Properties of the purified hypothalamic pituitary NA/K-ATPase inhibitor.

Previously we described the isolation and final purification of an endogenous sodium-pump inhibitor from the CNS, mainly from bovine hypothalamus and pituitary. The purification protocol consisted of lipophilic chromatography, followed by lipid extraction, and semipreparative and analytical reverse-phase high-pressure liquid chromatography. The bioassays used were in vitro Na+/K(+)-ATPase inhibition, and 3H-ouabain displacement from its specific binding site in the enzyme structure, as well as inhibition of 86Rb uptake from human red blood cells. We have obtained, from both tissues, a low-molecular-weight, nonpeptidic, nonlipidic substance that elutes as a single peak highly pure according to criteria of coincidence of its spectra properties. When rechromatographed in two different chromatographic systems, the same homogeneous peak is obtained suggesting complete purity. This pure substance can be isolated from other bovine tissues as well as from human plasma and human placenta. It shows a very distinctive fluorescence spectrum and it behaves as a potent inhibitor of the Ca2+ pump of synaptosomal plasma membrane.

Effect of ouabain and hypothalamic, hypophysary inhibitory factor on rat mesangial cell proliferation.

The mitogenic effect of a low-molecular-weight, Na+,K(+)-ATPase inhibitory factor isolated from bovine hypothalamus and hypophysis (hypothalamic hypophysary inhibitory factor, HHIF) and ouabain on cultured rat mesangial cells was examined. Ouabain induced a potent stimulation of the [3H]thymidine incorporation to DNA, ranging from threefold for a dose of 10(-7) M to seven times for 10(-5) M. The amount of proteins per well also increased in a dose-dependent way, which was already significant from basal values for the lower dose used. HHIF also induced a dose-dependent stimulation of [3H]thymidine incorporation into DNA, ranging from 1.7 times for a concentration of 0.02 U/ml to 4.5 times for the concentration of 2 U/ml. The amount of protein per well also increased with HHIF, but the increase was significant only for the higher dose used. These data allow us to conclude that HHIF could play a role in the vascular hypertrophy that is associated with the pathogenesis of hypertension.

Complete purification of two identical Na(+)-pump inhibitors isolated from bovine hypothalamus and hypophysis.

We have completely purified, in parallel, a low molecular weight, non-specific, non-lipidic, Na+,K(+)-ATPase inhibitory factor from bovine hypothalamic and pituitary tissues. In the final purification step we obtain, from both tissues, a single, homogeneous peak, with a maximal absorbance at 247 nm. This factor, at physiological concentrations of potassium (5-25 mM), inhibits in a dose-response manner Na+,K(+)-ATPase and displaces ouabain from its receptor at the enzyme structure. The factor isolated from both tissues is identical, being the specific activity per weight of tissue higher in hypophysis. No factor was found in cerebral cortex, used as tissue control.

Partial purification of a sodium pump inhibitor from bovine adenohypophysis. Its comparison with the natriuretic factor isolated from hypothalamus.

Adenohypophysis and hypothalamic bovine tissues were homogenized, lipid extracted, salt removed and loaded onto 2 successive mu Bondapak HPLC columns, semipreparative and analytic, respectively. In vitro sodium-pump inhibitory activity, recovered from each tissue, showed similar chromatographic patterns, but hypothalamus seems to contain a major hydrophobic material which appears at the end of the run, when acetonitrile gradient raised 40% approximately. Digitalis-like activity disappears along the purification procedure, and this fact suggests a clear dissociation between (Na/K)ATPase inhibition and digoxin-like activity, measured as crossing with digoxin antibodies.