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 Ca2+ indicator fura-2 (1-[2-(5-carboxyoxal-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2'-a mino-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 microM 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 microM. 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 microM and that of fura-2 AM to 90 microM. 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 microM, and the concentration of fura-2 AM was 58 microM. The half-time for loss of fura-2 from washed HPAE cells was 74 min.(ABSTRACT TRUNCATED AT 250 WORDS)
Oakes
S G
SG
Department of Pharmacology, Mayo Clinic and Foundation, Rochester, Minnesota 55905.
Martin
W J
WJ
Lisek
C A
CA
Powis
G
G
D000818:Animals; D001572:Benzofurans / Q000378:metabolism*; D002118:Calcium / Q000032:analysis; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D016257:Fura-2 / Q000031:analogs & derivatives*; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D008658:Inactivation, Metabolic; D008099:Liver / Q000378:metabolism; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D014407:Tumor Cells, Cultured
D001572:Benzofurans; D005456:Fluorescent Dyes; C049925:fura-2-am; D002118:Calcium; D016257:Fura-2