The volume of mitochondria-rich cells of frog skin epithelium

Summary The pathway for movement of chloride ions across frog skin is not well understood. Mitochondria-rich (MR) cells have been proposed as the route for chloride across the skin. To test this hypothesis we studied the MR cells of the skin of the frog,Rana pipiens, by quantitative light microscopic determination of cell volume. MR cell volume was influenced by changes in the chloride concentration or osmolality of the outside bathing solution. MR cells shrank about 23% when all chloride was removed from the outside (mucosal) bathing solution. MR cells were also shown to be responsive to changes in the osmolality of either the mucosal or serosal bath. Osmotically-induced swelling caused by dilution of the serosal bath resulted in volume regulatory decrease. These results are consistent with the hypothesis that MR cells constitute the pathway for chloride movement across frog skin.     

Cell volume regulation by Amphiuma red blood cells. The role of Ca+2 as a modulator of alkali metal/H+ exchange

In response to osmotic perturbation, the Amphiuma red blood cell regulates volume back to "normal" levels. After osmotic swelling, the cells lose K, Cl, and osmotically obliged H2O (regulatory volume decrease [RVD] ). After osmotic shrinkage, cell volume is regulated as a result of Na, Cl, and H2O uptake (regulatory volume increase [RVI] ). As previously shown (Cala, 1980 alpha), ion fluxes responsible for volume regulation are electroneutral, with alkali metal ions obligatorily counter-coupled to H, whereas net Cl flux is in exchange for HCO3. When they were exposed to the Ca ionophore A23187, Amphiuma red blood cells lost K, Cl, and H2O with kinetics (time course) similar to those observed during RVD. In contrast, when cells were osmotically swollen in Ca-free media, net K loss during RVD was inhibited by approximately 60%. A role for Ca in the activation of K/H exchange during RVD was suggested from these experiments, but interpretation was complicated by the fact that an increase in cellular Ca resulted in an increase in the membrane conductance to K (GK). To determine the relative contributions of conductive K flux and K/H exchange to total K flux, electrical studies were performed and the correspondence of net K flux to thermodynamic models for conductive vs. K/H exchange was evaluated. These studies led to the conclusion that although Ca activates both conductive and electroneutral K flux pathways, only the latter pathways contribute significantly to net K flux. On the basis of observations that A23187 did not activate K loss from cells during RVI (when the Na/H exchange was functioning) and that amiloride inhibited K/H exchange by swollen cells only when cells had previously been shrunk in the presence of amiloride, I concluded that Na/H and K/H exchange are mediated by the same membrane transport moiety.     

Mitochondria-rich cells in anuran amphibia: chloride conductance and regional distribution over the body surface

The distribution and density (D(mrc)) of mitochondria-rich cells (MR cells) in skin epithelium, were determined over the whole body surface in nine species of anuran Amphibia that live in a variety of habitats. It was found that the more terrestrial species (beginning with Hyla arborea) have a higher density of MR cells in their pelvic region. In the skin of aquatic (Xenopus laevis) or fossorial (Pelobates syriacus) species, D(mrc) is evenly distributed over the whole body surface. In dorsal skin pieces of H. arborea that lack detectable MR cells, transepithelial voltage activation did not induce Cl(-) conductance as it did in ventral pieces. Skins from Bufo viridis and X. laevis, both have MR cells in their skin, differ markedly in their biophysical properties: a Cl(-) specific current conductance is predominant in the skin epithelium of B. viridis, and is absent in X. laevis. In the latter, anionic conductance is due to glandular secretion. The biophysical properties cannot therefore be related solely to the presence or density of MR cells. Mitochondria-rich cells are sites of Cl(-) conductance across the skin of those amphibians that show this property, but must have different function(s) in other species. It is suggested that the specific zonal distribution of MR cells in the species that were examined in this study could be due to ion exchange activity and water conservation in more terrestrial environments.     

