Development of 19F NMR for measurement of [Ca2+]i and [Pb2+]i in cultured osteoblastic bone cells.
Lead (Pb) has been shown to perturb cellular calcium (Ca) homeostasis, altering sizes and flux rates of cellular pools of exchangeable Ca and impairing Ca-mediated cell processes. To date, however, a direct effect of Pb on intracellular-free Ca2+ has not yet been demonstrated. Heavy metals bind to the commonly used fluorescent Ca ion indicators with greater affinity than does Ca and thereby interfere with the expected Ca-dependent fluorescence. In this study, the fluorinated Ca ion indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane N,N,N',N'-tetraacetic acid (5F-BAPTA), and 19F NMR were used to measure the free intracellular Ca ion concentration ([Ca2+]i) in the rat osteoblastic bone cell line, ROS 17/2.8. Both Pb and Ca bind to 5F-BAPTA with high affinity, but the Pb-5F-BAPTA comple produces a 19F NMR signal at a chemical shift distinct from 5F-BAPTA and the Ca-5F-BAPTA complex. The apparent dissociation constants for Pb-5F-BAPTA and Ca-5F-BAPTA are 2 X 10(-10) M and 5 X 10(-7) M, respectively, at 30 degrees C, pH 7.1, and Mg2+ (0.5 mM). Thus, this methodology allows for the simultaneous identification and quantification of free Pb and free Ca ion concentrations. Determinations of [Ca2+]i were based on 19F NMR measurements of 5F-BAPTA-loaded ROS 17/2.8 osteoblastic bone cells that were attached to collagen-coated microcarrier beads. Cells were continuously superfused with freshly oxygenated medium at 30 degrees C. Under these conditions, the [Ca2+]i of ROS 17/2.8 cells was observed to be 128 +/- 14 nM.(ABSTRACT TRUNCATED AT 250 WORDS)
Schanne
F A
FA
Department of Pediatrics, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, NY 10467.
Dowd
T L
TL
Gupta
R K
RK
Rosen
J F
JF
D002118:Calcium / Q000032:analysis*; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004533:Egtazic Acid / Q000031:analogs & derivatives; D005462:Fluorine Radioisotopes; D006801:Humans; D007854:Lead / Q000032:analysis*; D009682:Magnetic Resonance Spectroscopy; D010006:Osteoblasts / Q000032:analysis*
D005462:Fluorine Radioisotopes; D007854:Lead; D004533:Egtazic Acid; C039877:5,5'-difluoro-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium