Sodium Transport In Toad Bladder Research Articles

Amiloride-sensitive trypsinization of apical sodium channels. Analysis of hormonal regulation of sodium transport in toad bladder.

Incubation of the mucosal surface of the toad urinary bladder with trypsin (1 mg/ml) irreversibly decreased the short-circuit current to 50% of the initial value. This decrease was accompanied by a proportionate decrease in apical Na permeability, estimated from the change in amiloride-sensitive resistance in depolarized preparations. In contrast, the paracellular resistance was unaffected by trypsinization. Amiloride, a specific blocker of the apical Na channels, prevented inactivation by trypsin. Inhibition of Na transport by substitution of mucosal Na, however, had no effect on the response to trypsin. Trypsinization of the apical membrane was also used to study regulation of Na transport by anti-diuretic hormone (ADH) and aldosterone. Prior exposure of the apical surface to trypsin did not reduce the response to ADH, which indicates that the ADH-induced Na channels were inaccessible to trypsin before addition of the hormone. On the other hand, stimulation of short-circuit current by aldosterone or pyruvate (added to substrate-depleted, aldosterone-repleted bladders) was substantially reduced by prior trypsinization of the apical surface. Thus, the increase in apical Na permeability elicited by aldosterone or substrate involves activation of Na channels that are continuously present in the apical membrane in nonconductive but trypsin-sensitive forms.

Insulin stimulates active sodium transport in toad bladder by two mechanisms

INSULIN has been shown to increase sodium and potassium fluxes in a series of tissues including muscle1, and colon2 and the isolated perfused kidney3. Attempts to define the exact mechanisms by which insulin stimulates ion transport in these tissues have been hindered by their anatomical complexity and heterogeneous cell populations. Tracer flux studies, using 22Na, in frog muscle1 and toad bladder4 have suggested a direct stimulatory action of insulin on the sodium pump. Preincubation of intact frog muscle with insulin for 1 h has been shown to increase the amount of ouabain binding by this tissue suggesting that insulin unmasks latent sodium pumps in this structure5. The urinary bladder of the toad (Bufo marinus) has been shown to transport sodium actively from the urinary (mucosal) side to the blood (serosal) side and has been used extensively to study cation transport and the effects and mode of action of numerous hormones and drugs6–8. We have now examined the mechanisms by which insulin increases short circuit current (a measure of active sodium transport) in the toad bladder.

Regulation of protein phosphorylation and sodium transport in toad bladder.

It is well established that active sodium-ion transport and water flow across isolated toad bladder are increased by antidiuretic hormone (ADH) and by cAMP. These agents were also observed in previous studies to cause changes in the amount of radioactive phosphate in a specific protein in the toad bladder. This protein, found by SDS-polyacrylamide gel electrophoresis of toad bladder epithelial preparations, had an apparent molecular weight of 49,000 daltons. In the present study, a correlation was found between the ability of a variety of substances to affect the amount of radioactive phosphate in this 40,000-dalton protein and their ability to alter the rate of sodium transport. Thus several agents (ADH, cAMP, theophylline, adenine, prostaglandin E1, and Mn Cl-2) caused a decrease in the amount of radioactive phosphate in the 49,000-dalton protein and also stimulated active sodium transport across the bladder. Conversely, ZnCl-2 produced an increase in the amount of radioactive phosphate in this protein and an inhibition of sodium transport. With each of these agents, the time-course of change in phosphorylation of this protein was, in general, similar to that for sodium transport. A second phosphoprotein, with an apparent molecular weight of about 42,000 daltons, showed changes in parallel with, but less extensive than, those observed in the 49,000 dalton protein. There was no consistent relationship between changes in level of phosphorylation of either in the 49,000- or 42,000- dalton protein and changes in osmotic water permeability. The results are compatible with the possibility that regulation by ADH and by cAMP of sodium transport in the toad bladder epithelium may be mediated through regulation of the amount of phosphate in a specific protein.

Potassium dependence of base-line and ADH-stimulated sodium transport in toad bladder.

