Transient potassium fluxes in toad skin

Summary Experiments were carried out in the isolated short-circuited skin of the toadBufo marinus ictericus.⁴²K influx and efflux experiments were carried out with skins bathed on both sides by NaCl-Ringer's solution. Those fluxes showed very similar kinetics of equilibration with time and the results could be fitted by equations of a model of two intraepithelial compartments and the bathing solutions. In the steady state K influx is 3.99 ±0.36 nmol cm^–2 hr^–1 (n=7) and efflux 3.62±0.38 nmol cm^– hr^–1 (n=7) and are not statistically different, indicating that no net K flux is present across the epithelium. Different kinds of perturbations affecting the rates of⁴²K discharge into the bathing solutions were studied. Immediately after addition of amiloride (10^–4 m) to the outer solution, a sharp decline is observed in the rate of⁴²K discharge into the bathing solution,J ₂₁ ^K , which falls from 3.62±0.38 nmol cm^–2 hr^–1 to 2.02±0.04 nmol cm^–2 hr^–1 (n=7) 2 min after addition of the drug, followed by a partial recuperation with time. A complete Na by K substitution in the outer bathing solution induces a prompt and marked decline inJ ₂₁ ^K which is similar to that induced by amiloride. Increase in the outer bathing solution Na concentration from zero Na concentration induces a nonlinear increase inJ ₂₁ ^K and a linear relationship was observed betweenJ ₂₁ ^K and short-circuit current in the range of 0 to 115mm external Na concentration. The decline inJ ₂₁ ^K induced by amiloride or by lowering external Na concentration was interpreted as being caused by electrical hyperpolarization of the external barrier of the epithelium induced by these procedures. Depolarization of the epithelial barriers by inner Na by K substitution in the short-circuited state (when the potential barriers are equal) drastically interfere with the rate of⁴²K discharge from the epithelium into the bathing solutions. Thus, transient increases are observed both in the rate of⁴²K discharge to the outer and to the inner bathing solutions upon depolarization of the barriers. These results indicate that at least the most important component of transepithelial K unidirectional fluxes goes through a transcellular route with a negligible paracellular component. Addition of ouabain (10^–3 m) to the inner bathing solution induces a transient rise in the rate of⁴²K discharge to the outer bathing solution with a peak on the order of 200% of the stationary value previous to the action of the inhibitor, followed by a return to new stationary values not statistically different from those observed previously to the effect of ouabain. The behavior ofJ ₂₁ ^K upon the effect of ouabain, as suggested by comparison with predictions from computer simulation, strongly supports the notion of a rheogenic Na pump in the inner barrier of the epithelium against the notion of a nonrheogenic 11 Na–K pump.     

Cation selectivity of the apical membrane of the turtle colon: sodium entry in the presence of lithium

Exposure of the apical surface of the isolated turtle colon to Li produced a marked transient in short-circuit current (ISC) and total tissue conductance (GT) which was abolished by amiloride but was unaffected by ouabain or by removing Na or Cl from the mucosal bathing solution. Despite marked changes in Isc, Na uptake across the apical membrane was a linear function of time during exposure to Li-containing solutions, and except at very high Li concentrations, the initial rate of Na uptake, JiNa, was identical to its pre-Li value. In the presence of Li, however, JiNa was significantly less than the total Isc. The apparent "transference number" for Na in the apical membranes was a function of the Li:Na concentration ratio in the mucosal bathing solution. These results suggest that Li can carry substantial amounts of current through amiloride-sensitive channels in the apical membrane of the colon without having any effect on the rate coefficient for Na entry. This behavior is not consistent with "competition" of Na and Li for a membrane "carrier" but rather suggests that the Na entry mechanism may be a population of pores or channels through which Na and Li may pass with negligible interaction.     

