The antiepileptic drug phenytoin affects sodium transport in toad epithelium

The effects of phenytoin on isolated Pleurodema thaul toad skin were investigated. Low (micromolar) concentrations of the antiepileptic agent applied to the outside surface of the toad epithelium increased the electrical parameters (short-circuit current and potential difference) by over 40%, reflecting stimulation of Na(+) transport, whereas higher (millimolar concentrations, outside and inside surface) decreased both electric parameters, the effect being greater at the inside surface (40% and 80% decrease, respectively). The amiloride test showed that the stimulatory effect was accompanied by an increase and the inhibitory effect by a decrease in the sodium electromotive force (ENa). It is concluded that the drug interaction with membrane lipid bilayers might result in a distortion of the lipid-protein interface contributing to disturbance of Na(+) epithelial channel activity. After applying the Na(+)-K(+)-ATPase blocker ouabain and replacing the Na(+) ions in the outer Ringer's solution by choline, it was concluded that both active and passive transport are involved in sodium absorption, although active transport predominates.     

Sodium arsenite affects Na+ transport in the isolated skin of the toad Pleurodema thaul

Arsenic, applied as sodium arsenite (As(III)) to either inner or outer surfaces of the isolated toad skin, dose-dependently decreased the short-circuit current (Isc), potential difference (PD) and sodium conductance (G(Na)) in the concentration range 1-1000 microM, with effects often lasting over 3 h. Maximal inhibitory effect was over 90% with an IC(50) of about 34 microM. Applied during amiloride block, As(III) did not change this effect. However, an increase in electric parameters was noted during the initial 30 min in 22 experiments, indicating a possible translocation of cytosolic protein kinase C (PKC) to the membrane within 15 min, thus stimulating sodium transport; this is followed by a progressive inhibition of kinase activity. Comparative effects of amiloride (8 microM), As(III) (100 microM, outer surface) and noradrenaline (NA, 10 microM, inner surface) showed a significant increase in the stimulatory effect of NA on the electric parameters, which could be the result of arsenite clustering of cell surface receptors and activation of ensuing cellular signal transduction pathways. Ouabain 5 microM, followed by As(III) 100 microM, also stimulated the skin response to NA (10 microM), although the duration of the two phases of the response was markedly shortened. The exact mechanism is still in doubt: however, As(III) increases cerebral metabolites of NA and ouabain can increase NA efflux from tissue slices. The amiloride test, performed with As(III) in the outer surface, confirmed significant decrease in all the parameters: the driving force (E(Na)), sodium conductance (G(Na)), and importantly, shunt conductance (G(sh)), due to the known fact that arsenic inhibits gap junctional intercellular communication.     

Effects of Pb<Superscript>2+</Superscript> ions on Na<Superscript>+</Superscript> transport in the isolated skin of the toad <Emphasis Type="Italic">Pleurodema thaul</Emphasis>

The effects induced by lead ions on the short-circuit current (SCC) and on the potential difference (V) of the toad Pleurodema thaul skin were investigated. Pb²⁺ applied to the outer (mucosal) surface increased SCC and V and when applied to the inner (serosal) surface decreased both parameters. The stimulatory effect, but not the inhibitory action, was reversible after washout of the metal ion. The amiloride test showed that the increase 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 also by a significant decrease in skin resistance indicating possible disruption of membrane and/or cell integrity. The effect of noradrenaline was increased by outer and decreased by inner administration of Pb²⁺. The results suggest that mucosal Pb²⁺ activates toad skin ion transport by stimulating the V-E_Na+ and that serosal Pb²⁺, with easier access to membrane and cellular constituents, inactivates this mechanism, revealing greater toxicity when applied to the inner surface of the skin. Abbreviations: SCC – short-circuit current; V – potential difference; V-E_Na+– driving potential for Na⁺; ENaC – epithelial sodium channel; R_Na+– active sodium resistance; RS – passive or shunt resistance; G_Na– active sodium conductance; GS – passive or shunt conductance; Gmax – total conductance; EC₅₀– half-maximal excitatory concentration; IC₅₀– half maximal inhibitory concentration; NA – noradrenaline.     

