BIOSYNTHESIS OF DIGITALIS-LIKE COMPOUNDS IN RAT ADRENAL-CELLS - HYDROXYCHOLESTEROL AS POSSIBLE PRECURSOR

The biosynthesis of digitalis-like compounds (DLC) was determined in b ovine and rat adrenal homogenates, as well as in primary rat adrenal c ells, by following changes in the concentration of DLC using three ind ependent sensitive bioassays: inhibition of [H-3]-ouabain binding to r ed blood cells and competitive ouabain and bufalin ELISA. The amounts of DLC in bovine and rat adrenal homogenates, as measured by the two f irst bioassays, increased with time when the mixtures were incubated u nder tissue culture conditions. Rat primary adrenal cells were incubat ed in the presence of [1,2-H-3]-25-hydroxycholesterol, [26,27-H-3]-25- hydroxycholesterol or [7-H-3]-pregnenolone. The radioactive products, as well as the digitalis-like activity, were fractionated by three seq uential chromatography systems. When [1,2-H-3]-25-hydroxycholesterol o r [7-H-3]-pregnenolone was added to the culture medium, the radioactiv ity was co-eluted with digitalis-like activity, suggesting that at lea st one of the DLC might originate in hydroxycholesterol. In contrast, when the culture medium was supplemented with [26,27-H-3]-25-hydroxych olesterol, the radioactivity was not co-eluted with the digitalis-like activity, indicating that side chain cleavage is the first step in th e synthesis of digitalis-like compounds by rat adrenal.