J.F. Cloix

Measurement of endogenous Na+,K+-ATPase inhibitors in human plasma and urine using high-performance liquid chromatography.

This study was undertaken to assess endogenous Na+,K+‐ATPase inhibitors in both plasma and urine in the same subjects. Samples were chromatographed on reverse‐phase HPLC using an acetonitrile gradient and the eluent screened using Na+,K+‐ATPase inhibition and cross‐reaction with anti‐digoxin antibodies. The donors were divided into inhibiting and non‐inhibiting subjects using a previously described method, plasma action on ouabain binding and on Na+,K+‐ATPase activity. Three Na+,K+‐ATPase inhibitors (IP, 2P and 3P) were detectable in plasma; the antibodies cross‐reaction of the peaks 2P and 3P were larger than that of peak IP. The peaks 2P and 3P were significantly higher in inhibiting subjects as compared to non‐inhibiting subjects. The 24‐h urine is resolved into two peaks inhibiting Na+,K+‐ATPase activity (1U and 2U). Peak 2U cross‐reacted with anti‐digoxin antibodies to a greater extent than peak 1U and is significantly larger in inhibiting subjects in terms of Na+,K+‐ATPase inhibition. These data support the heterogeneity of human Na+,K+‐ATPase inhibitor in both plasma and urine.

Circulating Digitalis-Like Compounds in Essential Hypertension

Inhibitors of the Na+ pump have been proposed as participating in sodium excretion, extracellular fluid regulation, and in the rise of blood pressure. The presence of digitalis-like compounds in human plasma has been investigated by comparing the effects of plasma extracts to those of ouabain in 4 tests. - competition with ouabain for binding to the Na+ pump, - inhibition of Na+ and K+ dependent hydrolysis - inhibition of serotonin uptake by human platelets - central hypertensive effect Plasma fractions exhibited digitalis-like properties in the 4 tests. The effects of plasma extracts of 42 normotensive subjects (21 with family history of hypertension) and 38 patients with essential hypertension (15 with antihypertensive treatment) and 9 patients with chronic renal failure were compared. Plasma from Forty per cent of untreated hypertensive patients and normotensives with hypertensive heredity had a high inhibition level. Inhibition was enhanced in beta-blocker treated patients and decreased in those on diuretics. No digitalis-like activity was observed in uremic plasma. These observations strongly suggest the presence of digitalis-like compound(s) in human plasma and point to its possible association with hypertension.

Purification from human plasma of endogenous dodium transport inhibitor(s)

Na+, K+-ATPase inhibitors extracted from plasma of healthy human subjects displaced3H-ouabain binding to human erythrocytes and inhibited the Na+ efflux catalyzed by the Na+, K+-pump and unexpectedly the Na+, K+-cotransport system without alteration of the Na+, Na+-exchange or the Na+ passive permeability. This suggests the presence in healthy human plasma of endogenous factors with ouabain-like and furosemide-like activities.

Affinity chromatography for human Na+, K+-ATPase inhibitors in plasma and urine

Semi-purified dog kidney Na+,K+-ATPase cross-linked with ovalbumin was used in batch-wise affinity chromatography for the detection of endogenous Na+,K+-ATPase inhibitor in human plasma and urine. Ammonium acetate 1 M washed off the endogenous inhibitor from the immobilized enzyme. The inhibitory activity of the eluate from hypertensive plasma and urine was significantly higher (p less than 0.0025, n = 5 and p less than 0.005, n = 6 respectively) than that of normotensive. This latter was correlated with the ability of plasma from the same subjects to compete with ouabain binding to erythrocytes. Plasma and urine extracts inhibited the activity of Na+, K+-ATPase in a dose-dependent manner as ouabain does and were shown to contain 3 or 4 active compounds by high pressure liquid chromatography. The activity of some of these compounds was lost after peptidase treatment. These data support the heterogeneity of endogenous inhibitors of Na+,K+-ATPase activity in plasma and urine.

