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)
Murphy
E
E
Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Steenbergen
C
C
Levy
L A
LA
Gabel
S
S
London
R E
RE
D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002614:Chelating Agents; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D005456:Fluorescent Dyes; D005461:Fluorine; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009206:Myocardium / Q000378:metabolism*; D010477:Perfusion; D010758:Phosphorus; D051381:Rats
D002614:Chelating Agents; D005456:Fluorescent Dyes; C087511:1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid; D010758:Phosphorus; D005461:Fluorine; D004533:Egtazic Acid; D002118:Calcium