Effect of amiloride on the apical cell membrane cation channels of a sodium-absorbing,potassium-secreting renal epithelium

Summary The effect of the K-sparing diuretic amiloride was assessed electrophysiologically in the isolated cortical collecting tubule of the rabbit, a segment which absorbs Na and secretes K. Low concentrations of amiloride in the perfusate caused a rapid, reversible, decrease in the magnitude of the lumen negative transepithelial potential difference,V _te, transepithelial conductanceG _te, and equivalent short-circuit current,I _sc, with an apparentK _1/2 of approximately 7×10^–8 m. The effects of a maximum inhibitory concentration of amiloride (10^–5 m) were identical to those observed upon Na removal from lumen and bath (Na removal from the bath alone has no effect). Removal of Na in the presence of 10^–5 m amiloride had no affect onV _te,G _te, orI _sc, and is consistent with the view that amiloride blocks the Na conductive pathways of the apical cell membrane. Further, in the absence of Na, the subsequent addition of amiloride had no influence. In tubules where active Na absorption was either spontaneously low, or abolished by removal of Na from lumen and bath, the elevation of K from 5 to 155 meq/liter in the perfusate caused a marked change of theV _te in the negative direction and an increase in theG _te. These effects could be attributed to a high K permeability of the apical cell membrane and not of the tight junctions. Amiloride (10^–5 m) had no effect on these responses to K. It is concluded that amiloride selectively blocks the apical cell membrane Na channels but has no effect on the K conductive pathways(s). This selective nature of amiloride may indicate that Na and K are transported across the apical cell membrane via separate conductive pathways.     

Kinetics of the effect of amiloride on the permeability of the apical membrane of rabbit descending colon to sodium

Summary The effects of the addition of graded concentrations of amiloride, (A) _m , to the mucosal bathing solution on the permeability of the apical membrane of rabbit descending colon to Na (P _Na ^m ) were determined when the Na activity in the mucosal bathing solution, (Na) _m , was 18, 32 or 100mm.P _Na ^m was obtained from current-voltage relations determined on tissues bathed with a high-K serosal solution before and after the addition of a maximally inhibitory concentration of amiloride to the mucosal solution as described by Turnheim et al. (Turnheim, K., Thompson, S.M., Schultz. S.G. 1983.J. Membrane Biol. 76:299–309).The results indicate that: (1) As demonstrated previously (Turnheim et al., 1983),P _Na ^m decreases with increasing (Na) _m . (2)P _Na ^m also decreases hyperbolically with increasing (A) _m . Kinetic analyses of the effect of amiloride onP _Na ^m are consistent with the conclusions that: (i) the stoichiometry between the interaction of amiloride with apical membrane receptors that results in a decrease inP _Na ^m is one-for-one; (ii) there is no evidence for cooperativity between amiloride and these binding sites; (iii) the value of (A) _m needed to halveP _Na ^m at a fixed (Na) _m is 0.6–1.0 m; and, (iv) this value is independent of (Na) _m over the fivefold range studied.These findings are consistent with the notion that the sites with which amiloride interacts to bring about closure of the channels through which Na crosses the apical membrane arekinetically distinct from the sites with which (Na) _m interacts to bring about closure (i.e., self-inhibition). In short, the effects of (Na) _m and (A) _m onP _Na ^m in this tissue appear to be independent and additive.     