ARTICLESPotassium dependence of base-line and ADH-stimulated sodium transport in toad bladderSA MendozaSA MendozaPublished Online:01 Jul 1972https://doi.org/10.1152/ajplegacy.1972.223.1.120MoreSectionsPDF (1 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByTransport Across Amphibian Urinary BladderRelationships between serosal medium potassium concentration and sodium transport in toad urinary bladderThe Journal of Membrane Biology, Vol. 26, No. 1Relationships between serosal medium potassium concentration and sodium transport in toad urinary bladderThe Journal of Membrane Biology, Vol. 26, No. 1Relationships between serosal medium potassium concentration and sodium transport in toad urinary bladderThe Journal of Membrane Biology, Vol. 26, No. 1A new model for regulation of sodium transport in high resistance epitheliaMedical Hypotheses, Vol. 2, No. 3The sodium transport pool in toad urinary bladder epithelial cellsThe Journal of Membrane Biology, Vol. 20, No. 1The kinetics of sodium transport in the toad urinary bladderThe Journal of Membrane Biology, Vol. 17, No. 1 More from this issue > Volume 223Issue 1July 1972Pages 120-124 Copyright & PermissionsCopyright © 1972 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1972.223.1.120PubMed4339002History Published online 1 July 1972 Published in print 1 July 1972 Metrics

Variable factors affecting ultrafiltration of a humoral sodium transport inhibitor.

Convective ultrafiltration using commercially available artificial membranes with selective sieving properties is a widely used method of molecular separation. We had previously reported an ultrafilterable inhibitor of toad bladder sodium transport in whole canine plasma using this technique. In the present study, two variables which have large effects on ultrafiltration of the transport inhibitor were identified. A relationship between stirring speed during ultrafiltration and recovery of the inhibitor was found, such that detectable recovery of the inhibitor was shown only at high stirring speeds. These results interpreted in terms of the gel layer model of the concentration polarization phenomenon indicate a profound effect of concentration polarization on the sieving properties of the membrane. In addition, it was found that recovery of the transport inhibitor was reduced by the polypropylene membrane support supplied by the manufacturer of the ultrafiltration cell. The evidence indicates that the inhibitor adsorbs to polypropylene. The net effect of these two variables on ultrafiltration can result in both false positive and false negative bioassays for the humoral sodium transport inhibitor.

The association of a humoral sodium transport inhibitory activity with renal escape from chronic mineralocorticoid administration in the dog.

1. Previous studies in dogs given large intravenous saline infusions had shown the presence of an inhibitor of toad bladder sodium transport in jugular venous plasma. In the present study plasma of six dogs on a constant sodium intake was tested for this humoral sodium transport inhibitory (HSTI) activity before and during chronic administration of desoxycorticosterone acetate (DOCA). 2. Renal escape from mineralocorticoid antinatriuresis occurred 2–7 days after beginning treatment with DOCA. No HSTI activity was present in plasma drawn at 9 a.m. during a control period before DOCA administration but it was found in every case on the morning of the day on which escape occurred. A positive correlation was shown between HSTI activity of 9 a.m. plasma and the rate of sodium excretion for the subsequent 24 h period (r = 0·66, P < 0·002). 3. When DOCA administration was continued for several days after escape had occurred in three dogs, oscillations in HSTI activity correlated with oscillations in renal sodium excretion. 4. The degree of antinatriuresis on any given day correlated with the amount of HSTI activity the following morning (r = 0·61, P < 0·0002). This finding suggests that extracellular fluid volume expansion resulting from DOCA-induced antinatriuresis leads to HSTI activity. These results fit the hypothesis that HSTI activity is a natriuretic hormone which plays a role in the mechanism of DOCA escape.

Studies in a humoral sodium transport inhibitory activity in normal dogs and dogs with ligation of the inferior vena cava.

Plasma ultrafiltrates of dogs acutely expanded with saline inhibit toad bladder sodium transport. The present studies were performed to delineate further the chemical and physiological characterist...

Studies of a Humoral Sodium Transport Inhibitory Activity in Normal Dogs and Dogs with Ligation of the Inferior Vena Cava

Plasma ultrafiltrates of dogs acutely expanded with saline inhibit toad bladder sodium transport. The present studies were performed to delineate further the chemical and physiological characteristics of this inhibitory activity. Column chromatography with Sephadex G-10 showed the activity to be present in the fraction ahead of the salt peak. Sodium balance studies in normal dogs were performed to determine the relationship between factor activity and the daily fluctuations of renal sodium excretion. A significant correlation was observed between the presence of humoral inhibitory activity and spontaneous antinatriuresis. In the absence of antinatriuresis, inhibitory activity was absent, regardless of the level of sodium intake. In normal dogs given saline loads acutely, inhibitory activity was present 60 and 120 minutes after the beginning of saline loading. In dogs with constriction of the inferior vena cava, no activity was observed during saline loading for up to 120 minutes. The data are compatible with the interpretation that the humoral sodium transport inhibitory activity is a natriuretic hormone released in response to expansion of the extracellular fluid volume.

