Microelectrode characterization of the basolateral membrane of rabbit S3 proximal tubule

Summary The purpose of this study was to characterize the basolateral membrane of the S3 segment of the rabbit proximal tubule using conventional and ion-selective microelectrodes. When compared with results from S1 and S2 segments, S3 cells under control conditions have a more negative basolateral membrane potential (V _bl=–69 mV), a higher relative potassium conductance (t _K=0.6), lower intracellular Na⁺ activity (A _Na=18.4mm), and higher intracellular K⁺ activity (A _K=67.8mm). No evidence for a conductive sodium-dependent or sodium-independent HCO ₃ ^– pathway could be demonstrated. The basolateral Na–K pump is inhibited by 10^–4 m ouabain and bath perfusion with a potassium-free (0-K) solution. 0-K perfusion results inA _Na=64.8mm,A _K=18.5mm, andV _bl=–28 mV. Basolateral potassium channels are blocked by barium and by acidification of the bathing medium. The relative K⁺ conductance, as evaluated by increasing bath K⁺ to 17mm, is dependent upon the restingV _bl in both S2 and S3 cells. In summary, the basolateral membrane of S3 cells contains a pump-leak system with similar properties to S1 and S2 proximal tubule cells. The absence of conductive bicarbonate pathways results in a hyperpolarized cell and larger Na⁺ and K⁺ gradients across the cell borders, which will influence the transport properties and intracellular ion activities in this tubule segment.     

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.     

Mechanism of inhibition of net ion transport across frog corneal epithelium by calcium channel antagonists

Summary In the isolated bullfrog cornea, three calcium channel antagonists had dose-dependent inhibitory effects on the Cl-originated short-circuit current (SCC). Their order of decreasing potency was bepridil, verapamil and diltiazem. One millimolar diltiazem inhibited the SCC by 98% and subsequent incubation with the calcium ionophore A23187 had no restorative effect. Increasing the bathing solution Ca concentration from 0.05 to 15mm, however, decreased diltiazem's inhibitory efficacy. This antagonist depolarized the intracellular potential differenceV _m from –54 to –18 mV (tear: reference) and the voltage divider ratioFR ₀ decreased from 0.58 to 0.30, suggesting an increase in basolateral membrane electrical resistance. Additional indication of a basolateral membrane effect by the drug was that preincubation with 10⁵ m amphotericin B in Cl-free Ringer's did not eliminate the inhibitory effect of the drug on the Na- and K-elicited SCC. In the absence of amphotericin B in Cl-free Ringer's (SCC=0), 1 ×10³ m diltiazem depolarized theV _m from –78 to –9 mV suggesting that the increase in basolateral membrane resistance was due to K channel blockade. Diltiazem (1×10³ m) significantly decreased cyclic AMP content; however, isoproterenol in the presence of the drug increased cyclic AMP fourfold without having any restorative effect on the inhibited SCC. Therefore, the inhibition of the Cl-originated SCC resulting from an increase in basolateral membrane K resistance is not caused by a decline in cyclic AMP content. In plasma membrane-enriched fractions prepared from broken cell preparations of bovine corneal epithelium, 1×10³ m diltiazem had no inhibitory effects on either Na,K-ATPase or Ca,Mg-ATPase activities. These latter effects further point to the selectivity of diltiazem as an inhibitor of K-channel activity, but do not preclude a Ca-channel blocker effect by the drug in the micromolar range.     

Implications of an anomalous intracellular electrical response in bullfrog corneal epithelium

