RT info:eu-repo/semantics/article
T1 Dynamics of mitochondrial [Ca2+] measured with the low-Ca2+-affinity dye rhod-5N
A1 Fuente Pérez, Sergio De La
A1 Fonteriz García, Rosalba Inés
A1 Montero Zoccola, María Teresa
A1 Álvarez Martín, Javier
K1 Mitocondria
AB Available methods to measure mitochondrial [Ca2+] ([Ca2+]M) include both targeted proteins and fluorescentdyes. Targeted proteins usually report much higher [Ca2+]M values than fluorescent dyes, up to twoorders of magnitude. However, we show here that the low-Ca2+-affinity dye rhod-5N provides [Ca2+]Mvalues similar to those reported by targeted aequorin, suggesting that the discrepancies are mainly dueto the higher Ca2+-affinity of the fluorescent dyes used. We find rhod-5N has an apparent in situ intramitochondrialKd around 0.5 mM. Addition of Ca2+ buffers containing between 4.5 and 10  M [Ca2+] topermeabilized cells loaded with rhod-5N induced increases in calibrated [Ca2+]M up to the 100  M–1 mMrange, which were dependent on mitochondrial membrane potential. Ca2+ release from mitochondria waslargely dependent on [Na+]. We have then used rhod-5N loaded cells to investigate the [Ca2+]M responseto agonist stimulation at the single-cell and subcellular level.The [Ca2+]M peaks induced by histaminevaried by nearly 10-fold among different cells, with a mean about 25  M. In the presence of the Ca2+uniporter stimulator kaempferol, the [Ca2+]M peaks induced by histamine were also highly variable, andthe mean [Ca2+]M peak was 3-fold higher. Simultaneous measurement of cytosolic and mitochondrial[Ca2+] peaks showed little correlation among the heights of the peaks in both compartments. Studyingthe [Ca2+]M peaks at the subcellular level, we found significant heterogeneities among regions in thesame cell. In particular, the [Ca2+]M increase in mitochondrial regions close to the nucleus was more thandouble that of mitochondrial regions far from the nucleus.
PB Elsevier Ltd.
SN 0143-4160
YR 2012
FD 2012
LK http://uvadoc.uva.es/handle/10324/5993
UL http://uvadoc.uva.es/handle/10324/5993
LA eng
NO Cell Calcium, 2012, vol. 51, p. 65-71
NO Producción Científica
DS UVaDOC
RD 23-dic-2024