Article
Reference
Sustained Ca2+ transfer across mitochondria is Essential for mitochondrial Ca2+ buffering, sore-operated Ca2+ entry, and Ca2+
store refilling
MALLI, Roland, et al .
Abstract
Mitochondria have been found to sequester and release Ca2+ during cell stimulation with inositol 1,4,5-triphosphate-generating agonists, thereby generating subplasmalemmal microdomains of low Ca2+ that sustain activity of capacitative Ca2+ entry (CCE). Procedures that prevent mitochondrial Ca2+ uptake inhibit local Ca2+ buffering and CCE, but it is not clear whether Ca2+ has to transit through or remains trapped in the mitochondria. Thus, we analyzed the contribution of mitochondrial Ca2+ efflux on the ability of mitochondria to buffer subplasmalemmal Ca2+, to maintain CCE, and to facilitate endoplasmic reticulum (ER) refilling in endothelial cells. Upon the addition of histamine, the initial mitochondrial Ca2+
transient, monitored with ratio-metric-pericam-mitochondria, was largely independent of extracellular Ca2+. However, subsequent removal of extracellular Ca2+ produced a reversible decrease in [Ca2+]mito, indicating that Ca2+ was continuously taken up and released by mitochondria, although [Ca2+]mito had returned to basal levels. Accordingly, inhibition of the mitochondrial Na+/Ca2+ exchanger with CGP 37157 [...]
MALLI, Roland, et al . Sustained Ca2+ transfer across mitochondria is Essential for
mitochondrial Ca2+ buffering, sore-operated Ca2+ entry, and Ca2+ store refilling. Journal of Biological Chemistry , 2003, vol. 278, no. 45, p. 44769-79
DOI : 10.1074/jbc.M302511200 PMID : 12941956
Available at:
http://archive-ouverte.unige.ch/unige:30407
Disclaimer: layout of this document may differ from the published version.
1 / 1
Mitochondria have been found to sequester and re- lease Ca2ⴙ during cell stimulation with inositol 1,4,5- triphosphate-generating agonists, thereby generating subplasmalemmal microdomains of low Ca2ⴙ that sus- tain activity of capacitative Ca2ⴙ entry (CCE). Proce- dures that prevent mitochondrial Ca2ⴙuptake inhibit local Ca2ⴙbuffering and CCE, but it is not clear whether Ca2ⴙhas to transit through or remains trapped in the mitochondria. Thus, we analyzed the contribution of mitochondrial Ca2ⴙefflux on the ability of mitochondria to buffer subplasmalemmal Ca2ⴙ, to maintain CCE, and to facilitate endoplasmic reticulum (ER) refilling in en- dothelial cells. Upon the addition of histamine, the ini- tial mitochondrial Ca2ⴙtransient, monitored with ratio- metric-pericam-mitochondria, was largely independent of extracellular Ca2ⴙ. However, subsequent removal of extracellular Ca2ⴙ produced a reversible decrease in [Ca2ⴙ]mito, indicating that Ca2ⴙwas continuously taken up and released by mitochondria, although [Ca2ⴙ]mito had returned to basal levels. Accordingly, inhibition of the mitochondrial Naⴙ/Ca2ⴙexchanger with CGP 37157 increased [Ca2ⴙ]mito and abolished the ability of mito- chondria to buffer subplasmalemmal Ca2ⴙ, resulting in an increased activity of BKCachannels and a decrease in CCE. Hence, CGP 37157 also reversibly inhibited ER refilling during cell stimulation. These effects of CGP 37157 were mimicked if mitochondrial Ca2ⴙuptake was prevented with oligomycin/antimycin A. Thus, during cell stimulation a continuous Ca2ⴙ flux through mito- chondria underlies the ability of mitochondria to gener- ate subplasmalemmal microdomains of low Ca2ⴙ, to facilitate CCE, and to relay Ca2ⴙfrom the plasma mem- brane to the ER.
Mitochondria have been found to contribute to cellular Ca2⫹
homeostasis in many cell types by taking up and releasing Ca2⫹, thereby propagating and synchronizing Ca2⫹ signals (1– 4). Notably, mitochondria are often strategically located near Ca2⫹release channels on the ER1or Ca2⫹influx channels
on the plasma membrane. This close connection allows mito- chondria to capture a substantial fraction of the Ca2⫹flowing through release or influx channels, and to generate microdo- mains of low Ca2⫹ near the mouth of these channels (5– 8).
Paradoxically, despite this close contact to Ca2⫹sources, the mitochondrial free Ca2⫹ concentration ([Ca2⫹]mito) increases only transiently during cell stimulation and returns rapidly to base line, regardless of the long lasting elevations in free cyto- solic Ca2⫹concentration ([Ca2⫹]cyto) (9 –11). The transient na- ture of mitochondrial Ca2⫹ signals is surprising, as the sub- plasmalemmal microdomains of low Ca2⫹ generated around Ca2⫹influx pathways can be maintained for extended periods (6, 12–16). Such long lasting microdomains have been postu- lated to underlie mitochondrial modulation of capacitative Ca2⫹ entry (CCE), which is activated by emptying of the ER either by inositol 1,4,5-triphosphate (IP3) or by SERCA inhibi- tion (17, 18). Although the channel(s) responsible for CCE are still under investigation, CCE has been clearly demonstrated to be prevented by enhanced [Ca2⫹]cytoat the inner gate of the channel (6, 12–16). Inhibition of mitochondrial Ca2⫹uptake, either by mitochondrial depolarization or by inhibition of mi- tochondrial Ca2⫹uniporter, prevents the maintenance of CCE (12–14, 19), suggesting that mitochondria maintain a low sub- plasmalemmal Ca2⫹ at the mouth of this Ca2⫹-inhibitable, Ca2⫹-entry pathway (20). However, to account for the long lasting activation of CCE, the microdomain of low Ca2⫹gener- ated by mitochondria must be maintained for several minutes.