Noise analysis reveals K+ channel conductance fluctuations in the apical membrane of rabbit colon

Summary In this paper we describe current fluctuations in the mammalian epithelium, rabbit descending colon. Pieces of isolated colon epithelium bathed in Na⁺ or K⁺ Ringer's solutions were studied under short-circuit conditions with the current noise spectra recorded over the range of 1–200 Hz. When the epithelium was bathed on both sides with Na⁺ Ringer's solution (the mucosal solution contained 50 m amiloride), no Lorentzian components were found in the power spectrum. After imposition of a potassium gradient across the epithelium by replacement of the mucosal solution by K⁺ Ringer's (containing 50 m amiloride), a Lorentzian component appeared with an average corner frequency,f _c=15.6±0.91 Hz and a mean plateau valueS _o=(7.04±2.94)×10^–20 A² sec/cm². The Lorentzian component was enhanced by voltage clamping the colon in a direction favorable for K⁺ entry across the apical membrane. Elimination of the K⁺ gradient by bathing the colon on both sides with K⁺ Ringer's solutions abolished the noise signal. The Lorentzian component was also depressed by mucosal addition of Cs⁺ or tetraethylammonium (TEA) and by serosal addition of Ba²⁺. The one-sided action of these K⁺ channel blockers suggests a cellular location for the fluctuating channels. Addition of nystatin to the mucosal solution abolished the Lorentzian component. Serosal nystatin did not affect the Lorentzian noise. This finding indicates an apical membrane location for the fluctuating channels. The data were similar in some respects to K⁺ channel fluctuations recorded from the apical membranes of amphibian epithelia such as the frog skin and toad gallbladder. The results are relevant to recent reports concerning transcellular potassium secretion in the colon and indicate that the colon possesses spontaneously fluctuating potassium channels in its apical membranes in parallel to the Na⁺ transport pathway.     

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.     

Ion transport in ‘tight’ epithelial monolayers of MDCK cells

Summary Cultured monolayers of MDCK cells grown upon filter supports display many features ofin vivo epithelia. Previously reported values of transmonolayer resistance of 100 cm^–2 (Misfeldt, Hamamoto & Pitelka, 1976; Cereijido, Robbins, Dolan, Rotunno & Sabatini, 1978) indicate a leaky epithelium. This paper describes the properties of a strain of MDCK cells which displays entirely different electrophysiological properties. The results show that (i) the mean transmonolayer resistance is 4.16 k cm^–2, (ii) transmonolayer ion transport is of small magnitude since the mean spontaneous open circuit PD is only 2.17 mV basal surface positive and isotopic Na and Cl flux measurements fail to demonstrate a significant net flux, (iii) the action of ouabain, amiloride and ion substitutions are consistent with transmonolayer net Na movement being largely responsible for the spontaneous PD, and (iv) asymmetry in the localization of the Na-K ATPase is evident on the basis of³H-ouabain binding to cell monolayer.     

Effect of diuretics on intestinal transport of electrolytes, glucose, and amino acid.

The jejunal mucosal membrane of albino mice was used to study the electrical properties and ion transport. The membrane was bathed in Krebs-Ringer solution with or without glucose.When ethacrynic acid (EA), furosemide, or amiloride was added to the bathing fluid of both sides, a transient increase followed by a decrease of both potential difference (PD) and short circuit current (Isc) were observed. In glucose-containing bathing medium, EA inhibited both net Na and Cl flux and residual flux; however, EA had little effect on both Na and Cl flux in glucose-free bathing medium. Studies using everted intestinal sac technique showed that EA inhibited both glucose and L-tyrosine across the mucosal membrane against concentration gradients. Furosemide and amiloride were less potent than EA in inhibiting the Na and Cl flux when the bathing solution contained glucose. But these two compounds had no effect on glucose and L-tyrosine transport across the intestinal mucosa. Furthermore, they did inhibit Cl flux even in the condition of glucose-free bathing medium. It is postulated that all three diuretics act on the brush-border membrane of the intestine. EA probably inhibits the Na-glucose cotransporting system; furosemide and amiloride inhibit the simple diffusion process of Na entry of Cl exit by decreasing the conductance of the membrane.     

