Conductive chloride flux across amphibian skin: inhibition by indacrinone and cobalt ion.

When amphibian skin was incubated under conditions in which transepithelial sodium transport was abolished, a conductive transepithelial Cl- flux arose when Cl- was removed from one of the compartments. This flux was matched by short-circuit current and it accounted entirely for transepithelial conductance. Cl- influx was larger than efflux; it was linearly related to the magnitude of transepithelial Cl- concentration difference. When applied to the epithelial surface of the tissue, divalent metal cations such as Co2+, and the ethacrynic acid derivative, indacrinone, reduced rapidly and reversibly both transepithelial Cl- (in)flux and short-circuit current. Frog skin proved to be more sensitive to these inhibitors than toad skin. Further characterization of transepithelial Cl- pathway(s) should benefit from the fact that Cl- across amphibian skin can easily be monitored by the short-circuit current method, and from the availability of agents which inhibit this passive flux rapidly and reversibly.     

Active sodium transport by the isolated toad bladder

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, Na²⁴ and Na²², 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.     

The Electrical Characteristics of Active Sodium Transport in the Toad Bladder

The mechanism responsible for active sodium transport in the urinary bladder of the toad appears to be located at the serosal boundary of the epithelial cell layer of the bladder. Studies of the potential step observed at the serosal boundary in the open-circuited state were undertaken in an attempt to define the factors responsible for its production. Glass micropipettes were used to measure the serosal potential step in bladders exposed on the serosal side to solutions of high potassium or of high potassium and low chloride concentration. Observed potentials exceed the maximum values which would have been expected if the serosal potential step were a potassium or chloride diffusion potential. Measurements of net cation flux exclude the possibility of a diffusion potential at this border due to the passive movement of any anionic species. The observed independence of transbladder potential and short-circuit current from the pH of the serosal medium over a wide range of pH makes it unlikely that the observed serosal potential step is a hydrogen ion diffusion potential. We conclude that the active sodium transport mechanism in toad bladder is "electrogenic."     

Cellular lithium and transepithelial transport across toad urinary bladder

Summary Toad urinary bladders were exposed on either their mucosal or serosal surfaces, or on both surfaces, to medium in which sodium was replaced completely by lithium. With mucosal lithium Ringer's, serosal sodium Ringer's, short-circuit current (SCC) declined by about 50 percent over the first 60 min and was then maintained over a further 180 min. Cellular lithium content was comparable to the sodium transport pool. With lithium Ringer's serosa, SCC was abolished over 60 to 120 min whether the mucosal cation was sodium or lithium. Measurements of cellular ionic composition revealed that the epithelial cells gained lithium from both the mucosal and serosal media. With lithium Ringer's mucosa and serosa, cells lost potassium and gained lithium and a little chloride and water, but these changes in cellular ions could not account for the current flow across the tissue under these conditions, which must, therefore, have been carried by a transepithelial movement of lithium itself. The inhibition by serosal lithium of SCC was overcome by exposure of the mucosal surface of the bladders to amphotericin B. Thus it reflected, predominantly, an inhibition of lithium entry to the cells across the apical membrane. It is suggested that this inhibition is a consequence of cellular lithium accumulation.     

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.     

Regulation of the sodium permeability of the luminal border of toad bladder by intracellular sodium and calcium: role of sodium-calcium exchange in the basolateral membrane

Sodium movement across the luminal membrane of the toad bladder is the rate-limiting step for active transepithelial transport. Recent studies suggest that changes in intracellular sodium regulate the Na permeability of the luminal border, either directly or indirectly via increases in cell calcium induced by the high intracellular sodium. To test these proposals, we measured Na movement across the luminal membrane (th Na influx) and found that it is reduced when intracellular Na is increased by ouabain or by removal of external potassium. Removal of serosal sodium also reduced the influx, suggesting that the Na gradient across the serosal border rather than the cell Na concentration is the critical factor. Because in tissues such as muscle and nerve a steep transmembrane sodium gradient is necessary to maintain low cytosolic calcium, it is possible that a reduction in the sodium gradient in the toad bladder reduces luminal permeability by increasing the cell calcium activity. We found that the inhibition of the influx by ouabain or low serosal Na was prevented, in part, by removal of serosal calcium. To test for the existence of a sodium- calcium exchanger, we studied calcium transport in isolated basolateral membrane vesicles and found that calcium uptake was proportional to the outward directed sodium gradient. Uptake was not the result of a sodium diffusion potential. Calcium efflux from preloaded vesicles was accelerated by an inward directed sodium gradient. Preliminary kinetic analysis showed that the sodium gradient changes the Vmax but not the Km of calcium transport. These results suggest that the effect of intracellular sodium on the luminal sodium permeability is due to changes in intracellular calcium.     