The existence of such long lasting microdomains of low Ca2⫹ could be demonstrated directly under physiological conditions by using a combined approach of single channel recordings and high resolution confocal microscopy (21).
These studies suggest that, despite the short lasting [Ca2⫹]mito increase, mitochondria can generate sustained mi- crodomains of low Ca2⫹in the subplasmalemmal region. How- ever, it is unclear whether mitochondria act as a Ca2⫹sink or as a Ca2⫹relay mechanism, or whether the increase in mito- chondrial Ca2⫹per seis an essential step in the activation of the CCE as suggested recently (22). Furthermore, inter-or- ganelle Ca2⫹cross-talk between the ER and the mitochondria
* This work was supported by the Austrian Science Funds P14586- PHA and SFB 714 (to W. F. G.) and the Swiss National Funds 31- 56902.99 (to N. D.). 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.
¶To whom correspondence should be addressed: Dept. Medical Bio- chemistry and Medical Molecular Biology, University of Graz, Harrach- gasse 21/III, A-8010 Graz, Austria. Tel.: 43-316-380-7560; Fax: 43-316- 380-9615; E-mail: wolfgang.graier@uni-graz.at.
1The abbreviations used are: ER, endoplasmic reticulum; BKCa, large
conductance Ca2⫹-activated K⫹channels; BHQ, 2,5-di-tert-butylhydro- quinone; [Ca2⫹]cyto, free cytosolic Ca2⫹; [Ca2⫹]pm, Ca2⫹concentration at the inner side of the patch membrane; CCE, capacitative Ca2⫹entry;
CGP 37157, 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin- 2(3H)-one; FCCP, carbonyl cyanide-4-trifluoromethoxyphenylhyrazone;
IP3, inositol 1,4,5-triphosphate; mtDsRed, mitochondrial-targeted DsRed;
Po, open state probability of single BKCachannels; RP-mt, mitochondrial- targeted ratiometric-pericam;Vapplied, applied holding potential;Vpm, ef- fective potential of the patch;Vwc, whole cell membrane potential; YC4- ER, ER-targeted yellow chameleon 4; GFP, green fluorescent protein;
SERCA, sarco/endoplasmic reticulum calcium ATPase.
This paper is available on line at http://www.jbc.org
44769
has also been described in both non-excitable and excitable cells (23–28), suggesting that different cellular Ca2⫹ pools might cooperatively control cellular Ca2⫹homeostasis.
Therefore, the goal of this work is to elucidate the impact of mitochondrial Ca2⫹ signaling on cellular Ca2⫹ homeostasis during cell stimulation in the human umbilical vein endothelial cell-derived cell line EA.hy926.
EXPERIMENTAL PROCEDURES
Materials—Cell culture chemicals were from Invitrogen, and fetal calf serum was obtained from PAA Laboratories (Linz, Austria). Fura- 2/AM and JC-1 were from Molecular Probes (Leiden, Netherlands).
CGP 37157 (7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiaz- epin-2(3H)-one) was purchased from Tocris Cookson Ltd. (Northpoint, Avonmouth, Bristol, UK). Oligomycin, antimycin A, 2,5-di-tert-butylhy- droquinone (BHQ), carbonyl cyanide-4-trifluoromethoxyphenylhyra- zone (FCCP), and histamine were from Sigma. Restriction enzymes and T4 DNA ligase were from New England Biolabs (Frankfurt, Germany), and the EndoFree Plasmid Maxi Kit was from Qiagen (Hilden, Germa- ny). All other chemicals were from Roth (Karlsruhe, Germany).
Cell Culture—The human umbilical vein endothelial cell line, EA.hy926 (29) passageⱖ45, was used for this study. Cells were cul- tured in Dulbecco’s minimum essential medium containing 10% fetal calf serum and 1% HAT (5 mMhypoxanthine, 20Maminopterin, 0.8 mMthymidine). For experiments cells were grown on glass coverslips.
Plasmids and Transfection—For double transfection YC4-ER (Cam4- ER) (30, 31) and mtDsRed were cloned into the two multiple cloning sites of the transfection vector pBudCE4.1 (Invitrogen). Alternatively, RP-mt (32), YC4-ER, and mtDsRed were inserted into pcDNA 3 for single transfection (Invitrogen). Cells (⬃80% confluency) were tran- siently transfected with 1.5–3g of purified plasmid DNA using Trans- FastTMTransfection Reagent (Promega, Mannheim, Germany).
Organelle Visualization—Organelle organization was visualized in cells transiently transfected with YC4-ER, mtDsRed, or RP-mt as de- scribed previously (33). Three-dimensional scans for visualization of the distribution of RP-mt was performed using a Zeiss Axiovert 200M (Zeiss Microsystems, Jena, Germany) equipped with VoxCell Scan® (Visi- Tech, Sunderland, UK) and controlled by Metamorph 5.0 (Universal Imaging, Visitron Systems, Puchheim, Germany). Image deconvolution was done using the quick maximum likelihood estimation with Huy- gens 2.4. (SVI, Hilversum, Netherlands), and image restoration was performed using Imaris 3.3. (Bitplane AG., Zu¨ rich, Switzerland).
Ca2⫹ Measurements—Cytosolic, ER, and mitochondrial free Ca2⫹ concentrations ([Ca2⫹]cyto, [Ca2⫹]ER, and [Ca2⫹]mito) were measured us- ing fura-2, YC4-ER, and RP-mt with high resolution imaging system as described previously (33, 34). To monitor [Ca2⫹]cyto, [Ca2⫹]ER, or [Ca2⫹]mito, cells were illuminated alternatively at 340⫾15 and 380⫾ 15 nm (fura-2, 340HT15 and 380HT15; Omega Optical, Brattleboro, VT), 440 nm (YC4-ER, 440AF21; Omega Optical) and 410/433 and 485 nm (RP-mt, 433DF15 and 485DF15; Omega Optical), respectively.
