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

• Published in: American journal of physiology. Heart and circulatory physiology Vol. 285; no. 5; pp. H2118 - H2124
• Main Authors: Scaduto, Russell C., Jr, Grotyohann, Lee W
• Format: Journal Article
• Language: English
• Published: United States 01.11.2003
• Subjects: Aniline Compounds - pharmacokinetics; Animals; Calcium - metabolism; Calibration; Cytosol - metabolism; Fluorescent Dyes - pharmacokinetics; Fura-2 - pharmacokinetics; Heterocyclic Compounds, 3-Ring; Hydrolysis; In Vitro Techniques; Microscopy, Fluorescence - methods; Mitochondria - metabolism; Myocardium - metabolism; Perfusion; Rats; Xanthenes - pharmacokinetics
• ISSN: 0363-6135; 1522-1539
• DOI: 10.1152/ajpheart.00881.2001

Department of Cellular and Molecular Physiology, College of Medicine, The Pennsylvania State University, Hershey, Pennsylvania 17033 Submitted 10 October 2001 ; accepted in final form 9 June 2003 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. rhod 2; indo 1; fluo 3; fura 2; mitochondria; surface fluorescence Address for reprint requests and other correspondence: R. C. Scaduto, Jr., Dept. of Cellular and Molecular Physiology, MS 166, Milton Hershey Medical Center, Hershey, PA 17033 (E-mail: rscaduto{at}psu.edu ).