Mucosal acidification and an acid microclimate in the hen colon in vitro

Experiments were performed on isolated, stripped colonic epithelia of low-salt-adapted hens (Gallus domesticus) in order to characterize acid secretion by this tissue. With symmetric, weak buffer solutions, colonic epithelia acidified both mucosal and serosal sides. Titration measurements of the mucosal acidification rate (pH-stat technique) averaged 1.63±0.25 Eq·cm^-2·h^-1. Mucosal acidification was also evident in colons from high-salt-adapted birds and in low-salt-adapted coprodeum, but was completely abolished in the high-salt coprodeum. Mucosal acidification by low-salt-adapted colonic epithelium was unaffected by sodium replacement, mucosal amiloride (10^-3 mol·l^-1), and serosal ouabain (5x10^-4 mol·l^-1), although all three treatments significantly reduced or reversed the short-circuit current. Acetazolamide (10^-3 mol·l^-1, serosal) reduced mucosal acidification by 15% and simultaneously increased short-circuit current by a similar amount. Colonic epithelia incubated in glucose-free solutions had significantly lower acidification rates (0.59±0.13 Eq·cm^-2·h^-1, P<0.002 versus controls) and addition of glucose (15 mmol·l^-1), but not galactose, partially restored acidification to control levels. Anoxia (N₂ gassing) completely inhibited short-circuit current, but reduced acidification by only 30%. A surface microclimate pH, nearly 2 pH units more acidic than the bath pH of 7.1–7.4 was measured in low-salt-adapted colon and coprodeum. The acid microclimate of both tissues was partially attenuated by adaptation to a high-salt diet. Colonic microclimate pH was dependent on the presence of glucose and sensitive to the bath pH. Histochemical staining for carbonic anhydrase localized this enzyme to cytoplasm and lateral margins of one subfraction of colonic cells, and to cytoplasm in a second subpopulation Intense staining was also evident in subepithelial capillaries. These results suggest that a large part of mucosal acidification and maintenance of the acid microclimate in hen colon may be dependent on glycolysis and metabolic acid production, although a smaller, electrogenic and acetazolamidesensitive component also appears to exist. This latter component may become more prominent under conditions of cellular acidification.Abbreviations CA carbonic anhydrase - I _SC short circuit current - NFM N-ethylmaleimide - PD transepithelial potential - SCFA short chain fatty acids     

Transport-dependent alterations of membrane properties of mammalian colon measured using impedance analysis

Summary Direct current (DC) measurement methods have been commonly used to characterize the conductance properties of the mammalian colon. However, these methods provide no information concerning the effects of tissue morphology on the electrophysiological properties of this epithelium. For example, distribution of membrane resistances along narrow fluid-filled spaces such as the lateral intercellular spaces (LIS) or colonic crypts can influence DC measurements of apical and basolateral membrane properties. We used impedance analysis to determine the extent of such distributed resistance effects and to assess the conductance and capacitance properties of the colon. Because capacitance is proportional to membrane area, this method provides new information concerning membrane areas and specific ionic conductances for these membranes.We measured transepithelial impedance under three conditions: (1) control conditions in which the epithelium was opencircuited and bathed on both sides with NaCl–HCO₃ Ringer's solutions, (2) amiloride conditions which were similar to control except that 100 m amiloride was present in the mucosal bathing solution, and (3) mucosal NaCl-free conditions in which mucosal Na and Cl were replaced by potassium and sulfate or gluconate (K⁺ Ringer's). Three morphologically-based equivalent circuit models were used to evaluate the data: (1) a lumped model (which ignores LIS resistance), (2) a LIS distributed model (distributed basolateral membrane impedance) and (3) a crypt-distributed model (distributed apical membrane impedance). To estimate membrane impedances, an independent measurement of paracellular conductance (G _s ) was incorporated in the analysis. Although distributed models yielded improved fits of the data, the distributed and lumped models produced similar estimates of membrane parameters. The predicted effects of distributed resistances on DC microelectrode measurements were largest for the LIS-distributed model. LIS-distributed effects would cause a 12–15% underestimate of membrane resistance ratio (R _a /R _b ) for the control and amiloride conditions and a 34% underestimate for the K Ringer's condition. Distributed resistance effects arising from the crypts would produce a 1–2% overestimate ofR _a /R _b .Apical and basolateral membrane impedances differed in the three different experimental conditions. For control conditions, apical membrane capacitance averaged 21 F/cm² and the mean apical membrane specific conductance (G _a-norm) was 0.17 mS/F. The average basolateral membrane capacitance was 11 F/cm² with a mean specific conductance (G _b-norm) of 1.27 mS/F.G _a-norm was decreased by amiloride or K⁺ ringer's to 0.07 mS/gmF and 0.06 mS/F, respectively. Basolateral conductance was also reduced by amiloride, whereas capacitance was unchanged (G _b-norm=0.97 mS/F). For the K⁺ Ringer's condition, both basolateral conductance and capacitance were greatly increased such thatG _b-norm was not significantly different from the control condition.     

