Cell membrane and transepithelial voltages and resistances in isolated rat hepatocyte couplets

Summary The basic electrical properties of an isolated rat hepatocyte couplet (IRHC) system have been analyzed using classical techniques of epithelial electrophysiology, including measurement of electric potentials, resistances and intracellular ion activities. Applications of these techniques are discussed with respect to their limitations in small isolated cells. Mean intracellular and intracanalicular membrane potentials ranged from –23.7 to –46.7 and –4.3 to –5.9 mV, respectively. Membrane resistances were determined using an equivalent circuit analysis modified according to the geometry of the IRHC system. Resistances of the sinusoidal (basolateral) and canalicular (luminal) cell membranes and tight junctions averaged 0.15 and 0.78 G and 25m, respectively. The cells are electrically coupled via low resistance intercellular communications (58 M). Intracellular ion activities for Na⁺, K⁺ and Cl^– averaged 12.2, 88.1 and 17.7 mmol/liter, respectively. The basolateral membrane potential reveals a permeability sequence ofP _K>P _Cl>P _Na. The luminal potential showed minimal dependence on changes in transjunctional ion gradients, indicating a poor ion selectivity of the paracellular pathway. The electrogenic (Na⁺–K)-ATPase contributes little to the luminal and cellular negative electric potential. Therefore, the luminal potential probably results from the secretion of impermeant ions and a Donnan distribution of permeant ions, a mechanism which provides the osmotic driving force for bile formation. By providing the unique opportunity to measure luminal potentials, this isolated hepatocyte system permits study of secretory mechanisms for the first time in a mammalian gland using electrophysiologic techniques.     

Electrophysiological studies of forskolin-induced changes in ion transport in the human colon carcinoma cell line HT-29 cl.19A: Lack of evidence for a cAMP-activated basolateral K+ conductance

Summary Forskolin (i.e, cAMP)-modulation of ion transport pathways in filter-grown monolayers of the Cl^–-secreting subclone (19A) of the human colon carcinoma cell line HT29 was studied by combined Ussing chamber and microimpalement experiments.Changes in electrophysiological parameters provoked by serosal addition of 10^–5 m forskolin included: (i) a sustained increase in the transepithelial potential difference (3.9±0.4 mV). (ii) a transient decrease in transepithelial resistance with 26±3 · cm² from a mean value of 138±13 · cm² before forskolin addition, (iii) a depolarization of the cell membrane potential by 24±1 mV from a resting value of –50±1 mV and (iv) a decrease in the fractional resistance of the apical membrane from 0.80±0.02 to 0.22±0.01. Both, the changes in cell potential and the fractional resistance, persisted for at least 10 min and were dependent on the presence of Cl^– in the medium. Subsequent addition of bumetanide (10^–4 m), an inhibitor of Na/K/2Cl cotransport, reduced the transepithelial potential, induced a repolarization of the cell potential and provoked a small increase of the transepithelial resistance and fractional apical resistance. Serosal Ba²⁺ (1mm), a known inhibitor of basolateral K⁺ conductance, strongly reduced the electrical effects of forskolin. No evidence was found for a forskolin (cAMP)-induced modulation of basolateral K⁺ conductance.The results suggest that forskolin-induced Cl^– secretion in the HT-29 cl.19A colonic cell line results mainly from a cAMP-provoked increase in the Cl^– conductance of the apical membrane but does not affect K⁺ or Cl^– conductance pathways at the basolateral pole of the cell. The sustained potential changes indicate that the capacity of the basolateral transport mechanism for Cl^– and the basal Ba²⁺-sensitive K⁺ conductance are sufficiently large to maintain the Cl^– efflux across the apical membrane. Furthermore, evidence is presented for an anomalous inhibitory action of the putative Cl^– channel blockers NPPB and DPC on basolateral conductance rather than apical Cl^– conductance.     

