Direct measurement of uptake of sodium at the outer surface of the frog skin

A combination of the methods described by Schultz et al. (6) and by Ussing and Zerahn (9) was used to measure directly the unidirectional uptake of sodium from the outside solution into the frog skin, under short-circuit conditions. The sodium uptake was determined at six sodium concentrations ranging from 3.4 to 114 mM. NaCl was replaced by choline chloride in the solutions bathing both sides of the skin. Sodium uptake is not a linear function of sodium concentration but appears to be composed of two components, a saturating one and one that varies linearly with concentration. The sodium uptake is inhibited by the addition of lithium to the outside solution. The effect appears to be primarily on the saturating component and has the characteristics of competitive inhibition. In addition, lithium uptake by the skin is inhibited by sodium. The effects of lithium cannot be ascribed to changes in electrical potential difference. Measurements with microelectrodes indicate that under short-circuit condition there is no change in the intracellular potential when lithium chloride is added to the outside solution.     

Sodium influx at the outer surface of frog skin

Summary The unidirectional sodium influx across the outside surface of the frog skin epithelium was measured. The method was identical to the one described by Biber and Curran [4] except that mannitol instead of inulin was used as the indicator for the amount of tracer containing test solution remaining on the surface of the skin after blotting. The space of distribution for³H-mannitol is about twice as large as the corresponding space for inulin after 32-sec exposure to the tracer. At a sodium concentration of 6.7mm the sodium influx determined with inulin as marker for the test solution is 0.146 Equiv hr^–1cm^–2 larger than the influx measured in the same preparation with mannitol. This difference increases in proportion to the sodium concentration and can be ascribed to diffusion of sodium into a space which is accessible to mannitol but not to inulin during a 30-sec interval. Hence, nearly two-thirds of the previously described linear component of the sodium influx proceeds, as was suspected previously, into a compartment which is not directly connected to net sodium transport across the skin. The nature of the remaining one-third of this linear compartment is not clear but previous experiments suggest that it is also not involved in net sodium transport.     

Effect of changes in transepithelial transport on the uptake of sodium across the outer surface of the frog skin

The unidirectional sodium, uptake at the outer surface of the frog skin was measured by the method described by Biber and Curran (8). With bathing solutions containing 6 mM NaCl there is a good correlation between sodium uptake and short-circuit current (SCC) measured simultaneously except that the average uptake is about 40% higher than the average SCC. The discrepancy between uptake and SCC increases approximately in proportion to an increase in sodium concentration of the bathing solutions. Amiloride inhibits the unidirectional sodium uptake by 21 and 69% at a sodium concentration of 115 and 6 mM, respectively. This indicates that amiloride acts on the entry step of sodium but additional effects cannot be excluded. The sodium, uptake is not affected by 10^-4 M ouabain at a sodium concentration of 115 mM but is inhibited by 40% at a sodium concentration of 6 mM. Replacement of air by nitrogen leads to a 40% decrease of sodium uptake at a sodium concentration of 6 mM. The results support the view proposed previously (8) that the sodium uptake is made up of two components, a linear component which is, essentially, not involved in transepithelial movement of sodium and a saturating component which reflects changes in transepithelial transport. Amiloride, seems largely to affect the saturating component.     

Does intracellular sodium regulate sodium transport across the mucosal surface of frog skin?

A method has been devised to functionally remove the serosal membrane of frog skin. Skins treated in this way have no spontaneous potential. However, if sodium gradients are placed across the tissues diffusion potentials and hence short-circuit currents of either sign, depending on the direction of the gradient, could be recorded. These short-circuit currents were completely imhibited by amiloride only from the mucosal face. However, the concentration of amiloride causing 50% inhibition of the short-circuit curent (Km) in treated skins was 2.3 . 10(-3)M, when a sodium gradient was applied from serosa to mucosa, whereas both in untreated skins without a sodium gradient and in treated skins with a mucosal to serosal sodium gradient, the Km of amiloride was 2 . 10(-7)-4 . 10(-7)M. The mechanism by which amiloride is able to inhibit the short-circuit currents of either sign is discussed.     

Chemical stimulation of Na+ current through the outer surface of frog skin epithelium

A number of organic molecules were found to increase the Na⁺ permeability of the Na⁺-selective membrane in frog skin epithelium quickly and reversibly when added to the outer bathing solution. The most effective was benzoylimidazole-guanidine. This substance stimulates the Na⁺ current by preventing the decrease of Na⁺ permeability which is normally caused by Na⁺ at the outer surface of the Na⁺-selective membrane.     

