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Secretion of a Lactone-Hydrogenated Ouabain-Like Effector of Sodium, Potassium-Adenosine Triphosphatase Activity by Adrenal Cells¹

Departments of Pathology and Laboratory Medicine (H.M.A.M.Q., M.A.E.-M., R.V.) and Biochemistry and Molecular Biology (R.V.), University of Louisville School of Medicine, Louisville, Kentucky 40292

Address all correspondence and requests for reprints to: Dr. Roland Valdes, Jr., Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, Kentucky 40292. E-mail: rvaldes{at}louisville.edu

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 phosphor-ylation 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 = 1700 nM). Using antiouabain and antidihydroouabain antisera we estimate that 3 x 10⁷ 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.

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