Isolated Frog Skin Epithelium Research Articles

Ussing's “Little Chamber”: 60 Years+ Old and Counting

GENERAL COMMENTARY article Front. Physiol., 03 March 2011Sec. Renal and Epithelial Physiology https://doi.org/10.3389/fphys.2011.00006

Alloxan decreases intracellular potassium content of the isolated frog skin epithelium

Alloxan has been widely used to provoke diabetes mellitus. This compound induces necrosis of the β-pancreatic cells and the renal tubules. However, the mechanism of this action has not been fully established. There is some evidence that this drug may act by an alteration of several ionic transport mechanisms. Nevertheless, there is scant information on the effect of alloxan on these ionic transport mechanisms of the membrane in epithelial cells. We reported that this drug induces a decrease in sodium transport in the frog skin. In order to obtain information about the mechanism involved in the sodium transport diminution provoked by alloxan, in this study the function of Na +–K +ATPase enzyme on transepithelial sodium transport altered by alloxan is explored. We measured changes in the short circuit current and in the intracellular content of sodium and potassium under conditions of maximally stimulated enzyme activity. Short circuit current was not modified by the treatment with alloxan during the period of highest activity of the enzyme, suggesting a site of action independent of this ATPase. Cell potassium was reduced in alloxan-treated epithelia, without significant changes in Na + content. This finding points out the existence of an alteration induced by alloxan of some modulator mechanisms of the intracellular K + concentration. The treatment of the frog skin with cesium chloride, a K + channel blocker, prevented the decrease of Na + transport produced by alloxan. This result suggests an action of this diabetogenic drug on the K + channels of the frog skin epithelium.

Alpha2-adrenoceptors inhibit antidiuretic hormone-stimulated Na+ absorption across tight epithelia (Rana esculenta).

In the present study we examined the effect of alpha-adrenergic regulation of active transepithelial Na+ absorption across the isolated frog skin epithelium. alpha-Adrenergic stimulation was achieved by addition of the adrenergic agonist noradrenaline in the presence of the beta-adrenergic blocker propranolol. alpha-Adrenergic stimulation inhibited basal as well as antidiuretic hormone (ADH)-stimulated Na+ transport. The ADH-induced increase in Na+ transport was accompanied by a membrane depolarisation due to an increase in the apical Na+ permeability. The subsequent application of noradrenaline inhibited the Na+ transport and repolarised the membrane potential, suggesting that alpha-adrenergic stimulation had reduced the apical Na+ permeability. The inhibition was abolished by the alpha2-adrenergic antagonist yohimbine whereas it was insensitive to the alpha1-adrenergic antagonist prazosin. alpha-Adrenergic stimulation had no effect on the cytosolic free [Ca2+] ([Ca2+]i). Incubation of the epithelium in the presence of ADH increased the cellular adenosine 3',5'-cyclic monophosphate (cAMP) content, an increase which was abolished by alpha-adrenergic activation. The effect of alpha-adrenergic stimulation on cAMP production was abolished by the alpha2-adrenergic antagonist yohimbine. We conclude that the noradrenaline-induced inhibition of the ADH-stimulated Na+ absorption and cAMP content is mediated by activation of alpha2-adrenoceptors. The data further indicate that the principal cells of the epithelium do not express alpha1-adrenoceptors. The noradrenaline-induced inhibition of the ADH-stimulated Na+ transport was concentration dependent, with 0.24+/-0.03 microM eliciting a half-maximal response. This alpha2-adrenergic-mediated down-regulation of Na+ absorption is achieved at a concentration of noradrenaline which begins to activate the NaCl secretion via the skin glands. The alpha2-adrenoceptors therefore appear to have considerable physiological importance.

EP3 receptors inhibit antidiuretic-hormone-dependent sodium transport across frog skin epithelium.

