Different sensitivity to amiloride of body and tail skins of Rana catesbeiana tadpoles during metamorphosis

Regional differences in potential difference and short-circuit current between the body (dorsal) and the tail skin during metamorphosis of Rana catesbeiana tadpoles were investigated. In body skin, the potential difference and the short-circuit current across the skin develop in two successive steps. At stage XX, the potential difference and the short-circuit current across the body skins were amiloride-insensitive (1st step). At stage XXII, however, amiloride-sensitive potential difference and the short circuit current appeared (2nd step). By contrast, in tail skin the potential difference and the short-circuit current remained amiloride-insensitive (1st step) even at stage XXIII. Since the tail regresses after stage XXIII, the appearance of the second step could not be followed in vivo. To determine whether or not the second step can be induced in the tail, tail skin was cultured under conditions where the skin survives for a much longer period than it does in normally developing tadpoles. Such cultured tail skin generated the amiloride-sensitive potential difference and the short-circuit current and cultured body skin also generated them. Therefore, development of the 2nd step in the tail skin may be delayed in vivo. To characterize the differences between body and tail skin, skins were mutally grafted between body and tail at stage XIII–XV. The body skin grafted on the tail underwent both the 1st and 2nd steps by stage XXII, whereas the tail skin grafted on the body only showed the 1st step by the same stage. These results suggest that the regional specificity of the skin is already established before the prometamorphic stage.Abbreviations CMFS Ca²⁺- and Mg²⁺-free saline - CTS charcoal-treated serum - EDTA ethylene diamine tetra-acetate - I current - PD potential difference - R skin resistance - SCC short-circuit current     

Sodium-dependent short-circuit current across the yolk sac membrane during embryonic development in normal and shell-less cultured chicks

Summary The transepithelial electrical characteristics of the isolated yolk sac membrane of normal in ovo or shell-less cultured chick embryos were investigated. In normal chicks the potential difference (blood side positive relative to yolk side) and short-circuit current of the membrane increased during development. Ouabain (10^-4 M) on the blood side (basolateral side, serosal side) significantly decreased potential difference and short-circuit current but was without effect on the yolk side (brush border side, mucosal side). Substitution of choline for Na⁺ in the bathing solutions abolished the potential difference and the short-circuit current; when Na⁺ replaced choline this effect was reversed. Amiloride added to both sides of the yolk sac membrane had no effect on potential difference or short-circuit current. Injection of aldosterone (50 g) and T₃ (10 M) into yolk did not induce amiloride sensitivity. The short-circuit current was not altered by addition of either glucose or alanine to the bath. The short-circuit current of the yolk sac membrane of shell-less cultured embryos was significantly lower than that of normal controls. Addition of Ca²⁺ to the serosal bathing medium did not reverse the foregoing condition, but decreased the short-circuit current. It is concluded that the yolk sac short-circuit current is Na⁺ dependent and increases with developmental age in the chick embryo.Abbreviations Hepes N-2-hydroxyethylpiperazine-N-2-ethaneoulphonic acid - PD potential difference - R resistance - SCC short-circuit current - TRIS tris-hydroxymethyl aminomethane - T₃ 3,3-5-triiodo-l-thyronine     

Sodium transport by isolated bullfrog small intestine. Effect of prostaglandin E1.

Addition of 446 micron prostaglandin E1 (PGE1) to the serosal medium of isolated short-circuited bullfrog small intestine elicited small increases transmural potential difference and short-circuit current while addition of PGE1 to the mucosal medium caused no change in the electrical parameters. Addition of 100 micron indomethacin to the mucosal medium inhibited both potential difference and short-circuit current with a resultant increase in steady-state tissue resistance. In the presence of mucosal 100 micron indomethacin, serosal 60 micron PGE1 markedly stimulated transmural potential difference and short-circuit current with a resultant decrease in steady-state tissue resistance. Serosal arachidonic acid (330 micron) stimulated transmural potential difference and short-circuit current and this effect was abolished by the addition of 100 micron indomethacin to the mucosal medium. Serosal 60 micron PGE1 only stimulated the M (mucosa) leads to S (serosa) unidirectional flux of sodium. These results strongly suggest that the PGE1 action is mediated either via a series of metabolic reactions which possibly increase the permeability of the mucosal membrane to sodium or via direct stimulation of rheogenic sodium pump activity.     