Summary The ionic dependencies of the transepithelial and intracellular electrical parameters were measured in the isolated frog cornea. In NaCl Ringer's the intracellular potential differenceV _sc measured under short-circuit conditions depolarized by nearly the same amount after either increasing the stromal-side KCl concentration from 2.5 to 25mm or exposure to 2mm BaCl₂ (K⁺ channel blocker). With Ba²⁺ the depolarization of theV _sc by 25mm K⁺ was reduced to one-quarter of the control change. If the Cl-permselective apical membrane resistanceR _o remained unchanged, the relative basolateral membrane resistanceR _i, which includes the lateral intercellular space, increased at the most by less than twofold after Ba²⁺. These effects in conjunction with the depolarization of theV _sc by 62 mV after increasing the stromal-side K⁺ from 2.5 to 100mm in Cl-free Ringer's as well as the increase of the apparent ratio of membrane resistances (a=R _o/R_i) from 13 to 32 are all indicative of an appreciable basolateral membrane K⁺ conductance. This ratio decreased significantly after exposure to either 25mm K⁺ or Ba²⁺. The decline ofR _o/R_i with 25mm K⁺ appears to be anomalous since this decrease is not consistent with just an increase of basolateral membrane conductance by 25mm K⁺, but rather perhaps a larger decrease ofR _o thanR _iAlso an increase of lateral space resistance may offset the effect of decreasingR _i with 25mm K⁺. In contrast,R _o/R_i did transiently increase during voltage clamping of the apical membrane potential differenceV _o and exposure to 25mm K⁺ on the stromal side. This increase and subsequent decrease ofR _o/R_i supports the idea that increases in stromal K⁺ concentration may produce secondary membrane resistance changes. These effects onR _o/R_i show that the presence of asymmetric ionic conductance properties in the apical and basolateral membranes can limit the interpretative value of this parameter. The complete substitution of Na⁺ withn-methyl-glucamine in Cl-free Ringer's on the stromal side hyperpolarized theV _sc by 6 mV whereas 10^–4 m ouabain depolarized theV _sc by 7 mV. Thus the basolateral membrane contains K⁺, Na⁺ and perhaps Cl^– pathways in parallel with the Na/K pump component.     

The electrogenic sodium pump of the frog retinal pigment epithelium

Summary It was previously shown that ouabain decreases the potential difference across anin vitro preparation of bullfrog retinal pigment epithelium (RPE) when applied to the apical, but not the basal, membrane and that the net basal-to-apical Na⁺ transport is also inhibited by apical ouabain. This suggested the presence of a Na⁺–K⁺ pump on the apical membrane of the RPE. In the present experiments, intracellular recordings from RPE cells show that this pump is electrogenic and contributes approximately –10 mV to the apical membrane potential (V _AP). Apical ouabain depolarizedV _AP in two phases. The initial, fast phase was due to the removal of the direct, electrogenic component. In the first one minute of the response to ouabain,V _AP depolarized at an average rate of 4.4±0.42 mV/min (n=10, mean ±sem), andV _AP depolarized an average of 9.6±0.5 mV during the entire fast phase. A slow phase of membrane depolarization, due to ionic gradients running down across both membranes, continued for hours at a much slower rate, 0.4 mV/min. Using a simple diffusion model and K⁺-specific microelectrodes, it was possible to infer that the onset of the ouabain-induced depolarization coincided with the arrival of ouabain molecules at the apical membrane. This result must occur if ouabain affects an electrogenic pump. Other metabolic inhibitors, such as DNP and cold, also produced a fast depolarization of the apical membrane. For a decrease in temperature of 10°C, the average depolarization of the apical membrane was 7.1±3.4 mV (n=5) and the average decrease in transepithelial potential was 3.9±0.3 mV (n=10). These changes in potential were much larger than could be explained by the effect of temperature on anRT/F electrodiffusion factor. Cooling the tissue inhibited the same mechanism as ouabain, since prior exposure to ouabain greatly reduced the magnitude of the cold effect. Bathing the tissue in 0mm [K⁺] solution for 2 hr inhibited the electrogenic pump, and subsequent re-introduction of 2mm [K⁺] solution produced a rapid membrane hyperpolarization. We conclude that the electrogenic nature of this pump is important to retinal function, since its contribution to the apical membrane potential is likely to affect the transport of ions, metabolites, and fluid across the RPE.     