Intracellular potential and K+ activity in rat kidney proximal tubular cells in acidosis and K+ depletion

Summary Techniques were developed for the measurement of intracellular potentials and potassium activities in rat proximal tubule cells using double barreled K⁺ liquid-ion-exchanger microelectrodes. After obtaining measurements of stable and reliable control values, the effects of K⁺ depletion and metabolic and respiratory acidosis on the intracellular potential and K⁺ activity in rat kidney proximal tubular cells were determined. At a peritubular membrane potential of –66.3±1.3 mV (mean±se), intracellular K⁺ activity was 65.9±2.0 mEq/liter in the control rats. In metabolic acidosis [70 mg NH₄ Cl/100 g body wt) the peritubular membrane potential was significantly reduced to –47.5±1.9 mV, and cellular K⁺ activity to 53.5±2.0 mEq/liter. In contrast, in respiratory acidosis (15% CO₂) the peritubular membrane potential was significantly lowered to –46.1±1.39 mV, but the cellular K⁺ activity was maintained at an almost unchanged level of 63.7±1.9 mEq/liter. In K⁺ depleted animals (6 weeks on low K⁺ diet), the peritubular membrane potential was significantly higher than in control animals, –74.8±2.1 mV, and cellular K⁺ activity was moderately but significantly reduced to 58.1±2.7 mEq/liter. Under all conditions studied, cellular K⁺ was above electrochemical equilibrium. Consequently, an active mechanism for cellular K⁺ accumulation must exist at one or both cell membranes. Furthermore, peritubular HCO₃ ^– appears to be an important factor in maintaining normal K⁺ distribution across the basolateral cell membrane.     

The regulation of total creatine content in a myoblast cell line

Total cellular creatine content is an important bioenergetic parameter in skeletal muscle. To understand its regulation we investigated creatine transport and accumulation in the G8 cultured skeletal myoblast line. Like other cell types, these contain a creatine transporter, whose activity, measured using a radiolabelling technique, was saturable (Km = 110 ± 25 M) and largely dependent on extracellular [Na⁺]. To study sustained influences on steady state creatine concentration we measured total cellular creatine content using a fluorimetric method in 48 h incubations. We found that the total cellular creatine content was relatively independent of extracellular creatine concentration, consistent with high affinity sodium-dependent uptake balanced by slow passive efflux. Accordingly, in creatine-free incubations net creatine efflux was slow ( 5 ± 1 % of basal creatine content per day over 6 days), while creatine content in 48 h incubations was reduced by 28 ± 13% of control by the Na⁺,K⁺-ATPase inhibitor ouabain. Creatine accumulation after 48 h was stimulated by treatment with the mixed - and -adrenergic agonist noradrenaline, the -adrenergic agonist isoproterenol, the ₂-agonist clenbuterol and the cAMP analogue N⁶,2-O-dibutyryladenosine 3,5-cyclic monophosphate, but was unaffected by the ₁ adrenergic agonist methoxamine. The noradrenaline enhancement of creatine accumulation at 48 h was inhibited by the mixed - and -antagonist labetalol and by the -antagonist propranolol, but was unaffected by the ₂ antagonist phentolamine; greater inhibition was caused by the ₂ antagonist butoxamine than the ₁ antagonist atenolol. Creatine accumulation at 48 h was increased to 230 ± 6% of control by insulin and by 140 ± 13% by IGF-I (both at 3 nM). Creatine accumulation at 48 h was also increased to 280 ± 40% of control by 3,3,5-triiodothyronine (at 70 M) and to 220 ± 35% of control by amylin (60 nM). As 3,3,5-triiodothyronine, amylin and isoproterenol all stimulate the Na⁺,K⁺-ATPase, we suggest that they stimulate Na⁺-creatine cotransport indirectly by increasing the transmembrane [Na⁺] concentration gradient and membrane potential.Abbreviations IGF-I insulin-like growth factor I - IGF-II insulin-like growth factor II - T₃ 3,3,5-triiodothyronine - CGRP calcitonin gene-related peptide     

Ba2+-sensitive potassium permeability of the apical membrane in newt kidney proximal tubule

