AJP - Cell Physiology, Vol 253, Issue 5 C731-C743, Copyright © 1987 by American Physiological Society
Na-K pump site density and ouabain binding affinity in cultured chick heart cells
L. A. Lobaugh and M. Lieberman
Department of Physiology, Duke University Medical Center, Durham, North Carolina 27710.
The possible existence of multiple [3H]ouabain binding sites and the
relationship between ouabain binding and Na-K pump inhibition in cardiac
muscle were studied using cultured embryonic chick heart cells. [3H]ouabain
bound to a single class of sites in 0.5 mM K (0.5 Ko) with an association
rate constant (k+1) of 3.4 X 10(4) M-1.s-1 and a dissociation rate constant
(k-1) of 0.0095 s-1 [corrected]. Maximal specific [3H]ouabain binding RT to
myocyte-enriched cultures is 11.7 pmol/mg protein and Kd is 0.43 microM in
0.5 Ko, whereas Kd,apparent is 6.6 microM in 5.4 Ko. The number of binding
sites per myocyte was calculated by correcting for the contribution of
fibroblasts in myocyte-enriched cultures using data from homogeneous
fibroblast cultures (RT = 3.3 pmol/mg protein; Kd = 0.19 microM in 0.5 Ko).
Equivalence of [3H]ouabain binding sites and Na-K pumps was implied by
agreement between maximal specific binding of [3H]ouabain and 125I-labeled
monoclonal antibody directed against Na+-K+-ATPase (approximately 2 X 10(6)
sites/cell). However, [3H]ouabain binding occurred at lower concentrations
than inhibition of ouabain-sensitive 42K uptake in 0.5 Ko. Further studies
in both 0.5 K and 5.4 Ko showed that ouabain caused cell Na content Nai to
increase over the same range of concentrations that binding occurred,
implying that increased Nai may stimulate unbound Na-K pumps and prevent a
proportional decrease in 42K uptake rate. The results show that Na-K pump
inhibition occurs as a functional consequence of specific ouabain binding
and indicate that the Na-K pump is the cardiac glycoside receptor in
cultured heart cells.
