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).     

Regulation of intracellular chloride activity during perfusion with hypertonic solutions in theNecturus proximal tubule

Summary In a previous study we presented evidence that chloride transport across the basolateral membrane inNecturus proximal tubule cells occurs predominantly via exchange for both Na⁺ and HCO ₃ ^– . In this study the regulation of intracellular chloride was further examined in the doubly-perfused kidney preparation using conventional and chloride-sensitive microelectrodes. Application of hypertonic basolateral solutions containing 80mm raffinose stimulated an efflux of chloride such that chloride activity remained unchanged at control levels. Membrane potential did not change in these experiments. Inhibition of Cl^– exit across the basolateral cell membrane by removal of either HCO ₃ ^– or Na⁺ from the perfusion solution resulted in a significant increase in intracellular chloride activity,a _Cl ⁱ , when basolateral osmolarity was raised. Hypertonic basolateral solutions also produced a significant rise ina _Cl ⁱ in the presence of SITS.This study provides further evidence that chloride is transported across the basolateral cell membrane in exchange for both Na⁺ and HCO ₃ ^– . Since this exchange mechanism is activated in response to hypertonic solutions, these studies suggest a functional role for this exchanger in the regulation ofa _Cl ⁱ in theNecturus proximal tubule cell during volume changes.     

pH-Dependent electrical properties and buffer permeability of theNecturus renal proximal tubule cell

Summary Necturus kidneys were perfused with Tris-buffered solutions at three different pH values, i.e. 7.5, 6.0 and 9.0. A significant drop in fluid absorption occurred at pH 6.0, whereas pH 9.0 did not increase volume flow significantly. When acute unilateral, i.e. either in the lumen or the peritubular capillaries, and bilateral pH changes were elicited in both directions from 7.5 to 9.0 at a constant Tris-butyrate buffer concentration, both peritubular membrane potential differenceV ₁ and transepithelial potential differenceV ₃ hyperpolarized, independently of the side where the change in pH was brought about. Acid perfusions at pH 6.0 caused a similar response but of opposite sign. Analysis of the potential changes shows that pH influences not only the electromotive force and resistance of the homolateral membrane, but also the electrical properties of the paracellular path. Interference of pH with Na, Cl or K conductance was assessed. Any appreciable role for sodium or chloride was excluded, whereas the potassium transference number (t _K) of the peritubular membrane increased 16% in alkaline pH. However, this increase accounts only for 19 to 36% of the observed hyperpolarization. Since changes in Tris-butyrate buffer concentration at constant pH do not affect V₁ or V₃ considerably, the hyperpolarization in pH 9 cannot be explained by an elevation in internal pH only, or by a Tris-H⁺ ion diffusion potential only. The role of the permeability of the buffers: bicarbonate, butyrate and phosphate, in determining electrical membrane parameters was evaluated. Transport numbers of the buffer anions ranked as follows:t _HCO3>t _butyrate>t _phosphate. It is concluded that modulation of membrane potential by extracellular pH is mediated primarily by a change in peritubular cell membranet _K and additionally by membrane currents carried by buffer anions.     

Cell pH and luminal acidification inNecturus proximal tubule

Summary Cellular potential and pH measurements (pH _i ) were carried out in the perfused kidney ofNecturus on proximal tubules with standard and recessed-tip glass microelectrodes under control conditions and after stimulation of tubular bicarbonate reabsorption. Luminal pH and net bicarbonate reabsorption were measured in parallel experiments with recessed-tip glass or antimony electrodes, both during stationary microperfusions as well as under conditions of isosmotic fluid transport. A mean cell pH of 7.15 was obtained in control conditions. When the luminal bicarbonate concentration was raised to 25 and 50mm, pH _i rose to 7.44 and 7.56, respectively. These changes in pH _i were fully reversible. Under all conditions intracellular H⁺ was below electrochemical equilibrium. Thus the maintenance of intracellular pH requires active H⁺ extrusion across one or both of the cell membranes. The observed rise in pH _i and the peritubular depolarization after stimulation of bicarbonate reabsorption are consistent with enhanced luminal hydrogen ion secretion and augmentation of peritubular bicarbonate exit via an anion-conductive transport pathway.     