Summary The apical membrane K⁺ permeability of the newt proximal tubular cells was examined in the doubly perfused isolated kidney by measuring the apical membrane potential change (V _a change) during alteration of luminal K⁺ concentration and resultant voltage deflections caused by current pulse injection into the lumen.V _a change/decade for K⁺ was 50 mV at K⁺ concentration higher than 25mm, and the resistance of the apical membrane decreased bt 58% of control when luminal K⁺ concentration was increased from 2.5 to 25mm. Ba²⁺ (1mm in the lumen) reducedV _a change/decade to 24 mV and increased the apical membrane resistance by 70%. These data support the view that Ba²⁺-sensitive K⁺ conductance exists in the apical membrane of the newt proximal tubule. Furthermore, intracellular K⁺ activity measured by K⁺-selective electrode was 82.4 ± 3.6 meq/liter, which was higher than that predicted from the Nernst equation for K⁺ across both cell membranes. Thus, it is concluded that cell K⁺ passively diffuses, at least in part, through the K⁺ conductive pathway of the apical membrane.     

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.     

Electrophysiology of cultured human lens epithelial cells

Summary The lens epithelial K⁺ conductance plays a key role in maintaining the lens ionic steady state. The specific channels responsible for this conductance are unknown. We used cultured lens epithelia and patch-clamp technology to address this problem. Human lens epithelial explants were cultured and after 1–4 passages were dissociated and used in this study. The cells from which we measured had a mean diameter of 31±1 m (sem,n=26). The resting voltage was –19±4 mV (sem,n=10) and the input resistance was 2.5±0.5 G (sem,n=17) at –60 mV. Two currents were prominent in whole-cell recordings. An outwardly rectifying current was seen in nearly every cell. The magnitude of this current was a function of K⁺ concentration and was blocked by 3mm tetraethylammonium. The instantaneous current-voltage relationship was linear in symmetric K⁺, implying that the outward rectificiation was due to gating. The current showed complex activation and inactivation kinetics. The second current seen was a transient inward current. This current had kinetics very similar to the traditional Na⁺ current of excitable cells and was blocked by 0.1 m tetrodotoxin. In single-channel recordings, a 150-pS K⁺ channel and a 35-pS nonselective cation channel were seen but neither account for the macroscopic currents measured.     

K+ transport in ‘Tight’ epithelial monolayers of MDCK cells

Summary Bidirectional transepithelial K⁺ flux measurements across high-resistance epithelial monolayers of MDCK cells grown upon millipore filters show no significant net K⁺ flux.Measurements of influx and efflux across the basal-lateral and apical cell membranes demonstrate that the apical membranes are effectively impermeable to K⁺.K⁺ influx across the basal-lateral cell membranes consists of an ouabain-sensitive component, an ouabain-insensitive component, an ouabain-insensitive but furosemide-sensitive component, and an ouabain-and furosemide-insensitive component.The action of furosemide upon K⁺ influx is independent of (Na⁺–K⁺)-pump inhibition. The furosemide-sensitive component is markedly dependent upon the medium K⁺, Na⁺ and Cl^– content. Acetate and nitrate are ineffective substitutes for Cl^–, whereas Br^– is partially effective. Partial Cl^– replacement by NO₃ gives a roughly linear increase in the furosemide-sensitive component. Na⁺ replacement by choline abolishes the furosemide-sensitive component, whereas Li⁺ is a partially effective replacement. Partial Na⁺ replacement with choline gives an apparent affinity of 7mm Na, whereas variation of the external K⁺ content gives an affinity of the furosemide-sensitive component of 1.0mm.Furosemide inhibition is of high affinity (K _1/2=3 m). Piretanide, ethacrynic acid, and phloretin inhibit the same component of passive K⁺ influx as furosemide; amiloride, 4,-aminopyridine, and 2,4,6-triaminopyrimidine partially so. SITS was ineffective.Externally applied furosemide and Cl^– replacement by NO ₃ ^– inhibit K⁺ efflux across the basal-lateral membranes indicating that the furosemide-sensitive component consists primarily of KK exchange.     