On the mechanism of the amiloride-sodium entry site interaction in anuran skin epithelia

The steady-state transport kinetics of the interaction between external sodium and the diuretic drug, amiloride, was studied in isolated anuran skin epithelia. We also investigated the effect of calcium on the amiloride-induced inhibition of short-circuit current (Isc) in these epithelial preparations. The major conclusions of this study are: (a) amiloride is a noncompetitive inhibitor of Na entry in bullfrog and grassfrog skin, but displays mixed inhibition in R. temporaria and the toad. A hypothesis which states that the interaction sites for amiloride and Na on the putative entry protein are spatially distinct in all of these species is proposed. (b) The stoichiometry of interaction between amiloride and the Na entry mechanism is not necessarily one-to-one. (c) The external Ca requirement for the inhibitory effect of amiloride is not absolute. Amiloride, at all concentrations, is equally effective in inhibiting Isc of bullfrog skin independently from the presence or absence of external Ca.     

Characterization of Na/Ca exchange in plasmalemmal vesicles from zona fasciculata cells of the bovine adrenal gland

The presence of an Na/Ca exchange system in fasciculata cells of the bovine adrenal gland was tested using isolated plasmalemmal vesicles. In the presence of an outwardly Na(+) gradient, Ca(2+) uptake was about 2-fold higher than in K(+) condition. Li(+) did not substitute for Na(+) and 5 mM Ni(2+) inhibited Ca(2+) uptake. Ca(2+) efflux from Ca(2+)-loaded vesicles was Na(+)-stimulated and Ni(2+)-inhibited. The saturable part of Na(+)-dependent Ca(2+) uptake displayed Michaelis-Menten kinetics. The relationship of Na(+)-dependent Ca(2+) uptake versus intravesicular Na(+) concentration was sigmoid (apparent K(0.5) approximately 24 mM; Hill number approximately 3) and Na(+) acted on V(max) without significant effect on K(m). Na(+)-stimulated Ca(2+) uptake was temperature-dependent (apparent Q(10) approximately 2.2). The inhibition properties of several divalent cations (Cd(2+), Sr(2+), Ni(2+), Ba(2+), Mn(2+), Mg(2+)) were tested and were similar to those observed in kidney basolateral membrane. The above results indicate the presence of an Na/Ca exchanger located on plasma membrane of zona fasciculata cells of bovine adrenal gland. This exchanger displays similarities with that of renal basolateral cell membrane.     

Sodium fluxes through nonselective cation channels in the plasma membrane of protoplasts from Arabidopsis roots

The aim of the present work was to characterize Na(+) currents through nonselective cation channels (NSCCs) in protoplasts derived from root cells of Arabidopsis. The procedure of the protoplast isolation was modified to increase the stability of Arabidopsis root protoplasts in low external Ca(2+) by digesting tissue in elevated Ca(2+). Experiments in whole-cell and outside-out modes were carried out. We found that Na(+) currents in Arabidopsis root protoplasts were mediated by cation channels that were insensitive to externally applied tetraethylammonium(+) and verapamil, had no time-dependent activation (permanently opened or completely activated within 1-2 ms), were voltage independent, and were weakly selective for monovalent cations. The selectivity sequence was as follows: K(+) (1.49) > NH(4)(+) (1.24) > Rb(+) (1.15) approximately equal to Cs(+) (1.10) approximately equal to Na(+) (1.00) > Li(+) (0.73) > tetraethylammonium(+) (0.47). Arabidopsis root NSCCs were blocked by H(+) (pK approximately equal to 6.0), Ca(2+) (K(1/2) approximately equal to 0.1 mM), Ba(2+), Zn(2+), La(3+), Gd(3+), quinine, and the His modifier diethylpyrocarbonate. They were insensitive to most organic blockers (nifedipine, verapamil, flufenamate, and amiloride) and to the SH-group modifier p-chloromercuriphenyl sulfonic acid. Voltage-insensitive, Ca(2+)-sensitive single channels were also resolved. Properties of Arabidopsis root NSCCs are discussed and compared with characteristics of similar conductances studied previously in plants and animals. It is suggested that NSCCs present a distinct group of plant ion channels, mediating toxic Na(+) influx to the cell and probably having other important roles in physiological processes of plants.     

