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) -N,N,N',N'-tetraacetic acid (TF BAPTA), a new high-dissociation constant (K-D) (65 mu M) 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 P-31-nuclear magnetic resonance (NMR). Cytosolic free Ca2+ (Ca-i(2+)) can be monitored in TF-BAPTA-loaded perfused rat heart using F-19-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 less than or equal to 8.5 T. Thus the shift difference between the 5F and 6F resonances is a measure of Ca-i(2+). Given the high KD 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.