Alpha-1 adrenergic receptors stimulate secretion of endogenous ouabain from human and bovine adrenocortical cells

The Na, K-pump inhibitor ouabain or a closely related isomer (EO) is present in the human circulation. We investigated the role of a-adrenergic receptors in the control of EO secretion from human CRL7050 and bovine adrenocortical cells in culture. EO accumulated in the medium conditioned by either human or bovine cells. In CRL cells, cortisol secretion (166 pmole/mg) was detected after prolonged exposure (24hr) to ACTH. Under serum-free conditions, EO was secreted constitutively in amounts ranging from 0.2 to 1.8 pmoles/mg cell protein/30 minutes. In CRL cells, phenylephrine stimulated EO secretion with an EC50 of 2.4 nM mediated by a single class of receptor sites and this effect was blocked specifically by the alpha-1 adrenergic antagonist prazosin. Reverse phase high performance chromatography of the medium bathing the stimulated cells revealed one peak of ouabain immunoreactivity whose retention characteristics were isopolar with commercial ouabain. Irrespective of the amount of EO secreted, the cell lysate EO remained constant suggesting replacement by de novo synthesis. Similar effects of phenylephrine were found in bovine cells and were blocked by doxazosin. These results show that the secretion of EO by human and bovine adrenocortical cells in culture is regulated by alpha-1 adrenergic receptors. These results raise the possibility that the plasma levels of EO are regulated by the sympathetic nervous system.