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J. Biol. Chem., Vol. 275, Issue 45, 35402-35407, November 10, 2000

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Intracellular Sodium Modulates Mitochondrial Calcium Signaling in Vascular Endothelial Cells^*

Marina Sedova and Lothar A. Blatter

From the Loyola University Chicago, Stritch School of Medicine, Department of Physiology, Maywood, Illinois 60153

Received for publication, July 10, 2000, and in revised form, August 22, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have investigated the role of extramitochondrial Na⁺ for the regulation of mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) in permeabilized single vascular endothelial cells. [Ca²⁺]_m was measured by loading the cells with the membrane-permeant Ca²⁺ indicator fluo-3/AM and subsequent removal of cytoplasmic fluo-3 by surface membrane permeabilization with digitonin. An elevation of extramitochondrial Ca²⁺ resulted in a dose-dependent increase in the rate of Ca²⁺ accumulation into mitochondria (k_0.5 = 3 µM) via the mitochondrial Ca²⁺ uniporter. In the presence of 10 mM extramitochondrial Na⁺ ([Na⁺]_em), repetitive application of brief pulses of high Ca²⁺ (2-10 µM) to simulate cytoplasmic [Ca²⁺] oscillations caused transient increases of [Ca²⁺]_m characterized by a fast rising phase that was followed by a slow decay. Removal of extramitochondrial Na⁺ or inhibition of mitochondrial Na⁺/Ca²⁺ exchange with clonazepam blocked mitochondrial Ca²⁺ efflux and resulted in a net accumulation of Ca²⁺ by the mitochondria. Half-maximal activation of mitochondrial Na⁺/Ca²⁺ exchange occurred at [Na⁺]_em = 4.4 mM, which is well within the physiological range of cytoplasmic [Na⁺]. This study provides evidence that Ca²⁺ efflux from the mitochondria in vascular endothelial cells occurs solely via Na⁺/Ca²⁺ exchange and emphasizes the important role of intracellular Na⁺ for mitochondrial Ca²⁺ regulation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Agonist stimulation of electrically non-excitable cells causes an increase in cytosolic calcium concentration ([Ca²⁺]_i) by releasing Ca²⁺ from IP₃¹-sensitive endoplasmic reticulum stores and subsequent activation of store-operated calcium influx (Ref. 1; for review see Refs. 2-6). Elevations of [Ca²⁺]_i mediate the signal transduction from the plasma membrane agonist receptors to the Ca²⁺-sensitive intracellular targets, modulating a variety of cellular functions, including secretion, enzyme activation, gene expression, and proliferation. At submaximal agonist concentrations the elevation of [Ca²⁺]_i often occurs in the form of [Ca²⁺]_i oscillations propagating throughout the cell as calcium waves (2, 7, 8). These oscillatory [Ca²⁺]_i changes have been suggested to result from a synchronized periodic activation/deactivation of endoplasmic reticulum Ca²⁺ release channels or changes in IP₃ concentration (9, 10) and are important for encoding the specificity of cellular responses (11-13).

Cytoplasmic Ca²⁺ signals are also important for the regulation of mitochondrial functions. It has been shown that the IP₃-mediated increase of [Ca²⁺]_i can be transferred rapidly to the mitochondrial matrix (14-18), leading to the activation of Ca²⁺-sensitive dehydrogenases that participate in oxidative ATP synthesis (19-23). In this way intracellular Ca²⁺ may provide an efficient mechanism to increase ATP production (24) and to compensate for increased ATP utilization by processes that are regulated by Ca²⁺ (e.g. muscle contraction and secretion) or regulate [Ca²⁺]_i itself, such as Ca²⁺ ATPases for example. Conversely, the accumulation of Ca²⁺ by mitochondria affects cytosolic Ca²⁺ levels; thus mitochondria participate actively in cytosolic Ca²⁺ signaling (for reviews see Refs. 25 and 26).