Effect of 5-hydroxytryptamine on sodium transport across bullfrog skin

Summary 5-hydroxytryptamine, when present in the solution bathing the inside surface of bullfrog skin at concentrations of 0.25–25.0 mM, reduced both electrical potential difference and short-circuit current across the skin. The magnitude of reduction in potential difference and short-circuit current was dependent on 5 HT concentration. Reduction in sodium influx entirely accounted for the reduction in short-circuit current. Preliminary evidence suggested a competition between 5 HT and vasopressin in the production of their effects on sodium transport across the skin, while high Ca⁺⁺ concentrations and 5 HT seemed to act independently of each other.Dr. Henry C. and Bertha H. Buswell Fellow.     

The Origin of the Short-Circuit Current in the Isolated Skin of the South American Frog Leptodactylus ocellatus

In isolated skins of Leptodactylus ocellatus the short-circuit current is smaller than the sodium net flux and this difference disappears when the skins are bathed in solutions in which the chloride ions have been replaced by sulfate or methylsulfate ions. There is a net movement of chloride ions from outside to inside of the skins in the short-circuit condition with chloride Ringer's solutions bathing the skins. The addition of ouabain to the inside solution markedly reduced not only sodium net flux but also the chloride net influx found. Copper ions added to the outside solutions produced a rise in short-circuit current, as well as the known increase in potential difference. In sodium-free Ringer's (sodium replaced by choline) the orientation of the potential difference across the skins was reversed, the inside being negative instead of positive. The results are interpreted as direct or indirect indications of the presence of a net transfer of chloride ions from outside to inside of these frog skins.     

THE EFFECT OF CALCIUM WITHDRAWAL ON THE STRUCTURE AND FUNCTION OF THE TOAD BLADDER

Previous reports have indicated that calcium is necessary to support active sodium transport by the toad bladder, and may be required as well in the action of vasopressin on both toad bladder and frog skin. The structure and function of the toad bladder has been studied in the absence of calcium, and a reinterpretation of the previous findings now appears possible. When calcium is withdrawn from the bathing medium, epithelial cells detach from one another and eventually from their supporting tissue. The short-circuit current (the conventional means of determining active sodium transport) falls to zero, and vasopressin fails to exert its usual effect on short-circuit current and water permeability. However, employing an indirect method for the estimation of sodium transport (oxygen consumption), it is possible to show that vasopressin exerts its usual effect on Q_oo2 when sodium is present in the bathing medium. Hence, it appears that the epithelial cells maintain active sodium transport when calcium is rigorously excluded from the bathing medium, and continue to respond to vasopressin. The failure of conventional techniques to show this can be attributed to the structural alterations in the epithelial layer in the absence of calcium. These findings may provide a model for the physiologic action of calcium in epithelia such as the renal tubule.     

Water and sodium transport: effects of calcium channel blocker and calmodulin antagonists on the apical and basolateral membranes of amphibian epithelia

Ca2+ channel blocker (sensit) and calmodulin antagonists (thioridazine, perphenazine, oxyprothepine) applied to the mucosal side of frog urinary bladder, weakened the response of epithelial cells to vasopressin. Thioridazine (2.7 X 10(-5) mol X l-1) and sensit (1.7 X 10(-4) mol X l-1) applied to the serosal side rapidly increased the permeability of the epithelia for sodium and potassium ions along the concentration gradient (from serosa to mucosa). The same concentrations of these blockers when applied to the mucosal side of frog urinary bladder selectively decreased vasopressin stimulated water permeability and did not influence ionic permeability. Both thioridazine and sensit decreased the short-circuit current across frog skin. The results show that the Ca2+ channel blocker and the calmodulin antagonists tested influenced water and ionic transport across the epithelial cell membranes, and had different effects upon the apical and the basolateral cell membranes.     

