Sodium transport and oxygen consumption in toad bladder. A thermodynamic approach.

The relationship between active sodium transport and oxygen consumption was investigated in toad urinary bladder exposed to identical sodium-Ringer's solution at each surface, while controlling the transepithelial electrical potential difference delta phi. Rates of sodium transport and oxygen consumption were measured simultaneously, both in the short-circuited state (delta phi = 0) and when delta phi was varied. Under short-circuit conditions, when the rates of active sodium transport changed spontaneously or were depressed with amiloride, the ratio of active sodium transport to the estimated suprabasal oxygen consumption Na/O2 was constant for each tissue, but varied among different tissues. Only when delta phi was varied did the ratio Na+/O2 change with the rate of active sodium transport; under these circumstances dNa+/dO2 was constant but exceeded the ratio measured at short-circuit [(Na+/O2)delta phi = 0[. This suggests that coupling between transport and metabolism is incomplete. The results are analyzed according to the principles of nonequilibrium thermodynamics, and intepreted in terms of a simple model of the transepithelial sodium transport system.     

Energetics of sodium-dependent α-aminoisobutyric acid transport in the moderate halophile Vibrio costicola

The energetics of α-aminoisobutyric acid transport were examined in Vibrio costicola grown in a medium containing the NaCl content (1 M) optimal for growth. Respiration rate, the membrane potential (Δψ) and α-aminoisobutyric acid transport had similar pH profiles, with optima at 8.5–9.0. Cells specifically required Na⁺ ions to transport α-aminoisobutyric acid and to maintain the highest Δψ (150–160 mV). Sodium was not required to sustain high rates of O₂-uptake. Δψ (and α-aminoisobutyric acid transport) recovered fully upon addition of Na⁺ to Na⁺-deficient cells, showing that Na⁺ is required in formation or maintenance of the transmembrane gradients of ions. Inhibitions by protonophores, monensin, nigericin and respiratory inhibitors revealed a close correlation between the magnitudes of Δψ and α-aminoisobutyric acid transport. Also, dissipation of Δψ with triphenylmethylphosphonium cation abolished α-aminoisobutyric acid transport without affecting respiration greatly. On the other hand, alcohols which stimulated respiration showed corresponding increases in α-aminoisobutyric acid transport, without affecting Δψ. Similarly, N,N′-dicyclohexylcarbodiimide (10 μM) stimulated respiration and α-aminoisobutyric acid transport and did not affect Δψ, but caused a dramatic decline in intracellular ATP content. From these, and results obtained with artificially established energy sources (Δψ and Na⁺ chemical potential), we conclude that Δψ is obligatory for α-aminoisobutyric acid transport, and that for maximum rates of transport an Na⁺ gradient is also required.     

Effect of temperature on electrolyte metabolism of isolated frog skin

A study is presented on the effect of temperature on unidirectional active ion transport, resting electrolyte equilibrium (electrolyte composition), and oxygen consumption in isolated frog skin. The aims were twofold: first, to find out whether the rate of active transport can be changed without affecting the Na⁺ and K⁺ balance of skin itself; second, to arrive at minimal ΔNa/ΔO₂ values by correlating quantitatively inhibition of active ion transport with inhibition of O₂ consumption. NaCl transport was maximal at 20°C. At 28° and at temperatures below 20°, rate of NaCl transport was diminished. In many instances NaCl transport was diminished in skins which maintained their normal Na⁺ and K⁺ content. In several cases, however, neither rate of transport nor resting electrolyte equilibrium was affected; in other cases, both were. O₂ consumption decreased when lowering the temperature over the range from 28 to 10°C. From a plot of log Q_OO2 against 1/T an activation energy of µ 13,700 cal. was calculated, valid for the range from 10 to 20°C. It appeared that µ was smaller for temperatures above 20°C. Working between 10 and 20°, it was found that, on the average, 4 to 5 equivalents of Na⁺ were transported for one mole of O₂ consumed in skins with undisturbed resting electrolyte equilibrium.     

