Transport In Toad Bladder Research Articles

Extracellular Protons Regulate Human ENaC by Modulating Na+ Self-inhibition

The epithelial Na(+) channel, ENaC, is exposed to a wide range of proton concentrations in the kidney, lung, and sweat duct. We, therefore, tested whether pH alters ENaC activity. In Xenopus oocytes expressing human alpha-, beta-, and gammaENaC, amiloride-sensitive current was altered by protons in the physiologically relevant range (pH 8.5-6.0). Compared with pH 7.4, acidic pH increased ENaC current, whereas alkaline pH decreased current (pH(50) = 7.2). Acidic pH also increased ENaC current in H441 epithelia and in human primary airway epithelia. In contrast to human ENaC, pH did not alter rat ENaC current, indicating that there are species differences in ENaC regulation by protons. This resulted predominantly from species differences in gammaENaC. Maneuvers that lock ENaC in a high open-probability state ("DEG" mutation, proteolytic cleavage) abolished the effect of pH on human ENaC, indicating that protons alter ENaC current by modulating channel gating. Previous work showed that ENaC gating is regulated in part by extracellular Na(+) ("Na(+) self-inhibition"). Based on several observations, we conclude that protons regulate ENaC by altering Na(+) self-inhibition. First, protons reduced Na(+) self-inhibition in a dose-dependent manner. Second, ENaC regulation by pH was abolished by removing Na(+) from the extracellular bathing solution. Third, mutations that alter Na(+) self-inhibition produced corresponding changes in ENaC regulation by pH. Together, the data support a model in which protons modulate ENaC gating by relieving Na(+) self-inhibition. We speculate that this may be an important mechanism to facilitate epithelial Na(+) transport under conditions of acidosis.

Effect of carbenoxolone on glucocorticoid metabolism and Na transport in toad bladder

In humans, diminished 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) enzyme activity has been associated with sodium retention and hypertension. These studies show that the toad bladder, another target tissue epithelium displaying steroid-induced sodium transport, possesses the enzyme 11 beta-OHSD. The toad urinary bladder rapidly transformed corticosterone (3 x 10(-8) M) (50% by 10 min and 90% by 180 min) with 11-dehydrocorticosterone being the major metabolite. The 11-dehydrocorticosterone produced reached an apparent plateau when the tissue incubations were repeated with higher concentrations of corticosterone (10(-7) and 10(-6) M). Carbenoxolone sodium (2.5 x 10(-5) M), a water soluble derivative of glycyrrhetinic acid, markedly inhibited the metabolism of corticosterone (3 x 10(-8) M) to 11-dehydrocorticosterone similar to previous observations in the mammalian kidney. Carbenoxolone sodium (2.5 x 10(-5) M) did not significantly affect short-circuit current (SCC) in toad bladders when added to either the serosal or mucosal bath. However, when carbenoxolone sodium was added to the mucosal bath and 60 min later corticosterone 10(-6) M was placed in the serosal bath, bladders generated a SCC 2.07 +/- 0.17 (mean +/- SE) times above base line at 360 min compared with 1.48 +/- 0.11 in bladders exposed to corticosterone alone (P less than 0.02). In parallel experiments, carbenoxolone sodium in the mucosal bath enhanced the rise in SCC induced by cortisol 10(-6) M; 1.66 +/- 0.16 times above base line at 360 min compared with 1.07 +/- 0.14 with cortisol alone (P less than 0.02). We conclude that the toad bladder contains 11 beta-OHSD and inhibition of this enzyme with carbenoxolone sodium is associated with amplification of glucocorticoid-induced transepithelial sodium transport in this tissue. However, since the quantity of 11-dehydro-product produced appears to be limited, other factors in addition to inhibition of 11 beta-OHSD may play a role in this amplification of sodium transport.

Aldosterone increases the apical Na+ permeability of toad bladder by two different mechanisms.

