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Human Adrenal Cells in Culture Produce Both Ouabain-like and Dihydroouabain-like Factors

Departments of
¹ Pathology and Laboratory Medicine and
² Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, KY 40292.

^aAddress correspondence to this author at: Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, KY 40292. Fax 502-852-1177; e-mail rvaldes{at}louisville.edu.

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 -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 x 10⁶ for ouabain and 8 x10⁵ 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.

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