Effect of oxytocin on transepithelial transport of water and Na+ in distinct ventral regions of frog skin (Rana catesbeiana)

Thoracic, abdominal, and pelvic fragments of ventral skin of Rana catesbeiana were analysed regarding the effect of oxytocin on: (1) transepithelial water transport; (2) short-circuit current; (3) skin conductance and electrical potential difference; (4) Na⁺ conductance and electrical potential difference; (4) Na⁺ conductance, the electromotive force of Na⁺ transport mechanism, and shunt conductance; (5) short-circuit current responses to fast Na⁺ by K⁺ replacement in the outer compartment, and (6) epithelial microstructure. Unstimulated water and Na⁺ permeabilities were low along the ventral skin. Hydrosmotic and natriferic responses to oxytocin increased from thorax to pelvis. Unstimulated Na⁺ conductance was greater in pelvis than in abdomen, the other electrical parameters being essentially similar in both skin fragments. Contribution of shunt conductance to total skin conductance was higher in abdominal than in pelvic skin. Oxytocin-induced increases of total skin conductance, Na⁺ conductance, and shunt conductance in pelvis were significantly larger than in abdomen. An oscillatory behaviour of the short-circuit current was observed only in oxytocin-treated pelvic skins. Decrease of epithelial thickness and increase of mitochondria-rich cell number were observed from thorax to pelvis. Oxytocin-induced increases of interspaces were more conspicuous in pelvis and abdomen than in thorax.Abbreviations E _Na electromotive force of sodium transport mechansim - G _KCI skin conductance with external KCI Ringer - G _Na sodium conductance (series conductance) - G _shunt shunt pathway conductance - G _total total skin conductance - J _v water flux (in units of volume per area per time) - MRC mitochondria-rich cells - PD potential difference across skin - R _shunt resistance of the shunt pathway - SCC short-circuit current     

Respiration and sodium transport in rabbit urinary bladder

Respiration of rabbit urinary bladder was measured in free-floating pieces and in short-circuited pieces mounted in an Ussing chamber. Ouabain, amiloride, and potassium-free saline inhibited respiration approx. 20%; sodium-free saline depressed respiration approx. 40–50%. The coupling ratio between respiration and transport in short-circuited tissues was about two sodium ions per molecule O₂. Chloride-free saline depressed mean oxygen consumption 21% in free-floating tissue pieces; 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) and furosemide had no effect. The effect of chloride-free saline in short-circuited tissues was variable; in tissues with low transport rates, respiration was stimulated about 21% while in tissue with high transport rates respiration was reduced about 24%. Nystatin and monensin, both of which markedly increase the conductance of cell membranes with a concomitant increase in sodium entry, stimulated respiration. These data indicate that 50–60% of the total oxygen consumption is not influenced by sodium, 20–25% is linked to (Na⁺ + K⁺)-ATPase transport, while the remaining 25–30% is sodium-dependent but not ouabain-inhibitable.     

Measurement of plasma membrane potentials of yeast cells with glass microelectrodes

Glass microelectrodes were used to measure the electrical potential difference (Δψ) across plasma membrane of the yeast Pichia humboldtii. The cells were captured in the neck of a glass microfunnel and impaled with a glass microelectrode. The measurements were reproducible and stable for several minutes. The highest Δψ values were obtained in cells metabolizing glucose at pH 6. Δψ in cells deenergized by uncouplers or in dead cells was reduced to about one third of the maximal value. This residual Δψ probably represented Donnan potential. Δψ also was reduced by increasing concentrations of K⁺ in the medium. Other monovalent cations were distinctly less effective: Li⁺ ⪡ Na⁺ < K⁺, and Ca²⁺ was without effect. These experiments prove the applicability of the electrophysiological technique on yeast cells and thus open the way for direct determination of the electrical component of the plasma membrane electrochemical proton gradient.     