Skin epithelial transport and structural relationships in naturally metamorphosing Pelobates syriacus

The onset of active Na(+) transport and activated Cl(-) conductance (G(Cl)) across the skin epithelium of Pelobates syriacus was investigated during natural ontogenetic development. Structural features, including band three and Peanut lectin bindings were tested in parallel and structure-function relationships were attempted. The 22 specimens studied were divided into two tadpole, three juvenile, and two adult stages, corresponding to the Taylor-Kollros standard table, in accordance with external morphology of their developmental stage. Onset of transepithelial electrical potential and drop in conductance occurred abruptly, coinciding with metamorphosis climax of tadpoles into juveniles at about stage XXI of development. Amiloride-sensitive Na(+) transport occurred a little later at stage XXIII, followed by the appearance of activated Cl(-) conductance, G(Cl). Parallel structural examination showed that skin MR cells occurred upon metamorphosis, as the tadpole integument transformed into the adult epithelium and could be associated with the occurrence of activated G(Cl). It was not related temporally with the appearance of band three protein in MR cells. Our findings support the association of G(Cl) with MR cells, whereas band three may only be a corollary of G(Cl) and not necessarily essential for its manifestation.     

Volume-activated chloride permeability can mediate cell volume regulation in a mathematical model of a tight epithelium

Cell volume regulation during anisotonic challenge is investigated in a mathematical model of a tight epithelium. The epithelium is represented as compliant cellular and paracellular compartments bounded by mucosal and serosal bathing media. Model variables include the concentrations of Na, K, and Cl, hydrostatic pressure, and electrical potential, and the mass conservation equations have been formulated for both steady-state and time-dependent problems. Ionic conductance is represented by the Goldman constant field equation (Civan, M.M., and R.J. Bookman. 1982. Journal of Membrane Biology. 65:63-80). A basolateral cotransporter of Na, K, and Cl with 1:1:2 stoichiometry (Geck, P., and E. Heinz. 1980. Annals of the New York Academy of Sciences. 341:57-62.) and volume-activated basolateral ion permeabilities are incorporated in the model. MacRobbie and Ussing (1961. Acta Physiologica Scandinavica. 53:348-365.) reported that the cells of frog skin exhibit osmotic swelling followed by a volume regulatory decrease (VRD) when the serosal bath is diluted to half the initial osmolality. Similar regulation is achieved in the model epithelium when both a basolateral cotransporter and a volume-activated Cl permeation path are included. The observed transepithelial potential changes could only be simulated by allowing volume activation of the basolateral K permeation path. The fractional VRD, or shrinkage as percent of initial swelling, is examined as a function of the hypotonic challenge. The fractional VRD increases with increasing osmotic challenge, but eventually declines under the most severe circumstances. This analysis demonstrates that the VRD response depends on the presence of adequate intracellular chloride stores and the volume sensitivity of the chloride channel.     

Response of the Frog Skin to Steady-State Voltage Clamping : I. The shunt pathway

Properties of the shunt pathway (a pathway in parallel to the Na transport system) in frog skin have been examined. The permeability of this shunt to urea increases markedly when the skin is depolarized to -100 mv (inside negative) but hyperpolarization to +100 mv produces no change in urea permeability compared to short-circuit conditions. The permeability increase at depolarizing potentials is dependent on the external solute concentration and is considerably reduced by the presence of external Ca. Neither urea permeability nor its response to changes in potential difference are affected by complete inhibition of Na transport by ouabain. In ouabain-poisoned skins, movements of Na, K, Cl, and mannitol through the shunt change in parallel with urea movements. Ion fluxes under these conditions and their response to potential can be described by the constant field equation. The selectivity of the shunt is in the order Cl > urea > K > Na > mannitol and this order does not appear to be affected by the absolute magnitude of the shunt permeability. Arguments are presented suggesting that the pathway is mainly between cells and that its permeability may be affected by cell swelling.     