Apical membrane K conductance in the toad urinary bladder

Summary The conductance of the apical membrane of the toad urinary bladder was studied under voltage-clamp conditions at hyperpolarizing potentials (mucosa negative to serosa). The serosal medium contained high KCl concentrations to reduce the voltage and electrical resistance across the basal-lateral membrane, and the mucosal solution was Na free, or contained amiloride, to eliminate the conductance of the apical Na channels. As the mucosal potential (V _m) was made more negative the slope conductance of the epithelium increased, reaching a maximum at conductance of the epithelium increased, reaching a maximum atV _m=–100 mV. This rectifying conductance activated with a time constant of 2 msec whenV _m was changed abruptly from 0 to –100 mV, and remained elevated for at least 10 min, although some decrease of current was observed. ReturningV _m to+100 mV deactivated the conductance within 1 msec. Ion substitution experiments showed that the rectified current was carried mostly by cations moving from cell to mucosa. Measurement of K flux showed that the current could be accounted for by net movement of K across the apical membrane, implying a voltage-dependent conductance to K (G _K). Mucosal addition of the K channel blockers TEA and Cs had no effect onG _K, while 29mm Ba diminished it slightly. Mucosal Mg (29mm) also reducedG _K, while Ca (29mm) stimulated it.G _K was blocked by lowering the mucosal pH with an apparent pK₁ of 4.5. Quinidine (0.5mm in the serosal bath) reducedG _K by 80%.G _K was stimulated by ADH (20 mU/ml), 8-Br-cAMP (1mm), carbachol (100 m), aldosterone (5×10^–7 m for 18 hr), intracellular Li and extracellular CO₂.     

Current-voltage analysis of apical sodium transport in toad urinary bladder: Effects of inhibitors of transport and metabolism

Summary The basal-lateral surface of the epithelium of the urinary bladder of the toad (Bufo marinus) was depolarized by exposure of the serosal surface to 85mm KCL and 50mm sucrose. The extent of depolarization appeared to be virtually complete, as evaluated by the invariance in the transepithelial electrical potential difference and conductance on addition of nystatin (a monovalent cation ionophore) to the serosal medium. The Na-specific current (I _Na) was defined as the current sensitive to the removal of Na from the mucosal medium or inhibitable by addition of amiloride to this medium. In the presence of the high K-sucrose serosal medium, rapid, serial, stepwise clamping of the transepithelial voltage (V) yielded a curvilinear dependence ofI _Na onV; which is taken to represent theI–V curve of the apical Na channels. The constant field equation (Goldman, D.E. 1943;J. Gen. Physiol. 27:37) fits theI–V data points closely, allowing estimates to be made of the permeability to Na of the apical membrane (P _Na) and of the intracellular Na activity (Na _c ). Exposure of the apical surface to amiloride (5×10^–7 m) decreasedP _Na in proportion to the decrease inI _Na (i.e., 70%) but decreased Na _c only 25%. In contrast, an equivalent lent reduction inI _Na elicited by exposure of the basallateral surface to ouabain was accompanied by only a 20% decrease inP _Na and a sixfold increase in Na _c . The effects of amiloride onP _Na and ouabain on Na _c are consistent with the primary pharmacological actions of these drugs. In addition,P _Na appears to be under metabolic control, in that 2-deoxyglucose, a specific inhibitor of glycolysis, decreasedI _Na andP _Na proportionately, and lowered Na _c marginally, effects indistinguishable from those obtained with amiloride.     

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.     

Flow-Dependent Activation of Maxi K+ Channels in Apical Membrane of Rabbit Connecting Tubule

The Ca²⁺-activated maxi K⁺ channel was found in the apical membrane of everted rabbit connecting tubule (CNT) with a patch-clamp technique. The mean number of open channels (NP _o ) was markedly increased from 0.007 ± 0.004 to 0.189 ± 0.039 (n= 7) by stretching the patch membrane in a cell-attached configuration. This activation was suggested to be coupled with the stretch-activation of Ca²⁺-permeable cation channels, because the maxi K⁺ channel was not stretch-activated in both the cell-attached configuration using Ca²⁺-free pipette and in the inside-out one in the presence of 10 mm EGTA in the cytoplasmic side. The maxi K⁺ channel was completely blocked by extracellular 1 μm charybdotoxin (CTX), but was not by cytoplasmic 33 μm arachidonic acid (AA). On the other hand, the low-conductance K⁺ channel, which was also found in the same membrane, was completely inhibited by 11 μm AA, but not by 1 μm CTX. The apical K⁺ conductance in the CNT was estimated by the deflection of transepithelial voltage (ΔV _t ) when luminal K⁺ concentration was increased from 5 to 15 mEq. When the tubule was perfused with hydraulic pressure of 0.5 KPa, the ΔV _t was only −0.7 ± 0.4 mV. However, an increase in luminal fluid flow by increasing perfusion pressure to 1.5 KPa markedly enhanced ΔV _t to −9.4 ± 0.9 mV. Luminal application of 1 μm CTX reduced the ΔV _t to −1.3 ± 0.6 mV significantly in 6 tubules, whereas no significant change of ΔV _t was recorded by applying 33 μm AA into the lumen of 5 tubules (ΔV _t =−7.2 ± 0.5 mV in control vs.ΔV _t =−6.7 ± 0.6 mV in AA). These results suggest that the Ca²⁺-activated maxi K⁺ channel is responsible for flow-dependent K⁺ secretion by coupling with the stretch-activated Ca²⁺-permeable cation channel in the rabbit CNT. Received: 21 August 1997/Revised: 20 March 1998     

