Modulation of active potassium transport by aldosterone

• 1.1. The role of aldosterone on active potassium transport across lizard colon under voltage-clamped conditions has been investigated.
• 2.2. Control colons exhibited no net potassium flux (J^k_net) despite of the existence of active opposite unidi ectional fluxes.
• 3.3. An important net secretory potassium flux was found in short-circuited aldosterone-stimulated colons.
• 4.4. Mucosal amiloride did not change (J^k_net) either in control or aldosterone-stimulated colons.
• 5.5. Luminal barium alters K ⁺ transport in a manner consistent with the presence of barium-sensitive conductances at the apical membrane of both control and aldosterone-treated colons.
• 6.6. The effects of ouabain and barium on control and aldosterone-induced potassium flows were consistent with a model involving basolateral uptake by an Na ⁺-K ⁺-ATPase and conductive exit across the apical membrane.
• 7.7. The stimulatory effect of aldosterone on potassium secretion is associated with parallel increases of both basolateral K ⁺ entry and the apical conductive pathway.

     

KCl transport across and insect epithelium: I. Tracer fluxes and the effects of ion substitutions

Summary Models for active Cl transport across epithelia are often assumed to be universal although they are based on detailed studies of a relatively small number of epithelia from vertebrate animals. Epithelial Cl transport is also important in many invertebrates, but little is known regarding its cellular mechanisms. We used short-circuit current, tracer fluxes and ion substitutions to investigate the basic properties of Cl absorption by locust hindgut, an epithelium which is ideally suited for transport studies. Serosal addition of 1mm adenosine 35-cyclic monophosphate (cAMP), a known stimulant of Cl transport in this tissue, increased short-circuit current (I _sc) and net reabsorptive³⁶Cl flux (J _net ^Cl ) by 1000%. Cl absorption did not exhibit an exchange diffusion component and was highly selective over all anions tested except Br. Several predictions of Na- and HCO₃-coupled models for Cl transport were tested: Cl-dependentI _sc was not affected by sodium removal (<0.05mm) during the first 75 min. Also, a large stimulation ofJ _net ^Cl was elicited by cAMP when recta were bathed for 6 hr in nominally Na-free saline (<0.001 to 0.2mm) and there was no correlation between Cl transport rate and the presence of micromolar quantities of Na contamination. Increased unidirectional influx of³⁶Cl into rectal tissue during cAMP-stimulation was not accompanied by a comparable uptake of²²Na.J _net ^Cl was independent of exogenous CO₂ and HCO₃, but was strongly dependent on the presence of K. These results suggest that the major fraction of Cl transport across this insect epithelium occurs by an unusual K-dependent mechanism that does not directly require Na or HCO₃.     

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

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

Volume regulatory activity of the Ehrlich ascites tumor cell and its relationship to ion transport

Summary The volume regulatory response of the Ehrlich ascites tumor was studied in KCl-depleted, Na⁺-enriched cells. Subsequent incubation in K⁺-containing NaCl medium results in the reaccumulation of K⁺, Cl^–, water and the extrusion of Na⁺. The establishment of the physiological steady state is due primarily to the activity of 2 transport systems. One is the Na/K pump (K _M for K ₀ ⁺ =3.5mm;J _max=30.1 mEq/kg dry min), which in these experiments was coupled 1K⁺/1 Na⁺. The second is the Cl^–-dependent (Na⁺+K⁺) cotransport system (K _M for K ₀ ⁺ =6.8mm;J _max=20.8 mEq/kg dry min) which mediates, in addition to net ion uptake in the ratio of 1K⁺1Na⁺2Cl^–, the exchange of K _i ⁺ for K ₀ ⁺ . The net passive driving force on the cotransport system is initially inwardly directed but does not decrease to zero at the steady state. This raises the possibility of the involvement of an additional source of energy. Although cell volume increases concomitant with net ion uptake, this change does not appear to be a major factor regulating the activity of the cotransport system.     

