Effects of standard diuretics and ortho-vanadate on sodium transport across isolated frog skin

Frog (Rana temporaria) skins were studied in an Ussing type lucite chamber adapted to diminish tissue edge damage. The transepithelial electrical potential difference, short circuit current and direct current (DC) resistance of skins mounted in this chamber were 56, 20 and 24% higher, respectively, than those of skins mounted in a conventional chamber. Amiloride, triamterene, ouabain and ortho-vanadate inhibited short circuit current and net mucosal to serosal flux of 22Na. Amiloride and triamterene had rapid onsets of action and were effective only when administered to the mucosal (pond) side of the skin. Ouabain and ortho-vanadate had slower onsets of action and were effective only when administered to the serosal side of the skin. Steady state of effects of these drugs was not reached within the three-hour period of the experiments. The inhibitory effect of ortho-vanadate was blocked by adding a disulfonic stilbene derivative (DIDS) to the serosal side of the skin. Serosal prostaglandin E2 stimulated the short-circuit current and decreased the DC resistance. Thiazides, acetazolamide and loop diuretics had no effects on Na+ transport by frog skin. Thus, frog skin seems to be a useful model only in studies of the mode of action and the structure-activity relationship of diuretic which act by inhibiting sodium entry or Na+-K+-ATPase activity.     

Endogenous prostaglandins, adenosine 3':5'-monophosphate and sodium transport across isolated frog skin.

1. Sodium transport across isolated frog skin, as measured by the short-circuit current, was decreased by acetylsalicylic acid, mefenamic acid, paracetamol and phenylbutazone. Indomethacin (6 X 10(-6) M) had a biphasic effect on the short-circuit current: a transient increase followed by a sustained decrease. 2. The release of prostaglandin-like material from the skin was reduced by acetylsalicylic acid and indomethacin. Paracetamol caused a significant reduction in the short-circuit current response of the skin to low doses of arachidonic acid, but the response to the highest dose tested was not significantly altered. 3. Indomethacin (6 X 10(-6) M) increased the sensitivity of the skin to applied prostaglandin E1. The other prostaglandin synthetase inhibitors did not have this effect. Indomethacin (6 X 10(-6) M) also enhanced the effect of antidiuretic hormone on the short-circuit current. 4. Indomethacin (30 X 10(-6) M) increased the short-circuit current and diminished the response to applied prostaglandin E1. 5. In sulphate Ringer, theophylline increased the short-circuit current and diminished the response to prostaglandin E1. 6. Prostaglandin E1 increased the levels of cyclic AMP in frog skin and these increases preceded the increases in short-circuit current. There was a seasonal variation in the level of cyclic AMP in the skin: the levels in winter exceeded those in summer. There was also a seasonal variation in the cyclic AMP response to prostaglandin E1: the winter response was greater than that in summer. 7. Indomethacin (6 X 10(-6) M) had a biphasic effect on cyclic AMP levels in the skin, an initial increase followed by a decrease. Indomethacin also potentiated prostaglandin E1 stimulated cyclic AMP accumulation. 8. Theophylline increased cyclic AMP levels in the skin and potentiated prostaglandin E1 stimulated cyclic AMP accumulation. 9. Pre-treatment of the skin with theophylline reversed the effects of cyclic AMP on the short-circuit current and open-circuit potential. 10. It is concluded that endogenous prostaglandins help to maintain sodium transport across isolated frog skin and that the effects of E-type prostaglandins on the short-circuit current are mediated by increased cyclic AMP levels. The transient increase in short-circuit current and the increased skin sensitivity caused by indomethacin (6 X 10(-6) M) are attributed to inhibition of phosphodiesterase activity. The failure of theophylline to potentiate the short-circuit current response of the skin to prostaglandin E1 is attributed to alteration of cyclic AMP action on the skin by theophylline.     

