Document Detail

Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics.
MedLine Citation:
PMID:  12767887     Owner:  NLM     Status:  MEDLINE    
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.
C M Loughrey; K E MacEachern; J Cooper; G L Smith
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Cell calcium     Volume:  34     ISSN:  0143-4160     ISO Abbreviation:  Cell Calcium     Publication Date:  2003 Jul 
Date Detail:
Created Date:  2003-05-27     Completed Date:  2004-03-04     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8006226     Medline TA:  Cell Calcium     Country:  Scotland    
Other Details:
Languages:  eng     Pagination:  1-9     Citation Subset:  IM    
Institute of Comparative Medicine, Veterinary School, G61 1QH, Glasgow, UK.
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MeSH Terms
Aniline Compounds / metabolism,  pharmacokinetics*
Calcium / metabolism*
Calcium Signaling / drug effects*,  physiology
Cell Membrane Permeability / drug effects,  physiology
Cells, Cultured
Diffusion / drug effects
Escin / pharmacology
Heart Ventricles / metabolism
Intracellular Fluid / drug effects,  metabolism
Membrane Potentials / drug effects,  physiology
Muscle Contraction / physiology
Myocytes, Cardiac / drug effects*,  metabolism*
Strophanthidin / pharmacology
Xanthenes / metabolism,  pharmacokinetics*
Reg. No./Substance:
0/Aniline Compounds; 0/Xanthenes; 121714-13-4/Fluo-3; 66-28-4/Strophanthidin; 6805-41-0/Escin; 7440-70-2/Calcium

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