The Origin of the Short-Circuit Current in the Isolated Skin of the South American Frog Leptodactylus ocellatus

In isolated skins of Leptodactylus ocellatus the short-circuit current is smaller than the sodium net flux and this difference disappears when the skins are bathed in solutions in which the chloride ions have been replaced by sulfate or methylsulfate ions. There is a net movement of chloride ions from outside to inside of the skins in the short-circuit condition with chloride Ringer's solutions bathing the skins. The addition of ouabain to the inside solution markedly reduced not only sodium net flux but also the chloride net influx found. Copper ions added to the outside solutions produced a rise in short-circuit current, as well as the known increase in potential difference. In sodium-free Ringer's (sodium replaced by choline) the orientation of the potential difference across the skins was reversed, the inside being negative instead of positive. The results are interpreted as direct or indirect indications of the presence of a net transfer of chloride ions from outside to inside of these frog skins.     

Modification of silver-enhanced sodium transport across toad skin

1. Silver stimulated short-circuit current and transepithelial potential difference. 2. Cysteine inhibited the silver-induced short-circuit current. 3. There was a dose-response inhibition of silver-induced short-circuit current by cysteine. 4. The silver-induced short-circuit current is carried by a net active sodium transfer from the outside to the inside bathing solution.     

Sodium transport by isolated bullfrog small intestine. Effect of Prostaglandin E1

Addition of 446 μM prostaglandin E₁ (PGE₁) to the serosal medium of isolated short-circuited bullfrog small intestine elicited small increases in transmural potential difference and short-circuit current while addition of PGE₁ to the mucosal medium caused no change in the electrical parameters. Addition of 100 μM indomethacin to the mucosal medium inhibited both potential difference and short-circuit current with a resultant increase in steady-state tissue resistance. In the presence of mucosal 100 μM indomethacin, serosal 60 μM PGE₁ markedly stimulated transmural potential difference and short-circuit current with a resultant decrease in steady-state tissue resistance. Serosal arachidonic acid (330μM) stimulated transmural potential difference and short-circuit current and this effect was abolished by the addition of 100 μM indomethacin to the mucosal medium. Serosal 60 μM PGE₁ only stimulated the M (mucosa) → S (serosa) unidirectional flux of sodium. These results strongly suggest that the PGE₁ action is mediated either via a series of metabolic reactions which possibly increase the permeability of the mucosal membrane to sodium or via direct stimulation of rheogenic sodium pump activity.     

The effect of ATP and acetylcholine on the sodium transport in bullfrog large intestine (author's transl)]

By Ussing's flux chamber method the effect of ATP and acetylcholine (ACh) on the sodium transport was studied in bullfrog colon. The results obtained are as follows; 1. ATP added to the mucosal medium caused biphasic changes in the transmural potential difference (P.D.) and short-circuit current (S.C.C.), although serosal ATP was ineffective. After an initial rapid and transient rise, both the P.D. and S.C.C. increased in parallel to reach a peak in about 10 min suggesting that the tissue conductance is little affected by ATP. Addition of ouabain to the serosal fluid depressed both the P.D. and S.C.C. and abolished the electrical responses to ATP. The application of ouabain to the mucosal side did not cause any significant depression. These results can be explained in terms of stimulation of sodium pump by ATP added to the mucosal medium. 2. ACh added to either the mucosal or the serosal medium caused increased in the P.D. and the S.C.C. The serosal application was more effective than the mucosal application. The increase in S.C.C. was more remarkable than that in the P.D., indicating an increase in the tissue conductance. It is suggested that ACh stimulates ion transport systems by changing the membrane permeability of the colon.     

Ethanol and silver effects on ion transport across toad skin

Silver stimulated short-circuit current and transepithelial potential difference. Ethanol inhibited transpithelial potential difference. Ethanol had no effect on short-circuit current. Ethanol stimulated unidirectional movements of chloride from outside to inside and from inside to outside.     

