Purification and properties of endogenous ouabain-like substances from hemofiltrate and adrenal glands

Chick embryo heart cells in culture were found to contain two cell types differing in their [Ca2+]i. Elevation of [Ca2+]i in cells with 99.4 +/- 7.1 nM [Ca2+]i was induced half-maximally at 2 x 10(-9) M ouabain and of cells with 27.9 +/- 4.4 nM [Ca2+]i at 4 x 10(-8) M ouabain. The localization of a sodium pump with differing sensitivity for ouabain in different cell types of the heart raised the question of the existence of endogenously formed ouabain. From 6,000 I hemofiltrate of uremic patients, a substance of 582 Da cross-reacting with ouabain antibodies was purified 42,000-fold. The substance was slightly more hydrophobic on a C-18 reversed-phase high-performance liquid chromatography (HPLC) than ouabain was. It inhibited 86Rb+ uptake into red blood cells and raised like ouabain [Ca2+]i in both types of chick heart cells. From pig adrenal glands, a substance inhibiting 86Rb+ uptake into erythrocytes was purified by affinity chromatography on a column containing antibodies against ouabain. A major part of the eluted substances chromatographed on a C-18-HPLC column as ouabain did, but a minor part was somewhat more hydrophobic. It is presumed that more than a single endogenous ouabain-like factor exists in mammals.