Emission was monitored at 510 nm (510WB40, Omega Optical) for fura-2, 480 and 535 nm for YC4-ER (480AF30, Omega Optical), or at 535 nm for RP-mt (535AF26, Omega Optical). For YC4-ER measure- ments an optical beam splitter (480 and 535 nm; Dual-View Micro- ImagerTM, Optical Insights, Visitron Systems) was used in order to allow simultaneous rationing with one given camera. The following buffers were used: Ca2⫹/Hepes-buffered solution containing (in mM) 145 NaCl, 5 KCl, 2 Ca2Cl, 1 MgCl2, and 10 Hepes acid; pH was adjusted to 7.4, and a nominal Ca2⫹-free solution containing (in mM) 145 NaCl, 5 KCl, 1 MgCl2, 1 EGTA and 10 Hepes acid, pH adjusted to 7.4.
Patch Clamp Recordings—The cell-attached configuration of the patch clamp technique was used as described previously (21, 33) using a pipette solution containing (in mM) 130 KCl, 1 MgCl2, 10 Hepes (pH 7.4 with KOH). Current recordings were performed with an EPC-7 amplifier (List Medical, Darmstadt, Germany) and were filtered at 1 kHz (900C9L8L, Frequency Devices, Haverhill, MA), and currents were sampled by a personal computer running with pClamp 8.0 (Axon In- struments, Union City, CA) at 5 kHz. Single currents were analyzed with Fetchan and pStat (Axon Instruments, Union City, CA). ThePo
was normalized in respect to the number of channels in the patch (N) as follows: normalizedPo⫽(to1⫹2to2⫹3to3⫹. . .⫹NtoN)/Nt, wheretoN
is the time spent by a channel at the open levelN. The BKCachannels were utilized to monitor subplasmalemmal Ca2⫹concentration as de- scribed previously (21, 33). In this work the physiological patch protocol was used that allowed free change of the cell membrane potential during stimulation. The bath solutions contained (in mM) 130 NaCl, 5.6
KCl, 1 MgCl2, 2 CaCl2, 8 Hepes (pH 7.45 with NaOH). As explained in detail previously (21), the Ca2⫹concentration at the mouth of the BKCa
channel ([Ca2⫹]pm) was calculated under physiological conditions (i.e.
no clamp of the cell membrane potential;Vpm) according to Equation 1,
log[Ca2⫹]pm⫽log EC50Ca⫺log冉PPo(max)o⫹P⫺o(min)Po冊 (Eq. 1)
where log EC50
Cais⫺5.566 (EC50
Cais Ca2⫹sensitivity of the BKCachan- nel),Po(max)is the maximalPo, andPo(min)is the minimalPomeasured in the respective experiments.
Measurement of Mitochondrial Membrane Potential—Mitochondrial membrane potential (m) was measured fluorometrically with JC-1 as described previously (34). Briefly, cells were loaded with 5MJC-1 for 10 min at room temperature in the dark, washed twice, and mounted in a customized superfusion chamber. JC-1 fluorescence was alternatively monitored at 485 (485DF15; Omega Optical) and 575 nm (575DF25) and 528 and 633 nm emission (528 – 633DBEN; Omega Optical).
Statistics—Analysis of variance and Scheffe’spost hoc Ftest were used for evaluation of the statistical significance.p⬍0.05 was deter- mined to be significant.
FIG. 1.Comparison of histamine-induced Ca2ⴙsignals in the cytosol ([Ca2ⴙ]cyt), the ER ([Ca2ⴙ]er), and the mitochondria ([Ca2ⴙ]mito) of endothelial cells. Representative tracings of Ca2⫹
signal elicited by histamine (100M) in the cytosol (A,n⫽89), the ER (B,n⫽47), and the mitochondria (C,n⫽53). Cultured endothelial cells were either loaded with fura-2/AM (A) or transiently transfected with YC4-ER (B) or RP-mt (C).
Trans-mitochondrial Ca Flux
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RESULTS
Measurements of Mitochondrial Ca2⫹Signals with the Tar- geted Ca2⫹Sensor RP-mt in Endothelial Cell—Mitochondrial Ca2⫹signaling in response to an IP3-generating agonist, such as histamine (100M), markedly differs from that obtained in the cytoplasm and the ER. In the cytosol and the ER, the Ca2⫹
concentrations rapidly change in response to cell stimulation and stay high (cytosol, [Ca2⫹]cyto) or low (ER, [Ca2⫹]ER) as long as the stimulus is present (Fig. 1,AandB). In contrast to these
long lasting changes in [Ca2⫹]cytoand [Ca2⫹]ER, mitochondrial Ca2⫹([Ca2⫹]mito) increases only transiently even if the agonist has not been washed out (Fig. 1C).
These experiments were conducted by transfection of the mitochondria-targeted Ca2⫹-sensory protein RP-mt (32) that revealed a distinct structure of the mitochondrial network in endothelial cells (Fig. 2A). Stimulation with 100Mhistamine transiently increased [Ca2⫹]mito to levels that were similar regardless of the presence or absence of extracellular Ca2⫹(Fig.
FIG. 2.Mitochondria distribution and measurement of mitochondrial Ca2ⴙ signals in endothelial cells. A, three-dimensional distribution of RP-mt visualized using a Nipkow laser disc scanning system (Visitron and VisiTech, Sunderland, UK).B,left panel, typical mitochondrial Ca2⫹signals elicited by 100Mhistamine recorded in cells transiently transfected with RP-mt in 2 mMCa2⫹containing (n⫽23) or Ca2⫹-free buffer (n⫽10).Right panel, statistical evaluation of the effect of extracellular Ca2⫹removal.n.s., not significant.C, removal of extracellular Ca2⫹during stimulation with 100Mhistamine decreased [Ca2⫹]mitoin a reversible manner (n⫽8). *,p⬍0.05versusin the presence of extracellular Ca2⫹.