Voltage dependence of the Ca2+-activated K+ conductance of human red cell membranes is strongly dependent on the extracellular K+ concentration

Summary The conductance of the Ca²⁺-activated K⁺ channel (g _K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V _m ) and extracellular K⁺ concentration ([K⁺]_ex). ATP-depleted cells, with fixed values of cellular K⁺ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K⁺ channels, to buffer-free salt solutions with given K⁺ concentrations. Outward-current conductances were calculated from initial net effluxes of K⁺, correspondingV _m , monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E _K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of⁴²K at (V _m –E _K)0, using a single-file flux ratio exponent of 2.7. Within a [K⁺]_ex range of 5.5 to 60mm and at (V _m –E _K) 20 mV a basic conductance, which was independent of [K⁺]_ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm² between 0 and –100 mV. As (V _m –E _K) decreased from 20 towards zero mVg _K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm². The zero-current conductance was not significantly dependent on [K⁺]_ex (30 to 156mm) corresponding toV _m (–50 mV to 0). A further increase ing _K(Ca) symmetrically aroundE _K is suggested as (V _m –E _K) becomes positive. Increasing the extracellular K⁺ concentration from zero and up to 3mm resulted in an increase ing _K(Ca) from 50 to 70 S/cm². Since the driving force (V _m –E _K) was larger than 20 mV within this range of [K⁺]_ex this was probably a specific K⁺ activation ofg _K(Ca). In conclusion: The Ca²⁺-activated K⁺ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel.     

Regulation of K+ channels in the basolateral membrane ofNecturus oxyntic cells

Summary Patch-clamp methods were used to study single-channel events in isolated oxyntic cells and gastric glands fromNecturus maculosa. Cell-attached, excised inside-out and outside-out patches from the basolateral membrane frequently contained channels which had conductances of 67±21 pS in 24% of the patches and channels of smaller conductance, 33±6 pS in 56% of the patches. Channels in both classes were highly selective for K⁺ over Na⁺ and Cl^–, and shared linear current-voltage relations. The 67-pS channel was activated by membrane depolarization, whereas the activity of the 33-pS channel was relatively voltage independent. The larger conductance channels were activated by intracellular Ca²⁺ in the range between 5 and 500nm, but unaffected by cAMP. The smaller conductance channels were activated by cAMP, but not Ca²⁺. The presence of K⁺ channels in the basolateral membrane which are regulated by these known second messengers can account for the increase in conductance and the hyperpolarization of the membrane observed upon secretagogue stimulation.     

Lipid peroxidation as the mechanism of modification of the affinity of the Na+, K+-ATPase active sites for ATP,K+, Na+, and strophanthidin in vitro

The effect of lipid peroxidation on the affinity of specific active sites of Na⁺, K⁺-ATPase for ATP (substrate), K⁺ and Na⁺ (activators), and strophanthidin (a specific inhibitor) was investigated. Brain cell membranes were peroxidized in vitro in the presence of 100M ascorbate and 25M FeCl₂ at 37°C for time intervals from 0–20 min. The level of thiobarbituric acid reactive substances and the activity of Na⁺, K⁺-ATPase were determined. The enzyme activity decreased by 80% in the first min. from 42.0±3.8 to 8.8±0.9 mol Pi/mg protein/hr and remained unchanged thereafter. Lipid peroxidation products increased to a steady state level from 0.2±0.1 to 16.5 ±1.5 nmol malonaldehyde/mg protein by 3 min. In peroxidized membranes, the affinity for ATP and strophanthidin was increased (two and seven fold, respectively), whereas affinity for K⁺ and Na⁺ was decreased (to one tenth and one seventh of control values, respectively). Changes in the affinity of active sites will affect the phosphorylation and dephosphorylation mechanisms of Na⁺, K⁺-ATPase reaction. The increased affinity for ATP favors the phosphorylation of the enzyme at low ATP concentrations whereas, the decreased affinity for K⁺ will not favor the dephosphorylation of the enzyme-P complex resulting in unavailability of energy for transmembrane transport processes. The results demonstrate that lipid peroxidation alters Na⁺, K⁺-ATPase function by modification at specific active sites in a selective manner, rather than through a non-specific destructive process.     