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.     

Electrical properties of chloride transport across theNecturus proximal tubule

Summary The chloride conductance of the basolateral cell membrane of theNecturus proximal tubule was studied using conventional and chloride-sensitive liquid ion exchange microelectrodes. Individual apical and basolateral cell membrane and shunt resistances, transepithelial and basolateral, cell membrane potential differences, and electromotive forces were determined in control and after reductions in extracellular Cl^–. When extracellular Cl^– activity is reduced in both apical and basolateral solutions the resistance of the shunt increases about 2.8 times over control without any significant change in cell membrane resistances. This suggests a high Cl^– conductance of the paracellular shunt but a low Cl^– conductance of the cell membranes. Reduction of Cl^– in both bathing solutions or only on the basolateral side hyperpolarizes both the basolateral cell membrane potential difference and electromotive force. Hyperpolarization of the basolateral cell membrane potential difference after low Cl^– perfusion was abolished by exposure to HCO ₃ ^– -free solutions and SITS treatment. In control conditions, intracellular Cl^– activity was significantly higher than predicted from the equilibrium distribution across both the apical and basolateral cell membranes. Reducing Cl^– in only the basolateral solution caused a decrease in intracellular Cl^–. From an estimate of the net Cl^– flux across the basolateral cell membrane and the electrochemical driving force, a Cl^– conductance of the basolateral cell membrane was predicted and compared to measured values. It was concluded that the Cl^– conductance of the basolateral cell membrane was not large enough to account for the measured flux of Cl^– by electrodiffusion alone. Therefore these results suggest the presence of an electroneutral mechanism for Cl^– transport across the basolateral cell membrane of theNecturus proximal tubule cell.     

Cellular and paracellular resistances of theNecturus proximal tubule

Summary Individual resistances of the apical cell membrane,R _a , the basolateral cell membrane,R _bl , and the paracellular shunt,R _s , were determined in theNecturus proximal tubule using a set of three electrical parameters. Four electrical parameters were measured: the transepithelial resistance, (R _te ), the apical and basolateral cell membrane resistance in parallel, (R _Z free-flow tubules), the basolateral cell membrane resistance in oil-filled tubules, (R _Z oil-filled), and the ratio of apical and basolateral cell membrane resistance (R _a /R _bl ).R _te was determined from an analysis of the spatial decay of luminal voltage following luminal current injection.R _Z free-flow andR _Z oil-filled were measured by the analysis of the spatial decay of intracellular voltage deflections following cellular current injection in free flow and oil-filled tubules, respectively.R _a /R _bl was estimated from the ratio of voltage deflections across the apical and basolateral cell membranes following transepithelial current injection. In addition, the magnitude of cellular and luminal cable interactions was evaluated, by comparing the spatial decay of voltage deflections in the cell and in the lumen following intracellular current injection. The combined cell membrane resistance (R _a +R _bl ) is between one to two orders of magnitude greater than the paracellular resistance. This result supports the view that theNecturus proximal tubule is a leaky epithelium.     

Phosphate transport across the basolateral membrane of chick kidney proximal tubule cells

Characterization of the phosphate transport system across the basolateral membrane of renal proximal tubule has been attempted using isolated proximal tubule cells prepared from chicks. The Pi efflux system is independent of Na+ ions and is not influenced by the nature of the chief anion present in the bathing medium. Pi efflux is not sensitive to DIDS and it is concluded that a generalized anion transporter of band III type is not the chief agent for facilitating Pi exit from the cell across the basolateral membrane. Inhibition of efflux by vanadate is evidence for a specific carrier protein in the membrane. The carrier probably possesses thiol group(s) that are essential for activity. The carrier may effect electroneutral transport of Pi possibly in exchange for OH- ions. The activity of the transport process is not stimulated by depleting the cells of phosphate or inhibited by rearing the chicks on a vitamin D-deficient diet. The system is unlikely to be of great importance for the expression of various regulatory mechanisms that act on the kidney to control the excretion of Pi. The activity declines as the chicks mature however.     