The relationship of K+ efflux at the inner surface of the isolated frog skin epithelium to the short circuit current

Isolated frog skins (without chorion) were incubated with ⁴²K⁺ Ringer's solution, bathing the internal surface for 2 h.All the K⁺ contained in the frog skin was equilibrated in specific activity with external ⁴²K⁺.The kinetics of the washout of ⁴²K⁺ from the internal surface of the skin exhibits one fast and one slow exponential component.Amiloride reduces the release of ⁴²K⁺ corresponding to both components without affecting the K⁺ content of the skin.Ouabain increases the loss of ⁴²K⁺ of the slow component by 200%. Since the total K⁺ in the skin decreases to 25% of its original value both compartments are affected.The results suggest that two distinct functional compartments exist defined by two ⁴²K⁺ release ratios and that because of the large K⁺ contents of these compartments both are intracellular.The relation with the transepithelial Na⁺ transport and the morphological identification of these compartments is discussed.     

Cationic selectivity and competition at the sodium entry site in frog skin

The cation selectivity of the Na entry mechanism located in the outer membrane of the bullfrog (Rana catesbeiana) skin epithelium was studied. This selectivity was determined by measuring the short-circuit current when all of the external sodium was replaced by another cation and, also, by noting the relative degree of inhibition that the alkali metal cations produced on Na influx. The ability of the Group Ia cations to permeate the apical membrane was determined from the tracer uptake experiments. The results demonstrate that (a) only Li and Na are actively transported through the epithelium; (b) the alkali cations K, Rb, and Cs do not enter the epithelium through the apical border and, therefore, Na and Li are the only alkali cations translocated through this membrane; (c) these impermeable cations are competitive inhibitors of Na entry; (d) the cations NH4 and Tl exhibit more complex behavior but, under well-defined conditions, also inhibit Na entry; and (e) the selectivity of the cation binding site is in the sequence Li congruent to Na > Tl > NH4 congruent to K > Rb > Cs, which corresponds to a high field strength site with tetrahedral symmetry.     

The relationship of K+ efflux at the inner surface of the isolated frog skin epithelium to the short circuit current.

Isolated frog skins (without chorion) were incubated with 42K+ Ringers' solution, bathing the internal surface for 2 h. All the K+ contained in the frog skin was equilibrated in specific activity with external 42K+. The kinetics of the washout of 42K+ from the internal surface of the skin exhibits one fast and one slow exponential component. Amiloride reduces the release of 42K+ corresponding to both components without affecting the K+ content of the skin. Ouabain increases the loss of 42K+ of the slow component by 200%. Since the total K+ in the skin decreases to 25% of its original value both compartments are affected. The results suggest that two distinct functional compartments exist defined by two 42K+ release ratios and that because of the large K+ contents of these compartments both are intracellular. The relation with the transepithelial Na+ transport and the morphological identification of these compartments is discussed.     

Amiloride and calcium effect on the outer barrier of the frog skin

Summary Amiloride (0.1mm) as well as Ca⁺⁺ (10mm) inhibit Na⁺ transport across frog skin by blocking Na⁺ entrance across the outer barrier of the epithelium. The inhibition produced by amiloride consists of an early and a late phase which together account for almost a total inhibition of the short-circuit current (SCC). The analysis of the time course indicates that the two phases are due to the inhibition of superficially and deeply located Na sites, respectively. Ca⁺⁺, instead, only blocks a fraction of the SCC, and this fraction seems to correspond to the inhibition of the same population of Na sites blocked by the late phase of amiloride effect. The location of the two populations of Na sites as well as the possible relationship between them are discussed in terms of maturation of the outermost cell layer.     

Cell sodium activity and sodium pump function in frog skin

Summary Cell Na activity,a _Na ^c , was measured in the short-circuited frog skin by simulaneous cell punctures from the apical surface with open-tip and Na-selective microelectrodes. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular conductance, with NaNO₃ Ringer on the apical surface. Under control conditionsa _Na ^c averaged 8±2mm (n=9,sd). Apical addition of amiloride (20 m) or Na replacement reduceda _Na ^c to 3mm in 6–15 min. Sequential decreases in apical [Na] induced parallel reductions ina _Na ^c and cell current,I _c . On restoring Na after several minutes of exposure to apical Na-free solutionI _c rose rapidly to a stable value whilea _Na ^c increased exponentially, with a time constant of 1.8±0.7 min (n=8). Analysis of the time course ofa _Na ^c indicates that the pump Na flux is linearly related toa _Na ^c in the range 2–12mm. These results indicate thata _Na ^c plays an important role in relating apical Na entry to basolateral active Na flux.     

