The role of mitochondria-rich cells in the chloride current conductance across toad skin

In early studies of salt transport across frog and toad skin, it was assumed that chloride movement is extracellular. However, later studies suggested that chloride movement is largely transcellular. Chloride transport across toad skin is greatly diminished in skins of salt-acclimated toads (Bufo viridis) and was correlated with the number of mitochondria-rich (m.r.) cells in the epithelium. The activated chloride conductance could be recovered upon in vitro incubation with theophylline. It was found that the short-circuit current (Isc) and the chloride conductance (Gcl) in toad skin could be separated experimentally by selective use of synthetic oxytocin (Syntocinon) or theophylline, and by substituting impermeable anions for chloride. With the use of the vibrating probe we demonstrated directly that chloride-dependent peak currents are localized only over m.r. cells, under hyperpolarized (V = -100 mV) conditions. It is concluded that the m.r. cells form the principal site for passive chloride movement across amphibian skin. This cellular pathway is regulated through a cyclic AMP-mediated process. It is suggested that the spatial separation of the sodium and chloride channels is essential to maintain the granulosum cells which are engaged in sodium transport hyperpolarized, and thus providing the driving force for the sodium entry into the cells.     

The effect of ethanol on ion transport in frog skin.

Frog skin has been used as a model epithelial sodium-transporting system to study the effect of ethanol on ion transport. Treatment of the outside of frog skin with ethanol decreased the net sodium transport due to inhibition of 22Na+ influx. Ethanol did not alter sodium outflux when bathin the outside of the skin. The inhibition was in proportion to the concentration of ethanol, 0.25 M resulting in 50% inhibition. The chloride permeability of the skin was increased several-fold when the skin was exposed to ethanol in either bathing solution. With 0.4 M ethanol in the inner bathing solution, all the unidirectional fluxes of Na+ and C1- were increased. The movement of C1- was evaluated by comparison of C1- flux with urea flux, since urea is thought to move passively across frog skin via an extracellular (shunt) pathway. Chloride flux was increased to a greater extent than urea flux. These experiments indicate that ethanol affects chloride permeability beyond an increase in extracellular ion flow and independent of its effect of Na+ transport.     

Effects of cadmium on Na+ transport in the isolated skin of the toad Pleurodema thaul

Cadmium ions applied to either (outer or inner) surface of the isolated toad skin dose-dependently increased the short-circuit current (SCC), the potential difference (V) and the active sodium conductance (G(Na)) in the concentration range 0.07-0.50mM. Maximal stimulatory effect was over 30% with an EC(50) of about 0.1mM. The effect of the highest concentration used (0.75mM) decreased considerably, and when it was applied to the inner surface (10 experiments), induced between 30% and 40% inhibition of the electric parameters in four experiments. Pretreatment with amiloride inverted the stimulatory effect of externally applied Cd(2+), suggesting competitive action on the apical Na(+) channel. The effect of noradrenaline (NA) was increased after outer application of Cd(2+) and decreased after inner application of the metal: the latter effect might be due to cadmium inhibition of the activity of Na(+),K(+)-ATPase. On the other hand, pretreatment with amiloride was followed by partial although transient reversal of its effects by serosal Cd(2+), which might be explained by action of cadmium on cytoplasmic lysine residues concerned with Na(+) channel gating. The amiloride test showed that the increment of the electric parameters was due principally to stimulation of the driving potential for Na(+) (V-E(Na(+))) and that inhibition was accompanied by a reduction in the V-E(Na(+)) and by a significant decrease in skin resistance indicating possible disruption of membrane or cell integrity. These data are in favor of the possibility that externally applied Cd(2+) activates toad skin ion transport, partly by increasing apical sodium conductance and also by stimulating the V-E(Na(+)), and that internally applied Cd(2+), with easier access to membrane and cellular constituents, may inhibit the sodium pump.     

