Frog Skin Epithelium Research Articles

The effect of molixan, a disulfide-containing immunomodulator, on Na<sup>+</sup> transport across frog skin epithelium

The effect of molixan, a disulfide-containing immunomodulator which is an analogue of oxidized glutathione, on Na+ transport across frog skin epithelium has been studied. Using the voltage-clamp technique, the capacity of molixan to modulate Na+ transport across frog skin epithelium has been revealed for the first time. It was also found that when molixan is applied to the apical surface of frog skin, it suppresses Na+ transport, while molixan added on the basolateral surface may mimic the effect of insulin and stimulate Na+ transport. The results obtained indicate that oxidized glutathione and its analogs molixan and glutoxim unidirectionally modulate Na+ transport across frog skin.

The Effect of Molixan, a Disulfide-Containing Immunomodulator, on Na+ Transport across Frog Skin Epithelium

The Effect of Molixan, a Disulfide-Containing Immunomodulator, on Na+ Transport across Frog Skin Epithelium

ЦИСТАМИН И ЦИСТИН ПОДАВЛЯЮТ ТРАНСПОРТ Na+ В ЭПИТЕЛИИ КОЖИ ЛЯГУШКИ

Amphibian skin and other isolated epithelial systems are classical model objects for studying the mechanisms of transepithelial ion transport. Na+ transport in osmoregulatory epithelium is a complex, multicomponent system, which involves Na+ transporting proteins and signaling cascades localized in various cell membranes. The protein components of this system may be a target for oxidative stress. At the same time, the effect of oxidizing and reducing agents on Na+ transport in native epithelial systems, such as frog skin epithelium, practically has not been studied. Using voltage-clamp technique the effect of disulfide-containing oxidizing agents cystine and cystamine on Na+ transport in frog skin was investigated. It has been shown for the first time that the treatment of the skin of the Rana temporaria frog with cystamine or cystine at a concentration of 10 μg/ml suppresses the transepithelial Na+ transport. It was also found that the preincubation with dithiothreitol, which reduces disulfide bonds in proteins, prevents the inhibitory effect of cystamine and cystine. The obtained results indicate that Na+ transport in the frog skin is sensitive to oxidative stress and is modulated by disulfide-containing oxidizing agents, such as cystamine or cystine, and also that the effect of cystamine and cystine on Na+ transport in the frog skin epithelium is mediated by their interaction with functionally important cysteine residues of Na+-transporting proteins.

The Sigma-1 Receptor Ligands Chlorpromazine and Trifluoperazine Inhibit Na+ Transport in Frog Skin Epithelium

Using the voltage-clamp technique, it has been shown for the first time that the neuroleptic drugs chlorpromazine and trifluoperazine, which act as sigma-1 receptor antagonists, attenuate Na+ transport in the frog skin epithelium. The results suggest that sigma-1 receptors may be involved in the regulation of transepithelial Na+ transport in frog skin.

Sigma-1 receptor antagonists haloperidol and chlorpromazine modulate the effect of glutoxim on NA+ transport in frog skin

Using voltage-clamp technique, the involvement of sigma-1 receptors in immunomodulatory drug glutoxim regulation of Na+ transport in frog skin was investigated. We have shown for the first time that preincubation of the frog skin with sigma-1 receptor antagonists – haloperidol or chlorpromazine – attenuates the stimulatory effect of glutoxim on Na+ transport. The results suggest the possible involvement of the sigma-1 receptors in glutoxim effect on Na+transport in frog skin epithelium.

Sigma-1 Receptor Antagonists Haloperidol and Chlorpromazine Modulate the Effect of Glutoxim on Na+ Transport in Frog Skin.

Using voltage-clamp technique, the involvement of sigma-1 receptors in the regulation of Na+ transport in frog skin by the immunomodulatory drug glutoxim was investigated. We have shown for the first time that preincubation of the frog skin with the sigma-1 receptor antagonists haloperidol and chlorpromazine attenuates the stimulatory effect of glutoxim on the Na+ transport. The results suggest the possible involvement of the sigma-1 receptors in the regulation of Na+ transport in frog skin epithelium by glutoxim.

Epoxygenase Inhibitors Attenuate the Stimulatory Effect of Glutoxim on Na+ Transport in Frog Skin.

Using voltage-clamp technique, the involvement of epoxygenases in immunomodulatory drug glutoxim regulation of Na+ transport in frog skin was investigated. We have shown for the first time that preincubation of the frog skin with epoxygenase inhibitors econazole or proadifen almost completely inhibits the stimulatory effect of glutoxim on Na+ transport. The data suggest the involvement of the enzymes and/or products of epoxygenase oxidation pathway of arachidonic acid metabolism in glutoxim effect on Na+ transport in frog skin epithelium.

