Measurement of cytosolic free calcium in perfused rat heart using TF-BAPTA
Murphy, E.; Steenbergen, C.; Levy, L.A.; Gabel, S.; London, R.E.
American Journal of Physiology 266(5 Pt 1): C1323-C1329
1994
ISSN/ISBN: 0002-9513 PMID: 8203497 Document Number: 431398
The feasibility and usefulness of loading 1,2-bis(2-amino-5,6difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (TF-BAPTA), a new high-dissociation constant (K-D) (65 mu-M) Ca-2+ 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 Ca-2+ (Ca-i-2+) 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 Ca-2+ binding with fast exchange kinetics at magnetic fields ltoreq 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 Ca-2+ 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.