There are two independent pathways for Ca²⁺ transport in and out of mitochondria (for reviews see Refs. 27-30). Ca²⁺ uptake into the mitochondria occurs via an electrogenic uniporter driven by the mitochondrial membrane potential. Ca²⁺ efflux is mediated by either Na⁺/Ca²⁺ (e.g. in heart and brain cells) or H⁺/Ca²⁺ exchange (e.g. in liver and smooth muscle). The activity of both Ca²⁺ influx and efflux mechanisms determines how spatio-temporal patterns of cytoplasmic [Ca²⁺] would be translated into mitochondrial [Ca²⁺] and metabolic responses.

It has been established previously that agonist stimulation of endothelial cells results in an increase of [Ca²⁺]_i, which leads to subsequent accumulation of Ca²⁺ by mitochondria (14, 31, 32). The mechanisms, however, that are involved in mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) regulation in endothelial cells are not well characterized.

In the present study we have used permeabilized calf pulmonary artery endothelial (CPAE) cells to examine the role of cytoplasmic Na⁺ in mitochondrial calcium signaling and to test the hypothesis that mitochondrial Na⁺/Ca²⁺ exchange plays a crucial role in endothelial [Ca²⁺]_m regulation. The results of our experiments show that 1) the mitochondria of permeabilized CPAE cells rapidly accumulate Ca²⁺ in response to an increase in extramitochondrial Ca²⁺ concentration, 2) the extrusion of Ca²⁺ from mitochondria is primarily mediated by Na⁺/Ca²⁺ exchange and is thereby controlled by cytoplasmic [Na⁺], and 3) inhibition of mitochondrial Na⁺/Ca²⁺ exchange shapes the mitochondrial calcium transients and converts oscillatory changes of [Ca²⁺]_m into sustained increases of [Ca²⁺]_m. The results indicate the important role of mitochondrial Na⁺/Ca²⁺ exchange as well as cytoplasmic [Na⁺] for the translation of cytoplasmic Ca²⁺ signals into the mitochondrial matrix and the regulation of Ca²⁺-dependent mitochondrial processes. A previous account of this work has been presented in abstract form (33).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Preparation-- The CPAE cell line was obtained from American Type Culture Collection (ATCC, CCL-209, Manassas, VA). The cells were cultured in Eagle's minimal essential medium supplemented with 20% fetal bovine serum (Life Technologies, Inc.) and 2 mM L-glutamine in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. Once a week the cells were dispersed using a Ca²⁺-free (0.1% EDTA) 0.25% trypsin solution and plated onto glass coverslips for further experiments. Cells from passages 2-6 were used. All experiments were performed at room temperature (20-22 °C) on single cells in non-confluent cultures.

Fluorescence Measurements-- Spatially averaged photometric [Ca²⁺] measurements from single endothelial cells were performed with the fluorescent Ca²⁺ indicator fluo-3. Cells were loaded with 25 µM membrane-permeant acetoxymethyl ester of the dye (fluo-3/AM; Molecular Probes, Inc., Eugene, OR) for 40 min at 37 °C in standard Tyrode solution containing (in mM concentrations) 135 NaCl, 4 KCl, 1 MgCl₂, 2 CaCl₂, 10 glucose, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.3). The fluo-3 fluorescence was excited at 485 nm, and emitted fluorescence was recorded at 535 nm. Fluorescence signals were low-pass filtered at 1 kHz and sampled at 5 Hz. [Ca²⁺]_m is expressed as F/F₀, i.e. as fluo-3 fluorescence (F) normalized to the basal fluorescence levels (F₀) measured after permeabilization of the surface membrane.

Cells were permeabilized by exposure to 10 µM digitonin (see Fig. 1) added to the "intracellular" solution consisting of (in mM concentrations) 135 KCl, 10 NaCl, 20 HEPES, 5 pyruvate, 2 glutamate, 2 malate, 0.5 KH₂PO₄, 1 MgCl₂, 5 EGTA, and 1.86 CaCl₂ to yield a free [Ca²⁺] of ~100 nM.

Laser scanning confocal microscopy (LSM 410, Carl Zeiss, Germany) was used for the co-localization of mitochondrial marker (TMRE) and mitochondrial Ca²⁺ signals (fluo-3). The potentiometric fluorescent cationic dye tetramethylrhodamine ethyl ester (TMRE, Molecular Probes) was added to the extracellular solution in a concentration of 0.2 µM. Both indicators were excited with the 488 nm line of the argon ion laser, and the emitted fluorescence signals were measured simultaneously at 510-525 nm (fluo-3) and >610 nm (TMRE).

