An endogenous digitalis-factor derived from the adrenal gland: studies of adrenocortical tumor cells.
The present studies demonstrate that the murine adrenocortical tumor cell line Y-1 releases a digoxin-like immunoreactive material into both serum-supplemented nutrient medium and minimal Krebs-Ringer bicarbonate medium. Release of pregnenolone into minimal medium from these cells was consistently inhibited by addition of the cholesterol side-chain cleavage inhibitor aminoglutethimide. However, release of digoxin-like immunoreactivity (DLI) was not similarly affected. To exclude the possibility that DLI could be accounted for by cross-reaction with another known adrenal steroid, aminoglutethimide inhibition was accompanied by inhibition of 17 alpha-hydroxylase with SU-10603 and inhibition of 3 beta-hydroxysteroid dehydrogenase with cyanoketone. Once again, pregnenolone release was effectively inhibited, but no similar pattern of inhibition of DLI release was observed. Increasing the time of the incubation periods from 1 to 2 h did not change the pattern of secretion of pregnenolone or DLI. HPLC analysis of DLI released over prolonged culture periods into serum-supplemented nutrient medium showed high levels of DLI in a single major and several adjacent peaks. Analysis of the ability of extracts of Y-1-conditioned medium to compete with tritiated ouabain for binding to erythrocytes indicates that conditioned medium contained highly enriched levels of ouabain-like activity. On HPLC analysis, the distribution of this activity showed partial correlation with the distribution of DLI. These observations indicate that Y-1 cells produce and release significant quantities of a material with cardiac glycoside-like properties reflected in the cross-reactivity with antidigoxin antibodies and the ability to compete with ouabain for binding to erythrocytes. In substantiation of previous findings in chopped adrenal cultures, the cardiac glycoside-like activity does not appear to result from cholesterol side-chain cleavage or pregnenolone production, since inhibition of side-chain cleavage as well as subsequent 17 alpha-hydroxylation and 3 beta-dehydrogenation did not result in consistent inhibition of DLI release.
Doris
P A
PA
Department of Cell Biology, Texas Tech University Health Sciences Center, Lubbock 79430.
Kilgore
M W
MW
Durham
D
D
Alberts
D
D
Stocco
D M
DM
D000306:Adrenal Cortex Neoplasms / Q000378:metabolism*; D000616:Aminoglutethimide / Q000494:pharmacology; D000818:Animals; D001798:Blood Proteins / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D002298:Cardenolides; D002460:Cell Line; D004077:Digoxin / Q000032:analysis; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D010042:Ouabain / Q000378:metabolism; D011284:Pregnenolone / Q000378:metabolism; D011863:Radioimmunoassay; D012503:Saponins
D001798:Blood Proteins; D002298:Cardenolides; D012503:Saponins; C034504:digoxin-like factors; D000616:Aminoglutethimide; D010042:Ouabain; D004077:Digoxin; D011284:Pregnenolone