title: Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics.
abstract: 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.
authors.affiliation: Institute of Comparative Medicine, Veterinary School, G61 1QH, Glasgow, UK.
authors.forename: C M
authors.initials: CM
authors.lastname: Loughrey
authors.forename: K E
authors.initials: KE
authors.lastname: MacEachern
authors.forename: J
authors.initials: J
authors.lastname: Cooper
authors.forename: G L
authors.initials: GL
authors.lastname: Smith
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism / Q000493:pharmacokinetics*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004058:Diffusion / Q000187:drug effects; D004928:Escin / Q000494:pharmacology; D006352:Heart Ventricles / Q000378:metabolism; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009119:Muscle Contraction / Q000502:physiology; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism*; D011817:Rabbits; D013327:Strophanthidin / Q000494:pharmacology; D014966:Xanthenes / Q000378:metabolism / Q000493:pharmacokinetics*
chemical_list: D000814:Aniline Compounds; D014966:Xanthenes; C059715:Fluo-3; D013327:Strophanthidin; D004928:Escin; D002118:Calcium