The effect of noradrenaline on the toad skin potential

1. The electrical response of isolated toad skins to the presence of 4 x 10(-5)M noradrenaline in the inner medium has been studied.2. When skins were bathed in Ringer solution, noradrenaline initiated a partial depolarization of the skin potential (inside surface becoming less positive) followed by a hyperpolarization; however, noradrenaline depolarized skins in sulphate Ringer.3. The origin of the hyperpolarizing phase of the response to noradrenaline was studied by comparing the size of perturbations in the skin potential, produced by identical changes in external sodium, external chloride or internal potassium concentrations, before and during the response to noradrenaline.4. Measurements of skin conductance were made in different sulphate media in order to estimate the magnitudes of the conductance of the shunt pathway through the skin and the conductance of the pathway for actively transported sodium ions.5. Interpretation of both the variations in the perturbations of skin potential and the skin conductance measurements led to the conclusion that the hyperpolarizing phase of the response to noradrenaline was generated by an increase in the sodium to chloride permeability ratio for the outer barrier. It was considered that other evidence was compatible with this view.6. Similar experimental methods were employed to study the action of antidiuretic hormone (ADH) and an elevated external concentration of calcium on the outer barrier. It was found that ADH increased the sodium to chloride permeability ratio whereas calcium decreased it. The separate actions of ADH and calcium on the sodium permeability of the outer barrier did not interfere apparently with the subsequent ability of noradrenaline to increase the sodium to chloride permeability ratio for this barrier in the skin.     

A role for endogneous prostaglandins in the short-circuit current responses to osmolal changes in isolated frog skin.

1. Reduction in osmolality of the Ringer solution bathing the morphological inside of frog skin (by lowering the NaCl concentration) caused a significant increase in sodium transport as measured by the short-circuit current. Pretreatment of the skin with acetylsalicylic acid (2.5 x 10(-4)M) abolished the short-circuit current and open-circuit potential responses to osmolal change.2. The output of prostaglandin-like material from isolated frog skin was increased by incubating the skin in hypotonic Ringer solution.3. The cyclic AMP levels of isolated frog skin were also increased by a reduction in the osmolality of the Ringer fluid bathing the skin.4. Prostaglandin-like material was released both by the separated epithelial and dermal layers of frog skin and the output from both layers, on a unit wet weight basis, did not differ.5. The output of prostaglandin-like material from the separated layers of the skin was substantially greater than from whole skin. Indomethacin (6 x 10(-6)M) reduced the output of this material by more than 90% from both layers.6. The release of prostaglandin-like material from the separated layers of the skin was not altered by a reduction in osmolality of the bathing medium.7. It is concluded that a reduction in the osmolality of the solution bathing frog skin stimulates prostaglandin production and that the increased level of prostaglandins stimulates transepithelial sodium transport by stimulating cyclic AMP accumulation. It is also concluded that the response to osmolal change can only occur in intact skin since separation of the epithelial and dermal layers abolished the increase in the release of prostaglandin-like material to osmolal change. The site of the increased prostaglandin production and the exact nature of the stimulus remain to be determined.     

An upper limit to the number of sodium channels in frog skin epithelium

1. Binding of [(14)C]amiloride to the mucosal surface of frog skin epithelium has been measured in the presence and absence of an excess of unlabelled amiloride. Simultaneous records were obtained of the sodium transport through the skin as indicated by the short circuit current.2. The number of binding sites with affinities for amiloride of 5 x 10(7) l./mole was around 400/mum(2).3. The relation between the binding sites and sodium channels is discussed.4. Making certain assumptions it is concluded that when frog skin epithelium is bathed in Ringer solution each sodium channel can handle 8000 sodium ions/sec.     

A comparative study of the effects of norepinephrine and vasopressin on Na transport and O2 consumption in frog skin

Summary Experiments were designed to compare the effects of two hormones-vasopressin and norepinephrine-on the energetics of Na transport in frog skin. Simultaneous measurements of O₂ consumption and net Na flux were performed in the same skins by means of O₂ cathodes and the short circuit current technique. The results showed that both hormones induced similar increments in Na transport. In contrast, there was a conspicuous difference in O₂ consumption values, norepinephrine having a very small stimulatory effect compared to the one induced by vasopressin. Thus, despite the fact that both hormones increase Na permeability of frog skin by similar mechanisms and to a similar extent, they appear to exert very different effects on cell metabolism.     

The mechanism of lithium accumulation in the isolated frog skin epithelium

Summary The intracellular concentration of Li ([Li]c) of epithelia isolated from frog skins was determined after a one hour exposure to external Na free Choline Ringer containing from 1 to 25 mM of LiCl. In both open and short circuit conditions, [Li]c was found to have values up to ten times that of the outside Li concentration, indicating accumulation of Li in the epithelia. A loss of an approximately equivalent amount of cellular K was associated with this Li accumulation, whereas no significant change in epithelial content of Na was observed. This accumulation of Li is reduced if Na is present together with Li in the outer solution. Preincubation of the epithelia with Amiloride (10^–4 M) suppressed short circuit current and potential difference changes consecutive to externali solutions exposure, prevented Li accumulation and K loss and reduced transepithelial movements of Li. The entry of Li into the cell was speeded up by oxytocin (20 mU/ml). In cyanide (10^–4 M) pretreated epithelia, Li uptake was reduced and accumulation failed to occur. 2,4 dinitrophenol (5×10^–4 M) also lowered the Li uptake. It is concluded that the main mechanism for monovalent cations entry into the epithelium is a process for which Na and Li compete. The existence of an active transport step at the level of the outward facing membrane of the frog skin epithelium is proposed.     