Chemicals-- The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), ruthenium red, and clonazepam were obtained from Sigma.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Measurements of Intramitochondrial Free Ca²⁺ in Single Permeabilized CPAE Cells-- To measure mitochondrial Ca²⁺ signals we took advantage of the fact that the acetoxymethyl ester form of fluorescent Ca²⁺ indicators sequester into intracellular organelles, including mitochondria. For our experiments we exposed intact CPAE cells to fluo-3/AM and subsequently used the non-ionic detergent digitonin to permeabilize the plasma membrane and to remove fluo-3 from the cytosol and nucleus. The effect of digitonin and elevation of extramitochondrial Ca²⁺ ([Ca²⁺]_em) on the time course of the spatially averaged fluo-3 signal from a single cell is shown in Fig. 1A. After digitonin treatment fluo-3 fluorescence decreased to 20.0 ± 1.3% of the initial level (mean ± S.E., n = 40). This remaining level of fluorescence represents the contribution of mitochondria to the total fluorescence signal. This fluorescence level (F₀) was used to normalize the fluo-3 signal. An increase of [Ca²⁺]_em from 0.1 to 2 µM resulted in an increase in fluo-3 fluorescence, suggesting Ca²⁺ accumulation into the mitochondria.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Measurements of [Ca2+]m in single permeabilized CPAE cells with compartmentalized fluo-3. A, changes in spatially averaged fluo-3 signal following digitonin addition and increase of extramitochondrial [Ca2+]. Permeabilization of the surface membrane with digitonin resulted in a decrease of cellular fluo-3 fluorescence by ~80% due to loss of cytoplasmic fluo-3. The rapid increase of fluo-3 fluorescence upon increasing [Ca2+]em from 0.1 to 2 µM was due to Ca2+ uptake into the mitochondrial matrix. a.u., arbitrary fluorescence units. B, simultaneous confocal images of fluo-3 (green, emitted fluorescence recorded at 510-525 nm) and TMRE (red, > 610 nm) fluorescence after permeabilization with digitonin and increase in [Ca2+]em from 0.1 to 2 µM. Fluo-3 and TMRE were excited at 488 nm. The right panel shows the overlay of the two individual images. Colocalization of fluo-3 and TMRE is represented in shades of yellow. Pixel size, 284 nm; calibration bar, 20 µm.

Visualization of Mitochondria with TMRE-- We have used laser scanning confocal microscopy in combination with the potentiometric fluorescent dye TMRE to visualize the mitochondria in CPAE cells. The large potential gradient across the inner mitochondrial membrane results in the accumulation of this cationic dye within the matrix compartment and allows mitochondrial labeling (34, 35). TMRE was added to extramitochondrial solution in a concentration of 0.2 µM. In Fig. 1B simultaneously acquired TMRE (red) and fluo-3 (green) images of CPAE cells are shown, following digitonin treatment and the increase of [Ca²⁺]_em to 2 µM. These images are overlaid in the right panel. The TMRE signal is highly co-localized with fluo-3 fluorescence, indicating that the measured fluo-3 signals originated from the mitochondria and report [Ca²⁺]_m.

Effect of FCCP and Ruthenium Red on [Ca²⁺]_m Increase-- Fig. 2A shows the effect of the dissipation of the mitochondrial membrane potential () with FCCP on mitochondrial Ca²⁺ accumulation. Mitochondrial Ca²⁺ uptake was initiated by elevating [Ca²⁺]_em from 0.1 to 2 µM and measured with fluo-3 trapped inside mitochondria of permeabilized CPAE cells. In the presence of 1 µM FCCP the amplitude of F/F₀ measured at 100 s after the elevation of [Ca²⁺]_em amounted to 1.9 ± 0.5 versus 8.5 ± 1.2 in control (n = 6; p < 0.0005, Student's t test). In the presence of 10 µM ruthenium red, an inhibitor of the mitochondrial Ca²⁺ uniporter, there were no changes in [Ca²⁺]_m following the increase in [Ca²⁺]_em (Fig. 2B, trace I; n = 4). Ruthenium red applied during the rising phase of the [Ca²⁺]_m transient (arrow) caused the F/F₀ signal to decline to the basal level, presumably due to inhibition of Ca²⁺ uptake and active Ca²⁺ extrusion (Fig. 2B, trace II; n = 4). These data indicate that the increase of the fluo-3 signal following the elevation of [Ca²⁺]_em represents a Ca²⁺ uniporter-mediated, -dependent Ca²⁺ uptake into the mitochondria.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Pharmacological inhibition of mitochondrial Ca2+ uptake. A, dissipation of the mitochondrial membrane potential with the uncoupler FCCP (1 µM) inhibited mitochondrial Ca2+ uptake. B, inhibition of the mitochondrial Ca2+ uniporter with ruthenium red (10 µM) prevents (trace I) or rapidly stops (trace II) mitochondrial Ca2+ accumulation. Arrows mark ruthenium red (RR) addition for traces I and II.

