This assay was used to measure the effect of OLF and Dh-OLF on phosphate release in the hydrolysis of ATP, based on the method of Chan and Swaminathan (33) with minor modifications (25), as we detailed previously, to increase the sensitivity of the assay for samples of small volume.
Thus, the antiserum developed to measure Dh-OLF seem more specific than the one developed to measure OLF for structural differences in the lactone ring.
SECRETION OF Dh-OLF AND OLF FROM HUMAN ADRENAL CELL CULTURES
Human adrenal cells (H295R-1) grown in serum-free medium for 24 h produced 0.18 [+ or -] 0.03 pmol of ouabainequivalent OLF and 0.39 [+ or -] 0.04 pmol of dihydroouabainequivalent Dh-OLF per [10.sup.6] cells (n = 3 separate experiments; Table 2).
INHIBITION OF [Na.sup.+],[K.sup.+]-ATPase BY Dh-OLF AND OLF
Both OLF and Dh-OLF isolated from H295R-1 cells inhibited the catalytic activity of porcine cerebrocortical [Na.sup.+], [K.sup.+]-ATPase in vitro.
STIMULATION OF OLF AND Dh-OLF SECRETION BY [(Bu).sub.2]cAMP
Progesterone, as well as other common steroids, is readily separable from the OLF or Dh-OLF in our HPLC isolation procedures (unpublished data).
The objective of this study was to investigate the production of OLF and Dh-OLF by the human adrenal cell line H295R-1 and to develop immunoassays sufficiently specific to distinguish between OLF and Dh-OLF.
Using plant-derived ouabain and dihydroouabain as immunogens, we developed antisera to detect OLF and Dh-OLF and confirmed the presence of OLF and Dh-OLF by use of EIAs in combination with several chromatographic separation steps.