Identification and regulation of whole-cell chloride currents in airway epithelium

We used the whole-cell patch-clamp technique to study membrane currents in human airway epithelial cells. The conductive properties, as described by the instantaneous current-voltage relationship, rectified in the outward direction when bathed in symmetrical CsCl solutions. In the presence of Cl concentration gradients, currents reversed near ECl and were not altered significantly by cations. Agents that inhibit the apical membrane Cl conductance inhibited Cl currents. These conductive properties are similar to the conductive properties of the apical membrane Cl channel studied with the single-channel patch-clamp technique. The results suggest that the outwardly rectifying Cl channel is the predominant Cl-conductive pathway in the cell membrane. The steady-state and non-steady-state kinetics indicate that current flows through ion channels that are open at hyperpolarizing voltages and close with depolarization. These Cl currents were regulated by the cAMP-dependent protein kinase: when the catalytic subunit of cAMP-dependent protein kinase was included in the pipette solution, Cl channel current more than doubled. We also found that reducing extracellular osmolarity by 30% increased Cl current, suggesting that cell-swelling stimulated Cl current. Studies of transepithelial Cl transport in cell monolayers suggest that a reduction in solution osmolarity activates the apical Cl channel: reducing extracellular osmolarity stimulated a short-circuit current that was inhibited by Cl-free solution, by mucosal addition of a Cl channel antagonist, and by submucosal addition of a loop diuretic. These results suggest that apical membrane Cl channels may be regulated by cell volume and by the cAMP-dependent protein kinase.     

Volume-responsive sodium and proton movements in dog red blood cells

Shrinkage of dog red blood cells (RBC) activates a Na transport pathway that is Cl dependent, amiloride sensitive, and capable of conducting Na- proton counterflow. It is possible to establish transmembrane gradients for either Na or protons and to demonstrate that each cation species can drive reciprocal movements of the other. The nature of the coupling between Na and proton movements was investigated using the fluorescent probe diS-C3(5) and also by an indirect method in which K movements through valinomycin channels were used to draw inferences about the membrane potential. No evidence was found to suggest that the Na-proton pathway activated by shrinkage of dog RBC is a conductive one. By exclusion, it is presumed that the coupling between the counterflow of Na and protons is electroneutral. The volume-activated Na-proton fluxes in dog RBC have certain properties that distinguish them from similar transport pathways in other cell types.     

Morphological changes in the skin of Rana pipiens in response to metabolic acidosis

The skin of Rana pipiens excretes H+ and this excretion is increased by metabolic acidosis. The mitochondria-rich (MR) cells of the skin have been found to mediate this H+ transport. The purpose of this study was to determine if there is a change in the MR cells of the skin during metabolic acidosis and if the isolated split epithelia of frog skin maintains its capacity to excrete H+. Metabolic acidosis was induced by injecting 120 mM NH4Cl (0.025 ml/g body wt) into the dorsal lymph sac three times a day for 2 days. The frogs were sacrificed and collagenase-split skins from the abdomen of normal and metabolic acidotic frogs were mounted between 2-ml chambers. H+ fluxes into both the mucosal and serosal media were measured and reported in units of (nmol) (cm2)-1 (min)-1. An increase in H+ flux was seen on both the mucosal and serosal sides of the acidotic split skins. The isolated epithelia were fixed, postosmicated, and dehydrated in the chamber. They were then embedded in Spurr's resin and 1-micron sections were cut and stained with Paragon multiple stain. Coded slides were used to count various cell types. Sections were randomly selected and approximately 40,000 cells were counted. Four basic cell types were noted and confirmed by TEM photomicrographs; basal (B) cells, granular (G) cells, keratinized cells, and MR cells. The ratio of G + B cells:MR cells in the normal skins was 1.0:0.021. The ratio in acidotic skins was 1.0:0.34. The average percentage of cell population of MR cells in the normal skins was 2.08 + 0.18 and in acidotic skins 3.20 + 0.36 (P less than 0.005). We conclude that the split skin maintains the capacity to acidify the mucosal fluid. Additionally, during metabolic acidosis there is an increased number of MR cells in the skin and this increase may be an adaptive mechanism of the skin to excrete excess H+ during acidosis.     