Na+,K+-ATPase activity in cultured C6 glioma cells

Rat C₆ glioma cells were cultured for 4 days in MEM medium supplemented with 10% bovine serum and Na⁺,K⁺-ATPase activity was determined in homogenates of harvested cells. Approximately 50% of enzyme activity was attained at 1.5 mM K⁺ and the maximum (2.76±0.13 mol Pi/h/mg protein) at 5 mM K⁺. The specific activity of Na⁺,K⁺-ATPase was not influenced by freezing the homogenates or cell suspensions before the enzyme assay. Ten minutes' exposure of glioma cells to 10^–4 or 10^–5 M noradrenaline (NA) remained without any effect on NA⁺,K⁺-ATPase activity. Neither did the presence of NA in the incubation medium, during the enzyme assay, influence the enzyme activity. The nonresponsiveness of Na⁺,K⁺-ATPase of C₆ glioma cells to NA is consistent with the assumption that (+) form of the enzyme may be preferentially sensitive to noradrenaline. Na⁺,K⁺-ATPase was inhibited in a dose-dependent manner by vanadate and 50% inhibition was achieved at 2×10^–7 M concentration. In spite of the fact that Na⁺,K⁺-ATPase of glioma cells was not responsive to NA, the latter could at least partially reverse vanadate-induced inhibition of the enzyme. Although the present results concern transformed glial cells, they suggest the possibility that inhibition of glial Na⁺,K⁺-ATPase may contribute to the previously reported inhibition by vanadate of Na⁺,K⁺-ATPase of the whole brain tissue.     

Basolateral membrane potassium conductance of A6 cells

Summary To study the properties of the basolateral membrane conductance of an amphibian epithelial cell line, we have adapted the technique of apical membrane selective permeabilization (Wills, N.K., Lewis, S.A., Eaton, D.C., 1979b, J. Membrane Biol. 45:81–108). Monolayers of A6 cells cultured on permeable supports were exposed to amphotericin B. The apical membrane was effectively permeabilized, while the high electrical resistance of the tight junctions and the ionic selectivity of the basolateral membrane were preserved. Thus the transepithelial current-voltage relation reflected mostly the properties of the basolateral membrane. Under basal conditions, the basolateral membrane conductance was inward rectifying, highly sensitive to barium but not to quinidine. After the induction of cell swelling either by adding chloride to the apical solution or by lowering the osmolarity of the basolateral solution, a large out-ward-rectifying K⁺ conductance was observed, and addition of barium or quinidine to the basolateral side inhibited, respectively, 82.4±1.9% and 90.9±1.0% of the transepithelial current at 0 mV. Barium block was voltage dependent; the half-inhibition constant (K _i) varied from 1499±97 m at 0 mV to 5.7±0.5 m at –120 mV.Cell swelling induces a large quinidine-sensitive K⁺ conductance, changing the inward-rectifying basolateral membrane conductance observed under basal conditions into a conductance with outward-rectifying properties.     

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.     

pH i -Dependent membrane conductance of proximal tubule cells in culture (OK): Differential effects on K+- and Na+-conductive channels

