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Incomplete hydrolysis of the calcium indicator precursor fura-2 pentaacetoxymethyl ester (fura-2 AM) by cells

1988; Elsevier BV; Volume: 169; Issue: 1 Linguagem: Inglês

10.1016/0003-2697(88)90267-9

1096-0309

S G Oakes, William Martin, Carol A. Lisek, Garth Powis,

Nitric Oxide and Endothelin Effects

Fura-2 AM is an esterified cell-permeant form of the Ca2+ indicator fura-2 (1-[2-(5-carboxyoxal-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2′-amino-5′-methylphenoxy)-ethane-N,N,N′,N′-tetraacetic acid). Fura-2 AM has been reported to be completely cleaved by cellular esterases to fura-2 which is trapped within cells and is used to measure intracellular free Ca2+ concentration by a fluorescence ratio method. Successful application of the method requires that fura-2 be the major cellular fluorescent metabolite of fura-2 AM. We have used high-performance liquid chromatography to study fura-2 AM hydrolysis by cells. Murine N1E-115 neuroblastoma cells incubated with 10 μm fura-2 AM formed fura-2 at a rate of 9.7 pmol/min/106 cells. The concentration of fura-2 in the cells after 60 min, assuming uniform distribution, was 137 μm. Smaller amounts of at least four other metabolites were present, as well as large amounts of unhydrolyzed fura-2 AM. Washing the cells with medium containing 2% bovine serum albumin decreased the concentration of fura-2 to 40 μm and that of fura-2 AM to 90 μm. The half-time for loss of fura-2 from neuroblastoma cells after washing was 34 min. Human pulmonary artery endothelial (HPAE) cells formed fura-2 at a rate of 2.6 nmol/min/106 cells and the concentration of fura-2 after 60 min of incubation and washing with albumin containing medium was 130 μm, and the concentration of fura-2 AM was 58 μm. The half-time for loss of fura-2 from washed HPAE cells was 74 min. Rat hepatocytes formed fura-2 at a rate of 77.9 pmol/min/106 cells. Fura-2 achieved a maximum concentration in hepatocytes of 82 μm after 15 min of incubation and was almost completely lost by washing the hepatocytes with medium containing albumin. It is concluded that the presence of unhydrolyzed fura-2 AM and, presumably, Ca2+-insensitive fluorescent fura-2 AM metabolites in cells in addition to fura-2 after fura-2 AM loading, will complicate measurements of intracellular free Ca2+ by the fluorescence ratio method.