Net CI− flux in short-circuited skin of Rana pipiens: Ouabain sensitivity and Na+ + K+ dependence

Skin of Rana pipiens, like many other species, is considered to actively trassport only Na⁺ when bathed in Ringer's solution on both sides. However, net CI^? influx was previously described by us in short-circuited skin of Rana pipiens, in the summer season. The skins were of low PD (5–30 mV) and high CI^? conductance. Comparison of these findings with other series indicated inverse seasonal variation between PD and CI^? conductance. It was postulated that active CI^? transport exists at all CI^?-conductance levels but at higher PD is too small to be be easily detected. This report evaluates CI^? transport across skins of higher PD (and lower conductance) in winter and further characterizes the system. Net CI^? influx was demonstrated over a wide PD range under short-circuit conditions and was inversely related to the magnitude of the open-circuit PD. It was inhibited by ouabain and dependent upon Na⁺ in the outside medium and K⁺ in the inside medium. It is concluded that this is the same system present in skin of Leptodactylus ocellatus and may not be unique to these two species. It appears to be distinctly different from the acetazolamide-sensitive influx system seen in “low-CI^?” solutions in skins of numerous species. The findings are discussed in terms of (1) parallel Na⁺ and CI^? active transport mechanisms and (2) a neutral NaCI-pump model, recently proposed by Rehm.     

Prostaglandin E2 enhances the sodium conductance of exocrine glands in isolated frog skin (Rana esculenta)

Summary Prostaglandins are known to stimulate the active transepithelial Na⁺ uptake and the active secretion of Cl^– from the glands of isolated frog skin. In the present work the effect of prostaglandin E₂ (PGE₂) on the glandular Na⁺ conductance was examined. In order to avoid interference from the Na⁺ uptake and the glandular Cl^– secretion the experiments were carried out on skins where the Cl^– secretion was inhibited (the skins were bathed in Cl^– Ringer's solution in the presence of furosemide, or in NO ₃ ^– Ringer's solution), and the active Na⁺ uptake was blocked by the addition of amiloride. Transepithelial current, water flow and ion fluxes were measured. A negative current was passed across the skins (the skins were clamped at –100 mV, basolateral solution was taken as reference). When PGE₂, was added to the skins under these experimental conditions, the current became more negative; this was mainly due to an increase in the Na⁺ efflux. Together with the increase in Na⁺ efflux a significant increase of the water secretion was observed. The water secretion was coupled to the efflux of Na⁺, and when one Na⁺ was pulled from the basolateral to the apical solution via this pathway 230 molecules of water follwed. From the data presented it is suggested that this pathway for Na⁺ is confined to the exocrine glands.     

Acidification and sodium entry in frog skin epithelium

Acidification of the external medium by isolated frog skin epithelium (Rana catesbeiana, Rana temporaria, and Caudiververa caudiververa) and its relationship to Na⁺ uptake was studied. Acidification was measured by the pH-stat technique under short-circuit or open-circuit conditions. The results of this study demonstrate that (a) acidification by these species of in vitro frog skins is not directly coupled to Na⁺ or anion transport; (b) acidification can be inhibited by the diuretic drug amiloride, but only at high external Na⁺ concentrations; (c) acidification rate in these species of frog skin is controlled in part by the metabolic production of CO₂; and (d) the positive correlation between net Na⁺ absorption and net acidification observed in whole animal studies could not be replicated in the in vitro skin preparation, even when the frogs were first chronically stressed by salt depletion, a physiological state comparable to that used in the in vivo experiments.     

