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

Loughrey, C.M. , MacEachern, K.E. , Cooper, J. and Smith, G.L. (2003) Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics. Cell Calcium, 34(1), pp. 1-9. (doi:10.1016/S0143-4160(03)00012-5) (PMID:12767887)

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Abstract

The Ca2+ dissociation constant (Kd) 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 β-escin (0.1 mg/ml) and equilibration with 10 μM Fluo-3. The Kd 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 [Ca2+], the minimum [Ca2+] values between Ca2+ waves was relatively constant despite changes in wave frequency. After loading intact cardiomyocytes with Fluo-3 by incubation with the −AM, spontaneous Ca2+ waves were produced by incubation with strophanthidin (10 μM). By assuming a common minimum [Ca2+] in permeabilised and intact cells, the intracellular Kd of Fluo-3 in intact myocytes was estimated to be 898±64 nM (n=6). Application of this Kd to fluorescence records shows that Ca2+ 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 Ca2+ transient with a maximum and minimum [Ca2+] of 1190±200 and 158±30 nM (n=11), respectively.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Smith, Professor Godfrey and MacEachern, Dr Karen and Loughrey, Professor Christopher and Cooper, Professor Jonathan
Authors: Loughrey, C.M., MacEachern, K.E., Cooper, J., and Smith, G.L.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
College of Medical Veterinary and Life Sciences > School of Veterinary Medicine
College of Science and Engineering > School of Engineering > Biomedical Engineering
Journal Name:Cell Calcium
Publisher:Elsevier
ISSN:0143-4160
ISSN (Online):1532-1991
Published Online:06 March 2003

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