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Originally published In Press as doi:10.1074/jbc.M409353200 on January 19, 2005

J. Biol. Chem., Vol. 280, Issue 13, 12114-12122, April 1, 2005
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The Role of Mitochondria for Ca2+ Refilling of the Endoplasmic Reticulum*

Rolland Malli{ddagger}, Maud Frieden§, Michael Trenker{ddagger}, and Wolfgang F. Graier{ddagger}

From the {ddagger}Department of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University Graz, 8010 Graz, Austria and §Department of Cell Physiology and Metabolism, University of Geneva, Medical Center, 1211 Geneva 4, Switzerland

Received for publication, August 16, 2004 , and in revised form, January 18, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Endoplasmic reticulum (ER) Ca2+ refilling is an active process to ensure an appropriate ER Ca2+ content under basal conditions and to maintain or restore ER Ca2+ concentration during/after cell stimulation. The mechanisms to achieve successful ER Ca2+ refilling are multiple and built on a concerted action of processes that provide a suitable reservoir for Ca2+ sequestration into the ER. Despite mitochondria having been found to play an essential role in the maintenance of capacitative Ca2+ entry by buffering subplasmalemmal Ca2+, their contribution to ER Ca2+ refilling was not subjected to detailed analysis so far. Thus, this study was designed to elucidate the involvement of mitochondria in Ca2+ store refilling during and after cell stimulation. ER Ca2+ refilling was found to be accomplished even during continuous inositol 1,4,5-trisphosphate (IP3)-triggered ER Ca2+ release by an agonist. Basically, ER Ca2+ refilling depended on the presence of extracellular Ca2+ as the source and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) activity. Interestingly, in the presence of an IP3-generating agonist, ER Ca2+ refilling was prevented by the inhibition of trans-mitochondrial Ca2+ flux by CGP 37157 (7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one) that precludes the mitochondrial Na+/Ca2+ exchanger as well as by mitochondrial depolarization using a mixture of oligomycin and antimycin A. In contrast, after the removal of the agonist, ER refilling was found to be largely independent of trans-mitochondrial Ca2+ flux. Under these conditions, ER Ca2+ refilling took place even without an associated Ca2+ elevation in the deeper cytosol, thus, indicating that superficial ER domains mimic mitochondrial Ca2+ buffering and efficiently sequester subplasmalemmal Ca2+ and consequently facilitate capacitative Ca2+ entry. Hence, these data point to different contribution of mitochondria in the process of ER Ca2+ refilling based on the presence or absence of IP3, which represents the turning point for the dependence or autonomy of ER Ca2+ refilling from trans-mitochondrial Ca2+ flux.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
For a large number of receptors, the binding of the respective agonist initiates the generation of inositol 1,4,5-trisposphate (IP3),1 which in turn triggers Ca2+ release from the endoplasmic reticulum (ER), leading to an instant rise of the cytosolic free Ca2+ concentration ([Ca2+]cyto). This elevation in [Ca2+]cyto is subsequently maintained by adjusting the activity of Ca2+-permeable ion channels, pumps, exchangers, and buffer systems (for review see Refs. 1 and 2). To ensure that, even under repetitive stimulations a suitable intracellular Ca2+ release is possible refilling of the main intracellular Ca2+ store, the ER has to be accomplished either while balancing high levels of [Ca2+]cyto during cell stimulation or at least during the decay of cell stimulation.

The mechanisms to achieve successful ER Ca2+ refilling are multiple and built on a concerted action of many processes that provide a suitable reservoir for Ca2+ sequestration via the ER Ca2+ pumps (SERCAs). Because elevated cytosolic Ca2+ is also substrate for Ca2+ extrusion mechanisms such as the plasma membrane Ca2+ ATPases or the Na+/Ca2+ exchanger (NCXpm), Ca2+ entry through the plasma membrane takes place in order to preclude a life-threatening loss of cellular Ca2+. At least in non-excitable cells, the firmly established phenomenon of capacitative Ca2+ entry (CCE) (3), which is activated by the emptying of intracellular Ca2+ stores, accounts for Ca2+ transit through the plasma membrane (4–6).

Although the mechanisms of activation of the non-voltage gated Ca2+-permeable ion channel(s) that account for CCE (e.g. Refs. 7–11) and its/their molecular nature are still under debate (12–14), recent investigations point to a fundamental role of mitochondria in the maintenance (15–19) and modulation (20) of the CCE in many cell types. Notably, the intriguing aptitude of mitochondria to effectively impound subplasmalemmal Ca2+ (i.e. mitochondrial Ca2+-buffering function) was found to be pivotal for the maintenance of the CCE pathway as they prevent Ca2+-dependent inactivation of the CCE channel(s) (15, 19, 21). Recently, we demonstrated that superficial mitochondria are able to efficiently buffer the subplasmalemmal Ca2+ concentration ([Ca2+]pm) beneath the plasma membrane (22) despite a strong agonist-evoked cytosolic Ca2+ elevation and the generation of high Ca2+ levels between the plasma membrane and superficial ER domains (i.e. subplasmalemmal Ca2+ control unit, SCCU) (22–24). Hence, mitochondrial Ca2+ uptake goes along with vectorial Ca2+ release via mitochondrial Na+/Ca2+ exchanger (NCXmito) toward the ER and the cytosol that becomes visible by ER Ca2+ refilling and the elevation in [Ca2+]cyto, respectively (19).