2B). Notably, while in the presence of extracellular Ca2⫹ the [Ca2⫹]mitoreturned to basal level, and in the nominal absence of free extracellular Ca2⫹, [Ca2⫹]mitodecreased under the base line even in the presence of the agonist (Fig. 2B). Moreover, removal of extracellular Ca2⫹after the initial Ca2⫹transient caused a decrease in [Ca2⫹]mito, whereas the subsequent readdition of Ca2⫹raised [Ca2⫹]mitoback to basal levels (Fig. 2C). Because the removal of extracellular Ca2⫹ did not initiate such a drop in [Ca2⫹]mitoin resting cells (Fig. 2B), these data indicate that Ca2⫹
fluxes across the mitochondria increased during stimulation, ren- dering [Ca2⫹]mitostringently dependent on the presence of extra- cellular Ca2⫹. This suggests that, during cell stimulation, part of the Ca2⫹entering the cell transits through mitochondria.
Trans-mitochondrial Ca2⫹ Flux Occurs during Cell Stimu- lation—To test whether Ca2⫹ transits through mitochondria during stimulation, we used 20MCGP 37157, an inhibitor of the mitochondrial Na⫹/Ca2⫹exchanger (NCXmito). In the pres- ence of 20MCGP 37157, 100Mhistamine initiated a long lasting elevation of [Ca2⫹]mitothat did not return to base line upon agonist washout (Fig. 3A), indicating that Ca2⫹was re- tained in the mitochondria. In resting cells 20MCGP 37157 failed to increase [Ca2⫹]mito within 3 min (data not shown), indicating that Ca2⫹uptake into mitochondria was increased during agonist stimulation. More importantly, addition of 20
M CGP 37157 after the transient increase, at a time when [Ca2⫹]mitohad returned to basal levels, resulted in a slow but FIG. 3.Modulation of mitochondrial Ca2ⴙsignals by inhibition of NCXmwith CGP 37157 and reduction of extracellular Naⴙ concentration.A, in the presence of 20MCGP 37157 100Mhistamine induced long lasting elevation in [Ca2⫹]mito(control,n⫽13; CGP 37157, n⫽13). In these experiments RP-mt was excited at 480 and 410 nm using a monochromator (DeltaRam, Photon Technology International Inc., Monmouth Junction, NJ), and emission was collected at 535 nm (505DCXR dichroic mirror and 535RDF45 emission filter; Omega Optical). All experiments were carried out in the presence of 2 mMextracellular Ca2⫹.B, inhibition of NCXmitowith 20MGCP 37157 after a stimulation with 100Mhistamine yielded slow and long lasting elevation of [Ca2⫹]mito(control,n⫽6; CGP 37157,n⫽17).Left panelsillustrate representative tracings, andright panelsshow the statistical evaluation. *,p⬍0.05versusthe absence of CGP 37157.C, endothelial cells expressing RT-mt were stimulated with 100Mhistamine in normal (i.e.130 mM) and low (i.e.5 mM) extracellular Na⫹buffer (Na⫹was substituted by choline chloride) that contains 2 mMextracellular Ca2⫹. As indicated extracellular Na⫹concentration was increased to 130 mM. *,p⬍0.05versusin control buffer (control,n⫽8; low Na⫹,n⫽10).
Trans-mitochondrial Ca Flux
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long lasting rise in [Ca2⫹]mitothat reached a plateau after⬃3 min in the presence of extracellular Ca2⫹(Fig. 3B). In contrast, in the absence of extracellular Ca2⫹, CGP 37157 failed to elevate [Ca2⫹]mitowhen applied after the initial transient (data not shown). These data indicate that in the presence of extra- cellular Ca2⫹, Ca2⫹ uptake into mitochondria remained in- creased throughout the agonist application but was balanced by an increased efflux of Ca2⫹from mitochondria. The effects of CGP 37157 were not due to alteration in mitochondrial mem- brane potential (⌬m) as 20M CGP 37157 did not affect the fluorescence of the potentiometric indicator JC-1 (data not shown).
Trans-mitochondrial Ca2⫹Flux Critically Depends on Extra- cellular Na⫹—Because inhibition of the NCXmitoby CGP 37157
prevented Ca2⫹transfer across the mitochondria, the impor- tance of extracellular Na⫹ as a source for Na⫹ influx that facilitates NCXmito activity was tested. In low extracellular Na⫹conditions (i.e.5 mM), the decline of [Ca2⫹]mitoafter the initial increase in response to 100Mhistamine was slow and was accelerated by increasing the extracellular Na⫹concentra- tion to normal values (i.e.130 mM; Fig. 3C).
Inhibition of NCXmito Prevents the Generation of Subplas- malemmal Microdomains of Low Ca2⫹—The ability of mito- chondria to generate local microdomains of low Ca2⫹(i.e.mi- tochondrial Ca2⫹ buffering) was found to depend on mitochondrial Ca2⫹ uptake (22). To determine whether con- comitant Ca2⫹extrusion is also required, we investigated the effect of CGP 37157 on mitochondrial “Ca2⫹buffering” under FIG. 4. Inhibition of NCXmito pre-
vents mitochondrial buffering in physiological conditions. Recordings of single BKCa channels were used to monitor subplasmalemmal Ca2⫹concen- tration ([Ca2⫹]pm) in the vicinity of mito- chondria under physiological conditions where the cell membrane potential was not clamped (21). This technique utilizes the activity of Ca2⫹-activated BKCachan- nel (Po) to estimate the whole cell mem- brane potential (Vwc) and [Ca2⫹]pm.A, il- lustration of the pipette location in the proximity of mitochondria in cells double transfected to express DsRed in the mito- chondria (red) and YC4-ER in the ER (green). Analysis ofPo,Vwc, and [Ca2⫹]pm
in the vicinity of mitochondria in response stimulation with 100Mhistamine in the absence (B) and presence (C) of 20 M CGP 37157. The [Ca2⫹]pm estimated to occur during the time periods P1 and P2 and a detailed statistical evaluation are given in Table I.
physiological patch conditions where hyperpolarization-facili- tated Ca2⫹entry occurs (21). By using the physiological patch technique the cell is allowed to manipulate its membrane po- tential freely as the cell membrane potential is not clamped but a compensatory patch potential is applied that facilitates mon- itoring of single BKCachannels at around ⫹40 mV patch po- tential (for further details see Ref. 21). As a result of this approach, the local subplasmalemmal Ca2⫹concentration un- der the patch (patch Ca2⫹concentration; [Ca2⫹]pm) in the vi- cinity of mitochondria can be estimated by the activity of BKCa channels, whereas the cell hyperpolarization upon stimulation occurs that in turn provides the driving force to facilitate CCE.
