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.
Paredes-Gamero
E J
EJ
Department of Biophysics, Federal University of São Paulo, Caixa Postal 20372, São Paulo 04034-060, Brazil. jean@biofis.epm.br
França
J P
JP
Moraes
A A F S
AA
Aguilar
M O
MO
Oshiro
M E
ME
Ferreira
A T
AT
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001854:Bone Marrow Cells / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism*; D016257:Fura-2 / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D066298:In Vitro Techniques; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D012394:Rosaniline Dyes / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; C546468:P2RX7 protein, human; C546469:P2rx7 protein, mouse; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D012394:Rosaniline Dyes; C017199:2',3'-dialdehyde ATP; D000255:Adenosine Triphosphate; D008274:Magnesium; C004692:coomassie Brilliant Blue; D002118:Calcium; D016257:Fura-2