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00925njm 22002777a 4500 dc Vay, Laura author Hernández San Miguel, Esther author Domínguez Lobatón, María Carmen author Moreno Díaz-Calderón, Alfredo author Montero Zoccola, María Teresa author Álvarez Martín, Javier author 2009 Mitochondrial Ca2+ 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 [Ca2+] ([Ca2+]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 [Ca2+]M from increasing above 2–3 M. Instead, using low-Ca2+-affinity aequorin probes, we have measured [Ca2+]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 [Ca2+]M to levels of 0.5–1mM was actually observed at any phosphate concentration (0–10mM) during continuous perfusion of 3.5–100 MCa2+-buffers. In spite of this high and maintained (>10 min) [Ca2+]M, mitochondria retained functionality and the [Ca2+]M drop induced by a protonophore was fully reversible. In addition, this high [Ca2+]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 [Ca2+]M. In conclusion [Ca2+]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. Cell Calcium, 2009, vol. 45, p. 243-250 0143-4160 http://uvadoc.uva.es/handle/10324/5985 10.1016/j.ceca.2008.10.007 243 250 Cell Calcium 45 Calcio - Metabolismo Mitochondrial free [Ca2+] levels and the permeability transition