Antidiuretic hormone effect on transport of non-charged solutes (urea and glycerol) across the epithelium

Background: The frog skin epithelium is used as a model to demonstrate the transport of various charged and non-charged solutes from the mucosal to serosal side through channels and transporters. The mucosal side, otherwise termed as the apical side or outer side, is the side which is exposed to the external environment (towards pond water). The serosal side, also known as the basolateral side, faces the internal environment of the organism. Aims and Objectives: The objective of the present study is to determine the action of antidiuretic hormonal (ADH) on the movement of non-charged solutes such as urea and glycerol across the frog skin epithelium. Materials and Methods: For this study, the ventral abdominal skin of frog, species Rana hexadactyla was mounted in an Ussing’s type chamber. Normal Ringer’s solutions containing urea (5 mM) and glycerol (90 mM) each were placed on the mucosal side, and ADH was added either on the mucosal side or the serosal side. After the addition of ADH, the colorimetric method was used to measure the transport of urea at the absorbance of 540nM and glycerol absorbance of 505 nM using a spectrophotometer; statistical analysis was done by Wilcoxon-Signed Ranks Test, all values were expressed as a mean ± standard error of means, n = 4. Results: Addition of ADH (40 nM) on serosal side increased the urea transport from control 29.3 ± 5.1 μmol/dL to 52.3 ± 6.8 μmol/dl and increased glycerol transport from 34.8 ± 6.0 μmol/dL to 45.3 ± 6.6 μmol/dL, whereas addition of ADH on the mucosal side did not increase the glycerol transport, the control value was 14.6 ± 4.3 μmol/dL and with ADH that the value was 9.6 ± 2.9 μmol/dL. Conclusion: The results concluded that the addition of ADH on serosal side enhanced the transport of urea and glycerol. Addition of ADH on the mucosal side did not show any effect on transport of glycerol.

Transepithelial sodium transport across frog skin.

amphibian skin serves as an extra renal organ for the regulation of ion transport across the epithelium ([2][1]). Ion channels and transporters present in the epithelium of frog skin assist in the regulation of the same. These channels and transporters are necessary for the maintenance of

Lipoxygenases modulate the effect of glutoxim on Na+ transport in the frog skin epithelium

Using voltage-clamp technique, the involvement of lipoxygenases in the effect of immunomodulatory drug glutoxim on Na+ transport in frog skin was investigated. It was shown for the first time that preincubation of the skin with lipoxygenase inhibitors caffeic acid, baicalein, and nordihydroguaiaretic acid significantly decreases the stimulatory effect of glutoxim on Na+ transport. The data suggest the involvement of lipoxygenase oxidation pathway of arachidonic acid metabolism in the glutoxim effect on Na+ transport in frog skin epithelium.

The effect of glutoxim on Na+ transport in frog skin: The role of cytoskeleton

Using voltage-clamp technique, the possible role of the cytoskeleton in the effect of pharmacological analogue of oxidized glutathione (GSSG), drug glutoxim, on Na+ transport in the frog Rana temporaria skin was investigated. It was shown for the first time that preincibation of the skin with the microtubular disrupter, nocodazole, actin filament disrupter, cytochalasin D or protein phosphatase PP1/PP2A inhibitor, calyculin A, significantly decrease the stimulatory effect of glutoxim on Na+ transport. The data suggest the involvement of microtubules and microfilaments in the regulatory effect of glutoxim on Na+ transport in frog skin and that reorganization of actin filaments or microtubules leads to inhibition of stimulatory effect of glutoxim on Na+ transport in frog skin epithelia.

Immunohistological classification of ionocytes in the external gills of larval Japanese black salamander, Hynobius nigrescens Stejneger

In this cytological and immunohistological study, we clarified the localization of the membrane transporters Na(+) , K(+) -ATPase (NKA), vacuolar-type H(+) -ATPase (VHA), and epithelial sodium channel (ENaC) and distinguished ionocyte subtypes in the gill of the Japanese salamander (Hynobius nigrescens). In larvae (IY stages 43-65), NKA immunoreactivity was observed on the basolateral plasma membrane in more than 60% cells and less than 20% cells in the primary filaments and secondary lamellae of the external gills, respectively. VHA immunoreactivity was observed on the apical membrane of some epithelial cells in the secondary lamellae of the external gills. High ENaCα immunoreactivity was widely observed on the apical cell membrane of a population of squamous cells, presumably pavement cells (PVCs), and mitochondria-rich cells (MRCs), in the primary filaments and secondary lamellae of the external gills. Using double immunofluorescence microscopy, epithelial cell types involved in ionic regulation were characterized and divided into three ionocyte types: NKA-, NKA- and ENaC-, and VHA-positive cells. VHA-immunoreactive cells as well as NKA-positive cells were observed during IY stages 43-65 of the salamander larvae. During late stages of metamorphosis, NKA, VHA, and ENaCα immunoreactivities in the external gills decreased and finally disappeared during the completion of metamorphosis (IY stage 68). PVCs and MRCs in the external gills are probably involved in acid-base balance regulation and osmoregulation in urodele amphibian larvae. The results are discussed in relation to the ionocytes previously reported in fish gills and the frog skin epithelium.

