Open AccessChick 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
Author(s) -
AHLEMEYER Barbara,
WEINTRAUT Horst,
ANTOLOVIĆA Roberto,
SCHONER Wilhelm
Publication year - 1992
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1992.tb16777.x
Subject(s) - calcium , ouabain , intracellular , digoxigenin , fura 2 , chemistry , calcium in biology , biophysics , ionophore , microbiology and biotechnology , biochemistry , sodium , biology , cytosol , enzyme , gene , in situ hybridization , gene expression , organic chemistry
Digital‐imaging fluorescence microscopy with fura‐2 allows the determination of intracellular calcium concentration ([Ca 2+ ] i ) in single cells. At a cell density of 10 5 cells/petri dish 44% of the chick embryo heart cells had a high [Ca 2+ ] i of 99.4 ± 7.1 nM and 56% of the cells a low [Ca 2+ ] i of 27.8 ± 4.4 nM (mean ± SE). This laboratory previously reported that high‐[Ca 2+ ] i and low‐[Ca 2+ ] i cells from chick embryo hearts differ in their sensitivity to cardiac glycosides, as shown by measuring the increase in [Ca 2+ ] 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‐ε‐aminocaproate (HDMA) which binds irreversibly to amino groups of the Na + /K + ‐ATPase, and sheep anti‐digoxigenin F ab fragments coupled with fluorescein isothiocyanate to identify different cardiac glycoside‐binding sites. Half‐maximal labelling of high‐[Ca 2+ ] i cells was obtained at 0.36 nM HDMA, and at 12.0 nM with the low‐[Ca 2+ ] i cells. Specific labelling of the cells by HDMA was 91% and 80% in high‐[Ca 2+ ] i and low‐[Ca 2+ ] i cells, respectively, as revealed by competition experiments with a 1000‐fold excess of ouabain. HDMA half‐maximally elevated the [Ca 2+ ] i of high‐[Ca 2+ ] i cells at a concentration of 50 pM and that of low‐[Ca 2+ ] i cells at 8.0 nM. Concentrations higher than 0.1 μM 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 μM. The data suggest that different isoforms of the α‐subunit of Na + /K + ‐ATPase may exist in low‐[Ca 2+ ] i and high‐[Ca 2+ ] i cells of chick embryo heart.