption of oxygen that is mediated by mitochondrial depolarization in mammalian central neurons. This process might function as a rapid feed-forward mechanism in homeostatic control of the cytosolic ATP concentration.     

Requirement for aralar and its Ca2+-binding sites in Ca2+ signal transduction in mitochondria from INS-1 clonal beta-cells

Aralar, the mitochondrial aspartate-glutamate carrier present in beta-cells, is a component of the malate-aspartate NADH shuttle (MAS). MAS is activated by Ca2+ in mitochondria from tissues with aralar as the only AGC isoform with an S0.5 of approximately 300 nm. We have studied the role of aralar and its Ca2+-binding EF-hand motifs in glucose-induced mitochondrial NAD(P)H generation by two-photon microscopy imaging in INS-1 beta-cells lacking aralar or expressing aralar mutants blocked for Ca2+ binding. Aralar knock-down in INS-1 beta-cell lines resulted in undetectable levels of aralar protein and complete loss of MAS activity in isolated mitochondria and in a 25% decrease in glucose-stimulated insulin secretion. MAS activity in mitochondria from INS-1 cells was activated 2-3-fold by extramitochondrial Ca2+, whereas aralar mutants were Ca2+ insensitive. In Ca2+-free medium, glucose-induced increases in mitochondrial NAD(P)H were small (1.3-fold) and unchanged regardless of the lack of aralar. In the presence of 1.5 mm Ca2+, glucose induced robust increases in mitochondrial NAD(P)H (approximately 2-fold) in cell lines with wild-type or mutant aralar. There was a approximately 20% reduction in NAD(P)H response in cells lacking aralar, illustrating the importance of MAS in glucose action. When small Ca2+ signals that resulted in extremely small mitochondrial Ca2+ transients were induced in the presence of glucose, the rise in mitochondrial NAD(P)H was maintained in cells with wild-type aralar but was reduced by approximately 50% in cells lacking or expressing mutant aralar. These results indicate that 1) glucose-induced activation of MAS requires Ca2+ potentiation; 2) Ca2+ activation of MAS represents a larger fraction of glucose-induced mitochondrial NAD(P)H production under conditions where suboptimal Ca2+ signals are associated with a glucose challenge (50 versus 20%, respectively); 3) inactivation of EF-hand motifs in aralar prevents activation of MAS by small Ca2+ signals. The results suggest a possible role for aralar and MAS in priming the beta-cell by Ca2+-mobilizing neurotransmitter or hormones.     

Intracellular sodium modulates mitochondrial calcium signaling in vascular endothelial cells

We have investigated the role of extramitochondrial Na(+) for the regulation of mitochondrial Ca(2+) concentration ([Ca(2+)](m)) in permeabilized single vascular endothelial cells. [Ca(2+)](m) was measured by loading the cells with the membrane-permeant Ca(2+) indicator fluo-3/AM and subsequent removal of cytoplasmic fluo-3 by surface membrane permeabilization with digitonin. An elevation of extramitochondrial Ca(2+) resulted in a dose-dependent increase in the rate of Ca(2+) accumulation into mitochondria (k(0.5) = 3 microm) via the mitochondrial Ca(2+) uniporter. In the presence of 10 mm extramitochondrial Na(+) ([Na(+)](em)), repetitive application of brief pulses of high Ca(2+) (2-10 microm) to simulate cytoplasmic [Ca(2+)] oscillations caused transient increases of [Ca(2+)](m) characterized by a fast rising phase that was followed by a slow decay. Removal of extramitochondrial Na(+) or inhibition of mitochondrial Na(+)/Ca(2+) exchange with clonazepam blocked mitochondrial Ca(2+) efflux and resulted in a net accumulation of Ca(2+) by the mitochondria. Half-maximal activation of mitochondrial Na(+)/Ca(2+) exchange occurred at [Na(+)](em) = 4.4 mm, which is well within the physiological range of cytoplasmic [Na(+)]. This study provides evidence that Ca(2+) efflux from the mitochondria in vascular endothelial cells occurs solely via Na(+)/Ca(2+) exchange and emphasizes the important role of intracellular Na(+) for mitochondrial Ca(2+) regulation.     

