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

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.