Effects of bafilomycin A1 and amiloride on the apical potassium and proton gradients in Drosophila Malpighian tubules studied by X-ray microanalysis and microelectrode measurements

The intracellular distribution of potassium in Malpighian tubules from Drosophila larva was measured by electron probe X-ray microanalysis of freeze-dried cryosections. Application of amiloride alone to the haemolymph space had no effect on the intracellular potassium concentration in the region of intermediate cytoplasm (between the basal region of basal membrane infoldings and the apical brush border), whereas a potassium increase as well as a chloride increase was observed after simultaneous blocking of the potassium conductance of the basal membrane with barium. Injected bafilomycin and amiloride applied in the haemolymph caused an increase of the potassium content in the basal cytoplasm but not in the microvilli. In addition, the intracellular water portion was decreased by bafilomycin. pH measurements in isolated larval anterior tubules with proton-selective microelectrodes showed that bafilomycin added to the bathing solution caused a decrease in intracellular pH. Addition of amiloride had no significant effect on intracellular pH, but the pH of the luminal fluid was decreased within 1 min by 0.5 pH units. The amiloride-induced luminal pH decrease could be inhibited by the metabolic blocker KCN as well as by bafilomycin. Furthermore, removing potassium from the bathing saline caused a slow luminal acidification, which could be blocked by KCN. Our results support the hypothesis of a functionally coupled transport system in the apical membrane consisting of a bafilomycin-sensitive V-ATPase and a K⁺-dependent, amiloride-sensitive K⁺/H⁺ exchange system.Abbreviation C _a element concentration related to water - C _d element content related to dry weight - dw dry weight - DMSO dimethylsulphoxide - emf electromotive force - NBD-Cl 7-chloro-4-nitrobenz-2-oxa-1,3-diazole - NEM N-ethylmaleimide - NMDG⁺ N-methyl-d-glucamine - PD potential difference - pH_i intracellular pH value - pH_lu luminal pH value - pmf protonmotive force - SD standard deviation - SE standard error - STEM scanning transmission electron microscopy - V _a apical potential difference - V _b basal potential difference - V _t transepithelial potential difference     

Relationships among sodium current,permeability, and Na activities in control and glucocorticoid-stimulated rabbit descending colon