Mitochondrial Ca(2+)-induced Ca(2+) release mediated by the Ca(2+) uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca(2+) during cell stimulation. The present study focuses on the pathways for mitochondrial Ca(2+) efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca(2+) uptake and increased the cytosolic [Ca(2+)] ([Ca(2+)](c)) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca(2+)](c) kinetics and inhibited Ca(2+) release from Ca(2+)-loaded mitochondria. This effect was due to inhibition of mitochondrial Na(+)/Ca(2+) exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca(2+)](c) triggered fast Ca(2+) release from these depolarized Ca(2+)-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca(2+)-induced Ca(2+) release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca(2+) uniporter. This novel kind of mitochondrial Ca(2+)-induced Ca(2+) release might contribute to Ca(2+) clearance from mitochondria that become depolarized during Ca(2+) overload.     

Muscarinic receptor activation determines the effects of store-operated Ca(2+)-entry on excitability and energy metabolism in pyramidal neurons

In various cell types, depletion of intracellular Ca(2+)-stores results in store-operated Ca(2+)-entry (SOCE) across the cellular membrane. However, the effects of SOCE on neuronal membrane excitability and mitochondrial functions in central neurons are not well defined. We investigated such cellular downstream effects in pyramidal neurons of rat organotypic hippocampal slice cultures by applying electrophysiological and fluorescence imaging techniques. We report that SOCE is associated with (i) elevations of Ca(2+)-concentration in individual neuronal mitochondria ([Ca(2+)](m)). In addition, SOCE can result in (ii) hyperpolarizing neuronal membrane currents, (iii) increase in extracellular K(+)-concentration ([K(+)](o)), (iv) mitochondrial membrane depolarization, and (v) changes in intracellular redox state (NAD(P)H and FAD fluorescence), the latter reflecting responses of energy metabolism. These additional downstream effects of SOCE required concomitant muscarinic receptor activation by carbachol or acetylcholine, and were suppressed by agonist washout or application of antagonist, atropine. We conclude that muscarinic receptor activation determines the downstream effects of SOCE on neuronal membrane excitability and energy metabolism. This mechanism might have significant impact on information processing and neurometabolic coupling in central neurons.     

Glucose induces synchronous mitochondrial calcium oscillations in intact pancreatic islets

Mitochondria shape Ca(2+) signaling and exocytosis by taking up calcium during cell activation. In addition, mitochondrial Ca(2+) ([Ca(2+)](M)) stimulates respiration and ATP synthesis. Insulin secretion by pancreatic beta-cells is coded mainly by oscillations of cytosolic Ca(2+) ([Ca(2+)](C)), but mitochondria are also important in excitation-secretion coupling. Here, we have monitored [Ca(2+)](M) in single beta-cells within intact mouse islets by imaging bioluminescence of targeted aequorins. We find an increase of [Ca(2+)](M) in islet-cells in response to stimuli that induce either Ca(2+) entry, such as extracellular glucose, tolbutamide or high K(+), or Ca(2+) mobilization from the intracellular stores, such as ATP or carbamylcholine. Many cells responded to glucose with synchronous [Ca(2+)](M) oscillations, indicating that mitochondrial function is coordinated at the whole islet level. Mitochondrial Ca(2+) uptake in permeabilized beta-cells increased exponentially with increasing [Ca(2+)], and, particularly, it became much faster at [Ca(2+)](C)>2 microM. Since the bulk [Ca(2+)](C) signals during stimulation with glucose are smaller than 2 microM, mitochondrial Ca(2+) uptake could be not uniform, but to take place preferentially from high [Ca(2+)](C) microdomains formed near the mouth of the plasma membrane Ca(2+) channels. Measurements of mitochondrial NAD(P)H fluorescence in stimulated islets indicated that the [Ca(2+)](M) changes evidenced here activated mitochondrial dehydrogenases and therefore they may modulate the function of beta-cell mitochondria. Diazoxide, an activator of K(ATP), did not modify mitochondrial Ca(2+) uptake.     