Na+ and K+ transport at basolateral membranes of epithelial cells. I. Stoichiometry of the Na,K-ATPase

The stoichiometry of pump-mediated Na/K exchange was studied in isolated epithelial sheets of frog skin. 42K influx across basolateral membranes was measured with tissues in a steady state and incubated in either beakers or in chambers. The short-circuit current provided estimates of Na+ influx at the apical membranes of the cells. 42K influx of tissues bathed in Cl- or SO4-Ringer solution averaged approximately 8 microA/cm2. Ouabain inhibited 94% of the 42K influx. Furosemide was without effect on pre-ouabain-treated tissues but inhibited a ouabain-induced and Cl--dependent component of 42K influx. After taking into account the contribution of the Na+ load to the pump by way of basolateral membrane recycling of Na+, the stoichiometry was found to increase from approximately 2 to 6 as the pump-mediated Na+ transport rate increased from 10 to 70 microA/cm2. Extrapolation of the data to low rates of Na+ transport (less than 10 microA/cm2) indicated that the stoichiometry would be in the vicinity of 3:2. As pump-mediated K+ influx saturates with increasing rates of Na+ transport, Na+ efflux cannot be obligatorily coupled to K+ influx at all rates of transepithelial Na+ transport. These results are similar to those of Mullins and Brinley (1969. Journal of General Physiology. 53:504-740) in studies of the squid axon.     

Effect of oleamide on water and sodium ion transport in osmoregulatory organs of vertebrates

In the frog Rana temporaria L., oleamide solution (10 μmole/L) applied to the isolated basal surface of the skin augmented the short-circuit current (SCC) from 59.8 ± 2.5 to 78.2 ± 1.4 μA/cm². When applied to the serous membrane of the urinary bladder, oleamide (1 μmole/L) induced more than a 30-fold increase in osmotic water permeability. The addition of argininevasotocin against the background of oleamide further increased SCC across the skin and osmotic water permeability in the bladder. In Wistar rats, intraperitoneal injection of oleamide (0.1 μmole/L per 100 g of body weight) to non-anesthetized animals after water load reduced diuresis by 22% and increased solute-free water reabsorption and urinary sodium excretion by 31% and 55%, respectively, but did not affect urinary potassium excretion. These findings provide evidence of the similarity between the effects of oleamide and nonapeptide neurohypophyseal hormones on water and ion transport in epithelial cells of osmoregulatory organs in vertebrates.     

Interactive effects of ethanol and silver on sodium transport across toad skin

Both ethanol and silver ions have been shown to affect ion transport across various epithelia. This investigation was principally undertaken to further define mechanisms of silver ions and ethanol, and their possible interactions, on sodium transport across toad skin. Isolated toad skin, mounted between identical oxygenated amphibian bicarbonate Ringer solutions, maintained stable transepithelial potential differences (serosa positive) and short-circuit currents for several hours at 25 degrees C. It was observed that (1) ethanol inhibited the active transcellular component of sodium absorption and this effect was reversible; (2) inhibition of sodium transport by ethanol was directly proportional to the applied concentration; (3) pretreatment with silver ions prevented any ethanol effects; and (4) pretreatment with ethanol prevented any silver ion effects. It was concluded from these results that ethanol induced its inhibitory effects on membrane phospholipids thereby perturbing the function of a sulfhydryl ligand, while silver ion or silver chloride complex binding to this ligand would maintain its function in sodium transport despite the presence of ethanol.     