Cytotoxic effects of FK506 on human renal proximal tubule cells in culture

Summary FK506 has been used as the primary immunosuppressive agent administered after a variety of organ transplants, with less reported nephrotoxicity than that of cyclosporine. This study examined in vitro cytotoxicity of FK506 on normal human renal proximal tubule cells. Cytotoxicity was assessed by neutral red inclusion and trypan blue exclusion; morphology was assessed by light and transmission electron microscopy. Neutral red inclusion decreased to less than 10% of the control after 3 days exposure to 200μg/ml FK506. Forty microgram per milliliter FK506 caused a decrease in neutral red inclusion to 61% of the control on Day 7, with recovery to 86% on Day 12. Similarly, trypan blue exclusion decreased to 66% of the control following 7 days exposure to 40μg/ml FK506, and confluency of the monolayer was reduced to 50% as evidenced by phase contrast microscopy. After a 12-day exposure, treated monolayers became more confluent. On ultrastructural examination, FK506-treated cells exhibited increased cytoplasmic vacuolation and lipid inclusion. These data suggest that FK506 is reversibly and mildly toxic to monolayers of human renal proximal tubule cells and are consistent with clinical reports of reversible nephrotoxicity.     

Cyclic AMP-induced changes in membrane conductance ofNecturus gallbladder epithelial cells

Summary Enhanced cellular cAMP levels have been shown to increase apical membrane Cl^– and HCO ₃ ^– conductances in epithelia. We found that the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) increases cAMP levels inNecturus gallbladder. We used conventional open-tip and double-barreled Cl^–-selective microelectrodes to study the effects of IBMX on membrane conductances and intracellular Cl^– activities in gallbladders mounted in a divided chamber and bathed with Ringer's solutions at 23°C and pH 7.4. In HCO ₃ ^– -free media, 0.1mM IBMX added to the mucosal medium depolarized the apical membrane potentialV _a , decreased the fractional resistanceF _R , and significantly reduced intracellular Cl^– activity (a _Cl ⁱ ). Under control conditions,a _Cl ⁱ was above the value corresponding to passive distribution across the apical cell membrane. In media containing 25mM HCO ₃ ^– , IBMX caused a small transient hyperpolarization ofV _a followed by a depolarization not significantly different from that observed in HCO ₃ ^– -free Ringer's. Removal of mucosal Cl^–, Na⁺ or Ca²⁺ did not affect the IBMX-induced depolarization inV _a . The basolateral membrane ofNecturus gallbladder is highly K⁺ permeable. Increasing serosal K⁺ from 2.5 to 80mM, depolarizedV _a . Mucosal IBMX significantly reduced this depolarization. Addition of 10mM Ba²⁺, a K⁺ channel blocker, to the serosal medium depolarizedV _a and, essentially, blocked the depolarization induced by IBMX. These results indicate that mucosal IBMX increases apical HCO ₃ ^– conductance and decreases basolateral K⁺ conductance in gallbladder epithelial cells via a cAMP-dependent mechanism. The latter effect, not previously reported in epithelial tissues, appears to be the major determinant of the IBMX-induced depolarization ofV _a .     

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.     