Basolateral Mg2+/Na+ exchange regulates apical nonselective cation channel in sheep rumen epithelium via cytosolic Mg2+

High potassium diets lead to an inverse regulation of sodium and magnesium absorption in ruminants, suggesting some form of cross talk. Previous Ussing chamber experiments have demonstrated a divalent sensitive Na(+) conductance in the apical membrane of ruminal epithelium. Using patch-clamped ruminal epithelial cells, we could observe a divalent sensitive, nonselective cation conductance (NSCC) with K(+) permeability > Cs(+) permeability > Na(+) permeability. Conductance increased and rectification decreased when either Mg(2+) or both Ca(2+) and Mg(2+) were removed from the internal or external solution or both. The conductance could be blocked by Ba(2+), but not by tetraethylammonium (TEA). Subsequently, we studied this conductance measured as short-circuit current (I(sc)) in Ussing chambers. Forskolin, IBMX, and theophylline are known to block both I(sc) and Na transport across ruminal epithelium in the presence of divalent cations. When the NSCC was stimulated by removing mucosal calcium, an initial decrease in I(sc) was followed by a subsequent increase. The cAMP-mediated increase in I(sc) was reduced by low serosal Na(+) and serosal addition of imipramine or serosal amiloride and depended on the availability of mucosal magnesium. Luminal amiloride had no effect. Flux studies showed that low serosal Na(+) reduced (28)Mg fluxes from mucosal to serosal. The data suggest that cAMP stimulates basolateral Na(+)/Mg(2+) exchange, reducing cytosolic Mg. This increases sodium uptake through a magnesium-sensitive NSCC in the apical membrane. Likewise, the reduction in magnesium uptake that follows ingestion of high potassium fodder may facilitate sodium absorption, as observed in studies of ruminal osmoregulation. Possibly, grass tetany (hypomagnesemia) is a side effect of this useful mechanism.     

Changes in ionic conductances and in sensitivity to amiloride during the natural moulting cycle of toad skin (Bufo viridis,L.)

Summary The resistance of the apical membranes of toad skin (Bufo viridis) was measured during its natural moulting cycle using a fast flow technique. The skin behaved in all periods of the moulting cycle as a nearly perfect sodium electrode. In the presence of amiloride (10^–4 M), the total resistance of the same skin was identical with solutions which contained either sodium or potassium. The resistance of the skin with potassium was sensitive to amiloride in the period just after moulting. The resistance of skins which were made shunted by treating them with urea on the outside was insensitive to amiloride in solutions containing potassium; a small effect was still observed with sodium. It is suggested that the transient sensitivity to amiloride, with potassium, is the result of differentiation of the sodium specific sites at the apical membranes of the skin.     

Effect of sodium, lithium and amiloride on the K+-dependent phosphatase activity of membrane preparations of Na,K-ATPase

Effects of sodium, lithium and amiloride on the ATPase reaction and on its potassium-dependent step were studied using membrane preparations of Na,K-ATPase. It was established that the addition of 70 mM NaCl or LiCl to the reaction medium diminished the hydrolysis of para-nitrophenyl phosphate (pNPP) by 70 and 40%, respectively. Amiloride (0.8 mM) inhibited activities of Na,K-ATPase and pNPPase by 50 and 15%, respectively. The higher concentrations of amiloride produced a more prominent inhibition of Na,K-ATPase, but not of pNPPase. There was no correlation between the effect of amiloride on the pNPP hydrolysis and potassium concentration in the medium. There was the additivity in the inhibition of pNPPase by 0.8 mM amiloride and sodium or lithium ions up to the concentrations of ions as high as 30 mM. A conclusion is made that the inhibition of Na,K-ATPase by amiloride is mediated through the modification of the sensitivity of the enzyme to sodium.     

Mechanism of apical K+ channel modulation in principal renal tubule cells. Effect of inhibition of basolateral Na(+)-K(+)-ATPase

