Interdependence between sodium transport, external chloride, and sodium/calcium exchanger in the isolated skin of the Rana pipiens

The aim of this study was to analyze the relationship of the Na+/Ca2+ exchanger, cytosolic calcium, and chloride to the transepithelial transport of sodium in isolated frog skin. Sodium transport was measured as amiloride-inhibitable short circuit current (SCC). We studied the effect of variations in the concentrations of external chloride and of the manipulation of calcium on sensitive amiloride SCC. Modifications in the movement of Ca2+ were induced by an ionophore, A23187, and a Ca2+ channel blocker, nifedipine. Calcium ionophore A23187 (5 and 20 microM), in a normal Ringer's solution, increased SCC and transepithelial potential difference (PD). In contrast, nifedipine (20 microM) reduced SCC and PD. The role of the Na+/Ca2+ exchanger was studied using dichlorobenzamil (DCB, 50 microM) and quinacrine (1 mM), inhibitors of this exchanger. They selectively increased SCC and PD on the mucosal side of the skin, with no effect on the serosal side. This response occurred only in the presence of extracellular calcium. Replacement of NaCl by sodium methanesulfonate or the addition of furosemide (1 mM) at the serosal compartment, decreased basal SCC and PD and blocked the response to A23187 and the mucosal effect of DCB and quinacrine. These results suggest the presence of an Na+/Ca2+ exchanger located on the mucosal side of the frog skin, which participates in the transepithelial sodium transport. The action of this exchanger may be modulated by external chloride and calcium. J. Exp. Zool. 289:23-32, 2001.     

Electrical parameters of the toad skin: effects of forskolin.

Forskolin stimulated short-circuit current (SCC) and transepitelial electrical conductance (G) in the isolated skin of the toad Bufo arenarum in a concentration-dependent manner, between 1.0 x 10(-6) and 2.4 x 10(-5) M. At the latter concentration, glandular secretion appeared to be stimulated also. The increase in G was considerably greater in skins bathed in Ringer solution than in solutions containing no chloride. The increased SCC was abolished by amiloride, a specific blocker of sodium transport in amphibian membranes, irrespective of the anion present in the solution bathing the skin. G was also decreased by amiloride to control values in skins bathed in solutions without chloride, but remained elevated in the presence of Cl-. The increase in SCC following exposure to forskolin, 4.4 x 10(-6) M, was not altered when furosemide, a specific blocker of chloride transport, was present in the Ringer solution bathing the dermal side of the skin. The response to forskolin, 2.4 x 10(-5) M, however, was significantly decreased by dermal furosemide; the inhibitor was ineffective in the absence of chloride. The data indicate that forskolin acts on at least two sites: stratum granulosum cells (the main pathway for sodium transport, and an alternate site, responsible for the increase in permeability to chloride. In addition, at high concentration of the agent, glandular secretion is also stimulated. The data suggest that the adenylate cyclase-cyclic AMP system is involved in the regulation of the permeability of the toad skin to sodium and chloride, probably by separate cell types.     

Sodium transport and distribution of electrolytes in frog skin

The objective of this study on frog skin was to examine correlations between transepidermal active Na-transport and intracellular [Na]c, [K]c, [Cl]c homeostasis. Isolated, whole skins, and "split skins" were used in measurements of short-circuit current (SCC) and open skin potential (PD). Water and ion contents were estimated on split skins. Absolute [Na]c and [K]c varied over the range of 18 to 46, and 113 to 80 mM, respectively (Figure 7), but a complementary relationship existed between Na and K, such that [Na]c + [K]c remained approximately equal to 129 mM. Average values for [Na]c and [K]c were approximately equal to 31 and approximately equal to 96 mM, respectively. [Cl]c remained constant at approximately equal to 38 mM. This complementary relationship does not seem to be an artifact, caused by collagenase, used in the preparation of split skins. Whole skins and split skins in Ringer's solution, when treated with fluoroacetate (FAc), ouabain (Ou), or vanadate (Va) over wide ranges of concentrations, showed that FAc greatly depressed the SCC and the PD, without changing [Na]c, [K]c, [Cl]c. FAc acted only from the corium side of the skin. The decreasing SCC remained a Na-current, as in control skins. By comparison, such a separation of cellular functions could not be established with Ou, or Va. These inhibitors either affected SCC, PD, and cellular ion concentration, or they had no effect on any of these parameters. The complementary relationship between [Na]c and [K]c, with [Cl]c remaining again at approximately equal to 38 mM, was also found in tissues exposed to inhibitors. These results indicate that transcellular active Na transport and electrolyte homeostasis are not always rigidly coupled, suggesting that these processes may not be uniformly distributed within the epithelial cells, or among the interconnected cell layers of the frog skin epidermis.     