Summary Effects of a potent synthetic glucocorticoid, methylprednisolone (MP), on transepithelial Na transport were examined in rabbit descending colon. Current-voltage (I–V) relations of the amiloride-sensitive apical Na entry pathway were measured in colonic tissues of control and MP-treated (40 mg im for 2 days) animals. Tissues were bathed mucosally by solutions of various Na activities, (Na)_m, ranging from 6.2 to 75.6mm, and serosally by a high K solution. TheseI–V relations conformed to the constant field flux equation permitting determination of the permeability of the apical membrane to Na,P _Na ^m , and the intracellular Na activity, (Na)_c. The following empirical relations were observed for both control and MP-treated tissues: (i) Na transport increases hyperbolically with increasing (Na)_m obeying simple Michaelis-Mentin kinetics; (ii)P _Na ^m decreased hyperbolically with increasing (Na)_m, but was unrelated to individual variations in (Na)_c; (iii) (Na)_c increased hyperbolically with (Na)_m; (iv) both spontaneous and steroid-stimulated variations in Na entry rate could be attributed entirely to parallel variations inP _Na ^m at each mucosal Na activity. Comparison of these empirical, kinetic relations between control and MP-treated tissues revealed: (i) maximal Na current andP _Na ^m were greater in MP tissues, but the (Na)_m's at which current andP _Na ^m were half-maximal were markedly reduced; (ii) (Na)_c was significantly increased in MP tissues at each (Na)_m while the (Na)_m at half-maximal (Na)_c was unchanged. These results provide direct evidence that glucocorticoids cause marked stimulation of Na absorption across rabbit colon primarily by increasing the Na permeability of the apical membrane. While the mechanism for the increased permeability remains to be determined, the altered relation betweenP _Na ^m and (Na)_m suggests possible differences in the conformation or environment of the Na channel in MP-treated tissues.     

Electrogenic H<Superscript>+</Superscript> Transport and pH Gradients Generated by a V-H<Superscript>+</Superscript>-ATPase in the Isolated Perfused Larval <Emphasis Type="Italic">Drosophila</Emphasis> Midgut

A method for microperfusion of isolated segments of the midgut epithelium of Drosophila larvae has been developed to characterize cellular transport pathways and membrane transporters. Stereological ultrastructural morphometry shows that this epithelium has unusually long tight junctions, with little or no lateral intercellular volume normally found in most epithelia. Amplification of the apical and basal aspects of the cells, by ≈ 17-fold and ≈ 7-fold, respectively, predicts an almost exclusively transcellular transport system for solutes. This correlates with the high lumen-negative transepithelial potential (V_t) of 38 to 45 mV and high resistance (R_t) of 800 to 1400 Ω • cm² measured by terminated cable analysis, in contrast to other microperfused epithelia like the renal proximal tubule. Several blockers (amiloride 10⁻⁴ M, ouabain 10⁻⁴ M, bumetanide 10⁻⁴ M), K⁺-free solutions, or organic solutes such as D-glucose 10 mM or DL-alanine 0.5 mM failed to affect V_t or R_t. Bafilomycin-A₁ (3 to 5 μM) decreased V_t by ≈ 40% and short-circuit current (I_sc) by ≈ 50%, and decreased intracellular pH when applied from the basal side only, consistent with an inhibition of an electrogenic V-H⁺-ATPase located in the basal membrane. Gradients of H⁺ were detected by pH microelectrodes close to the basal aspect of the cells or within the basal extracellular labyrinth. The apical membrane is more conductive than the basal membrane, facilitating secretion of base (presumably HCO₃⁻), driven by the basal V-H⁺-ATPase.     

Role of Cl− in Electrogenic H+ Secretion by Cortical Distal Tubule

The presence of an electrogenic H⁺-ATPase has been described in the late distal tubule, a segment which contains intercalated cells. The present paper studies the electrogenicity of this transport mechanism, which has been demonstrated in turtle bladder and in cortical collecting duct. Transepithelial PD (V _t ) was measured by means of Ling-Gerard microelectrodes in late distal tubule of rat renal cortex during in vivo microperfusion. The tubules were perfused with electrolyte solutions to which 2 × 10⁻⁷ m bafilomycin or 4.6 × 10⁻⁸ m concanamycin were added. No significant increase in lumen-negative V _t upon perfusion with these inhibitors as compared to control, was observed as well as when 10⁻³ m amiloride, 10⁻⁵ m benzamil or 3 mm Ba²⁺ were perfused alone or in combination. The effect of an inhibition of electrogenic H⁺ secretion, i.e., increase in lumen-negative V _t by 2–4 mV, was observed only when Cl⁻ channels were blocked by 10⁻⁵ m 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). This blocker also reduced the rate of bicarbonate reabsorption in this segment from 1.21 ± 0.14 (n= 8) to 0.62 ± 0.03 (8) nmol.cm⁻².sec⁻¹ as determined by stationary microperfusion and pH measurement by ion-exchange resin microelectrodes. These results indicate that: (i) the participation of the vacuolar H⁺ ATPase in the establishment of cortical late distal tubule V _t is minor in physiological conditions, but can be demonstrated after blocking Cl⁻ channels, thus suggesting a shunting effect of this anion; and, (ii) the rate of H⁺ secretion in this segment is reduced by a Cl⁻ channel blocker, supporting coupling of H⁺-ATPase with Cl⁻ transport. Received: 6 July 1996/Revised: 27 December 1996     

