Variant HeLa cells selected for their resistance to ouabain.

Abstract

The cardiac glyoside, ouabain, normally kills HeLa cells at concentrations of about 10-7 M or greater. By treating a population of HeLa cells with increasingly higher concentrations of the drug, a vaiant population was obtained of HeLa cells capable of growing in medium containing 10-4 M ouabain. Inhibition of volume regulation of cells subjected to hypotonic shock was used as a measure of inhibition of active transport of Na across the plasma membrane. In that way of dose-response curves for the rapid effects of ouabain and other inhibitors of active Na transport were obtained with both the original, ouabain-sensitive (OS) and the variant, ouabain-resistant (OR) cells. Three other cardiac glycosides (digoxin, digtoxin and hellebrin) and two aglycones (digitoxigenin and strophanthidin) were found to be equally as effective as ouabain in inhibiting volume regulation of the OS cells; the concentration whichproduced half-maximum inhibition, I(max/2), was about 6X 10-7 M in each case. Similar inhibition of the OR population by ouabain was observed only when the concentration exceeded 10-4 M [I(max/2-2.5 X 10-4 M], and the other steroid compounds had no effect on the variant cells at the highest concentrations tested (-2 X 10-5 M). OR and OS cells different also in their sensitivities to its cardoactive erythrophleum alkaloid, coumingine; I(max/2) for OS and OR cells was 5 X 10-8 M and 6 X 10-7 M, respectively. These results in addition to results of ouabain binding experiments and measurements of the rates of reversal of inhibition of volume regulation, suggest that a major reason for the differential sensitivities of the two phenotypes to these drugs is different affinities of their sodium pumps for inhibitors of active transport.