title: Dynamics of mitochondrial [Ca(2+)] measured with the low-Ca(2+)-affinity dye rhod-5N.
abstract: Available methods to measure mitochondrial [Ca(2+)] ([Ca(2+)](M)) include both targeted proteins and fluorescent dyes. Targeted proteins usually report much higher [Ca(2+)](M) values than fluorescent dyes, up to two orders of magnitude. However, we show here that the low-Ca(2+)-affinity dye rhod-5N provides [Ca(2+)](M) values similar to those reported by targeted aequorin, suggesting that the discrepancies are mainly due to the higher Ca(2+)-affinity of the fluorescent dyes used. We find rhod-5N has an apparent in situ intramitochondrial Kd around 0.5mM. Addition of Ca(2+) buffers containing between 4.5 and 10μM [Ca(2+)] to permeabilized cells loaded with rhod-5N induced increases in calibrated [Ca(2+)](M) up to the 100μM-1mM range, which were dependent on mitochondrial membrane potential. Ca(2+) release from mitochondria was largely dependent on [Na(+)]. We have then used rhod-5N loaded cells to investigate the [Ca(2+)](M) response to agonist stimulation at the single-cell and subcellular level. The [Ca(2+)](M) peaks induced by histamine varied by nearly 10-fold among different cells, with a mean about 25μM. In the presence of the Ca(2+) uniporter stimulator kaempferol, the [Ca(2+)](M) peaks induced by histamine were also highly variable, and the mean [Ca(2+)](M) peak was 3-fold higher. Simultaneous measurement of cytosolic and mitochondrial [Ca(2+)] peaks showed little correlation among the heights of the peaks in both compartments. Studying the [Ca(2+)](M) peaks at the subcellular level, we found significant heterogeneities among regions in the same cell. In particular, the [Ca(2+)](M) increase in mitochondrial regions close to the nucleus was more than double that of mitochondrial regions far from the nucleus.
authors: Instituto de Biología y Genética Molecular (IBGM), Departamento de Bioquímica y Biología Molecular y Fisiología, Universidad de Valladolid, Valladolid, Spain.
authors: Sergio
authors: S
authors: de la Fuente
authors: Rosalba I
authors: RI
authors: Fonteriz
authors: Mayte
authors: M
authors: Montero
authors: Javier
authors: J
authors: Alvarez
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002138:Calibration; D002463:Cell Membrane Permeability / Q000187:drug effects; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D006367:HeLa Cells; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D044949:Kaempferols / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D012235:Rhodamines / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D044949:Kaempferols; C574804:Rhod-5N; D012235:Rhodamines; C006552:kaempferol; D006632:Histamine; D002118:Calcium