Isotonic secretion via frog skin glands in vitro. Water secretion is coupled to the secretion of sodium ions

In isolated frog skin at least three different types of cells are engaged in the transepithelial ion and water transport; these are the granular cells, the mitochondria-rich cells and the glandular cells. The experiments presented were carried out on isolated frog skin bathed in Cl- or NO3- Ringer's solution, where the active transepithelial Na+ uptake via the granular cells was blocked by amiloride. Transepithelial current and water flow were measured. When a negative current was passed across the skins (the skins were clamped at -100 mV), the current was mainly carried by a net influx of Cl- via the mitochondria-rich cells. The current had no effect on the transepithelial water movement. This finding indicates that there is nearly no coupling between the Cl- flux and the movement of water via the mitochondria-rich cells. Prostaglandin E2 activates the glandular cells of the exocrine glands in the skin. When prostaglandin E2 was added under these experimental conditions (the skins were clamped at -100 mV, with amiloride in the apical bathing solution, and the glandular secretion of ions was blocked by the use of NO3- Ringer's solution), then the transepithelial current became more negative. This change in current was mainly due to an increase in the Na+ efflux via the glands. Thus PGE2 increase the Na+ conductance of the skin glands. Together with this increase in the Na+ efflux a highly significant increase in the water secretion was observed. The water movement (secretion) across the skin was under these conditions coupled to the PGE2-induced efflux of Na+, and when one Na+ was pulled from the basolateral to the apical solution via this pathway 215 molecules of water followed. This must be due to electro-osmosis (friction between ions and water) or current-induced local osmosis.     

The isolated frog skin epithelium: Permeability characteristics and responsiveness to oxytocin,cyclic AMP and theophylline

Summary The combined treatment of the frog skin with collagenase and hydrostatic pressure enables complete separation of the epithelial layer from the supporting dermis. The separation entirely preserves the epithelium's passive permeability and active sodium transport capacity. The short circuit current, d.c. resistance, unidirectional fluxes of Na⁺ and Cl^– ions, and osmotic water permeability were found identical on series of isolated epitheliums and intact skins obtained from the same groups of animals. Furthermore, the isolated epithelium is fully responsive to oxytocin, cyclic AMP and theophylline—three agents known to enhance active sodium transport and water permeability of the intact skin.     

The effect of sodium concentration on the content and distribution of sodium in the frog skin

1. The content and distribution of sodium in the epithelium of the frog skin (Leptodactylus ocellatus L.) have been studied.2. The inulin space, the (22)Na exchange, and the amounts of water and sodium were measured in samples of connective tissue. The results indicate that the necessary assumptions generally made to calculate the sodium and water contents of the epithelial cells as the difference between the total content in the tissue and the amounts contained in the inulin space are not valid in the frog skin.3. The mean concentration of sodium in the epithelium has been obtained from direct measurements of sodium and water in samples of epithelium. To measure the water content of the epithelium a new technique has been developed. When the skin is bathed with Ringer solution containing 115 mM-Na on both sides, the mean concentration of sodium in the epithelium is 79 mM. When the concentration of sodium in the Ringer is 1 mM the mean concentration in the epithelium is 25 mM. When the skin is bathed with Ringer with 1 mM-Na on the outside and 115 mM-Na on the inside-a situation which resembles the natural condition in the skin-the mean concentration of sodium in the epithelium is 52 mM.4. The compartmentalization of Na was studied by comparing the sodium content and the degree of exchange with (22)Na in the bathing solutions. In these experiments the skins were exposed to Ringer solutions with different concentrations of sodium, and (22)Na on one or both sides.5. The results indicate that the epithelium has a compartment of sodium which is not exchangeable in 40-80 min and whose size is not appreciably changed by a threefold change in the Na content in the epithelium and a hundredfold change in the concentration of the bathing solution.6. Sodium exchangeable in 40-80 min seems to be contained in two different compartments: (a) a large one that contains fixed sodium is mainly connected to the inside, and does not appear to participate directly in sodium transport across the frog skin; (b) a small one, that is bounded on the inside by a Na-impermeable barrier, and that seems to comprise the sodium involved in active transport. When the skin is bathed with Ringer solutions with 115 mM-Na on the inside and 1 mM-Na on the outside, the transporting compartment contains some 13% of the total sodium in the epithelium.7. The results are interpreted on the basis of a model recently proposed by Cereijido & Rotunno (1968). The major feature of this model is that the sodium transporting compartment is confined to the plasma membrane of the epithelial cells.     