Rabit distal colon epithelium: II. Characterization of (Na+, K+, Cl−)-cotransport and [3H]-bumetanide binding

Summary Loop diuretic-sensitive (Na⁺,K⁺,Cl^–)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of grandients of all three ions was essential for optimal transport activity (Na⁺,K⁺) gradien-driven³⁶Cl fluxes weree half-maximally inhibited by 0.14 m bumetanide and 44 m furosimide. While⁸⁶Rb uptake rates showed hyperbolic dependencies on Na⁺ and K⁺ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl^– concentration, Hill coefficient 1.8, indicating a 1 Na⁺: 1 K⁺:2 Cl^– stoichiometry of ion transport.The interaction of putative (Na⁺, K⁺, Cl^–)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [³H]-bumetanide. The requirement for the simulataneous presence of Na⁺,K⁺, and Cl^–, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na⁺, K⁺, Cl^–)-cotransporter. [³H]-bumetanide recognizes a minimum of two classes of diuretic receptors sites. high-affinity (K _D1=0.13 m;B _max1 =6.4 pmol/mg of protein) and low-affinity (K _D2=34 m;B _max2=153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl^– (K ₁=60mm), whereas low-affinity sites seem to be unaffected by Cl^–. We have shown that only high-affinity [³H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.     

Short-chain fatty acids and CO2 as regulators of Na+ and Cl− absorption in isolated sheep rumen mucosa

Summary Unidirectional ²²Na⁺ and ³⁶Cl^– fluxes were determined in short-circuited, stripped rumen mucosa from sheep by using the Ussing chamber technique. In both CO₂/HCO _– ³ -containing and CO₂/HCO _– ³ -free solutions, replacement of gluconate by short-chain fatty acids (SCFA, 39 mM) significantly enhanced mucosal-toserosal Na⁺ absorption without affecting the Cl^– transport in the same direction. Short-chain fatty acid stimulation of Na⁺ transport was at least partly independent of Cl^– and could almost completely be abolished by 1 mM mucosal amiloride, while stimulation of Na⁺ transport was enhanced by lowering the mucosal pH from 7.3 to 6.5. Similar to the SCFA action, raising the PCO₂ in the mucosal bathing solution led to an increase in the amiloride-sensitive mucosal-to-serosal Na⁺ flux. Along with its effect on sodium transport, raising the PCO₂ also stimulated chloride transport. The results are best explained by a model in which undissociated SCFA and/or CO₂ permeate the cell membrane and produce a raise in intracellular H⁺ concentration. This stimulates an apical Na⁺/H⁺ exchange, leading to increased Na⁺ transport. The stimulatory effect of CO₂ on Cl^– transport is probably mediated by a Cl^–/HCO _– ³ exchange mechanism in the apical membrane. Binding of SCFA anions to that exchange as described for the rat distal colon (Binder and Mehta 1989) probably does not play a major role in the rumen.Abbreviations DIDS 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid - G _t transepithelial conductance (mS·cm^-2) - HSCFA undissociated short-chain fatty acids - J _ms mucosal-to-serosal flux (Eq · cm^-2 · h^-1) - J _net net flux (Eq · cm^-2 · h^-1) - J _sm serosal-to-mucosal flux (Eq · cm^-2 · h^-1) - PD transepithelial potential difference (mV) - SCFA dissociated short-chain fatty acids - SCFA short-chain fatty acids     

Evaluation of ion gradient-dependent H+ transport systems in isolated enterocytes from the chick

Summary The experiments reported here evaluate the capability of isolated intestinal epithelial cells to accomplish net H⁺ transport in response to imposed ion gradients. In most cases, the membrane potential was kept constant by means of a K⁺ plus valinomycin voltage clamp in order to prevent electrical coupling of ion fluxes. Net H⁺ flux across the cellular membrane was examined at pH 6.0 (the physiological lumenal pH) and at pH 7.4 using methylamine distribution or recordings of changes in media pH. Results from both techniques suggest that the cells have an Na⁺/H⁺ exchange system in the plasma membrane that is capable of rapid and sustained changes in intracellular pH in response to an imposed Na⁺ gradient. The kinetics of the Na⁺/H⁺ exchange reaction at pH 6.0 [K _t for Na⁺=57mm,V _max=42 mmol H⁺/liter 3OMG (3-O-methylglucose) space/min] are dramatically different from those at pH 7.4 (K _t for Na⁺=15mm,V _max=1.7 mmol H⁺/liter 3OMG space/min). Experiments involving imposed K⁺ gradients suggest that these cells have negligible K⁺/H⁺ exchange capability. They exhibit limited but measurable H⁺ conductance. Anion exchange for base equivalents was not detected in experiments performed in media nominally free of bicarbonate.     