However, it is not clear to what extent the transfer of entering Ca2+ across the mitochondria accounts for ER Ca2+ refilling and under which conditions trans-mitochondrial Ca2+ flux is prerequisite for intracellular Ca2+ store-refilling processes. In addition, the kinetics of ER Ca2+ refilling during cell stimulation, the impact of IP3, and how mitochondria participate to the process of ER Ca2+ refilling need to be investigated in more detail. Thus, we monitored changes in the free Ca2+ concentration in the cytoplasm, the mitochondrial matrix, and within the lumen of the ER ([Ca2+]er) upon cell stimulation and correlated the Ca2+ dynamics in the cytosol and these organelles during Ca2+ release and refilling processes in single cells of the human umbilical vein endothelial cell-derived cell line EA.hy926 (25).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Cell culture chemicals were from Invitrogen, and fetal calf serum was obtained from PAA Laboratories (Linz, Austria). Fura-2/AM was from Molecular Probes Europe (Leiden, Netherlands). CGP 37157 was purchased from Tocris Cookson Ltd. (Northpoint, Avonmouth, Bristol, United Kingdom). Histamine, BHQ, EGTA, oligomycin, and antimycin A were from Sigma-Aldrich (Vienna, Austria). Restriction enzymes and T4 DNA ligase were from New England BioLabs (Frankfurt, Germany), and the EndoFree Plasmid Maxi Kit was from Qiagen. All of the other chemicals were from Roth (Karlsruhe, Germany).

Cell Culture—The human umbilical vein endothelial cell line, EA.hy926 passage ≥45, was used for this study. Cells were cultured in Dulbecco's minimum essential medium containing 10% fetal calf serum and 1% HAT (5 mM hypoxanthine, 20 µM aminopterin, 0.8 mM thymidine). For experiments, cells were grown on glass coverslips (24 or 30 mm).

Plasmids and Transfection—For transfection, an improved version of the YC4-ER (26, 27), vYC4-ER (28), and RP-mt (29) in pcDNA 3 (Invitrogen) was used. Cells (~80% confluency) were transiently transfected with 1.5–3 µg of purified plasmid DNA using TransFastTM transfection reagent (Promega, Mannheim, Germany). Between 24 and 36 h after transfection, cells were used for the experiments. To monitor [Ca2+]mito, an EA.hy926 cell line that stably expresses RP-mt was also used, which provided virtually identical results to those obtained in cells transiently transfected with RP-mt.

Solutions—Dye loading was performed with a loading buffer solution containing 2 mM CaCl2, 135 mM NaCl, 1 mM MgCl2, 5 mM KCl, 10 mM Hepes, 2.6 mM NaHCO3, 0.44 mM KH2PO4, 10 mM D-glucose, 0.1% vitamins, 0.2% essential amino acids, 1% penicillin/streptomycin, and 1% fungizone, pH adjusted to 7.4, with NaOH. The Ca2+ containing experimental buffer was composed of (in mM) 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 D-glucose, and 10 Hepes acid, pH adjusted to 7.4, with NaOH. In experiments where a Ca2+-free solution was applied to the cells, Ca2+-free experimental buffer containing 1 mM EGTA was used.

Imaging Device—Our setup for monitoring cytosolic and organelle Ca2+ signaling was described previously (24). All of the Ca2+ measurements were performed at room temperature on a Nikon inverted microscope (Eclipse 300 TE, Nikon, Vienna, Austria) equipped with an epifluorescence system (150 W XBO, Optiquip, Highland Mills, NY) using the Plan Fluor x40 oil immersion objective from Nikon. Excitation filters were changed with a Ludl filter-wheel device (Ludl Electronic Products, Hawthorne, NY). Fluorescence was monitored with a cooled CCD camera (–30 °C; Quantix KAF 1400G2, Roper Scientific, Acton, MA). All of the devices were controlled by the MetaFluor® 4.0 software (Universal Imaging, Visitron Systems, Puchheim, Germany). The glass coverslips were mounted into an experimental chamber, and cells were perfused with experimental buffer at a rate of ~1.5 ml/min. The perfusion system allowed fast buffer exchange from seven reservoirs.

Cytoslic Ca2+ Measurements—Changes in [Ca2+]cyto were monitored using the Ca2+-sensitive dye fura-2 (24). Cells were loaded for 45 min at room temperature in the dark in loading buffer containing 2 µM Fura-2/AM. Prior experiments, cells were washed twice with loading buffer and equilibrated for an additional 30 min in the same buffer in the dark. Cells were illuminated alternatively at 340 ± 15 and 380 ± 15 nm (340HT15 and 380HT15; Omega Optical, Brattleboro, VT), and emission was monitored at 510 nm (510WB40, Omega Optical). [Ca2+]cyto was expressed as (F340/F380)/F0, where F0 was calculated for each single cell according to the ratio F340/F380 collected at the beginning of each experiment.