Acid pH and weak acids induce Na−Cl contransport in the rabbit urinary bladder

We have described a coupled Na--Cl entry step at the apical membrane of a tight epithelium, the rabbit urinary bladder. Mucosal pH values, more acid than 4.6, stimulate a 20 to 40-fold increase in mucosal-to-serosal Na+ and Cl- flux. The flux increase is almost completely blocked by low concentrations of of bumetanide. The transepithelial movement of Na+ and Cl- is normally electroneutral; however, when weak acids (such as acetate) are present in the mucosal solution, the acid-induced increase in flux is accompanied by a large increase in short-circuit current. Besides blockage by bumetanide, both the increase in flux and short-circuit current are blocked by: (1) Na+-free solutions on the mucosa; (2) Cl--free solutions on the mucosa; (3) phosphodiesterase inhibitors; (4) ouabain in the serosal solution; (5) K+-free solutions on the serosa; and (6) HCO3--free solutions on the serosa. The increase in the fluxes and the short-circuit current is unaffected by: (1) amiloride application in the mucosal solution; (2) mucosally applied stilbene derivatives which block Cl-/HCO3- exchange (SITS); and (3) Cl--free solutions applied to the serosa. We interpret these results to imply a coupled Na--Cl uptake step at the apical membrane which is stimulated by intracellular acetate (or (pH). The uptake step leads to a movement of Na+ and Cl- across the basolateral membrane, which is mediated by the Na+, K+-ATPase and a Na/Cl/HCO3- exchange mechanism. Our results demonstrate that "tight" epithelia may, under appropriate circumstances, demonstrate mechanisms of ion movement which are similar to "leaky" epithelia.     

Energetics of coupled active transport of sodium and chloride

A Clark electrode was used to measure oxygen consumption by the gall bladder, in which there is a direct and one-to-one linkage between active Na and active Cl transport. O2 uptake was reversibly depressed when Cl in the mucosal bathing solution was replaced by a poorly transported anion, such as sulfate. This effect of Cl was abolished by ouabain or in Na-free solutions. When the anion was chloride, treatment with ouabain or replacement of Na by a poorly transported cation depressed QO2 more than did replacement of Cl. However, ouabain or removal of Na also depressed QO2 in Na2SO4 solutions, in which salt transport is minimal. It is concluded that oxygen uptake in the gall bladder consists of three fractions: 9% requires both Na and Cl, is inhibited by ouabain, and is linked to the NaCl pump; 36% requires Na but not Cl, is inhibited by ouabain, and possibly is linked to the cellular K uptake mechanism; and 55% represents basal uptake. If the extra oxygen uptake observed during transport supplies all the energy for transport, then 25 Na + 25 Cl ions are transported actively per O2 consumed; i.e., twice as many ions as in epithelia which transport only Na actively. This extra uptake is more than sufficient to supply the energy for overcoming internal membrane resistance under the experimental conditions used.     

Ion selectivity of the apical membrane Na channel in the toad urinary bladder

Summary The ion selectivity of the apical membrane Na channel in the toad urinary bladder was investigated. The electrical potential difference and resistance across the basal-lateral membrane were reduced using high concentrations of KCl in the serosal bathing medium, and gradients for various ions were imposed across the apical membrane by altering the composition of the mucosal bathing medium. Ion fluxes through the channel were measured as the transepithelial current inhibited by amiloride, a specific blocker of the channel's Na conductance. The selectivity sequence for alkali metal cations was H>Li>NaK. K, permeability was barely detectable; the selectivity for Na over K was about 1000:1. Ammonium, hydroxyl ammonium and hydrazinium ions were, like K, virtually impermeant. The results suggest that the size of the unhydrated ion is an important factor in determining permeability in this channel.     

The capacitance of skeletal muscle fibers in solutions of low ionic strength

The capacitance of skeletal muscle fibers was measured by recording with one microelectrode the voltage produced by a rectangular pulse of current applied with another microelectrode. The ionic strength of the bathing solution was varied by isosmotic replacement of NaCl with sucrose, the [K] [Cl] product being held constant. The capacitance decreased with decreasing ionic strength, reaching a value of some 2 µF/cm² in solutions of 30 mM ionic strength, and not decreasing further in solutions of 15 mM ionic strength. The capacitance of glycerol-treated fibers did not change with ionic strength and was also some 2 µF/cm². It seems likely that lowering the ionic strength reduces the capacitance of the tubular system (defined as the charge stored in the tubular system), and that the 2 µF/cm² which is insensitive to ionic strength is associated with the surface membrane. The tubular system is open to the external solution in low ionic strength solutions since peroxidase is able to diffuse into the lumen of the tubules. Twitches and action potentials were also recorded from fibers in low ionic strength solutions, even though the capacitance of the tubular system was very small in these solutions. This finding can be explained if there is an action potential—like mechanism in the tubular membrane.     