Vasopressin-dependent coupling between sodium transport and water flow in a mouse cortical collecting duct cell line

Water balance is achieved through the ability of the kidney to control water reabsorption in the connecting tubule and the collecting duct. In a mouse cortical collecting duct cell line (mCCD(c11)), physiological concentrations of arginine vasopressin increased both electrogenic, amiloride-sensitive, epithelial sodium channel (ENaC)-mediated sodium transport measured by the short-circuit current (Isc) method and water flow (Jv apical to basal) measured by gravimetry with similar activation coefficient K(1/2) (6 and 12 pM, respectively). Jv increased linearly according to the osmotic gradient across the monolayer. A small but highly significant Jv was also measured under isoosmotic conditions. To test the coupling between sodium reabsorption and water flow, mCCD(c11) cells were treated for 24 h under isoosmotic condition with either diluent, amiloride, vasopressin or vasopressin and amiloride. Isc, Jv, and net chemical sodium fluxes were measured across the same monolayers. Around 30% of baseline and 50% of vasopressin-induced water flow is coupled to an amiloride-sensitive, ENaC-mediated, electrogenic sodium transport, whereas the remaining flow is coupled to an amiloride-insensitive, nonelectrogenic sodium transport mediated by an unknown electroneutral transporter. The mCCD(c11) cell line is a first example of a mammalian tight epithelium allowing quantitative study of the coupling between sodium and water transport. Our data are consistent with the 'near isoosmotic' fluid transport model.

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

Analysis of time dependent states of solute coupled water transport

At transepithelial equilibrium water uptake by toad skin epithelium is linearly correlated with the active Na+ flux. The present study aimed at analyzing the coupling between these fluxes during transition from one steady state to another. The short‐circuited isolated toad skin epithelium was studied in an Ussing setup with a horizontal capillary tube on the inside chamber. The volume was clamped by keeping the position of the meniscus in the capillary tube constant via a motor‐driven syringe the position of which was continuously recorded. When the isoproterenol stimulated short‐circuit current (ISC) and volume flow (JV) were stationary, 100 μM amiloride was added on the outside while the time courses of ISC and JV were followed until a new steady state was obtained. Initially, ISC = 18±4.1 μA/cm2 and JV = 1.3±0.4 nl/cm2/s (mean ± SEM, n = 9). Amiloride produced a relatively fast decrease of ISC with T1/2 = 1.6± 0.1 min, and a slow decrease of JV with T1/2 = 14.1± 2.3 min towards the new steady states of, ISC = ‐0.1±0.4 μA/cm2 and JV = 0.39±0.11 nl/cm2/s. Theoretical quantitative analysis was performed by solving the set of equations of our mathematical‐physical model for time dependent states, indicating that also during the imposed transient state JV is driven by the lateral Na/K‐pumps governed by the rate at which the cellular Na+ pool is emptied. Supported by The Danish Natural Science Research Council 272‐05‐0417.

The lateral intercellular space as osmotic coupling compartment in isotonic transport

Solute-coupled water transport and isotonic transport are basic functions of low- and high-resistance epithelia. These functions are studied with the epithelium bathed on the two sides with physiological saline of similar composition. Hence, at transepithelial equilibrium water enters the epithelial cells from both sides, and with the reflection coefficient of tight junction being larger than that of the interspace basement membrane, all of the water leaves the epithelium through the interspace basement membrane. The common design of transporting epithelia leads to the theory that an osmotic coupling of water absorption to ion flow is energized by lateral Na(+)/K(+) pumps. We show that the theory accounts quantitatively for steady- and time dependent states of solute-coupled fluid uptake by toad skin epithelium. Our experimental results exclude definitively three alternative theories of epithelial solute-water coupling: stoichiometric coupling at the molecular level by transport proteins like SGLT1, electro-osmosis and a 'junctional fluid transfer mechanism'. Convection-diffusion out of the lateral space constitutes the fundamental problem of isotonic transport by making the emerging fluid hypertonic relative to the fluid in the lateral intercellular space. In the Na(+) recirculation theory the 'surplus of solutes' is returned to the lateral space via the cells energized by the lateral Na(+)/K(+) pumps. We show that this theory accounts quantitatively for isotonic and hypotonic transport at transepithelial osmotic equilibrium as observed in toad skin epithelium in vitro. Our conclusions are further developed for discussing their application to solute-solvent coupling in other vertebrate epithelia such as small intestine, proximal tubule of glomerular kidney and gallbladder. Evidence is discussed that the Na(+) recirculation theory is not irreconcilable with the wide range of metabolic cost of Na(+) transport observed in fluid-transporting epithelia.