Effects of anions on cellular volume and transepithelial Na+ transport across toad urinary bladder

Summary The effects of complete substitution of gluconate for mucosal and/or serosal medium Cl^– on transepithelial Na⁺ transport have been studied using toad urinary bladder. With mucosal gluconate, transepithelial potential difference (V _T) decreased rapidly, transepithelial resistance (R _T) increased, and calculated short-circuit current (I _sc) decreased. CalculatedE _Na was unaffected, indicating that the inhibition of Na⁺ transport was a consequence of a decreased apical membrane Na⁺ conductance. This conclusion was supported by the finding that a higher amiloride concentration was required to inhibit the residual transport. With serosal gluconateV _T decreased,R _T increased andI _sc fell to a new steady-state value following an initial and variable transient increase in transport. Epithelial cells were shrunken markedly as judged histologically. CalculatedE _Na fell substantially (from 130 to 68 mV on average). Ba²⁺ (3mm) reduced calculatedE _Na in Cl^– Ringer's but not in gluconate Ringer's. With replacement of serosal Cl^– by acetate, transepithelial transport was stimulated, the decrease in cellular volume was prevented andE _Na did not fall. Replacement of serosal isosmotic Cl^– medium by a hypo-osmotic gluconate medium (one-half normal) also prevented cell shrinkage and did not result in inhibition of Na⁺ transport. Thus the inhibition of Na⁺ transport can be correlated with changes in cell volume rather than with the change in Cl^– per se. Nystatin virtually abolished the resistance of the apical plasma membrane as judged by measurement of tissue capacitance. With K⁺ gluconate mucosa, Na⁺ gluconate serosa, calculated basolateral membrane resistance was much greater, estimated basolateral emf was much lower, and the Na⁺/K⁺ basolateral permeability ratio was much higher than with acetate media. It is concluded the decrease in cellular volume associated with substitution of serosal gluconate for Cl^– results in a loss of highly specific Ba²⁺-sensitive K⁺ conductance channels from the basolateral plasma membrane. It is possible that the number of Na⁺ pump sites in this membrane is also decreased.     

Measurement and significance of the membrane potential in Methanoba cterium bryantii

The membrane potential (Δψ) of Methanobacterium bryantii was 133–142 mV as measured from the distribution of ⁸⁶Rb⁺ in valinomycin-treated cells, and was considerably higher than that obtained using triphenylmethylphosphonium in the presence of tetraphenylboron. The Δψ measured using the Rb⁺/valinomycin method was sensitive to certain ionophores including gramicidin, nigericin, carbonyl cyanide m-chlorophenylhydrazone and 3,3′,4′,5-tetrachlorosalicylanilide. It was also dissipated by 1 mM tetraphenylphosphonium and was abolished in heat-treated or permeabilized cells. The Δψ could be varied by adjusting the extracellular potassium concentration in valinomycin-treated cells. Monensin-treated cells possessed a significantly increased Δψ, as monitored by the Rb⁺ / valinomycin method. Tetraphenylphosphonium cation (1 mM) abolished methane synthesis, intracellular ATP and Δψ, supporting a role for Δψ in ATP and CH₄ synthesis. However, lower concentrations of the lipophilic cation (50 μM) greatly elevated both the intracellular ATP concentration and Δψ but decreased the rate of CH₄ synthesis by almost 50%. Thus, tetraphenylphosphonium cation exerts a primary inhibitory effect on CH₄ synthesis which cannot be attributed to the loss of Δψ or ATP.     

The interrelationships between sodium ion,calcium transport and oxygen utilization in the isolated chick chorioallantoic membrane

