Molecular Pharmacology, Vol 13, 161-171, Copyright © 1977 by the American Society for Pharmacology and Experimental Therapeutics

Analysis of Intracellular Drug (Ouabain) Sequestration as a Mechanism of Detoxification

¹ Cancer and Toxicology Program, Biology Division, Oak Ridge National Laboratory, and the University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, Tennessee 37830

The cardiac glycoside ouabain, bound in a brief pulse to growing HeLa cells, is released over a period of days in a nonexponential manner that indicates heterogeneity of the dissociation processes. On release, the drug is chromatographically indistinguishable from authentic ouabain. We describe a model for release as the sum of three first-order processes: (a) simple dissociation from its membrane binding site [(Na⁺+K⁺)-ATPase] into the medium; (b) sequestration or internalization of the drug, possibly together with its binding site, as part of membrane turnover; and (c) externalization of the internalized drug. Processes (a) and (b) lead to physiological recovery, and together occur substantially more rapidly than the sum of processes (a) and (c), which lead to release of drug from the cell. A program devised by Chandler et al. [(1972) Comp. Biomed. Res., 5, 515-534] is used to compute from drug release data the optimum least-squares fit as well as the compartment sizes and rate constants for the three processes. The analyses, based on data from intact cells, yield results that closely resemble those from data obtained from more disruptive cell fractionation procedures. These results show that ouabain is neither degraded nor stored in the cell. It can be found free in the cytoplasmic fraction, but it is not cytotoxic in that location. It is eventually released intact. Thus ouabain is not converted to an innocuous form by the cell, but rather is translocated to an innocuous location within the cell, with concomitant physiological recovery of normal functions at the cell surface prior to drug release.

Note:
ACKNOWLEDGMENTS The authors thank Margarita Churchich for assistance with the computations involving CRICF, and D. G. Wilson and R. J. Brake for constructive comments on the manuscript.

This article has been cited by other articles:

				T Hayashi, Y Ijiri, H Toko, H Shimomura, M Okabe, F Terasaki, Y Kitaura, and K Kawamura Increased digitalis-like immunoreactive substances in patients with hypertrophic cardiomyopathy Eur. Heart J., February 2, 2000; 21(4): 296 - 305. [Abstract] [PDF]