Benzodiazepines stimulate sodium ion transport in frog skin epithelium

Benzodiazepine binding sites are present in a variety of non-neuronal tissues including the kidney where they are localized to distal nephron segments. It is postulated that renal binding sites are involved in modulating ion transport. This study examined the effects of two benzodiazepines on sodium transport in frog skin epithelium, a model system for sodium transport in renal collecting duct. Treatment of short-circuited frog skin with diazepam (a non-selective benzodiazepine agonist) stimulated amiloride-sensitive short-circuit current, reflecting stimulation of active sodium transport. The diazepam response was equally effective with either serosal or mucosal application of the drug. Maximal stimulation of the current (42 +/- 8%) was achieved with 10 microM diazepam (serosal). Short-circuit current was similarly augmented by serosal or mucosal addition of Ro5-4864, a benzodiazepine agonist with selective activity at peripheral (non-neuronal) receptors. The natriferic response to diazepam was additive to that of vasopressin or cyclic AMP suggesting that the mode of action of benzodiazepines is probably distinct from the cyclic AMP pathway. Thus, frog skin appears to be a useful model to examine the epithelial effects of benzodiazepines. Whether stimulation of sodium transport, however, involves peripheral-type benzodiazepine receptors in this tissue requires further studies.     

Movement of Sodium Across the Mucosal Surface of the Isolated Toad Bladder and its Modification by Vasopressin

Studies have been made on the isolated urinary bladder of the toad, Bufo marinus, in an attempt to evaluate gradients of chemical activity across the mucosal surfaces of the epithelial cells which would serve to maintain a net movement of sodium from the mucosal medium into the cells. The likelihood of such chemical gradients has been established by the demonstration of lower contents of sodium within the tissue, expressed as microequivalents per gram of tissue water, than of concentrations of sodium in the mucosal medium at all levels of the latter examined. The transepithelial transport of sodium and the sodium content of the tissue were found to increase rapidly with rise in concentration of sodium in the mucosal medium up to values of 30 to 60 meq per liter. Further increase in concentration of the medium above this value failed to induce further stimulation of sodium transport or increase in the sodium content of the tissue. Vasopressin increased the rate of transport of sodium at every concentration of sodium in the mucosal medium without altering this relationship. Although entry of sodium across the mucosal surface of the epithelial cells may be passive it is not by free diffusion but involves some considerable interaction with the mucosal surface of the bladder and constitutes the major determinant of the rate of transepithelial transport of sodium. Vasopressin acts to enhance this initial step in the transport of sodium.     

Dependence of ion transport across the plasma membrane on cell culture density. II. Active and passive cation transport during the growth of L cell cultures

Rubidium and lithium influxes as well as intracellular potassium and sodium contents were investigated in L cells during the culture growth. In sparse culture over the cell densities 0.5-3 X 10(4) cells/cm2 ouabain-sensitive rubidium influx is small and ouabain-resistant lithium influx in high. With the increase in culture density up to 4-5 X 10(4) cells/cm2 the active rubidium influx, mediated by ouabain-sensitive component, is enhanced, and ion "leakage" tested by lithium influx is diminished. Simultaneously with the exponential growth of culture the intracellular potassium content is increased and the intracellular sodium content is decreased resulting in the higher K/Na ratio in cell. During the further transition to dense culture and in stationary state (10-17 X 10(4) cells/cm2) the sodium content and lithium influx do not change significantly, but the potassium content is decreased. The decrease in intracellular potassium is correlated with that in the portion of cells in S-phase from 27-30 to 12%. Thus, in transformed cells the density-dependent alterations in membrane cation transport are observed.     

Thyroid hormone. Aldosterone antagonism in cultured epithelial cells

Thyroid hormone (T3) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have examined the effect of T3 on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5 X 10(-8) M T3 to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T3 completely inhibits the maximal increase in short-circuit current in response to 1 X 10(-7) M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T3 and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5 X 10(-9) M T3. T3 has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T3 on nuclear binding of [3H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K'd1 = (0.82 +/- 0.36) X 10(-10) M and K'd2 = (3.2 +/- 0.60) X 10(-8) M. The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1 X 10(-8) M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5 X 10(-8) M T3 showed a K'd1 = (0.15 +/- 0.10) X 10(-10) M and K'd2 = (3.5 +/- 0.10) X 10(-8) M. We conclude that T3 inhibits the action of aldosterone on sodium transport at a site after receptor binding in the nucleus.     