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     

Na+-H+ exchange and Na+ entry across the apical membrane of Necturus gallbladder

The role of Na+-H+ exchange in Na+ transport across the apical membrane was evaluated in Necturus gallbladder epithelium by means of intracellular Na+ activity (aNai) and 22Na+ uptake measurements. Under control conditions, complete replacement of Na+ in the mucosal solution with tetramethylammonium reduced aNai from 14.0 to 6.9 mM in 2 min (P less than 0.001). Mucosal addition of the Na+-H+ exchange inhibitor amiloride (10(-3) M) reduced aNai from 15.0 to 13.3 mM (P less than 0.001), whereas bumetanide (10(-5) and 10(-4) M) had no effect. Na+ influx across the apical membrane was studied by treating the tissues with ouabain, bathing them in Na-free solutions, and suddenly replacing the mucosal solution with an Na-containing solution. When the mucosal solution was replaced with Na-Ringer's, aNai increased at approximately 11 mM/min. This increase was inhibited by 54% by amiloride (10(-3) M, P less than 0.001) and was unaffected by bumetanide (10(-5) M). Amiloride-inhibitable Na+ fluxes across the apical membrane were also induced by the imposition of pH gradients. Na+ influx was also examined in tissues that had not been treated with ouabain. Under control conditions, 22Na+ influx from the mucosal solution into the epithelium was linear over the first 60 s and was inhibited by 40% by amiloride (10(-3) M, P less than 0.001) and by 19% by bumetanide (10(-5) M, P less than 0.025). We conclude that Na+-H+ exchange is a major pathway for Na+ entry in Necturus gallbladder, which accounts for at least half of apical Na+ influx both under transporting conditions and during exposure to ouabain. Bumetanide-inhibitable Na+ entry mechanisms may account for only a smaller fraction of Na+ influx under transporting conditions, and cannot explain influx in ouabain-treated tissues. These results support the hypothesis that NaCl entry results primarily from the operation of parallel Na+-H+ and Cl--HCO-3 exchangers, and not from a bumetanide-inhibitable NaCl cotransporter.     

Effect of changes in transepithelial transport on the uptake of sodium across the outer surface of the frog skin

The unidirectional sodium, uptake at the outer surface of the frog skin was measured by the method described by Biber and Curran (8). With bathing solutions containing 6 mM NaCl there is a good correlation between sodium uptake and short-circuit current (SCC) measured simultaneously except that the average uptake is about 40% higher than the average SCC. The discrepancy between uptake and SCC increases approximately in proportion to an increase in sodium concentration of the bathing solutions. Amiloride inhibits the unidirectional sodium uptake by 21 and 69% at a sodium concentration of 115 and 6 mM, respectively. This indicates that amiloride acts on the entry step of sodium but additional effects cannot be excluded. The sodium, uptake is not affected by 10^-4 M ouabain at a sodium concentration of 115 mM but is inhibited by 40% at a sodium concentration of 6 mM. Replacement of air by nitrogen leads to a 40% decrease of sodium uptake at a sodium concentration of 6 mM. The results support the view proposed previously (8) that the sodium uptake is made up of two components, a linear component which is, essentially, not involved in transepithelial movement of sodium and a saturating component which reflects changes in transepithelial transport. Amiloride, seems largely to affect the saturating component.     

Characterization of esophageal desalination in the seawater eel,Anguilla japonica