We examined the effect of prostaglandin E2 (PGE2) on antidiuretic hormone (ADH)-dependent Na+ transport and cAMP production in isolated frog skin epithelium. ADH caused an increase in transepithelial Na+ transport and a decrease in cellular potential, indicating an increase in apical Na+ permeability. Subsequent addition of PGE2 decreased Na+ transport and repolarised the cells. The PGE2 receptor EP1/3-selective analogue sulprostone and the PGE2 receptor EP2/3-selective analogue misoprostol were able to mimic the effect of PGE2. ADH increased cellular cAMP levels, whereas PGE2, sulprostone and misoprostol were able to reduce the ADH-dependent cAMP production. Measurements of intracellular Ca2+ concentration ([Ca2+]i) revealed that it was unaffected by both PGE2 and sulprostone. The inhibitory effect of PGE2 on ADH-dependent Na+ transport was also observed in Ca2+-depleted epithelia. We conclude that ADH stimulates transepithelial Na+ transport by increasing cellular cAMP levels, whereas PGE2 inhibits ADH-dependent Na+ transport by activating EP3-type receptors, which decrease cellular cAMP levels. We have found no evidence that [Ca2+]i is involved in the regulation of ADH-dependent Na+ transport by PGE2.

The H+ pump in frog skin (Rana esculenta): identification and localization of a V-ATPase.

We here report on studies on the frog skin epithelium to identify the nature of its excretory H+ pump by comparing transport studies, using inhibitors highly specific for V-ATPases, with results from immunocytochemistry using V-ATPase-directed antibodies. Bafilomycin A1 (10 microM) blocked H+ excretion (69 +/- 8% inhibition) and therefore Na+ absorption (61 +/- 17% inhibition after 60 min application, n = 6) in open-circuited skins bathed on their apical side with a 1 mm Na2SO4 solution, "low-Na+ conditions" under which H+ and Na+ fluxes are coupled 1:1. The electrogenic outward H+ current measured in absence of Na+ transport (in the presence of 50 microM amiloride) was also blocked by 10 microM bafilomycin A1 or 5 microM concanamycin A. In contrast, no effects were found on the large and dominant Na+ transport (short-circuit current), which develops with apical solutions containing 115 mm Na+ ("high-Na+ conditions"), demonstrating a specific action on H+ transport. In immunocytochemistry, V-ATPase-like immunoreactivity to the monoclonal antibody E11 directed to the 31-kDa subunit E of the bovine renal V-ATPase was localized only in mitochondria-rich cells (i) in their apical region which corresponds to apical plasma membrane infoldings, and (ii) intracellularly in their neck region and apically around the nucleus. In membrane extracts of the isolated frog skin epithelium, the selectivity of the antibody binding was tested with immunoblots. The antibody labeled exclusively a band of about 31 kDa, very likely the corresponding subunit E of the frog V-ATPase. Our investigations now deliver conclusive evidence that H+ excretion is mediated by a V-ATPase being the electrogenic H+ pump in frog skin.

Trapping of 134Cs+ in frog skin epithelium as a function of short circuit current

In the sodium/potassium pump of the isolated frog skin epithelium, 134CS+ can to a certain degree replace potassium. If 134CS+ in almost a carrier-free amount is added to the solution bathing the inside of the frog skin in an Ussing chamber, there will be an uptake of 134 CS+ into the epithelium cells which is proportional to the short circuit current. Transepithelial flux of 134CS+ in leaky epithelia, for instance small intestine and frog skin glands, indicates a paracellular pathway. The consequences of these findings for the recycling-theory are discussed.

Cross-talk between ATP-regulated K+ channels and Na+ transport via cellular metabolism in frog skin principal cells.

Isolated frog skin epithelium, mounted in an Ussing chamber and bathed in standard NaCl Ringer solution, recycles K+ across the basolateral membrane of principal cells through an inward-rectifier K+ channel (Kir) operating in parallel with a Na+-K+-ATPase pump. Here we report on the metabolic control of the Kir channel using patch clamping, short-circuit current measurement and enzymatic determination of cellular (ATP (ATPi). 2. The constitutively active Kir channel in the basolateral membrane has the characteristics of an ATP-regulated K+ channel and is now classed as a KATP channel. In excised inside-out patches the open probability (Po) of KATP channels was reduced by ATPi with half-maximum inhibition at an ATPi concentration of 50 microM. 3. ATPi measured (under normal Na+ transport conditions) with luciferin-luciferase was 1.50 +/- 0.23 mM (mean +/- S.E.M.; range, 0.4-3.3 mM n = 11). Thus the KATP channel would be expected to be inactive in intact cells if ATPi was the sole regulator of channel activity. KATP channels which were inactivated by 1 mM ATPi in excised patches could be reactivated by addition of 100 microM ADP on the cytosolic side. When added alone, ADP blocks this channel with half-maximal inhibition at [ADPi] > 5 mM. 4. Sulphonylureas inhibit single KATP channels in cell-attached patches as well as the total basolateral K+ current measured in frog skin epithelia perforated with nystatin on the apical side. 5. Na+-K+-ATPase activity is a major determinant of cytosolic ATP. Blocking the pump activity with ouabain produced a time-dependent increase in ATPi and reduced the open probability of KATP channels in cell-attached membranes. 6. We conclude that the ratio of ATP/ADP is an important metabolic coupling factor between the rate of Na+-K+ pumping and K+ recycling.

