Frog Skin Epithelium Research Articles

An increase in [Ca 2+ ] i activates basolateral chloride channels and inhibits apical sodium channels in frog skin epithelium

The aim of this study was to investigate the mechanisms by which increases in free cytosolic calcium ([Ca2+]i) cause a decrease in macroscopic sodium absorption across principal cells of the frog skin epithelium. [Ca2+]i was measured with fura-2 in an epifluorescence microscope set-up, sodium absorption was measured by the voltage-clamp technique and cellular potential was measured using microelectrodes. The endoplasmic reticulum calcium-ATPase inhibitor thapsigargin (0.4 microM) increased [Ca2+]i from 66 +/- 9 to 137 +/- 19 nM (n = 13, P = 0.002). Thapsigargin caused the amiloride-sensitive short circuit current (Isc) to drop from 26.4 to 10.6 microA cm-2 (n = 19, P<0.001) concomitant with a depolarization of the cells from -79 +/- 1 to -31 +/- 2 mV (n = 18, P<0.001). Apical sodium permeability (PaNa) was estimated from the current/voltage (I/V) relationship between amiloride-sensitive current and the potential across the apical membrane. PaNa decreased from 8.01.10(-7 )to 3.74.10(-7) cm s-1 (n = 7, P = 0.04) following an increase in [Ca2+]i. A decrease in apical sodium permeability per se would tend to decrease Isc and result in a hyperpolarization of the cell potential and not, as observed, a depolarization. Serosal addition of the chloride channel inhibitors 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), diphenylamine-2-carboxylate (DPC), indanyloxyacetic acid 94 (IAA-94) and furosemide reversed the depolarization induced by thapsigargin, indicating that chloride channels were activated by the increase in [Ca2+]i. This was confirmed in wash-out experiments with 36Cl where it was shown that thapsigargin increased the efflux of chloride from 32.49 +/- 5.01 to 62.63 +/- 13.3 nmol.min-1 cm-2 (n = 5, P = 0.04). We conclude that a small increase in [Ca2+]i activates a chloride permeability and inhibits the apical sodium permeability. The activation of chloride channels and the closure of apical sodium channels will tend to lower the macroscopic sodium absorption.

Regulation of epithelial Na+ permeability by protein kinase C is tissue specific.

Protein kinase C (PKC) is a major regulator of a broad range of cellular functions. Activation of PKC has been reported to stimulate Na+ transport across frog skin epithelium by increasing the apical Na+ permeability. This positive natriferic response has not been observed with other epithelial preparations, and could reflect the specific experimental conditions of different laboratories, or species or organ specificity of the response to PKC. In the present study, measurements were conducted with skins and urinary bladders from the same animals of two different species. The PKC activator TPA uniformly increased the transepithelial Na+ transport (measured as amiloride-sensitive short-circuit current, ISC, across skins from Rana temporaria and Bufo marinus, and inhibited ISC across bladders from the same animals. Inhibitors of PKC (staurosporine, H-7 and chelerythrine) partially blocked the TPA-induced stimulation of ISC across frog skin. The specificity of the PKC response by amphibian skin could have reflected an induction of moulting, similar to that observed with aldosterone. However, light micrographs of paired areas of frog skin revealed no evidence of the putative moulting. Separation of stratum corneum from the underlying stratum granulosum could be detected following application of aldosterone. We conclude that the effect of PKC on epithelial Na+ channels is organ, and not species specific. The stimulation of Na+ permeability in amphibian skin does not arise from sloughing of the stratum corneum. These observations are consistent with the hypothesis that the natriferic action arises from the calcium-independent isozyme of PKC previously detected 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.