Alteration of membrane permeability by amphotericin B

Amphotericin B (AmB) increased unidirectional Na transport and net transcellular sodium movements across the skin of the frog, Rana pipiens, when added to the solution bathing the corium side, but not from the outer epidermal surface. The AmB response was prevented with pretreatment with amiloride, ouabain and mucosal sodium substitution. Alteration in pH markedly reduced the permeability changes induced by AmB. AmB did not interfere with the increase in sodium transport induced by antidiuretic hormone. The present study demonstrates that AmB interacts with the skin of the frog, Rana pipiens, from the corium side specifically increasing transepithelial sodium transport. The increase in transport apparently occurs through the existing sodium pathway.     

The effect of ethanol on ion transport in frog skin

Frog skin has been used as a model epithelial sodium-transporting system to study the effect of ethanol on ion transport. Treatment of the outside of frog skin with ethanol decreased the net sodium transport due to inhibition of ²²Na⁺ influx. Ethanol did not alter sodium outflux when bathing the outside of the skin. The inhibition was in proportion to the concentration of ethanol, 0.25 M resulting in 50% inhibition. The chloride permeability of the skin was increased several-fold when the skin was exposed to ethanol in either bathing solution. With 0.4 M ethanol in the inner bathing solution, all the unidirectional fluxes of Na⁺ and Cl^? were increased. The movement of Cl^? was evaluated by comparison of Cl^? flux with urea flux, since urea is thought to move passively across frog skin via an extracellular (shunt) pathway. Chloride flux was increased to a greater extent than urea flux. These experiments indicate that ethanol affects chloride permeability beyond an increase in extracellular ion flow and independent of its effect on Na⁺ transport.     

Effect of sodium taurocholate and sodium cholate on short-circuit current on amphibian membranes

The effects of the bile salts, sodium taurocholate (NaTc) and sodium cholate (NaCh), and toad bile gallbladder (bile) on short-circuit current (SCC) across isolated skin, and sodium taurocholate (NaTc) on isolated bladder of Bufo arenarum toads were tested. Sodium taurocholate (NaTc), sodium cholate (NaCh) and toad bile gallbladder (bile) promoted an increase in SCC, when added to the external side. The stimulatory effect was reversible after rinsing the preparation for 60 min. Implications on in vivo renal function of these results are discussed.     

The antiepileptic drug phenytoin affects sodium transport in toad epithelium

The effects of phenytoin on isolated Pleurodema thaul toad skin were investigated. Low (micromolar) concentrations of the antiepileptic agent applied to the outside surface of the toad epithelium increased the electrical parameters (short-circuit current and potential difference) by over 40%, reflecting stimulation of Na(+) transport, whereas higher (millimolar concentrations, outside and inside surface) decreased both electric parameters, the effect being greater at the inside surface (40% and 80% decrease, respectively). The amiloride test showed that the stimulatory effect was accompanied by an increase and the inhibitory effect by a decrease in the sodium electromotive force (ENa). It is concluded that the drug interaction with membrane lipid bilayers might result in a distortion of the lipid-protein interface contributing to disturbance of Na(+) epithelial channel activity. After applying the Na(+)-K(+)-ATPase blocker ouabain and replacing the Na(+) ions in the outer Ringer's solution by choline, it was concluded that both active and passive transport are involved in sodium absorption, although active transport predominates.     

Effects of metabolic inhibitors and drugs on ion transport and oxygen consumption in isolated frog skin

Active ion (NaCl) transport across isolated frog skin is discussed in relation to sodium and potassium composition and to O(2) consumption of skin. A distinction is made between processes in skin related to "unidirectional active ion transport" and processes related to "maintenance electrolyte equilibrium;" i.e., ionic composition of skin. Several metabolic inhibitors were found that could be used in separating maintenance electrolyte equilibrium from unidirectional active ion transport. Fluoroacetate (up to 1 x 10(-2)M/liter) did not affect maintenance electrolyte equilibrium, but severely diminished the rate of active ion transport. This could also be accomplished with azide and diethyl malonate when 1 x 10(-3) molar concentrations were used. When applied in higher concentrations, these two inhibitors, and several others, diminished active ion transport, but this was associated with changes in maintenance electrolyte equilibrium (gain of Na(+) by and loss of K(+) from skin). Similar observations were made when skins were subjected to K(+)-deficient media. Mersalyl and theophylline, in low concentrations, stimulated active ion transport without leading to changes in maintenance electrolyte equilibrium. Inhibition of active ion transport was found accompanied by decrease, increase, and unaltered over-all O(2) consumption, depending on the kind of chemical agent used. A provisional scheme of the mechanism of unidirectional active ion transport is proposed. It is conceived as a process of metabolically supported ion exchange adsorption, involving a carrier, forming complexes with K(+) and Na(+), a trigger, K(+) ions, and two spatially separated metabolic pathways.     