Distribution of actin microfilaments in frog skin epithelial granular cells

Microfilaments were localised by immunofluorescence and immunogold cytochemistry to examine their distribution in granular cells of the isolated frog skin epithelium. Strongly fluorescent bundles of actin were observed beneath the plasma membrane with little evidence for actin in the central regions. Higher resolution offered by cytochemistry revealed that bundles of actin filaments comprised a substantial portion of the cortical cytoskeleton. Quantitative analysis of the frequency of gold label revealed an extremely rich array of filaments beneath the apical membrane of granular cells, with markedly less babel along the basolateral membrane and in the central cytoplasm. Treating cells with cytochalasin B or arginine vasopressin caused an apparent disruption of the apical actin fibres, concurrent with a decrease in gold label density. Assumably these signs are indicative of depolymerization of the filaments. Although the significance of this distribution is unknown, the apical polarisation of actin is consistent with a role in regulating the Na + permeability of the apical membrane. The data are discussed in relation to possible roles of the cytoskeleton in the regulation of transepithelial sodium transport by vasopressin.

Cytochemical localization of adenylate cyclase in the sodium-transporting epithelium isolated from frog skin

A modified cytochemical technique with 5'-adenylylimidodiphosphate as substrate, was used to examine the distribution of adenylate cyclase in cells comprising the transepithelial Na+ transport pathway in isolated frog skin epithelium. Particular attention was paid to the effects of fixation on the activity and localization of adenylate cyclase. Fixation in glutaraldehyde alone or in combination with paraformaldehyde reduced the amount of reaction product, while better results were obtained using unfixed tissues. Optimum results were obtained following stimulation of adenylate cyclase with forskolin and in the presence of specific metabolic inhibitors. Adenylate cyclase was localized in the basolateral membranes of the principal cells which constitute a functional syncytium for Na+ transport and was absent from the apical membranes of the outermost granulosum cells. This distribution is consistent with the transepithelial Na+ transport model and defines the functional morphology of the cells involved in Na+ transport across frog skin. The results are compatible with the process of Na+ re-absorption across other epithelial cells, verifying that frog skin is a convenient model-tissue to study Na+ transport mechanisms. Adenylate cyclase was also found in membranes of the mitochondria-rich cells, a minor and parallel Na+ transporting pathway.

PHi determines rate of sodium transport in frog skin: results of a new method to determine pHi.

The pH of the isolated frog skin epithelium was determined on a cellular and subcellular level based on the distribution of a weak organic acid, 4-bromobenzoic acid. The indicator is detectable by X-ray microanalysis due to the presence of an element label. The results show that the pH of principal cells, but not the Na concentration, is closely correlated with the rate of transepithelial Na transport. Acidification leads to an inhibition of Na transport, regardless of whether the change was spontaneous or experimentally induced. Under the conditions of this study, the pH of principal cells was not well regulated. At a bath pH of 7.0, large pH differences between the cell layers were detectable. In mitochondria-rich cells, the pH was a function of the intracellular Cl concentration but not the Na transport rate. The cytoplasmic pH consistently exceeded the nuclear pH. The nuclear-cytoplasmic pH differential in principal cells amounted to 0.3 pH units, which is equivalent to a nuclear potential of -17 mV. The results support the view that the intracellular pH (pHi) is an important regulator of transepithelial Na transport. Regulation is primarily achieved at the level of the apical Na channel, making the Na influx the rate-limiting step in Na reabsorption.

Intracellular ion concentrations in the isolated frog skin epithelium: evidence for different types of mitochondria-rich cells.