Time- and dose-dependent effects of protein kinase C on proximal bicarbonate transport

Summary Activation of protein kinase C has been shown to cause both stimulation and inhibition of transport processes in the brush-border membrane and renal tubule. This study was designed to examine the dose-response nature and time-dependent effect of 4 -phorbol-12-myristate-13-acetate (PMA) on the rates of bicarbonate absorption (J _HCO3) and fluid absorption (J _v) in the proximal convoluted tubule (PCT) of rat kidney. Bicarbonate flux was determined by total CO₂ changes between the collected fluid and the original perfusate as analyzed by microcalorimetry. Luminal perfusion of PMA (10^–10 10^–5 M) within 10 min caused a significant increase ofJ _HCO3 andJ _v. A peaked curve of the dose response was observed with maximal effect at 10^–8 M PMA on both bicarbonate and fluid reabsorption, which could be blocked completely by amiloride (10^–3 m) and EIPA (10^–5 M). On the other hand, with an increase of perfusion time beyond 15 min, PMA (10^–8 and 10^–6 M) could inhibitJ _HCO3 andJ _v. Amiloride (10^–3 M) or EIPA (10^–5 M) significantly inhibitsJ _HCO3 andJ _v, while there is no additive effect of PMA and amiloride or EIPA on PCT transport. An inactive phorbol-ester, 4-phorbol, that does not activate protein kinase C, had no effects onJ _HCO3 andJ _v. Capillary perfusion of PMA (10^–8 M) significantly stimulate bothJ _HCO3 andJ _v; however, PMA did not affect glucose transport from either the luminal side or basolateral side of the PCT. These results indicate that activation of endogenous protein kinase C by PMA could either stimulate or inhibit both bicarbonate and fluid reabsorption in the PCT dependent on time and dose, and these effects are through the modulation of Na⁺/H^– exchange mechanism.     

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.     

Variation in the electrical properties of cultured human proximal tubule cells

Summary Monolayers of human proximal tubule (HPT) cells, when grown on permeable supports and mounted in Ussing chambers, spontaneously display a transepithelial potential difference (PD), short-circuit current (Isc), and transepithelial specific resistance (R_T). These electrical parameters were used to determine the degree of heterogeneity among independent isolates of human proximal tubule cell cultures. Seventeen independent isolates of cells were assessed, totaling 260 individual determinations of spontaneous electrical properties. On average, these cell monolayers displayed an apicalnegative PD of 1.5 ± 0.1 mV, an Isc of 2.7 ± 0.2 μA/cm², and an R_T of 480 ± 19 ohms × cm². Each independent cell isolate, however, displayed electrical values within a narrow range, in some cases allowing isolates to be distinguished from one another. The individual isolates were also assessed for Na-coupled glucose transport, Na⁺,K⁺-ATPase activity, cAMP stimulation by parathyroid hormone (PTH), forskolin stimulation of Isc, and ouabain inhibition. With the exception of a strong correlation between Na⁺,K⁺-ATPase activity and Isc, these parameters, in contrast to electrical properties, were found to be consistent and did not reveal distinctions among the isolates. HPT cell cultures seem to consistently retain important features of proximal tubule differentiation while maintaining the variability, as demonstrated by electrical properties, that might be expected of cells isolated from a random population.     

Electrophysiology ofNecturus urinary bladder: II. Time-dependent current-voltage relations of the basolateral membranes

Summary As reported previously (S.R. Thomas et al.,J. Membrane Biol. 73:157–175, 1983) the current-voltage (I–V) relations of the Na-entry step across the apical membrane of short-circuitedNecturus urinary bladder in the presence of varying mucosal Na concentrations are (i) time-independent between 20–90 msec and (ii) conform to the Goldman-Hodgkin-Katz constant field flux equation for a single cation over a wide range of voltages.In contrast, theI–V relations of the basolateral membrane under these conditions are (i) essentially linear between the steady-state, short-circuited condition and the reversal potential (E ^s ); and (ii) are decidedly time-dependent withE ^s increasing and the slope conductance,E ^s , decreasing between 20 and 90 msec after displacing the transepithelial electrical potential difference. Evidence is presented that this time-dependence cannot be attributed entirely to the electrical capacitance of the tissue.The values ofg ^s determined at 20 msec are linear functions of the short-circuit current,I _sc, confirming the relations reported previously, which were obtained using a more indirect approach.The values ofE ^s determined at 20 msec are significantly lower than any reasonable estimate of the electromotive force for K across the basolateral membrane, indicating that this barrier possesses a significant conductance to other ions which may exceed that to K. In addition, these values increase linearly with decreasingI _sc and approach the value of the electrical potential difference across the basolateral membrane observed when Na entry across the apical membrane is blocked with amiloride or when Na is removed from the mucosal solution.A possible explanation for the time-dependence ofE ^s andg ^s is offered and the implications of these findings regarding the interpretation of previous microelectrophysiologic studies of epithelia are discussed.     

