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.

Evidence for a circulating sodium transport inhibitor in essential hypertension.

The active sodium transport of white cells and red cells obtained from patients with essential hypertension was impaired. Incubating white cells from normotensive subjects in serum obtained from patients with essential hypertension caused an impairment in sodium transport in the white cells of normotensive subjects similar to that found in the white cells of hypertensive patients. The impairment in sodium transport was due to a fall in the ouabain-sensitive component of the total sodium efflux rate constant. These results show that the serum of patients with essential hypertension contains a substance which influences sodium transport and that it has ouabain-like activity. They also suggest that it is this substance which causes the impairment in sodium transport in the leucocytes of patients with essential hypertension. These findings support the hypothesis that the rise in blood pressure in patients with essential hypertension is due to an increased concentration of a circulating sodium transport inhibitor which is continuously correcting a tendency for sodium retention by the kidney.

Further observations on a low-molecular-weight natriuretic substance in the urine of normal man.

Large quantities of the low-molecular-weight natriuretic material (F4), which appears after the salts when fractionated on G-25 Sephadex column, were obtained from the urine of normal man on a normal diet. The natriuretic substance in F4 was (1) untrafiltrable through a membrane with a claimed molecular-weight cut-off of 500 daltons (Amicon UMO5); (2) soluble in more polar organic solvents; (3) totally soluble in 95% acetone when specific activity was doubled; (4) relatively resistant to heating at 100 degrees C for 1 hour at a pH of 10, and to heating at 110 degrees C in 6 N hydrochloric acid for up to 90 hours under anaerobic conditions, and treatment with nitrous acid; it was less resistant to these procedures when extracted into 95% acetone; (5) not destroyed by trypsin, chymotrypsin, pronase, pepsin, leucine aminopeptidase, and subtilysin, nor was it destroyed by pepsin, leucine aminopeptidase, subtilysin, carboxypeptidase A and B, and aminopeptidase M, or by monoamine oxidase, aryl sulphatase, and beta-glucuronidase when extracted into 95% acetone. The natriuretic substance in the 95% acetone-soluble F4 was totally destroyed by incubation with prolidase. The least amount of 95% acetone-soluble F4 required to produce a significant natriuresis in the bioassay rat was that derived from a 7-min sample of urine. The maximal response was obtained from a 30-min sample of urine. Continuous i.v. infusion of the 95% acetone-soluble F4 for 40 min produced a sustained natriuresis, whereas a greater amount injected as a bolus produced an effect which was not sustained beyond 20 min.

Two natriuretic substances in extracts of urine from normal man when salt-depleted and salt-loaded.

When the extra-cellular fluid volume is expanded, the subsequent rise in urinary sodium excretion that occurs is due in part to a change in the concentration of some circulating natriuretic substance. Two natriuretic substances with different characteristics on Sephadex chromatography have been previously been identified separately by different workers. Extracts prepared from the urine of 31 normal subjects were tested for these two natriuretic materials in the normal conscious water-loaded rat. Two natriuretic fractions were found. The larger of the two was prepared on G50 Sephadex, and the smaller on G25 Sephadex. The natriuresis produced by the larger material was slow to develop and persisted for two hours. The natriuresis produced by the smaller material was maximal in the first 20 min, declined rapidly within the next 40 min, and tended to rise again during the subsequent 60 min. The amount of natriuretic activity that could be extracted from the freeze-dried urine was diminished by high concentrations of sodium chloride. The natriuretic activity of both materials was greater in the urine of the subjects when they were salt-loaded than when they were salt-depleted. The urine of salt-depleted subjects contained significant amounts of natriuretic material.

Slow sodium: an oral slowly released sodium chloride preparation.

The use of a slowly released oral preparation of sodium chloride is described. It was given to patients and athletes to treat or prevent acute and chronic sodium chloride deficiency. Gastrointestinal side effects were not encountered after the ingestion of up to 500 mEq in one day or 200 mEq in 10 minutes.