Differential effects of extracellular ATP on chloride transport in cortical collecting duct cells

Extracellular ATP in the cortical collecting duct can inhibit epithelial sodium channels (ENaC) but also stimulate calcium-activated chloride channels (CACC). The relationship between ATP-mediated regulation of ENaC and CACC activity in cortical collecting duct cells has not been clearly defined. We used the mpkCCD(c14) cortical collecting duct cell line to determine effects of ATP on sodium (Na(+)) and chloride (Cl(-)) transport with an Ussing chamber system. ATP, at a concentration of 10(-6) M or less, did not inhibit ENaC-mediated short-circuit current (I(sc)) but instead stimulated a transient increase in I(sc). The macroscopic current-voltage relationship for ATP-inducible current demonstrated that the direction of this ATP response changes from positive to negative when transepithelial voltage (V(te)) is clamped to less than -10 mV. We hypothesized that this negative V(te) might be found under conditions of aldosterone stimulation. We next stimulated mpkCCD(c14) cells with aldosterone (10(-6) M) and then clamped the V(te) to -50 mV, the V(te) of aldosterone-stimulated cells under open-circuit conditions. ATP (10(-6) M) induced a transient increase in negative clamp current, which could be inhibited by flufenamic acid (CACC inhibitor) and BAPTA-AM (calcium chelator), suggesting that ATP stimulates Cl(-) absorption through CACC. Together, our findings suggest that the status of ENaC activity, by controlling V(te), may dictate the direction of ATP-stimulated Cl(-) transport. This interplay between aldosterone and purinergic signaling pathways may be relevant for regulating NaCl transport in cortical collecting duct cells under different states of extracellular fluid volume.     

Effects of calcitonin gene-related peptide on airway epithelial functions in dogs

We studied the effects of calcitonin gene-related peptide (CGRP) on ciliary beat frequency (CBF) and electrical properties of canine tracheal epithelium by a photoelectric method and Ussing's short-circuit technique, respectively. CGRP dose dependently increased CBF, an effect that was accompanied by elevation of intracellular cyclic AMP but not affected by blockade of either Ca2+-influx or arachidonic acid metabolism. In contrast, CGRP elicited only a small and transient increase in short-circuit current without significant alterations in transepithelial potential difference or tissue conductance. These results suggest that CGRP may play a role in regulating airway mucociliary transport function.     

Mechanisms of transport of Na+ and Cl- in the lizard colon

1. Ionic fluxes of sodium and chloride across lizard colon mucosa were measured and compared with the electrical characteristics of the tissue under voltage-clamped conditions. 2. In a Ringer-bicarbonate solution there was both a net sodium flux (JNanet) and a net chloride flux (JClnet) from mucosa to serosa. The net flux residual (JR) was near zero, indicating that net sodium and chloride transport is the result of an electrically neutral transport mechanism. 3. In the presence of sodium, the net chloride flux was abolished and the short-circuit current (Isc) and the electrical potential difference (PD) were unchanged. In the absence of chloride the net sodium flux was abolished and the short-circuit current and electrical potential difference were not modified. 4. From an analysis of the effects of the inhibitors, furosemide, amiloride and disulfonic stilbene (DIDS), a plausible model was developed to explain the characteristics of sodium and chloride absorption.     

Alterations in electrophysiology of isolated amphibian small intestine produced by removing the muscle layers

Isolated segments of Amphiuma small intestine bathed in chloride or sulfate buffer generate a greater short-circuit current and a larger change in current in response to galactose when the serosal muscle layers are stripped from the mucosa. Intact (unstripped) segments are not apparently anoxic since stripped segments exposed to serosal N₂ for 3 h display normal short-circuit currents but a reduced potential response to galactose, while the presence of muscle layers tends to reduce the short-circuit current but does not alter the potential response to galactose. Bullfrog small intestine also generates greater short-circuit current following removal of the muscle layers. The enhancing effect of stripping appears to be related to removal of a resistance to ion flow across the tissue.     