Measurement of Free Mitochondrial Ca2+ ([Ca2+]mito)—EA.hy926 cells expressing RP-mt were used to follow [Ca2+]mito as described previously (19, 28). Notably, RP-mt was demonstrated to be a reliable Ca2+ sensor when excited at 410–440 nm, whereas the fluorescence of the permutated yellow fluorescent protein reflects mainly changes in the H+ concentration (30) when excited at 480 nm. Therefore, in contrast to our previous work (19), in this study, RP-mt was only excited at 433 nm (433DF15, Omega Optical) and emission was collected at 535 nm (535AF26, Omega Optical). Thus, [Ca2+]mito was expressed as 1 – F430/F0 as previously shown (30).

[Ca2+]er Measurements—The improved version of the original YC4-ER (26, 27), vYC4-ER (28), was used to monitor the free Ca2+ concentration within the ER lumen. This Ca2+ sensor was excited at 440 ± 21 nm (440AF21, Omega Optical), and emission was collected simultaneously at 535 and 480 nm with one given camera using an optical beam splitter (535 and 480 nm, Dual-View MicroImagerTM, Optical Insights, Visitron Systems) as described previously (19, 24). To correct the decay in the F535/F480 ratio during the experiments, which was probably due to photobleaching or photochromism of vYC4-ER, the changes of the ER Ca2+ concentration were expressed as (F535/F480)/F0.

Statistics—Analysis of variance (ANOVA) and Scheffe's post hoc F test were used for evaluation of the statistical significance. p < 0.05 was defined to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
ER Ca2+ Refilling Is Only Moderately Affected by IP3—As published recently (19), the removal of extracellular Ca2+ during stimulation with histamine accelerated the rate of ER Ca2+ depletion in endothelial cells, whereas Ca2+ re-addition yielded a rapid Ca2+ refilling of the ER (Fig. 1). Notably, the kinetics of ER Ca2+ refilling upon the addition of extracellular Ca2+ was similar in the presence and absence of the IP3-generating autacoid. However, under conditions where histamine was washed out prior to Ca2+ re-addition, the ER Ca2+ content was completely restored to initial values, whereas in the presence of histamine, the ER refilled up to the level prior to the removal of extracellular Ca2+ (Fig. 1). These data indicate that, in endothelial cells, a strong and efficient machinery exists that ensures ER Ca2+ refilling even under conditions of continuous intracellular Ca2+ release. As the ER Ca2+ refilling critically depends on Ca2+ influx, which is maintained by mitochondrial Ca2+ buffering (15–19), the question on the impact of mitochondrial function for the ER Ca2+ refilling was assessed.



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  		FIG. 1.
The kinetics of ER refilling upon Ca2+ re-addition is affected slightly by the presence of an IP3-generating agonist. Endothelial cells transiently expressing vYC4-ER were stimulated with 100 µM histamine in the presence of 2 mM extracellular Ca2+. As indicated, the extracellular Ca2+ was substituted by 1 mM EGTA followed by the re-addition of extracellular Ca2+ in the absence (dotted line, filled circles; n = 7) or presence of 100 µM histamine (continuous line, open circles; n = 10). Tracings indicate the average curves, and the circles express the mean ± S.E. at the given time point. *, p < 0.05 versus in the absence of histamine.

 
Inhibition of NCXmito Yields Ca2+ Accumulation in the Mitochondria and Spoils Their Capacity to Sequester Entering Ca2+ Independently of the Presence or Absence of an Agonist— The impact of Ca2+ influx on mitochondrial Ca2+ dynamics was investigated by the removal and subsequent re-addition of extracellular Ca2+ during and after cell stimulation with histamine. Upon stimulation with histamine (100 µM), [Ca2+]mito elevated transiently despite cytosolic Ca2+ levels remaining elevated (Fig. 2A, both panels). Subsequent removal of extracellular Ca2+ accelerated the decrease of mitochondrial free Ca2+ until the initial value of basal [Ca2+]mito was reached (Fig. 2A, both panels).



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  		FIG. 2.
NCXmito contributes to mitochondrial and cytosolic Ca2+ homeostasis particularly during the Ca2+ entry phase. Mitochondrial (A) and cytosolic (B)Ca2+ signals were recorded using RP-mt and fura-2, respectively. The same protocols were applied in A and B. Left panels, in the continuous presence of 100 µM histamine in the absence (continuous line, open circles; A, n = 23; B, n = 9) or presence (dotted line, filled circles; A, n = 23; B, n = 31) of 20 µM CGP 37517, a specific inhibitor of the NCXmito. Extracellular Ca2+ was removed and re-added as indicated. Right panels, histamine washout prior to the re-addition of extracellular Ca2+, whereas CGP 37157 was absent (continuous line, open circles; A, n = 25; B, n = 11) or present (dotted line, filled circles; A, n = 63; B, n = 37). *, p < 0.05 versus in the absence of CGP 37157. C, the kinetics of the [Ca2+]cyto elevation upon Ca2+ re-addition shown in B were further analyzed by time-resolved differentiation of the respective ratiometric signals. The dotted lines indicate no changes in [Ca2+]cyto. *, p < 0.05 versus in the absence of CGP 37157. D, schematic illustration of the estimated Ca2+ fluxes toward the cytosol in the presence (left panel) and absence (right panel) of histamine during Ca2+ re-addition. Numbers represent the percentage of the contribution of the individual path of Ca2+ toward deeper cytosol for cytosolic Ca2+ elevation upon re-addition of Ca2+ in the presence (left panel) or absence (right panel) of histamine and were calculated according to the traces shown in B.