PH modulates cAMP-induced increase in Na + transport across frog skin epithelium

Apical membrane potential ( V a), fractional apical membrane resistance (FR a), and/or intracellular pH (pH i) were measured in principal cells of isolated frog ( Rana pipiens) skin with microelectrodes under short-circuit conditions. Apical exposure to 0.33 mM 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (cAMP) depolarized V a, decreased FR a and increased short-circuit current ( I sc). cAMP-induced 50% larger effects on V a and I sc at external pH (pH o) of 8.0 than at pH o 6.4. Increasing pH o from 6.4 to 8.0 in presence of cAMP further depolarized V a and increased I sc. cAMP-induced effects on V a and I sc were observed in the absence of Cl − and HCO 3 − and in the presence of 1 mM 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) or 10 μM 5-( N-ethyl- N-isopropyl)amiloride (EIPA) or 1 μM 5-( N-methyl- N-isobutyl)amiloride (MIA). These data indicate that Na +-H + exchange, Cl −-HCO 3 − exchange, and electrogenic Na +-(HCO 3 −) n cotransport are not involved in cAMP-induced increase in I sc. Apical exposure to 2 mM Cd 2+ or Zn 2+ depolarized V a, decreased FR a, increased I sc and increased pH i. In HCO 3 −-free solutions containing DIDS, unilateral replacement of apical Cl − by NO 3 − induced a fast transient depolarization of V a and an increase in I sc. These data suggest that potential-dependent changes in pH i are involved in increases in I sc. However, when changes in V a were minimized by pretreating the basolateral membrane with 25 or 75 mM K +, the cAMP-induced increase in I sc was not blocked. These data indicate that changes in pH i do not play a strict regulatory role but are only permissive in cAMP-induced effects on I sc.

Cross-talk between sodium and potassium channels in tight epithelia

The barrier epithelia of amphibians such as frog skin and toad urinary bladder play an essential role in electrolyte, acid/base and water balance of the whole animal. These epithelia have junctions of high electrical resistance between the outermost living cells which render the epithelium tight to paracellular flow of Na + or K + , thus allowing vectorial transepithelial transport of these ions. The Na + absorption function is carried out by granular or principal cells via Na + channels in the apical membranes and an Na,K-ATPase pump in the basolateral membranes; K + is recyled across this barrier via K + -selective channels. The multilayered epithelium of frog skin functions as a syncytial Na + transport compartment [1, 2]. The granular cells, along with the deeper spinosum and germinativum cells, form the syncytium. The cells in deeper layers are coupled to the first reactive cell layer by gap junctions and although the cells in the different layers share the Na transport load, only the granular cell layer has a polarized apical membrane containing amiloride-sensitive Na + channels. The amphibian skin and urinary bladder actively secrete hydrogen ions under appropriate electrochemical gradients [3, 4]. Active H + secretion is restricted to the mitochondria-rich cells [5–8]. The amphibian skin and urinary bladder share common Na + and H + transport properties with distal renal tubule, and have been used for the past 50 years as highly successful models in studies of the mechanisms and regulation of ion transport across tight-junction epithelia [9]. A vast amount of information is available on the mechanisms and regulation of Na + , K + , Cl - , H + and water transport systems in amphibian skin and urinary bladder [10–13]. Indeed, many of the first descriptions of epithelial transport mechanisms and the cellular action of natriferic and anti-diuretic hormones and diuretic drugs were first described in these epithelia.

Development of technical approaches to studing of placental macrophages cultivated In vitro

The theory of Na+ recirculation for isosmotic fluid absorption follows logically from Hertz's convection–diffusion equation applied to the exit of water and solutes from the lateral intercellular space. Experimental evidence is discussed indicating Na+ recirculation based upon the following approaches: (i) An isotope tracer method in small intestine. Simultaneous measurement of water flow and ion transport in toad skin epithelium demonstrating, (ii) occasional hyposmotic absorbates, and (iii) reduced fluid absorption in the presence of serosal bumetanide. (iv) Studies of the metabolic cost of net Na+ absorption demonstrating an efficiency that is lower than the 18 Na+ per O2 consumed given by the stoichiometry of the Na+/K+-pump. Mathematical modeling predicts a significant range of observations such as isosmotic transport, hyposmotic transport, solvent drag, anomalous solvent drag, the residual hydraulic permeability in proximal tubule of AQP1(−/−) mice, the adverse relationship between hydraulic permeability and the concentration difference needed to reverse transepithelial water flow, and in a non-contradictory way the wide range of metabolic efficiencies from above to below 18 Na+/O2. Certain types of observations are poorly or not at all reproduced by the model. It is discussed that such lack of agreement between model and experiment is due to cellular regulations of ion permeabilities that are not incorporated in the modeling. Clarification of these problems requires further experimental studies.