Summary Confluent monolayers of the established opossum kidney cell line were exposed to NH₄Cl pulses (20 mmol/liter) during continuous intracellular measurements of pH, membrane potential (PD _m ) and membrane resistance (R _m) in bicarbonate-free Ringer. The removal of extracellular NH₄Cl leads to an intracellular acidification from a control value of 7.33±0.08 to 6.47±0.03 (n=7). This inhibits the absolute K conductance (g _K+), reflected by a decrease of K⁺ transference number from 71±3% (n=28) to 26±6% (n=5), a 2.6±0.2-fold rise ofR _m, and a depolarization by 24.2±1.5 mV (n=52). In contrast, intracellular acidification during a block ofg _K+ by 3 mmol/liter BaCl₂ enhances the total membrane conductance, being shown byR _m decrease to 68±7% of control and cell membrane depolarization by 9.8±2.8 mV (n=17). Conversely, intracellular alkalinization under barium elevatesR _m and hyperpolarizes PD _m . The replacement of extracellular sodium by choline in the presence of BaCl₂ significantly hyperpolarizes PD _m and increasesR _m, indicating the presence of a sodium conductance. This conductance is not inhibited by 10^–4 mol/liter amiloride (n=7). Patch-clamp studies at the apical membrane (excised inside-out configuration) revealed two Na⁺-conductive channels with 18.8±1.4 pS (n=10) and 146 pS single-channel conductance. Both channels are inwardly rectifying and highly selective towards Cl^–. The low-conductive channel is 4.8 times more permeable for Na⁺ than for K⁺. Its open probability rises at depolarizing potentials and is dependent on the pH of the membrane inside (higher at pH 6.5 than at pH 7.8).     

The diabetogenic agent alloxan increases K+ permeability by a mechanism involving activation of ATP-sensitive K+-channels in mouse pancreatic β-cells

The effects of the diabetogenic agent, alloxan, on membrane potential, input resistance and electrical activity of normal mouse pancreatic -cells were studied. Tetraethylammonium (TEA), quinine and Glyburide were used to block K⁺-channels and to elucidate the mechanisms underlying alloxan's effects on -cell membrane potential. Exposure of the islet to alloxan (75–100 M) in the presence of glucose (11 mM), produced a rapid (15 sec), transient inhibition of electrical activity, often accompanied by hyperpolarization of the membrane, and this was followed by recovery of the burst pattern. This early effect of alloxan was followed after approximately 15 min by a complete inhibition of electrical activity and hyperpolarization. The inhibition accompanied by hyperpolarization was associated with a decrease in input resistance, indicating increased K⁺-conductance. Both the transient and delayed effects of alloxan were blocked by glucose (33 mM), quinine and glyburide but not by other conditions which induced continuous electrical activity such as elevated external [K⁺] (10 mM), ouabain, K⁺ removal, or TEA (20 mM). The transient inhibition induced by alloxan may be due to a direct competition with glucose transport/metabolism since it did not occur when alpha-keto isocaproic acid (KIC) was used to induce electrical activity. The delayed inhibition may reflect indirect effects of accumulation of this agent or its metabolites within the cell. Since both effects of alloxan are blocked by glyburide they appear to involve activation of the ATP-sensitive K⁺-channel (K-ATP).     

Na+ and K+ fluxes stimulated by Na+-coupled glucose transport: Evidence for a Ba2+-insensitive K+ efflux pathway in rabbit proximal tubules

Summary Addition of glucose or the nonmetabolizable analogue -methyl-d-glucoside to rabbit proximal tubules suspended in a glucoseand alanine-free buffer caused a sustained increase in intracellular Na⁺ content (+43±7 nmol · (mg protein)^–1) and a concomitant but larger decrease in K⁺ content (–72±11 nmol· (mg protein)^–1). A component of the net K⁺ efflux was Ba²⁺ insensitive, and was inhibited by high (1mm) but not low (10 m) concentrations of the diuretics, furosemide and bumetanide. The increase in intracellular Na⁺ content is consistent with the view that the increased rates of Na⁺ and water transport seen in the proximal tubule in the presence of glucose can be attributed (at least in part) to a stimulation of basolateral pump activity by an increased [Na⁺] _i .     

