An ouabain-like factor is secreted from immortalized hypothalamic cells in an aldosterone-dependent manner

Ouabain-like factor (OLF) modulates blood pressure via sodium pump inhibition in the central nervous system and in the peripheral circulation. Ouabain-like factor (OLF) is thought to be produced in the adrenal gland and hypothalamus, and it may relate locally to the renin-angiotensin-aldosterone system. However, the evidence for the latter was obtained from in vivo experiments using animals. In the present study, we investigated ouabain production in the immortalized hypothalamic cell line N1. First, cell culture supernatant was collected from the immortalized hypothalamic cell line N1 at 0.5, 4, 8, and 24 h. A newly developed enzyme-linked immunosorbent assay (ELISA) that used anti-ouabain antibody showed that immunoreactivity in the supernatant was increased significantly at 24 vs. 0.5 h (0.01±0.004 vs. 0.16±0.033 pmol/mg protein, p<0.01). A combination of HPLC and ELISA was used to characterize N1 cell-derived OLI, showing that the highest peak of OLI had the same retention time as authentic ouabain. Thereafter, N1 cells were cultured with (1-10 μM) aldosterone, and supernatant was collected after 24 h of culture. In addition, N1 cells were cultured with 5 μM eplerenone, a mineralocorticoid receptor blocker, plus aldosterone. OLI was significantly increased in the supernatant of the cells cultured with 10 μM aldosterone (0.40±0.078 pmol/mg protein), and this increase was abolished by the addition of the aldosterone antagonist eplerenone (0.12±0.030 pmol/mg protein). These data suggest that the immortalized hypothalamic N1 cells secrete OLF and that aldosterone stimulates its secretion via mineralocorticoid receptors.