Intracellular ion concentrations were determined in split skins of Rana pipiens using the technique of electron microprobe analysis. Under control conditions, principal cells and mitochondria-rich cells (MR cells) had a similar intracellular ion composition, only the Cl concentration in MR cells was significantly lower. Inhibition of transepithelial Na transport by low concentrations of ouabain (2 x 10(-6) M, inner bath) resulted in a Na concentration increase of principal cells from 10.9 to 54.3 mmol/kg wet wt. The increase was completely abolished by simultaneous application of amiloride (10(-4) M, outer bath). Amiloride alone resulted in a significant decrease of the Na concentration to 6.1 mmol/kg. w.w. Among MR cells, two different groups of cells could be distinguished; cells that showed a Na increase after ouabain which was even larger than that in principal cells and cells that did not respond to ouabain. In about half of all ouabain-sensitive MR cells the Na increase could be prevented by amiloride. According to these results, a subpopulation of MR cells displays the transport characteristics expected for a transepithelial Na transport compartment, an apical amiloride-sensitive Na influx and a basal ouabain-inhibitable Na efflux. Given the small number of cells, however, it is unlikely that this subtype of MR cells contributes significantly to the overall rate of transepithelial Na transport.

Effect of 12‐O‐tetradecanoyl phorbol 13‐acetate on solute transport and production of cAMP in isolated frog skin

In the present study we have examined the action of the phorbol diester tetradecanoyl phorbol acetate, an activator of protein kinase C, on the transepithelial transport of sodium, chloride and water and the production of cAMP in the isolated frog skin epithelium (Rana esculenta). Addition of tetradecanoyl phorbol acetate to the mucosal solution resulted initially in an increase in the short-circuit current, which was followed by a progressive decrease. If the short-circuit current was first activated by addition of the antidiuretic hormone, arginine vasotocin, then the addition of tetradecanoyl phorbol acetate resulted only in a pronounced inhibition. The changes in the short-circuit current were the result of changes in the active influx of Na+. The effect of tetradecanoyl phorbol acetate on the intracellular potential measured under short-circuited conditions (Vscc) was time-dependent. Just after addition of tetradecanoyl phorbol acetate to the mucosal solution, Vscc depolarized; this was followed by a slight hyperpolarization, after which Vscc continued to decline. The inhibition of the Na+ transport by tetradecanoyl phorbol acetate was associated with a decline in the response to the antidiuretic hormone (arginine vasotocin), but the ability of arginine vasotocin to increase the cellular level of cAMP and to stimulate the osmotic water flow was not affected by the presence of tetradecanoyl phorbol acetate. In skin halves in which the short-circuit current was stimulated with arginine vasotocin, addition of tetradecanoyl phorbol acetate resulted in a dose-dependent inhibition of the short-circuit current, but only minor changes in Vscc were observed. The results presented suggest that the addition of tetradecanoyl phorbol acetate to the isolated frog skin first increases and then decreases the arginine vasotocin-sensitive sodium permeability of the apical membrane. This might be due to a stimulating effect of tetradecanoyl phorbol acetate on both the activation and deactivation (turnover) of the sodium channels.

Intracellular pH controls cell membrane Na+ and K+ conductances and transport in frog skin epithelium.

We determined the effects of intracellular respiratory and metabolic acid or alkali loads, at constant or variable external pH, on the apical membrane Na+-specific conductance (ga) and basolateral membrane conductance (gb), principally due to K+, in the short-circuited isolated frog skin epithelium. Conductances were determined from the current-voltage relations of the amiloride-inhibitable cellular current pathway, and intracellular pH (pHi) was measured using double barreled H+-sensitive microelectrodes. The experimental set up permitted simultaneous recording of conductances and pHi from the same epithelial cell. We found that due to the asymmetric permeability properties of apical and basolateral cell membranes to HCO3- and NH+4, the direction of the variations in pHi was dependent on the side of addition of the acid or alkali load. Specifically, changing from control Ringer, gassed in air without HCO3- (pHo = 7.4), to one containing 25 mmol/liter HCO3- that was gassed in 5% CO2 (pHo = 7.4) on the apical side caused a rapid intracellular acidification whereas when this maneuver was performed from the basolateral side of the epithelium a slight intracellular alkalinization was produced. The addition of 15 mmol/liter NH4Cl to control Ringer on the apical side caused an immediate intracellular alkalinization that lasted up to 30 min; subsequent removal of NH4Cl resulted in a reversible fall in pHi, whereas basolateral addition of NH4Cl produced a prolonged intracellular acidosis. Using these maneouvres to change pHi we found that the transepithelial Na+ transport rate (Isc), and ga, and gb were increased by an intracellular alkalinization and decreased by an acid shift in pHi. These variations in Isc, ga, and gb with changing pHi occurred simultaneously, instantaneously, and in parallel even upon small perturbations of pHi (range, 7.1-7.4). Taken together these results indicate that pHi may act as an intrinsic regulator of epithelial ion transport.