Dependence of Mitochondrial Ca²⁺ Uptake on [Ca²⁺]_em-- To examine the [Ca²⁺]_em dependence of the mitochondrial Ca²⁺ uptake, permeabilized cells were exposed to various concentrations of extramitochondrial Ca²⁺ in the absence of Na⁺ (Fig. 3A). At the end of each experiment ionomycin (2 µM) together with high Ca²⁺ (100 µM) were applied (arrow) to obtain a maximum response and to normalize mitochondrial Ca²⁺ uptake. Fig. 3 shows an overlay of normalized traces of mitochondrial Ca²⁺ uptake obtained at [Ca²⁺]_em of 0.8, 1, 1.2, and 10 µM, indicating that increasing [Ca²⁺]_em resulted in a concentration-dependent increase in the rate of mitochondrial Ca²⁺ accumulation. We used the rate of increase of the normalized [Ca²⁺]_m transients to characterize the dose-dependence of mitochondrial Ca²⁺ uptake in CPAE cells (Fig. 3B). To minimize potential problems arising from the non-linear relationship between [Ca²⁺] and fluo-3 fluorescence, the rate of Ca²⁺ uptake was determined from the initial linear phase (Fig. 3B, inset) of the [Ca²⁺]_m increase. Data were fitted with the Hill equation, yielding a Hill coefficient (n_H) of 2.7. The [Ca²⁺]_em resulting in a half-maximal rate (k_0.5) of Ca²⁺ accumulation was 3 µM. There was no activation of the Ca²⁺ uniporter at [Ca²⁺]_em < 0.8 µM. n_H > 1 suggested cooperativity of the Ca²⁺ uptake process.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   [Ca2+]em dependence of mitochondrial Ca2+ uptake. A, permeabilized cells were exposed to various concentrations of extramitochondrial Ca2+ in Na+-free solution. [Ca2+]em was elevated from 0 (1 mM EGTA solution) to 0.5-10 µM. The arrows indicate the addition of 2 µM ionomycin together with 100 µM Ca2+ at the end of the experiment to obtain the maximum fluorescence signal (Fmax). Individual traces shown were recorded from different cells and were normalized to Fmax. B, the rates of fluorescence increase (expressed as % Fmax/s) were determined from the linear part of the traces shown in panel A and plotted against [Ca2+]em. The inset shows the early part of the traces in panel A on an extended time scale. The line is the fit to the Hill equation with k0.5 = 3.0 µM and nH = 2.7. Data represent the mean ± S.E. Numbers in parentheses indicate the number of individual cells exposed to a given [Ca2+]em.