Ion transport across the skin of a larval salamander

The transport characteristics of the skin of neotenic Ambystoma tigrinum were investigated using ion substitution and circuit analysis. When bathed with sodium Ringer solution on both sides, a transepithelial potential of up to 50 mV (inside positive) and a short-circuit current (I_sc) of up to 10 μA/cm² were observed. When amiloride was added or Na⁺ was replaced by tetramethylammonium in the apical solution, I_sc was decreased from 3.7 ± 0.4 to 1.5 ± 0.2 μA/cm² (n = 10). When K⁺ replaced Na⁺, there was a smaller change in I_sc from 5.8 ± 0.6 to 3.7 ± 0.5 μA/cm² (n = 10). Although barium had no effect when added to 100 K Ringer on normal skin, it inhibited I_sc on skins taken from K⁺-loaded animals. Nystatin caused substantial increases in I_sc with either Na⁺ or K⁺ as the dominant cation in the apical solution. Current voltage analysis using amiloride was used to estimate the resistances and electromotive forces (EMF) associated with ion transport. The EMF for ion transport was partially dependent on K⁺ in the basolateral solution and it was similar to that observed in other epithelia. The resistance of the transport pathway was high, consistent with the low I_sc. These results suggest that there is an amiloride-sensitive Na⁺ channel in parallel with a small K⁺ conductance in the apical membrane of this preparation.     

The H+ Pump in Frog Skin (Rana esculenta): Identification and Localization of a V-ATPase

We here report on studies on the frog skin epithelium to identify the nature of its excretory H⁺ pump by comparing transport studies, using inhibitors highly specific for V-ATPases, with results from immunocytochemistry using V-ATPase-directed antibodies. Bafilomycin A₁ (10 μm) blocked H⁺ excretion (69 ± 8% inhibition) and therefore Na⁺ absorption (61 ± 17% inhibition after 60 min application, n= 6) in open-circuited skins bathed on their apical side with a 1 mm Na₂SO₄ solution, ``low-Na<sup>+</sup> conditions' under which H<sup>+</sup> and Na<sup>+</sup> fluxes are coupled 1:1. The electrogenic outward H<sup>+</sup> current measured in absence of Na<sup>+</sup> transport (in the presence of 50 μm amiloride) was also blocked by 10 μm bafilomycin A<sub>1</sub> or 5 μm concanamycin A. In contrast, no effects were found on the large and dominant Na<sup>+</sup> transport (short-circuit current), which develops with apical solutions containing 115 mm Na<sup>+</sup> (``high-Na⁺ conditions'), demonstrating a specific action on H⁺ transport. In immunocytochemistry, V-ATPase-like immunoreactivity to the monoclonal antibody E11 directed to the 31-kDa subunit E of the bovine renal V-ATPase was localized only in mitochondria-rich cells (i) in their apical region which corresponds to apical plasma membrane infoldings, and (ii) intracellularly in their neck region and apically around the nucleus. In membrane extracts of the isolated frog skin epithelium, the selectivity of the antibody binding was tested with immunoblots. The antibody labeled exclusively a band of about 31 kDa, very likely the corresponding subunit E of the frog V-ATPase. Our investigations now deliver conclusive evidence that H⁺ excretion is mediated by a V-ATPase being the electrogenic H⁺ pump in frog skin. Received: 21 May 1996/Revised: 24 December 1996     

Lithium transport across isolated frog skin epithelium

Summary Transepithelial Li⁺ influx was studied in the isolated epithelium from abdominal skin ofRana catesbeiana. With Na⁺-Ringer's as inside medium and Li⁺-Ringer's as outside medium, the Li⁺ influx across the epithelium was 15.6 A/cm². This influx was considerably reduced by removal of either Na⁺ or K⁺ from the inside bath or by the addition of ouabain or amiloride. Epithelial K⁺ or Na⁺ concentration was respectively lower in epithelia bathed in K⁺-free Ringer's or Na⁺-free Ringer's. In conditions of negligible Na⁺ transport, a 20mm Li⁺ gradient (outin) produced across the short-circuited epithelium a Li⁺ influx of 11.8 A/cm² and a mean short-circuit current of 10.2 A/cm². The same Li⁺ gradient in the opposite direction produced a Li⁺ outflux of only 1.9 A/cm². With equal Li⁺ concentration (10.3 and 20.6mm) on both sides of the epithelium, plus Na⁺ in the inside solution only, a stable Li⁺-dependent short-circuit current was observed. Net Li⁺ movement (outin) was also indirectly determined in the presence of an opposing Li⁺ gradient. Although Li⁺ does not substitute for Na⁺ as an activator of the (Na⁺+K⁺)-ATPase from frog skin epithelium, Li⁺ influx appears to be related to Na⁺–K⁺ pump activity. It is proposed that the permeability of the outer barrier to Na⁺ and Li⁺ is regulated by the electrical gradient produced by electrogenic Na⁺–K⁺ pumps located in the membrane of the deeper epithelial cells.     

