The effect of dialysates and ultrafiltrates of plasma of saline-loaded dogs on toad bladder sodium transport
01 May 1970-Journal of Clinical Investigation (American Society for Clinical Investigation)-Vol. 49, Iss: 5, pp 926-935
TL;DR: In this article, the authors used selective molecular filters which permit passage of substances on the basis of molecular weight and three dimensional configuration to obtain direct evidence for the existence of a natriuretic hormone.
Abstract: In order to obtain direct evidence for the existence of a natriuretic hormone, dialysates and ultrafiltrates of plasma of dogs expanded with saline were tested for effects on sodium transport by the toad urinary bladder. Dialysate was obtained by dialysis of blood in vivo in a clinical dialyzer and by dialysis in vitro of small volumes of blood using a miniature model of the clinical dialyzer. Ultrafiltrates were prepared using selective molecular filters which permit passage of substances on the basis of molecular weight and three dimensional configuration.
Dialysates and ultrafiltrates of hydropenic dogs caused a change in toad bladder potential difference of + 1% and in short circuit current of - 5%. In contrast, dialysates and ultrafiltrates from expanded dogs caused a change in potential difference of - 23% and in short circuit current of - 32%, a highly significant difference. Onset of reduction of short circuit current occurred within 3-5 min, reaching a maximum in 10-20 min. The effect was rapidly reversible, was specific for the serosal surface of the bladder, and could not be explained on the basis of nonspecific alterations in ionic composition or by dilutional effects. Ultrafiltrates of jugular vein plasma caused significantly more reduction of short circuit current than ultrafiltrates of femoral vein plasma. The data indicate the presence in plasma of saline-loaded dogs of a dialyzable inhibitor of toad bladder sodium transport. Ultrafiltrate studies using membranes of appropriate selectivity suggest the factor has a molecular weight of less than 3000.
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TL;DR: It is concluded that the urine from the subjects when salt-loaded contains a natriuretic substance.
1,756 citations
TL;DR: In this article, a human ouabain-like compound (OLC) was identified by mass spectroscopy as an endogenous substance from human plasma that binds with high affinity to the digitalis glycosides and their aglycones.
Abstract: The plasma membrane sodium-potassium pumps that regulate intracellular sodium in most animal cells have specific, high-affinity receptors for the digitalis glycosides and their aglycones. This has fostered speculation that there is an endogenous ligand. We have purified and structurally identified by mass spectroscopy an endogenous substance from human plasma that binds with high affinity to this receptor and that is indistinguishable from the cardenolide ouabain. This human ouabain-like compound (OLC) displaces [3H]ouabain from its receptor, inhibits Na,K-ATPase and ouabain-sensitive 86Rb+ uptake, and has cardiotonic actions quantitatively similar to commercial ouabain. Immunoreactive OLC was detected in the plasma of many mammals, and high concentrations were found in the adrenals. The circulating OLC may modulate intracellular Na+ and affect numerous Na+ gradient-dependent processes including intracellular Ca2+ and pH homeostasis in many tissues. Furthermore, altered circulating levels of OLC may be associated with the pathogenesis of certain forms of hypertension.
749 citations
TL;DR: The physiological interactions between CTS and other regulatory systems that may be important in the pathophysiology of essential hypertension, preeclampsia, end-stage renal disease, congestive heart failure, and diabetes mellitus are focused on.
Abstract: Endogenous cardiotonic steroids (CTS), also called digitalis-like factors, have been postulated to play important roles in health and disease for nearly half a century. Recent discoveries, which include the specific identification of endogenous cardenolide (endogenous ouabain) and bufadienolide (marinobufagenin) CTS in humans along with the delineation of an alternative mechanism by which CTS can signal through the Na+/K+-ATPase, have increased the interest in this field substantially. Although CTS were first considered important in the regulation of renal sodium transport and arterial pressure, more recent work implicates these hormones in the regulation of cell growth, differentiation, apoptosis, and fibrosis, the modulation of immunity and of carbohydrate metabolism, and the control of various central nervous functions and even behavior. This review focuses on the physiological interactions between CTS and other regulatory systems that may be important in the pathophysiology of essential hypertension, preeclampsia, end-stage renal disease, congestive heart failure, and diabetes mellitus. Based on our increasing understanding of the regulation of CTS as well as the molecular mechanisms of these hormone increases, we also discuss potential therapeutic strategies.
470 citations
TL;DR: The preliminary isolation of a substance in plasma of dogs which competes with digoxin for two specific digoxin antibodies and is an inhibitor of (Na+ + K+) ATPase activity is reported, suggesting it is the putative NH.
Abstract: Considerable evidence supports the hypothesis that renal sodium excretion is partly regulated by a humoral agent released or activated by extracellular fluid volume (ECFY) expansion1, including the demonstration of biological activities in extracts of plasma and urine which support the natriuretic hormone (NH) hypothesis2–4. NH is a low molecular weight peptide formed from a precursor in plasma5, which inhibits sodium transport (anti-natriferic activity) in isolated toad bladder, an analogue of the distal renal tubule and collecting duct. Other evidence indicates that NH can inhibit renal (Na++ K+) ATPase6, which suggests that it might be an endogenous digitalis-like substance. Although an immunoreactive ouabain-like substance has been demonstrated in amphibian plasma7, none has been reported in mammals. To identify an endogenous digitalis-like substance, we used an approach suggested by the work of Spector and his collaborators, who demonstrated that biological substances which bind to the same receptors as drugs, such as endogenous opioids8 and benzodiazepines9, may compete with antibodies specific for the drugs. Hough and Edwardson10 showed that antibodies to thaumatin, a sweet-tasting plant protein, also bind non-protein sweet substances in a linearly related fashion to the sweetness of the compound, suggesting that the antibody binding site may be similar to the sweet taste receptor. Based on these observations, we postulated that if NH were an endogenous digitalis-like substance, it might bind to antibodies specific for digitalis glycosides. We report here the preliminary isolation of a substance in plasma of dogs which competes with digoxin for two specific digoxin antibodies and is an inhibitor of (Na+ + K+) ATPase activity. Increased amounts of this factor are found in plasma of volume-expanded dogs, suggesting it is the putative NH.
