Secretion of a lactone-hydrogenated ouabain-like effector of sodium, potassium-adenosine triphosphatase activity by adrenal cells.

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.

Two biologically active isomers of dihydroouabain isolated from a commercial preparation.

Ouabain is a plant-derived cardiac glycoside that inhibits the catalytic activity of Na(+),K(+)-ATPase (sodium pump; NKA). Dihydroouabain, a derivative of ouabain with a reduced lactone ring, is commonly used as a sodium pump antagonist. It has been assumed that commercially available dihydroouabain is homogeneous. We now report that preparations of dihydroouabain contain two components each with a different potency for inhibition of sodium pump activity. We used reverse-phase HPLC chromatography, UV spectrophotometry, electrospray ionization-mass spectrometry (ESI-MS), nuclear magnetic resonance (NMR) spectroscopy and two independent bioassays to characterize these compounds. The two dihydroouabain fractions (Dho-A and Dho-B) resolved by 3 min chromatographically, had UV absorbance maxima at 196 nm, and comprised 37% and 63% of the stock dihydroouabain, respectively. The molar potency of each component for inhibition of NKA from porcine cerebral cortex differed by 4. 4-fold (Dho-A, IC(50) = 7.13 +/- 0.8 microM; Dho-B, IC(50) = 1.63 +/- 0.12 microM). The relative potencies were 9% and 40% of those of ouabain, respectively. A similar pattern for phosphorylation of NKA was observed. Mass spectrometry (ESI-MS) and fragmentation patterns are consistent with Dho-A and Dho-B being isomers of identical molecular mass (587 Da) and each with six hydroxyl groups, a deoxyhexose sugar moiety and a lactone ring. Furthermore, NMR spectroscopy revealed structural differences between Dho-A and Dho-B by displaying noticeably different chemical shifts at only two groups of proton resonances assigned to H-21 and H-22. The ESI-MS and NMR results confirm the presence of the isomerism at C20 of the lactone ring. Our results demonstrate the existence of two molecular forms of dihydroouabain, each with a different biological potency. These findings underscore the importance of characterizing the purity of dihydroouabain commercial preparations. It also provides possible molecular models for investigating the metabolism of endogenous ouabain-like factors recently reported in mammals.