Zn(2+) induces permeability transition pore opening and release of pro-apoptotic peptides from neuronal mitochondria

Rapid entry of Ca(2+) or Zn(2+) kills neurons. Mitochondria are major sites of Ca(2+)-dependent toxicity. This study examines Zn(2+)-initiated mitochondrial cell death signaling. 10 nm Zn(2+) induced acute swelling of isolated mitochondria, which was much greater than that induced by higher Ca(2+) levels. Zn(2+) entry into mitochondria was dependent upon the Ca(2+) uniporter, and the consequent swelling resulted from opening of the mitochondrial permeability transition pore. Confocal imaging of intact neurons revealed entry of Zn(2+) (with Ca(2+)) to cause pronounced mitochondrial swelling, which was far greater than that induced by Ca(2+) entry alone. Further experiments compared the abilities of Zn(2+) and Ca(2+) to induce mitochondrial release of cytochrome c (Cyt-c) or apoptosis-inducing factor. In isolated mitochondria, 10 nm Zn(2+) exposures induced Cyt-c release. Induction of Zn(2+) entry into cortical neurons resulted in distinct increases in cytosolic Cyt-c immunolabeling and in cytosolic and nuclear apoptosis-inducing factor labeling within 60 min. In comparison, higher absolute [Ca(2+)](i) rises were less effective in inducing release of these factors. Addition of the mitochondrial permeability transition pore inhibitors cyclosporin A and bongkrekic acid decreased Zn(2+)-dependent release of the factors and attenuated neuronal cell death as assessed by trypan blue staining 5-6 h after the exposures.     

Mitochondrial Ca2+ uptake with and without the formation of high-Ca2+ microdomains

The mitochondrial Ca(2+) uniporter has low affinity for Ca(2+), therefore it has been assumed that submicromolar Ca(2+) signals cannot induce mitochondrial Ca(2+) uptake. The close apposition of the plasma membrane or the endoplamic reticulum (ER) to the mitochondria and the limited Ca(2+) diffusion in the cytoplasm result in the formation of perimitochondrial high-Ca(2+) microdomains (HCMDs) capable of activating mitochondrial Ca(2+) uptake. The possibility of mitochondrial Ca(2+) uptake at low submicromolar [Ca(2+)](c) has not yet been generally accepted. Earlier we found in permeabilized glomerulosa, luteal and pancreatic beta cells that [Ca(2+)](m) increased when [Ca(2+)](c) was raised from 60 nM to less than 200 nM. Here we report data obtained from H295R (adrenocortical) cells transfected with ER-targeted GFP. Cytoplasmic Ca(2+) response to angiotensin II was different in mitochondrion-rich and mitochondrion-free domains. The mitochondrial Ca(2+) response to angiotensin II correlated with GFP fluorescence indicating the vicinity of ER. When the cells were exposed to K(+) (inducing Ca(2+) influx), no correlation was found between the mitochondrial Ca(2+) signal and the vicinity of the plasma membrane or the ER. The results presented here provide evidence that mitochondrial Ca(2+) uptake may occur both with and without the formation of HCMDs within the same cell.     