 
In the presence of histamine, the re-addition of Ca2+ yielded a fast and instant rise in [Ca2+]mito (Fig. 2A, left panel), whereas in the absence of histamine, mitochondrial Ca2+ elevation upon Ca2+ re-addition developed at a slower pace (Fig. 2A, right panel).

To investigate the impact of the major Ca2+ export pathway from mitochondria (the NCXmito), CGP 37157, a specific inhibitor of NCXmito (31, 32), was used. CGP 37157 (20 µM) had no effect on [Ca2+]mito under basal conditions. In contrast, upon cell stimulation, NCXmito inhibition lead to a long-lasting elevation of [Ca2+]mito that could be reduced neither by the removal of extracellular Ca2+ (Fig. 2A, both panels) nor by agonist washout (Fig. 2A, right panel). In the presence of CGP 37157, the re-addition of Ca2+ had no further effect on [Ca2+]mito independently of the agonist (Fig. 2A, both panels), indicating that the capacity of mitochondria to sequester Ca2+ has been saturated under these conditions. This is in line with our previous report (19) where we described that CGP 37157 prevented Ca2+-inhibitable capacitative Ca2+ entry due to the lack of subplasmalemmal Ca2+ buffering by mitochondria. Thus, during cell stimulation, the inhibition of NCXmito resulted in the accumulation of Ca2+ in the mitochondria that is not reversible either by the removal of extracellular Ca2+ or by the washout of the agonist.

Mitochondria Differentially Contribute to Cytosolic Ca2+ Elevation upon CCE Activity in the Presence and Absence of an Agonist—To elucidate whether or not the presence of an agonist modulates the impact of mitochondria on cytosolic Ca2+ elevation upon Ca2+ entry, similar time course protocols as described above were conducted while [Ca2+]cyto was monitored. Cell stimulation with histamine in the presence of extracellular Ca2+ caused a rapid and stable increase of [Ca2+]cyto that was immediately reduced upon the removal of extracellular Ca2+ (data not shown). In the presence of histamine, subsequent re-addition of extracellular Ca2+ rapidly and permanently enhanced [Ca2+]cyto (Fig. 2B, left panel). However, cytosolic Ca2+ elevation upon Ca2+ re-addition after histamine washout was fast but transient (Fig. 2B, right panel). Thus, in the presence of histamine, Ca2+ entry yielded a similar profile of Ca2+ elevation in the cytosol and the mitochondria, whereas in the absence of the agonist, the response differed between both compartments.

Remarkably, the importance of trans-mitochondrial Ca2+ flux for cytosolic Ca2+ elevation also depended on the presence of histamine. Notably, inhibition of NCXmito with CGP 37157 reduced the cytosolic Ca2+ elevation upon re-addition of extracellular Ca2+ in the presence of histamine by ~40% (Fig. 2B, left panel). A detailed analysis of the Ca2+ entry kinetics in the presence of histamine revealed that CGP 37157 reduced the rate of the initial Ca2+ elevation upon Ca2+ re-addition by ~90%, whereas a continuous cytosolic Ca2+ elevation developed slowly (Fig. 2C, left panel). However, the inhibitory effect of CGP 37157 was much more pronounced if histamine was removed prior to Ca2+ re-addition (Fig. 2B, right panel) and no subsequent slow continuous Ca2+ elevation occurred (Fig. 2C, right panel). These data indicate that, in the presence of IP3, blocking NCXmito modestly affects cytosolic Ca2+ elevation upon Ca2+ re-addition (Fig. 2D, left panel), which might be due to Ca2+ release from partially refilled ER in the presence of histamine. However, after histamine washout, trans-mitochondrial Ca2+ flux is required to allow cytosolic Ca2+ elevation (Fig. 2D, right panel) and, thus, under these conditions the ER does not contribute to cytosolic Ca2+ elevation.

To confirm this hypothesis, SERCA activity was blocked with 15 µM BHQ during Ca2+ re-addition in the presence of histamine. In the absence of CGP 37157, SERCA inhibition slightly enhanced cytosolic Ca2+ elevation upon Ca2+ re-addition (Fig. 3). However, in the presence of the NCXmito inhibitor, cytosolic Ca2+ elevation upon Ca2+ re-addition was strongly affected by BHQ (Fig. 3). These data point to a Ca2+ cycling across the ER in the presence of IP3 and partially filled ER stores and/or to the contributory role of ER Ca2+ buffering to the regulation of plasma membrane Ca2+ entry.



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  		FIG. 3.
SERCA inhibition further reduced Ca2+ entry in the presence of CGP 37157. In the absence (open symbols) or presence of 20 µM CGP 37157 (filled symbols), cells were stimulated with 100 µM histamine in the absence of extracellular Ca2+ at time 2 min. To prevent ER Ca2+ refilling, the SERCA inhibitor BHQ (15 µM) was added at time 7.5 min (without CGP 37157: open square symbols, dotted line, n = 10; with CGP 37157: filled square symbols, dotted line, n = 11) followed by the addition of 2 mM extracellular Ca2+. #, p < 0.05 versus in the absence of BHQ. Experiments without BHQ are shown as round symbols full line (without CGP 37157: open round symbols, n = 17; with CGP 37157: filled round symbols, n = 20). *, p < 0.05 versus in the absence of CGP 37157.