Endothelial cells were transiently transfected with the transfection vector pBudCE4.1 (Invitrogen) encoding YC-4ER and mtDsRed, and the patch pipette was placed in the proxim- ity and within distance to the mitochondria (Fig. 4A). In control cells, 100 M histamine initiated a biphasic activation of plasma membrane BKCachannels (Fig. 4B,upper panel) that was accompanied by a long lasting cell hyperpolarization (Fig.
4B,middle panel). For further analysis, the initial transient phase (P1), which reflects Ca2⫹release and early CCE, and the long lasting plateau phase (P2), which reflects mainly influx at the plasma membrane, were separately evaluated (Table I).
Therefore, mitochondrial Ca2⫹buffering became visible by the very limited elevation of [Ca2⫹]pmof about 0.25 and 0.10Min P1 and P2, respectively (Fig. 4B,lower panel; Table I).
In the presence of 20MCGP 37157, histamine induced an
⬃7 times higher activation of BKCachannels than that in the absence of CGP 37157 (Fig. 4C,upper panel; Table I). The cell hyperpolarization was similar to that in the controls (Fig. 4C, middle panel). The lack of mitochondrial Ca2⫹ buffering was strikingly shown by the large elevations in [Ca2⫹]pmto 1.34 and 0.40Min P1 and P2, respectively (Fig. 4C,lower panel; Table I; Fig. 4D,left panel).
In contrast, if the pipette was located away from superficial mitochondria domains, 20MCGP 37157 failed to affect BKCa channel activation/[Ca2⫹]pm elevation in response to 100 M histamine compared with experiments carried out in the ab- sence of CGP 37157 (Table I). Moreover, CGP 37157 (20M) had no effect if Ca2⫹ influx through the CCE pathway was prevented by cell depolarization using the standard patch clamp technique (23) (data not shown). Finally, 20M CGP 37157 had no effect on Ca2⫹-triggered BKCachannel activation/
activity in inside-out patches (data not shown). Overall, these data indicate that CGP 37157 does not affect BKCaactivation/
activity but prevents mitochondrial Ca2⫹buffering by inhibi- tion of NCXmito.
Trans-mitochondrial Ca2⫹Flux Is Essential for Maintenance of CCE Activity—To elucidate the importance of trans-mito- chondrial Ca2⫹flux for CCE, we assessed the effects of uncou- pling agents such as carbonyl cyanide p-chlorophenylhydra- zone and FCCP (6, 12). In previous studies, these compounds
initiated depolarization of the mitochondria (35), induced Ca2⫹
release from the ER (36), reduced histamine-evoked ER deple- tion, and prevented any mitochondrial Ca2⫹signaling in endo- thelial cells (37). To test whether FCCP/oligomycin or oligomy- cin/antimycin A also affected CCE, we utilized the classical Ca2⫹readdition protocol for visualization of CCE (e.g.Refs. 18, 22, and 38 – 42), and we measured the elevation of cytosolic Ca2⫹in response to the re-addition of Ca2⫹to cells stimulated with 100Mhistamine. The peak Ca2⫹levels achieved after Ca2⫹readdition to histamine-induced Ca2⫹entry were dimin- ished by 63 and 74% in the presence of either 2MFCCP/2M oligomycin or 2Moligomycin/10Mantimycin A, respectively (Fig. 5,AandB). Although the Ca2⫹readdition protocol reflects the combined action of Ca2⫹entry, Ca2⫹-induced Ca2⫹release, and Ca2⫹ extrusion and re-uptake, the observed inhibition suggests that Ca2⫹entry is significantly reduced. Thus, these data indicate that under conditions where mitochondria are depolarized and the mitochondrial Ca2⫹ signal is abolished, histamine-stimulated CCE was largely attenuated.
To assess whether Ca2⫹extrusion from mitochondria is also of importance for modulating CCE, NCXmitowas inhibited by CGP 37157 to promote accumulation of Ca2⫹into mitochondria without affecting ⌬m. Although CGP 37157 (20 M) did not affect the magnitude of the Ca2⫹transient elicited by 100M histamine, which reflects Ca2⫹release, the subsequent eleva- tion in [Ca2⫹]cyto upon Ca2⫹ readdition was reduced by 81%
(Fig. 5C). Furthermore, CGP 37157 reversibly decreased the histamine-initiated long lasting plateau phase (Fig. 5D) with an IC50of 16.0M(6.4 – 40.1) (n⫽7–16; Fig. 5E). In line with these findings, Ca2⫹elevation upon Ca2⫹readdition to cells prestimulated with the SERCA inhibitor BHQ (15M) was also attenuated in the presence of 20MCGP 37157 by 75% (Fig.
5F). In contrast, Ca2⫹entry in cells prestimulated with 100 nM
ionomycin, which allowed Ca2⫹ efflux from the mitochondria even in the presence of 20MCGP 37157 (data not shown), was not affected by 20 M CGP 37157 (Fig. 5F). These findings indicate that both mitochondrial Ca2⫹influx and Ca2⫹efflux are required to sustain CCE.
Trans-mitochondrial Ca2⫹Flux Is Essential for ER Refilling during Agonist Stimulation—To assess the impact of mitochon- dria on ER Ca2⫹levels, we followed the depletion and refilling of the ER by transfecting cells with the GFP-based Ca2⫹indi- cator YC4-ER (30, 31) (Fig. 6A). In the presence of extracellular Ca2⫹, histamine initiated a significant reduction of the ER Ca2⫹content (Fig. 6B). Removal of extracellular Ca2⫹acceler- ated the rate of ER depletion, whereas Ca2⫹ readdition re- sulted in a partial refilling of the ER even in the presence of the agonist (Fig. 6B).
