Measurement of cytosolic free calcium in perfused rat heart using TF- BAPTA

E. Murphy, C. Steenbergen, L. A. Levy, S. Gabel, R. E. London

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)C1323-C1329
JournalAmerican Journal of Physiology - Cell Physiology
Volume266
Issue number5 35-5
DOIs
StatePublished - 1994
Externally publishedYes

Keywords

  • 5-fluoro-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
  • ischemia
  • left ventricular developed pressure

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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