 
To approve our data with CGP 37157, identical Ca2+ readdition protocols were accomplished in the presence of 2 µM oligomycin and 10 µM antimycin A. As shown in Fig. 4A, cell treatment with the mixture of oligomycin/antimycin A reduced histamine-induced [Ca2+]mito elevation by ~83%. Nevertheless, mitochondrial Ca2+ decreased upon the removal of extracellular Ca2+ even in the presence of oligomycin/antimycin A, indicating that Ca2+ still moved through mitochondria despite their depolarization (Fig. 4A). However, similar to the data with CGP 37157 presented above (i.e. Fig. 2B), the inhibitory effect of oligomycin/antimycin A on cytosolic Ca2+ elevation upon Ca2+ re-addition was more pronounced after histamine washout (Fig. 4, B and C).



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  		FIG. 4.
{psi}mito contributes to mitochondrial and cytosolic Ca2+ homeostasis particularly during the Ca2+ entry phase. Mitochondrial (A) and cytosolic (B) Ca2+ signals were monitored using RP-mt and fura-2, respectively. Identical protocols were used in A and B. Left panels, in the continuous presence of 100 µM histamine in the absence (continuous line, open circles; A, n = 17; B, n = 21) or presence (dotted line, filled circles; A, n = 18; B, n = 17) of oligomycin/antimycin A (2 µM/10 µM). Extracellular Ca2+ was removed and re-added as indicated. Right panels, histamine was removed prior to the re-addition of extracellular Ca2+ in the absence (continuous line, open circles; A, n = 15; B, n = 34) or presence of oligomycin/antimycin A (dotted line, filled circles; A, n = 18; B, n = 26). C, the kinetics of the [Ca2+]cyto elevation upon Ca2+ readdition shown in B were further analyzed by time-resolved differentiation of the ratiometric signals. *, p < 0.05 versus in the absence of oligomycin/antimycin A.

 
Overall, these data suggest that the presence of an IP3-generating agonist not only affects mitochondrial Ca2+ handling of entering Ca2+ but also modulates the contribution of trans-mitochondrial Ca2+ flux for cytosolic Ca2+ elevation. Considering such striking differences in the pathways for cytosolic Ca2+ elevation upon Ca2+ re-addition in prestimulated cells, the modus operandi to accomplish ER Ca2+ refilling in the presence and absence of IP3 were investigated.

Route and Source of Ca2+ for ER Refilling Differ Depending on the Presence or Absence of an Agonist—Inhibition of NCXmito strongly reduced ER Ca2+ refilling in the presence of the agonist (Fig. 5, A, left panel, and C). In contrast, the inhibition of NCXmito in the absence of the agonist only marginally reduced ER Ca2+ refilling (Fig. 5, B, left panel, and C), despite the strong inhibition (~83%) of the cytosolic Ca2+ signaling by CGP 37157 (Fig. 2D, right panel). In line with these findings, oligomycin/antimycin A attenuated ER Ca2+ refilling more efficiently in the presence of histamine than in its absence (Fig. 5C, right panel).



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  		FIG. 5.
In the presence of histamine, ER Ca2+ refilling requires trans-mitochondrial Ca2+ flux, whereas in the absence of the agonist, the ER refills independently of mitochondria. Endothelial cells were transiently transfected with vYC4-ER to monitor [Ca2+]er. ER depletion was achieved by stimulation with 100 µM histamine in Ca2+-free solution in the absence (continuous line, open circles; A, n = 18; B, n = 10) or presence of 20 µM CGP 37157 (dotted line, filled circles; A, n = 11; B, n = 10) followed by Ca2+ re-addition in the presence (A) or absence (B) of histamine. *, p < 0.05 versus in the absence of CGP 37157. C, statistical evaluation of ER Ca2+ refilling in the presence of 20 µM CGP 37157 (left panel) or oligomycin/antimycin A (2 µM/10 µM) (right panel). Data are expressed in relation to the basal ER Ca2+ content (=100%) and after maximal ER depletion with 100 µM histamine in the absence of extracellular Ca2+ (=0%). Left panel: same as A and B; right panel, control plus histamine, n = 10; oligomycin/antimycin A plus histamine, n = 7; control without histamine, n = 11; oligomycin/antimycin A minus histamine, n = 8. *, p < 0.05 versus control; #, p < 0.05 versus in the presence of histamine.

 
These data suggest that, in the presence of IP3, trans-mitochondrial Ca2+ flux equally contributes to cytosolic Ca2+ elevation and ER Ca2+ refilling upon Ca2+ re-addition to prestimulated cells. However, in the absence of IP3, mitochondrial Ca2+ transit is pivotal for cytosolic Ca2+ elevation upon Ca2+ re-addition, whereas ER Ca2+ refilling primarily occurs independently from the mitochondrial Ca2+ signaling.