To characterize mechanisms of esophageal desalination, osmotic water permeability and ion fluxes were measured in the isolated esophagus of the seawater eel. The osmotic permeability coefficient in the seawater eel esophagus was 2·10^-4 cm·s^-1. This value was much lower than those in tight epithelial, although the eel esophagus is a leaky epithelium with a tissue resistance of 77 ohm·cm^-2. When the esophagus was bathed in normal Ringer solutions on both sides no net ion and water fluxes were observed. However, when mucosal NaCl concentration was increased by a factor of 3, Na⁺ und Cl^- ions were transferred from mucosa to serosa (desalination). If only Na⁺ or Cl^- concentration in the mucosal fluid was increased by a factor of 3, net Na⁺ and Cl^- fluxes were reduced to 30–40%, indicating that 60–70% of the net Na⁺ and Cl^- fluxes are coupled mutually. The coupled NaCl transport seems to be effective in desalting the luminal high NaCl. The remaining 30–40% of the total Na⁺ and Cl^- fluxes seems to be due to a simple diffusion, because these components are independent of each other and follow their electrochemical gradients, and also because these fluxes remain even after treatment with NaCN or ouabain. A half of the coupled NaCl transport could be explained by a Na⁺/H⁺–Cl^-/HCO ₃ ^- double exchanger on the apical membrane of the esophageal epithelium, because mucosal amiloride and 4.4-diisothiocyanatostilbene-2,2-disulphonic acid inhibited the net Na⁺ and Cl^- fluxes by approximately 30%. The other half of the coupled NaCl transport, which follows their electrochemical gradients, still remains to be explained.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonic acid - NMDG N-methyl-d-glucosamine - P _Cl Cl^- permeability coefficient - PD transepithelial potential difference - P _Na Na⁺ permeability coefficient - P _osm osinotic permeability coefficient - TALH thick ascending limb of Henle's loop     

Evaluation of the rate of basal oxygen consumption in the isolated frog skin and toad bladder.

In the study of active transport it is important to distinguish between oxygen consumption sustaining transepithelial transport and that responsible for other tissue functions (basal metabolism). Since amiloride blocks transepithelial active sodium transport and the associated oxygen consumption in the frog skin and toad bladder, we and others have employed this agent to evaluate the rate of basal metabolism. This technique has recently been criticized in a report that amiloride (and ouabain) increased oxygen consumption when no sodium was available for transport. We have been unable to corroborate these observations. With magnesium-Ringer as external bathing solutions, amiloride and ouabain failed to stimulate oxygen consumption. With sodium-Ringer as external bathing solution amiloride reduced oxygen consumption about 30%, to a level indistinguishable from that found on external substitution of magnesium-Ringer for sodium-Ringer. We conclude that the use of amiloride permits evaluation of the rate of basal metabolism with acceptable accuracy; a possible slight depressant effect of ouabain on basal metabolism remains to be investigated.     

Evaluation of the rate of basal oxygen consumption in the isolated frog skin and toad bladder

In the study of active transport it is important to distinguish between oxygen consumption sustaining transepithelial transport and that responsible for other tissue functions (basal metabolism). Since amiloride blocks transepithelial active sodium transport and the associated oxygen consumption in the frog skin and toad bladder, we and others have employed this agent to evaluate the rate of basal metabolism. This technique has recently been criticized in a report that amiloride (and ouabain) increased oxygen consumption when no sodium was available for transport. We have been unable to corroborate these observations.With magnesium-Ringer as external bathing solutions, amiloride and ouabain failed to stimulate oxygen consumption. With sodium-Ringer as external bathing solution amiloride reduced oxygen consumption about 30%, to a level indistinguishable from that found on external substitution of magnesium-Ringer for sodium-Ringer. We conclude that the use of amiloride permits evaluation of the rate of basal metabolism with acceptable accuracy; a possible slight depressant effect of ouabain on basal metabolism remains to be investigated.     

Sodium uptake across the apical border of the isolated turtle colon: Confirmation of the two-barrier model

Summary The initial rate of Na uptake by the turtle colon from the mucosal bathing solution consists of two operationally distinct components. One component is a linear function of mucosal Na concentration, is unaffected by amiloride, and appears to represent Na uptake into the paracellular shunt path. The major component of Na uptake is abolished by amiloride and is virtually equal to the short-circuit current over a wide range of. mucosal Na concentrations, suggesting that this portion of Na uptake represents Na movement into Na-transporting cells of the colon. The amiloride-sensitive component of Na uptake, at low mucosal Na concentrations, was unaffected if net Na transport was abolished by ouabain. Similarly, at low mucosal Na concentrations the amiloride-sensitive conductance of the colon was identical in the presence and in the absence of net Na transport.These results show that the isolated turtle colon behaves, as two distinct barriers to transmural Na transport, an apical barrier blocked by amiloride and a more basal-lying barrier where active, transmural Na transport is blocked by ouabain. In addition, these experiments appear to provide the first unambiguous demonstration that the initial-rate isotope uptake technique can provide adirect measure of the properties of the amiloridesensitive barrier to transmural Na movement, presumably the apical membranes of the Na-transporting cells. The results are consistent with the notion that the rate of transmural active Na transport and the conductance of the active Na-transport path are determined by the properties of the apical membrane.