Summary The interrelationships between sodium ion, calcium transport and oxygen utilization have been investigated in the chick chorioallantoic membrane. The oxygen uptakes of the two surface layers of the tissue, the ectoderm and the endoderm, were separated into their basal, Na⁺ dependent and Ca⁺⁺ dependent components. The endoderm has a basal rate of respiration of 3.6 liters O₂/cm²/hr and a Na⁺ dependent component of 1.4 liters O₂/cm²/hr. The ectoderm has a basal rate of respiration of about 3.5 liters O₂/cm²/hr, and Na⁺ and Ca⁺⁺ dependent components of 1.1 and 3.6 liters O₂/cm²/hr, respectively. The rate of ectodermal calcium transport and calcium-stimulated oxygen uptake is strictly dependent on the presence of sodium in the bathing medium, and complex kinetics are observed as a function of sodium concentration. On the other hand, in 140mm Na⁺ the rate of calcium transport exhibits simple saturation kinetics as a function of calcium concentration. Ca⁺⁺/O₂ ratios determined for many different rates of transport give a ratio of about 0.5, a value much lower than similar ratios determined for other transport mechanisms. The calcium transport mechanism in the ectoderm responds to changes in transport rate very sluggishly, taking 30 to 50 min to give a maximum response. The differences between the calcium transport mechanism in this membrane and other known transport systems are discussed and it is suggested that these differences may represent the adaptations necessary for transcellular calcium transport.     

Effect of amiloride,ouabain and Ba++ on the nonsteady-state Na−K pump flux and short-circuit current in isolated frog skin epithelia

Summary Effect of amiloride, ouabain, and Ba⁺⁺ on the nonsteady-state Na–K pump flux and short-circuit current in isolated frog skin epithelia.The active Na⁺ transport across isolated frog skin occurs in two steps: passive diffusion across the apical membrane of the cells followed by an active extrusion from the cells via the Na⁺–K⁺ pump at the basolateral membrane. In isolated epithelia with a very small Na⁺ efflux, the appearing Na⁺-flux in the basolateral solution is equal to the rate of the pump, whereas the short-circuit current (SCC) is equal to the active transepithelial Na⁺ transport. It was found that blocking the passive diffusion of Na⁺ across the apical membrane (addition of amiloride) resulted in an instantaneous inhibition of the SCC (the transepithelial Na⁺ transport, whereas the appearing flux (the rate of the Na⁺–K⁺ pump) decreased with a halftime of 1.9 min. Addition of the Na⁺–K⁺ pump inhibitor ouabain (0.1mm) resulted in a faster and bigger inhibition of the appearing flux than of the SCC. Thus, by simultaneous measurement of the SCC and the appearing Na⁺ flux one can elucidate whether an inhibitor exerts its effect by inhibiting the pump or by decreasing the passive permeability. Addition of the K⁺ channel inhibitor Ba⁺⁺, in a concentration which gave maximum inhibition of the SCC, had no effect on the appearing flux (the rate of the Na–K pump) in the first 2 min, although the inhibition of the SCC was already at its maximum.It is argued that in the short period, where the Ba⁺⁺-induced inhibition of SCC is at its maximum and the appearing flux in unchanged, the decrease in the SCC (SCC) is equal to the net K⁺ flux via the Na⁺–K⁺ pump, and the coupling ratio () of the Na⁺–K⁺ pump can be calculated from the following equation =SCC _t=0/SCC where SCC _t=0 is the steady-state SCC before the addition of Ba⁺⁺.     

EGF and its related growth factors mediate sodium transport in mpkCCDc14 cells via ErbB2 (neu/HER‐2) receptor

Amiloride‐sensitive sodium entry, via the epithelial sodium channel (ENaC), is the rate‐limiting step for Na⁺ absorption. Epidermal growth factor (EGF) is involved in the regulation of Na⁺ transport and ENaC activity. However it is still controversial exactly how EGF regulates ENaC and Na⁺ absorption. The aim of the present study was to characterize the EGF regulation of Na⁺ transport in cultured mouse renal collecting duct principal mpkCCD_c14 cells, a highly differentiated cell line which retains many characteristics of the cortical collecting duct (CCD). EGF dose dependently regulates basal transepithelial Na⁺ transport in two phases: an acute phase (<4 h) and a chronic phase (>8 h). Similar effects were observed with TGF‐α, HB‐EGF, and amphiregulin which also belong to the EGF‐related peptide growth factor family. Inhibition of MEK1/2 by PD98059 or U0126 increased acute effects and disrupted chronic effects of EGF on Na⁺ reabsorption. Inhibition of PI3‐kinase with LY294002 abolished acute effect of EGF. As assessed by Western blotting, ErbB2 is the most predominant member of the ErbB family detected in mpkCCD_c14 cells. Immunohistochemistry analysis revealed localization of ErbB2 in the CCD in Sprague–Dawley rat kidneys. Both acute and long‐term effects of EGF were abolished when cells were treated with tyrphostin AG‐825 and ErbB2 inhibitor II, chemically dissimilar selective inhibitors of the ErbB2 receptor. Thus, we conclude that EGF and its related growth factors are important for maintaining transepithelial Na⁺ transport and that EGF biphasically modulates sodium transport in mpkCCD_c14 cells via the ErbB2 receptor. J. Cell. Physiol. 223: 252–259, 2010. © 2009 Wiley‐Liss, Inc.     