In the absence of IP3, ER Ca2+ Refilling Is Achieved Independently from Cytosolic Ca2+ Elevation and Mitochondrial Ca2+ Homeostasis—To further investigate the mechanisms of ER refilling, in the continuous presence or absence of CGP 37157 or the mixture of oligomycin/antimycin A, ER Ca2+ was released twice by histamine in Ca2+-free solution with an intermediate refilling period in Ca2+-containing solution. In line with the data presented above, interruption of transmitochondrial Ca2+ flux by either CGP 37157 or oligomycin/antimycin A had no or little impact on the amount of Ca2+ that could be released by a second stimulation with histamine, although [Ca2+]cyto elevation upon Ca2+ re-addition was reduced by >80% in the presence of CGP 37157 (20 µM) (Fig. 6, A and B) or oligomycin/antimycin A (2 µM/10 µM) (Fig. 6B).



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  		FIG. 6.
In the absence of an agonist, the ER refills independently of the Ca2+ signal in the mitochondria and the deeper cytosol. A, cytosolic Ca2+ signals were measured in fura-2-loaded cells. Similar to the protocols depicted in Fig. 3B (left panel), 100 µM histamine was applied a second time in the absence of extracellular Ca2+ after 4 min of Ca2+ re-addition in the absence (continuous line, open circles; n = 41) or presence of 20 µM CGP 37157 (dotted line, filled circles; n = 26). n.s., not significant versus in the absence of CGP3157; *, p < 0.05 versus in the absence of CGP 37157. B, statistical evaluation of protocol given in A (filled bars), in the presence of oligomycin/antimycin A (gray bars, n = 29), and without a re-addition of Ca2+ (lined bar, n = 64). Data are expressed as percentage of the effect that was recorded in the same protocol in the absence of any mitochondrial effecting compound (control, n = 57). *, p < 0.05 versus control; #, p < 0.05 versus without re-addition of extracellular Ca2+.

 
These data indicate that, in the absence of the agonist, ER Ca2+ refilling is achieved independently of trans-mitochondrial Ca2+ flux and cytosolic Ca2+ elevation. Thus, in the absence of IP3, ER refills directly via the ER-plasma membrane junction (i.e. SCCU) (33, 34) and, thus, represents a mitochondria-independent phenomenon. However, in the presence of IP3, IP3-initiated Ca2+ release elevates the Ca2+ concentration in the SCCU, which prevents Ca2+-inhibitable Ca2+ entry at that location, and in turn, no direct ER Ca2+ refilling is achieved. Consequently, under these conditions, ER Ca2+ refilling essentially depends on mitochondrial Ca2+ flux.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
ER Ca2+ refilling is an active process where SERCAs sequester Ca2+ against a concentration gradient into the ER to ensure an appropriate ER Ca2+ content under basal conditions and to maintain or restore ER Ca2+ concentration during/after cell stimulation. In this paper, the mechanisms of such Ca2+ store-refilling procedures and the contribution of mitochondria to these processes were elucidated (Fig. 7). Remarkably, ER Ca2+ refilling was found to be efficiently accomplished even during continuous IP3-triggered ER Ca2+ release in the presence of an agonist. ER Ca2+ refilling depended essentially on the presence of extracellular Ca2+ as the source and SERCA activity, which sequesters entering Ca2+ into the ER (19). Moreover, the present data point to differences in the mitochondrial contribution to ER Ca2+ refilling that are based on IP3, which represents the turning point for the dependence or autonomy of ER Ca2+ refilling from trans-mitochondrial Ca2+ flux. Notably, in the presence of IP3, which continuously achieves elevation of subplasmalemmal Ca2+ concentration in the vicinity of the ER (i.e. SCCU) (22, 24) and, thus, prevents the Ca2+-inhibitable CCE pathway/channel, ER Ca2+ refilling crucially depends on transmitochondrial Ca2+ flux as this organelle efficiently buffers subplasmalemmal Ca2+ and therefore facilitates CCE (Fig. 7A). In contrast, after the removal of the IP3-generating agonist, ER-refilling was found to be independent of mitochondrial Ca2+-buffering capacity and cytosolic Ca2+ elevation but might occur directly at the junctions between the ER and the plasma membrane (Fig. 7B).



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  		FIG. 7.
Schematic diagram showing the refilling process of the ER depending on the presence or absence of an IP3-generating agonist. A, in the presence of the agonist, superficial ER domains generate high Ca2+ gradients that prevent Ca2+ entry due to the inhibitory effect of Ca2+ on capacitative Ca2+ entry channels (CCECs). Thus, Ca2+ entry occurs in the vicinity of subplasmalemmal mitochondria that buffer entering Ca2+ and, thus, facilitate maintenance of CCE. Subsequently, mitochondrial Ca2+ is vectorially transferred via NCXmito toward SERCA that accomplishes ER Ca2+ refilling even in the presence of IP3. B, if extracellular Ca2+ is re-added to cells after agonist washout (i.e. under conditions where no IP3-triggered Ca2+ release from the ER occurs), the superficial domains of the empty ER mimic mitochondrial Ca2+ buffering by sequestration of entering Ca2+. This ER Ca2+ buffer function results in ER Ca2+ refilling that is independent of mitochondrial Ca2+ handling.