Amiloride-induced stimulation of HCO3− reabsorption in turtle bladder

Amiloride in the mucosal fiuid (at concentrations of 5 · 10^?6 M to 10^?4 M) reversibly stimulates the HCO₃^?-dependent moiety of the short-circuiting current (I_sc) in ouabain-treated turtle bladders bathed by Na-free Ringer solutions with or without Cl_?.This effect is uniquely different from the known inhibitory effect of this agent on Na⁺ transport. Thus, any comprehensive hypothesis on the action of amiloride over a wide dosage-response fange should take into account its effect on HCO₃^? transport.     

The excretion of hydrogen ion by the isolated amphibian skin: Effects of antidiuretic hormone and amiloride

H⁺ extrusion by the isolated skins of two amphibia, Rana ridibunda and Bufo bufo was studied in order to test for the presence of exchange mechanisms of the type Na⁺/H⁺ and Cl^?/HCO₃^?, which have been described in several epithelial structures. The preparations were mounted in chambers of the Ussing type, so that the short-circuit current could be used as a function of Na⁺ transport and the pH-stat technique was utilized to determine the rates of H⁺ extrusion under different experimental conditions. These conditions were either the withdrawal of the ions intervening in the mentioned exchanges (Cl^- or Na⁺, or the addition of drugs with well-known effects on Na⁺ uptake and transport (antidiuretic hormone and amiloride).In the frog skin, H⁺ excretion was detected in solutions containing either Cl^? or SO₄^2?, with identical rates. Again, Na⁺ substitution by Mg²⁺ had no effect on H⁺ excretion rates, neither did the suppression of Na⁺ influx by amiloride or its stimulation by antidiuretic hormone. These experiments were repeated with similar results in gland-free preparations of the epidermis of frog skin separated from the corion by the action of collagenase.Experiments in toad skin showed that H⁺ excretion could not be detected when Cl^? was present in the outer medium, but became apparent if an impermeant anion, SO₄^2?, was used. This observation is compatible with the existence of an exchange mechanism of the type Cl^?/HCO₃^?. Secondly, in these preparations H⁺ extrusion increased after stimulation with antidiuretic hormone and decreased when amiloride was used or when Na⁺ was substituted by Mg²⁺, suggesting that at least a fraction of the total H⁺ efflux is linked to Na⁺ influx. In the isolated frog skin this mechanism does not seem to be operative.     

The Hyperpolarizing Region of the Current-Voltage Curve in Frog Skin

Excitability (action potential and refractory period) has been described by A. Finkelstein in the depolarizing region of the current-voltage (I-V) curve of the isolated frog skin. Recently Fishman and Macey interpreted this phenomenon as a consequence of a region with negative resistance that confers to the I-V curve an N shape. We have studied the I-V relation of the isolated frog skin in the hyperpolarizing region with a current-ramp system. It was found that in Na₂SO₄ Ringer's, the resistance continuously increases in the hyperpolarizing direction. When hyperpolarization reaches 300 mv an electrical breakdown occurs, occasionally followed by a region of negative resistance. In NaCl Ringer's the breakdown was also found although the I-V relation was reasonably linear. Unidirectional Na⁺ outflux was measured at different levels of voltage clamping across the skin and with different Na⁺ concentrations in the solutions. The Na⁺ outflux was found to be relatively independent of these parameters. Based on these results a Na⁺ rectifying structure is postulated. An electrical model for active Na⁺ transport including a diode and an oscillator is proposed. The effects of CO₂, nitrogen, amiloride, and ouabain on the I-V relation are described.     