In order to investigate the impact of trans-mitochondrial Ca2⫹flux for Ca2⫹refilling of the ER, NCXmitowas prevented by CGP 37157, and the ER Ca2⫹content was monitored. As shown in Fig. 6C, the Ca2⫹depletion of the ER upon histamine TABLE I
Inhibition of trans-mitochondrial Ca2⫹flux by CGP 37157 weakened mitochondrial Ca2⫹buffering
No. exp. No.
channels
P1 P2
NormalizedPo VWC [Ca2⫹]pm NormalizedPo VWC [Ca2⫹]pm
mV M mV M
Close to mitochondria
8 2.3⫾0.6 0.073⫾0.024 ⫺59.2⫾6.0 0.25⫾0.07 0.038⫾0.017 ⫺57.2⫾6.1 0.10⫾0.03 Close to mitochondria⫹CGP 37157
4 2.0⫾1.0 0.419⫾0.092a ⫺53.9⫾4.8 1.34⫾0.58a 0.184⫾0.050a ⫺52.5⫾5.0 0.40⫾0.18a Far from mitochondria
14 3.2⫾0.5 0.444⫾0.044a ⫺58.6⫾1.7 4.51⫾1.07a 0.280⫾0.046a ⫺57.1⫾1.9 1.21⫾0.20a Far from mitochondria⫹CGP 37157
6 2.8⫾1.0 0.420⫾0.108a ⫺59.9⫾0.9 2.08⫾0.87a 0.308⫾0.103a ⫺50.7⫾0.7 0.48⫾0.25a
ap⬍0.05versusnext to mitochondria.
Trans-mitochondrial Ca Flux
44774
in Ca2⫹-containing buffer was more pronounced in the pres- ence of 20MCGP 37157. Furthermore, the Ca2⫹refilling of the ER by readdition of extracellular Ca2⫹was reversibly pre- vented by 20MCGP 37157 (Fig. 6,CandD). Similar to CGP 37157, the combination of 2 M oligomycin and 10 M anti- mycin A augmented histamine-induced Ca2⫹depletion of the ER in Ca2⫹-containing solution and prevented Ca2⫹refilling of the ER upon Ca2⫹ readdition in the presence of histamine (Fig. 6D).
Inhibition of SERCA Decreases Mitochondrial Ca2⫹Extru- sion—Similar to the removal of extracellular Ca2⫹, SERCA
inhibition with BHQ in cells stimulated with histamine further decreased ER Ca2⫹content in a reversible manner (Fig. 7A).
Remarkably, an inhibition of SERCA by BHQ (15M), after the cells have been stimulated with 100 M histamine and [Ca2⫹]mitoalready returned back to basal level, resulted in an instant rise in [Ca2⫹]mito, indicating that the lack of ER Ca2⫹ sequestration reduces mitochondrial Ca2⫹efflux (Fig. 7B). In the absence of extracellular Ca2⫹, BHQ failed to elevate [Ca2⫹]mito, thus suggesting that the net increase of the passive Ca2⫹ leak out from the ER in the presence of BHQ was not responsible for the maintained increase of [Ca2⫹]mito(Fig. 7B).
FIG. 5.Inhibition of trans-mitochondrial Ca2ⴙflux prevents capacitative Ca2ⴙentry.CCE was assessed as the increase in [Ca2⫹]cytoin response to the addition of 2 mMextracellular Ca2⫹to cells stimulated with 100Mhistamine in nominal Ca2⫹free buffer. Histamine-initiated Ca2⫹entry was diminished in the presence of either 2MFCCP plus 2Moligomycin (to prevent ATP depletion under FCCP) (A,Control,n⫽13;
FCCP,n⫽13;p⬍0.05versuscontrol), 2Moligomycin plus 10Mantimycin A (B,Control,n⫽16; oligomycin/antimycin A,n⫽17;p⬍0.05 versuscontrol), or 20MCGP 37157 (C, Control,n⫽36; CGP 37157,n⫽37;p⬍0,05versuscontrol).Pointsrepresent the mean⫾S.E.D, if cells were stimulated with 100Mhistamine in the presence of 2 mMextracellular Ca2⫹, 20MCGP 37157 reduced the long lasting plateau in a reversible manner (n⫽43).E, concentration-response curve of CGP 37157 on histamine-evoked CCE (n⫽7–16) measured using the same protocol as shown inD.F, comparison on the effect of CGP 37157 on CCE evoked by 100Mhistamine (Control,n⫽36; CGP 37157,n⫽37;p⬍0.05versus control), 15MBHQ (Control,n⫽48; CGP 37157,n⫽41;p⬍0.05versuscontrol), and 100 nMionomycin (Control,n⫽30; CGP 37157,n⫽45;
p⬍0.05versuscontrol).
In line with these findings, an inhibition of SERCA simulta- neously to stimulation with histamine yielded long lasting el- evation of [Ca2⫹]mitoin the presence (Fig. 7C) but not absence (Fig. 7D) of extracellular Ca2⫹.
DISCUSSION
Our data describe that mitochondrial Ca2⫹uptake and re- lease (i.e.trans-mitochondrial Ca2⫹flux) represent crucial phe- nomena for (sub-)cellular Ca2⫹ handling during cell stimula- tion. By using high resolution fluorescence microscopy of organelle-targeted Ca2⫹probes and electrophysiology for spa- tially defined single channel recordings, we show that this trans-mitochondrial Ca2⫹ flux is essential for the ability of mitochondria to buffer Ca2⫹ locally, to facilitate Ca2⫹ entry through the CCE pathway, and to promote ER refilling.
