Development of sup 19 F NMR for measurement of (Ca2+)i and (Pb2+)i in cultured osteoblastic bone cells

Journal Article · Thu Mar 01 00:00:00 EST 1990 · Environmental Health Perspectives; (USA)

DOI:https://doi.org/10.1289/ehp.908499· OSTI ID:6693354

Schanne, F A; Dowd, T L; Gupta, R K; Rosen, J F ^[1]

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1. Albert Einstein College of Medicine, Bronx, NY (USA)

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.

• OSTI ID: 6693354
• Journal Information: Environmental Health Perspectives; (USA), Vol. 84; ISSN 0091-6765
• Country of Publication: United States
• Language: English

Related Subjects

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
59 BASIC BIOLOGICAL SCIENCES
BONE CELLS
NMR SPECTRA
CALCIUM
LEAD
CELL CULTURES
CHEMICAL SHIFT
FLUORESCENCE
FLUORINE 19
HOMEOSTASIS
MAN
NUCLEAR MAGNETIC RESONANCE
TRACER TECHNIQUES
ALKALINE EARTH METALS
ANIMAL CELLS
ANIMALS
CONNECTIVE TISSUE CELLS
ELEMENTS
FLUORINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LUMINESCENCE
MAGNETIC RESONANCE
MAMMALS
METALS
NUCLEI
ODD-EVEN NUCLEI
PRIMATES
RESONANCE
SOMATIC CELLS
SPECTRA
STABLE ISOTOPES
VERTEBRATES
560300* - Chemicals Metabolism & Toxicology
550201 - Biochemistry- Tracer Techniques