Opposite actions of different doses of arginine-vasotocin and 1-deamino-arginine-vasotocin on sodium ion transport in skin of the frog Rana temporaria

Active transport of sodium ions across the isolated abdominal skin of the frog Rana temporaria after application of arginine-vasotocin (AVT) and 1-deamino-arginine-vasotocin (1dAVT) was studied by measurement of the short-circuit current (SCC). The maximal increase in the SCC values (26 and 19 microA/cm2) was observed after addition of 10 nM AVT or 100 nM 1dAVT, respectively, to the frog skin basal surface. An increase of concentration of AVT to 100 nM and of IdAVT to 1 microM terminated the sodium transport in the frog skin. A preliminary addition of an antagonist of arginine-vasopressin V1a-receptors to the Ringer's solution at the frog skin basal surface led to a rise in the SCC values in response to administration of ineffective doses of AVT or 1dAVT. V2-receptor antagonists did not affect the frog skin reaction to administration of these doses of AVT or IdAVT.     

Oppositely directed actions of different doses of arginine-vasotocin and 1-deamino-arginine-vasotocin on sodium ion transport in skin of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Active transport of sodium ions across the isolated abdominal skin of the frog Rana temporaria after application of arginine-vasotocin (AVT) and 1-deamino-arginine-vasotocin (1dAVT) was studied by measurement of the short-circuit current (SCC). The maximal increase in the SCC values (26 and 19 mk/cm²) was observed after addition of 10 nM AVT or 100 nM 1dAVT, respectively, to the frog skin basal surface. An increase of concentration of AVT to 100 nM and of 1dAVT to 1 μM terminated the sodium transport in the frog skin. A preliminary addition of an antagonist of arginine-vasopressin Vi_a-receptors to the Ringer’s solution at the frog skin basal surface led to a rise in the SCC values in response to administration of ineffective doses of AVT or 1dAVT. V₂-receptor antagonists did not affect the frog skin reaction to administration of these doses of AVT of 1dAVT.     

A specific alkaline phosphatase of amphibia integument levamisole effect on short circuit current (SCC).

Using the Ussing chamber technique we have measured the short-circuit current (SCC), and so the ion transport, in the ventral skin of samples of Rana esculenta complex. The animals were not exposed to experimental treatment, and on SCC we have observed the effect of levamisole, administered either on external or internal side. Levamisole 0.0025 mM was ineffective; higher concentrations (0.025 mM, 2.5 mM), which inhibit alPase activity in tissue extracts and sections, induced an increase in SCC measurements and the effect was proportional to the concentration. Levamisole 25 mM produced a rapid and transitory increase of SCC, followed by a very quick decrement of it. Because of the action of Levamisole, "specific inhibitor of alPase activity", on ion transport in Rana skin, we propose that the alPase enzyme is probably involved in ion cutaneous transport and thus in the adaptative osmoregulation in the integument of amphibia.     

Effect of luminal Na+ on the kinetics of intestinal absorption of sugars in vivo

The effect of sodium concentration on the absorption kinetics of glucose, galactose and 3-o-methyl-glucose in rat and hamster jejunum in vivo has been studied. In consecutive 1 min periods the total absorption and absorption in presence of 0.5 mM phlorizin were measured. The difference between them was taken as the active transport rate. The perfusion rate value was 5.6 ml X min-1 and sugar concentrations in the perfusion solution ranged from 1 to 10 mM. The results for the different sodium concentrations show a nearly common Vmax for the same sugar and animal species, while the apparent KT values increase when the sodium concentration in the lumen decreases, mimicking a pure affinity-type activation system. The absorption of sugar when solutions without Na+ are perfused, is greater than that entering passively in the presence of phlorizin. An explanation may be that appreciable amounts of endogenous Na+ find their way to the intestinal lumen in favour of the gradient, making Na+-sugar cotransport possible.     