The aldosterone-induced augmentation of Na+ transport in toad bladder was analyzed by comparing the hormonal actions on the transepithelial short-circuit current and on the amiloride-sensitive 22Na+ uptake in isolated membrane vesicles. Incubating bladders with 0.5 microM aldosterone for 3 hr evoked more than a 2-fold increase of the short-circuit current (because of the activation or insertion of apical amiloride-blockable channels) but had no effect on the amiloride-sensitive Na+ transport in apical vesicles derived from the treated tissue. A longer incubation (e.g., 6 hr) produced an additional augmentation of the short-circuit current, which was accompanied by about a 3-fold increase of the channel activity in isolated membranes. The stimulatory effect of aldosterone sustained in vesicles was inhibited by the antagonist spironolactone (present at 1000-fold excess) and the protein synthesis inhibitor cycloheximide (1 microM). In addition, triiodothyronine and butyrate, previously reported to partly inhibit the aldosterone-induced increase in short-circuit current, blocked the hormonal effect in vesicles. It is suggested that aldosterone elevates the apical Na+ permeability of target epithelia by two different mechanisms: a relatively fast effect (less than or equal to 3 hr), which is insensitive to triiodothyronine or butyrate and is not sustained by the isolated membrane, and a slower or later (greater than 3 hr) response blocked by these reagents, which is preserved by the isolated membrane. The data also indicate that these processes are mediated by different nuclear receptors.

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.

Activation energy for water transport in toad bladder.

Activation energies (Ea) for water movement across vasopressin-(ADH) sensitive epithelia have been reported to be about 10 kcal/mol (1, 12). The present study shows that measurements of Ea for osmotic water flow across toad bladders are unreliable, because a temperature change induces marked alterations in membrane permeability to water within a 2.5-min interval. Thus bladders equilibrated with ADH either at room temperature or at 33 degrees C and then suddenly subjected to a lower temperature were found to exhibit a marked increase in membrane permeability to water. This observation suggests that there is a rapid turn-over of water permeability sites and that sudden exposure to cold inhibits the removal more than the induction of sites by ADH. To stabilize ADH-induced water channels for Ea measurements, bladders were exposed to ADH at room temperature, fixed with glutaraldehyde, and subjected to osmotic gradients at different temperatures. The Ea values for osmotic water flow across these ADH-permeabilized, glutaraldehyde-fixed bladders were 5.1 (4-12 degrees C), 4.3 (12-21 degrees C), 3.6 (21-36 degrees C), and 3.6 kcal/mol (30-38 degrees C). Ea values for shear viscosity of water in these temperature ranges were calculated to be 4.7, 4.2, 4.1, and 3.6 kcal/mol, respectively. The close correlation between Ea values for bulk water viscosity and osmotic water flow across the bladder wall suggests that an equivalent number of hydrogen bonds must be broken to achieve an increase in water flow through ADH-induced channels and an increase in fluidity of water in bulk solution.

Quercetin enhances water transport in toad bladder.

A highly significant enhancement of the hydrosmotic actions both of vasopressin and of exogenous cAMP was seen in the presence of quercetin. The hypothesis is advanced that quercetin affects the intracellular coupling between Ca++ in cAMP.

Comparison of effects of standard diuretics and indanone in isolated toad cornea and bladder.

Short-circuit current (SCC) techniques were used to monitor effects of various diuretic agents on Na+ transport in toad bladder and Cl- transport in toad cornea. In bladder, various agents from different "classes" of diuretics inhibited SCC whereas in cornea only "loop diuretics," i.e., those with a primary site of action in the ascending limb of the loop of Henle, inhibited SCC. Classification of the diuretics based on sensitivity in cornea and bladder revealed that diuretics with a common renal site of action gravitated into common classification groups. Thus, our studies suggested that cornea may be a suitable model of the thick ascending limb of the loop of Henle and that cornea and bladder, studied jointly, may serve as an in vitro system for predicting potential renal sites of action of new diuretics. This system, when used to characterize the new diuretic indanone, revealed that this agent, in all respects, displayed the characteristics of a loop diuretic.