Electron microscopical observations on the brush border of proximal tubule cells of mammalian kidney

Summary The ultrastructure of the plasma membrane and the core of microvilli of proximal tubule cells has been investigated by electron microscopy using sectioned and negatively stained material. By the technique of negative staining, a particulated coat is disclosed on the outside of the plasma membrane of microvilli of brush borders isolated from rat, rabbit and ox. This coat is composed of 30 to 60 Å particles and is 150 to 300 Å thick and appears to be a distinguishing feature for the luminal plasma membrane (brush border) of proximal tubule cells. The plasma membrane of the basal part of tubule cells is found to be smooth. By thin sectioning, an axial bundle of 50 to 70 Å diameter filaments regularly arranged in an 1+6 configuration, one axially located filament being surrounded by a ring of six, is disclosed. The distance from the ring of filaments to the inner surface of the plasma membrane is 250–300 Å, the diameter of the ring 300 Å and the center-to-center distance between filaments 120 Å. Negative staining also discloses 60 Å filaments in microvilli of isolated brush borders. Broken off, single microvilli (fingerstalls) are observed with thin filaments projecting from their broken ends. Filaments up to 1 in length are seen. At high magnification, the filaments appear beaded and show strong resemblance with actin filaments isolated from skeletal muscle. Based on present evidence, it is postulated that microvilli constituting renal brush borders possess contractile properties, which may play a role in the absorption process operating at the luminal part of the cells.The authors are indebted to Miss Kirsten Sjöberg for skilled technical assistance, and to the Danish State Research Foundation and the Tuborg Foundation for financial support.     

Normal rat kidney proximal tubule cells in primary and multiple subcultures

Summary Anin vitro model to establish primary and subcultures of rat kidney proximal tubule (RPT) cells is described. After excising the kidneys and separating the cortex, the cortical tissue is digested with the enzyme DNAse-collagenase (Type I) resulting in a high yield of viable RPT Cells. The isolated RPT cells are then seeded onto rat tail collagen-coated surfaces and grown to confluency in a serum-free, hormonally defined medium. The cell yield can be increased by transfering the conditioned medium on Day 1 to more rat tail collagen-coated surfaces. RPT cell attachment and morphology was better on rat tail collagen-coated surfaces than on bovine collagen Type I coated surfaces. The culture medium was a 1∶1 mixture of Ham’s F-12 and Dulbecco’s modified Eagle’s medium supplemented with bovine serum albumin, insulin, transferrin, selenium, hydrocortisone, triiodothyronine, epidermal growth factor, and glutamine. The RPT cells became confluent in 7–10 d, at which point they could be subcultured by trypsinizing and growth in the same medium. In some studies, 10 ng/ml cholera toxin was added to the culture medium. We could passage the RPT cells up to 14 times in the presence of cholera toxin. The cells were investigated for activity of several markers. The cells were histochemically positive for alkaline phosphatase and γ-glutamyl transpeptidase activity and synthesized the intermediate filament pankeratin. The RPT cells displayed apically directed sodium-dependent active glucose transport in culture. Hence, the RPT cells retain structural and functional characteristics of transporting renal epithelia in culture. This rat cell culture model will be a valuable tool for substrate uptake and nephrotoxicity studies.     