Bioelectric properties and ion flow across excised human bronchi

Bioelectric properties and ion transport of excised human segmental/subsegmental bronchi were measured in specimens from 40 patients. Transepithelial electric potential difference (PD), short-circuit current (Isc), and conductance (G), averaged 5.8 mV (lumen negative), 51 microA X cm-2, and 9 mS X cm-2, respectively. Na+ was absorbed from lumen to interstitium under open- and short-circuit conditions. Cl- flows were symmetrical under short-circuit conditions. Isc was abolished by 10(-4) M ouabain. Amiloride inhibited Isc (the concentration necessary to achieve 50% of the maximal effect = 7 X 10(-7) M) and abolished net Na+ transport. PD and Isc were not reduced to zero by amiloride because a net Cl- secretion was induced that reflected a reduction in Cl- flow in the absorptive direction (Jm----sCl-). Acetylcholine (10(-4) M) induced an electrically silent, matched flow of Na+ (1.7 mueq X cm-1 X h-1) and Cl- (1.9 mueq X cm-12 X h-1) toward the lumen. This response was blocked by atropine. Phenylephrine (10(-5) M) did not affect bioelectric properties or unidirectional ion flows, whereas isoproterenol (10(-5) M) induced a small increase in Isc (10%) without changing net ion flows significantly. We conclude that 1) Na+ absorption is the major active ion transport across excised human bronchi, 2) Na+ absorption is both amiloride and ouabain sensitive, 3) Cl- secretion can be induced by inhibition of the entry of luminal Na+ into the epithelia, and 4) cholinergic more than adrenergic agents modulate basal ion flow, probably by affecting gland output.     

Thiocyanate inhibition of active chloride absortion in Aplysia intestine

This investigation was principally undertaken to examine the mechanism of active chloride absorption across the Aplysia californica intestine by using various inhibitors of ion transport. Isolated intestine, mounted between identical oxygenated sodium-free seawater solutions, maintained stable transmural potential differences (serosa negative) and short-circuit currents for several hours at 25°C. The metabolic inhibitors, 2,4-dinitrophenol and flouride, reduced both transmural potential difference and short-circuit current; however, the electrical characteristics were predominantly dependent upon glycolytic energy. The addition of thiocyanate to the mucosal solution inhibited both electrical characteristics in parallel, and this inhibition could be titrated according to the thiocyanate concentration. The short-circuit current was carried wholly by a net active chloride transfer from mucosa to serosa as determined by flux measurements. These results suggest that active chloride absorption may be mediated by a primary active transport process.     

Effect of 5-hydroxytryptamine on sodium transport across bullfrog skin

Summary 5-hydroxytryptamine, when present in the solution bathing the inside surface of bullfrog skin at concentrations of 0.25–25.0 mM, reduced both electrical potential difference and short-circuit current across the skin. The magnitude of reduction in potential difference and short-circuit current was dependent on 5 HT concentration. Reduction in sodium influx entirely accounted for the reduction in short-circuit current. Preliminary evidence suggested a competition between 5 HT and vasopressin in the production of their effects on sodium transport across the skin, while high Ca⁺⁺ concentrations and 5 HT seemed to act independently of each other.Dr. Henry C. and Bertha H. Buswell Fellow.     

Thiocyanate inhibition of active chloride absorption in Aplysia intestine

This investigation was principally undertaken to examine the mechanism of active chloride absorption across the Aplysia californica intestine by using various inhibitors of ion transport. Isolated intestine, mounted between identical oxygenated sodium-free seawater solutions, maintained stable transmural potential differences (serosa negative) and short-circuit currents for several hours at 25 degrees C. The metabolic inhibitors, 2,4-dinitrophenol and fluoride, reduced both transmural potential difference and short-circuit current; however, the electrical characteristics were predominantly dependent upon glycolytic energy. The addition of thiocyanate to the mucosal solution inhibited both electrical characteristics in parallel, and this inhibition could be titrated according to the thiocyanate concentration. The short-circuit current was carried wholly by a net active chloride transfer from mucosa to serosa as determined by flux measurements. These results suggest that active chloride absorption may be mediated by a primary active transport process.     

Effect of basolateral adenosine triphosphate on chloride secretion by bovine oviductal epithelium

The composition of the fluid within the oviduct is largely determined by the secretory and absorptive activities of the oviduct epithelium. The present study explored the effects of basolateral nucleotide stimulation on ion transport in the bovine oviduct using the chamber short-circuit current technique. Basolateral application of ATP induced a rapid transient increase in ion secretion by oviduct epithelial monolayers in a concentration-dependent manner. The ATP-induced short-circuit current (I(SC)) response was preserved in the presence of amiloride, whereas it was reduced in the absence of extracellular chloride or in the presence of bumetanide. The channels underlying the chloride secretory response were identified as Ca(2+)-activated Cl(-) channels and CFTR. The ATP-induced Cl(-) secretory response was largely preserved in the absence of extracellular Ca(2+) but was significantly reduced in the presence of BAPTA-am (1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid-acetomethoxy ester), thapsigargin, or 2-APB (2-aminoethoxydiphenylborate), demonstrating an important role for intracellular Ca(2+) signaling in mediating these effects. A nucleotide potency profile of ATP = UTP (uridine triphosphate) > ADP, sensitivity to suramin, and cross-desensitization by basolateral UTP suggests that ATP exerted its effects on chloride secretion through the purinergic receptor P2Y, G protein-coupled 2, and the presence of the P2RY2 gene was confirmed by RT-PCR. These results provide strong evidence that purinergic signaling constitutes a key mechanism of regulating chloride secretion and thus fluid formation in the bovine oviduct.     