Stimulation of Transepithelial Na<Superscript>+</Superscript> Current by Extracellular Gd<Superscript>3+</Superscript> in <Emphasis Type="Italic">Xenopus laevis</Emphasis> Alveolar Epithelium

In the present study we investigated the effect of extracellular gadolinium on amiloride-sensitive Na⁺ current across Xenopus alveolar epithelium by Ussing chamber experiments and studied its direct effect on epithelial Na⁺ channels with the patch-clamp method. As observed in various epithelia, the short-circuit current (I _sc) and the amiloride-sensitive Na⁺ current (I _ami) across Xenopus alveolar epithelium was downregulated by high apical Na⁺ concentrations. Apical application of gadolinium (Gd³⁺) increased I _sc in a dose-dependent manner (EC ₅₀ = 23.5 µM). The effect of Gd³⁺ was sensitive to amiloride, which indicated the amiloride-sensitive transcellular Na⁺ transport to be upregulated. Benz-imidazolyl-guanidin (BIG) and p-hydroxy-mercuribenzonic-acid (PHMB) probably release apical Na⁺ channels from Na⁺-dependent autoregulating mechanisms. BIG did not stimulate transepithelial Na⁺ currents across Xenopus lung epithelium but, interestingly, it prevented the stimulating effect of Gd³⁺ on transepithelial Na⁺ transport. PHMB increased I _sc and this stimulation was similar to the effect of Gd³⁺. Co-application of PHMB and Gd³⁺ had no additive effects on I _sc. In cell-attached patches on Xenopus oocytes extracellular Gd³⁺ increased the open probability (NP _o) of Xenopus epithelial sodium channels (ENaC) from 0.72 to 1.79 and decreased the single-channel conductance from 5.5 to 4.6 pS. Our data indicate that Xenopus alveolar epithelium exhibits Na⁺-dependent non-hormonal control of transepithelial Na⁺ transport and that the earth metal gadolinium interferes with these mechanisms. The patch-clamp experiments indicate that Gd³⁺ directly modulates the activity of ENaCs.     

Oxygen-Dependent Exclusion of Sodium Ions from Shoots by Roots of Zea mays (cv Pioneer 3906) in Relation to Salinity Damage

Using radio-tracers, we measured Na⁺ and K⁺ accumulation in roots and transport to shoots in Zea mays (cv Pioneer 3906) as a function of NaCl concentration and O₂ partial pressure in the nutrient solution. Under fully aerobic conditions, roots partially excluded Na⁺ from the shoots over a wide range of NaCl concentration (0.2-200 millimolar). With root anoxia, the exclusion mechanism broke down so that much greater amounts of Na⁺ reached the shoots, with simultaneous inhibition of K⁺ transport. The ratio Na⁺/K⁺ entering the shoot consequently increased 90 to 200 times. Increases in Na⁺ transport were first detected when the O₂ partial pressure was reduced from ambient (21% v/v) to 15%, whereas K⁺ transport was not inhibited until O₂ concentrations were <5%. Since soil O₂ deficiency can often accompany high salinity in irrigation agriculture, failure of the Na⁺ exclusion mechanism may be a contributory factor in salinity damage of salt-sensitive glycophytes.     