Dynamics and density of mitochondria-rich cells in toad skin epithelium

The dynamics of change in mitochondria-rich (MR) cells density in the skin epithelium of Bufo viridis was studied on skin biopsies taken in vivo, throughout experimental periods lasting up to 3 months. When the bathing solution contained Cl-, MR cells' density (Dmrc) greatly decreased. There was one exception, when the acclimation solution was KCl, Dmrc in the skin increased. The rate of decrease in Dmrc depended on the mode of acclimation. When bath NaCl concentration was elevated slowly in small increments, the change in Dmrc was very slow. A regression line was calculated for the rate of decrease in the density of MR cells. An equation in the form of y = 1574 - 10.23x (where x = days; R2 = 0.626) was obtained with bath NaCl that was elevated from 30 to 200 mmol/l, in 45 days. Oxytocin (60 mU/ml) increased sodium transport, independently and without effect on Cl- conductance. Theophylline (1 mmol/l), which leads also to elevation of cellular cAMP in contrast, increased Na+ transport, but elevated Cl- conductance 3-4 times as well. Cl- conductance that is activated by transepithelial potential was much lower in skin from hyperosmotic NaCl-acclimated toads, as compared with that in skin from tap water-acclimated animals. Our experiments confirm that MR cells are a major pathway for Cl- conductance, as suggested earlier. However, the density of these cells in the skin epithelium of B viridis depends not only on bath NaCl concentration, but also on the mode of acclimation of the animals. Since transport functions other than gCl reside in the amphibian skin MR cells, the density of MR cells must also depend on these functions. These functions, and the mechanisms responsible for the down and up regulation of MR cells' density, remain to be established.

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.

Effect of crude extract of roots of Bredemeyera floribunda Willd. III. Effect on hormone-stimulated water transport in isolated frog skin

The diuretic activity of the crude extract of roots of Bredemeyera floribunda Willd. (Polygalaceae) was investigated on basal and hormone-stimulated absorptive water transport in the isolated frog skin. The extract, added to the mucosal skin surface, at the peak of oxytocin (Oxy) action induces steady and dose-dependent reduction of water transport. Pre-treatment of the skins during 2 h with the extract, added to mucosal skin surface, renders the epithelium nonresponsive to oxytocin. These effects are not observed when the extract is added to the serosal skin surface. Basal water transport is not altered by the extract. The results, apart from indicating inhibition of the oxytocin-stimulated water transport in frog skin epithelium, support the folk therapeutic use of infusion of the root extract as a diuretic.

Chloride currents of single mitochondria-rich cells of toad skin epithelium.

