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

Fura-2 AM is an esterified cell-permeant form of the Ca 2+ 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 Ca 2+ 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 μ m fura-2 AM formed fura-2 at a rate of 9.7 pmol/min/10 6 cells. The concentration of fura-2 in the cells after 60 min, assuming uniform distribution, was 137 μ m. 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 μ m and that of fura-2 AM to 90 μ m. 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/10 6 cells and the concentration of fura-2 after 60 min of incubation and washing with albumin containing medium was 130 μ m, and the concentration of fura-2 AM was 58 μ m. 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/10 6 cells. Fura-2 achieved a maximum concentration in hepatocytes of 82 μ m 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, Ca 2+-insensitive fluorescent fura-2 AM metabolites in cells in addition to fura-2 after fura-2 AM loading, will complicate measurements of intracellular free Ca 2+ by the fluorescence ratio method.