Effects of tryptamine on active sodium and chloride transport in the isolated bullfrog cornea

The effects of the serotonin analogue, tryptamine, on the active transepithelial transport of Na⁺ and Cl⁻ in the in vitro bullfrog cornea were studied. Tryptamine, 1 mM, inhibited both the short-circuit current (I_sc) and potential difference (PD) of corneas transporting either Na⁺ alone or both Na⁺ and Cl⁻. The electrical resistance, R, increased in all cases. Both unidirectional Na⁺ and Cl⁻ fluxes were decreased by tryptamine and these changes accounted for the inhibitory effects on the I_sc. The effects of tryptamine were considered along with with those of 2 mM theophylline and 0.1 mM ouabain. Tryptamine inhibited the I_sc and both undirectional Cl⁻ fluxes which were previously stimulated by theophylline. Theophyline addition, after tryptamine preincubation, increased the Cl⁻ undirectional fluxes but did not restore the inhibited I_sc. The inhibitory effects of tryptamine on active Na⁺ and Cl⁻ transport were different from those of ouabain. While both drugs inhibited the forward Na⁺ and Cl⁻ fluxes, their backfluxes decreased with tryptamine and increased with ouabain. The addition to the bathing solution of tryptamine after ouabain preincubation reduced the ouabain-increased backward Cl⁻ flux and further increased the electrical resistance. These results are analyzed in terms of an electrical model from which it appears that tryptamine's mechanism of action was to decrease cellular permeability to the transepithelial movement of Na⁺ and Cl⁻.     

Transport of lithium and rectification by frog skin

The isolated frog skin, bathed with Li⁺-Ringer (Na⁺-free) on the outside and Na⁺-Ringer on the inside, can maintain a normal potential difference (PD) and short-circuit current (s.c.c.) for more than 6. h. The s.c.c. correspondended to the Li⁺ influx. The Na⁺ efflux was 4% of the s.c.c. 10⁻⁵ M ouabain depressed Li⁺ influx and s.c.c. 10¹⁰⁻⁵ M amiloride abolished the Li⁺ s.c.c., while 0.1 unit/ml oxytocin stimulated it. When the inside of the skin was bathed with Li⁺-Ringer, PD and s.c.c. fell to zero within 2 h. The oxygen consumption of skin slices bathed in Li⁺-Ringer was 29% lower than controls bathed in Na⁺-Ringer.When the isolated frog skin is bathed in Na₂SO₄-Ringer it shows electrical rectification which has been correlated with the active transport of Na⁺. In skins transporting Li⁺, rectification characteristics are similar to those of skins transporting Na⁺. When the inner face of the skin is bathed with Li⁺-Ringer, rectification, PD and s.c.c. decline in a parallel fashion.It is concluded that: (1) Li⁺ can be transported when Na⁺ is present at the inner face. (2) Amiloride, ouabain and oxytocin affect Li⁺ and Na⁺ transport in a similar manner. (3) Li⁺ transport, like Na⁺ transport, is associated with rectification. (4) Active transport of Na⁺ and Li⁺ seems to depend on two different but associated proceses; one taking place at the external barrier (where rectification occurs) as shown by the effect of amiloride; and the other of an inner site related to energy requirements and affected by ouabain and Li⁺. (5) The cation being transported is not necessarily activating the (Na⁺-K⁺-ATPase.     

Coupled transepithelial sodium and potassium transport across isolated frog skin: Effect of ouabain,amiloride and the polyene antibiotic filipin

Summary Addition of the polyene antibiotic filipin (50 m) to the outside bathing solution (OBS) of the isolated frog skin resulted in a highly significant active outward transport of K⁺ because filipinper se increases the nonspecific Na⁺ and K⁺ permeability of the outward facing membrane. The K⁺ transport was calculated from the chemically determined changes in K⁺ concentrations in the solution bathing the two sides of the skin. The active transepithelial K⁺ transport required the presence of Na⁺ in the OBS, but not in the inside bathing solution (IBS), and it was inhibited by the Na⁺, K⁺-ATPase inhibitor ouabain. The addition of Ba⁺⁺ to the IBS in the presence of filipin in the OBS resulted in an activation of the transepithelial K⁺ transport and in an inhibition of the active Na⁺ transport. This is in agreement with the notion that Ba⁺⁺ decreases the passive K⁺ permeability of the inward facing membrane. In the presence of amiloride (which blocks the specific Na permeability of the outward facing membrane) and Ba⁺⁺ there was a good correlation between the active Na⁺ and K⁺ transport. It is concluded that the active transepithelial K⁺ transport is carried out by a coupled electrogenic Na–K pump, and it is suggested that the pump ratio (Na/K) is 1.5.     

