Hydrolysis of Ca2+-sensitive fluorescent probes by perfused rat heart.
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.
Scaduto
Russell C
RC
Department of Cellular and Molecular Physiology, Milton Hershey Medical Center, College of Medicine, The Pennsylvania State University, Hershey, PA 17033, USA. rscaduto@psu.edu
Grotyohann
Lee W
LW
D000814:Aniline Compounds / Q000493:pharmacokinetics; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002138:Calibration; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D016257:Fura-2 / Q000493:pharmacokinetics; D006575:Heterocyclic Compounds, 3-Ring; D006868:Hydrolysis; D066298:In Vitro Techniques; D008856:Microscopy, Fluorescence / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D010477:Perfusion; D051381:Rats; D014966:Xanthenes / Q000493:pharmacokinetics
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; D014966:Xanthenes; C068483:rhod-2; C059715:Fluo-3; D002118:Calcium; D016257:Fura-2