Chick heart cells with high intracellular calcium concentration have a higher affinity for cardiac glycosides than those with low intracellular calcium concentration, as revealed by affinity labelling with a digoxigenin derivative.
Digital-imaging fluorescence microscopy with fura-2 allows the determination of intracellular calcium concentration ([Ca2+]i) in single cells. At a cell density of 10(5) cells/petri dish 44% of the chick embryo heart cells had a high [Ca2+]i of 99.4 +/- 7.1 nM and 56% of the cells a low [Ca2+]i of 27.8 +/- 4.4 nM (mean +/- SE). This laboratory previously reported that high-[Ca2+]i and low-[Ca2+]i cells from chick embryo hearts differ in their sensitivity to cardiac glycosides, as shown by measuring the increase in [Ca2+]i to reach a new steady state [Ahlemeyer, B., Weintraut, H., Seibold, G. & Schoner, W. (1991) in The sodium pump: recent developments (Kaplan, J. H. & De Weer, P., eds) pp. 653-656, Rockefeller University Press, New York]. This time we used N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate (HDMA) which binds irreversibly to amino groups of the Na+/K(+)-ATPase, and sheep anti-digoxigenin Fab fragments coupled with fluorescein isothiocyanate to identify different cardiac glycoside-binding sites. Half-maximal labelling of high-[Ca2+]i cells was obtained at 0.36 nM HDMA, and at 12.0 nM with the low-[Ca2+]i cells. Specific labelling of the cells by HDMA was 91% and 80% in high-[Ca2+]i and low-[Ca2+]i cells, respectively, as revealed by competition experiments with a 1000-fold excess of ouabain. HDMA half-maximally elevated the [Ca2+]i of high-[Ca2+]i cells at a concentration of 50 pM and that of low-[Ca2+]i cells at 8.0 nM. Concentrations higher than 0.1 microM produced signs of intoxication. When the labelled cells were subjected to a SDS/PAGE, a 100-kDa band was found to contain HDMA. The electrophoretic mobility of a protein labelled at 10 nM HDMA was slightly higher than that of a protein labelled at 1.0 microM. The data suggest that different isoforms of the alpha-subunit of Na+/K(+)-ATPase may exist in low-[Ca2+]i and high-[Ca2+]i cells of chick embryo heart.
Ahlemeyer
B
B
Institut für Biochemie und Endokrinologie, Justus-Liebig-Universität Giessen, Federal Republic of Germany.
Weintraut
H
H
Antolović
R
R
Schoner
W
W
D000345:Affinity Labels; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002301:Cardiac Glycosides / Q000378:metabolism*; D002413:Cations, Divalent; D002478:Cells, Cultured; D002642:Chick Embryo; D004076:Digoxigenin / Q000031:analogs & derivatives / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016257:Fura-2; D008856:Microscopy, Fluorescence; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D013388:Succinimides
D000345:Affinity Labels; D002301:Cardiac Glycosides; D002413:Cations, Divalent; D013388:Succinimides; C073892:N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate; D004076:Digoxigenin; D002118:Calcium; D016257:Fura-2