Induction of reverse flow of Na+ through the active transport pathway in toad urinary bladder

When active Na⁺ transport across the toad urinary bladder was abolished by ouabain, a ’reversed‘ short circuit current could be induced by an Na⁺ concentration gradient. This reversed current was increased by vasopressin and inhibited by amiloride and appears to represent net Na⁺ movement ‘backwards’ through epithelial cells which normally participate in active Na⁺ transport across the bladder.     

Active and passive Na+ fluxes across the basolateral membrane of rabbit urinary bladder

Summary The apical membrane of rabbit urinary bladder can be functionally removed by application of nystatin at high concentration if the mucosal surface of the tissue is bathed in a saline which mimics intracellular ion concentrations. Under these conditions, the tissue is as far as the movement of univalent ions no more than a sheet of basolateral membrane with some tight junctional membrane in parallel. In this manner the Na⁺ concentration at the inner surface of the basolateral membrane can be varied by altering the concentration in the mucosal bulk solution. When this was done both mucosal-to-serosal²²Na flux and net change in basolateral current were measured. The flux and the current could be further divided into the components of each that were either blocked by ouabain or insensitive to ouabain. Ouabain-insensitive mucosal-to-serosal Na⁺ flux was a linear function of mucosal Na⁺ concentration. Ouabain-sensitive Na⁺ flux and ouabain-sensitive, Na⁺-induced current both display a saturating relationship which cannot be accounted for by the presence of unstirred layers. If the interaction of Na⁺ with the basolateral transport process is assumed to involve the interaction of some number of Na⁺ ions,n, with a maximal flux,M _max, then the data can be fit by assuming 3.2 equivalent sites for interaction and a value forM _max of 287.8pm cm^–2 sec^–1 with an intracellular Na concentration of 2.0mm Na⁺ at half-maximal saturation. By comparing these values with the ouabain-sensitive, Na⁺-induced current, we calculate a Na⁺ to K⁺ coupling ratio of 1.40±0.07 for the transport process.     

Effect of amiloride,ouabain and Ba++ on the nonsteady-state Na−K pump flux and short-circuit current in isolated frog skin epithelia

Summary Effect of amiloride, ouabain, and Ba⁺⁺ on the nonsteady-state Na–K pump flux and short-circuit current in isolated frog skin epithelia.The active Na⁺ transport across isolated frog skin occurs in two steps: passive diffusion across the apical membrane of the cells followed by an active extrusion from the cells via the Na⁺–K⁺ pump at the basolateral membrane. In isolated epithelia with a very small Na⁺ efflux, the appearing Na⁺-flux in the basolateral solution is equal to the rate of the pump, whereas the short-circuit current (SCC) is equal to the active transepithelial Na⁺ transport. It was found that blocking the passive diffusion of Na⁺ across the apical membrane (addition of amiloride) resulted in an instantaneous inhibition of the SCC (the transepithelial Na⁺ transport, whereas the appearing flux (the rate of the Na⁺–K⁺ pump) decreased with a halftime of 1.9 min. Addition of the Na⁺–K⁺ pump inhibitor ouabain (0.1mm) resulted in a faster and bigger inhibition of the appearing flux than of the SCC. Thus, by simultaneous measurement of the SCC and the appearing Na⁺ flux one can elucidate whether an inhibitor exerts its effect by inhibiting the pump or by decreasing the passive permeability. Addition of the K⁺ channel inhibitor Ba⁺⁺, in a concentration which gave maximum inhibition of the SCC, had no effect on the appearing flux (the rate of the Na–K pump) in the first 2 min, although the inhibition of the SCC was already at its maximum.It is argued that in the short period, where the Ba⁺⁺-induced inhibition of SCC is at its maximum and the appearing flux in unchanged, the decrease in the SCC (SCC) is equal to the net K⁺ flux via the Na⁺–K⁺ pump, and the coupling ratio () of the Na⁺–K⁺ pump can be calculated from the following equation =SCC _t=0/SCC where SCC _t=0 is the steady-state SCC before the addition of Ba⁺⁺.     

