The Role of Mitochondria for Ca2+ Refilling of the Endoplasmic Reticulum
2005; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês
10.1074/jbc.m409353200
ISSN1083-351X
AutoresRoland Malli, Maud Frieden, Michael Trenker, Wolfgang F. Graier,
Tópico(s)Ion Channels and Receptors
ResumoEndoplasmic 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. 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. For a large number of receptors, the binding of the respective agonist initiates the generation of inositol 1,4,5-trisposphate (IP3), 1The 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; ψmito, 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. 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.Berridge M.J. J. Physiol. (Lond.). 1997; 499: 291-306Crossref Scopus (916) Google Scholar and 2.Berridge M.J. Bootman M.D. Roderick H.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 517-529Crossref PubMed Scopus (4213) Google Scholar). 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.Putney Jr., J.W. Cell Calcium. 1990; 11: 611-624Crossref PubMed Scopus (1261) Google Scholar), which is activated by the emptying of intracellular Ca2+ stores, accounts for Ca2+ transit through the plasma membrane (4.Putney Jr., J.W. Nature. 2001; 410: 648-649Crossref PubMed Scopus (32) Google Scholar, 5.Groschner K. Graier W.F. Kukovetz W.R. Circ. Res. 1994; 75: 304-314Crossref PubMed Google Scholar, 6.Nilius B. Viana F. Droogmans G. Annu. Rev. Physiol. 1997; 59: 145-170Crossref PubMed Scopus (259) Google Scholar). 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(2004) Science's STKE http://stke.sciencemag.org/cgi/content/full/sigtrans;2004/36/pe1Google Scholar, 14.Nilius B. Droogmans G. Wondergem R. Endothelium. 2003; 10: 5-15Crossref PubMed Scopus (158) Google Scholar), recent investigations point to a fundamental role of mitochondria in the maintenance (15.Hoth M. Button D.C. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10607-10612Crossref PubMed Scopus (235) Google Scholar, 16.Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (205) Google Scholar, 17.Gilabert J.A. Bakowski D. Parekh A.B. EMBO J. 2001; 20: 2672-2679Crossref PubMed Scopus (104) Google Scholar, 18.Hofer A.M. Fasolato C. Pozzan T. J. Cell Biol. 1998; 140: 325-334Crossref PubMed Scopus (202) Google Scholar, 19.Malli R. Frieden M. Osibow K. Zoratti C. Mayer M. Demaurex N. Graier W.F. J. Biol. Chem. 2003; 278: 44769-44779Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) and modulation (20.Parekh A.B. News Physiol. Sci. 2003; 18: 252-256PubMed Google Scholar) 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.Hoth M. Button D.C. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10607-10612Crossref PubMed Scopus (235) Google Scholar, 19.Malli R. Frieden M. Osibow K. Zoratti C. Mayer M. Demaurex N. Graier W.F. J. Biol. Chem. 2003; 278: 44769-44779Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 21.Hoth M. Fanger C.M. Lewis R.S. J. Cell Biol. 1997; 137: 633-648Crossref PubMed Scopus (464) Google Scholar). Recently, we demonstrated that superficial mitochondria are able to efficiently buffer the subplasmalemmal Ca2+ concentration ([Ca2+]pm) beneath the plasma membrane (22.Malli R. Frieden M. Osibow K. Graier W.F. J. Biol. Chem. 2003; 278: 10807-10815Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) 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.Malli R. Frieden M. Osibow K. Graier W.F. J. Biol. Chem. 2003; 278: 10807-10815Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 23.Frieden M. Graier W.F. J. Physiol. (Lond.). 2000; 524: 715-724Crossref Scopus (30) Google Scholar, 24.Frieden M. Malli R. Samardzija M. Demaurex N. Graier W.F. J. Physiol. (Lond.). 2002; 540: 73-84Crossref Scopus (36) Google Scholar). 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.Malli R. Frieden M. Osibow K. Zoratti C. Mayer M. Demaurex N. Graier W.F. J. Biol. Chem. 2003; 278: 44769-44779Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). 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.Edgell C.J. McDonald C.C. Graham J.B. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3734-3737Crossref PubMed Scopus (1360) Google Scholar). 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.Miyawaki A. Llopis J. Heim R. McCaffery J.M. Adams J.A. Ikura M. Tsien R.Y. Nature. 1997; 388: 882-887Crossref PubMed Scopus (2620) Google Scholar, 27.Miyawaki A. Griesbeck O. Heim R. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2135-2140Crossref PubMed Scopus (727) Google Scholar), vYC4-ER (28.Zoratti C. Kipmen-Korgun D. Osibow K. Malli R. Graier W.F. Br. J. Pharmacol. 