Basolateral K channel activated by carbachol in the epithelial cell line T84

Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the electrical gradient favoring apical Cl efflux and allows K to recycle at the basolateral membrane. We have used transepithelial short-circuit current (I _SC), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T₈₄ cells. Carbachol had little effect on I _SC when added alone but produced large, transient currents if added to monolayers prestimulated with cAMP. cAMP also enhanced the subsequent I _SC response to calcium ionophores. Carbachol (100 m) transiently elevated intracellular free calcium ([Ca²⁺] _i ) by 3-fold in confluent cells cultured on glass coverslips with a time course resembling the I _sc response of confluent monolayers that had been grown on porous supports. In parallel patch clamp experiments, carbachol activated an inwardly rectifying potassium channel on the basolateral aspect of polarized monolayers which had been dissected from porous culture supports. The same channel was transiently activated on the surface of subconfluent monolayers during stimulation by carbachol. Activation was more prolonged when cells were exposed to calcium ionophores. The conductance of the inward rectifier in cell-attached patches was 55 pS near the resting membrane potential (–54 mV) with pipette solution containing 150 mm KCl (37°C). This rectification persisted when patches were bathed in symmetrical 150 mm KCl solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na Cs based on permeability ratios under bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively insensitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, and was unaffected by external 10 mm barium. It is referred to as the K_BIC channel based on its most distinctive properties (Ba-insensitive, inwardly rectifying, Ca-activated). Like single K_BIC channels, the carbachol-stimulated I _SC was relatively insensitive to several blockers on the basolateral side and was unaffected by barium. These comparisons between the properties of the macroscopic current and single channels suggest that the K_BIC channel mediates basolateral membrane K conductance in T₈₄ cell monolayers during stimulation by cholinergic secretagogues.We thank Dr. Marcel Crest (Laboratoire de Neurobiologie, CNRS, Marseille) for providing a sample of kaliotoxin. This work was supported by the Canadian Cystic Fibrosis Foundation and the Respiratory Health Network of Centres of Excellence. J.W.H. is a Chercheur-Boursier of the Fonds de la recherche en santé du Québec.     

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.     

Regulation of the basolateral potassium conductance of theNecturus proximal tubule

Summary Two methods, the measurement of the response of the basolateral membrane potential (V _bl) of proximal tubule cells ofNecturus to step changes in basolateral K⁺ concentration, and cellular cable analysis, were used to assess the changes in basolateral potassium conductance (G _K) caused by a variety of maneuvers. The effects of some of these maneuvers on intracellular K⁺ activity (a _K ⁱ ) were also evaluated using double-barreled ion-selective electrodes. Perfusion with 0mm K⁺ basolateral solution for 15 min followed by 45 min of 1mm K⁺ solution resulted in a fall in basolateral potassium (apparent) transference number (t _K),V _bl anda _K ⁱ . Results of cable analysis showed that total basolateral resistance,R _b , rose. The electrophysiological effects of additional manipulations, known to inhibit net sodium reabsorption across the proximal tubular epithelium ofNecturus, were also investigated. Ouabain caused a fall int _K accompanied by large decreases ina _K ⁱ andV _bl. Lowering luminal sodium caused a fall int _K and a small reduction inV _bl. Selective reduction of peritubular sodium, a maneuver that has been shown to block sodium transport from lumen to peritubular fluid, also resulted in a significant decrease int _K. These results suggest thatG _K varies directly with rate of transport of the sodium pump, irrespective of the mechanism of change in pump turnover.Part of this material has been presented at the 10th International Conference on Biological Membranes (Cohen & Giebisch, 1984).     

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.     

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.     

Effects of the anion transport inhibitor,SITS, on the proximal straight tubule of the rabbit perfusedin vitro

Summary Conventional microelectrodes were used to study the effects of SITS (4-acetamido-4-isothiocyanostilbene-2,2-disulfonate) on the basolateral membrane potentialVbl of the superficial proximal straight tubule (PST) of the rabbit kidney perfusedin vitro. Addition of 0.1mm SITS to the bathing solution resulted in a slow and irreversible hyperpolarization ofVbl from –42.5±1.17 (37) mV to –77.3±0.83 (52) mV. The new steady-state potential was reached in 10 to 15 min and was accompanied by visible cell swelling. Associated with thisVbl hyperpolarization was: 1) an increased steady-state depolarization (from 6.2±0.77 (17) mV to 25.7±0.83 (29) mV) in response to increasing bath potassium concentration from 5 to 16.7mm (HK); 2) a decreased transient depolarization (from 19.8±1.88 (8) mV to 0.43±0.37 (8) mV) in response to decreasing bath bicarbonate concentration from 22 to 6.6mm at constant bath pH (L-HCO₃); and 3) inhibition of a depolarizing overshoot and a decreased steady-state depolarization (from 35.9±1.84 (12) mV to 4.7±1.37 (13) mV) in response to reducing bath sodium concentration from 144 to zero (0-Na). Sodium, chloride and NMDG (N-methyl-d-glucamine) were used as the substituting ions, respectively. These results are consistent with the presence of a coupled sodium-bicarbonate carrier in the basolateral membrane which is electrogenic and SITS inhibitable. Comparison of the time course of SITS effects on these ion-substitution responses suggests that the inhibition of the bicarbonate exit pathway(s) is the primary event and that the changes inVbl and in the steady-stateVbl responses to HK and 0-Na are secondary events which may be related to changes in intracellular composition and/or basolateral membrane properties.     