Voltage dependence of the basolateral membrane conductance in theAmphiuma collecting tubule

Summary The basolateral potassium conductance of cells of most epithelial cells plays an important role in the transcellular sodium transport inasmuch as the large negative equilibrium potential of potassium across this membrane contributes to the electrical driving force for Na⁺ across the apical membrane. In the present study, we have attempted to establish, theI-V curve of the basolateral membrane of theAmphiuma collecting tubule, a membrane shown to be K⁺ selective. TransepithelialI-V curves were obtained in short, isolated perfused collecting tubule segments. The shunt conductance was determined using amiloride to block the apical membrane Na⁺ conductance. In symmetrical solutions, the shuntI-V curve was linear (conductance: 2.2±0.3 mS·cm^–2). Transcellular current was calculated by subtracting the shunt current from the transepithelial current in the absence of amiloride. Using intracellular microelectrodes, it was then possible to measure the basolateral membrane potential simultaneously with the transcellular current. The basolateral conductance was found to be voltage dependent, being activated by hyperpolarization: conductance values at –30 and –80 mV were 3.6±1.0 and 6.6±1.0 mS·cm^–2, respectively. BasolateralI-V curves were thus clearly different from that predicted by the constant field model. These results indicate that the K⁺-selective basolateral conductance of an amphibian collecting tubule shows inward (anomalous) rectification. Considering the electrogenic nature basolateral Na–K-pump, this may account for coupling between pump-generated potential and basolateral K⁺ conductance.     

Noise analysis of the K+ current through the apical membrane ofNecturus gallbladder

Summary Current noise power spectra of the voltage-clamped (V=0)Necturus gallbladder, exposed to NaCl-Ringer's on both sides contained a relaxation noise component, which overlapped with a 1/f noise component, with being about 2. Substitution of all Na⁺ by K⁺ on either the serosal or mucosal side increased the relaxation as well as the 1/f noise component considerably. InNecturus gallbladder both noise components are reduced by addition of 10mm 2,4,6-triaminopyrimidine (TAP) or 5mm of tetraethylammonium (TEA⁺) added to ification of the mucosal solution to pH 5 and lower. Fivemm of tetraethylammonium (TEA⁺) added to the mucosal solution, abolished K⁺ relaxation noise and decreased the 1/f noise component. Applying a Cs⁺ concentration gradient across the epithelium did not yield relaxation noise. However, if Rb⁺ was substituted for all Na⁺ on one side, a Lorentzian noise component appeared in the spectrum. Its plateau was smaller than with KCl-Ringer's on the respective side. These data confirm the existence of fluctuating K⁺ channels in the apical membrane of theNecturus gallbladder. Furthermore it can be concluded that these channels have the permeability sequence K⁺>Rb⁺>Sc⁺. The inhibition of the fluctuations by mucosal acidification indicates the existence of acidic sites in the channel. The single-channel conductance was estimated to be between 6.5 and 40 pS.     

Regulation of gluconeogenesis in swine kidney proximal tubule cells

Summary Proximal tubule cells were isolated from swine kidney and cultured for periods of more than 30 days. The cells formed confluent monolayers after plating on a collagen surface and they were passaged more than 5 times on this matrix. The cells maintain several metabolic functions of proximal tubule cells, including gluconeogenesis and the ability to respond to epinephrine and parathyroid hormone. Gluconeogenesis, a principal metabolic pathway in proximal tubule cells, was examined as a function of days in culture. The isolated cells showed a nearly constant rate of gluconeogenesis from ¹⁴C-lactate, ¹⁴C-alkaine and 14C-glycerol with no significant loss of activity for at least 30 days in culture. Likewise, the activities of several cytosolic and membrane associated enzymes including, alkaline phosphatase, -glutamyltransferase, fructose-1,6-bisphosphatase and phosphofructokinase were nearly constant over the same time period.The cells responded to treatment with epinephrine and parathyroid hormone, and the rate of gluconeogenesis from ¹⁴C-lactate doubled in the presence of these hormones. The morphological and biochemical evidence obtained in these studies show that the proximal tubule cells isolated from swine kidney provide an excellent well defined system for studying the hormonal regulation of carbohydrate metabolism in this tissue.Abbreviations PTH Parathyroid Hormone - cAMP cyclic 3,5-adenosine Monophosphate