The Effect of the Removal of Extramitochondrial Na⁺ on [Ca²⁺]_m-- Stimulation of endothelial cells with a low concentration of agonists has been reported to generate [Ca²⁺]_i oscillations (36-38). To simulate these oscillatory changes of [Ca²⁺]_i, brief (5-10 s) pulses of high extramitochondrial Ca²⁺ (2-10 µM) were applied to the cells every 100-200 s. Fig. 4 shows the pattern of [Ca²⁺]_m following repetitive exposure to 2 µM Ca²⁺ for 5 s. In the presence of 10 mM extramitochondrial Na⁺, repetitive increases of [Ca²⁺]_em caused transient elevations of [Ca²⁺]_m characterized by a fast rising phase followed by a slow decay. Removal of extramitochondrial Na⁺ abolished the decay of [Ca²⁺]_m, suggesting an inhibition of mitochondrial Ca²⁺ efflux that resulted in a net accumulation of Ca²⁺ into mitochondria with each [Ca²⁺]_em pulse.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   The effect of extramitochondrial Na+ ([Na+]em) on the kinetics of [Ca2+]m. Brief (5 s) pulses of high [Ca2+]em (2 µM) were applied to simulate [Ca2+]i oscillations. In the presence of 10 mM Na+, extramitochondrial Ca2+ pulses produced oscillatory changes in [Ca2+]m. Removal of extramitochondrial Na+ inhibited Ca2+ efflux from mitochondria and resulted in a net accumulation of mitochondrial Ca2+.

Na⁺ Dependence of Ca²⁺ Extrusion from Mitochondria-- The data from the above experiments (Fig. 4) indicate that Ca²⁺ removal from endothelial mitochondria is a Na⁺-dependent process. Fig. 5 directly shows the effect of extramitochondrial Na⁺ concentration ([Na⁺]_em) on [Ca²⁺]_m recovery. Ca²⁺ uptake into the mitochondria was activated by increasing [Ca²⁺]_em from 0.1 to 2 µM for 10 s, and the decline of [Ca²⁺]_m was recorded in the presence of different [Na⁺]_em (Fig. 5A). The value of 1/t_1/2 (t_1/2 is the time required for the fluo-3 signal to decrease to half-maximal amplitude) was used to characterize the rate of decline of [Ca²⁺]_m. Ca²⁺ removal in the presence of 20 mM Na⁺ was determined to be maximal and served as the reference for normalization, i.e. for each cell the rates of [Ca²⁺]_m decrease for various values of [Na⁺]_em were normalized to the maximal rate of decline recorded in 20 mM Na⁺. An increase in [Na⁺]_em resulted in a sigmoidal increase in the rate of Ca²⁺ efflux (Fig. 5B). The concentration of Na⁺ giving a half-maximal rate of decrease of [Ca²⁺]_m was 4.4 mM, and the Hill coefficient was 2.2. 

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effect of Na+ on the rate of mitochondrial Ca2+ extrusion. A, Ca2+ uptake into the mitochondria was initiated by exposure to 2 µM Ca2+, and the recovery of [Ca2+]m was recorded in the presence of different [Na+]em varying from 1 to 20 mM. The continuous recording of [Ca2+]m was obtained from a single permeabilized CPAE cell. B, [Na+]em dependence of Ca2+ efflux. The time required for [Ca2+]m to recover to half-maximum (t1/2) was used to analyze the rate of Ca2+ extrusion. The data are expressed as a percentage of the maximum extrusion rate recorded in the presence of 20 mM Na+. The data were fitted by the Hill equation with k0.5 = 4.4 mM and nH = 2.2. Data represent the mean ± S.E. Numbers in parentheses indicate the number of individual cells exposed to a given [Na+]em.