Role of mitochondria in Ca(2+) homeostasis of mouse pancreatic acinar cells

The effects of mitochondrial Ca(2+) uptake on cytosolic Ca(2+) concentration ([Ca(2+)](c)) were investigated in mouse pancreatic acinar cells using cytosolic and/or mitochondrial Ca(2+) indicators. When calcium stores of the endoplasmic reticulum (ER) were emptied by prolonged incubation with thapsigargin (Tg) and acetylcholine (ACh), small amounts of calcium could be released into the cytosol (Delta[Ca(2+)](c)=46 +/- 6 nM, n=13) by applying mitochondrial inhibitors (combination of rotenone (R) and oligomycin (O)). However, applications of R/O, soon after the peak of Tg/Ach-induced Ca(2+) transient, produced a larger cytosolic calcium elevation (Delta[Ca(2+)](c)=84 +/- 6 nM, n=9), this corresponds to an increase in the total mitochondrial calcium concentration ([Ca(2+)](m)) by approximately 0.4 mM. In cells pre-treated with R/O or Ru360 (a specific blocker of mitochondrial Ca(2+) uniporter), the decay time-constant of the Tg/ACh-induced Ca(2+) response was prolonged by approximately 40 and 80%, respectively. Tests with the mitochondrial Ca(2+) indicator rhod-2 revealed large increases in [Ca(2+)](m) in response to Tg/ACh applications; this mitochondrial uptake was blocked by Ru360. In cells pre-treated with Ru360, 10nM ACh elicited large global increases in [Ca(2+)](c), compared to control cells in which ACh-induced Ca(2+) signals were localised in the apical region. We conclude that mitochondria are active elements of cellular Ca(2+) homeostasis in pancreatic acinar cells and directly modulate both local and global calcium signals induced by agonists.     

High- and low-calcium-dependent mechanisms of mitochondrial calcium signalling

The Ca(2+) coupling between endoplasmic reticulum (ER) and mitochondria is central to multiple cell survival and cell death mechanisms. Cytoplasmic [Ca(2+)] ([Ca(2+)](c)) spikes and oscillations produced by ER Ca(2+) release are effectively delivered to the mitochondria. Propagation of [Ca(2+)](c) signals to the mitochondria requires the passage of Ca(2+) across three membranes, namely the ER membrane, the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM). Strategic positioning of the mitochondria by cytoskeletal transport and interorganellar tethers provides a means to promote the local transfer of Ca(2+) between the ER membrane and OMM. In this setting, even >100 microM [Ca(2+)] may be attained to activate the low affinity mitochondrial Ca(2+) uptake. However, a mitochondrial [Ca(2+)] rise has also been documented during submicromolar [Ca(2+)](c) elevations. Evidence has been emerging that Ca(2+) exerts allosteric control on the Ca(2+) transport sites at each membrane, providing mechanisms that may facilitate the Ca(2+) delivery to the mitochondria. Here we discuss the fundamental mechanisms of ER and mitochondrial Ca(2+) transport, particularly the control of their activity by Ca(2+) and evaluate both high- and low-[Ca(2+)]-activated mitochondrial calcium signals in the context of cell physiology.     

Mode of mitochondrial Ca2+ clearance and its influence on secretory responses in stimulated chromaffin cells

To study the role of mitochondrial Ca(2+) clearance in stimulated cells, changes in free Ca(2+) concentration in the cytosol, [Ca(2+)](c) and that in mitochondria, [Ca(2+)](m) along with secretory responses were observed using chromaffin cells co-loaded with Fura-2 and Rhod-2 in the perfused rat adrenal medulla. When the cells were stimulated with 40 mM K(+) in the perfusate, the duration of [Ca(2+)](m) response markedly increased with prolongation of the stimulation period, exhibiting a mean half-decay time of 21 min with 30s stimulation, whereas its amplitude was not altered with stimulations of 10-30s. A computer simulation analysis showed that such a mode of [Ca(2+)](m) response can be produced if excess Ca(2+) taken up by mitochondria precipitates as calcium phosphate (Pi) salt. In the presence of 5 microM rotenone plus 10 microM oligomycin, a decrease in the duration of [Ca(2+)](m) response and a slight but significant increase (24%) in the secretory response to 30s stimulation with 40 mM K(+) were observed. Simulation analyses suggested that this effect of rotenone may be due to reduction in mitochondrial Ca(2+) uptake induced by rotenone-elicited partial depolarization of the mitochondrial membrane potential. In chromaffin cells transsynaptically stimulated through the splanchnic nerve, the intensity of NAD(P)H autofluorescence changed with time courses similar to those of [Ca(2+)](m) responses. The temporal profiles of those two responses were prolonged in a similar manner by application of an inhibitor of mitochondrial Na(+)/Ca(2+) exchanger, CGP37157. Thus, due to the unique Ca(2+) buffering mechanism, [Ca(2+)](m) responses associated with massive mitochondrial Ca(2+) uptake may occur within a limited concentration range in which Ca(2+)-sensitive dehydrogenases are activated to control the mitochondrial redox state in stimulated chromaffin cells.     