Cation selectivity of the resting membrane of squid axon

Summary Permeability constant ratios among monovalent cations were studied in the resting membrane of a giant axon of a Pacific squid,Loligo opalescens, by observing the relationship between the membrane potential and the ion concentration.The average permeability ratios are: Tl, 1.8; K, 1.0; Rb, 0.72; Cs, 0.16; Na, <0.08; Li, <0.08. These permeability ratios suggest that neither valinomycin nor nonactin are adequate models for the sites producing the resting permeability in the axonal membrane.Cyclic polyetherbis(t-butyl cyclohexyl) 18-crown-6 does not increase the permeability ratioP _Cs/P _K except when applied at concentrations (5×10^–5 m) at which the surfactant properties of this molecule may become significant.     

Calcium-mobilizing agonists stimulate anion fluxes in cultured endothelial cells from human umbilical vein

Intracellular ion concentrations were determined in split skins of Rana pipiens using the technique of electron microprobe analysis. Based on the 1 min Br uptake from the apical bath, two types of mitochondria-rich (MR) cells could be distinguished: active cells which rapidly exchanged their anions with the apical bath and inactive cells which did not. Br uptake and frequency of active MR cells were closely correlated with the skin conductance, g _t. Replacing Cl in the apical bath with an impermeant anion significantly lowered g _t and the Br uptake and Na concentration of active cells. Even larger reductions were observed after apical amiloride (0.1 mm). The inhibition of the Br uptake was reversible by voltage clamping (100 mV, inside positive). Cl removal and amiloride also led to some shrinkage of active cells. The results suggest that the active cell is responsible for a large part of g _t. Inactive MR cells had much lower Br and Na concentrations which were not significantly affected by Cl removal, amiloride, or voltage clamping. Principal cells, which represent the main cell type of the epithelium, showed only a minimal Br uptake from the apical side which was not correlated with g _t. Moreover, Cl removal had no effect on the Na, Br, and Cl concentrations of principal cells.I wish to thank Cathy Langford for her excellent technical assistance. Financial support was provided by National Institutes of Health grants DK35717 and 1S10-RR0-234501.     

Nonselective Cation and BK Channels in Apical Membrane of Outer Sulcus Epithelial Cells

The outer sulcus epithelium was recently shown to absorb cations from the lumen of the gerbil cochlea. Patch clamp recordings of excised apical membrane were made to investigate ion channels that participate in this reabsorptive flux. Three types of channel were observed: (i) a nonselective cation (NSC) channel, (ii) a BK (large conductance, maxi K or K _Ca ) channel and (iii) a small K⁺ channel which could not be fully characterized. The NSC channel found in excised insideout patch recordings displayed a linear current-voltage (I-V) relationship (27 pS) and was equally conductive for Na⁺ and K⁺, but not permeable to Cl⁻ or N-methyl-d-glucamine. Channel activity required the presence of Ca²⁺ at the cytosolic face, but was detected at Ca²⁺ concentrations as low as 10⁻⁷ m (open probability (P _o ) = 0.11 ± 0.03, n= 8). Gadolinium decreased P _o of the NSC channel from both the external and cytosolic side (IC₅₀∼ 0.6 μm). NSC currents were decreased by amiloride (10 μm− 1 mm) and flufenamic acid (0.1 mm). The BK channel was also frequently (38%) observed in excised patches. In symmetrical 150 mm KCl conditions, the I-V relationship was linear with a conductance of 268 pS. The Goldman-Hodgkin-Katz equation for current carried solely by K⁺ could be fitted to the I-V relationship in asymmetrical K⁺ and Na⁺ solutions. The channel was impermeable to Cl⁻ and N-methyl-d-glucamine. P _o of the BK channel increased with depolarization of the membrane potential and with increasing cytosolic Ca²⁺. TEA (20 mm), charybdotoxin (100 nm) and Ba²⁺ (1 mm) but not amiloride (1 mm) reduced P _o from the extracellular side. In contrast, external flufenamic acid (100 μm) increased P _o and this effect was inhibited by charybdotoxin (100 nm). Flufenamic acid inhibited the inward short-circuit current measured by the vibrating probe and caused a transient outward current. We conclude that the NSC channel is Ca²⁺ activated, voltage-insensitive and involved in both constitutive K⁺ and Na⁺ reabsorption from endolymph while the BK channel might participate in the K⁺ pathway under stimulated conditions that produce an elevated intracellular Ca²⁺ or depolarized membrane potential. Received: 14 October 1999/Revised: 10 December 1999     