2003; 140: 1351-1362Crossref PubMed Scopus (102) Google Scholar), and RP-mt (29.Nagai T. Sawano A. Park E.S. Miyawaki A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3197-3202Crossref PubMed Scopus (807) Google Scholar) in pcDNA 3 (Invitrogen) was used. Cells (∼80% confluency) were transiently transfected with 1.5–3 μg of purified plasmid DNA using TransFast™ 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.Frieden M. Malli R. Samardzija M. Demaurex N. Graier W.F. J. Physiol. (Lond.). 2002; 540: 73-84Crossref Scopus (36) Google Scholar). 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 ×40 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.Frieden M. Malli R. Samardzija M. Demaurex N. Graier W.F. J. Physiol. (Lond.). 2002; 540: 73-84Crossref Scopus (36) Google Scholar). 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.Malli R. Frieden M. Osibow K. Zoratti C. Mayer M. Demaurex N. Graier W.F. J. Biol. Chem. 2003; 278: 44769-44779Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 28.Zoratti C. Kipmen-Korgun D. Osibow K. Malli R. Graier W.F. Br. J. Pharmacol. 2003; 140: 1351-1362Crossref PubMed Scopus (102) Google Scholar). 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.Frieden M. James D. Castelbou C. Danckaert A. Martinou J.C. Demaurex N. J. Biol. Chem. 2004; 279: 22704-22714Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) when excited at 480 nm. Therefore, in contrast to our previous work (19.Malli R. Frieden M. Osibow K. Zoratti C. Mayer M. Demaurex N. Graier W.F. J. Biol. Chem. 2003; 278: 44769-44779Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), 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.Frieden M. James D. Castelbou C. Danckaert A. Martinou J.C. Demaurex N. J. Biol. Chem. 2004; 279: 22704-22714Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). [Ca2+]er Measurements—The improved version of the original YC4-ER (26.Miyawaki A. Llopis J. Heim R. McCaffery J.M. Adams J.A. Ikura M. Tsien R.Y. Nature. 1997; 388: 882-887Crossref PubMed Scopus (2620) Google Scholar, 27.Miyawaki A. Griesbeck O. Heim R. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2135-2140Crossref PubMed Scopus (727) Google Scholar), vYC4-ER (28.Zoratti C. Kipmen-Korgun D. Osibow K. Malli R. Graier W.F. Br. J. Pharmacol. 2003; 140: 1351-1362Crossref PubMed Scopus (102) Google Scholar), 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 MicroImager™, Optical Insights, Visitron Systems) as described previously (19.Malli R. Frieden M. Osibow K. Zoratti C. Mayer M. Demaurex N. Graier W.F. J. Biol. Chem. 2003; 278: 44769-44779Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 24.Frieden M. Malli R. Samardzija M. Demaurex N. Graier W.F. J. Physiol. (Lond.). 2002; 540: 73-84Crossref Scopus (36) Google Scholar). 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. ER Ca2+ Refilling Is Only Moderately Affected by IP3—As published recently (19.Malli R. Frieden M. Osibow K. Zoratti C. Mayer M. Demaurex N. Graier W.F. J. Biol. Chem. 2003; 278: 44769-44779Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), 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.Hoth M. Button D.C. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10607-10612Crossref PubMed Scopus (235) Google Scholar, 16.Gilabert J.A. Parekh A.B. EMBO J. 2000; 19: 6401-6407Crossref PubMed Scopus (205) Google Scholar, 17.Gilabert J.A. Bakowski D. Parekh A.B. EMBO J. 2001; 20: 2672-2679Crossref PubMed Scopus (104) Google Scholar, 18.Hofer A.M. Fasolato C. Pozzan T. J. Cell Biol. 1998; 140: 325-334Crossref PubMed Scopus (202) Google Scholar, 19.Malli R. Frieden M. Osibow K. Zoratti C. Mayer M. Demaurex N. Graier W.F. J. Biol. Chem. 2003; 278: 44769-44779Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), the question on the impact of mitochondrial function for the ER Ca2+ refilling was assessed. 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). 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.Cox D.A. Conforti L. Sperelakis N. Matlib M.A. J. Cardiovasc. Pharmacol. 1993; 21: 595-599Crossref PubMed Scopus (166) Google Scholar, 32.Cox D.A. Matlib M.A. J. Biol. Chem. 1993; 268: 938-947Abstract Full Text PDF PubMed Google Scholar), 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.Malli R. Frieden M. Osibow K. Zoratti C. Mayer M. Demaurex N. Graier W.F. J. Biol. Chem. 2003; 278: 44769-44779Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) 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. 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). 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). 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
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