Effect of Pharmacological Inhibition of Mitochondrial Na⁺/Ca²⁺ Exchange on Ca²⁺ Efflux from Mitochondria-- To determine the effect of inhibition of mitochondrial Na⁺/Ca²⁺ exchange on Ca²⁺ efflux from mitochondria, [Ca²⁺]_m recovery was studied in the presence of 10 mM extramitochondrial Na⁺ and increasing concentrations of clonazepam, an inhibitor of mitochondrial Na⁺/Ca²⁺ exchange (39). As shown in Fig. 6A, cell treatment with 50 µM clonazepam significantly slowed Ca²⁺ extrusion from the mitochondria. The decline of [Ca²⁺]_m during the 5-min time interval after the decrease in [Ca²⁺]_em back to 0.1 µM was fitted with the monoexponential function. The time constant  was used as a measure of the rate of Ca²⁺ extrusion. Dose-dependent inhibition of mitochondrial Ca²⁺ efflux by clonazepam is shown in Fig. 6B. Data for each cell are presented as percent inhibition of Ca²⁺ extrusion (percent inhibition = 100·(1  _control/)). Half-maximal inhibition of Ca²⁺ extrusion was observed at clonazepam concentrations of ~5 µM. 50 µM clonazepam almost completely (>95%) abolished Ca²⁺ extrusion. The inhibition of the [Na⁺]_em-dependent decrease of [Ca²⁺]_m by clonazepam supports the hypothesis that Na⁺-mediated efflux of Ca²⁺ from endothelial mitochondria occurs via the mitochondrial Na⁺/Ca²⁺ exchange mechanism.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   The effect of inhibition of mitochondrial Na+/Ca2+ exchange on Ca2+ efflux. A, treatment with clonazepam (50 µM) in the presence of 10 mM extramitochondrial Na+ significantly slowed Ca2+ extrusion from mitochondria. The time constant  of a monoexponential fit to the decline of [Ca2+]m (black line) served as a measure of the rate of Ca2+ efflux. B, dose dependence of inhibition of mitochondrial Ca2+ efflux with clonazepam. Data are presented as percent inhibition of extrusion = 100·(1  control/). Numbers in parentheses indicate numbers of cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mitochondria are capable of sequestering Ca²⁺ resulting from agonist-induced cytosolic Ca²⁺ elevations. This accumulation of Ca²⁺ by mitochondria has 2-fold consequences for cellular processes. It modulates the dynamics of cytoplasmic Ca²⁺ signals (40, 41; for reviews see Refs. 25 and 26), and it affects Ca²⁺-mediated mitochondrial functions such as mitochondrial oxidative metabolism (21-24). In the vascular endothelium, Ca²⁺ links mitochondrial functions to specific endothelial cell functions. For example the stimulated production of endothelial-derived relaxing factors (42, 43) requires ATP, which is derived from mitochondrial oxidative phosphorylation (44). Although Ca²⁺ accumulation by mitochondria has also been observed in endothelial cells (14, 31, 32), little is known about the uptake pathway involved, and no information is available on the mechanism and kinetics of Ca²⁺ extrusion from endothelial mitochondria.

Permeabilized Cells as a Model for Studying [Ca²⁺]_m Regulation-- The membrane-permeant acetoxymethyl ester form of a variety of fluorescent Ca²⁺ indicators tends to compartmentalize into cellular organelles such as the endoplasmic reticulum, lysosomes, and mitochondria. Although this is considered a disadvantage for cytosolic [Ca²⁺] measurements, this property can be used to measure ion concentrations in cellular subcompartments and organelles (e.g. Ref. 45). Quenching of the cytosolic portion of the dye with Mn²⁺ or removal of the cytosolic indicator by permeabilization of the plasma membrane has been used for fluorescence measurements originating solely from the mitochondrial matrix. For example, permeabilized cells were used to study the interaction between IP₃-sensitive Ca²⁺ release channels and mitochondria (17), for imaging the opening of the mitochondrial permeability transition pore (46), and to characterize mitochondrial signals involved in apoptosis (47). We used this approach to investigate the kinetics of mitochondrial Ca²⁺ uptake and release and to characterize the role of extramitochondrial Na⁺ for the regulation of [Ca²⁺]_m in vascular endothelial (CPAE) cells.

Mitochondrial Ca²⁺ Uptake-- Under physiological conditions the only relevant pathway of Ca²⁺ entry into mitochondria is the Ca²⁺ uniporter located in the inner mitochondrial membrane. Therefore, the rate of mitochondrial Ca²⁺ accumulation directly reflects the activity of the Ca²⁺ uniporter. Fig. 3 shows that raising extramitochondrial Ca²⁺ led to a dose-dependent increase in mitochondrial Ca²⁺ uptake rates. The [Ca²⁺]_em resulting in a half-maximal Ca²⁺ influx rate (k_0.5) was 3 µM. The sigmoidal dependence of the Ca²⁺ uptake rate with an n_H > 1 (Fig. 3B) indicates positive cooperativity of the uptake mechanism. Quantitative parameters of the mitochondrial Ca²⁺ uptake (V_max, k_0.5, n_H) reported in the literature vary with the experimental conditions and techniques used (for review see Ref. 27). Experimental approaches similar to ours applied to permeabilized HeLa cells with mitochondrially targeted aequorin (15) and rat basophilic leukemia cells loaded with the low affinity Ca²⁺ dye fura-2FF (17) yielded values for k_0.5 of ~4 and 10 µM, respectively.