Electrical effects of potassium and bicarbonate on proximal tubule cells ofNecturus

Summary The effects of stepwise concentration changes of K⁺ and HCO ₃ ^– in the basolateral solution on the basolateral membrane potential (V _bl) of proximal tubule cells of the doubly-perfusedNecturus kidney were examined using conventional microelectrodes. Apparent transference numbers were calculated from changes inV _bl after alterations in external K⁺ concentration from 1.0 to 2.5mm (t _{K, 1.0–2.5}), 2.5 to 10, and in external HCO ₃ ^– concentration (at constant pH) from 5 to 10mm (t _{HCO3, 5–10}), 10 to 20, or 10 to 50.t _{K, 2.5–10} was 0.38±0.02 under control conditions but was sharply reduced to 0.08±0.03 (P>0.001) by 4mm Ba⁺⁺. This concentration of Ba⁺⁺ reducedV _bl by 9±1 mV (at 2.5 external K⁺). Perfusion with SITS (5×10^–4 m) for 1 hr hyperpolarizedV _bl by 10±3 mV and increasedt _{K, 2.5–10} significantly to 0.52±0.01 (P<0.001). Ba⁺⁺ application in the presence of SITS depolarizedV _bl by 22±3 mV. In control conditionst _{HCO3, 10–50} was 0.63±0.05 and was increased to 0.89±0.07 (P<0.01) by Ba⁺⁺ but was decreased to 0.14±0.02 (P<0.001) by SITS. In the absence of apical and basolateral chloride, the response ofV _bl to bicarbonate was diminished but still present (t _{HCO3, 10–20} was 0.35±0.03). Intracellular pH, measured with liquid ion-exchange microelectrodes, increased from 7.42±0.19 to 7.57±0.17 (P<0.02) when basolateral bicarbonate was increased from 10 to 20mm at constant pH. These data show that the effects of bicarbonate onV _bl are largely independent of effects on the K⁺ conductance and that there is a significant current-carrying bicarbonate pathway in the basolateral membrane. Hence, both K⁺ and HCO ₃ ^– gradients are important in the generation ofV _bl, and their relative effects vary reciprocally.     

Sodium flux in the apical membrane of the toad skin: Aspects of its regulation and the importance of the ionic strength of the outer solution upon the reversibility of amiloride inhibition

Summary Injection of small pulses of concentrate solutions of salts or drugs into the outer bathing fluid led to sudden increases of its solute concentration. Vigorous stirring of the outer bathing solution was used to minimize the thickness of the unstirred layer adjacent to the outer skin surface. Pulses of 1m NaCl injected into the outer compartment induced sharp increases of the SCC following a time course variable with the magnitude of the pulse and the particular condition of each skin. Comparison of the spontaneous decline of the SCC with the decline induced by a small dose of amiloride, where an increase inR was observed, indicates that the spontaneous decline cannot be explained simply as a reduction of the Na permeability of the apical membrane by self-inhibition of feedback inhibition of the apical membrane Na channels. Reduction of the driving force for Na movement into the epithelial cells must play an important role in the process. Reversibility of the amiloride inhibition of the SCC was highly dependent upon the ionic strength of the solution used to rinse and wash out the inhibitor from the outer skin surface. With H₂O, the amiloride molecules washed out slowly as compared to NaCl or KCl solutions. Na or K have the same ability to dislodge the amiloride molecules from their binding sites. This effect is apparently of a purely electrostatic nature.     