Analysis of presteady-state Na+ fluxes across the rabbit corneal endothelium

Summary Instead of the conventional steady-state fluxes, the presteady-state fluxes of²²Na across the rabbit corneal endothelium were measured. In contrast to reports that there is no net Na⁺ movement across the corneal endothelium, we find a net transport of Na⁺ across this tissue. The direction of net Na⁺ flux is from the stromal to the aqueous side and the magnitude is 2.3±0.4 eq/cm²·hr (n=11,sem). Net Na⁺ transport is inhibited in the presence of ouabain (10^–4 m). Acetazolamide (10^–4 m) has only a slight inhibitory effect on the rate of Na⁺ transport but decreases the transendothelial potential difference by about 30%. The passive component of the Na⁺ transport has been estimated by analyzing the presteady-state influx and efflux curves and found to occur 10% via cellular and 90% via paracellular routes. The analysis for the separation of the pathways has been based on a recently proposed theory which holds that the flux ratio, regardless of its driving forces, is independent of time.     

Activation of Apical K<Superscript>+</Superscript> Conductances by Muscarinic Receptor Stimulation in Rat Distal Colon: Fast and Slow Components

In the epithelium of rat distal colon the acetylcholine analogue carbachol induces a transient increase of short-circuit current (I_sc) via stimulation of cellular K⁺ conductances. Inhibition of the turnover of inositol-1,4,5-trisphosphate (IP₃) by LiCl significantly reduced both the amplitude and the duration of this response. When the apical membrane was permeabilized with nystatin, LiCl nearly abolished the carbachol-induced activation of basolateral K⁺ conductances. In contrast, in epithelia, in which the basolateral membrane was bypassed by a basolateral depolarization, carbachol induced a biphasic increase in the K⁺ current across the apical membrane consisting of an early component carried by charybdotoxin- and tetraethylammonium-sensitive K⁺ channels followed by a sustained plateau carried by channels insensitive against these blockers. Only the latter was sensitive against LiCl or inhibition of protein kinases. In contrast, the stimulation of the early apical K⁺ conductance by carbachol proved to be resistant against inhibition of phospholipase C or protein kinases. However, apical dichlorobenzamil, an inhibitor of Na⁺/Ca²⁺ exchangers, or a Ca²⁺-free mucosal buffer solution significantly reduced the early component of the carbachol-induced apical K⁺ current. The presence of an apically localized Na⁺/Ca²⁺-exchanger was proven immunohistochemically. Taken together these experiments reveal divergent regulatory mechanisms for the stimulation of apical Ca²⁺-dependent K⁺ channels in this secretory epithelium, part of them being activated by an inflow of Ca²⁺ across the apical membrane.

Ion transport by rabbit colon: II. Unidirectional sodium influx and the effects of amphotericin B and amiloride

Summary The unidirectional influx of Na from the mucosal solution into the epithelium ofin vitro descending rabbit colon (J _me ^Na ) determined under short-circuit conditions, is comprised of two components: one represents entry of Na into transporting epithelial cells and is abolished by amiloride which also abolishes Na absorption (J _net ^Na ). The other represents diffusional Na entry into paracellular pathways traversing the epithelium. In all instances, exposure of the mucosal surface to amphotericin B increased tissue conductance andJ _me ^Na and elicited K secretion. Tissues showing a spontaneousI _sc of approximately 4 eq/cm²hr did not respond to amphotericin B with increasedI _sc andJ _net ^Na . However, in tissues characterized by a lowerI _sc under control conditions, amphotericin B increasedI _sc andJ _net ^Na to approximately 4eq/cm²hr. These findings suggest that amphotericin increasesJ _net ^Na and elicits K secretion by disrupting the normal permselectivity of the mucosal membrane. Under these conditions the extrusion of Na from cell-to-serosal solution becomes the rate limiting step in transepithelial Na transport. Finally, a close correlation betweenJ _me ^Na andJ _net ^Na was observed when the rate of Na absorption varied either spontaneously or experimentally with amiloride, suggesting that the backflux of Na from cell-to-mucosal solution is undetectably small.     