Transport numbers in transdermal iontophoresis

Parameters determining ionic transport numbers in transdermal iontophoresis have been characterized. The transport number of an ion (its ability to carry charge) is key to its iontophoretic delivery or extraction across the skin. Using small inorganic ions, the roles of molar fraction and mobility of the co- and counterions present have been demonstrated. A direct, constant current was applied across mammalian skin in vitro. Cations were anodally delivered from either simple M(+)Cl(-) solutions (single-ion case, M(+) = sodium, lithium, ammonium, potassium), or binary and quaternary mixtures thereof. Transport numbers were deduced from ion fluxes. In the single-ion case, maximum cationic fluxes directly related to the corresponding ionic aqueous mobilities were found. Addition of co-ions decreased the transport numbers of all cations relative to the single-ion case, the degree of effect depending upon the molar fraction and mobility of the species involved. With chloride as the principal counterion competing to carry current across the skin (the in vivo situation), a maximum limit on the single or collective cation transport number was 0.6-0.8. Overall, these results demonstrate how current flowing across the skin during transdermal iontophoresis is distributed between competing ions, and establish simple rules with which to optimize transdermal iontophoretic transport.     

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.     

NaCl adaptation in Rana ridibunda and a comparison with the euryhaline toad Bufo viridis.

The physiological adaptation of the frog Rana ridibunda to saline environment was studied. It was found that blood was always hypertonic to the external solution, but at the highest salinity tolerated (i.e. 300 mOsM) the osmotic gradient across the skin was nearly abolished. Water uptake by the living frog remained unchanged, whereas sodium transport across the skin decreased markedly. Neurohypophyseal hormone increased water uptake and sodium transport to levels similar to those in tap water frogs. Water content of the tissues was not affected by saline adaptation, although it varied appreciably under acute conditions. Oxygen consumption increased in dehydrated frogs, but not in adapted ones. The results are discussed and compared to the euryhaline toad Bufo viridis; the importance of high urea levels for high salt adaptation is stressed.     

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 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.     

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.     

Nonequilibrium Thermodynamic Analysis of the Coupling between Active Sodium Transport and Oxygen Consumption

Sodium transport and oxygen consumption have been simultaneously studied in the short-circuited toad skin. A constant stoichiometric ratio was observed in each skin under control condition (NaCl-Ringer's solution bathing both sides of the skin) and after block of sodium transport by ouabain. During alterations of sodium transport by removal and addition of K to the internal solution the stoichiometric ratio is constant although having a value higher than that observed in other untreated skins. The coupling between active sodium transport and oxygen consumption was studied after a theoretical nonequilibrium thermodynamic model. Studies were made of the influence of Na chemical potential difference across the skin on the rates of Na transport and oxygen consumption. A linear relationship was observed between the rates of Na transport and oxygen consumption and the Na chemical potential difference. Assuming the Onsager relationship to be valid, the three phenomenological coefficients which describe the system were evaluated. Transient increases in the rate of sodium transport and oxygen consumption were observed after a transitory block of sodium transport by removal of Na from the external solution. Cyanide blocks completely the rate of oxygen consumption in less than 2 min and the short-circuit current measured after that time decays exponentially with time, suggesting a depletion of ATP from a single compartment.     

