AJP - Heart and Circulatory Physiology, Vol 259, Issue 3 973-H981, Copyright © 1990 by American Physiological Society

ARTICLES

In situ calibration of fura-2 and BCECF fluorescence in adult rat ventricular myocytes

S. Borzak, R. A. Kelly, B. K. Kramer, Y. Matoba, J. D. Marsh and M. Reers
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.

Quantitation of Ca+ and H+ activities within cells using presently available fluorescent probes is optimal when the fluorescence signal is calibrated in situ after each experiment. Fura-2 and 2',7'-bis(2-carboxy-ethyl)-5,6-carboxyfluoroscein (BCECF) are difficult to calibrate in freshly dissociated adult cardiac myocytes because calibration procedures produce cellular hypercontracture. In situ calibration was accomplished in rat ventricular cells by saturating fura-2 with La3+, an agent known to produce myocardial relaxation. Since fura-2 has different spectral properties when complexed with La3+ than with Ca2+, scaling factors were defined in vitro and then verified by experiments in cultured neonatal myocytes. In adult rat myocytes using the La3+ method, intracellular Ca2+ concentration ([Ca2+]i) was 131 +/- 47 nM (n = 14) in quiescent cells; diastolic [Ca2+]i and systolic [Ca2+]i in myocytes stimulated at 1 Hz were 140 +/- 56 and 1,088 +/- 211 nM (n = 5), respectively. BCECF fluorescence was calibrated in situ by a method that prevented cellular hypercontracture and reported a pH value of 7.10 +/- 0.10 in cells stimulated at 1.5 Hz. An additional advantage of both methods is that the buffers employed prevented large changes in the redox state of intracellular pyridine nucleotides, thus preventing a change in cellular autofluorescence during the calibration procedure.

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