Correlation Between Transepithelial Na+ Transport and Transepithelial Water Movement Across Isolated Frog Skin (Rana esculenta)

In the present work the coupling under short-circuited conditions between the net Na⁺-influx across isolated frog skin and the transepithelial transport of water was examined i.e., the short-circuit current (I _sc ) and the transepithelial water movement (TEWM) were measured simultaneously. It has been shown repeatedly that the I _sc across isolated frog skin is equal to the net transepithelial Na⁺ transport. Furthermore the coupling between transepithelial uptake of NaCl under open-circuit conditions and TEWM was also measured. The addition of antidiuretic hormone (AVT) to skins incubated under short-circuited conditions resulted in an increase in the I _sc and TEWM. Under control conditions I _sc was 9.14 ± 2.43 and in the presence of AVT 45.9 ± 7.3 neq cm⁻² min⁻¹ (n= 9) and TEWM changed from 12.45 ± 4.46 to 132.8 ± 15.8 nL cm⁻² min⁻¹. The addition of the Na⁺ channel blocking agent amiloride resulted in a reduction both in I _sc and TEWM, and a linear correlation between I _sc and TEWM was found. The correlation corresponds to that 160 ± 15 (n= 7) molecules of water follow each Na⁺ across the skin. In another series of experiments it was found that there was a linear correlation between I _sc and the increase in apical osmolarity needed to stop the TEWM. The data presented indicate that the observed coupling between the net transepithelial Na⁺ transport and TEWM is caused by local osmosis. Received: 16 October 1996/Revised: 6 March 1997     

Ion transport across the skin of a larval salamander

The transport characteristics of the skin of neotenic Ambystoma tigrinum were investigated using ion substitution and circuit analysis. When bathed with sodium Ringer solution on both sides, a transepithelial potential of up to 50 mV (inside positive) and a short-circuit current (I_sc) of up to 10 μA/cm² were observed. When amiloride was added or Na⁺ was replaced by tetramethylammonium in the apical solution, I_sc was decreased from 3.7 ± 0.4 to 1.5 ± 0.2 μA/cm² (n = 10). When K⁺ replaced Na⁺, there was a smaller change in I_sc from 5.8 ± 0.6 to 3.7 ± 0.5 μA/cm² (n = 10). Although barium had no effect when added to 100 K Ringer on normal skin, it inhibited I_sc on skins taken from K⁺-loaded animals. Nystatin caused substantial increases in I_sc with either Na⁺ or K⁺ as the dominant cation in the apical solution. Current voltage analysis using amiloride was used to estimate the resistances and electromotive forces (EMF) associated with ion transport. The EMF for ion transport was partially dependent on K⁺ in the basolateral solution and it was similar to that observed in other epithelia. The resistance of the transport pathway was high, consistent with the low I_sc. These results suggest that there is an amiloride-sensitive Na⁺ channel in parallel with a small K⁺ conductance in the apical membrane of this preparation.     

