Identification of endogenous ouabain in culture supernatant of PC12 cells

Ouabain-like factor (OLF), assayed as ouabain-like immunoreactivity (OLI), is thought to represent an endogenous digitalis-like factor. We found increased plasma OLI during the surgical removal of a pheochromocytoma. The elution volume of the OLI extracted from plasma and the pheochromocytoma tissue was the same as that for authentic ouabain, using reverse phase high-performance liquid chromatography. The present study was performed to characterize OLF from the culture supernatant of a rat pheochromocytoma cell line, PC12 cells.

OLI from culture supernatant and chromatographic fractions were assayed by a sensitive enzyme-linked immunosorbent assay for ouabain. PC12 cells, subcultured in RPMI 1640 with 10% horse serum and 5% fetal bovine serum, were washed, and then cultured in Iscove's modified Dulbecco's medium (Life Technologies, Rockville, Maryland, USA) with 0.4% bovine serum albumin (without serum). Progesterone was added to augment the production or secretion of OLI. The conditioned medium was acidified to dissociate the binding protein, and OLI was purified by five steps of octadecylsilane (ODS) column chromatography. The structural identity of this OLI was determined by liquid chromatography and mass spectrometry (LC/MS).

OLI in the culture medium increased after addition of progesterone in a dose-dependent manner. The concentration in the culture medium was approximately double of that in homogenized PC12 cells. After five rounds of ODS column chromatography, approximately 100 ng of OLI was purified from 21 of culture supernatant, without fetal calf serum, in the presence of progesterone. The molecular size of purified OLI was found to be identical to authentic ouabain, based on analysis by LC/ MS.

Mammalian cells originating from a rat pheochromocytoma cell line were found to produce and/or secrete OLF by the addition of progesterone.