The conventional short-circuiting technique under-short-circuits most epithelia

Summary The relationships among ion current, membrane potential difference, and resistance of an epithelium are studied. The short-circuit technique introduced by Ussing and Zerahn does not completely short circuit the epithelium if the series resistance parallel to the cell layer between the voltage electrodes is not properly compensated. The residual potential difference across the epithelial cell layer in the short-circuit state is proportional to both the measured short-circuit current and the resistance of the diffusion barriers not compensated. In the conventionally short-circuited small intestinal mucosa the villus and crypt areas are hypo-polarized to different degrees rather than simultaneously hyper- and hypo-polarized. Short-circuiting the whole tissue reduces but does not abolish the passive net ion movement across the tissue. Measurements of the electrical properties of the whole and denuded rat distal small intestine in HCO₃-Ringer solution containing 10mm glucose reveal that the measured short-circuit current has under-estimated approximately 33% of the true short-circuit current and that the passive net Na flux from serosa to mucosa and Cl flux from mucosa to serosa are not negligible in the short-circuit state.     

Ion transport across the frog olfactory mucosa: the basal and odorant-stimulated states

The Ussing method was adapted to study the basal electrolyte transfer as well as the events that occur upon odorant stimulation in frog olfactory mucosa. The unstimulated short-circuit current was due mainly to a furosemide-sensitive ion transport system on the apical side of the olfactory mucosa. This current was not amiloride sensitive. The current-voltage relationship of the unstimulated state was linear. That of the odorant-evoked current was non-linear and amiloride-sensitive. Ouabain caused collapse of both the unstimulated and odorant-stimulated short-circuit current. In this case, voltage-clamping the tissue to non-zero values restored the odorant-evoked current with polarity depending on that of the clamping voltage. This suggested that the direction of the current is determined by that of the sodium electrochemical potential difference. Our results indicate that the unstimulated short-circuit current occurs through an apical sodium cotransport system, while the odorant-evoked current is due to odorant-activated, passive sodium channels that are amiloride sensitive.     

Role of luminal ATP in regulating electrogenic Na(+) absorption in guinea pig distal colon

Extracellular ATP regulates a variety of functions in epithelial tissues by activating the membrane P2-receptor. The purpose of this study was to investigate the autocrine/paracrine regulation by luminal ATP of electrogenic amiloride-sensitive Na(+) absorption in the distal colon from guinea pigs treated with aldosterone by measuring the amiloride-sensitive short-circuit current (I(sc)) and (22)Na(+) flux in vitro with the Ussing chamber technique. ATP added to the luminal side inhibited the amiloride-sensitive I(sc) and (22)Na(+) absorption to a similar degree. The concentration dependence of the inhibitory effect of ATP on amiloride-sensitive I(sc) had an IC(50) value of 20-30 microM, with the maximum inhibition being approximately 50%. The effects of different nucleotides and of a nucleoside were also studied, the order of potency being ATP = UTP > ADP > adenosine. The effects of ATP were slightly, but significantly, reduced in the presence of suramin in the luminal solution. The inhibitory effect of luminal ATP was more potent in the absence of both Mg2+ and Ca2+ from the luminal solution. Pretreatment of the tissue with ionomycin or thapsigargin in the absence of serosal Ca2+ did not affect the percent inhibition of amiloride-sensitive I(sc) induced by ATP. Mechanical perturbation with a hypotonic luminal solution caused a reduction in amiloride-sensitive I(sc), this effect being prevented by the presence of hexokinase, an ATP-scavenging enzyme. These results suggest that ATP released into the luminal side by hypotonic stimulation could exert an inhibitory effect on the electrogenic Na(+) absorption. This effect was probably mediated by a P2Y(2) receptor on the apical membrane of colonic epithelial cells, and a change in the intracellular Ca2+ concentration may not be necessary for this process.