Basolateral Cl channels in primary airway epithelial cultures

Salt and water absorption and secretion across the airway epithelium are important for maintaining the thin film of liquid lining the surface of the airway epithelium. Movement of Cl across the apical membrane involves the CFTR Cl channel; however, conductive pathways for Cl movement across the basolateral membrane have been little studied. Here, we determined the regulation and single-channel properties of the Cl conductance (G(Cl)) in airway surface epithelia using epithelial cultures from human or bovine trachea and freshly isolated ciliated cells from the human nasal epithelium. In Ussing chamber studies, a swelling-activated basolateral G(Cl) was found, which was further stimulated by forskolin and blocked by N-phenylanthranilic acid (DPC) = sucrose > flufenamic acid = niflumic acid = glibenclamide > CdCl(2) = 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) = DIDS = ZnCl(2) > tamoxifen > 4,4'-dinitro-2,2'-stilbene-disulfonate disodium salt (DNDS). In whole cell patch-clamp experiments, three types of G(Cl) were identified: 1) a voltage-activated, DIDS- (but not Cd-) blockable and osmosensitive G(Cl); 2) an inwardly rectifying, hyperpolarization-activated and Cd-sensitive G(Cl); and 3) a forskolin-activated, linear G(Cl), which was insensitive to Cd and DIDS. In cell-attached patch-clamp recordings, the basolateral pole of isolated ciliated cells expressed three types of Cl channels: 1) an outwardly rectifying, swelling-activated Cl channel; 2) a strongly inwardly rectifying Cl channel; and 3) a forskolin-activated, low-conductance channel. We propose that, depending on the driving force for Cl across the apical membrane, basolateral Cl channels confine Cl(-) secretion or support transcellular Cl(-) absorption.     

Interactions of sodium transport, cell volume, and calcium in frog urinary bladder

The volume of individual cells in intact frog urinary bladders was determined by quantitative microscopy and changes in volume were used to monitor the movement of solute across the basolateral membrane. When exposed to a serosal hyposmotic solution, the cells swell as expected for an osmometer, but then regulate their volume back to near control in a process that involves the loss of KCl. We show here that volume regulation is abolished by Ba++, which suggests that KCl movements are mediated by conductive channels for both ions. Volume regulation is also inhibited by removing Ca++ from the serosal perfusate, which suggests that the channels are activated by this cation. Previously, amiloride was observed to inhibit volume regulation: in this study, amiloride-inhibited, hyposmotically swollen cells lost volume when the Ca++ ionophore A23187 was added to Ca++-replete media. We attempted to effect volume changes under isosmotic conditions by suddenly inhibiting Na+ entry across the apical membrane with amiloride, or Na+ exit across the basolateral membrane with ouabain. Neither of these Na+ transport inhibitors produced the expected results. Amiloride, instead of causing a decrease in cell volume, had no effect, and ouabain, instead of causing cell swelling, caused cell shrinkage. However, increasing cell Ca++ with A23187, in both the absence and presence of amiloride, caused cells to lose volume, and Ca++-free Ringer's solution (serosal perfusate only) caused ouabain-blocked cells to swell. Finally, again under isosmotic conditions, removal of Na+ from the serosal perfusate caused a loss of volume from cells exposed to amiloride. These results strongly suggest that intracellular Ca++ mediates cell volume regulation by exerting a negative control on apical membrane Na+ permeability and a positive control on basolateral membrane K+ permeability. They also are compatible with the existence of a basolateral Na+/Ca++ exchanger.     

Calcium-mobilizing agonists stimulate anion fluxes in cultured endothelial cells from human umbilical vein

Intracellular ion concentrations were determined in split skins of Rana pipiens using the technique of electron microprobe analysis. Based on the 1 min Br uptake from the apical bath, two types of mitochondria-rich (MR) cells could be distinguished: active cells which rapidly exchanged their anions with the apical bath and inactive cells which did not. Br uptake and frequency of active MR cells were closely correlated with the skin conductance, g _t. Replacing Cl in the apical bath with an impermeant anion significantly lowered g _t and the Br uptake and Na concentration of active cells. Even larger reductions were observed after apical amiloride (0.1 mm). The inhibition of the Br uptake was reversible by voltage clamping (100 mV, inside positive). Cl removal and amiloride also led to some shrinkage of active cells. The results suggest that the active cell is responsible for a large part of g _t. Inactive MR cells had much lower Br and Na concentrations which were not significantly affected by Cl removal, amiloride, or voltage clamping. Principal cells, which represent the main cell type of the epithelium, showed only a minimal Br uptake from the apical side which was not correlated with g _t. Moreover, Cl removal had no effect on the Na, Br, and Cl concentrations of principal cells.I wish to thank Cathy Langford for her excellent technical assistance. Financial support was provided by National Institutes of Health grants DK35717 and 1S10-RR0-234501.     