Effect of hibernation on sodium and chloride ion transport in isolated frog skin

The aim of the study was to evaluate the effect of hibernation on electrophysiological parameters of isolated frog skin under control incubation (Ringer solution) and after inhibition of Na+ and CI- transepithelial transport by application of amiloride and bumetanide. The transepithelial electrical potential difference (PD in mV) was measured before and after mechanical stimulation of isolated frog skin. The tissues were mounted in a modified Ussing chamber. The results revealed a reduced PD of frog skin during hibernation. In February, as compared with November, PD of frog skin incubated in Ringer solution decreased by about 50%. Hibernation also affected hyperpolarization (dPD) of frog skin after mechanical stimulation. In November and December, dPD was about 50% and 30% lower, respectively, compared with the subsequent two months of the experiment. The incubation of frog skin with amiloride, a sodium ion channel blocker, resulted in reduced values of all measured electrophysiological parameters irrespective of the phase of hibernation. After application of chloride ion transport inhibitor (bumetanide), the PD in November and December decreased compared with the control incubation by about 80% and 75%, while in January and February by about 40% and 25%, respectively. In January and February dPD increased by four times and three times as compared with November and December. Hibernation reduces net ion flow in isolated frog skin. During the initial period of hibernation the sensitivity of the skin to mechanical stimulation also decreases. Towards the end of hibernation, on the other hand, excitation of mechanosensitive ion channels takes place.     

Effect of Ba2+ on the K+ conductance pathways in the frog cornea

Two types of transepithelial potential difference (PD) responses have been observed in the bullfrog, Rana catesbeiana, when the K+ concentration is changed in the aqueous solution. (1) A normal response, that is, a decrease in the positivity of the aqueous solution when the K+ is increased in this solution. (2) An anomalous response, that is, an increase in PD when K+ is increased from 0 to 4 mM in the aqueous solution. In present experiments 2 mM Ba2+ results in a significant decrease in transepithelial PD and an increase in resistance (R), consistent with the well-known effect of Ba2+ on the K+ conductance in other biological membranes. In the presence of Ba2+ compared to its absence the normal PD responses were decreased when K+ was increased from 4 to 20 or to 79 mM in the aqueous solution. Barium enhanced, but not significantly, the anomalous PD response (PD increase) when K+ was increased from 0 to 4 mM. An anomalous PD response (PD decrease) was obtained with Ba2+ when K+ was changed from 4 to 0 mM while in its absence the response was normal (PD increase) or did not change. These findings support the concept that anomalous PD responses as a result of the electrogenic (Na+ + K+)-ATPase may be obtained when the resistance of the simple K+ pathway is increased.     

Effect of sodium taurocholate and sodium cholate on short-circuit current on amphibian membranes

The effects of the bile salts, sodium taurocholate (NaTc) and sodium cholate (NaCh), and toad bile gallbladder (bile) on short-circuit current (SCC) across isolated skin, and sodium taurocholate (NaTc) on isolated bladder of Bufo arenarum toads were tested. Sodium taurocholate (NaTc), sodium cholate (NaCh) and toad bile gallbladder (bile) promoted an increase in SCC, when added to the external side. The stimulatory effect was reversible after rinsing the preparation for 60 min. Implications on in vivo renal function of these results are discussed.     

Isoosmotic transport of fluid across the hamster small intestine in the presence of phlorizin-induced inhibition of sugar transport.