Two-substrate kinetic analysis: a novel approach linking ion and acid-base transport at the gills of freshwater trout,Oncorhynchus mykiss

Summary The novel application of a two-substrate model (Florini and Vestling 1957) from enzymology to transport kinetics at the gills of freshwater trout indicated that Na⁺/acidic equivalent and Cl^-/basic equivalent flux rates are normally limited by the availability of the internal acidic and basic counterions, as well as by external Na⁺ and Cl^- levels. Adult rainbow trout fitted with dorsal aortic and bladder catheters were chronically infused (10–16 h) with isosmotic HCl to induce a persistent metabolic acidosis. Acid-base neutral infusions of isosmotic NaCl and non-infused controls were also performed. Results were compared to previous data on metabolic alkalosis in trout induced by either isosmotic NaHCO₃ infusion or recovery from environmental hyperoxia (Goss and Wood 1990a, b). Metabolic acidosis resulted in a marked stimulation of Na⁺ influx, no change in Cl^- influx, positive Na⁺ balance, negative Cl^- balance, and net H⁺ excretion at the gills. Metabolic alkalosis caused a marked inhibition of Na⁺ influx and stimulation of Cl^- influx, negative Na⁺ balance, positive Cl^- balance, and net H⁺ uptake (=base excretion). Mean gill intracellular pH qualitatively followed extracellular pH. Classical one-substrate Michaelis-Menten analysis of kinetic data indicated that changes in Na⁺ and Cl^- transport during acid-base disturbance are achieved by large increases and decreases in J_max, and by increases in K_m. However, one-substrate analysis considers only external substrate concentration and cannot account for transport limitations by the internal substrate. The kinetic data were fitted successfully to a two-substrate model, using extracellular acid-base data as a measure of internal HCO ₃ ^- and H⁺ availability. This analysis indicated that true J_max values for Na⁺/acidic equivalent and Cl^-/basic equivalent transport are 4–5 times higher than apparent J_max values by one-substrate analysis. Flux rates are limited by the availability of the internal counterions; transport K_m values for HCO ₃ ^- and H⁺ are far above their normal internal concentrations. Therefore, small changes in acid-base status will have large effects on transport rates, and on apparent J_max values, without alterations in the number of transport sites. This system provides an automatic, negative feedback control for clearance or retention of acidic/basic equivalents when acid-base status is changing.Abbreviations Amm total ammonia in water - DMO 55-dimethyl-24-oxyzolidine-dione - J_in unidirectional inward ion movement across the gill - J_out unidirectional outward ion movement across the gill - J_net net transfer of ions (sum of J_in and J_out) across the gill - J_max maximal transport rate for ion - K_m inverse of affinity of transporter for ion - PIO₂ partial pressure of oxygen in inspired water - P_aCO₂ partial pressure of carbon dixide in arterial blood - TAlk titratable alkalinity of the water - PEG polyethylene glycol - NEN New England Nuclear     

Evidence for two K+ currents activated upon hyperpolarization ofParamecium tetraurelia

Summary Hyperpolarization of voltage-clampedParamecium tetraurelia in K⁺ solutions elicits a complex of Ca²⁺ and K⁺ currents. The tail current that accompanies a return to holding potential (–40 mV) contains two K⁺ components. The tail current elicited by a step to –110 mV of 50-msec duration contains fast-decaying (3.5 msec) and slow-decaying (20 msec) components. The reversal potential of both components shifts by 55–57 mV/10-fold change in external [K⁺], suggesting that they represent pure K⁺ currents. The dependence of the relative amplitudes of the two tail currents on duration of hyperpolarization suggests that the slow K⁺ current activates slowly and is sustained, whereas the fast current activates rapidly during hyperpolarization and then rapidly inactivates. Iontophoretic injection of a Ca²⁺ chelator, EGTA, specifically reduces slow tail-current amplitude without affecting the fast tail component. Both K⁺ currents are inhibited by extracellular TEA⁺ in a concentration-dependent, noncooperative manner, whereas the fast K⁺ current alone is inhibited by 0.7mm quinidine.     