Effect of potassium on cell volume regulation in renal straight proximal tubules

Summary The present study was designed to assess for the influence of extracellular potassium and of inhibitors of potassium transport on cell volume regulatory decrease in isolated perfused straight proximal tubules of the mouse kidney. Volume regulatory decrease is virtually unaffected when bath potassium concentration is elevated from 5 to 20 mmol/liter, and still persists, albeit significantly retarded, in the presence of the potassium channel blocker barium on both sides of the epithelium and during virtually complete dissipation of the transmembrane potassium gradient by increasing extracellular potassium concentration to 40 mmol/liter. As evident from electrophysiologic observations, barium blocks the potassium conductance of the basolateral cell membrane. Reduction of bicarbonate concentration and increase of H⁺ concentration in the bath solution cannot compensate for enhanced potassium concentration and cell volume regulatory decrease is not affected in the presence of the K/H exchange inhibitor omeprazole. Similarly cell volume regulatory decrease is not affected by ouabain. In conclusion, potassium movements through potassium channels in the basolateral cell membrane are important determinants of cell volume and may participate in cell volume regulatory decrease. However, a powerful component of cell volume regulatory decrease in straight proximal tubules of the mouse kidney is apparently independent of potassium conductive pathways, K/H exchange and Na⁺/K⁺-ATPase.     

Proton pump-driven cutaneous chloride uptake in anuran amphibia

Krogh introduced the concept of active ion uptake across surface epithelia of freshwater animals, and proved independent transports of Na(+) and Cl(-) in anuran skin and fish gill. He suggested that the fluxes of Na(+) and Cl(-) involve exchanges with ions of similar charge. In the so-called Krogh model, Cl(-)/HCO(3)(-) and Na(+)/H(+) antiporters are located in the apical membrane of the osmoregulatory epithelium. More recent studies have shown that H(+) excretion in anuran skin is due to a V-ATPase in mitochondria-rich (MR) cells. The pump has been localized by immunostaining and H(+) fluxes estimated by pH-stat titration and mathematical modelling of pH-profiles in the unstirred layer on the external side of the epithelium. H(+) secretion is voltage-dependent, sensitive to carbonic-anhydrase inhibitors, and rheogenic with a charge/ion-flux ratio of unity. Cl(-) uptake from freshwater is saturating, voltage independent, and sensitive to DIDS and carbonic-anhydrase inhibitors. Depending on anuran species and probably on acid/base balance of the animal, apical exit of protons is coupled to an exchange of Cl(-) with base (HCO(3)(-)) either in the apical membrane (gamma-type of MR cell) or in the basolateral membrane (alpha-type MR cell). The gamma-cell model accounts for the rheogenic active uptake of Cl(-) observed in several anuran species. There is indirect evidence also for non-rheogenic active uptake accomplished by a beta-type MR cell with apical base secretion and basolateral proton pumping. Several studies have indicated that the transport modes of MR cells are regulated via ion- and acid/base balance of the animal, but the signalling mechanisms have not been investigated. Estimates of energy consumption by the H(+)-ATPase and the Na(+)/K(+)-ATPase indicate that the gamma-cell accomplishes uptake of NaCl in normal and diluted freshwater. Under common freshwater conditions with serosa-positive or zero V(t), the K(+) conductance of the basolateral membrane would have to maintain the inward driving force for Na(+) uptake across the apical membrane. With the K(+) equilibrium potential across the basolateral membrane estimated to -105 mV, this would apply to external Na(+) concentrations down to 40-120 micromol/l. NaCl uptake from concentrations down to 10 micromol/l, as observed by Krogh, presupposes that the H(+) pump hyperpolarizes the apical membrane, which would then have to be associated with serosa-negative V(t). In diluted freshwater, exchange of cellular HCO(3)(-) with external Cl(-) seems to be possible only if the proton pump has the additional function of keeping the external concentration of HCO(3)(-) low. Quantitative considerations also lead to the conclusion that with the above extreme demand, at physiological intracellular pH of 7.2, the influx of Cl(-) via the apical antiporter and the passive exit of Cl(-) via basolateral channels would be possible within a common range of intracellular Cl(-) concentrations.     