Electrogenic 2 Na+/1 H+ exchange in crustanceans

Summary Hepatopancreatic brush border membrane vesicles of the freshwater prawn,Macrobrachium rosenbergii and the marine lobster,Homarus americanus exhibited²²Na uptake which was Cl-independent, amiloride sensitive, and stimulated by a transmembrane H gradient (H _i >H _o ). Sodium influx by vesicles of both species were sigmoidal functions of [Na] _o , yielding Hill coefficients that were not significantly different (P>0.5) than 2.0. Estimations of half-saturation constants (K _Na) were 82.2mm (prawn) and 280.1mm (lobster), suggesting a possible adaptation of this transporter to environmental salinity.Trans-stimulation andcis-inhibition experiments involving variable [H] suggested that the exchangers in both species possessed single internal cation binding sites (pK 6.5–6.7) and two external cation binding sites (prawn, pK 4.0 and 5.7; lobster pK 3.5 and 6.1). Similarcis inhibition studies using amiloride as a competitive inhibitor of Na uptake supported the occurrence of dual external sites (prawn,K _i 50 and 1520 m; lobsterK _i 9 and 340 m). Electrogenic Na/H exchange by vesicles from both crustaceans was demonstrated using equilibrium shift experiments where a transmembrane potential was used as the only driving force for the transport event. Transport stoichiometries of the antiporters were determined using Static Head analysis where driving forces for cation transfer were balanced using a 101 Na gradient, a 1001 H gradient, and a stoichiometry of 2.0. These electrogenic 2 Na/1 H exchangers appear thermodynamically capable of generating sufficient gastric acidification for organismic digestive activities.     

Redistribution of hepatocyte chloride during l-alanine uptake

We used ion-sensitive, double-barrel microelectrodes to measure changes in hepatocyte transmembrane potential (V _m), intracellular K⁺, Cl^-, and Na⁺ activities (a ⁱ _k, a _Cl ⁱ and a _Na ⁱ ), and water volume during l-alanine uptake. Mouse liver slices were superfused with control and experimental Krebs physiological salt solutions. The experimental solution contained 20 m l-alanine, and the control solution was adjusted to the same osmolality (305 mOsm) with added sucrose. Hepatocytes also were loaded with 50 mm tetramethylammonium ion (TMA⁺) for 10 min. Changes in cell water volume during l-alanine uptake were determined by changes in intracellular, steady-state TMA⁺ activity measured with the K⁺ electrode. Hepatocyte control V _m was -33±1 mV. l-alanine uptake first depolarized V _m by 2±0.2 mV and then hyperpolarized V _m by 5 mV to-38±1 mV (n = 16) over 6 to 13 min. During this hyperpolarization, a _Na ⁱ increased by 30% from 19±2 to 25±3 mm (P < 0.01), and a _K ⁱ did not change significantly from 83±3 mm. However, with added ouabain (1 mm) l-alanine caused only a 2-mV increase in V _m, but now a _K ⁱ decreased from 61±3 to 54±5 mm (P < 0.05). Hyperpolarization of V _m by l-alanine uptake also resulted in a 38% decrease of a _Cl ⁱ from 20±2 to 12±3 mm (P < 0.001). Changes in V _m and V _Cl — V _m voltage traces were parallel during the time of l-alanine hyperpolarization, which is consistent with passive distribution of intracellular Cl^– with the V _m in hepatocytes. Added Ba²⁺ abolished the l-alanineinduced hyperpolarization, and a _Cl ⁱ remained unchanged. Hepatocyte water volume during l-alanine uptake increased by 12±3%. This swelling did not account for any changes in ion activities following l-alanine uptake. We conclude that hepatocyte a _K ⁱ is regulated by increased Na⁺-K⁺ pump activity during l-alanine uptake in spite of cell swelling and increased V _m due to increased K⁺ conductance. The hyperpolarization of V _m during l-alanine uptake provides electromotive force to decrease a _Cl ⁱ . The latter may contribute to hepatocyte volume regulation during organic solute transport.This work was supported by grant AA-08867 from the Alcohol, Drug Abuse, and Mental Health Association.     