We found that mitochondria of permeabilized CPAE cells can sequester large amounts of Ca²⁺ very rapidly at [Ca²⁺]_em  5 µM. Exposure to 10 µM Ca²⁺ led to a rapid rise of [Ca²⁺]_m, which reached a new steady state within 0.5 s (Fig. 3B). There was no further increase in fluo-3 signal upon subsequent addition of ionomycin and 100 µM Ca²⁺, indicating saturation of the dye (F_max, maximum fluorescence signal). Assuming that F_max corresponds to ~10 µM free Ca²⁺, we can estimate a rate of Ca²⁺ accumulation of about 20 µM/s. This value is clearly higher than the uptake rate of 0.8 µM/s (15) measured in HeLa cells with wild-type aequorin, a Ca²⁺-sensitive photoprotein that reliably reports changes of [Ca²⁺] between 0.3 and  100 µM (48). Using a mutated low affinity aequorin (Ca²⁺ sensitivity, 20 µM-1 mM), however, Montero et al. (18) measured a Ca²⁺ uptake rate of ~35 µM/s (with [Ca²⁺]_em = 10 µM) in chromaffin cells, similar to the value found in our study.

A rapid increase of [Ca²⁺]_m following elevations of [Ca²⁺]_i was also found in intact bovine vascular endothelial cells expressing aequorin targeted to mitochondria (14). In that study it was shown that an increase in cytosolic free Ca²⁺, induced by stimulation with ATP, was paralleled by a very rapid increase in [Ca²⁺]_m to over 5 µM. Possible explanations for this rapid accumulation of Ca²⁺ into the mitochondria were either 1) a close physical association of mitochondria with Ca²⁺ release sites, exposing these organelles to microdomains of high Ca²⁺ (15, 17, 49) or 2) a rapid mode (RAM) of Ca²⁺ uptake originally described in liver mitochondria (50). This rapid mode of mitochondrial Ca²⁺ uptake was activated by small (<0.4 µM) but fast elevations of extramitochondrial Ca²⁺. Because we did not see any accumulation of Ca²⁺ into mitochondria at [Ca²⁺]_em below 0.5 µM, it seems unlikely that in our experiments Ca²⁺ uptake occurred via RAM.

Mitochondrial Ca²⁺ Efflux in Vascular Endothelial Cells Occurs via Na⁺/Ca²⁺ Exchange-- The experiments illustrated in Fig. 2B show that mitochondria from vascular endothelial cells possess an efficient mechanism to extrude accumulated Ca²⁺. Inhibition of Ca²⁺ entry via Ca²⁺ uniporter by ruthenium red not only stopped mitochondrial Ca²⁺ accumulation but resulted in a decrease of [Ca²⁺]_m, suggesting that mitochondria are capable of extruding accumulated Ca²⁺. Two mitochondrial Ca²⁺ efflux mechanisms have been described that differ by their Na⁺ dependence. Both Na⁺-dependent and Na⁺-independent transport exist in a variety of cell types (51-53). The Na⁺-dependent Ca²⁺ extrusion mechanism is predominant in heart, brain, adrenal cortex, and skeletal muscle mitochondria (54, 55) and occurs via Na⁺/Ca²⁺ exchange (53, 56). In contrast, the Na⁺-independent Ca²⁺ extrusion dominates in liver, lung, and kidney tissue and is carried by H⁺/Ca²⁺ exchange, whereas the Na⁺-dependent mechanism is strongly inhibited by physiological (0.5-1 mM) levels of cytosolic Mg²⁺ (51, 57). Our study shows that the mitochondrial Na⁺/Ca²⁺ exchange mechanism is the major pathway for Ca²⁺ efflux from mitochondria in the vascular endothelium (Figs. 4-6; see also Ref. 58). Lowering extramitochondrial Na⁺ concentration or pharmacological inhibition of mitochondrial Na⁺/Ca²⁺ exchange turned [Ca²⁺]_m signals from transient or oscillatory into prolonged and continuous elevations. In that sense the activity of the mitochondrial Na⁺/Ca²⁺ exchange directly determines the time during which Ca²⁺-sensitive mitochondrial targets, such as Ca²⁺-dependent enzymes controlling mitochondrial metabolic activity, are exposed to elevated [Ca²⁺].