Aldosterone regulation of active sodium chloride transport in the lizard colon (Gallotia galloti)

Summary Bioelectrical parameters and unidirectional sodium and chloride fluxes were measured under voltageclamp conditions in groups of lizards submitted to single or chronic aldosterone treatment. Both acute (AT) and chronic (CT) treatment induced significant increases in the short-circuit current (I _sc), as well as in the mucosa-to-serosa (J _m-s ^Na ) and net sodium flux (J _net ^Na ). In AT tissues, aldosterone did not change net chloride flux (J _net ^Cl ) but did so in CT tissues. Amiloride reduced the aldosterone-increased I _sc in AT and CT tissues, inhibited J _net ^Na in AT tissues and abolished it in CT colons. J _net ^Cl was also reduced by the diuretic in the group of AT colons, whereas no changes were observed in the CT tissues. Addition of luminal DIDS reduced Na⁺ absorption and totally inhibited Cl^- absorption in the AT tissues, but did not change I _sc. However, in CT tissues neither Na⁺ nor Cl^- transport were affected by DIDS. A good relationship between I _sc and J _m-s ^Na was apparent after DIDS treatment in AT tissues. In this group, simultaneous addition of DIDS and amiloride totally abolished J _net ^Na and reduced I _sc to untreated control values. Addition of serosal ouabain abolished I _sc and Na⁺ absorption in AT and CT colons, but Cl^- absorption was only altered in AT tissues. These results support the hypothesis that aldosterone induces an electrogenic, amiloride-sensitive sodium absorption, and in a dose-dependent fashion suppresses electroneutral NaCl absorption in the lizard colon.Abbreviations AT acutely treated - CT chronically treated animals - DIDS 4-4-diisothiocyanatostibene-2-2-disulfonic acid - DMSO dimethylsulphoxide - G _t tissue conductance - I _sc short circuit current - PD transepithelial potential difference - SITS 4-acetamido-4-isothiocyanatostilbene-2-2-disulfonic acid - UC untreated controls Preliminary results of this paper were presented at the X ^th meeting of the European Intestinal Transport Group (EITG), Askov Hojskole, Denmark, 16–19 September 1990     

The effects of amiloride on electrolyte transport and acid-base balance in the larval salamander,Ambystoma tigrinum

Summary The responses of net and unidirectional fluxes of Na⁺ and acid-base balance to the drug amiloride were assessed during normocapnia and hypercapnia in larval salamanders, Ambystoma tigrinum. Isotope flux measurements demonstrated that 10^-4 M amiloride in the external medium inhibits Na⁺ influx during normocapnia and reverses the usual increase in influx of this ion during hypercapnia, causing a significant decrease instead. Measurements of blood-gas/acid-base balance conditions of artcrially cannulated salamanders demonstrated a significant metabolic acidosis in amiloridetreated animals that did not occur in untreated animals over the same period. the same concentration of amiloride also blocked the normal compensatory increase in [HCO _- ³ ] that follows a respiratory acidosis produced by a hypercapnic environment.Abbreviations IU international nnits - J _in influx - J _net net flux - PCO ₂ parial pressure of carbon dioxide     

Resistance properties of the diluting segment ofAmphiuma kidney: Influence of potassium adaptation

Summary Chronic exposure to high potassium stimulates K⁺-secretory mechanisms in the diluting segment of the amphibian kidney (K⁺ adaptation). Since K⁺ net flux depends critically on the passive cell membrane permeabilities for K⁺ ions, cable analysis and K⁺-concentration step changes were applied in this nephron segment to assess the individual resistances of the epithelium and the K⁺ conductance of the luminal cell membrane. Experiments were performed in the isolated, doubly-perfused kidney of both control and K⁺-adaptedAmphiuma. In control animals transepithelial resistance was 290±27 cm², which decreased significantly to 199±17 cm² after K⁺ adaptation. The resistance in parallel of the luminal and peritubular cell membrane decreased from a control value of 157±14 to 108±6 cm² after chronic K⁺ treatment. This was paralleled by a decrease of the ratio of the luminal to peritubular cell membrane resistance from 2.5±0.1 to 1.9±0.1, respectively. Estimation of the individual cell membrane resistances reveals that the combined resistance of the luminal and peritubular cell membrane is in the same order of magnitude as the paracellular shunt resistance in diluting segments of both control and K⁺-adapted animals. The luminal cell membrane is K⁺ selective under both conditions, but the absolute luminal K⁺ conductance increases by some 60% with K⁺ adaptation. This leads to an increased back-leak of K⁺ from cell to lumen and may explain stimulated K⁺ net secretion found after chronic K⁺ loading.     