Effects of Pb<Superscript>2+</Superscript> ions on Na<Superscript>+</Superscript> transport in the isolated skin of the toad <Emphasis Type="Italic">Pleurodema thaul</Emphasis>

The effects induced by lead ions on the short-circuit current (SCC) and on the potential difference (V) of the toad Pleurodema thaul skin were investigated. Pb²⁺ applied to the outer (mucosal) surface increased SCC and V and when applied to the inner (serosal) surface decreased both parameters. The stimulatory effect, but not the inhibitory action, was reversible after washout of the metal ion. The amiloride test showed that the increase was due principally to stimulation of the driving potential for Na⁺ (V-E_Na+) and that inhibition was accompanied by a reduction in the V-E_Na+ and also by a significant decrease in skin resistance indicating possible disruption of membrane and/or cell integrity. The effect of noradrenaline was increased by outer and decreased by inner administration of Pb²⁺. The results suggest that mucosal Pb²⁺ activates toad skin ion transport by stimulating the V-E_Na+ and that serosal Pb²⁺, with easier access to membrane and cellular constituents, inactivates this mechanism, revealing greater toxicity when applied to the inner surface of the skin. Abbreviations: SCC – short-circuit current; V – potential difference; V-E_Na+– driving potential for Na⁺; ENaC – epithelial sodium channel; R_Na+– active sodium resistance; RS – passive or shunt resistance; G_Na– active sodium conductance; GS – passive or shunt conductance; Gmax – total conductance; EC₅₀– half-maximal excitatory concentration; IC₅₀– half maximal inhibitory concentration; NA – noradrenaline.     

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.     

Metabolic evidence that serosal sodium does not recycle through the active transepithelial transport pathway of toad bladder

Summary The possibility that sodium from the serosal bathing medium back-diffuses into the active sodium transport pool within the mucosal epithelial cell of the isolated toad bladder was examined by determining the effect on the metabolism of the tissue of removing sodium from the serosal medium. It was expected that if recycling of serosal sodium did occur through the active transepithelial transport pathway of the isolated toad bladder, removal of sodium from the serosal medium would reduce the rate of CO₂ production by the tissue and enhance the stoichiometric ratio of sodium ions transported across the bladder per molecule of sodium transport dependent CO₂ produced simultaneously by the bladder (J _Na/J _{CO 2}). The data revealed no significant change in this ratio (17.19 with serosal sodium and 16.13 after replacing serosal sodium with choline). Further, when transepithelial sodium transport was inhibited (a) by adding amiloride to the mucosal medium, or (b) by removing sodium from the mucosal medium, subsequent removal of sodium from the serosal medium, or (c) addition of ouabain failed to depress the basal rate of CO₂ production by the bladder [(a) rate of basal, nontransport related, CO₂ production (J _CO2 ^b ) equals 1.54±0.52 with serosal sodium and 1.54±0.37 without serosal sodium; (b)J _CO2 ^b equals 2.18±0.21 with serosal sodium and 2.09±0.21 without serosal sodium; (c) 1.14±0.26 without ouabain and 1.13±0.25 with ouabain; unite ofJ _CO2 ^b are nmoles mg d.w.^–1 min^–1]. The results support the hypothesis that little, if any, recycling of serosal sodium occurs in the toad bladder.     

Variation in the effects of antidiuretic hormone on the isolated skin of the toad, Scaphiopus couchi.

The rate of active sodium transport as measured by short-circuit current across the isolated skin of the toad, Scaphiopus couchi, was elevated following vasopressin (0.2 units/ml) or arginine vasotocin (0.1 units/ml) treatment of skins from active animals at all times of the year tested. Skins from dormant animals showed no such elevation at any time of the year. The rate of active sodium transport was elevated following treatment with dibutyryl cyclic AMP (2.5mM) plus theophylline (10 mM) in all skins tested. The hydraulic conductivity of isolated skins from both active and dormant animals showed no significant change following treatment with vasopressin (0.2 units/ml) or arginine vasotocin (0.1 units/ml except on the first day following emergence from dormancy in the field. A correlation was, therefore, observed between the occurrence of a hydroosmotic response to antidiuretic hormones and the seasonal exposure of S. couchi to standing water. A small but significant elevation of hydraulic conductivity was observed across the skins of dormant toads following treatment with dibutyryl cyclic AMP (2.5 mM) plus theophylline (10 mM) whereas a substantial elevation was observed with the skins of active animals.