Related Experiment Videos
Na-K pump site density and ouabain binding affinity in cultured chick heart cells
1Department of Physiology, Duke University Medical Center, Durham, North Carolina 27710.
Abstract:
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.
Insights
This study shows that the Na-K pump is the cardiac glycoside receptor in heart cells. Ouabain binding specifically inhibits Na-K pump function, impacting cardiac muscle.
Area of Science:
- Cardiovascular Physiology
- Cellular Biology
- Pharmacology
Background:
- Cardiac glycosides, like ouabain, are crucial for treating heart failure.
- Understanding the precise mechanism of ouabain's action on cardiac cells is essential for optimizing its therapeutic use.
- Previous research suggested potential multiple binding sites for ouabain, necessitating clarification.
Purpose of the Study:
- To investigate the existence of multiple [3H]ouabain binding sites in cardiac muscle.
- To determine the relationship between ouabain binding and Na-K pump inhibition.
- To identify the specific receptor for cardiac glycosides in cultured heart cells.
Main Methods:
- Utilized cultured embryonic chick heart cells for experiments.
- Quantified [3H]ouabain binding kinetics (association and dissociation rates, Kd).
- Measured ouabain-sensitive 42K uptake and intracellular sodium content (Nai).
- Employed 125I-labeled monoclonal antibody against Na+-K+-ATPase to quantify pump sites.
Main Results:
- [3H]ouabain bound to a single class of sites in low potassium conditions (0.5 Ko).
- Maximal binding (RT) and dissociation constant (Kd) were determined for myocyte-enriched and fibroblast cultures.
- Na-K pump inhibition correlated with ouabain binding concentrations, with increased intracellular sodium potentially modulating pump activity.
- Equivalence was found between [3H]ouabain binding sites and Na+-K+-ATPase sites.
Conclusions:
- The Na-K pump acts as the primary cardiac glycoside receptor in cultured heart cells.
- Ouabain binding directly leads to Na-K pump inhibition.
- The study clarifies the mechanism of cardiac glycoside action at the cellular level.