Inflammation alters regional mitochondrial Ca²⁺ in human airway smooth muscle cells

Regulation of cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in airway smooth muscle (ASM) is a key aspect of airway contractility and can be modulated by inflammation. Mitochondria have tremendous potential for buffering [Ca(2+)](cyt), helping prevent Ca(2+) overload, and modulating other intracellular events. Here, compartmentalization of mitochondria to different cellular regions may subserve different roles. In the present study, we examined the role of Ca(2+) buffering by mitochondria and mitochondrial Ca(2+) transport mechanisms in the regulation of [Ca(2+)](cyt) in enzymatically dissociated human ASM cells upon exposure to the proinflammatory cytokines TNF-α and IL-13. Cells were loaded simultaneously with fluo-3 AM and rhod-2 AM, and [Ca(2+)](cyt) and mitochondrial Ca(2+) concentration ([Ca(2+)](mito)) were measured, respectively, using real-time two-color fluorescence microscopy in both the perinuclear and distal, perimembranous regions of cells. Histamine induced a rapid increase in both [Ca(2+)](cyt) and [Ca(2+)](mito), with a significant delay in the mitochondrial response. Inhibition of the mitochondrial Na(+)/Ca(2+) exchanger (1 μM CGP-37157) increased [Ca(2+)](mito) responses in perinuclear mitochondria but not distal mitochondria. Inhibition of the mitochondrial uniporter (1 μM Ru360) decreased [Ca(2+)](mito) responses in perinuclear and distal mitochondria. CGP-37157 and Ru360 significantly enhanced histamine-induced [Ca(2+)](cyt). TNF-α and IL-13 both increased [Ca(2+)](cyt), which was associated with decreased [Ca(2+)](mito) in the case of TNF-α but not IL-13. The effects of TNF-α on both [Ca(2+)](cyt) and [Ca(2+)](mito) were affected by CGP-37157 but not by Ru360. Overall, these data demonstrate that in human ASM cells, mitochondria buffer [Ca(2+)](cyt) after agonist stimulation and its enhancement by inflammation. The differential regulation of [Ca(2+)](mito) in different parts of ASM cells may serve to locally regulate Ca(2+) fluxes from intracellular sources versus the plasma membrane as well as respond to differential energy demands at these sites. We propose that such differential mitochondrial regulation, and its disruption, may play a role in airway hyperreactivity in diseases such as asthma, where [Ca(2+)](cyt) is increased.     

Identification of a ryanodine receptor in rat heart mitochondria

Recent studies have shown that, in a wide variety of cells, mitochondria respond dynamically to physiological changes in cytosolic Ca(2+) concentrations ([Ca(2+)](c)). Mitochondrial Ca(2+) uptake occurs via a ruthenium red-sensitive calcium uniporter and a rapid mode of Ca(2+) uptake. Surprisingly, the molecular identity of these Ca(2+) transport proteins is still unknown. Using electron microscopy and Western blotting, we identified a ryanodine receptor in the inner mitochondrial membrane with a molecular mass of approximately 600 kDa in mitochondria isolated from the rat heart. [(3)H]Ryanodine binds to this mitochondrial ryanodine receptor with high affinity. This binding is modulated by Ca(2+) but not caffeine and is inhibited by Mg(2+) and ruthenium red in the assay medium. In the presence of ryanodine, Ca(2+) uptake into isolated heart mitochondria is suppressed. In addition, ryanodine inhibited mitochondrial swelling induced by Ca(2+) overload. This swelling effect was not observed when Ca(2+) was applied to the cytosolic fraction containing sarcoplasmic reticulum. These results are the first to identify a mitochondrial Ca(2+) transport protein that has characteristics similar to the ryanodine receptor. This mitochondrial ryanodine receptor is likely to play an essential role in the dynamic uptake of Ca(2+) into mitochondria during Ca(2+) oscillations.     