Cellular and Subcellular Ca²⁺ Signaling: The Role of [Na⁺]_i-- Under the premise that Ca²⁺ extrusion from endothelial mitochondria occurs solely via mitochondrial Na⁺/Ca²⁺ exchange, it becomes obvious that [Na⁺]_i plays a crucial role in the regulation of mitochondrial Ca²⁺ homeostasis in endothelial cells. Whether changes in Na⁺/Ca²⁺ exchange activity occur in intact cells in response to changes in cytosolic [Na⁺] depends on the affinity (k_m) of the exchanger for extramitochondrial Na⁺ and the concentration of free cytosolic Na⁺. In our study the [Na⁺]_em that resulted in a half-maximal rate of Ca²⁺ efflux from mitochondria was 4.4 mM. This [Na⁺] is comparable with k_m values of 2.6-9.4 mM for the Na⁺ dependence determined for heart and liver mitochondria (39, 51, 54, 57, 59). The level of cytosolic free Na⁺ in a variety of non-excitable cells was estimated to be in the range of 4-12 mM (60-62). These levels of [Na⁺]_i together with the measured [Na⁺] dependence of the mitochondrial Na⁺/Ca²⁺ exchange suggest that the exchanger actively controls [Ca²⁺]_m and that relatively small changes of [Na⁺]_i (as they may occur under a variety of physiological and pathophysiological conditions) might be significant enough to alter mitochondrial Na⁺/Ca²⁺ exchange activity and [Ca²⁺]_m. We found that Ca²⁺ extrusion via Na⁺/Ca²⁺ exchange was maximal at [Na⁺]_em of 20 mM, a value very similar to that found in liver, kidney, and lung mitochondria (52, 59).

The dependence of mitochondrial Na⁺/Ca²⁺ exchange on [Na⁺]_i further emphasizes the role of cellular mechanisms that regulate [Na⁺]_i itself (i.e. the plasmalemmal Na⁺/Ca²⁺ and Na⁺/H⁺ exchange mechanisms, the Na⁺/K⁺ pump, and Na⁺-permeable channels) for mitochondrial function. Control of Ca²⁺ efflux from mitochondria might be a physiological role of the [Na⁺]_i increase seen in agonist-stimulated cells (60-63). Furthermore, it becomes apparent that [Na⁺]_i plays a critical dual role in the regulation of cytoplasmic and mitochondrial [Ca²⁺]. For example, it has been demonstrated that [Na⁺]_i gradients are important for the regulation of the Ca²⁺-dependent nitric-oxide synthase activity and agonist-evoked Ca²⁺ oscillations through local increases of [Ca²⁺]_i mediated by the plasmalemmal Na⁺/Ca²⁺ exchange mechanism (63, 64). The present study established further evidence for the importance of intracellular sodium for the regulation of both cytoplasmic and mitochondrial Ca²⁺ levels and the control of Ca²⁺-dependent processes in both of these cellular compartments.

	ACKNOWLEDGEMENTS

We thank Katherine A. Sheehan for critically reading the manuscript. Holly R. Gray and Rachel L. Gulling provided expert technical expertise, and John J. Payne and Vezetter Whitaker made invaluable contributions by building custom-made equipment.

	FOOTNOTES

^* Financial support was provided by grants from the National Institutes of Health (HL-51941 and HL-62231) and the American Heart Association National Center (Established Investigator Award 95002520).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Physiology, Loyola University Chicago, 2160 S. First Ave., Maywood, IL 60153. Tel.: 708-216-1182; Fax: 708-216-6308; E-mail: LBLATTE@LUMC.EDU.

Published, JBC Papers in Press, August 24, 2000, DOI 10.1074/jbc.M006058200

	ABBREVIATIONS

The abbreviations used are: IP₃, inositol 1,4,5-trisphosphate; CPAE, calf pulmonary artery endothelial; TMRE, tetramethylrhodamine ethyl ester; FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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