Stimulation of specific binding of [H-3]-progesterone to bovine luteal cell-surface membranes: specificity of digitonin

Non-genomic actions of progesterone have been described in the ovary, and l uteal membranes of several species have been shown to possess specific bind ing sites for [H-3]-progesterone. However, binding of radiolabelled progest erone to luteal membranes was demonstrable only in the presence of digitoni n. Digitonin is a non-ionic detergent which is thought to act by forming on e-to-one complexes with certain sterols. It is also a cardiotonic agent, in hibiting (Na+-K+) ATPase activity by interaction with the extracellular (ou abain/K+) binding site. We therefore investigated which properties of digit onin were responsible for its stimulatory actions on progesterone binding t o bovine luteal membranes. A range of compounds with detergent, cardiotonic and/or cholesterol-complexing activities were tested for their effects on [H-3]-progesterone binding to bovine luteal membrane fractions, and on haem olysis of rat erythrocytes. Stimulation of progesterone binding to luteal m embranes was highly specific for digitonin, and a number of ionic and non-i onic detergents, cardenolides, saponins and cholesterol-complexing reagents tested failed either to stimulate [H-3]-progesterone binding to bovine lut eal membranes in the absence of digitonin, or to inhibit binding specifical ly in the presence of digitonin. When digitonin was first reacted with exce ss cholesterol or pregnenolone to form the respective digitonides, stimulat ory activity was greatly reduced, suggesting that the ability of digitonin to interact with (an) endogenous steroid(s) may be important in its action. High performance liquid chromatography (HPLC)-mass spectrometry of commerc ially available digitonin preparations indicated the presence of numerous m inor impurities in most commercial digitonin preparations. Three major UV-a bsorbing peaks were isolated and characterised by mass spectrometry: all st imulated progesterone binding to bovine luteal membrane receptors in a dose -dependent manner, though to differing extents. Our data suggest that the u nique action of digitonin on luteal membrane progesterone receptors is not related to its detergent or cardiotonic properties, but appears to be relat ed to its ability to complex with membrane sterols. (C) 1999 Elsevier Scien ce Ireland Ltd. All rights reserved.