Problems caused by high concentration of ATP on activation of the P2X7 receptor in bone marrow cells loaded with the Ca2+ fluorophore fura-2

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, ATPyS and UTP, but not alphabetaATP, 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 P2X7 inhibitors, Mg2+ (5 mM), OxATP (300 microM) 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 P2X7 receptors 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+]i in these cells in the presence of a higher concentration of ATP which activated the P2X7 receptor.