De novo biosynthesis and radiolabeling of mammalian digitalis-like factors

Digoxin-like immunoreactive factors (DLIFs) are endogenous mammalian cardenolides with structural features similar to those of the plant-derived digitalis compounds. DLIFs and their structurally related forms (Dh-DLIFs) may serve as effectors of ion-transport activity mediated by their interaction with Na,K-ATPase and thus play a role as a new hormonal axis. Although some evidence implicates the adrenal gland as a tissue source for the DLIFs, little is known about the biosynthetic pathway producing these compounds. We now demonstrate de novo biosynthesis of DLIF by incorporation of radioactive carbon ((14)C) into the structures of both DLIF and Dh-DLIF.

We used a combination of reversed-phase HPLC techniques to separate the radioactive DLIF components after incorporation of (14)C into their structure by use of either [1,2-(14)C]acetic acid or [4-(14)C]cholesterol as precursors and a Y-1 mouse adrenocortical tumor cell line. We also stimulated and suppressed production of steroidogenesis by use of cAMP analogs and Mevastatin, respectively, to demonstrate the dependence of DLIF production on the cholesterol-dependent biosynthetic pathway. A combination of chromatographic mobility, immunoassays specific for digoxin and dihydrodigoxin, and deglycosylation using 5-sulfosalicylic acid were used to identify the DLIF and Dh-DLIF components.

With cholesterol as precursor, the cells produced DLIF (7.5 mCi/mmol) with a labeling efficiency of 10%, whereas with acetate the cells produced DLIF (72.2 mCi/mmol) with a labeling efficiency of 0.08% of the total DLIF produced. The radiolabeled DLIF and Dh-DLIF molecules had identical chromatographic mobilities and stoichiometric removal of sugars as the previously characterized DLIFs isolated from different mammalian species and tissues. With radioactive cholesterol as precursor, the (14)C was incorporated into the DLIF-genin portion of the compounds and not the sugars. Interestingly, treatment of Y-1 cells with 8-bromoadenosine 3':5'-cAMP to stimulate steroidogenesis did not increase production of DLIF or Dh-DLIF but did increase production of progesterone. Mevastatin (5 micromol), an inhibitor of the enzyme hydroxymethylglutaryl-CoA reductase and thus of cholesterol biosynthesis, gave an 85% decrease in the production of (14)C-DLIF and progesterone, but only a modest 15% decrease in (14)C-Dh-DLIF production.

These data demonstrate that the adrenal cell has the cellular machinery necessary for de novo biosynthesis of DLIF and Dh-DLIF starting from a simple carbon pool and also support the concept that cholesterol is a major precursor of the DLIF compounds. This cell culture model provides a source of radiolabeled DLIF compounds for future experimental work.