1. Mitochondria-rich (MR) cells in free suspension were obtained by collagenase and trypsin treatments of toad skin epithelium and studied by whole-cell voltage clamp and membrane current fluctuation analysis. 2. Cells studied with a 100 microM amiloride and 5 mM Ba2+ Ringer solution on the outside and 10 mM Cs+ in the pipette generated large membrane currents with reversal potentials varying in a Nernstian way with pipette [Cl-]. 3. The membrane chloride currents were activated in excess of Goldman-Hodgkin-Katz rectification by cell depolarization and clamping to positive cell potentials (VC). The resulting Cl- permeability was presented as an S-shaped function of membrane potential with half-maximal activation in the range 0 mV < VC < 50 mV. 4. The power density spectrum of Cl- current fluctuations could be fitted with a single Lorentzian component with a corner frequency, fc, of 34.9 +/- 2.6 Hz, and a low frequency asymptote, S(o), or 14.6 +/- 1.3 pA2 s per cell (mean +/- S.E.M.; VC = 25 mV, ECl = 0 mV, n = 6). 5. The lower-limit single channel conductance, gamma Cl(1 - Po), was 128 +/- 9 pS (VC = 25 mV, n = 6), where Po is the open channel probability. With Po = 0.5, this result indicates that Cl- channels of large unitary conductance (200-300 pS) are present in the mitochondria-rich cell membrane.

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.

Control of Na+ and H+ transports by exocytosis/endocytosis phenomena in a tight epithelium.

The relationship linking Na+ and H+ transports and exocytosis/endocytosis located in the apical membranes of the frog skin epithelium was investigated under various conditions of ion transport stimulation. The exocytosis process, indicating insertion of intracellular vesicles, which were preloaded with fluorescent FITC-dextran (FD), was measured by following the FD efflux in the apical bathing solution. Na+ transport stimulators such as serosal hypotonic shock (replacement of serosal Ringer solution by half-Ringer or 4/5-Ringer), apical PCMPS (10(-3) M) and amphotericin-B (20 micrograms/ml), were also found to stimulate the exocytotic rates of FD. Acidification of the epithelium by CO2 or post NH4 load, conditions which increase the proton secretion also stimulated the FD release in the apical bathing solution. On the other hand, alkalization of the epithelial cells increased the endocytosis rate. Hypotonic shock, acid load and PCMPS induced an increase in cell calcium which is probably the signal within the cell for exocytosis. In addition, quantitative spectrofluorimetric measurements of F-actin content after rhodamine-phalloidin staining, indicated a decrease in the F-actin content as a result of cell acidosis, hypotonic conditions and amphotericin additions. It is proposed that the insertion/retrieval of intracytoplasmic vesicles containing H+ pumps plays a key role in the regulation of proton secretion in tight epithelia. In addition, it is suggested that cytoskeleton depolymerization of F-actin filaments facilitates H+ pump insertion. A comparable working hypothesis for the control of Na+ transport is proposed.

Effects of anions and/or cell volume on the permeance of an apical water pathway induced by Hg in toad skin epithelium.

Hg compounds block membrane transport units behaving as water channels. Here we show that Hg induces an apical water pathway in toad skins pretreated with 10(-3) M CH3ClHg or HgCl2, added to the outer bathing medium. Washing with SO4-Ringer caused a several-fold increase in net water flow (Jw) and osmotic permeability coefficient (Pf) that was reversed by re-exposure to Cl- or NO3-Ringer and mimicked by gluconate-Ringer. These Pf changes could be elicited repeatedly and were present if, and only if, anion replacements took place in the inner bathing solution. Such inner polarity was related to the anion permeability of the epidermal basolateral membrane: impermeant anions (SO4, gluconate) increased Pf; permeant anions (Cl, NO3) did not change basal Pf but reversed the high Pf induced by impermeant anions. Hg induced the appearance of aggregates that persisted despite repeated washings of the skins during 4-5 h, and whether Pf was high (SO4-Ringer) or low (Cl-Ringer) before skin fixation. The Hg-induced apical water pathway in toad skin appears to be a unique model for studying the interplay between cell volume, cell ionic composition and water permeability.

Role of proton pump of mitochondria-rich cells for active transport of chloride ions in toad skin epithelium.

