Measurement of cytosolic free calcium in perfused rat heart using TF-BAPTA

The feasibility and usefulness of loading 1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (TF-BAPTA), a new high-dissociation constant (KD) (65 microM) Ca2+ indicator, into perfused rat heart is demonstrated. TF-BAPTA-loaded perfused rat heart showed less than a 10% reduction in left ventricular developed pressure. In addition, loading perfused rat heart with TF-BAPTA had no effect on cell high-energy phosphates measured by 31P-nuclear magnetic resonance (NMR). Cytosolic free Ca2+ (Ca2+i) can be monitored in TF-BAPTA-loaded perfused rat heart using 19F-NMR. TF-BAPTA has a Ca(2+)-insensitive resonance (6F) and a Ca(2+)-sensitive fluorine (5F) that responds to changes in Ca2+ binding with fast exchange kinetics at magnetic fields < or = 8.5 T. Thus the shift difference between the 5F and 6F resonances is a measure of Ca2+i. Given the high KD and the slight differences in intra- vs. extracellular fluorine shifts, TF-BAPTA is not well suited for measuring basal Ca2+i, but it is useful for measuring increases in Ca2+i above this level. For studies in which intracellular pH changes are significant, e.g., during ischemia, pH-dependent corrections must be made to obtain an accurate Ca2+i value. Given its fast exchange kinetics, TF-BAPTA is also useful for measurement of free Ca2+ in different compartments or cells with different Ca2+i. We show that the rise in Ca2+i is not uniform during prolonged global ischemia (60 min); several different Ca2+i values are present. Thus TF-BAPTA is a useful new indicator for measuring elevations in Ca2+i or compartmentation of Ca2+i.(ABSTRACT TRUNCATED AT 250 WORDS)