Ion transport by mitochondria-rich cells in toad skin

Summary The optical sectioning video imaging technique was used for measurements of the volume of mitochondria-rich (m.r.) cells of the isolated epithelium of toad skin. Under short-circuit conditions, cell volume decreased by about 14% in response to bilateral exposure to Cl-free (gluconate substitution) solutions, apical exposure to ouabain resulted in a large increase in volume, which could be prevented either by the simultaneous application of amiloride in the apical solution or by the exposure of the epithelium to bilateral Cl-free solutions. Unilateral exposure to a Cl-free solution did not prevent ouabain-induced cell swelling. It is concluded that m.r. cells have an amiloride-blockable Na conductance in the apical membrane, a ouabain-sensitive Na pump in the basolateral membrane, and a passive Cl permeability in both membranes. From the initial rate of ouabain-induced cell volume increase the active Na current carried by a single m.r. cell was estimated to be 9.9±1.3 pA. Voltage clamping of the preparation in the physiological range of potentials (0 to –100 mV, serosa grounded) resulted in a cell volume increase with a time course similar to that of the stimulation of the voltage-dependent activation were prevented by exposure of the tissue to a Cl-free apical solution. The steady-state volume of the m.r. cells increased with the clamping voltage, and at –100 mV the volume was about 1.15 times that under short-circuit conditions. The rate of volume increase during current passage was significantly decreased by lowering the serosal K concentration (K _i ) to 0.5mm, but was independent of whether K _i was 2.4, 5, or 10mm. This indicates that the K conductance of the serosal membrane becomes rate limiting for the uptake of KCl when K _i is significantly lower than its physiological value. It is concluded that the voltage-activated Cl currents flow through the m.r. cells and that swelling is caused by an uptake of Cl ions from the apical bath and K ions from the serosal bath. Bilateral exposure of the tissue to hypo- or hypertonic bathing solutions changed cell volume without detectable changes in the Cl conductance. The volume response to external osmotic perturbations followed that of an osmometer with an osmotically inactive volume of 21%. Using this value and the change in cell volume in response to bilateral Cl-free solutions, we calculated an intracellular steady-state Cl concentration of 19.8±1.7mm (n=6) of the short-circuited cell.     

On the distribution of Na+ pump sites in the frog skin

Exposure of the outside of the isolated frog skin to a Ringer's solution, made hypertonic by the addition of mannitol, causes a rapid and sustained increase in transepithelial permeability through a structural distortion-a focal blistering-of the "tight" junctions of the outermost living cell layer. [(3)H]ouabain, used as an autoradiographic marker for the Na+-pump (Na+-K+-adenosine triphosphatase), is usually unable to penetrate the frog skin from the outside solution, but when added to a hypertonic mannitol- Ringer's solution in the outside bath it readily penetrates the epithelium, presumably through the opened shunt pathway. Radioautographic analysis of [(3)H]ouabain binding sites revealed that most of ouabain enters from the outside solution binds to the sites on the cell membranes of the stratum spinosum, as was the case when it was applied from the inside bath in an earlier study. The outer living cell layer, the first to be exposed to ouabain, does not appear to be the major site for the Na+-pump, and therefore, is not likely to be responsible for most of the active pumping of Na+. This result demonstrates that previous failure to show a high density of Na+-pump sites on the cells of the outermost layer, when [(3)H]ouabain was applied from the inside solution, was not due to the inability of the marker to reach these cells at a sufficient concentration to reveal all pump sites. These results provide further support for a model of Na+-transport across the frog skin which distributes the active pump step on the inward facing membranes of all living cells.     

