Background: Ouabain-like factor (OLF) and its newly discovered reduced species, dihydroouabain-like factor (Dh-OLF), are mammalian cardenolides whose structural and functional characteristics are similar to the plant-derived compounds ouabain and dihydroouabain. These endogenous compounds are believed to be produced by the adrenals and to constitute part of an hormonal axis that may regulate the catalytic activity of the α-subunit of Na+,K+-ATPase. We developed antibodies sufficiently specific to distinguish between OLF and Dh-OLF, and in this study demonstrate the selective secretion of OLF and Dh-OLF from human H295R-1 adrenocortical cells in culture. Methods: We used reversed-phase HPLC, inhibition of Na+,K+-ATPase catalytic activity, and two enzyme immunoassays developed with antibodies specific to ouabain and dihydroouabain to purify and characterize the secretion of these two compounds by human adrenal cells in culture. Purified antisera had high titers (1 × 106 for ouabain and 8 ×105 for dihydroouabain) and were specific to their corresponding antigens. Results: Human H295R-1 cells grown in serum-free medium secreted 0.18 ± 0.03 pmol of OLF and 0.39 ± 0.04 pmol of Dh-OLF per 106 cells in 24 h. Both OLF and Dh-OLF inhibited the ouabain-sensitive catalytic activity of the sodium pump (0.03 μmol/L OLF inhibited 29% of the catalytic activity; 0.07 μmol/L Dh-OLF inhibited 17%). Stimulation of the cell culture by dibutryl cAMP increased the secretion of Dh-OLF 50% over control (unstimulated), whereas the secretion of OLF did not increase significantly. Conclusions: OLF and Dh-OLF are secreted by human adrenal cells, and antibodies specific to these two compounds can be developed, using the plant-derived counterparts as antigens. The secretion of Dh-OLF is responsive to a cAMP-dependent stimulation mechanism, whereas OLF is not. Our data suggest that either the secretory or biosynthetic pathways for production of these two compounds by human adrenal cells may have different control mechanisms or that they may be linked via a precursor–product relationship.

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. Results: 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

Digoxin-like immunoreactive factor (DLIF) from adrenal cortex is an endogenous molecule with structural features remarkably similar to those of digoxin, a plantderived cardiac glycoside (Shaikh, I. M., Lau, B. W. C., Siegfried, B. A., and Valdes, R., Jr. (1991) J. Biol. Chem. 266, 13672-13678). Two characteristic structural and functional features of digoxin are a lactone ring and three digitoxose sugars attached to a steroid nucleus. Digoxin is known to undergo deglycosylation during metabolism in humans. We now demonstrate the existence of several naturally occurring deglycosylated components of DLIF in human serum. The components are identified as DLIF-genin, DLIF-mono, and DLIF-bis, corresponding to the aglycone, and the aglycone with one and two sugars, respectively. Similar components are produced by acid-induced deglycosylation of DLIF isolated from bovine adrenal cortex. The elution pattern and sequence of DLIF-deglycosylation was identical to that of digoxin suggesting identical sugar stoichiometry. However, analysis of these newly discovered congeners by reverse-phase chromatography, spectrophotometry, antibody reactivity, and kinetics of deglycosylation, demonstrates that subtle structural and physical differences do exist when compared to digoxin. DLIF was chromatographically distinct from digoxin, and interestingly, the mobility of the DLIF-genin was shifted toward increased polarity relative to digoxigenin. DLIF and DLIF-bis, -mono, and -genin congeners have absorbance maxima at 216 nm, whereas digoxin and its congeners absorb at 220 nm. Reaction with specific antibodies directed at the lactone portion of these molecules shows DLIF and its deglycosylated congeners to be 10 3 -fold less reactive than digoxin. Kinetics of sugar removal suggests that DLIF is 8-fold more susceptible to deglycosylation than is digoxin. Two less polar DLIF components produced from the DLIF-genin have max at 196 nm and are 4-fold less immunoreactive than DLIF. Our data suggest that subtle structural differences exist between DLIF and digoxin at or near the lactone ring as well as in the nature of the sugars. The presence of deglycosylated congeners of DLIF in human serum, including the less polar components, suggests in vivo deglycosylation of these factors. This is the first demonstration of the existence of naturally occurring deglycosylated derivatives of DLIF and establishes the likelihood of active metabolism of DLIF in mammals.Endogenous digoxin-like immunoreactive factors (DLIFs)

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Ouabain-like factor (OLF), a mammalian cardenolide, is a counterpart to plant-derived ouabain and is found in the adrenal, hypothalamus, and blood of several mammalian species. We now report the existence of a mammalian lactone-hydrogenated ouabain-like factor (dihydro-OLF) in secretions from cultured mouse adrenal Y-1 cells. Dihydro-OLF structurally and functionally mimics plant-derived dihydroouabain. We measured both OLF and the newly discovered dihydro-OLF using five independent techniques: immunoreactivity with two specific antisera, one against ouabain and one against dihydroouabain; chromatographic mobility; spectral absorbance characteristics; and concentration-dependent inhibition and phosphorylation of Na,K-adenosine triphosphatase. All measured physical attributes of dihydro-OLF mimic those of plant-derived dihydroouabain, including a spectral shift maxima, 220 nm (OLF) to 196 nm (dihydro-OLF), with appropriately decreased molar absorptivity. Dihydro-OLF (IC50 = 590 nM) is a 10-fold less potent Na+,K+-adenosine triphosphatase inhibitor than its oxidized mammalian counterpart OLF (IC50 = 60 nM), just as dihydroouabain is less potent than ouabain. Dihydro-OLF is also 3-fold more potent than a recently identified isomer of plant-derived dihydroouabain (IC50 = 1,700 nM). Using antiouabain and antidihydroouabain antisera we estimate that 3 x 10(7) mouse adrenal Y-1 cells secreted 1.3 ng OLF and 8.9 ng dihydro-OLF. The relative abundance of dihydro-OLF is consistently greater than that of its oxidized form, OLF, in bovine adrenals (22-fold), human serum (13-fold), and secretions from cultured mouse Y-1 cells (5-fold). The discoveries of OLF, OLF-genin, and now dihydro-OLF constitute an intriguing structural polymorphism probably involved in the synthesis, regulation, and metabolic control of these new hormone-like compounds.