Expression of RP-mt, a circularly permutated GFP (32), re- vealed an impressive mitochondrial network in endothelial cells and a transient elevation in [Ca2⫹]mitoto histamine. Such kinetics of [Ca2⫹]mitowas reported in permeabilized endothe- lial cells (37) and in other cell types by various techniques (e.g.
mitochondria-targeted aequorin and chameleon) (9, 23). Thus, mitochondrial Ca2⫹ signal markedly differs from that in the cytosol and the ER where sustained cytosolic Ca2⫹elevation mirrors long lasting ER Ca2⫹ reduction. Notably, like most GFP mutants RP-mt is pH-sensitive especially at excitation wavelength 480 nm, while virtually pH-insensitive at excita- tion wavelengths 410/430 nm.2By utilizing these properties of RP-mt, we could ensure that during all experiments (except
that with FCCP), no large alterations in mitochondrial pH took place (data not shown) and changes mainly reflect alterations in [Ca2⫹]mito.
Because the initial mitochondrial Ca2⫹transient remained unchanged even in the absence of extracellular Ca2⫹, it is tempting to speculate that this Ca2⫹spike is predominantly due to uptake of Ca2⫹released from the ER. Similar data have been described recently in HeLa cells (43). Our findings that histamine-initiated elevation in [Ca2⫹]mito turned out to be long lasting when NCXmitowas inhibited with CGP 37157 (44) is in line with reports demonstrating that NCXmitois the main mechanism of mitochondrial Ca2⫹ extrusion in endothelial cells (37).
Our data suggest that a decrease in extracellular Na⫹ de- layed the decline in [Ca2⫹]mitoafter initial elevation (Fig. 3C) and that Na⫹influx contributes to mitochondrial Ca2⫹home- ostasis. Moreover, despite the obvious independence of the initial Ca2⫹transient from extracellular Ca2⫹, Ca2⫹entry may contribute to long lasting mitochondrial Ca2⫹ homeostasis.
This was indicated by our findings that in the presence of extracellular Ca2⫹, [Ca2⫹]mitoreturned to basal levels after the initial transient, whereas in the absence of extracellular Ca2⫹, [Ca2⫹]mitoreturned to levels lower than base line. Hence, re- moval of extracellular Ca2⫹after the initial Ca2⫹spike revers- ibly decreased [Ca2⫹]mitobelow basal levels. Because neither EGTA nor CGP 37157 had similar effects on [Ca2⫹]mito in resting cells, these data support the concept that even if [Ca2⫹]mitoremains at base-line levels, mitochondrial Ca2⫹se- questration and efflux continuously occur and are in a long term equilibrium during cell stimulation. This suggestion is
2R. Malli, M. Frieden, K. Osibow, C. Zoratti, M. Mayer, N. Demau- rex, and W. F. Graier, unpublished results.
FIG. 6.ER refilling during agonist stimulation requires sustained trans-mitochondrial Ca2ⴙ fluxes. A, typical staining of an endothelial cell transiently transfected with YC4-ER.B, the importance of extracellular Ca2⫹for ER refilling during agonist stimulation was demonstrated by removal of extracellular Ca2⫹.C, the Ca2⫹content of the ER was monitored in the presence of the NCXmitoinhibitor CGP 37157.
In the presence of 2 mMextracellular Ca2⫹, the ER was depleted with 100Mhistamine. To achieve a pronounced ER depletion, extracellular Ca2⫹
was removed as indicated. ER refilling by the readdition of Ca2⫹-containing buffer (2 mM) in the presence of histamine was prevented by CGP 37157 (20M) in a reversible manner. Tracings are representative curves.D, statistical evaluation of the ER depletion and ER refilling in the presence of 100Mhistamine under control (n⫽19) and in the presence of 20MCGP 37157 (n⫽6) or 2Moligomycin and 10Mantimycin A (n⫽7).
Data are expressed in relation to the basal ER Ca2⫹content (⫽100%) and after addition of EGTA in the presence of 100Mhistamine (⫽0%).
*,p⬍0.05versuscontrol.
Trans-mitochondrial Ca Flux
44776
further supported by our findings that addition of CGP 37157 after the initial Ca2⫹ transient resulted in a slow but long lasting rise in [Ca2⫹]mitoonly in the presence of extracellular Ca2⫹.
In line with a report regarding a calf pulmonary artery endothelial cell line (37), depolarization of mitochondria with oligomycin/antimycin A prevented mitochondrial Ca2⫹ eleva- tion and thus trans-mitochondrial Ca2⫹ flux in response to histamine (data not shown). Thus, mitochondrial Ca2⫹signal- ing during stimulation is a biphasic phenomenon where a tran- sient and rapid [Ca2⫹]mito increase triggered by intracellular Ca2⫹release is accompanied by a continuous trans-mitochon- drial Ca2⫹flux that originates from the extracellular space.
Our data on trans-mitochondrial flux of Ca2⫹ entering the cells during stimulation are in agreement with the recent hy- pothesis that mitochondrial Ca2⫹uptake facilitates CCE activ- ity (6, 12–16) and complement our own data on Ca2⫹buffering by superficial mitochondria (21). In this work, the physiological
patch protocol to estimate subplasmalemmal Ca2⫹concentra- tion by monitoring Ca2⫹-activated BKCachannels under con- ditions where cell membrane potential is not clamped was used to demonstrate that during stimulation only a very limited subplasmalemmal Ca2⫹ elevation occurred in the vicinity of superficial mitochondria (⬃0.25M), whereas close to the ER and far from any organelle subplasmalemmal Ca2⫹exceeded 3.5M. Considering the importance of this mitochondrial Ca2⫹
buffering that required Ca2⫹uptake (21), the contribution of Ca2⫹efflux on the ability of mitochondria to buffer Ca2⫹was assessed similarly. Our findings that in the presence of CGP 37157 a large subplasmalemmal Ca2⫹elevation occurred close to superficial mitochondria domains upon addition of hista- mine suggest that inhibition of the NCXmitowith CGP 37157 prevented the ability of mitochondria to buffer Ca2⫹and dem- onstrate that not only Ca2⫹uptake but also trans-mitochon- drial Ca2⫹flux is essential for mitochondrial Ca2⫹buffering.