The effects of inhibition of the basolateral Na(+)-K(+)-ATPase (pump) on the apical low-conductance K+ channel of principal cells in rat cortical collecting duct (CCD) were studied with patch-clamp techniques. Inhibition of pump activity by removal of K+ from the bath solution or addition of strophanthidin reversibly reduced K+ channel activity in cell-attached patches to 36% of the control value. The effect of pump inhibition on K+ channel activity was dependent on the presence of extracellular Ca2+, since removal of Ca2+ in the bath solution abolished the inhibitory effect of 0 mM K+ bath. The intracellular [Ca2+] (measured with fura-2) was significantly increased, from 125 nM (control) to 335 nM (0 mM K+ bath) or 408 nM (0.2 mM strophanthidin), during inhibition of pump activity. In contrast, cell pH decreased only moderately, from 7.45 to 7.35. Raising intracellular Ca2+ by addition of 2 microM ionomycin mimicked the effect of pump inhibition on K+ channel activity. 0.1 mM amiloride also significantly reduced the inhibitory effect of the K+ removal. Because the apical low-conductance K channel in inside-out patches is not sensitive to Ca2+ (Wang, W., A. Schwab, and G. Giebisch, 1990. American Journal of Physiology. 259:F494-F502), it is suggested that the inhibitory effect of Ca2+ is mediated by a Ca(2+)-dependent signal transduction pathway. This view was supported in experiments in which application of 200 nM staurosporine, a potent inhibitor of Ca(2+)- dependent protein kinase C (PKC), markedly diminished the effect of the pump inhibition on channel activity. We conclude that a Ca(2+)- dependent protein kinase such as PKC plays a key role in the downregulation of apical low-conductance K+ channel activity during inhibition of the basolateral Na(+)-K(+)-ATPase.     

Effects of amiloride analogues on Na+ transport in toad bladder membrane vesicles. Evidence for two electrogenic transporters with different affinities toward pyrazinecarboxamides

Most of the electrical potential-driven 22Na+ uptake in toad bladder membrane vesicles can be blocked by the diuretic amiloride. Analysis of the amiloride inhibition curve indicates the presence of two pathways with low and high affinities to the diuretic (Garty, H. (1984) J. Membr. Biol. 82, 269-279). The selectivity of these pathways to amiloride was explored by comparing the inhibition curve of this diuretic with those of 10 of its structural analogues. The relative potencies of various amiloride-like compounds as blockers of the flux component with high affinity to amiloride were in good agreement with the structure-activity relationships elucidated from transepithelial short-circuit current measurements. Thus, this pathway is most probably the apical Na+-specific channel. The other pathway with lower affinity to the diuretic was relatively insensitive to modifications of the amiloride molecule, and the structure-activity relationships measured for the inhibition of this pathway were different from those reported for any other amiloride-blockable process. Other experiments have established that the Na+ flux with low affinity to amiloride is electrogenic and is not mediated by a Na+/H+ or Na+/Ca2+ exchanger, Na+-hexose cotransporter, or the Na+/K+-ATPase. The data indicate that tracer flux measurements in toad bladder membrane vesicles monitor, in addition to the well-characterized apical Na+ channels, another amiloride-blockable electrogenic Na+ transporter. This pathway could be responsible for the basolateral amiloride-blockable Na+ conductance recently observed in nystatin-treated bladders (Garty, H., Warncke, J., and Lindemann, B. (1987) J. Membr. Biol. 95, 91-103).     

Stimulation of Ca2+ uptake into epididymal bull spermatozoa by analogues of amiloride

Certain amiloride analogues 3',4'-dichlorobenzamil 2',4'-dimethylbenzamil and alpha',2'-benzobenzamil hydrochloride (ATBB) stimulate calcium accumulation and motility by epididymal bovine spermatozoa. This stimulation can be seen at a range of 0.1-0.4 mM, while at higher concentration there is inhibition of calcium uptake by these amiloride analogues. The amiloride derivative 5-(4-chlorobenzyl)-2',4'-dimethylbenzamil (CBDMB), which bears a 4-chlorobenzyl substituent on the 5-amino nitrogen atom, did not stimulate calcium uptake. The amiloride analogue 3',4'-dichlorobenzamil inhibits the Na+/Ca2(+)-exchange activity in isolated plasma membrane vesicles, and the stimulatory effect of 3',4'-dichlorobenzamil on calcium uptake into epididymal sperm could be seen in Na(+)-free medium. Thus, the stimulation of Ca2+ accumulation in the cells caused by 3',4'-dichlorobenzamil is not a result of inhibiting the Na(+)-dependent Ca2+ clearance. There is no stimulation of Ca2+ uptake into ejaculated cells by adding 3',4'-dichlorobenzamil, which is not due to the presence of the calcium-transport inhibitor (caltrin) in these cells [Rufo, G.A., Schoff, P.K. & Lardy, H.A. (1984) J. Biol. Chem. 259, 2547-2552]. The stimulatory effect of 3',4'-dichlorobenzamil on Ca2+ uptake is inhibited by the voltage-dependent Ca2(+)-channel blockers nifedipin and diltiazem. This indicates that the stimulation of Ca2+ uptake by the amiloride analogues is due to the activation of a voltage-dependent Ca2+ channel of the plasma membrane.     