Plasma membrane potential oscillations in insulin secreting Ins-1 832/13 cells do not require glycolysis and are not initiated by fluctuations in mitochondrial bioenergetics

Oscillations in plasma membrane potential play a central role in glucose-induced insulin secretion from pancreatic β-cells and related insulinoma cell lines. We have employed a novel fluorescent plasma membrane potential (Δψ(p)) indicator in combination with indicators of cytoplasmic free Ca(2+) ([Ca(2+)](c)), mitochondrial membrane potential (Δψ(m)), matrix ATP concentration, and NAD(P)H fluorescence to investigate the role of mitochondria in the generation of plasma membrane potential oscillations in clonal INS-1 832/13 β-cells. Elevated glucose caused oscillations in plasma membrane potential and cytoplasmic free Ca(2+) concentration over the same concentration range required for insulin release, although considerable cell-to-cell heterogeneity was observed. Exogenous pyruvate was as effective as glucose in inducing oscillations, both in the presence and absence of 2.8 mM glucose. Increased glucose and pyruvate each produced a concentration-dependent mitochondrial hyperpolarization. The causal relationships between pairs of parameters (Δψ(p) and [Ca(2+)](c), Δψ(p) and NAD(P)H, matrix ATP and [Ca(2+)](c), and Δψ(m) and [Ca(2+)](c)) were investigated at single cell level. It is concluded that, in these β-cells, depolarizing oscillations in Δψ(p) are not initiated by mitochondrial bioenergetic changes. Instead, regardless of substrate, it appears that the mitochondria may simply be required to exceed a critical bioenergetic threshold to allow release of insulin. Once this threshold is exceeded, an autonomous Δψ(p) oscillatory mechanism is initiated.     

Effect of ebselen on Ca2+ transport in mitochondria

The seleno-organic compound ebselen mimics the glutathione-dependent, hydroperoxide reducing activity of glutathione peroxidase. The activity of glutathione peroxidase determines the rate of hydroperoxide-induced Ca2+ release from mitochondria. Ebselen stimulates Ca2+ release from mitochondria, accelerates mitochondrial respiration and uncoupling, and induces mitochondrial swelling, indicating a deterioration of mitochondrial function. These manifestations are abolished by cyclosporine A, a potent inhibitor of the mitochondrial permeability transition. However, when ebselen-induced Ca2+ cycling is prevented with ruthenium red, an inhibitor of the Ca2+ uniporter, or by chelation of extramitochondrial Ca2+ by EGTA, no detectable elevation of swelling or uncoupling is observed. The release of Ca2+ from mitochondria is delayed in the absence of rotenone, i.e. when pyridine nucleotides are maintained in the reduced state due to succinate-driven reversed electron flow. We suggest that ebselen induces Ca2+ release from intact mitochondria via an NAD+ hydrolysis-dependent mechanism.     

Impaired regulation of brain mitochondria by extramitochondrial Ca2+ in transgenic Huntington disease rats

