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-difluoro phenoxy) -N,N,N',N'-tetraacetic acid (TF BAPTA), a new high-dissociati on constant (K-D) (65 mu M) Ca2+ indicator, into perfused rat heart is demonstrated. TF-BAPTA-loaded perfused rat heart showed less than a 1 0% reduction in left ventricular developed pressure. In addition, load ing perfused rat heart with TF-BAPTA had no effect on cell high-energy phosphates measured by P-31-nuclear magnetic resonance (NMR). Cytosol ic free Ca2+ (Ca-i(2+)) can be monitored in TF-BAPTA-loaded perfused r at heart using F-19-NMR. TF-BAPTA has a Ca2+-insensitive resonance (6F ) and a Ca2+-sensitive fluorine (5F) that responds to changes in Ca2binding with fast exchange kinetics at magnetic fields less than or eq ual to 8.5 T. Thus the shift difference between the 5F and 6F resonanc es is a measure of Ca-i(2+). Given the high KD and the slight differen ces in intra- vs, extracellular fluorine shifts, TF-BAPTA is not well suited for measuring basal Ca-i(2+), but it is useful for measuring in creases in Ca-i(2+) above this level. For studies in which intracellul ar pH changes are significant, e.g., during ischemia, pH-dependent cor rections must be made to obtain an accurate Ca-i(2+) value. Given its fast exchange kinetics, TF-BAPTA is also useful for measurement of fre e 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 ,2-bis(2-amino phenoxy)ethane-N,N,N',N'-tetraacetic acid (5F-BAPTA), introduction of TF-BAPTA into perfused rat heart has minimal effects on contractility.