Ion Secretion and Isotonic Transport in Frog Skin Glands

The aim of this study was to clarify the mechanism of isotonic fluid transport in frog skin glands. Stationary ion secretion by the glands was studied by measuring unidirectional fluxes of ²⁴Na⁺, ⁴²K⁺, and carrier-free ¹³⁴Cs⁺ in paired frog skins bathed on both sides with Ringer's solution, and with 10⁻⁵ m noradrenaline on the inside and 10⁻⁴ m amiloride on the outside. At transepithelial thermodynamic equilibrium conditions, the ¹³⁴Cs⁺ flux ratio, J ^out _Cs/J ⁱⁿ _Cs, varied in seven pairs of preparations from 6 to 36. Since carrier-free ¹³⁴Cs⁺ entering the cells is irreversibly trapped in the cellular compartment (Ussing & Lind, 1996), the transepithelial net flux of ¹³⁴Cs⁺ indicates that a paracellular flow of water is dragging ¹³⁴Cs⁺ in the direction from the serosal- to outside solution. From the measured flux ratios it was calculated that the force driving the secretory flux of Cs⁺ varied from 30 to 61 mV among preparations. In the same experiments unidirectional Na⁺ fluxes were measured as well, and it was found that also Na⁺ was subjected to secretion. The ratio of unidirectional Na⁺ fluxes, however, was significantly smaller than would be predicted if the two ions were both flowing along the paracellular route dragged by the flow of water. This result indicates that Na⁺ and Cs⁺ do not take the same pathway through the glands. The flux ratio of unidirectional K⁺ fluxes indicated active secretion of K⁺. The time it takes for steady-state K⁺ fluxes to be established was significantly longer than that of the simultaneously measured Cs⁺ fluxes. These results allow the conclusion that — in addition to being transported between cells — K⁺ is submitted to active transport along a cellular pathway.Based on the recirculation theory, we propose a new model which accounts for stationary Na⁺, K⁺, Cl⁻ and water secretion under thermodynamic equilibrium conditions. The new features of the model, as compared to the classical Silva-model for the shark-rectal gland, are: (i) the sodium pumps in the activated gland transport Na⁺ into the lateral intercellular space only. (ii) A barrier at the level of the basement membrane prevents the major fraction of Na⁺ entering the lateral space from returning to the serosal bath. Thus, Na⁺ is secreted into the outside bath. It has to be assumed then that the Na⁺ permeability of the basement membrane barrier (P ^BM _Na) is smaller than the Na⁺ permeability of the junctional membrane (P ^JM _Na), i.e., P ^JM _Na/P ^BM _Na > 1. The secretory paracellular flow of water further requires that the Na⁺ reflection coefficients (σ_Na) of the two barriers are governed by the conditions, σ^BM _Na > 0, and σ^BM _Na > σ^JM _Na. (iii) Na⁺ channels are located in the apical membrane of the activated gland cells, so that a fraction of the Na⁺ outflux appearing downstream the lateral intercellular space is recirculated by the gland cells. Based on measured unidirectional fluxes, a set of equations is developed from which we estimate the ion fluxes flowing through major pathways during stationary secretion. It is shown that 80% of the sodium ions flowing downstream the lateral intercellular space is recycled by the gland cells. Our calculations also indicate that under the conditions prevailing in the present experiments 1.8 ATP molecule would be hydrolyzed for every Na⁺ secreted to the outside bath. Received: 30 January 1996/Revised: 12 March 1996     

Development of Na+ transport in the chicken colon

To evaluate the developmental changes in colonic Na⁺ transport, Na, K-ATPase activity and the sensitivity of the short-circuit current to amiloride were investigated. The amiloride-sensitive short-circuit current which represents the electrogenic, amiloride-sensitive Na⁺ transport through Na⁺ channels, was not present in chicken embryos but rose significantly after hatching in chicks which were kept on a low-salt diet. Amiloride-sensitive short-circuit current increased gradually but the plateau was not reached during the first 15 days of life. Drinking of 0.9% NaCl totally inhibited the induction of amiloride-sensitive Na⁺ transport. Na⁺, K⁺-ATPase activity increased during development but was not influenced by changes in salt intake. Na⁺ transport in chicken colon therefore undergoes profound developmental changes. The increase of Na⁺ transport refleets not only the adaptation of colonocytes to low salt intake but also the maturation of Na⁺ absorption in colon. The possible role of aldosterone in the adaptation to low-salt intake is discussed.Abbreviations LS low-salt - HS high-salt - I _sc short-circuit current