Control of sodium permeability of the outer barrier in toad skin

Summary The²⁴Na efflux (J _eff ^Na ) (i.e., the rate of appearance of²⁴Na in the outer compartment) in the isolated short-circuited toad skin bathed by NaCl-Ringer's solution on both sides is composed of para- and transcellular components of almost equal magnitudes. This relies on the assumption that amiloride acts on the transcellular component only and could block it completely.Ouabain induces a large transient increase of the transcellular component. This increase, which starts within a few minutes after the addition of ouabain, is due to electrical depolarization of the outer barrier, rather than a consequence of blocking Na recirculation across the inner barrier. The subsequent decline ofJ _eff ^Na , which takes place after the ouabain-inducedJ _eff ^Na peak, is due to a progressive block of outer barrier Na channels with time, which can eventually be complete, depending on the duration of action of ouabain. As the external Na concentration was always kept high and constant in these experiments, the results indicate that a rise in cell Na concentration, and not in the outer bathing solution, is the signal that triggers the reduction of outer barrier Na permeability (P ₀ ^Na ).Ouabain has no effect uponJ _eff ^Na with Na-free solution bathing the outer and NaCl-Ringer's solution the inner skin surface, showing the importance of Na penetration across the outer barrier, and not across the inner barrier due to its low Na permeability, in the process of closing the Na channels of this structure.Step changes from Na 115mm to Na-free external solution, or vice-versa, may affect both the outer barrier electrical potential difference (PD₀) and cell Na concentration (Na) _c . Therefore, the behavior ofJ _eff ^Na depends on which variable (if PD₀ or (Na) _c regulated outer barrier Na permeability) is most affected by step changes in outer bathing solution Na concentration.Amiloride in the control condition blocks the transcellular component ofJ _eff ^Na . However, in the condition of approximate short-circuiting of the outer barrier and high cellular Na concentration induced by long term effects of ouabain, when the Na channels of the outer barrier are already blocked by elevated cell Na concentration, amiloride may induce the opposite effect, increasing Na permeability of the outer barrier.With outer barrier Na channels completely blocked by high cell Na concentration, PCMB in the outer bathing medium induces a large increase ofJ _eff ^Na , rendering these channels again amiloride sensitive.The results are consistent with the notion that Na efflux from cell compartment to the outer bathing solution goes through the amiloride-sensitive Na channels of the apical border of the superficial cell layer of toad skin, with an apparent Na permeability modulated by cell ionic environment, most probably the cell Na concentration.The ensemble of the present results are consistent with Na permeability regulation taking place at the outer barrier level. However, this precise location could only be made unambiguously by measurements across the individual outer cell membranes.     

Lithium transport by fibroblastic mouse cells: Characterization and stimulation by serum and growth factors in quiescent cultures

Serum enhances the rate of Li⁺ entry and exit in quiescent cultures of mouse fibroblasts by 2- to 3-fold. Tertiary cultures of whole mouse embryos as well as established fibroblast lines (3T3, 3T6) show the increase in Li⁺ permeability when serum is added to cultures whose growth has been arrested by serum deprivation. Growing cells are only slightly more permeable to Li⁺ in the presence of serum. Purified compounds which initiate DNA synthesis also rapidly increase Li⁺ entry; mitogenic levels of thrombin and the combination of epidermal growth factor, insulin, and bovine serum albumin were the most effective ones tested. The effect of serum on Li⁺ uptake occurs within a few minutes, is not affected by inhibitors of macromolecular synthesis, and appears mainly to increase the Vmax of entry. Inhibitors of energy production partially reduce Li⁺ entry but do not block the activation by serum. One portion of Li⁺ uptake (?40%), which is inhibited by ouabain, phloretin, or Na⁺ deprivation, is mediated by the Na⁺/K⁺ pump in the plasma membrane. A second mechanism of Li⁺ entry which is blocked by Na⁺ or amiloride appears to be a Na⁺ specific “porter.” The activity of both components is stimulated by serum. The increased activity of the putative Na⁺ porter would increase Na⁺ availability to the Na⁺ pump and may account for its enhancement by serum, which was also noted previously (Rozengurt and Heppel, '75).     

