Observations on a low molecular weight natriuretic and Na-K-ATPase inhibitory material in urine.

Natriuretic and Na-K-ATPase inhibitory material prepared from urine by gel filtration on G25 Sephadex was previously found to be of low molecular weight, polar and non-peptide. Although activity appeared to depend on an amino group, tests and radioenzymatic assays for catecholamines suggested that these were not implicated in the natriuretic activity. Further purification of the material included solvent extraction, cation exchange and high performance liquid chromatography. At each stage, fractions were assayed for natriuretic activity, stimulation of G6PD and inhibition of Na-K-ATPase in cytochemical assays, and for digoxin-like activities i.e. inhibition of dog kidney Na-K-ATPase (Sigma), displacement of 3H ouabain bound to cell membranes and cross reaction with antidigoxin antibody. The crude material possessed all activities, but with successive purifications the activities separated from each other and were thus due to different substances. Analyses for catecholamines with HPLC and electrochemical detection revealed that the natriuretic activity was due to dopamine.