 
ER Ca2+ Dynamics in Endothelial Cells—In the endothelial cells used in this study, the ER Ca2+ content remained unchanged even in the absence of extracellular Ca2+. In contrast, in HeLa cells, the removal of extracellular Ca2+ results in an instant decline in [Ca2+]er because of passive Ca2+ leak from the ER that cannot be compensated under these conditions (35). Thus, while in HeLa cells, a constant transfer of extracellular Ca2+ toward the ER is necessary to maintain the filling state of the ER under basal condition, endothelial cells, at least for a time period of up to 5 min, are able to preserve the ER Ca2+ content independently of extracellular Ca2+ (36). However, our findings that histamine-induced ER Ca2+ depletion was much more pronounced in the absence of extracellular Ca2+ than in its presence indicate that, during agonist stimulation, ER Ca2+ refilling in endothelial cells crucially depends on the influx of Ca2+ from the extracellular compartment. These data are in agreement with those found in HeLa cells2 but in contrast to those found in human embryonic kidney 293 cells where the extent of agonist-induced ER depletion was equal in the presence or absence of extracellular Ca2+ (37). However, while in these cell lines, large ER depletion became visible only in the presence of a SERCA inhibitor, whereas agonists had only little impact on the [Ca2+]er in endothelial cells, the extent of agonist-achievable ER depletion in the absence of extracellular Ca2+ is far higher and equals that of a SERCA inhibitor (19).

Interestingly, the kinetics of ER Ca2+ refilling did not differ if the agonist was present or not, and upon addition of extracellular Ca2+, the ER Ca2+ content was restored up to the level prior to its removal (Fig. 1). Although these data support our hypothesis that Ca2+ entry is essential for ER Ca2+ refilling, they further indicate that the refilling process was affected only slightly by simultaneous ER Ca2+ release. Thus, during cell stimulation with an IP3-generating agonist, a steady-state balance between ER Ca2+ turnover and Ca2+ entry exists.

Mitochondrial Ca2+ Dynamics during Cell Stimulation—Recently, the pivotal role of mitochondria for CCE has been highlighted in many cell types (15–18, 38) including the endothelial cell line used in this study (19). Namely, the properties of superficial mitochondria to sequester entering Ca2+ and to release it vectorially toward the ER/cytosol (i.e. trans-mitochondrial Ca2+ flux) have been found to represent a key process in cellular Ca2+ homeostasis.

In line with our previous findings (19), inhibition of transmitochondrial Ca2+ flux with either CGP 37157 or a mixture of oligomycin/antimycin A diminished cytosolic Ca2+ elevation upon re-addition of extracellular Ca2+ (i.e. the classical protocol for visualization of CCE) (39). However, the impact of an interruption of trans-mitochondrial Ca2+ flux on cytosolic Ca2+ elevation upon Ca2+ re-addition markedly differed depending whether or not the agonist was present.

The Contribution of Mitochondria to Cellular Ca2+ Homeostasis in the Presence of an Agonist—In the presence of histamine, mitochondrial Ca2+ rise upon Ca2+ re-addition was fast and long-lasting and resembled the cytosolic Ca2+ elevation. Thus, it significantly differed from that found in the absence of the agonist. Remarkably, despite a continuous IP3-mediated Ca2+ release, ER refilling is almost fully accomplished as indicated in our ER Ca2+ measurements. Because inhibition of NCXmito strongly diminished ER Ca2+ refilling under these conditions, it is tempting to speculate that, in the presence of an agonist, ER Ca2+ refilling is achieved by a Ca2+ cross-talk with mitochondria, which sequester entering Ca2+ and deliver it toward the SER-CA(s) (Figs. 5 and 6) (19). These assumptions are further supported by our recent data indicating that, under agonist stimulation, the subplasmalemmal Ca2+ concentration between the cell membrane and superficial domains of the ER raises higher than 6 µM and, thus, does not allow the Ca2+-inhibitable Ca2+ entry pathways to be activated in this location (19). Moreover, since we have shown in this work that the continuous ER-refilling process in the presence of an agonist essentially depends on extracellular Ca2+, our data propose that, even if the ER Ca2+ store is refilled by ~80%, CCE still occurs.

Furthermore, it has been shown that Ca2+, which has been released from the ER by a continuous IP3 generation, is sequestered by mitochondria and, thus, yields elevation in [Ca2+]mito (40). Considering this convincing report, we suggest that, in the presence of an agonist, [Ca2+]mito closely follows [Ca2+]cyto as a consequence of IP3-mediated Ca2+ release. Thus, our data indicate that, during continuous IP3 generation, a bidirectional intraorganelle Ca2+ cycle between the mitochondria and the ER occurs where mitochondria contribute essentially to ER Ca2+ refilling, which subsequently elevates [Ca2+]cyto, and promote elevation of [Ca2+]mito (Fig. 7A).

Hence, such continuous Ca2+ release from a partially refilled ER in the presence of histamine would explain the slow cytosolic Ca2+ elevation upon Ca2+ re-addition in the presence of CGP 37157 (Fig. 2B, left panel). This suggestion is further supported by our findings that an inhibition of SERCA by BHQ prevented this slow rise of [Ca2+]cyto (Fig. 3). In addition, these findings may reflect the contribution of ER Ca2+ buffering to the maintenance of the activity of Ca2+ entry.