Flux ratio of valinomycin-mediated K+ fluxes across the human red cell membrane in the presence of the protonophore CCCP

Summary The ratio of valinomycin-mediated unidirectional K⁺ fluxes across the human red cell membrane, has been determined in the presence of the protonophore carbonylcyanidem-chlorophenylhydrazone, CCCP, using the K⁺ net efflux and⁴²K influx. The driving force for the net efflux (V _m –E _K ⁺) has been calculated from the membrane potential, estimated by the CCCP-mediated proton distribution and the Nernst potential for potassium ions across the membrane. An apparent driving potential for the K⁺ net efflux has been calculated from the K⁺ flux ratio, determined in experiments where the valinomycin and CCCP concentrations were varied systematically. This apparent driving force, in conjunction with the actual driving force calculated on basis of the CCCP estimated membrane potential, is used to calculate a flux ratio exponent, which represents an estimate of the deviation of valinomycin-mediated K⁺ transport from unrestricted electrodiffusion, when protonophore is present.In the present work, the flux ratio exponent is found to be 0.90 when the CCCP concentration is 5.0 m and above, while the exponent decreases to about 0.50 when no CCCP is present. The influence of CCCP upon the rate constants in the valinomycin transport cycle is discussed. The significance of this result is that red cell membrane potentials are overestimated, when calculated from valinomycin-mediated potassium isotope fluxes, using a constant field equation.     

ATP is required for K+ active transport in the archaebacterium Haloferax volcanii

The Archaebacterium Haloferax volcanii concentrates K⁺ up to 3.6 M. This creates a very large K⁺ ion gradient of between 500- to 1,000-fold across the cell membrane. H. volcanii cells can be partially depleted of their internal K⁺ but the residual K⁺ concentration cannot be lowered below 1.5 M. In these conditions, the cells retain the ability to take up potassium from the medium and to restore a high internal K⁺ concentration (3 to 3.2 M) via an energy dependent, active transport mechanism with a K _m of between 1 to 2 mM. The driving force for K⁺ transport has been explored. Internal K⁺ concentration is not in equilibrium with m suggesting that K⁺ transport cannot be accounted for by a passive uniport process. A requirement for ATP has been found. Indeed, the depletion of the ATP pool by arsenate or the inhibition of ATP synthesis by N,N-dicyclohexylcarbodiimide inhibits by 100% K⁺ transport even though membrane potential m is maintained under these conditions. By contrast, the necessity of a m for K⁺ accumulation has not yet been clearly demonstrated. K⁺ transport in H. volcanii can be compared with K⁺ transport via the Trk system in Escherichia coli.Abbreviations CCCP Carbonylcyanide m-chlorophenyl-hydrazone - DCCD N,N-dicyclohexylcarbodiimide - MES 2-[N-morpholino] ethane sulfonic acid - MOPS 3-[N-morpholino] propane sulfonic acid - TRIS Tris (hydroxymethyl) aminomethane - TPP tetraphenyl phosphonium     

Intracellular potassium activity in mammalian proximal tubule: Effect of perturbations in transepithelial sodium transport