The kinetics of sodium transport in the toad bladder. II. Dual effects of vasopressin.

In the accompanying paper, a compartmental model for the toad bladder sodium transport system was developed. In the present paper, the model is tested by determining the effects of antidiuretic hormone on the pools and fluxes. It is shown that this hormone affects only that sodium pool previously designated as the transport pool, and that the effects are on two separate sites. In the first place, the hormone stimulates entry at the mucosal side of the transport compartment, and by this means brings about an increase in the amount of sodium contained in the compartment. Second, the hormone has a distinct stimulatory effect on the rate coefficient for efflux across the serosal boundary, the pump rate coefficient. Evidence is presented that under control conditions, the pump rate coefficient is a decreasing function of the pool size, a characteristic feature of a saturating system. Therefore, the effect of vasopressin in increasing both the pool size and the pump rate coefficient must be construed as a direct effect on the pump, and not one which is secondary to the increase in the pool size. Furthermore, it is shown that the effect of the hormone on the sodium pump is not dependent on the presence of sodium in the serosal medium.

The effect of dialysates and ultrafiltrates of plasma of saline-loaded dogs on toad bladder sodium transport.

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.

Relevance of sodium transport pool measurements in toad bladder tissue for the elucidation of the mechanism whereby hormones stimulate active sodium transport.

The size of toad bladder sodium transport pool, defined as that amount of sodium of apical origin and recovered in tissue at equilibrium, was compared with sodium transport rate, derived from short-circuit current read immediately before tissue analysis. Provided certain precautions be taken, the relationship between both variables can be described by a curve starting at the intersect ofX (pool, in μEq) andY (transport, in μEq/hr) axes, with a tendency forX to increase faster thanY. Assuming sodium transport pool forms one compartment, its calculated half-life averages 2–3 min. sodium transport pool measurements are thought to shed light on mechanism of sodium transport by toad bladder because pool size was large with respect to transport rate when tissue was exposed to several inhibitors of sodium transport. Conversely, upon stimulation of activity of (substrate — depleted) preparations by glucose, a relative reduction of pool size was observed. Aldosterone, vasopressin (and adenosine 3′,5′-phosphate) increased sodium pool size and transport rate, proportionately; insulin stimulated sodium transport more than it increased pool size. Thus, insulin presumably exerts its effect at the sodium “pump” while such a site of action need not be postulated for aldosterone and vasopressin: these 2 hormones would instead induce, permeability changes faciliting sodium movement at the apical border of toad bladder epithelial cells.

The site of the stimulatory action of vasopressin on sodium transport in toad bladder.

Vasopressin increases the net transport of sodium across the isolated urinary bladder of the toad by increasing the mobility of sodium ion within the tissue. This change is reflected in a decreased DC resistance of the bladder; identification of the permeability barrier which is affected localizes the site of action of vasopressin on sodium transport. Cells of the epithelial layer were impaled from the mucosal side with glass micropipettes while current pulses were passed through the bladder. The resulting voltage deflections across the bladder and between the micropipette and mucosal reference solution were proportional to the resistance across the entire bladder and across the mucosal or apical permeability barrier, respectively. The position of the exploring micropipette was not changed and vasopressin was added to the serosal medium. In 10 successful impalements, the apical permeability barrier contributed 54% of the initial total transbladder resistance, but 98% of the total resistance change following vasopressin occurred at this site. This finding provides direct evidence that vasopressin acts to increase ionic mobility selectively across the apical permeability barrier of the transporting cells of the toad bladder.

Inhibition of toad bladder sodium transport and Na+ plus K+ stimulated adenosinetriphosphatase by extracts of human urine: studies of patients with virilizing adrenal hyperplasia.