438 citations
TL;DR: New physiological approaches using genetically engineered mice are being used to define the biological significance of the "receptor function" of the Na,K-ATPase and its regulation by potential endogenous cardiotonic steroid-like compounds.
Abstract: The Na,K-ATPase is the membrane "pump" that generates the Na(+) and K(+) gradients across the plasma membrane that drives many physiological processes. This enzyme is highly sensitive to inhibition by cardiotonic steroids, most notably the digitalis/ouabain class of compounds, which have been used for centuries to treat congestive heart failure and arrhythmias. The amino acids that constitute the ouabain-binding site are highly conserved across the evolutionary spectrum. This could be fortuitous or could result from this site being conserved because it has an important biological function. New physiological approaches using genetically engineered mice are being used to define the biological significance of the "receptor function" of the Na,K-ATPase and its regulation by potential endogenous cardiotonic steroid-like compounds. These studies extend the reach of earlier studies involving the biochemical purification of endogenous regulatory ligands.
276 citations
References
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TL;DR: The present author has been able to obviate the difficulties of the Hawkins and Van Slyke method and yet retain the principle involved, and the resulting method is simple, easy, and accurate.
1,428 citations
624 citations
TL;DR: The short-circuit current of the isolated toad bladder was regularly stimulated with pure oxytocin and vasopressin when applied to the serosal surface under aerobic and anaerobic conditions.
Abstract: Studies were made of the active ion transport by the isolated urinary bladder of the European toad, Bufo bufo, and the large American toad, Bufo marinus. The urinary bladder of the toad is a thin membrane consisting of a single layer of mucosal cells supported on a small amount of connective tissue. The bladder exhibits a characteristic transmembrane potential with the serosal surface electrically positive to the mucosal surface. Active sodium transport was demonstrated by the isolated bladder under both aerobic and anaerobic conditions. Aerobically the mean net sodium flux across the bladder wall measured with radioactive isotopes, Na(24) and Na(22), just equalled the simultaneous short-circuit current in 42 periods each of 1 hour's duration. The electrical phenomenon exhibited by the isolated membrane was thus quantitatively accounted for solely by active transport of sodium. Anaerobically the mean net sodium flux was found to be slightly less than the short-circuit current in 21 periods of observation. The cause of this discrepancy is not known. The short-circuit current of the isolated toad bladder was regularly stimulated with pure oxytocin and vasopressin when applied to the serosal surface under aerobic and anaerobic conditions. Adrenaline failed to stimulate the short-circuit current of the toad bladder.
308 citations
TL;DR: A completely automated method for the simultaneous or individual determination of calcium and phosphorus in serum and urine without preliminary sample treatment is reported and Reproducibility and recovery data as well as comparisons with manual methods are presented.
Abstract: A completely automated method for the simultaneous or individual determination of calcium and phosphorus in serum and urine without preliminary sample treatment is reported. Separation of calcium and phosphorus from interfering materials is achieved by dialysis under acid conditions. The dialyzed calcium is determined colorimetrically using a metal complexing dye (cresolphthalein complexone) in an alkaline medium. Magnesium, phosphate, citrate, sulfate, etc., did not introduce significant errors under the test conditions. Inorganic phosphorus is determined by a modified Fiske-SubbaRow procedure. Reproducibility and recovery data as well as comparisons with manual methods are presented.
287 citations
TL;DR: Evidence is afforded that peritubular capillary colloid osmotic pressure is one important determinant of proximal sodium reabsorption, and it is apparent that mechanisms other than or in addition to this must be invoked to explain the delayed inhibition of re absorption that accompanies expansion of extracellular fluid volume by colloid solutions.
Abstract: The relationship between peritubular capillary protein concentration and rate of sodium reabsorption by the rat proximal tubule was examined using free-flow recollection micropuncture techniques. Tubule fluid-to-plasma inulin ratios were measured before, during, and at successive intervals after brief (15-25 sec) intra-aortic injections (at the level of the renal artery) of colloid-free, isoncotic, and hyperoncotic solutions. Arterial hematocrit and protein concentrations were measured simultaneously in these rats. In other rats, total protein concentration of peritubular capillary blood plasma was determined before, during, and after these same infusions with a newly described submicroliter fiber-optic colorimeter.
In the 15-25 sec interval necessary to infuse 2 ml of these test solutions, fractional and absolute sodium reabsorption varied directly with peritubular capillary colloid osmotic pressure, declining during infusion of colloid-free solutions, increasing during hyperoncotic infusions, and remaining unchanged during isoncotic infusions.
In the subsequent 20-min interval after intra-aortic injection of these test solutions, capillary protein concentration remained at (isoncotic infusions) or returned to (colloid-free and hyperoncotic fluids) control values. Whereas reabsorption after colloid-free solutions returned to base line levels in parallel with the return in capillary protein concentration, after colloid infusions (which resulted in continued expansion of extracellular fluid volume), a progressive decline in reabsorption was observed.
These results afford strong evidence that peritubular capillary colloid osmotic pressure is one important determinant of proximal sodium reabsorption. Nevertheless it is apparent that mechanisms other than or in addition to this must be invoked to explain the delayed inhibition of reabsorption that accompanies expansion of extracellular fluid volume by colloid solutions.
285 citations