Ion transport by mitochondria-rich cells in toad skin

Summary The optical sectioning video imaging technique was used for measurements of the volume of mitochondria-rich (m.r.) cells of the isolated epithelium of toad skin. Under short-circuit conditions, cell volume decreased by about 14% in response to bilateral exposure to Cl-free (gluconate substitution) solutions, apical exposure to ouabain resulted in a large increase in volume, which could be prevented either by the simultaneous application of amiloride in the apical solution or by the exposure of the epithelium to bilateral Cl-free solutions. Unilateral exposure to a Cl-free solution did not prevent ouabain-induced cell swelling. It is concluded that m.r. cells have an amiloride-blockable Na conductance in the apical membrane, a ouabain-sensitive Na pump in the basolateral membrane, and a passive Cl permeability in both membranes. From the initial rate of ouabain-induced cell volume increase the active Na current carried by a single m.r. cell was estimated to be 9.9±1.3 pA. Voltage clamping of the preparation in the physiological range of potentials (0 to –100 mV, serosa grounded) resulted in a cell volume increase with a time course similar to that of the stimulation of the voltage-dependent activation were prevented by exposure of the tissue to a Cl-free apical solution. The steady-state volume of the m.r. cells increased with the clamping voltage, and at –100 mV the volume was about 1.15 times that under short-circuit conditions. The rate of volume increase during current passage was significantly decreased by lowering the serosal K concentration (K _i ) to 0.5mm, but was independent of whether K _i was 2.4, 5, or 10mm. This indicates that the K conductance of the serosal membrane becomes rate limiting for the uptake of KCl when K _i is significantly lower than its physiological value. It is concluded that the voltage-activated Cl currents flow through the m.r. cells and that swelling is caused by an uptake of Cl ions from the apical bath and K ions from the serosal bath. Bilateral exposure of the tissue to hypo- or hypertonic bathing solutions changed cell volume without detectable changes in the Cl conductance. The volume response to external osmotic perturbations followed that of an osmometer with an osmotically inactive volume of 21%. Using this value and the change in cell volume in response to bilateral Cl-free solutions, we calculated an intracellular steady-state Cl concentration of 19.8±1.7mm (n=6) of the short-circuited cell.     

Effect of Dicyclohexylcarbodiimide (DCCD) on Transport Parameters in the Frog Cornea Epithelium

Dicyclohexylcarbodiimide (DCCD) is a carboxyl group modifier and it is an inhibitor of various ATPases. Present experiments, using an in vitro preparation, were designed to study whether DCCD affected the transporters of the bullfrog cornea epithelium, specifically, the Na⁺/K⁺ ATPase pump located in the basolateral membrane. For this purpose, corneas were impaled with microelectrodes and experiments were done under short-circuit current (I _sc ) conditions. Addition of DCCD to a concentration of 10⁻⁴ m to the tear solution gave a marked decrease in I _sc ; a marked depolarization of the intracellular potential, V _o ; and a significant decrease in the apical membrane fractional resistance, fR _o . There were small and variable although significant changes in the transepithelial conductance, g _t . The effects may be explained by a decrease in the basolateral membrane K⁺ conductance, in combination with a partial inhibition of the Na⁺/K⁺-ATPase pump located in the basolateral membrane. There is also evidence for an increase in the apical membrane Cl⁻ conductance. Received: 12 August 1999/Revised: 16 November 1999     

Intracellular potassium activity and the role of potassium in transepithelial salt transport in the human reabsorptive sweat duct