Acidification and sodium entry in frog skin epithelium

Acidification of the external medium by isolated frog skin epithelium (Rana catesbeiana, Rana temporaria, and Caudiververa caudiververa) and its relationship to Na⁺ uptake was studied. Acidification was measured by the pH-stat technique under short-circuit or open-circuit conditions. The results of this study demonstrate that (a) acidification by these species of in vitro frog skins is not directly coupled to Na⁺ or anion transport; (b) acidification can be inhibited by the diuretic drug amiloride, but only at high external Na⁺ concentrations; (c) acidification rate in these species of frog skin is controlled in part by the metabolic production of CO₂; and (d) the positive correlation between net Na⁺ absorption and net acidification observed in whole animal studies could not be replicated in the in vitro skin preparation, even when the frogs were first chronically stressed by salt depletion, a physiological state comparable to that used in the in vivo experiments.     

Influx and efflux by leaves ofVallisneria spiralis

Summary A critical survey is given of the active uptake processes in order to have a basis for comparing the passage through the plasmatic membranes by non-electrolytes and by electrolytes (aminoacids and salts).According to Höfler's Two pathways theory the lipid path introduces lipophilic substances by diffusion through the lipids and lipoproteins of the membranes and the cytoplasm into the vacuoles. The other pathway leads non-lipophilic substances through pores in the membranes and is more susceptible to outer and inner conditions influencing the rate of permeation through the pores.Collander assumed that as a consequence of the presence of lipids in the cell membranes their permeability is so small that non-lipophilic substances enclosed in the different compartments of the cell do not show a noticeable efflux.The object of this investigationwas to establish whether inVallisneria leaves an influx of salts (chloride ions) occurs without a simultaneous efflux.In normal healthy tissue permeability of the plasma-membranes is so low that no efflux can be shown during 22 hours' uptake.For measuring the presence or absence of efflux of chloride ions during uptake a method was developed comparing the amount of labelled chloride ions absorbed with the increase of the total amount of chloride ions estimated by chemical methods.     

Effect of acetazolamide on sodium transport through the isolated frog skin at low and high external sodium concentrations.

The action of acetazolamine on sodium transport in $\text{Rana esculenta}$ skin was studied with the external face bathed in dilute (2mMM) or concentrated (Ringer) solutions of sodium chloride.The absorption of Na⁺ from a dilute solution is inhibited at an acetazolamide concentration of 10⁻⁵M. This is due to an inhibition of the influx: the efflux remains unchanged. Acetazolamide has no effect, however, on transport from Ringer solution.The graphic determination of the Na⁺ transport pool at the 2 mM NaCl concentration showed that acetazolamide diminished the pool without affecting the $\text{t}\text{1}\text{2}$. The inhibitor had no effect on the pool at the higher (Ringer) concentration.These results indicate that acetazolamide acts on the external barrier of the sodium transport compartment without affecting the active pump of this ion when it is being transported from a dilute sodium chloride solution.     

Barriers to sodium movement across frog skin

Summary The aim of this paper is to obtain information on the number, nature and location of the barriers to Na movement across the frog skin, and on the size and location of the Na-pool that might be contained between these barriers. On the basis that Na penetrates passively across an outer barrier, and is actively extruded across an inner barrier which is impermeable to passive movements of Na, we expected to detect at least the Na-pool of a single cell layer containing some 10^–8 moles per cm² of epithelium (i.e., in a cell layer 5 thick and with 21mm Na). Yet no Na-pool with these characteristics was found. The method employed could have detected a Na-pool at least an order of magnitude smaller than the one expected. It is concluded that either a Na-pool does not exist (except for the Na bound to the mechanisms operating the translocation), or else that the Na-pool is contained between barriers with different characteristics than the ones assumed above. In the first case, Na transportacross the epithelium would consist of a translocation across a single asymmetrical functional barrier. In the second case, the experimental results would require that ouabain either directly (by inhibiting an active step) or indirectly (through a mediated decrease of the Na permeability of the outer barrier) prevents Na penetration at the outer border.