Voltage- and time dependence of apical membrane conductance during current clamp inNecturus gallbladder epithelium

Summary The effects of short (1 sec) and long (1 min) transepithelial current clamps on membrane voltages and resistances ofNecturus gallbladder were investigated. Transepithelial and cell membrane current-voltage relationships determined from 1-sec clamps revealed that: a) depolarization of the apical membrane voltage (V _mc) results in a marked decrease in apical membrane fractional resistance (fR _a), whereas hyperpolarization ofV _mc results in either no change infR _a or a small increase, and b) the voltage-dependent changes infR _a are essentially complete within 500 msec. Exposure of the tissue to 5mm TEA⁺ on the mucosal side caused no significant change in baselineV _mc (–69±2 mV) and yet virtually abolished the voltage dependence offR _a. A possible interpretation of these results is that two types of K⁺ channels exist in the apical membrane, with different voltage dependencies and TEA⁺ sensitivities. Acidification or Ba²⁺ addition to the mucosal solution also reduced the voltage-dependent changes infR _a. The time courses of the changes infR _a and in the cable properties of the epithelium were assessed during 1-min transepithelial current clamps (±200 A/cm²). No secondary change infR _a was observed with mucosa-to-serosa currents, but a slow TEA⁺-sensitive decrease infR _a (half-time of seconds) was evident with serosa-to-mucosa currents. Cable analysis experiments demonstrated that the initial (<500 msec) voltage-dependent decrease infR _a is due to a fall in apical membrane resistance. The later decrease infR _a is due to changes in both cell membrane resistances attributable to the increase in transcellular current flow resulting from a fall in paracellular conductance. The voltage dependence of the apical membrane conductance is a more significant problem in estimatingfR _a than the current-induced effects on the lateral intercellular spaces. In principle, TEA⁺ can be used to prevent the nonlinear behavior ofR _a during measurements of the voltage divider or membrane resistance ratio.     

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.     

Modifications of current properties by expression of a foreign potassium channel gene in Xenopus embryonic cells

The pH-sensitivity of transepithelial K⁺ transport was studied in vitro in isolated vestibular dark cell epithelium from the gerbil ampulla. The cytosolic pH (pH _iwas measured microfluorometrically with the pH-sensitive dye 2,7-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF) and the equivalent short-circuit current (I _sc), which is a measure for transepithelial K⁺ secretion, was calculated from measurements of the transepithelial voltage (V _t)and the transepithelial resistance (R _t) in a micro-Ussing chamber. All experiments were conducted in virtually HCO ₃ ^– -free solutions. Under control conditions, pH _iwas 7.01±0.04 (n=18), V _twas 9.1±0.5 mV, R _t16.7±0.09 cm², and I _sc was 587±30 A/cm² (n=49). Addition of 20 mm propionate^– caused a biphasic effect involving an initial acidification of pH _i, increase in V _tand I _sc and decrease in R _tand a subsequent alkalinization of pH _i, decrease of V _tand increase of R _t. Removal of propionate^– caused a transient effect involving an alkalinization of pH _i, a decrease of V _tand I _sc and an increase in R _t. pH _iin the presence of propionate^– exceeded pH _iunder control conditions. Effects of propionate ^– on V _t, R _tand I _sc were significantly larger when propionate^– was applied to the basolateral side rather than to the apical side of the epithelium. The pH _i-sensitivityof I _sc between pH 6.8 and 7.5 was –1089 A/(cm² · pH-unit) suggesting that K⁺ secretion ceases at about pH _i7.6. Acidification of the extracellular pH (pH _o)caused an increase of V _tand I _sc and a decrease of R _tmost likely due to acidification of pH _i. Effects were significantly larger when the extracellular acidification was applied to the basolateral side rather than to the apical side of the epithelium. The pH _osensitivity of I _sc between pH 7.4 and 6.4 was –155 A/(cm² · pH unit). These results demonstrate that transepithelial K⁺ transport is sensitive to pH _iand pH _oand that vestibular dark cells contain propionate^– uptake mechanism. Further, the data suggest that cytosolic acidification activates and that cytosolic alkalinization inactivates the slowly activating K⁺ channel (I _sK)in the apical membrane. Whether the effect of pH _ion the I _sK channel is a direct or indirect effect remains to be determined.The authors wish to thank Drs. Daniel C. Marcus, Zhjiun Shen and Hiroshi Sunose for helpful discussions. This work was supported by grants NIH-R29-DC01098 and NIH-R01-DC00212.