1. Active Cl- currents were studied in short-circuited toad skin epithelium in which the passive voltage-activated Cl- current is zero. Under visual control double-barrelled microelectrodes were used for impaling principal cells from the serosal side, or for measuring the pH profile in the solution bathing the apical border. 2. The net inward (active) 36Cl- flux of 27 +/- 8 pmol s-1 cm-2 (16) (mean +/- S.E.M (number of observation)) was abolished by 2 mM-CN- (6.3 +/- 3.5 pmol s-1 cm-2 (8)). The active flux was maintained in the absence of active Na+ transport when the latter was eliminated by either 100 microM-mucosal amiloride, replacement of mucosal Na+ with K+, or by 3 mM-serosal ouabain. 3. In Ringer solution buffered by 24 mM-HCO3- -5% CO2 mucosal amiloride reversed the short circuit current (ISC). The outward ISC was maintained when gluconate replaced mucosal Cl-, and it was reversibly reduced in CO2-free 5 mM-Tris-buffered Ringer solution (pH = 7.40) or by the proton pump inhibitor oligomycin. These observations indicate that the source of the outward ISC is an apical proton pump. 4. Amiloride caused principal cells to hyperpolarize from a basolateral membrane potential, Vb, of -73 +/- 3 (22) to -93 +/- 1 mV (26), and superfusion with CO2-free Tris-buffered Ringer solution induced a further hyperpolarization (Vb = -101 +/- 1 mV (26)) which could be blocked by Ba2+. The CO2-sensitive current changes were null at Vb = EK (potassium reversal potential, -106 +/- 2 mV (55)) implying that they are carried by K+ channels in the basolateral membrane. Such a response cannot account for the inhibition of the outward ISC which by default seems to be located to mitochondria-rich (MR) cells. 5. In the absence of mucosal Cl- a pH gradient was built up above MR cells with pH = 7.02 +/- 0.04 (42) and pH increasing to 7.37 +/- 0.02 (10) above principal cells (pH = 7.40 in bulk solution buffered by 0.1 mM-Tris). This observation localizes a proton pump to the apical membrane of MR cells. Using the integrated diffusion equation it was shown that the measured external pH gradient would account within an order of magnitude for measured currents. 6. Standing gradients of protons were eliminated in the presence of mucosal Cl- suggesting that active uptake of Cl- is associated with the exit of base equivalents across the apical membrane of MR cells.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Effects of intracellular signals on Na +/K +-ATPase pump activity in the frog skin epithelium

The effects of intracellular signals (pH i, Na i +, Ca i 2+, and the electrical membrane potential), on Na + transport mediated by the Na +/K + pump were investigated in the isolated Rana esculenta frog skin. In particular we focussed on pH i sensitivity since protons act as an intrinsic regulator of transepithelial Na + transport ( J Na) by a simultaneous control of the apical membrane Na + conductance ( g Na) and the basolateral membrane K + conductance ( g K), pH i changes which modify J Na, g Na and g K, do not affect the Na + transport mediated by the pump as shown by kinetic and electrophysiological studies. In addition, no changes were observed in the number of 3H-ouabain binding sites in acid-loaded epithelia. Our attempts to modify cellular Ca 2+ (by using Ca 2+-free/EGTA Ringer solution or A23187 addition) also failed to produce any significant effects in the Na + pump turnover rate or the number of 3H-ouabain binding sites. The Na + pump current was found to be sensitive to the basolateral membrane potential, saturating for very positive (cell) potentials and a reversal potential of − 160 mV was calculated from I–V relationships of the pump. Changes in Na i + considerably affected the Na + pump rate. A saturating relationship was found between pump rate and Na i + with maximal activation at Na i + > 40 mmol/l; a high dependence of the pump rate and of the number of 3H-ouabain binding sites was observed in the physiological range of Na i +. We conclude that protons (in the physiological pH range) which act directly and simultaneously on the passive transport pathways ( g Na and g K), have no direct effect on the Na +/K + pump rate. After an acid load, the inhibition of J Na is primarily due to the reduction of g Na. This results in a reduction of Na i and the pump turnover rate then becomes dependent on other pathways of Na + entry such as the basolateral membrane Na +/H + exchanger.

