Hydrolysis of Ca2+-sensitive fluorescent probes by perfused rat heart

Russell C. Scaduto, Lee W. Grotyohann

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

Rat hearts were loaded with the fluorescent calcium indicators fura 2, indo 1, rhod 2, or fluo 3 to determine cytosolic calcium levels in the perfused rat heart. With fura 2, however, basal tissue fluorescence increased above anticipated levels, suggesting accumulation of intermediates of fura 2-AM deesterification. To examine this process, we separated the intermediates of the deesterification process using HPLC after incubation of fura 2-AM with tissue homogenates and after loading in the rat heart. Loading of hearts with fura 2-AM resulted in tissue levels of fura 2 free acid that were only 5% of the total heart dye content of all fura 2 species. The parent fura 2-AM form accumulated without accumulation of intermediate products. Similar results were obtained with indo 1-AM. Fluo 3 loaded very poorly in perfused hearts. Unlike other indictors, rhod 2 rapidly loaded in perfused hearts and was completely converted to the free acid form. To determine the subcellular localization of the free acid form of these indictors, mitochondria from indicator-loaded hearts were assayed for the free acid form. Approximately 75% of the total amount of rhod 2 in hearts could be recovered in isolated mitochondria. Subcellular localization of indo 1 and fura 2 was more evenly distributed between mitochondria and nonmitochondrial compartments. We conclude that measurement of calcium in the perfused rat heart using surface fluorescence with either indo 1 or fura 2 is complicated by an inconsistent accumulation of the parent ester and that the resulting signal cannot be easily calibrated using "in situ" methods using the free acid form. Rhod 2 does not display this shortcoming, but like other indicators, it also loads into the mitochondrial matrix.

Original languageEnglish (US)
Pages (from-to)H2118-H2124
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume285
Issue number5 54-5
DOIs
StatePublished - Nov 2003

All Science Journal Classification (ASJC) codes

  • Physiology
  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

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