Ion transport across the frog olfactory mucosa: the basal and odorant-stimulated states

The Ussing method was adapted to study the basal electrolyte transfer as well as the events that occur upon odorant stimulation in frog olfactory mucosa. The unstimulated short-circuit current was due mainly to a furosemide-sensitive ion transport system on the apical side of the olfactory mucosa. This current was not amiloride sensitive. The current-voltage relationship of the unstimulated state was linear. That of the odorant-evoked current was non-linear and amiloride-sensitive. Ouabain caused collapse of both the unstimulated and odorant-stimulated short-circuit current. In this case, voltage-clamping the tissue to non-zero values restored the odorant-evoked current with polarity depending on that of the clamping voltage. This suggested that the direction of the current is determined by that of the sodium electrochemical potential difference. Our results indicate that the unstimulated short-circuit current occurs through an apical sodium cotransport system, while the odorant-evoked current is due to odorant-activated, passive sodium channels that are amiloride sensitive.     

Characteristics of rat jejunal transport of tryptophan.

The parameters of rat jejunal transport of tryptophan have been examined. The interactions between tryptophan and lysine or methionine have been reexamined, and some aspects of the trans effects of cellularly accumulates amino acids have been studied. It has been demonstrated that: (1) The influx of tryptophan across the jejunal brush border (Jmc-Trp) can be accounted for by the carrier of alpha-aminomonocarboxylic acids alone. (2) Tryptophan competes with lysine for the carrier of basic amino acids across the brush border membrane without itself being transported by this carrier. (3) Lysine has neither cis nor trans effects on Jmc-Trp, whereas intracellular tryptophan is highly inhibitory to Jmd-Lys. (4) The intracellular concentration of lysine and of tryptophan, [Lys]c and [Trp]c, are unaffected by tryptophan and lysine, respectively, although the transmural fluxes, from the mucosal side to the serosal side, Jms, of lysine, Jms-Lys, and of tryptophan, Jms-Trp, are inhibited by tryptophan and lysine, respectively. The latter effects thus represent inhibitory interactions at the basolateral membrane. (5) Methionine is a potent cis and transinhibitor of Jmc-Trp, but stimulated Jms-Trp and reduces [Trp]c. (6) Methionine causes trans acceleration of the influx of lysine across the brush border membrane, Jmc-Lys, but has no effect on the influx of galactose, Jmc-Gal. (7) Leucine causes trans inhibition of Jmc-Leu. (8) Tryptophan does not cause cis inhibition of Jmc-Gal, but is a strongtransinhibitor of Jmc-Gal. (9) Cellularly accumulated tryptophan appears to accelerate the eventual decline in transepithelial potential difference and short-circuit current. These results are consistent with the conclusions that: (1) Tryptophan is transported across the brush border membrane by the carrier of neutral amino acids alone, but leaves the cell across the basolateral membrane by a mechanism used by lysine also. (2) Leucine, methionine and probably tryptophan have a transeffect on the transport of neutral amino acids across the brush border membrane which may represent a phenomenon which can appropriately be termed decelerating exchange diffusion. (3) Cellularly accumulated tryptophan has a strong and indiscriminate depressive effect on all transport functions of rat jejunal epithelium.     

Muscosal and serosal effects of insulin on ion contents of the frog urinary bladder.

When applied in vitro at the serosal border of the bladder of Rana temporaria insulin (1.7.10(-5) M) brings about a decrease in the tissue sodium content, suggesting a stimulation of the pump extruding sodium ions from epithelial cells. On the other hand, the application of insulin at the two sides of the bladder results in a significant increase of the sodium content of the tissue. It is hence concluded that the contact of the hormone with the mucosal membranes of the epithelial cells of the bladder enhances sodium entry across the membranes. The effect if so pronounced that it obscures the stimulation of the pumps localized at the opposite pole of the cells.