Effects of oxytocin on cation content and electrophysiology of frog skin epithelium

We measured effects of oxytocin on current-voltage (I-V) relations of frog (Rana catesbeiana) skins impaled with an intracellular microelectrode. In both Cl- and Cl(-)-free (SO4(2-) solutions, oxytocin caused an approximate doubling short-circuit current (Isc) and a depolarization of the cell membrane. Increase in apical membrane slope conductance, chord conductance, and permeability after oxytocin correlated with the increase in amiloride-sensitive Isc. Oxytocin also increased basolateral membrane conductance (gb). In Cl-, the shift in the voltage intercept of the apical membrane I-V relation (Ea) implied increased intracellular Na+ activity (a(Na)c) after oxytocin. In isolated frog skin epithelia, a similar increase in intracellular [Na+] after oxytocin was demonstrated by flame photometry. In SO4(2-), changes caused by oxytocin in both Ea and in flame photometrically determined cell [Na+] were minimal. The voltage intercept of the basolateral membrane I-V relations (Eb) was shifted by oxytocin in both Cl- and SO4(2-) solutions. Assuming that the basolateral membrane is selectively permeable to K+, changes in K+ obtained from Eb were in disagreement with those obtained by flame photometry. These results suggest that 1) the increase in a(Na)c caused by oxytocin is not essential to produce either the increase in gb or Isc and 2) ions other than K+ make an important contribution to basolateral membrane conductance.

Evidence for a Na+/H+ exchanger at the basolateral membranes of the isolated frog skin epithelium: effect of amiloride analogues.

We have investigated the possible existence of a Na+/H+ ion exchanger in the frog skin epithelium by using isotopic methods and two amiloride analogues:5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and phenamil. We found phenamil to be a specific blocker of sodium entry to its cellular transport compartment since it inhibited both the transepithelial Na+ influxes (J13) with a K1 of 4 X 10(-7) mol/l and the Na+ pool (control: 77 +/- 4 neq X h-1 X cm-2; phenamil: 21 +/- 1 neq X h-1 X cm-2). On the contrary EIPA (10(-5) mol/l) had no effect on J13 nor on the apical Na+ conductance. Acidification of the epithelium by passing from a normal Ringer (25 mmol/l HCO3-, 5% CO2, pH 7.34) to a HCO3(-)-free Ringer (5% CO2, pH 6.20) while blocking the Na+ conductance with phenamil, produced a large stimulation of Na+ influxes exclusively across the basolateral membranes (J32), after return to a normal Ringer (J32 = 706 +/- 76 and 1635 +/- 199 neq X h-1 X cm-2 in control and acid-loaded epithelia respectively). The stimulation of J32 was initiated when the epithelia were acid-loaded with Ringer of pH lower than 6.90 and was blocked by amiloride (KI = 7 X 10(-6) mol/l) and EIPA (KI = 5 X 10(-7) mol/l) whereas phenamil had no effect. In Na+-loaded epithelia (ouabain treated) the Na+ efflux across the basolateral membranes was stimulated by an inwardly directed proton gradient and was blocked by EIPA (10(-5) mol/l) or amiloride (10(-4) mol/l), a result suggesting reversibility of the mechanism. We conclude that a Na+ permeability mediated by a Na+/H+ ion exchanger exists in the basolateral membranes, which is stimulated by intracellular acidification and is sensitive to amiloride or EIPA. This exchanger is proposed to be involved in intracellular pH regulation.