Experiments were performed to investigate whether the fluid transported across the small intestine is isoosmotic with the mucosal solution when the active transport of glucose is partially inhibited. Everted hamster mid small intestine was incubated in one of the following four mucosal solutions: (1) Isotonic control, Krebs-Ringer bicarbonate solution containing 10 mM glucose (KRBSG), (2) Isotonic with phlorizin, KRBSG + 5X10-5 M phlorizin, (3) Hypertonic control, KRBSG + 50 mM mannitol, (4) Hypertonic with phlorizin, KRBSG + 50 MM mannitol + 5x10-5 M phlorizin. The serosal surface of the intestine was not bathed. Results indicate that the transported fluid was always isoosmotic with any of the mucosal solutions used. When the mucosal solution was made hypertonic with mannitol, the concentration of glucose and electrolytes in the absorbate increased, and as a result, the absorbate became hypertonic and isoosmotic with the mucosal solution. The presence of phlorizin either in the isotonic or in the hypertonic mucosal solution decreased the glucose concentration of the absorbate, but the transported fluid became isoosmotic with the mucosal solution due to a higher concentration of Na, K, and their associated anions. Phlorizin caused a decrease in the transmural potential difference. In spite of this, the presence of this glucoside in the mucosal solution increased the transport of sodium in relation to glucose transport. It is suggested that, at the concentrations used, phlorizin inhibits sodium movement through the electrogenic pathway, but increases the transport of this ion through the non-electrogenic route. This increase in neutral sodium transport seems to compensate for the low concentration of glucose in the absorbate, so that the absorbate becomes isoosmotic with the mucosal solution whether the latter is isotonic or hypertonic. It is suggested further that isoosmotic transport of fluid is an inherent property of the small intestine and that there may be an osmoregulatory mechanism in the gut which controls this process.     

Electrophysiological effect of ruthenium red and pH on the skin of the edible frog, Rana esculenta L

Amphibian skin is a sensitive interface between the organism and the environment. Metal ions from the external environment, some of them being trace elements, act on the amphibian skin. It had been shown that stimulation of tactile receptors affected Na+ transport in the frog skin and changed the potential difference, therefore the aim of this project was to study the effect of ruthenium complex, known as ruthenium red (RR), on the ion transport in this organ in vitro under control conditions, after mechanical stimulation and also in the presence of the Na+ transport inhibitor-amiloride. Three different concentrations of RR (0.12, 1.2, and 12.0 mM) in two different pH values (6.4 and 7.4) were studied in vitro in the Ussing apparatus. The measured electrophysiological parameters were the transepithelial electrical potential difference (PD) and the changes in PD after mechanical stimulation (dPD). The gentle mechanical stimulus was a jet of bath fluid from a peristaltic pump directed on the mucosal surface of isolated frog skin. After mechanical stimulation, transient hyperpolarization invariably occurred (dPD = 1.5 +/- 0.2 mV). In the presence of RR the hyperpolarization was smaller and this diminution was concentration dependent: 0.5 +/- 0.1 mV for 1.2 mM of RR and 0.1 +/- 0.1 mV for 1.2 mM of RR. At pH 6.4 the reactions of the skins on the mechanical and chemical stimuli were smaller, in the presence of amiloride disappearing completely, but after the washing away of amiloride from the experimental organ in pH 6.4 the action of RR was stimulatory. The natural defensive reactions of frog skin related to the ion transport and electrical potential difference are affected or disappear in the presence of ruthenium complex.     

Mechanism of basolateral membrane H+/OH-/HCO-3 transport in the rat proximal convoluted tubule. A sodium-coupled electrogenic process