Basolateral membrane potential and conductance in frog skin exposed to high serosal potassium

Summary In studies of apical membrane current-voltage relationships, in order to avoid laborious intracellular microelectrode techniques, tight epithelia are commonly exposed to high serosal K concentrations. This approach depends on the assumptions that high serosal K reduces the basolateral membrane resistance and potential to insignificantly low levels, so that transepithelial values can be attributed to the apical membrane. We have here examined the validity of these assumptions in frog skins (Rana pipiens pipiens). The skins were equilibrated in NaCl Ringer's solutions, with transepithelial voltageV _t clamped (except for brief perturbations V _t) at zero. The skins were impaled from the outer surface with 1.5m KCl-filled microelectrodes (R _el>30 M). The transepithelial (short-circuit) currentl _i and conductanceg _t=–I _t/V _t, the outer membrane voltageV _o (apical reference) and voltage-divider ratio (F _o=V _o/V _t), and the microelectrode resistanceR _el were recorded continuously. Intermittent brief apical exposure to 20 m amiloride permitted estimation of cellular (c) and paracellular (p) currents and conductances. The basolateral (inner) membrane conductance was estimated by two independent means: either from values ofg _i andF _o before and after amiloride or as the ratio of changes (–I _c/V _i) induced by amiloride. On serosal substitution of Na by K, within about 10 min,I _c declined andg _t increased markedly, mainly as a consequence of increase ing _p. The basolateral membrane voltage (V _i(=–V _o) was depolarized from 75±4 to 2±1 mV [mean±sem (n=6)], and was partially repolarized following amiloride to 5±2 mV. The basolateral conductance increased in high serosal K, as estimated by both methods. Essentially complete depolarization of the basolateral membrane and increase in its conductance in response to high [K] were obtained also when the main serosal anion was SO₄ or NO₃ instead of Cl. On clampingV _t over the range 0 to +125 mV in K₂SO₄-depolarized skins, the quasi-steady-stateV _o V _t relationship was linear, with a mean slope of 0.88±0.03. The above results demonstrate that, in a variety of conditions, exposure to high serosal K results in essentially complete depolarization of the basolateral membrane and a large increase in its conductance.     

Microelectrode studies of the effect of lanthanum on the electrical potential and resistance of outer and inner cell membranes of isolated frog skin

Summary Microelectrodes were used to investigate the effect of 0.5mm mucosal lanthanum (La³⁺) on the intracellular potential and the resistance of outer and inner isolated frog skin (Rana esculenta) cell membranes. Under short-circuit conditions, the transapical membrane potentialV _o ^sc (mean value=–65.4±3.2 mV, inside negative) hyperpolarized to –108.7±2.3 mV in control skins, after addition of the sodium blocker amiloride. Current-voltage curves for the outer and inner membranes were constructed from the amiloride-inhibitable current versus the outer membrane potentialV _o or the inner membrane potentialV _t . The outer, and to a lesser degree the inner, membrane showed a characteristic nonlinearity with two slope resistances. Addition of La³⁺ to the outer medium increased the short-circuit current to 190% of the control value.V _o ^sc concomitantly changed to –28±3.5 mV and outer and inner membrane resistances fell, considerably attenuating the nonlinearity seen in control skins. La³⁺ is suggested to raise the conductance by its effect on the surface potential. A secondary long-term inhibitory effect of La³⁺ on short-circuit current has been observed. It is ascribed to the penetration of La³⁺ into the sodium channels.