Tizolemide-induced changes of passive transport components across the basolateral membrane of isolated frog skin

The effect on transepithelial Na transport of tizolemide was investigated in isolated frog skin (Rana temporaria). It was found that tizolemide (2–5 mM, serosal side) decreased transepithelial Na transport (measured as short circuit current and as net sodium flux) within 60 min to 25–40% of the control level resulting from reduction of the unidirectional sodium influx. Intracellular recording with microelectrodes revealed that these changes were associated with depolarization of the intracellular space to less than 40% of the control values (averaging –71.7±5.1 mV) which is a consequence of a decrease in conductance of the basolateral border to about 25% of the control values. The conductance of the apical border was only slightly reduced. It is suggested that tizolemde blocks the partial conductance of potassium at the basolateral border which secondarily diminishes trans-epithelial Na transport due to a decrease of the driving force for apical border Na entry. A certain degree of inhibition of the Na-K-ATPase by tizolemide cannot be excluded. When vasopressin (ADH) was added to frog skin after treatment with tizolemide, the response was markedly reduced compared to that of untreated control preparations. Under these conditions, the conductance of the basolateral border increased while the apical border remained little influenced by the hormone — opposite to the response of frog skins under control conditions. It is concluded that the mode of action of ADH is more complex than has been recognized hitherto and includes effects at the basolateral border.Dedicated to Professor H. Schwiegk on the occassion of his 75th birthday.Some of these results have been presented at the 8. Int. Congr. Néphrology, Athens 1981.     

Sodium transport across the isolated epithelium of the frog skin

1. A method to separate the epithelium from the underlying layers of the frog skin is described. The method is based on the combined use of collagenase and hydrostatic pressures.2. The potential difference and the short-circuit current values of isolated epithelia and whole skins are similar. Na net flux and short-circuit current are equivalent.3. The time course of changes in potential following rapid changes in composition of the bathing solutions shows that the barrier to K diffusion at the internal surface of the isolated epithelium is larger than the barrier to Na diffusion at the external surface.4. In the isolated epithelium there are 133 m-mole K(+) and 24.7 m-mole Na/l. cellular water. The amount of extracellular water was considered to be equal to the inulin space.5. Arginine vasopressin (0.1 u./ml.) markedly increased short-circuit current and potential difference in isolated epithelia. The amount of Na in the epithelium that equilibrated with Na in the external solution was not increased by the hormone.6. Ouabain (10(-4)M) reduced short circuit current and potential difference to values close to zero. The ouabain treated epithelia contained an increased amount of Na originating in the internal solution. On the other hand the amount of Na that originated from the external solution was not increased.7. The amount of epithelial Na that equilibrated with Na in the external solution was 0.009 mu-equiv/cm(2). This figure is about ten times smaller than the values found in whole skins.     

Protective effect of hydrocortisone on vasopressin response in frog skin

The effect of microtubular-poisons, such as colchicine and vincristine, on frog skin permeability has been investigated. Three-hour treatment with the drugs has no effect on nonelectrolyte basal transepithelial permeability, but completely supresses the effect of ADH. Colchicine and vincristine, in addition, affect both basal sodium transport and the rise in short circuit current induced by vasopressin.The inhibition produced by microtubular-poisons disappears, however, when hydrocortisone, a glucocorticoid known to preserve junctional communications is used.Together with the results previously obtained with isolated epithelial cells (Svelto et al. 1979), these findings provide further support for our hypothesis that the microtubularmicrofilament-system, is involved in cell-to-cell exchange.     