Huntington disease (HD) is characterized by polyglutamine expansions of huntingtin (htt), but the underlying pathomechanisms have remained unclear. We studied brain mitochondria of transgenic HD rats with 51 glutamine repeats (htt(51Q)), modeling the adult form of HD. Ca(free)(2+) up to 2 mum activated state 3 respiration of wild type mitochondria with glutamate/malate or pyruvate/malate as substrates. Ca(free)(2+) above 2 mum inhibited respiration via cyclosporin A-dependent permeability transition (PT). Ruthenium red, an inhibitor of the mitochondrial Ca(2+) uniporter, did not affect the Ca(2+)-dependent activation of respiration but reduced Ca(2+)-induced inhibition. Thus, Ca(2+) activation was mediated exclusively by extramitochondrial Ca(2+), whereas inhibition was promoted also by intramitochondrial Ca(2+). In contrast, htt(51Q) mitochondria showed a deficient state 3 respiration, a lower sensitivity to Ca(2+) activation, and a higher susceptibility to Ca(2+)-dependent inhibition. Furthermore htt(51Q) mitochondria exhibited a diminished membrane potential stability in response to Ca(2+), lower capacities and rates of Ca(2+) accumulation, and a decreased Ca(2+) threshold for PT in a substrate-independent but cyclosporin A-sensitive manner. Compared with wild type, Ca(2+)-induced inhibition of respiration of htt(51Q) mitochondria was less sensitive to ruthenium red, indicating the involvement of extramitochondrial Ca(2+). In conclusion, we demonstrate a novel mechanism of mitochondrial regulation by extramitochondrial Ca(2+). We suggest that specific regulatory Ca(2+) binding sites on the mitochondrial surface, e.g. the glutamate/aspartate carrier (aralar), mediate this regulation. Interactions between htt(51Q) and distinct targets such as aralar and/or the PT pore may underlie mitochondrial dysregulation leading to energetic depression, cell death, and tissue atrophy in HD.     

Mitochondrial reactive oxygen species and calcium uptake regulate activation of phagocytic NADPH oxidase

Production of superoxide (O(2)(·-)) by NADPH oxidases contributes to the development of hypertension and atherosclerosis. Factors responsible for activation of NADPH oxidases are not well understood; interestingly, cardiovascular disease is associated with both altered NADPH oxidase activity and age-associated mitochondrial dysfunction. We hypothesized that mitochondrial dysfunction may contribute to activation of NADPH oxidase. The effect of mitochondrial inhibitors on phagocytic NADPH oxidase in human lymphoblasts and whole blood was measured at the basal state and upon PKC-dependent stimulation with PMA using extracellular 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium or mitochondria-targeted 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine spin probes and electron spin resonance (ESR). Intracellular cytosolic calcium [Ca(2+)](i) was measured spectrofluorometrically using fura-2 AM. Incubation of lymphoblasts with the mitochondrial inhibitors rotenone, antimycin A, CCCP, or ruthenium red (an inhibitor of mitochondrial Ca(2+) uniporter) did not significantly change basal activity of NADPH oxidase. In contrast, preincubation with the mitochondrial inhibitors prior to PMA stimulation of lymphoblasts resulted in two- to three-fold increase of NADPH oxidase activity compared with stimulation with PMA alone. Most notably, the intracellular Ca(2+)-chelating agent BAPTA-AM abolished the effect of mitochondrial inhibitors on NADPH oxidase activity. Cytosolic Ca(2+) measurements with fura-2 AM showed that the mitochondrial inhibitors increased [Ca(2+)](i), while BAPTA-AM abolished the increase in [Ca(2+)](i). Furthermore, depletion of cellular Ca(2+) with thapsigargin attenuated CCCP- and antimycin A-mediated activation of NADPH oxidase in the presence of PMA by 42% and 31%, correspondingly. Our data suggest that mitochondria regulate PKC-dependent activation of phagocytic NADPH oxidase. In summary, increased mitochondrial O(2)(·-) and impaired buffering of cytosolic Ca(2+) by dysfunctional mitochondria result in enhanced NADPH oxidase activity, which may contribute to the development of cardiovascular diseases.     