Summary We have measured the intracellular potassium activity, [K⁺]_i and the mechanisms of transcellular K⁺ transport in reabsorptive sweat duct (RSD) using intracellular ion-sensitive microelectrodes (ISMEs). The mean value of [K⁺]_i in RSD is 79.8±4.1mm (n=39). Under conditions of microperfusion, the [K⁺]_i is above equilibrium across both the basolateral membrane, BLM (5.5 times) and the apical membrane, APM (7.8 times). The Na⁺/K⁺ pump inhibitor ouabain reduced [K⁺]_i towards passive distribution across the BLM. However, the [K⁺]_i is insensitive to the Na⁺/K⁺/2 Cl^– cotransport inhibitor bumetanide in the bath. Cl^– substitution in the lumen had no effect on [K⁺]_i. In contrast, Cl^– substitution in the bath (basolateral side) depolarized BLM from –26.0±2.6 mV to –4.7^*±2.4 mV (n=3;^* indicates significant difference) and decreased [K⁺]_i from 76.0±15.2mm to 57.7^* ±12.7mm (n=3). Removal of K⁺ in the bath decreased [K⁺]_i from 76.3±15.0mm to 32.3^*±7.6mm (n=4) while depolarizing the BLM from –32.5±4.1 mV to –28.3^*±3.0 mV (n=4). Raising the [K⁺] in the bath by 10-fold increased [K⁺]_i from 81.7±9.0mm to 95.0^*±13.5mm and depolarized the BLM from –25.7±2.4 mV to –21.3^*±2.9 mV (n=4). The K⁺ conductance inhibitor, Ba²⁺, in the bath also increased [K⁺]_i from 85.8±6.7mm to 107.0^*±11.5mm (n=4) and depolarized BLM from –25.8±2.2 mV to –17.0^*±3.1 mV (n=4). Amiloride at 10^–6 m increased [K⁺]_i from 77.5±18.8mm to 98.8^*±21.6mm (n=4) and hyperpolarized both the BLM (from –35.5±2.6 mV to –47.8^*±4.3 mV) and the APM (from –27.5±1.4 mV to –46.0^* ±3.5 mV,n=4). However, amiloride at 10^–4 m decreased [K⁺]_i from 64.5±0.9mm to 36.0^*±9.9mm and hyperpolarized both the BLM (from –24.7±1.4 mV to –43.5^*±4.2 mV) and APM (from –18.3±0.9 mV to –43.5^*±4.2 mV,n=6). In contrast to the observations at the BLM, substitution of K⁺ or application of Ba²⁺ in the lumen had no effect on the [K⁺]_i or the electrical properties of RSD, indicating the absence of a K⁺ conductance in the APM. Our results indicate that (i) [K⁺]_i is above equilibrium due to the Na⁺/K⁺ pump; (ii) only the BLM has a K⁺ conductance; (iii) [K⁺]_i is subject to modulation by transport status; (iv) K⁺ is probably not involved in carrier-mediated ion transport across the cell membranes; and (v) the RSD does not secrete K⁺ into the lumen.     

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.     

Transient potassium fluxes in toad skin

Summary Experiments were carried out in the isolated short-circuited skin of the toadBufo marinus ictericus.⁴²K influx and efflux experiments were carried out with skins bathed on both sides by NaCl-Ringer's solution. Those fluxes showed very similar kinetics of equilibration with time and the results could be fitted by equations of a model of two intraepithelial compartments and the bathing solutions. In the steady state K influx is 3.99 ±0.36 nmol cm^–2 hr^–1 (n=7) and efflux 3.62±0.38 nmol cm^– hr^–1 (n=7) and are not statistically different, indicating that no net K flux is present across the epithelium. Different kinds of perturbations affecting the rates of⁴²K discharge into the bathing solutions were studied. Immediately after addition of amiloride (10^–4 m) to the outer solution, a sharp decline is observed in the rate of⁴²K discharge into the bathing solution,J ₂₁ ^K , which falls from 3.62±0.38 nmol cm^–2 hr^–1 to 2.02±0.04 nmol cm^–2 hr^–1 (n=7) 2 min after addition of the drug, followed by a partial recuperation with time. A complete Na by K substitution in the outer bathing solution induces a prompt and marked decline inJ ₂₁ ^K which is similar to that induced by amiloride. Increase in the outer bathing solution Na concentration from zero Na concentration induces a nonlinear increase inJ ₂₁ ^K and a linear relationship was observed betweenJ ₂₁ ^K and short-circuit current in the range of 0 to 115mm external Na concentration. The decline inJ ₂₁ ^K induced by amiloride or by lowering external Na concentration was interpreted as being caused by electrical hyperpolarization of the external barrier of the epithelium induced by these procedures. Depolarization of the epithelial barriers by inner Na by K substitution in the short-circuited state (when the potential barriers are equal) drastically interfere with the rate of⁴²K discharge from the epithelium into the bathing solutions. Thus, transient increases are observed both in the rate of⁴²K discharge to the outer and to the inner bathing solutions upon depolarization of the barriers. These results indicate that at least the most important component of transepithelial K unidirectional fluxes goes through a transcellular route with a negligible paracellular component. Addition of ouabain (10^–3 m) to the inner bathing solution induces a transient rise in the rate of⁴²K discharge to the outer bathing solution with a peak on the order of 200% of the stationary value previous to the action of the inhibitor, followed by a return to new stationary values not statistically different from those observed previously to the effect of ouabain. The behavior ofJ ₂₁ ^K upon the effect of ouabain, as suggested by comparison with predictions from computer simulation, strongly supports the notion of a rheogenic Na pump in the inner barrier of the epithelium against the notion of a nonrheogenic 11 Na–K pump.     