Removal of ambient K+ inhibits net Na+ transport in toad bladder by reducing Na+ permeability of the luminal border

Sodium crosses epithelia in two steps: an influx into the cell through the luminal membrane down an electrochemical gradient, and an energy-requiring efflux across the serosal border against a gradient. Koefoed-Johnsen and Ussing1 originally proposed that cellular efflux of Na+ at the serosal border of frog skin is coupled to K+ influx by an exchange pump. It was, therefore, assumed that the inhibition of active Na+ transport following the removal of K+ from the serosal side was due to a reduced pool of K+ available for the coupled exchange. Indirect evidence suggests that this is not correct. (1) K+ influx from the serosa does not correlate to any significant degree with active Na+ transport2–5. (2) Removal of Na+ as well as K+ from the serosal medium substantially reduces the inhibitory effect of K+ removal on active transport2,6. (3) One would predict that if K+ removal did inhibit active transport only at the serosal pump site, then as active transport decreased the ‘active transport pool’, that is, the mucosally derived cell Na+, would increase as it does with ouabain. Studies by Robinson and Macknight7, however, showed that the mucosally derived pool does not change on removal of external K+. Essig and Leaf2 suggested that removal of serosal K+ inhibited Na+ transport by decreasing the permeability of the luminal membrane. They showed that when K+ was removed from the serosa, there was a decrease in the amount of 22Na taken up into the tissue after exposure of the luminal surface to the isotope for 5 min. We have recently found that tissue uptake of 22Na after exposure of toad bladders to the isotope is linear with time for only up to 24 s. Because only the linear portion of the uptake is a measure of the Na+ flux across the luminal border8, it is clear that longer periods of uptake are more representative of the steady-state isotope distribution in the tissue. This value is controlled not only by the luminal permeability but also by the serosal permeability and the rate of net transport. These issues prompted us to re-investigate the effect of low external K+ on Na+ movement across the luminal border. We report here that the Na+ transport inhibition seems to be due to a reduced Na+ permeability at the luminal border.

Thyroid hormone-aldosterone interaction on Na+ transport in toad bladder.

Repeated administration of thyroxine (T4) in vivo (2 microgram/100 g body wt for 6 days) lowered by 2.3 times (P less than 0.025, df = 18) the basal rate of Na+ transport measured by the short-circuit current (SCC) in vitro in the urinary bladder of the toad (Bufo marinus). This difference was not accounted for by a change in the plasma aldosterone concentration. Moreover the response of the SCC to aldosterone in vitro was markedly inhibited in bladders from T4-treated animals (P less than 0.001, df = 18). These findings raised the possibility of a direct interaction between thyroid hormone and aldosterone in the target cell. The effects of L-triiodothyronine (T3) and aldosterone were examined in vitro. T3 alone (60 nM) had no significant effect on the base-line SCC (deltamuA = -14 +/- 11 (SE) muA per hemibladder; P greater than 0.3, n = 10). By contrast, T3 (60 nM) inhibited the response of the SCC to aldosterone from 6 to 8 h after its addition (deltamuA = -98 +/- 19 muA per hemibladder; P less than 0.001, n = 10). The inhibition by T3 was present at 6 nM (P less than 0.01, n = 10) and became not significant at 0.6 nM. T3 had no significant effect on base-line or aldosterone-stimulated H+ transport. Thyroid hormone might therefore regulate the late response of the SCC to aldosterone at the level of its target cell.

Barium inhibition of sodium ion transport in toad bladder

The effect of Ba 2+ on Na + transport and electrical characteristics of toad bladder was determined from change produced in short circuit current ( I sc, epithelial, apical and basal-lateral potentials ( ψ t, ψ a, ψ b), epithelial and membrane resistances ( R t, R a, R b) and shunt resistance ( R s). Mucosal Ba 2+ had no effect. Serosal Ba 2+ reduced I sc, ψ t, ψ a, and ψ b, but had no effect on R t, R a, R b and R s. Minimal effective Ba 2+ concentration was 5 · 10 −5 M. The phenomenon was reversed by Ba 2+ removal, but not by 86 mM serosal K +. Ba 2+ inhibition of I sc did not impair the response to vasopressin which was quantitatively the same as controls. ψ a with Ba 2+ equalled ψ b. After Ba 2+ inhibition, ouabain produced no further decrease in ψ t and I sc. Ba 2+ exposure after ouabain did not decrease ψ t and I sc. The results suggest that Ba 2+ inhibits the basal-lateral electrogenic Na + pump.

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.

