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 19F-nuclear magnetic resonance spectroscopy.
We have applied the 19F-nuclear magnetic resonance (NMR) calcium indicator 1,2-bis(2-amino-5-fluoro-phenoxy)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 degrees C using 2.4 mM extracellular Ca2+. Subcellular fractionation studies using [3H]5FBAPTA showed that after loading of its tetraacetoxymethyl ester, approximately 55% was de-esterified, with the other 45% remaining as the tetraester bound to membranes. Of the de-esterified 5FBAPTA, greater than 90% was in the cytosolic fractions, with less than 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+]i was constant over a 5-h period, although the scatter of individual results was +/- 20%. The [Ca2+]i 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 surpisingly resistant to changes in extracellular Ca2+ concentration.
Badar-Goffer
R S
RS
Department of Biochemistry, University of Cambridge, U.K.
Ben-Yoseph
O
O
Dolin
S J
SJ
Morris
P G
PG
Smith
G A
GA
Bachelard
H S
HS
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D001923:Brain Chemistry; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D002412:Cations; D002458:Cell Fractionation; D002462:Cell Membrane / Q000032:analysis; D002614:Chelating Agents; D003593:Cytoplasm / Q000032:analysis; D004533:Egtazic Acid / Q000031:analogs & derivatives; D006168:Guinea Pigs; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D011188:Potassium / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology
D002412:Cations; D002614:Chelating Agents; D000001:Calcimycin; D004533:Egtazic Acid; C039877:5,5'-difluoro-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D012964:Sodium; D008274:Magnesium; D011188:Potassium; D002118:Calcium