Mechanism of stimulation by epinephrine of active transepithelial Cl transport in isolated frog cornea

Summary In the isolated frog cornea, the effects of 0.1mm epinephrine were measured on both the transepithelial and intracellular electrical parameters. Epinephrine increased the short-circuit current (I _sc) and transepithelial electrical conductance (g _t) by 176 and 96%, respectively. The effective electromotive driving force for active transepithelial Cl transport (E _Cl) was 45 mV and agrees with the value forE _Cl calculated by a different technique in the isolated rabbit corneal epithelium (Klyce, S.D., Wong, R.K.S., 1977,J. Physiol. (London) 266: 777). With respect to the tear-side bathing solution, epinephrine caused the intracellular potential difference of shortcircuited frog corneas to decrease from –54 to –50 mV (P>0.05). The fractional resistance of the apical membrane {F(R _o)=(R_o/R_o+R_i)} whereR _o andR _i represent the resistances of the apical and basolateral membranes, respectively, decreased from 0.38±0.06 to 0.23±0.03. Using these values ofF(R _o) and the cellular conductances, the calculated Cl resistances ofR _o andR _i decreased 4.3- and 2.3-fold, respectively. However, the value forE _Cl calculated from the intracellular electrical measurements (48 mV) did not appear to change since this value was in close agreement with the value forE _Cl calculated from the effects of epinephrine on the transepithelial electrical parameters. Thus, the effects of epinephrine onI _sc andg _t can be accounted for by increases in the Cl conductance of both the apical and basolateral membranes. Epinephrine caused the potential difference across the basolateral membrane to hyperpolarize by 9 mV. All of these results are consistent with the notion that the steps in transepithelial Cl transport include uphill movement into the cell across the basolateral membrane followed by downhill movement across the apical membrane into the tear-side bathing solution.     

Basolateral membrane potential of a tight epithelium: Ionic diffusion and electrogenic pumps

Summary The contribution of specific ions to the conductance and potential of the basolateral membrane of the rabbit urinary bladder has been studied with both conventional and ion-specific microelectrode techniques. In addition, the possibility of an electrogenic active transport process located at the basolateral membrane was studied using the polyene antibiotic nystatin. The effect of ion-specific microelectrode impalement damage on intracellular ion activities was examined and a criterion set for acceptance or rejection of intracellular activity measurements. Using this criterion, we found (K⁺)=72mm and (Cl^–)=15.8mm. Cl^– but not K⁺ was in electrochemical equilibrium across the basolateral membrane. The selective permeability of the basolateral membrane was measured using microelectrodes, and the data analyzed using the Goldman, Hodgkin-Katz equation. The sodium to potassium permeability ratio (P _Na/P _K) was 0.044, and the chloride to potassium permeability ratio (P _Cl/P _K) was 1.17. Since K⁺ was not in electrochemical equilibrium, intracellular (K⁺) is maintained by active metabolic processes, and the basolateral membrane potential is a diffusion potential with K⁺ and Cl^– the most permeable ions. After depolarizing the basolateral membrane with high serosal potassium bathing solutions and eliminating the apical membrane as a rate limiting step for ion movement using the polyene antibiotic nystatin, we found that the addition of equal aliquots of NaCl to both solutions caused the basolateral membrane potential to hyperpolarize by up to 20 mV (cell interior negative). This popential was reduced by 80% within 3 min of the addition of ouabain to the serosal solution. This hyperpolarization most probably represents a ouabain sensitive active transport process sensitive to intracellular Na⁺. An equivalent electrical circuit for Na⁺ transport across rabbit urinary bladder is derived, tested, and compared to previous results. This circuit is also used to predict the effects that microelectrode impalement damage will have on individual membrane potentials as well as time-dependent phenomena; e.g., effect of amiloride on apical and basolateral membrane potentials.     