Reversed short-circuit current across isolated skin of the toadBufo arenarum

Short-circuit current (SCC) aross isolated pelvic skin of the toadBufo arenarum has been shown to be reflected by the algebraic sum of net sodium and chloride transport. After the animals had been maintained in tap water, amiloride — an apical sodium channel blockerled to a reversal of potential difference (rPD) across this preparation, to which corresponded a reversed short-circuit current (rSCC). Both rSCC and rPD were abolished by dermal treatment of skins with the metabolic inhibitor dinitrophenol, or by omission of chloride ion from the Ringer solution bathing both sides of the skin. There was a significant positive correlation between rSCC and isotopically determined net chloride transport after amiloride. An inhibitory action of amiloride on unidirectional chloride fluxes was detected, but only early after drug addition. rSCC was absent in skins of toads exposed to 110 mmol/l NaCl in tap water during 10 days. Together, our results suggest that amiloride addition —by inhibiting active sodium movement — can in certain conditions reveal the existence of an inward active chloride transport.     

Effect of Amiloride on sodium transport in frog skin

Summary The effect of Amiloride on several parameters of sodium transport was investigated on the isolated frog skin. Amiloride at a concentration of 10^–4 M/l decreased the sodium concentration in the sodium transport pool from 8.9 meq/kg cell water to 3.7 meq/kg cell water. No effect was observed on the intracellular sodium which is exchangeable from the corium side. Short circuit current and unidirectional sodium influx were diminished to the same extent whilst the unidirectional sodium efflux was not affected. In contrast to the short circuit current, which reaches a new steady state value within seconds, the unidirectional sodium influx reaches its new steady state with a half-time of 3.3 min. From the difference in the time courses of the decreases of short circuit current and unidirectional sodium influx, an amount of sodium could be calculated which agreed well with the directly measured fall in the sodium transport pool.The results provide further evidence for the concept that Amiloride inhibits the passive entrance of sodium into the sodium transport compartment, without influencing the transport capacity of the sodium pump.Supported by the Deutsche Forschungsgemeinschaft.     

Monensin can transport calcium across cell membranes in a sodium independent fashion in the crayfish Procambarus clarkii.

Monensin, a Na(+)-selective ionophore, enhances transmitter release when applied to crustacean and frog neuromuscular junctions. Monensin is believed to raise intracellular sodium ([Na+]i) which in turn elevates intracellular calcium ([Ca2+]i). Using the fluorescent indicator fura-2, we measured [Ca2+]i in crayfish Procambarus clarkii presynaptic terminals during monensin application in normal Ringer, zero-calcium Ringer and zero-sodium Ringer to determine if [Ca2+]i increases with monensin application and if so by what mechanism. In normal Ringer, monensin, 10 microM and 100 microM, elevated [Ca2+]i by 440 nM and 7 microM respectively. This rise in [Ca2+]i was dependent on external calcium, as [Ca2+]i did not increase in zero-calcium Ringer. However, in a zero-sodium Ringer, monensin (10 microM) elevated [Ca2+]i by 370 nM. It is important to recognize that monensin, thought to be a sodium-selective ionophore, can transport calcium across the cytoplasmic membrane in a sodium-independent manner.     

Sodium nitroprusside induced cGMP accumulation in isolated frog skin epithelium. Effect on CAMP,hydroosmotic and natriferic response to antidiuretic hormone

The cGMP content of isolated frog skin epithelia has been measured, and the basal level was found to be 14.3±1.7 fmol/mg dry weight. 0.1 mM sodium nitroprusside induced a 10-fold increase in the cGNP level within 5 min after which it rose more slowly. The maximum increase in cGMP level was obtained with 1 mM sodium nitroprusside, giving a 20–50-fold increase. 1 mM sodium nitroprusside per se had no effect on osmotic water flow or active sodium transport. On the other hand, the osmotic water flow response to arginine vasotocin was somewhat enhanced in skins which had been pretreated with 1 mM sodium nitroprusside; thus the water flow responses to 1 and 31 ng/ml arginine vasotocin were on the average 31 and 14% higher in skins exposed to sodium nitroprusside than in control skins. Sodium nitroprusside had no effect on the increment in sodium transport rate elicited by arginine vasotocin. Sodium nitroprusside alone increased the cAMP level slightly; the enhanced cAMP level, reached after 30 min incubation with 40 ng/ml arginine vasotocin, was 20 % higher in the presence of 1 mM sodium nitroprusside. In conclusion: cGMP has no effect on osmotic water flow nor on active sodium transport and is not involved in the regulation of sodium transport by antidiuretic hormone. However, cGMP (or sodium nitroprusside) has a moderate effect on the hormone-stimulated osmotic water flow.     