Mitochondrial Ca2⫹buffering was described to be crucial for FIG. 7.Inhibition of SERCA by BHQ affects mitochondrial Ca2ⴙsignaling upon stimulation with histamine.A, the contribution of SERCA for ER refilling during agonist stimulation was demonstrated by the inhibition of SERCA with 15MBHQ.B, the impact of SERCA for mitochondrial Ca2⫹homeostasis was tested by the addition of 15MBHQ to RP-mt-expressing endothelial cells that were already stimulated with 100Mhistamine in the presence and absence of 2 mMextracellular Ca2⫹(n⫽9). Mitochondrial Ca2⫹concentration in the presence (C) or absence (D) of 2 mMextracellular Ca2⫹in RP-mt-expressing cells stimulated simultaneously with 15MBHQ and 100Mhistamine (n⫽11). *,p⬍0.05 versuscontrol.
CCE activity (6, 12–16). In these studies, mitochondrial Ca2⫹
signaling was prevented either with uncouplers (6, 12–16) or with ruthenium red, an inhibitor of the uniporter (22), and CCE was monitored in whole cell recordings under high cyto- solic EGTA concentration (0.1–10 mM) and high extracellular Ca2⫹ (5–10 mM) or by following [Ca2⫹]cyto upon addition of extracellular Ca2⫹ to prestimulated cells. In agreement with these reports, FCCP diminished histamine-induced CCE in endothelial cells. This effect of FCCP might be due to mitochon- dria depolarization that prevents mitochondrial Ca2⫹ uptake and thus Ca2⫹ buffering, which results in high subplasmale- mmal Ca2⫹concentration that in turn inhibits CCE channels.
However, due to the reported effects of FCCP on nonmitochon- drial Ca2⫹pools, for example (36), data obtained with FCCP are ambiguous. Recent data (22) and our own findings with oligomycin/antimycin A, which did not evoke Ca2⫹release but prevented mitochondrial Ca2⫹ signaling, further support the concept of mitochondrial Ca2⫹buffering to be essential for CCE activity. However, both FCCP and oligomycin/antimycin A de- polarize mitochondria by interfering with the respiratory chain or ATP production, thus their action on mitochondrial Ca2⫹
signals is indirect.
In this study, mitochondrial Ca2⫹ signaling was directly manipulated by CGP 37157 that had no effect on ⌬m but efficiently prevented mitochondrial Ca2⫹extrusion and dimin- ished CCE activity. The IC50value of CGP 37157 for CCE was in the range of its effect on NCXmito (44). The observation that the time course of CCE inhibition by CGP 37157 corre- lates with its effect on mitochondrial Ca2⫹accumulation and the attenuation of its Ca2⫹buffering by CGP 37157 suggest that trans-mitochondrial Ca2⫹flux is vital for maintenance of CCE. This assumption was further supported by our data that CGP 37157 prevented CCE stimulated by BHQ but not by 100 nM ionomycin, which activates CCE (45) and allows free Ca2⫹ movements through all organelles even in the presence of CGP 37157. Thus, the inhibitory effect of CGP 37157 on CCE is not due to a direct inhibition of the CCE pathway.
To estimate the contribution of trans-mitochondrial Ca2⫹
flux for ER Ca2⫹ refilling, ER refilling was measured using ER-targeted chameleon (YC4-ER; see Refs. 30 and 31). As shown previously (23, 46), ER depletion induced by histamine was augmented by BHQ, indicating that, even under hista- mine, ER is continuously refilled by SERCA. Furthermore, ER refilling during histamine stimulation was reversibly pre- vented by removal of extracellular Ca2⫹, suggesting that ER Ca2⫹refilling essentially depends on Ca2⫹entry. This assump- tion is in line with the model of CCE activation in which Ca2⫹
entry finally terminates CCE by reloading ER after agonist washout (18, 39). Inhibition of mitochondrial Ca2⫹ efflux by CGP 37157 or prevention of mitochondrial Ca2⫹flux by oligo- mycin/antimycin A augmented histamine-evoked ER depletion in the presence of extracellular Ca2⫹and abolished ER refilling upon readdition of extracellular Ca2⫹. These data demonstrate that ER refilling is sensitive to disturbances in trans-mitochon- drial Ca2⫹signaling as this phenomenon ensures Ca2⫹entry.
Considering the close proximity of these organelles (Fig. 4A) (47) and the reported intra-organelle Ca2⫹cross-talk between ER and mitochondria in other cell types (23–26), trans-mito- chondrial Ca2⫹flux may also direct Ca2⫹influx toward the ER for its refilling during stimulation. This hypothesis was further supported by our findings that in the presence but not in the absence of extracellular Ca2⫹inhibition of SERCA yielded long lasting elevation of [Ca2⫹]mito (Fig. 7, B and C). Because in histamine-stimulated cells, BHQ failed to potentiate CCE (data not shown), these data point to a contribution of SERCA to
mitochondrial Ca2⫹ signaling, as SERCA may generate local negative Ca2⫹gradients (i.e.lower than [Ca2⫹]cyto) in the gap between mitochondria and ER that accelerates Ca2⫹ efflux from mitochondria and thus directs mitochondrial Ca2⫹efflux toward SERCA for ER refilling.
In conclusion, we show that Ca2⫹continuously transits mi- tochondria during cell stimulation. This trans-mitochondrial Ca2⫹flux is essential for the ability of mitochondria to generate subplasmalemmal microdomains of low Ca2⫹in order to main- tain CCE activity and to sustain the transfer of Ca2⫹from the extracellular space to the ER.
Acknowledgments—We thank Beatrix Petschar and Anna Schreilechner for their excellent technical assistance; Prof. R. Y. Tsien and Dr. A. Miyawaki for providing the chameleon and RP-mt con- structs; and Dr. C. J. S. Edgell for the EA.hy926 cells. The Department of Medical Biochemistry and Medical Molecular Biology is a member of the Institutes of Basic Medical Sciences at the University of Graz and was supported by the infrastructure programs UGP4 and UGP6 of the Austrian Ministry of Education, Science, and Culture.
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