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Problems Caused by High Concentration of ATP on Activation of the P2X7Receptor in Bone Marrow Cells Loaded with the Ca2+Fluorophore fura-2

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العنوان: Problems Caused by High Concentration of ATP on Activation of the P2X7Receptor in Bone Marrow Cells Loaded with the Ca2+Fluorophore fura-2
المؤلفون: Paredes-Gamero, E., França, J., Moraes, A., Aguilar, M., Oshiro, M., Ferreira, A.
المصدر: Journal of Fluorescence; November 2004, Vol. 14 Issue: 6 p711-722, 12p
مستخلص: Fura-2 is one of the most used fluorophore for measuring intracellular calcium concentration ([Ca2+]i). In mouse bone marrow cell suspensions ATP produces a biphasic effect: till 1 mM, ATP produces increases in [Ca2+]i; from 1 mM on an increase is observed, that is followed by the decrease in the 340/380 nm ratio (R340/380). At high ATP (4 mM) concentration fura-2 leaked from loaded bone marrow cell suspensions. We observed that ATP decreases fluorescence in the absorption and excitation spectra of fura-2, consequently the emitted one is decreased including the isobestic point (360 nm). ATP analogs: BzATP, ATPγS and UTP, but not αβATP, ADP or AMP, promote decrease of fluorescence in the isobestic point of fura-2. The physical/chemical process that reduces the absorption and excitation of fura-2 by ATP is unknown. The P2X7inhibitors, Mg2+(5 mM), OxATP (300 μM) and Brilliant Blue (100 nM), blocked the efflux of fura-2 and ATP-induced R340/380 decrease. The J774 cell line and mononuclear cells with a higher expression of P2X7receptors show the same decrease in R340/380 as that induced by ATP. In the HL-60 cell line, myeloid cells and erythroblasts extracted from bone marrow, such effect does not occur. It is concluded that the use of the fluorescent Ca2+indicator fura-2 does not allow the correct measurement of [Ca2+]iin these cells in the presence of a higher concentration of ATP which activated the P2X7receptor.
قاعدة البيانات: Supplemental Index
الوصف
تدمد:10530509
15734994
DOI:10.1023/B:JOFL.0000047221.51493.e3