Effects of basolateral ouabain, amphotericin B, cyanide and potassium on amiloride noise during voltage clamp of Rana pipiens skin support sodium-amiloride competition

In a previous study, the amiloride-induced corner frequency (fc) was found to decrease as apical sodium was increased. This effect was small or absent when the basolateral surface was exposed to high potassium. It has been suggested that the apical sodium effect may be indirect, due either to increased intracellular [Na+] which repelled amiloride or to an increased potential at the apical surface which reduced amiloride affinity. High basolateral K+ might then suppress the sodium effect either by preventing intracellular [Na+] from increasing or by allowing a better clamp of the apical membrane potential by reducing basolateral membrane resistance and potential. We checked the effects of basolateral [K+], of cyanide and of ouabain at concentrations known to increase intracellular [Na+]. We found only negligible effects on fc. In addition, amphotericin B added to the basolateral bathing solution either in 115 mM Na+ or in 120 mM K+ had no significant effect on fc. We found that relatively wide variation in clamp potential under all conditions, even with active transport severely inhibited, left fc virtually constant. Since the amiloride kinetics were independent of clamp potential, we were able to measure paracellular and transcellular conductances separately by examining the voltage dependence of clamp current (linear) and amiloride noise power (quadratic). This made possible estimation of channel density and single-channel current.     

Sodium-dependent copper uptake across epithelia: a review of rationale with experimental evidence from gill and intestine

The paper reviews the evidence for apparent sodium-dependent copper (Cu) uptake across epithelia such as frog skin, fish gills and vertebrate intestine. Potential interactions between Na(+) and Cu during transfer through epithelial cells is rationalized into the major steps of solute transfer: (i) adsorption on to the apical/mucosal membrane, (ii) import in to the cell (iii) intracellular trafficking, and (iv) export from the cell to the blood. Interactions between Na(+) and Cu transport are most likely during steps (i) and (ii). These ions have similar mobilities (lambda) in solution (lambda, Na(+), 50.1; Cu(2+), 53.6 cm(2) Int. ohms(-1) equiv(-1)); consequently, Cu(2+) may compete equally with Na(+) for diffusion to membrane surfaces. We present new data on the Na(+) binding characteristics of the gill surface (gill microenvironment) of rainbow trout. The binding characteristics of Na(+) and Cu(2+) to the external surface of trout gills are similar with saturation of ligands at nanomolar concentrations of solutes. At the mucosal/apical membrane of several epithelia (fish gills, frog skin, vertebrate intestine), there is evidence for both a Cu-specific channel (CTR1 homologues) and Cu leak through epithelial Na(+) channels (ENaC). Cu(2+) slows the amiloride-sensitive short circuit current (I(sc)) in frog skin, suggesting Cu(2+) binding to the amiloride-binding site of ENaC. We present examples of data from the isolated perfused catfish intestine showing that Cu uptake across the whole intestine was reduced by 50% in the presence of 2 mM luminal amiloride, with 75% of the overall inhibition attributed to an amiloride-sensitive region in the middle intestine. Removal of luminal Na(+) produced more variable results, but also reduced Cu uptake in catfish intestine. These data together support Cu(2+) modulation of ENaC, but not competitive entry of Cu(2+) through ENaC. However, in situations where external Na(+) is only a few millimoles (fish gills, frogs in freshwater), Cu(2+) leak through ENaC is possible. CTR1 is a likely route of Cu(2+) entry when external Na(+) is higher (e.g. intestinal epithelia). Interactions between Na(+) and Cu ions during intracellular trafficking or export from the cell are unlikely. However, effects of intracellular chloride on the Cu-ATPase or ENaC indicate that Na(+) might indirectly alter Cu flux. Conversely, Cu ions inhibit basolateral Na(+)K(+)-ATPase and may increase [Na(+)](i).     

Changes in cell membrane fluidity affect the sodium transport across frog skin and its sensitivity to amiloride

1. 1-5 mM n-hexanol added to the outer (mucosal) medium of isolated skin of the frog Rana temporaria increases the short circuit current (Isc) across it. 2. This effect shows a saturable dependency on the outer sodium concentration, also when NaCl is replaced by Na2SO4. 3. n-Hexanol at a concentration of 1 mM, and cold acclimation of the frogs, which increases the fluidity of epidermal cell membranes, do not affect the sensitivity of Isc to the inhibiting effect of amiloride. 4. n-Hexanol at a concentration (5 mM) which causes a fluidization of cell membrane preparations from isolated frog epidermis also increases the sensitivity of Isc to amiloride. 5. The effects of low concentrations of n-hexanol and of cold acclimation probably depend on an increase of the permeability of apical membranes of epidermal cells to sodium caused by membrane fluidization. At higher concentrations of n-hexanol, a further disordering of the membrane structure occurs with a better access of amiloride to its action sites.     

