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 (K(D)) (65 μM) 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+ (Ca(i)/2+) can be monitored in TF-BAPTA-loaded perfused rat heart using 19F-NMR. TF-BAPTA has a Ca2+-insensitive resonance (6F) and a Ca2+- sensitive fluorine (5F) that responds to changes in Ca2+ binding with fast exchange kinetics at magnetic fields ≤8.5 T. Thus the shift difference between the 5F and 6F resonances is a measure of Ca(i)/2+. Given the high K(D) and the slight differences in intra- vs. extracellular fluorine shifts, TF-BAPTA is not well suited for measuring basal Ca(i)/2+, but it is useful for measuring increases in Ca(i)/2+ 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 Ca(i)2+ value. Given its fast exchange kinetics, TF-BAPTA is also useful for measurement of free Ca2+ in different compartments or cells with different Ca(i)/2+. We show that the rise in Ca(i)/2+ is not uniform during prolonged global ischemia (60 min); several different Ca(i)/2+ values are present. Thus TF-BAPTA is a useful new indicator for measuring elevations in Ca(i)/2+ or compartmentation of Ca(i)/2+. In addition, unlike 5-fluoro-1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5F-BAPTA), introduction of TF-BAPTA into perfused rat heart has minimal effects on contractility.
- 5-fluoro-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
- left ventricular developed pressure
ASJC Scopus subject areas
- Cell Biology