Cyclic AMP-and beta-agonist-activated chloride conductance of a toad skin epithelium.

1. The control by intracellular cyclic AMP and beta-adrenergic stimulation of chloride conductance was studied in toad skin epithelium mounted in a chamber on the stage of an upright microscope. Impalement of identified principal cells from the serosal side with single-barrelled conventional or double-barrelled Cl(-)-sensitive microelectrodes was performed at x500 magnification. For blocking the active sodium current 50 microM-amiloride was present in the mucosal bath. 2. When clamped at transepithelial potential difference V = 0 mV, the preparations generated clamping currents of 0.9 +/- 1 microA/cm2 (mean +/- S.E.M.; number of observations n = 55). The intracellular potential of principal cells (Vb) was -96 +/- 2 mV with a fractional resistance of the basolateral membrane (fRb) of 0.016 +/- 0.003 (n = 54), and an intracellular Cl- activity of 40 +/- 2 mM (n = 24). 3. At V = 0 mV, serosal application of a cyclic AMP analogue, dibutyryl cyclic AMP (500 microM) or a beta-adrenergic agonist, isoprenaline (5 microM) resulted in a sixfold increase in transepithelial Cl- conductance identified by standard 36Cl- tracer technique. 4. The clamping current at V = 0 mV was unaffected by cyclic AMP (short-circuit current Isc = 0.1 +/- 0.3 microA/cm2, n = 16) indicating that subepidermal Cl(-)-secreting glands are not functioning in our preparations obtained by collagenase treatment. 5. Cyclic AMP- or isoprenaline-induced chloride conductance (Gcl) activation (V = 0 mV) was not reflected in membrane potential and intracellular Cl- activity in principal cells. Intracellular chloride activity was constant at approximately 40 mM at membrane potentials between -90 and -100 mV. Therefore, it can be concluded that the principal cells are not contributing to activated Cl- currents. 6. At V = -100 mV where the voltage-dependent chloride conductance of mitochondria-rich (MR) cells was already fully activated, GCl was unaffected by cyclic AMP or isoprenaline. The major effect of these treatments was a rightward displacement of the MR cell-generated GCl-V relationship along the V axis. 7. Our results indicate that the beta-adrenergically controlled cyclic AMP-mediated chloride conductance is localized to the mitochondria-rich cells.

Papaverine reduces the sodium permeability of the apical membrane and the potassium permeability of the basolateral membrane in isolated frog skin.

The effect of papaverine, an inhibitor of the phosphodiesterase responsible for breakdown of cAMP, on the transepithelial sodium transport across the isolated frog skin was investigated. Serosal addition of papaverine caused initially an increase in the short-circuit current (SCC), a doubling of the cellular cAMP content and a depolarization of the intracellular potential under SCC conditions (Vscc). The initial increase in the SCC was followed by a pronounced decrease both in the SCC and in the natriferic action of antidiuretic hormone (ADH), but papaverine had no inhibitory effect on the ability of ADH to increase the cellular cAMP content. As SCC declines, no hyperpolarization was observed. The I/V relationship across the apical membrane during the inhibitory phase, revealed that papaverine reduces the sodium permeability of the apical membrane (PNaa) as well as intracellular sodium concentration. These observations and the previously noted effect of papaverine on Vscc indicates that papaverine must have an effect on the cellular Cl or K permeability. The basolateral Na,K,2Cl cotransporter was blocked with bumetanide, which should bring the cellular chloride in equilibrium. Bumetanide had no effect on basal SCC and Vscc. When papaverine was added to skins preincubated with bumetanide, the effect of papaverine on SCC and Vscc was unchanged. Therefore, the depolarization of Vscc, observed during the papaverine-induced inhibition of the SCC, must be due to a reduction in the cellular K permeability. In conclusion, it is suggested that papaverine reduces the sodium permeability of the apical membrane and the potassium permeability of the basolateral membrane of the frog skin epithelium.