25-Hydroxycholecalciferol-binding protein: Partial purification from rat duodenal mucosa cells

Abstract The 25-hydroxycholecalciferol-binding protein has been partially purified (purification factor: 37) from rat duodenal cytosol, using chromatographic procedures on gel and ionic exchange resin. This partially purified protein bound 25-hydroxycholecalciferol with high affinity (KD = 5.7 × 10−9M) and low binding capacity (23 × 10−12 mole/mg of protein. Using a rabbit antiserum obtained against such partially purified protein, we demonstrated that 25-hydroxycholecalciferol cytosolic binder and 25-hydroxycholecalciferol plasmatic binding share common antigenic sites.

Chemical and Clinical Studies of Endogenous Digitalis‐Like Factor in Hypertension

Endogenous digitalis-like factor (endalin) was investigated by measuring the ability of rat and human plasma and urine to inhibit [3H]ouabain-specific binding, digoxin-antidigoxin antibodies interaction, and renal Na+, K+-ATPase activity. Endalin was detected in plasma (and urine) of one third of 112 patients with sustained and moderate hypertension (Na+ intake = 110 mmol/l). Endalin tended to be increased in the more pronounced hypertensives. No correlation with any other clinical and biological parameter could be detected. An activity to inhibit Na+, K+-ATPase was also detected in the rat after acute and chronic Na+ loading, in reduced renal mass-type hypertension and in SHRs as compared to WKY rats. Comparison of the plasma and urine inhibitory effects in the different tests revealed some chemical heterogeneity. However, a compound possessing the biochemical and pharmacological characteristics of digitaline was extracted from human urine. Chromatographic and spectral analysis of about 1,000 liters revealed a compound with apparent chemical homogeneity, molecular weight around 500, devoid of peptidic bound and of aliphatic structure.

Recent Advances on Endogenous Na+K+-ATPase Inhibitors: Clinical Investigation and Purification

Evidence exists which demonstrates the relationship between a Natriuretic Factor or Na+,K+-ATPase inhibitor and volemic expansion, both in man and animal. Patients having extracellular volume expansion have been studied for the effect of their plasma on erythrocytes 3H-ouabain binding. High levels of ouabain-like activity was found in plasma from acromegalic patients and patients with chronic renal failure. High levels were also observed in some hypertensive patients. A partial purification of such a compound was performed from urine of hypertensives. The partially purified compound inhibited to a greater extent the Na+,K+-ATPase semi-purified from dog kidney than that from sheep brain. The present data are consistent with the possible regulation of the activity or the secretion of plasma ouabain-like activity by extracellular volume.

Electrophoretical characterization of red blood cell membrane proteins and plasma protein in spontaneously hypertensive rat

Abstract The red blood cell membrane proteins and plasma proteins of normal and spontaneously hypertensive rats were studied by uni- and bidimensional polyacrylamide gel electrophoresis. The amount of band 3, the major intrinsic protein of the erythrocyte membrane, was observed to be significantly reduced in spontaneously hypertensive rats. Plasma from these rats contained two additional heat stable proteins, characterized by a molecular weight of 16,000 daltons and isoelectric points of 4.7 and 5.1, respectively. These proteins were not detected in normotensive control Wistar Kyoto rats, in normal Wistar rats, or in Wistar rats with experimentally induced hypertension.

Biochemical, Physiological and Pathological Properties of Endogenous Digitalis-Like Compounds

Sodium and water homeostasis are regulated by intrarenal physical and hormonal factors and by extrarenal hormones. The latter include factors retaining Na+ and water, and factors eliminating Na+ while the sodium balance is positive. There is a relationship between arterial blood pressure, Na+ homeostasis and body fluid volumes (1), and an increase in blood pressure may result from the incapacity of the kidney to eliminate a Na+ and water loading (2). This renal defect in the excretion of Na+ leads to an extracellular volume expansion which in turn, increases the activity or the concentration of a natriuretic hormone through hypothalamic stimulation (2).