Cellular pH and the ADH-induced hydrosmotic response in different ADH target epithelia

The hydrosmotic response elicited by oxytocin in the frog skin epithelium (Rana esculenta) was reversibly inhibited by 70% when the medium pH was reduced to 6.2 by CO2 bubbling on the serosal side. On the contrary, the response to 8-bromo cyclic AMP (8 Br-CAMP) was not affected by medium acidification, even after corion removal. In other experiments intracellular pH was measured, employing the dimetyl-oxazolidine-dione distribution technique, in frog urinary bladder and the isolated frog skin epithelium. As previously observed in the case of oxytocin, 8 Br-CAMP increased intracellular pH in frog urinary bladder. Incubation with oxytocin also augmented the intracellular pH in the isolated frog skin epithelium but 8 Br-CAMP did not modify cell proton concentration in this tissue. From previous and present results it can be summarized that: 1) The intracellular alkalinization effect elicited by oxytocin addition and the inhibition in the hydrosmotic response induced by medium acidification were qualitatively similar in both tested target epithelia. 2) On the contrary, a post cyclic AMP step sensitive to changes in intracellular pH was not observed in frog skin, as is the case in frog urinary bladder. 3) The 8 Br-CAMP induced intracellular alkalinization effect was only observed in frog urinary bladder.

Correlation between cAMP in isolated frog skin epithelium and stimulation of sodium transport and osmotic water flow by antidiuretic hormone and phosphodiesterase inhibitors

The cAMP level in isolated frog skin epithelia was stimulated by a range of concentrations of the antidiuretic hormone arginine vasotocin (AVT) and compared to active sodium transport measured as short-circuit current (SCC). The response of SCC and osmotic water flow (OWF) to AVT was investigated in a separate series. SCC was approximately three times more sensitive to AVT than OWF. Measurable increments of cAMP above the basal level were found only with AVT concentrations eliciting half-maximal response or more of SCC. Two models are offered to explain the findings: (A) Total SCC depends on epithelial cAMP with a sigmoidal relationship. (B) Epithelial cAMP exists in two separate pools of which only one is accessible to AVT, and SCC stimulation depends on cAMP in the AVT-controled pool in a simple saturable fashion. Correlation between cAMP and SCC after stimulation with theophylline resembled that after AVT. Papaverine (10 μ M) induced only small changes in SCC inspite of a substantial increase in cAMP level. Higher concentrations of papaverine inhibited both basal and AVT-stimulated SCC, while the cAMP level was further increased. This effect of papaverine may be due to a simultaneous block of another rate-determining process. Papaverine had no effect on basal or AVT-stimulated OWF despite a marked stimulation of the cAMP level. Thus, the role of cAMP as mediator of the hydroosmotic action of AVT must be questioned.

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

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

Enhanced sensitivity to stimulation of sodium transport and cyclic AMP by antidiuretic hormone after Ca2+ depletion of isolated frog skin epithelium.

The role of Ca2+ in the stimulation by antidiuretic hormone (ADH) of active sodium transport across the isolated epithelium of frog skin was investigated. This has been done by bathing the blood side with Ca2+-free solution containing 0.1 mM EGTA. This Ca2+ depletion halved the resistance but had no significant effect on the short-circuit current (SCC). The sensitivity of both cAMP- and SCC-stimulation to ADH was increased 40-fold by Ca2+ depletion. Sensitivity to stimulation by theophylline was only changed a little, while stimulation by exogenous cAMP was completely unaltered. The increase in sensitivity to ADH was dependent on the duration of preincubation in Ca2+-free solution, which indicates that a slowly exchanging Ca2+ pool is involved in the determination of sensitivity to ADH. We suggest this pool is of cellular origin and the increased sensitivity is due to the decrease of a Ca2+ inhibition of the ADH-stimulated adenylate cyclase. But a direct effect of Ca2+ on binding of ADH to the receptor cannot be excluded. Our results are not compatible with the hypothesis that entry of extracellular Ca2+ is an obligatory step in the natriferic action of ADH, although it may be so in the hydroosmotic action of ADH. We also found the maximal response to ADH to be higher after Ca2+ depletion. This is in agreement with the hypothesis of intracellular Ca2+ as a modulator of the sodium permeability of the outward-facing membrane.

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 CO 2; 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.