Evidence for an apical membrane effect in the regulation of the hexose monophosphate shunt pathway in toad bladder studies with amiloride

Aldosterone stimulates Na⁺ transport in toad bladder and, simultaneously with a coincident dose-response relationship, inhibits the hexose monophosphate shunt pathway. Amiloride, an acylguanidine diuretic, inhibits sodium transport when applied to the apical surface of the bladder. In this study amiloride was found to partially reverse the inhibitory effect of aldosterone on the hexose monophosphate shunt pathway. The amiloride effect upon glucose metabolism was detected when it was applied to both surfaces of the bladder simultaneously, in flask experiments, and when it was applied to the apical surface. No effect of amiloride on the shunt pathway was detected when it was applied to the serosal surface only, even at very high concentrations. It may be, but has not been proven, that the effects of aldosterone and amiloride on the hexose monophosphate shunt pathway are mediated by a common site at the apical membrane.     

Localization of the Na+/K+-ATPase and of an amiloride sensitive Na+ uptake on thyroid epithelial cells

The Na+/K+-ATPase was localized using purified specific antibodies, on the basolateral membranes of rat thyroid epithelial cells and of cultured porcine thyroid cells, by immunofluorescence and immunoelectron microscopy. No staining was observed on the apical membranes. When cultured cells formed monolayers, with their apical pole in contact with the culture medium, 22Na+ uptake was inhibited by amiloride. Inhibition was dependent upon extracellular Na+ concentration, half maximal inhibition was obtained with 0.7 microM amiloride in the presence of 5 mM Na+. Ouabain was ineffective on Na+ uptake into intact monolayers. A brief treatment of the monolayers with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) opened the tight junctions and allowed the access of ouabain to the basal pole of the cells. In this condition ouabain increased Na+ uptake. When cells were reorganized into follicle-like structures, with their basal pole in contact with the culture medium, Na+ uptake was not modified by amiloride but was increased by ouabain. We conclude that in thyroid cells, the Na+/K+-ATPase is present on the basolateral domain of the plasma membrane whereas an amiloride sensitive sodium uptake occurs at the apical surface.     

Aldosterone induction of electrogenic sodium transport in the apical membrane vesicles of rat distal colon

Na-H exchange is present in apical membrane vesicles (AMV) isolated from distal colon of normal rats. Because in intact tissue aldosterone both induces amiloride-sensitive electrogenic sodium transport and inhibits electroneutral sodium absorption, these studies with AMV were designed to establish the effect of aldosterone on sodium transport. An outward-directed proton gradient stimulated 22Na uptake in AMV isolated from distal colon of normal and dietary sodium depleted (with elevated aldosterone levels) experimental rats. Unlike normal AMV, proton gradient-dependent 22Na uptake in experimental AMV was inhibited when uptake was measured under voltage-clamped conditions. 10 microM amiloride inhibited the initial rate of proton gradient-dependent 22Na uptake in AMV of normal and experimental rats by 30 and 75%, respectively. In contrast, 1 mM amiloride produced comparable inhibition (90 and 80%) of 22Na uptake in normal and experimental AMV. Intravesicular-negative potential stimulated 22Na uptake in experimental but not in normal AMV. This increase was inhibited by 90% by 10 microM amiloride. An analogue of amiloride, 5-(N-ethylisopropyl) amiloride (1 microM), a potent inhibitor of electroneutral Na-H exchange in AMV of normal rat distal colon, did not alter potassium diffusion potential-dependent 22Na uptake. Increasing sodium concentration saturated proton gradient-dependent 22Na uptake in normal AMV. However, in experimental AMV, 22Na uptake stimulated by both proton gradient and potassium diffusion potential did not saturate as a function of increasing sodium concentration. We conclude from these results that an electrically sensitive conductive channel, not electroneutral Na-H exchange, mediates 22Na uptake in AMV isolated from the distal colon of aldosterone rats.