In order to examine the mechanism of basolateral membrane H+/OH-/HCO-3 transport, a method was developed for the measurement of cell pH in the vivo doubly microperfused rat proximal convoluted tubule. A pH-sensitive fluorescein derivative, (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein, was loaded into cells and relative changes in fluorescence at two excitation wavelengths were followed. Calibration was accomplished using nigericin with high extracellular potassium concentrations. When luminal and peritubular fluids were pH 7.32, cell pH was 7.14 +/- 0.01. Decreasing peritubular pH from 7.32 to 6.63 caused cell pH to decrease from 7.16 +/- 0.02 to 6.90 +/- 0.03. This effect occurred at an initial rate of 2.4 +/- 0.3 pH units/min, and was inhibited by 0.5 mM SITS. Lowering the peritubular sodium concentration from 147 to 25 meq/liter caused cell pH to decrease from 7.20 +/- 0.03 to 6.99 +/- 0.01. The effect of peritubular sodium concentration on cell pH was inhibited by 0.5 mM SITS, but was unaffected by 1 mM amiloride. In addition, when peritubular pH was decreased in the total absence of luminal and peritubular sodium, the rate of cell acidification was 0.2 +/- 0.1 pH units/min, a greater than 90% decrease from that in the presence of sodium. Cell depolarization achieved by increasing the peritubular potassium concentration caused cell pH to increase, an effect that was blocked by peritubular barium or luminal and peritubular sodium removal. Lowering the peritubular chloride concentration from 128 to 0 meq/liter did not affect cell pH. These results suggest the existence of an electrogenic, sodium-coupled H+/OH-/HCO-3 transport mechanism on the basolateral membrane of the rat proximal convoluted tubule.     

The effect of ethanol on ion transport in frog skin.

Frog skin has been used as a model epithelial sodium-transporting system to study the effect of ethanol on ion transport. Treatment of the outside of frog skin with ethanol decreased the net sodium transport due to inhibition of 22Na+ influx. Ethanol did not alter sodium outflux when bathin the outside of the skin. The inhibition was in proportion to the concentration of ethanol, 0.25 M resulting in 50% inhibition. The chloride permeability of the skin was increased several-fold when the skin was exposed to ethanol in either bathing solution. With 0.4 M ethanol in the inner bathing solution, all the unidirectional fluxes of Na+ and C1- were increased. The movement of C1- was evaluated by comparison of C1- flux with urea flux, since urea is thought to move passively across frog skin via an extracellular (shunt) pathway. Chloride flux was increased to a greater extent than urea flux. These experiments indicate that ethanol affects chloride permeability beyond an increase in extracellular ion flow and independent of its effect of Na+ transport.     

Effect of acetazolamide on sodium transport through the isolated frog skin at low and high external sodium concentrations.

The action of acetazolamine on sodium transport in $\text{Rana esculenta}$ skin was studied with the external face bathed in dilute (2mMM) or concentrated (Ringer) solutions of sodium chloride.The absorption of Na⁺ from a dilute solution is inhibited at an acetazolamide concentration of 10⁻⁵M. This is due to an inhibition of the influx: the efflux remains unchanged. Acetazolamide has no effect, however, on transport from Ringer solution.The graphic determination of the Na⁺ transport pool at the 2 mM NaCl concentration showed that acetazolamide diminished the pool without affecting the $\text{t}\text{1}\text{2}$. The inhibitor had no effect on the pool at the higher (Ringer) concentration.These results indicate that acetazolamide acts on the external barrier of the sodium transport compartment without affecting the active pump of this ion when it is being transported from a dilute sodium chloride solution.     

Effects of cadmium on Na+ transport in the isolated skin of the toad Pleurodema thaul

Cadmium ions applied to either (outer or inner) surface of the isolated toad skin dose-dependently increased the short-circuit current (SCC), the potential difference (V) and the active sodium conductance (G(Na)) in the concentration range 0.07-0.50mM. Maximal stimulatory effect was over 30% with an EC(50) of about 0.1mM. The effect of the highest concentration used (0.75mM) decreased considerably, and when it was applied to the inner surface (10 experiments), induced between 30% and 40% inhibition of the electric parameters in four experiments. Pretreatment with amiloride inverted the stimulatory effect of externally applied Cd(2+), suggesting competitive action on the apical Na(+) channel. The effect of noradrenaline (NA) was increased after outer application of Cd(2+) and decreased after inner application of the metal: the latter effect might be due to cadmium inhibition of the activity of Na(+),K(+)-ATPase. On the other hand, pretreatment with amiloride was followed by partial although transient reversal of its effects by serosal Cd(2+), which might be explained by action of cadmium on cytoplasmic lysine residues concerned with Na(+) channel gating. The amiloride test showed that the increment of the electric parameters was due principally to stimulation of the driving potential for Na(+) (V-E(Na(+))) and that inhibition was accompanied by a reduction in the V-E(Na(+)) and by a significant decrease in skin resistance indicating possible disruption of membrane or cell integrity. These data are in favor of the possibility that externally applied Cd(2+) activates toad skin ion transport, partly by increasing apical sodium conductance and also by stimulating the V-E(Na(+)), and that internally applied Cd(2+), with easier access to membrane and cellular constituents, may inhibit the sodium pump.     