ABSTRACT In order to detect the presence of a possible sodium transport inhibitor, urine extracts were tested directly in 2 in vitro systems: (A) sodium transport across the urinary bladder of the toad and (B) Na++K+ stimulated adenosine triphosphatase (Na-K-ATPase). The sodium transport across the toad bladder, which remained 60 min after urine extract was added, was compared to the sodium transport before the addition and expressed as a ratio, R60. The R60 in children with virilizing adrenal hyperplasia (VAH) (R60=.33±.05) and in normal adults (R60=.39±.08) was significantly lower than normal prepubertal children (R60=.79±.03), indicating more inhibitory activity in the former 2 groups. The effect of urine extracts on Na-K-ATPase was similar to the effect on the bladder (children with VAH-46.9±7.0% of control ethanol additions, normal adults 46.8±7.7% of control, and normal children 75.6±2.6% of control). Among the children with VAH, there was no difference in the inhibitory activity of urine extracts f...

Synergistic effect of cyclopropane and epinephrine on sodium transport in toad bladder.

Synergistic effect of cyclopropane and epinephrine on sodium transport in toad bladder.

Phospholipids and active sodium transport in toad bladder

A study has been made of the phospholipids of the urinary bladder of the toad, Bufo marinus, in relation to Na + transport. The phospholipids were found to contain some 22% of the total phosphate in this tissue. The major phospholipid fractions were identified chromatographically as phosphatidyl inositide, sphingomyelin, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and phosphatidic acid. The last named compound, although quantitatively one of the least of the phospholipids had the highest rate of incorporation of 32P i. Although Na + transport was varied over a considerable range by stimulating it with vasopressin or aldosterone, on the one hand, or by inhibiting it by removing Na + from the mucosal medium or K + from the serosal medium, on the other hand, no relation between Na + transport and the levels of the phospholipids or their rates of incorporation of 32P i was observed. However, a small fraction of the phosphatidic acid phosphate (some 0.1%) appeared to turn over at a rate comparable to that of the phosphate of ATP and the available evidence does not permit assessment of a possible role of this small moiety in the active transport of Na +.

ACTIVE SODIUM TRANSPORT IN TOAD BLADDER DESPITE REMOVAL OF SEROSAL POTASSIUM.

Previous studies have demonstrated that removal of potassium from sodium-Ringer solution bathing the serosal surface of the toad badder depressed net sodium transport to some 5% of control value, whereas with choline-Ringer solution as serosal medium removal of serosal potassium depressed net sodium transport only to some 55% of control value. Although transport is down a chemical potential gradient in the latter situation, it appears to be an active process, for it is depressed by anaerobiosis, and persists against an electrochemical potential gradient. The data suggest that the concentration of potassium at the serosal aspect of the sodium pump is not in itself the rate-determining factor for active sodium transport following removal of serosal potassium.

NA-K ACTIVATED ADENOSINE TRIPHOSPHATASE AND SODIUM TRANSPORT IN TOAD BLADDER.

The effect of several agents in vitro on sodium transport by the intact toad bladder and on sodium-potassium activated, magnesium-dependent ATPase (Na-K ATPase) activity has been examined in an attempt to elucidate the relationship between the agents, the Na-K ATPase system, and active sodium transport. Ouabain and erythrophleine inhibited sodium transport and Na-K ATPase activity, but had no effect on sodium-potassium independent, magnesium-dependent ATPase (Mg-ATPase) activity. Vasopressin, adenosine 3',5'-monophosphate (cyclic-AMP), and aldosterone had no effect on Na-K ATPase or Mg-ATPase activity. The results are compatible with the assumption that the Na-K ATPase system is closely related to the active sodium pump, and that ouabain and erythrophleine inhibit sodium transport by the bladder through inhibition of the Na-K ATPase system. The stimulatory effect on sodium transport of vasopressin, cyclic-AMP, and aldosterone is not due to direct stimulation of the Na-K ATPase system.

STIMULATION OF SODIUM TRANSPORT IN TOAD BLADDER BY ACIDIFICATION OF MUCOSAL MEDIUM.

The short-circuit current, sodium flux, and electrical potential across the isolated urinary bladder of the toad were stimulated by acidification of the medium bathing the mucosal or urinary surface of the bladder. By contrast the transport of chloride, potassium, urea, and water across the bladder was unaffected by similar acidification. The pH optimum for this action was approximately 5.5. The tissue pH, as determined by the distribution of DMO-C14, was not detectably affected by mucosal acidification.