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

Abstract

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.

Determination of Endogenous Bufalin in Serum of Patients With Hepatocellular Carcinoma Based on HPLC-MS/MS

“…The endogenous digitalis-like compounds (EDLC), a group of steroids, have been demonstrated to exist in mammals as potential Na + /K + -ATPase inhibitors (31)(32)(33)(34)(35). These compounds are postulated to play an essential role in the pathophysiology of hypertension, preeclampsia, end-stage renal disease, congestive heart failure, and diabetes mellitus (36).…”

Signaling function of Na,K-ATPase induced by ouabain against LPS as an inflammation model in hippocampus

“…Ouabain (OUA) is synthesized by the adrenal gland and hypothalamus [ 20 , 21 ] and is likely to have important physiological roles in both the central and peripheral nervous systems [ 22 , 23 ]. OUA binds to NKA in hippocampal astrocytes, activating inositol trisphosphate receptor (InsP3R), which generates calcium oscillations, thereby activating NF-κB [ 23 ].…”

The involvement of Na+/K(+)-ATPase in the development of platelet procoagulant response.

“…The endogenous blockers of Na + /K + -ATPase constitute a group of so-called endogenous cardiac glycosides, now recognized as a new class of steroid hormones (Kawamura et al, 1999;Dimitrieva & Doris, 2002;Schoner, 2002;el-Masri et al, 2002). Ouabain is believed to be the major endogenous cardiac glycoside, but several additional cardiotonic steroids have been isolated from the blood, adrenals, and hypothalamus (Hamlyn et al, 1991;Blaustein, 1993;Dimitrieva & Doris, 2002;Schoner, 2002).…”