Beta-adrenergic activation of solute coupled water uptake by toad skin epithelium results in near-isosmotic transport

Transepithelial potential (V(T)), conductance (G(T)), and water flow (J(V)) were measured simultaneously with good time resolution (min) in isolated toad (Bufo bufo) skin epithelium with Ringer on both sides. Inside application of 5 microM isoproterenol resulted in the fast increase in G(T) from 1.2+/-0.3 to 2.4+/-0.4 mS x cm(-2) and slower increases in equivalent short circuit current, I(SC)(Eqv) = -G(T) x V(T), from 12.7+/-3.2 to 33.1+/-6.8 microA cm(-2), and J(V) from 0.72+/-0.17 to 3.01+/-0.49 nL cm(-2) s(-1). Amiloride in the outside solution abolished I(SC)(Eqv) (-1.6+/-0.1 microA cm(-2)) while J(V) decreased to 0.50+/-0.15 nL cm(-2) x s(-1), which is significantly different from zero. Isoproterenol decreased the osmotic concentration of the transported fluid, C(osm) approximately 2 x I(SC)(Eqv)/J(V), from 351+/-72 to 227+/-28 mOsm (Ringer's solution: 252.8 mOsm). J(V) depicted a saturating function of [Na+]out in agreement with Na+ self-inhibition of ENaC. Ouabain on the inside decreased I(SC)(Eqv) from 60+/-10 to 6.1+/-1.7 microA cm(-2), and J(V) from 3.34+/-0.47 to 1.40+/-0.24 nL cm(-2) x s(-1). Short-circuited preparations exhibited a linear relationship between short-circuit current and J(V) with a [Na+] of the transported fluid of 130+/-24 mM ([Na+]Ringer's solution = 117.4 mM). Addition of bumetanide to the inside solution reduced J(V). Water was transported uphill and J(V) reversed at an excess outside osmotic concentration, deltaC(S,rev) = 28.9+/-3.9 mOsm, amiloride decreased deltaC(S,rev) to 7.5+/-1.5 mOsm. It is concluded that water uptake is accomplished by osmotic coupling in the lateral intercellular space (lis), and hypothesized that a small fraction of the Na+ flux pumped into lis is recirculated via basolateral NKCC transporters.

Application of the Na+ recirculation theory to ion coupled water transport in low- and high resistance osmoregulatory epithelia

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.

Clomipramine induces the opening of Na+ channels in the frog skin epithelium.

This paper studies the effect of clomipramine on the electrical parameters of the frog skin epithelium. We have found that clomipramine induces: 1) an increase in short circuit current that is equivalent to an increase in net Na+ flux, 2) an increase in total transepithelial conductance, 3) a depolarisation of the apical barrier, and 4) a decrease in the fractional resistance of the apical membrane. These results suggest that clomipramine promotes an increase in the sodium permeability of the apical barrier by increasing either the concentration of amiloride-sensitive Na+ channels or by increasing their individual conductance. Using kinetic models, values for the affinity constants for clomipramine and amiloride were estimated (K(Clo)=(18.l +/- l.04) x l0(-6) M; K(Amil) = 56 x 10(-8) M). Identical studies were performed for eel intestinal epithelium and no significant changes on the short circuit current were observed.

Effect of the putative Ca2+-receptor agonist Gd3+ on the active transepithelial Na+ transport in frog skin.

In this communication we show that Gd3+ acts as an activator of the apical sodium channel (ENaC) in frog skin epithelia. Application of Gd3+ to the apical solution of frog skin epithelia increased the Na+ absorption measured as the amiloride-inhibitable short-circuit current (Isc). The stimulation was dose dependent with a concentration for half-maximal stimulation (EC50) of 0.023 mM. The change in Isc was found to correlate with the net Na+ flux, confirming that Gd3+ enhances Na+ absorption. By monitoring the cellular potential (Vsc) with microelectrodes during addition of Gd3+, it was found that Vsc depolarized as Isc rose, indicating that Gd3+ affects apical Na+ permeability (PNa). This was confirmed by measuring the I/V relations of the apical membrane. In the presence of benzimidazolylguanidin (BIG), a drug known to abolish the Na+ self-inhibition, Gd3+ had no effect on Isc. The Na+ self-inhibition was investigated using fast changes of the apical Na+ concentration on K+-depolarized epithelia. BIG was found to abolish the Na+ self-inhibition and to activate the basal Na+ transport, whereas Gd3+ only activated the basal Na+ transport but had no effect on the self-inhibition. These results indicate the existence of an alternative nonhormonal mechanism to Na+ self-inhibition, via which both Gd3+ and BIG act, possibly components of the Na+ feedback inhibition system.