Resolution of parameters in the equivalent electrical circuit of the sodium transport mechanism across toad skin

Summary In amphibian epithelia, amiloride reduces net sodium transport by hindering the entry of sodium to the active transport mechanism, that is, by increasing the series resistance (R _ser ). Theoretically, therefore, analysis of amiloride-induced changes in potential differences and short-circuit current should yield numerical estimates of all the parameters in the equivalent electrical circuit of the sodium transport mechanism.The concept has been explored by analysis of such changes in toad skins (Xenopus laevis) bathed in hypotonic sulphate Ringer's, after exposure to varying doses of amiloride, or to amphotericin, dinitrophenol or Pitressin.The estimated values ofR _ser , of the electromotive force of the sodium pump (E _Na), and of the shunt resistance (R _sh ) were independent of the dose of amiloride employed. Skins bathed in hypotonic sulphate Ringer's exhibited a progressive rise inE _Na. Amphotericin produced a fall inR _ser , while dinitrophenol caused a fall inE _Na; washout of the drugs reversed these effects. Pitressin produced a fall in bothR _ser andR _sh , with a rise inE _Na. These results are in accord with earlier suggestions regarding the site(s) of action of these agents.     

Effects of sodium-transport inhibitors on diuresis and mid-gut (Na+ + K+)-ATPase in the tsetse fly Glossina morsitans

The effects of four inhibitors of specific sodium-transport mechanisms on diuresis in the tsetse fly Glossina morsitans, have been determined. Ouabain (1.0, 0.1 mM) and ethacrynic acid (1.0, 0.2 mM) reduced the rate of water loss, whereas amiloride (1.0 mM) and furosemide (1.0 mM) did not. The effects of ouabain, ethacrynic acid and meal size upon the anterior mid-gut (Na⁺ + K⁺)-ATPase activity were also determined. For ouabain, the negative logarithm causing 50% inhibition of (Na⁺ + K⁺)-ATPase (pI₅₀) was 6.0, whilst ethacrynic acid together with meal size did not affect the activity of this enzyme. These results show that diuresis in this insect involves the active transport of sodium ions by both electrogenic and $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ exchange pumps.     

Coupled transepithelial sodium and potassium transport across isolated frog skin: Effect of ouabain,amiloride and the polyene antibiotic filipin

Summary Addition of the polyene antibiotic filipin (50 m) to the outside bathing solution (OBS) of the isolated frog skin resulted in a highly significant active outward transport of K⁺ because filipinper se increases the nonspecific Na⁺ and K⁺ permeability of the outward facing membrane. The K⁺ transport was calculated from the chemically determined changes in K⁺ concentrations in the solution bathing the two sides of the skin. The active transepithelial K⁺ transport required the presence of Na⁺ in the OBS, but not in the inside bathing solution (IBS), and it was inhibited by the Na⁺, K⁺-ATPase inhibitor ouabain. The addition of Ba⁺⁺ to the IBS in the presence of filipin in the OBS resulted in an activation of the transepithelial K⁺ transport and in an inhibition of the active Na⁺ transport. This is in agreement with the notion that Ba⁺⁺ decreases the passive K⁺ permeability of the inward facing membrane. In the presence of amiloride (which blocks the specific Na permeability of the outward facing membrane) and Ba⁺⁺ there was a good correlation between the active Na⁺ and K⁺ transport. It is concluded that the active transepithelial K⁺ transport is carried out by a coupled electrogenic Na–K pump, and it is suggested that the pump ratio (Na/K) is 1.5.     