Responses of frog skin Na(+) and Cl(-) transport to guanylin

A possible role for the peptide hormone guanylin was investigated in frog skin (Rana pipiens) epithelium. Sodium and chloride fluxes in response to this peptide were evaluated in Ussing-type chambers. Net and unidirectional Na(+) fluxes were measured by using (22)Na(+) and atomic absorption analysis of total [Na(+)], whereas net Cl(-) fluxes were measured by using electrometric titration for [Cl(-)]. Mucosal application of guanylin (0.5-2.0 micromol/l) caused marked increases in serosal to mucosal net flux and efflux of Na(+). Serosal application of guanylin over the same dose range caused similar large increases in net serosal to mucosal (S-->M) Na(+) and Cl(-) flux as well as Na(+) efflux. Responses of Na(+) influx were small and inconsistent. When frog skin was bathed on the serosal side with Cl(-)-free Ringer's solution mucosal application of guanylin stimulated large efflux and S-->M net fluxes of Na(+). Serosal treatment yielded large Na(+) effluxes and S-->M Na(+) and Cl(-) net fluxes. When frog skin serosal surfaces were bathed with Na(+)- free Ringer's solution mucosal guanylin treatment had no effect but serosal treatment produced large S-->M Cl(-) net fluxes.     

Electron microprobe analysis of frog skin epithelium: Evidence for a syncytial sodium transport compartment

Summary For elucidation of the functional organization of frog skin epithelium with regard to transepithelial Na transport, electrolyte concentrations in individual epithelial cells were determined by electron microprobe analysis. The measurements were performed on 1-m thick freeze-dried cryosections by an energy-dispersive X-ray detecting system. Quantification of the electrolyte concentrations was achieved by comparing the X-ray intensities obtained in the cells with those of an internal albumin standard.The granular, spiny, and germinal cells, which constitute the various layers of the epithelium, showed an identical behavior of their Na and K concentrations under all experimental conditions. In the control, both sides of the skin bathed in frog Ringer's solution, the mean cellular concentrations (in mmole/kg wet wt) were 9 for Na and 118 for K. Almost no change in the cellular Na occurred when the inside bathing solution was replaced by a Na-free isotonic Ringer's solution, whereas replacing the outside solution by distilled water resulted in a decrease of Na to almost zero in all layers. Inhibition of the transepithelial Na transport by ouabain (10^–4 m) produced an increase in Na to 109 and a decrease in K to 16. The effect of ouabain on the cellular Na and K concentrations was completely cancelled when the Na influx from the outside was prevented, either by removing Na or adding amiloride (10^–4 m). When, after the action of ouabain, Na was removed from the outside bathing solution, the Na and K concentration in all layers returned to control values. The latter effect could be abolished by amiloride.The other cell types of the epithelium showed under some experimental conditions a different behavior. In the cornified cells and the light cells, which occurred occasionally in the stratum granulosum, the electrolyte concentrations approximated those of the outer bathing meium under all experimental conditions. In the mitochondria-rich cells, the Na influx after ouabain could not be, prevented by adding amiloride. In the gland cells, only a small change in the Na and K concentrations could be detected after ouabain.The results of the present study are consistent with a two-barrier concept of transepithelial Na transport. The Na transport compartment comprises all living epithelial layers. Therefore, with the exception of some epithelial cell types, the frog skin epithelium can be regarded as a functional syncytium for Na.     

Cytochemistry and ultrastructure of frog pancreatic exo- and endocrine epithelium under normal conditions]

The pancreatic epithelium of intact frog has been studied using morphometry, cytochemistry and electron microscopy. A low level of DNA synthesis was shown to be characteristic of pancreatic epithelium of frogs caught in winter. The B/A cell volume ratio is 3.68 +/- 0.16 and 2.55 +/- 0.36 in small and median pancreatic islets, resp. A-, B-, D-cells are found in pancreatic islets, and intermediate acinar A and acinar B cells in intact frog pancreas.