Calcium-dependent spontaneously reversible remodeling of brain mitochondria

An exposure of cultured hippocampal neurons expressing mitochondrially targeted enhanced yellow fluorescent protein to excitotoxic glutamate resulted in reversible mitochondrial remodeling that in many instances could be interpreted as swelling. Remodeling was not evident if glutamate receptors were blocked with MK801, if Ca(2+) was omitted or substituted for Sr(2+) in the bath solution, if neurons were treated with carbonylcyanide p-trifluoromethoxyphenylhydrazone to depolarize mitochondria, or if neurons were pretreated with cyclosporin A or N-methyl-4-isoleucine-cyclosporin (NIM811) to inhibit the mitochondrial permeability transition. In the experiments with isolated brain synaptic or nonsynaptic mitochondria, Ca(2+) triggered transient, spontaneously reversible cyclosporin A-sensitive swelling closely resembling remodeling of organelles in cultured neurons. The swelling was accompanied by the release of cytochrome c, Smac/DIABLO, Omi/HtrA2, and AIF but not endonuclease G. Depolarization with carbonylcyanide p-trifluoromethoxyphenylhydrazone or inhibition of the Ca(2+) uniporter with Ru360 prevented rapid onset of the swelling. Sr(2+) depolarized mitochondria but failed to induce swelling. Neither inhibitors of the large conductance Ca(2+)-activated K(+) channel (charybdotoxin, iberiotoxin, quinine, and Ba(2+)) nor inhibitors of the mitochondrial ATP-sensitive K(+) channel (5-hydroxydecanoate and glibenclamide) suppressed swelling. Quinine, dicyclohexylcarbodiimide, and Mg(2+), inhibitors of the mitochondrial K(+)/H(+) exchanger, as well as external alkalization inhibited a recovery phase of the reversible swelling. In contrast to brain mitochondria, liver and heart mitochondria challenged with Ca(2+) experienced sustained swelling without spontaneous recovery. The proposed model suggests an involvement of the Ca(2+)-dependent transient K(+) influx into the matrix causing mitochondrial swelling followed by activation of the K(+)/H(+) exchanger leading to spontaneous mitochondrial contraction both in situ and in vitro.     

Stimulatory effects of calcium on respiration and NAD(P)H synthesis in intact rat heart mitochondria utilizing physiological substrates cannot explain respiratory control in vivo

Mitochondrial TCA cycle dehydrogenase enzymes have been shown to be stimulated by Ca(2+) under various substrate and ADP incubation conditions in an attempt to determine and understand the role of Ca(2+) in maintaining energy homeostasis in working hearts. In this study, we tested the hypothesis that, at physiological temperature and 1 mM extramitochondrial free magnesium, Ca(2+) can stimulate the overall mitochondrial NAD(P)H generation flux in rat heart mitochondria utilizing pyruvate and malate as substrates at both subsaturating and saturating concentrations. In both cases, we found that, in the physiological regime of mitochondrial oxygen consumption observed in the intact animal and in the physiological range of cytosolic Ca(2+) concentration averaged per beat, Ca(2+) had no observable stimulatory effect. A modest apparent stimulatory effect (22-27%) was observable at supraphysiological maximal ADP-stimulated respiration at 2.5 mM initial phosphate. The stimulatory effects observed over the physiological Ca(2+) range are not sufficient to make a significant contribution to the control of oxidative phosphorylation in the heart in vivo.     

Mitochondrial Ca2+ influx and efflux rates in guinea pig cardiac mitochondria: low and high affinity effects of cyclosporine A

Ca(2+) plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca(2+) is controlled primarily by the mitochondrial Ca(2+) uniporter and the mitochondrial Na(+)/Ca(2+) exchanger, influencing NADH production through Ca(2+)-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca(2+)-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca(2+) release. Here we selectively measure Ca(2+) influx rate through the mitochondrial Ca(2+) uniporter and Ca(2+) efflux rates through Na(+)-dependent and Na(+)-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na(+)/Ca(2+) exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ(m) loss, Ca(2+) release, NADH oxidation, swelling) of high extramitochondrial Ca(2+) additions, allowing mitochondria to tolerate total mitochondrial Ca(2+) loads of >400nmol/mg protein. For Ca(2+) pulses up to 15μM, Na(+)-independent Ca(2+) efflux through the permeability transition pore accounted for ~5% of the total Ca(2+) efflux rate compared to that mediated by the mitochondrial Na(+)/Ca(2+) exchanger (in 5mM Na(+)). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na(+)/Ca(2+) exchanger-mediated Ca(2+) efflux at higher concentrations (IC(50)=2μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~40% at 10μM cyclosporine A, while having no effect on the mitochondrial Ca(2+) uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca(2+) load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.