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

Summary 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 (V _scc).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 (P _Na ^a )as well as intracellular sodium concentration. These observations and the previously noted effect of papaverine on V _scc 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 V _scc. When papaverine was added to skins preincubated with bumetanide, the effect of papaverine on SCC and V _scc was unchanged. Therefore, the depolarization of V _scc, 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.     

Characterization of Na-K-ATPase in dog tracheal epithelium: enzymatic and ion transport measurements

The dog tracheal epithelium actively secretes Cl and absorbs Na. The possible dependency of this electrolyte transport on a Mg-dependent, Na-K-activated adenosine triphosphatase (Na-K-ATPase, EC 3.6.1.3) was examined. The characteristics of this enzyme system were investigated using homogenates of tracheal epithelium. The electrical properties and ion fluxes of this epithelium were determined in tissues mounted in Ussing chambers. Addition of Na and K produced an approximate 50% activation of basal Mg-ATPase activity. The apparent Km values for ATP, Na, K, and Mg were 0.4, 12.7, 1.9, and 1.6 mM, respectively. The total specific ATPase activity was 8.1 +/- 0.4 and that of the Mg-ATPase 4.3 +/- 0.1 mumol Pi. mg protein -1.h-1. Addition of ouabain (1 muM) or omission of K from the submucosal bathing solution reduced potential difference (PD) and short-circuit current (SCC) significantly. Relatively low concentrations (0.1 mM or less) of ethacrynic acid, furosemide, or 2,4-dinitrophenol (2,4-DNP) depressed SCC and PD significantly, i.e., at concentrations that were without effect on the Na-K-ATPase activity. Ethacrynic acid inhibited Cl secretion, whereas 2,4-DNP lowered both Na and Cl transport. These data demonstrate that 1) the tracheal mucosa of dogs contains a Na-K-ATPase at relatively high specific activity, 2) this enzyme is likely contained in the basal aspect of this membrane, 3) it appears to be essential for maintenance of Cl secretion, and 4) Cl secretion can be reduced (by ethacrynic acid, furosemide, and 2,4-DNP) without Na-K-ATPase inhibition.     

Oxygen consumption by frog skin and its isolated epithelial layers as a function of their sodium-transporting activity.

The metabolic cost (in terms of oxygen consumption) of transcellular sodium transport was assessed on ventral frog skin and its isolated epithelial layers, by measuring the decrease in oxygen consumption by the tissue upon transient withdrawal of sodium from the outside solution. The same number of sodium ions was transported per molecule oxygen consumed whole skin (17.4 +/- 2.3) and its isolated epithelium (17.3 +/- 2.4). The metabolic cost of sodium transport could not be estimated properly when this process was blocked by amiloride or ouabain, as these drugs were found to bring about an increase in oxygen consumpton by the tissue when no sodium was available for transport.     

Oxygen consumption by frog skin and its isolated epithelial layers as a function of their sodium-transporting activity

The metabolic cost (in terms of oxygen consumption) of transcellular sodium transport was assessed on ventral frog skin and its isolated epithelial layers, by measuring the decrease in oxygen consumption by the tissue upon transient withdrawal of sodium from the outside solution. The same number of sodium ions was transported per molecule oxygen consumed by whole skin (17.4±2.3) and its isolated epithelium (17.3±2.4).The metabolic cost of sodium transport could not be estimated properly when this process was blocked by amiloride or ouabain, as these drugs were found to bring about an increase in oxygen consumption by the tissue when no sodium was available for transport.