title: Mitochondrial free [Ca2+] levels and the permeability transition.
abstract: Mitochondrial Ca(2+) activates many processes, from mitochondrial metabolism to opening of the permeability transition pore (PTP) and apoptosis. However, there is considerable controversy regarding the free mitochondrial [Ca(2+)] ([Ca(2+)](M)) levels that can be attained during cell activation or even in mitochondrial preparations. Studies using fluorescent dyes (rhod-2 or similar), have reported that phosphate precipitation precludes [Ca(2+)](M) from increasing above 2-3 microM. Instead, using low-Ca(2+)-affinity aequorin probes, we have measured [Ca(2+)](M) values more than two orders of magnitude higher. We confirm here these values by making a direct in situ calibration of mitochondrial aequorin, and we show that a prolonged increase in [Ca(2+)](M) to levels of 0.5-1mM was actually observed at any phosphate concentration (0-10mM) during continuous perfusion of 3.5-100 microM Ca(2+)-buffers. In spite of this high and maintained (>10 min) [Ca(2+)](M), mitochondria retained functionality and the [Ca(2+)](M) drop induced by a protonophore was fully reversible. In addition, this high [Ca(2+)](M) did not induce PTP opening unless additional activators (phenyl arsine oxide, PAO) were present. PAO induced a rapid, concentration-dependent and irreversible drop in [Ca(2+)](M). In conclusion [Ca(2+)](M) levels of 0.5-1mM can be reached and maintained for prolonged periods (>10 min) in phosphate-containing medium, and massive opening of PTP requires additional pore activators.
authors.affiliation: Instituto de Biología y Genética Molecular (IBGM), Departamento de Bioquímica y Biología Molecular y Fisiología, Universidad de Valladolid and Consejo Superior de Investigaciones Científicas (CSIC), Ramón y Cajal, 7, E-47005 Valladolid, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Vay
authors.forename: Esther
authors.initials: E
authors.lastname: Hernández-SanMiguel
authors.forename: Carmen D
authors.initials: CD
authors.lastname: Lobatón
authors.forename: Alfredo
authors.initials: A
authors.lastname: Moreno
authors.forename: Mayte
authors.initials: M
authors.lastname: Montero
authors.forename: Javier
authors.initials: J
authors.lastname: Alvarez
mesh_terms: D000331:Aequorin / Q000378:metabolism; D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D002021:Buffers; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D010477:Perfusion; D010539:Permeability / Q000187:drug effects; D010710:Phosphates / Q000494:pharmacology
chemical_list: D001152:Arsenicals; D002021:Buffers; D050505:Mutant Proteins; D010710:Phosphates; C029341:oxophenylarsine; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000331:Aequorin; D002118:Calcium