Electrical characteristics of stomatal guard cells: The contribution of ATP-dependent, “Electrogenic” transport revealed by current-voltage and difference-current-voltage analysis

Summary The steady-state, current-voltage (I–V) characteristics of stomatal guard cells fromVicia faba L. were explored by voltage clamp using conventional electrophysiological techniques, but with double-barrelled microelectrodes containing 50mm K⁺-acetate. Attention was focused, primarily, on guard cell response to metabolic blockade. Exposures to 0.3–1.0mm NaCN and 0.4mm salicylhydroxamic acid (SHAM) lead consistently to depolarizing (positive-going) shifts in guard cell potentials (V _m ), as large as +103 mV, which were generally complete within 60–90 sec (mean response half-time, 10.3±1.7 sec); values forV _m in NaCN plus SHAM were close or positive to –100 mV and well removed from the K⁺ equilibrium potential. Guard cell ATP content, which was followed in parallel experiments, showed a mean half-time for decay of 10.8±1.9 ([ATP] _t=0, 1.32±0.28mm; [ATP] _{t=60–180sec}, 0.29±0.40mm). In respiring cells, theI–V relations were commonly sigmoid aboutV _m or gently concave to the voltage axis positive toV _m . Inward- and outward-rectifying currents were also observed, especially near the voltage extremes (nominally –350 and +50 mV). Short-circuit currents (atV=0 mV) were typically about 200–500 mA m^–2. The principal effect of cyanide early on was to linearize theI–V characteristic while shifting it to the right along the voltage axis, to decrease the membrane conductance, and to reduce the short-circuit current by approx. 50–75%. The resulting difference-current-voltage (dI–V) curves (±cyanide) showed a marked sensitivity to voltages negative from –100 mV and, when clamp scans had been extended sufficiently, they revealed a distinct minimum near –300 mV before rising at still more negative potentials. The difference currents, along with changes in guard cell potential, conductance and ATP content are interpreted in context of a primary, ATP-consuming ion pump. FittingdI–V curves to reaction kinetic model for the pump [Hansen, U.-P., et al. (1981)J. Membrane Biol. 63:165; Blatt, M.R. (1986)J. Membrane Biol. 92:91] implicates a stoichiometry of one (+) charge transported outward for each ATP hydrolyzed, with pump currents as high as 200 mA m^–2 at the free-running potential. The analysis indicates that the pump can comprise more than half of the total membrane conductance and argues against modulations of pump activity alone, as an effective means to controlling K⁺ transport for stomatal movements.     

Whole-cell and single-channel currents across the plasmalemma of corn shoot suspension cells

Summary Whole-cell sealed-on pipettes have been used to measure electrical properties of the plasmalemma surrounding protoplasts isolated from Black Mexican sweet corn shoot cells from suspension culture. In these protoplasts the membrane resting potential (V _m ) was found to be –59±23 mV (n=23) in 1mm K _o ^– . The meanV _m became more negative as [K^–] _o decreased, but was more positive than the K⁺ equilibrium potential. There was no evidence of electrogenic pump activity. We describe four features of the current-voltage characteristic of the plasmalemma of these protoplasts which show voltagegated channel activity. Depolarization of the whole-cell membrane from the resting potential activates time- and voltage-dependent outward current through K⁺-selective channels. A local minimum in the outward current-voltage curve nearV _m =150 mV suggests that these currents are mediated by two populations of K⁺-selective channels. The absence of this minimum in the presence of verapamil suggests that the activation of one channel population depends on the influx of Ca²⁺ into the cytoplasm. We identify unitary currents from two K⁺-selective channel populations (40 and 125 pS) which open when the membrane is depolarized; it is possible that these mediate the outward whole-cell current. Hyperpolarization of the membrane from the resting potential produces time- and voltage-dependent inward whole-cell current. Current activation is fast and follows an exponential time course. The current saturates and in some cases decreases at membrane potentials more negative than –175 mV. This current is conducted by poorly selective K⁺ channels, whereP _Cl/P _K=0.43±0.15. We describe a low conductance (20 pS) channel population of unknown selectivity which opens when the membrane is hyperpolarized. It is possible that these channels mediate inward whole-cell current. When the membrane is hyperpolarized to potentials more negative than –250 mV large, irregular inward current is activated. A third type of inward whole-cell current is briefly described. This activates slowly and with a U-shaped current-voltage curve over the range of membrane potentials –90<V _m <0 mV.