The Contribution of Mitochondria to Cellular Ca2+ Homeostasis after Agonist Washout—Because in the absence of the agonist, [Ca2+]mito increased secondary to a transient cytosolic Ca2+ elevation, one might suggest that, in the initial phase after Ca2+ re-addition, entering Ca2+ rapidly crosses mitochondria to promote cytosolic Ca2+ elevation. This suggestion is further supported by our findings that inhibition of NCXmito with CGP 37157 strongly diminished cytosolic Ca2+ elevation upon re-addition of extracellular Ca2+. Confirmatory results were obtained with oligomycin/antimycin A. Moreover, because in the absence of the agonist, no further IP3-triggered Ca2+ release occurs, the transient nature of the cytosolic Ca2+ increase might point to a shut down of CCE due to ER Ca2+ refilling. These data indicate that, in the absence of an agonist, cytosolic Ca2+ elevation following re-addition of extracellular Ca2+ to prestimulated cells is predominantly due to entering Ca2+ that crosses mitochondria prior to reaching the major cytosolic compartment (Figs. 2, B and C, and 4, B and C, right panel). Mitochondrial Ca2+ extrusion has been found to depend largely on neighboring ER Ca2+ pumps that generate a negative Ca2+ gradient and, subsequently, facilitate mitochondrial Ca2+ efflux (19). Thus, once ER Ca2+ refilling is completed, the secondary rise in [Ca2+]mito might point to a reduced mitochondrial Ca2+ efflux due to the lack of this negative Ca2+ gradient.

However, our data that repetitive histamine stimulation achieved identical intracellular Ca2+ release independent of whether or not CGP 37157 or oligomycin/antimycin A was present during the Ca2+ re-addition phase (Fig. 6) indicate that ER Ca2+ refilling is accomplished independently of mitochondrial Ca2+ homeostasis. These surprising findings further suggest that, in the absence of an agonist, ER Ca2+ refilling is largely independent of the cytosolic Ca2+ concentration, and our findings were confirmed by the negligible effect of CGP 37157 or oligomycin/antimycin A on ER Ca2+ refilling measured with vYC4-ER (Fig. 5).

These data indicate that, after removal of the IP3-generating agonist, ER refilling is achieved independently of mitochondria and cytosolic Ca2+ elevation and may occur directly at the junction between the ER and the facing plasma membrane CCE channels (Fig. 7B). Thus, in agreement with previous reports (e.g. Ref. 41), in the absence of ER Ca2+ release, emptied superficial ER domains might mimic mitochondrial Ca2+ buffering and facilitate the maintenance of CCE by sequestering subplasmalemmal Ca2+ into the ER lumen in order to accomplish ER refilling independently from mitochondria and Ca2+ concentration in the deeper cytosol. These data are in line with the initial superficial Ca2+ buffer barrier concept, introduced by the VanBreemen group, in smooth muscle cells (42–45) and might indicate that this landmark concept is also valid in endothelial cells.

In conclusion, this work suggests that ER Ca2+ refilling is accomplished even in the presence of an IP3-generating agonist. Furthermore, the contribution of mitochondria to cellular Ca2+ homeostasis is modulated extensively by IP3 (Fig. 6). Herein we report that, in the absence of IP3, the ER refills upon Ca2+ re-addition independently of the mitochondria by sequestrating entering Ca2+ in the subplasmalemmal area between the ER and the plasma membrane. In the presence of IP3, subplasmalemmal ER domains generate high Ca2+ gradients that locally prevent activity of the Ca2+-inhibitable CCE channels. Thus, ER Ca2+ refilling essentially depends on a Ca2+ cross-talk between the ER and the mitochondria that delivers entering Ca2+ toward the ER and the cytoplasmic compartment.


   FOOTNOTES
 
* This work was supported by the Austrian Science Funds (P-16860-B9 and SFB714). The Department of Medical Biochemistry & Medical Molecular Biology is a member of the Institutes of Basic Medical Sciences at the Medical University of Graz and was supported by the infrastructure programs (UGP4) of the Austrian Ministry of Education, Science and Culture. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Medical Biochemistry & Medical Molecular Biology, University of Graz, Harrachgasse 21/III, A-8010 Graz, Austria. Tel.: 43-316-380-7560; Fax: 43-316-380-9615; E-mail: wolfgang.graier{at}meduni-graz.at.

1 The abbreviations used are: IP3, inositol 1,4,5-triphosphate; ER, endoplasmic reticulum; BHQ, 2,5-di-tert-butylhydroquinone; [Ca2+]cyto, free cytosolic Ca2+ concentration; [Ca2+]er, free intraluminal ER Ca2+ concentration; [Ca2+]mito, free Ca2+ concentration in the mitochondrial matrix; CCE, capacitative Ca2+ entry; CGP 37157, 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one; NCXmito, mitochondrial Na+/Ca2+, exchanger; NCXpm plasmalemmal Na+/Ca2+ exchanger; {psi}mito, mitochondrial membrane potential; RP-mt, mitochondrial-targeted ratiometric pericam; SERCA(s), sarcoplasmic/endoplasmic reticulum Ca2+ ATPase(s); SCCU, subplasmalemmal Ca2+ control unit; vYC4-ER, ER-targeted Venus cameleon 4; ANOVA, analysis of variance. Back

2 Prof. Nicolas Demaurex, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank Beatrix Petschar and Anna Schreilechner for their excellent technical assistance, Prof. R. Y. Tsien and Dr. A. Miyawaki for providing the cameleon, Venus, and RP-mt constructs, and Dr. C. J. S. Edgell for the EA.hy926 cells.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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