The effect of catecholamines on ion transport in the toad bladder

Noradrenalin (8 · 10 −6 M) and adrenalin (6 · 10 −6 and 6 · 10 −7 M) were found to cause marked stimulation of short-circuit current (S.C.C.) in isolated toad bladder, but isoprenalin (8 · 10 −7 M) was found to be without effect. The percentage rise in S.C.C. due to noradrenalin was found to be inversely proportional to the initial S.C.C. or total conductance of the bladder. Again in the case of noradrenalin the rise in S.C.C. was almost completely abolished by α-adrenergic blockade but not by β-blockade. This rise in S.C.C. was found not to be significantly different from the rise in net Na + flux. Bidirectional Cl − fluxes were estimated using 82Br as a companion radionuclide to 36Cl. No significant net Cl − flux was apparent, either before or after addition of any of the three catecholamines tested. However, in some cases the unidirectional Cl − fluxes rose markedly following addition of noradrenalin or of adrenalin and this change was not reflected in a change in total conductance. This anomaly was noted to occur in bladders whose initial conductance was of the order of 0.5 kΩ −1 · cm −2 or greater. The evidence presented suggests that two actions of catecholamines on ion transport in toad bladder are (a) to increase Na + transport via stimulation of α-adrenergic sites and (b) at the concentrations tested to cause an increase in passive Cl permeability in bladders whose initial conductance is high.

Evidence for an apical membrane effect in the regulation of the hexose monophosphate shunt pathway in toad bladder studies with amiloride

Aldosterone stimulates Na + transport in toad bladder and, simultaneously with a coincident dose-response relationship, inhibits the hexose monophosphate shunt pathway. Amiloride, an acylguanidine diuretic, inhibits sodium transport when applied to the apical surface of the bladder. In this study amiloride was found to partially reverse the inhibitory effect of aldosterone on the hexose monophosphate shunt pathway. The amiloride effect upon glucose metabolism was detected when it was applied to both surfaces of the bladder simultaneously, in flask experiments, and when it was applied to the apical surface. No effect of amiloride on the shunt pathway was detected when it was applied to the serosal surface only, even at very high concentrations. It may be, but has not been proven, that the effects of aldosterone and amiloride on the hexose monophosphate shunt pathway are mediated by a common site at the apical membrane.

Regulation of phosphorylation of a specific protein in toad-bladder membrane by antidiuretic hormone and cyclic AMP, and its possible relationship to membrane permeability changes.

Phosphorylation of a specific protein was decreased in intact toad bladders by exposure to either antidiuretic hormone or monobutyryl cyclic AMP. The decrease in phosphorylation caused by these agents preceded the change in electrical potential difference (an indicator of the rate of sodium ion transport) observed in response to the same compounds. The addition of cyclic AMP to homogenates of toad bladder led to a decrease in phosphorylation of the same, or a similar, protein. In subcellular fractionation studies, the effect of cyclic AMP on the phosphorylation of this protein was observed in those fractions rich in membrane fragments, but not in the nuclear or cell-sap fractions. These and other results are compatible with the possibility that the regulation by vasopressin and cyclic AMP of sodium and/or water transport in toad bladder may be mediated through regulation of the phosphorylation of this 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

On the similarity of effects of aldosterone and adenosine 3′,5′-phosphate on Na + transport and glucose metabolism in toad bladder

The stimulation of Na + transport in the toad bladder by aldosterone is associated with an increased utilization of oxygen and an increase in the evolution of 14CO 2 from [6- 14C]glucose in response to the increased energy requirement of stimulated Na + transport. These are secondary effects of aldosterone and are abolished by the removal of Na + from the mucosal bathing medium and consequent absence of Na + transport. At the same time, however, aldosterone decreases the evolution of 14CO 2 from [1- 14C]glucose. This effect persists even in the absence of Na + and has similar characteristics of time of onset, concentration response, steroid specificity, and sensitivity to actinomycin D and spirolactone. Both the effect of aldosterone to decrease the evolution of 14CO 2 from [1- 14C]-glucose and its ability to stimulate Na + transport can be reproduced by adenosine 3′,5′-phosphate (cyclic AMP). This has been shown for endogenous cyclic AMP produced by the action of theophylline or vasopressin in combination with theophylline and for exogenous cyclic AMP as seen with N 6-2′- O-dibutyryl adenosine 3′,5′-phosphate (dibutyryl cyclic AMP) or with cyclic AMP in the presence of low concentrations of theophylline. Although these results suggest that cyclic AMP might mediate the action of aldosterone, no change in tissue concentration of cyclic AMP was found even after prolonged incubation with the hormone.

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.

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 +.