Summary Intracellular potassium activity (a _{K i} ) was measured in control conditions in mid-cortical rabbit proximal convoluted tubule using two methods: (i) by determination of the K⁺ equilibrium potential (E _K) using Ba²⁺-induced variations in the basolateral membrane potential (V _BL) during transepithelial current injections and (ii) with double-barrel K-selective microelectrodes. Using the first method, the meanV _BL was –48.5±3.2 mV (n=16) and the meanE _K was –78.4±4.1 mV corresponding to aa _{K i} of 68.7mm. With K-selective microelectrodes,V _BL was –36.6±1.1 mV (n=19),E _K was –64.0±1.1 mV anda _{K i} averaged 40.6±1.7mm. While these lastE _K andV _BL values are significantly lower than the corresponding values obtained with the first method (P<0.001 andP<0.01, respectively), the electrochemical driving force for K transport across the basolateral membrane ( _K =V _BL –E _K) is not significantly different for both techniques (30.1±3.3 mV for the first technique and 27.6±1.8 mV for ion-selective electrodes). This suggests an adequate functioning of the selective barrel but an underestimation ofV _BL by the reference barrel of the double-barrel microelectrode. Such double-barrel microelectrodes were used to measure temporal changes ina _{K i} and _K in different experimental conditions where Na reabsorption rate (J _Na) was reduced.a _{K i} was shown to increase by 12.2±2.7 (n=5) and 14.1±4.4mm (n=5), respectively, whenJ _Na was reduced by omitting in the luminal perfusate: (i) 5.5mm glucose and 6mm alanine and (ii) glucose, alanine, other Na-cotransported solutes and 110mm Na. In terms of the electrochemical driving force for K exit across the basolateral membrane, _K, a decrease of 5.4±2.0 mV (P<0.05,n=5) was measured when glucose and alanine were omitted in the luminal perfusate while _K remained unchanged whenJ _Na was more severely reduced (mean change =–1.7±2.1 mV, NS,n=5). In the latter case, this means that the electrochemical driving force for K efflux across the basolateral membrane has not changed while both the active influx through the Na–K pump and the passive efflux in steady state are certainly reduced. If the main pathway for K transport is through the basolateral K conductance, this implies that this conductance must have decreased in the same proportion as that of the reduction in the Na–K pump activity.     

An Amiloride-sensitive Na+ conductance in the basolateral membrane of toad urinary bladder

Summary Exposing the apical membrane of toad urinary bladder to the ionophore nystatin lowers its resistance to less than 100 cm². The basolateral membrane can then be studied by means of transepithelial measurements. If the mucosal solution contains more than 5mm Na⁺, and serosal Na⁺ is substituted by K⁺, Cs⁺, or N-methyl-d-glucamine, the basolateral membrane expresses what appears to be a large Na⁺ conductance, passing strong currents out of the cell. This pathway is insensitive to ouabain or vanadate and does not require serosal or mucosal Ca²⁺. In Cl-free SO ₄ ^2– Ringer's solution it is the major conductive pathway in the basolateral membrane even though the serosal side has 60mm K⁺. This pathway can be blocked by serosal amiloride (K _i=13.1 m) or serosal Na⁺ ions (K _i 10 to 20mm). It also conducts Li⁺ and shows a voltage-dependent relaxation with characteristic rates of 10 to 20 rad sec^–1 at 0 mV.     

Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytesin vitro andin vivo

Summary To study the physiological role of the bidirectionally operating, furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes, the effect of furosemide on red cell cation and hemoglobin content was determined in cells incubated for 24 hr with ouabain in 145mm NaCl media containing 0 to 10mm K⁺ or Rb⁺. In pure Na⁺ media, furosemide accelerated cell Na⁺ gain and retarded cellular K⁺ loss. External K⁺ (5mm) had an effect similar to furosemide and markedly reduced the action of the drug on cellular cation content. External Rb⁺ accelerated the Na⁺ gain like K⁺, but did not affect the K⁺ retention induced by furosemide. The data are interpreted to indicate that the furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes mediates an equimolar extrusion of Na⁺ and K⁺ in Na⁺ media (Na⁺/K⁺ cotransport), a 1:1 K⁺/K⁺ (K⁺/Rb⁺) and Na⁺/Na⁺ exchange progressively appearing upon increasing external K⁺ (Rb⁺) concentrations to 5mm. The effect of furosemide (or external K⁺/Rb⁺) on cation contents was associated with a prevention of the cell shrinkage seen in pure Na⁺ media, or with a cell swelling, indicating that the furosemide-sensitive Na⁺/K⁺ transport system is involved in the control of cell volume of human erythrocytes. The action of furosemide on cellular volume and cation content tended to disappear at 5mm external K⁺ or Rb⁺. Thein vivo red cell K⁺ content was negatively correlated to the rate of furosemide-sensitive K⁺ (Rb⁺) uptake, and a positive correlation was seen between mean cellular hemoglobin content and furosemide-sensitive transport activity. The transport system possibly functions as a K⁺ and waterextruding mechanism under physiological conditiosin vivo. The red cell Na⁺ content showed no correlation to the activity of the furosemide-sensitive transport system.