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Use of 1,2‐Bis(2‐Amino‐5‐Fluorophenoxy)ethane‐ N,N,N′,N′ ‐Tetraacetic Acid (5FBAPTA) in the Measurement of Free Intracellular Calcium in the Brain by 19 F‐Nuclear Magnetic Resonance Spectroscopy
Author(s) -
BadarGoffer Ronnitte S.,
BenYoseph Oded,
Dolin Simon J.,
Morris Peter G.,
Smith Gerry A.,
Bachelard Herman S.
Publication year - 1990
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1990.tb04573.x
Subject(s) - extracellular , calcium , chemistry , cytosol , ionophore , intracellular , mole , nuclear magnetic resonance spectroscopy , analytical chemistry (journal) , nuclear chemistry , biochemistry , stereochemistry , chromatography , enzyme , organic chemistry
Abstract: We have applied the 19 F‐nuclear magnetic resonance (NMR) calcium indicator 1,2‐bis(2‐amino‐5‐fluorophenoxy)ethane‐ N,N,N′,N′ ‐tetraacetic acid (5FBAPTA) to the measurement of the free intracellular calcium concentration ([Ca 2+ ] i ) in superfused brain slices. A mean ± SD control value of 380 ± 71 n M (n = 18) was obtained at 37°C using 2.4 m M extracellular Ca 2+ . Subcellular fractionation studies using [ 3 H] 5FBAPTA showed that after loading of its tetraac‐etoxymethyl ester, ∼55% was de‐esterified, with the other 45% remaining as the tetraester bound to membranes. Of the de‐esterified 5FBAPTA, >90% was in the cytosolic fractions, with >1% in the mitochondria or microsomes. The NMR‐visible de‐esterified 5FBAPTA slowly disappeared from the tissue with a t 1/2 of 4 h. A time course after loading confirmed that the calculated [Ca 2+ ], was constant over a 5‐h period, although the scatter of individual results was ±20%. The [Ca 2+ ]; was increased by a high extracellular K + concentration ([K + ] e ), by a low extracellular concentration of Na + , and by the calcium ionophore A23187. On recovery from high [K + ] e . the [Ca 2+ ] i “overshot” to values lower than the original control value. The [Ca 2+ ] i was surprisingly resistant to changes in extracellular Ca 2+ concentration.