Trifluoperazine stimulated sodium transport by increased prostaglandin E2 synthesis in isolated frog skin (Rana esculenta)

Addition of trifluoperazine (TFP) to the inside bathing solution of the isolated frog skin resulted in a biphasic increase in the short-circuit current (SCC). The Na+-flux measurements showed that the TFP-induced increase of SCC was accounted for by active sodium transport. The intracellular voltage in short circuited skins changed after addition of TFP (100 microM) from a control value of -80.9 +/- 0.8 to -66.2 +/- 1.0 mV (n = 8). This depolarization indicates that TFP acts by increasing the Na+-permeability of the apical membrane. The biphasic increase in SCC is due to different mechanisms. The primary activation could be abolished by the calcium ionophore A23187, whereas the secondary activation could be abolished by the prostaglandin synthesis inhibitor indomethacin, PGE2 or A23187. Stimulation of the SCC by TFP and theophylline or antidiuretic hormone (AVT) was additive. Furthermore, TFP did not increase the cAMP level of isolated epithelia or theophylline-stimulated epithelia. These results indicate that the TFP-induced change in the Na+-permeability was not due to an enhanced cAMP level. The TFP simulated SCC requires Ca2+ in the inside bathing solution. Addition of TFP resulted in an increase in prostaglandin E2 release to the inside bathing solution from a control value of 0.31 +/- 0.04 to 5.4 +/- 1.4 pmol PGE2 h-1 cm-2 (n = 8). It is suggested that TFP induces a Ca2+-dependent PGE2 synthesis, leading to an increase in the intracellular PGE2 concentration which might increase the Na+-permeability of the apical membrane.     

Differentiation of the active sodium transport system during metamorphosis in Rana catesbeiana skin in relation to cadmium- and amiloride-induced responses

The differentiation of the sodium active transport system across Rana catesbeiana skin during metamorphosis was investigated and the system was analyzed by the effects of Cd2+ and amiloride. Active transport of Na in the frog skin first appeared at stage XXI of the tadpole, indicated by the appearance of a potential difference (PD) and a short circuit current (SCC) across the skin. The effects of epidermal application of Cd2+ and amiloride on the various indicators of active Na transport were as follows: (1) Cd2+ increased PD and SCC after stage XXII; (2) Cd2+ also increased the skin resistance (RM) from stage B to XXIII, but decreased it after stage XXV; and (3) amiloride decreased PD and SCC but increased RM after stage XXI. The adult frog skin can be expressed as an equivalent circuit by three parameters: ENa, the electromotive force of the active Na current, and RNa and R sigma, corresponding to the resistance to the active Na current and the resistance of the shunt pathway, respectively. These three parameters were calculated from the amiloride effect on PD and SCC. ENa was almost null and RNa was infinite until stage XX. ENa then gradually increased, while RNa was fairly constant between 10 and 20 k omega . cm2 after stage XXI. R sigma gradually increased during metamorphosis. These data suggest that there is not an active Na pathway but a passive one in the early stage of metamorphosis of the tadpole, and that the active Na transport system suddenly appears at stage XXI and gradually develops thereafter.     

Active transepithelial potassium transport in frog skin via specific potassium channels in the apical membrane

In frog skin bathed in Cl--Ringer's solution the short circuit current (SCC) is equal to the net Na+ flux. In the present study Na+ and K+ transport across frog skin have been investigated in skins bathed in a solution where all Cl- has been substituted by the impermeable anion gluconate. In this solution the net Na+ flux (9.22 +/- 0.72 nmole/cm2/min) was significantly higher than the SCC (7.61 +/- 0.63) nmole/cm2/min). Measurement of the transepithelial K+ influx and K+ efflux showed that the discrepancy between the net Na+ flux and the SCC was caused by an active outwards going transepithelial K+ transport. The K+ but not the Na+ transport could be blocked by adding the K+ channel blocking agent Ba++ to the apical solution. Thus, the K+ transport occurs via a K+ specific pathway in the apical membrane. Ouabain blocked both the Na+ and the K+ transport, whereas the presence of the Na+ channel blocking agent amiloride in the apical solution blocked the Na+ transport and reduced the K+ transport. In the presence of amiloride in the apical solution the SCC and the transepithelial potential difference (PD) reversed so that the outside (the apical side) of the frog skin became positive with respect to the basolateral side. The inverted SCC was carried by an active transepithelial K+ transport, this K+ transport required the presence of Na+ in the basolateral solution. The experiments show that frog skin can insert or activate K+ channels in the apical membrane, indicating that the frog may regulate its K+ content by varying the K+ permeability of the apical membrane.