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 F-19-NUCLEAR MAGNETIC-RESONANCE SPECTROSCOPY

Abstract: We have applied the 19F‐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 ([Ca2+] i) in superfused brain slices. A mean ± SD control value of 380 ± 71 nM(n = 18) was obtained at 37°C using 2.4 mM extracellular Ca2+. 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 t1/2 of 4 h. A time course after loading confirmed that the calculated [Ca2+], was constant over a 5‐h period, although the scatter of individual results was ±20%. The [Ca2+]; 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 [Ca2+]i“overshot” to values lower than the original control value. The [Ca2+]i was surprisingly resistant to changes in extracellular Ca2+ concentration. Copyright © 1990, Wiley Blackwell. All rights reserved