Transport Pathways for Therapeutic Concentrations of Lithium in Rat Liver

Although both amiloride- and phloretin-sensitive Na⁺/Li⁺ exchange activities have been reported in mammalian red blood cells, it is still unclear whether or not the two are mediated by the same pathway. Also, little is known about the relative contribution of these transport mechanisms to the entry of therapeutic concentrations of Li⁺ (0.2–2 mm) into cells other than erythrocytes. Here, we describe characteristics of these transport systems in rat isolated hepatocytes in suspension. Uptake of Li⁺ by hepatocytes, preloaded with Na⁺ and incubated in the presence of ouabain and bumetanide, comprised three components. (a) An amiloride-sensitive component, with apparent K _m 1.2 mm Li⁺, V _max 40 μmol · (kg dry wt · min)⁻¹, showed increased activity at low intracellular pH. The relationship of this component to the concentration of intracellular H⁺ was curvilinear suggesting a modifier role of [H⁺] _i . This system persisted in Na⁺-depleted cells, although with apparent K _m 3.8 mm. (b) A phloretin-sensitive component, with K _m 1.2 mm, V _max 21 μmol · (kg · min)⁻¹, was unaffected by pH but was inactive in Na⁺-depleted cells. Phloretin inhibited Li⁺ uptake and Na⁺ efflux in parallel. (c) A residual uptake increased linearly with the external Li⁺ concentration and represented an increasing proportion of the total uptake. The results strongly suggest that the amiloride-sensitive and the phloretin-sensitive Li⁺ uptake in rat liver are mediated by two separate pathways which can be distinguished by their sensitivity to inhibitors and intracellular [H⁺]. Received: 8 April 1999/Revised: 19 July 1999     

Ion pathways in renal brush border membranes

The absorbance change of the weak base dye probe, Acridine orange, was used to monitor alterations of pH gradients across renal brush border membrane vesicles. The presence of Na⁺/H⁺ or Li⁺/H⁺ exchange was demonstrated by diluting Na₂SO₄ or Li₂SO₄ loaded vesicles into Na⁺- or Li⁺-free solutions, which caused dye uptake. About 20% of the uptake was abolished by lipid permeable cations such as valinomycin-K⁺ or tetraphenylphosphonium, indicating perhaps the presence of a finite Na⁺ conductance smaller than electroneutral Na⁺/H⁺ exchange. The protonophore tetrachlorosalicylanilide raised the rate of dye uptake under these conditions, hence the presence of an Na⁺ conductance greater than the H⁺ conductance was suggested. K⁺ gradients also induced changes of pH, at about 10% of the Na⁺ or Li⁺ rate. Partial inhibition (21%) was seen with 0.1 mM amiloride indicating that K⁺ was a low affinity substrate for the Na⁺/H⁺ exchange. Acceleration both by tetrachlorosalicylanilide (2-fold) and valinomycin (4-fold) suggested the presence of 2 classes of vesicles, those with high and those with low K⁺ conductance. The larger magnitude of the valinomycin dependent signal suggested that 75% of the vesicles had a low K⁺ conductance. Inward Cl^? gradients also induced acidification, partially inhibited by the presence of tetraphenylphosphonium, and accelerated by tetrachlorosalicylanilide. Thus both a Cl^? conductance greater than the H⁺ conductance and a Cl^?/OH^? exchange were present. The rate of Na⁺/H⁺ exchange was amiloride sensitive with a pH optimum of 6.5 and an apparent K_m for Na⁺ or Li⁺ of about 10 mM and an E_A of 14.3 kcal per mol. A 61-fold Na₂SO₄ gradient resulted in a pH gradient of 1.64 units which increased to 1.8 with gramicidin. An equivalent NaCl gradient gave a much lower ΔpH even in the presence of gramicidin showing that the H⁺ and Cl^? pathways could alter the effects of the Na⁺/H⁺ exchange.