Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics

The CA(2+) dissociation constant (K(d)) of Fluo-3 was determined using confocal fluorescence microscopy in two different situations: (i) within the cytosol of a permeabilised cardiomyocyte; and (ii) in an intact cardiomyocyte after incubation with the acetoxymethyl ester form of Fluo-3 (AM). Measurements were made on isolated rabbit ventricular cardiomyocytes after permeabilisation by a brief treatment with beta-escin (0.1mg/ml) and equilibration with 10 microM Fluo-3. The K(d) of Fluo-3 within the cytosol was not significantly different from that in free solution (558 +/- 15 nM, n=6). Over a range of cytoplasmic [CA(2+)], the minimum [CA(2+)] values between CA(2+) waves was relatively constant despite changes in wave frequency. After loading intact cardiomyocytes with Fluo-3 by incubation with the -AM, spontaneous CA(2+) waves were produced by incubation with strophanthidin (10 microM). By assuming a common minimum [CA(2+)] in permeabilised and intact cells, the intracellular K(d) of Fluo-3 in intact myocytes was estimated to be 898 +/-64 nM (n=6). Application of this K(d) to fluorescence records shows that CA(2+) waves in intact cells have similar amplitudes to those in permeabilised cells. Stimulation of cardiac myocytes at 0.5 Hz in the absence of strophanthidin (room temperature) resulted in a CA(2+) transient with a maximum and minimum [CA(2+)] of 1190 +/- 200 and 158 +/- 30 nM (n=11), respectively.