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.     

Coupled Na+/H+ exchange in rat parotid basolateral membrane vesicles

Summary pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H⁺ gradient (pH_in=6.0, pH_out=8.0)²²Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pH_in=pH_out=6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K ₁ =1.6 m) while the transport inhibitors furosemide (1mm), bumetanide (1mm) SITS (0.5mm) and DIDS (0.1mm) were without effect. This transport activity copurified with the basolateral membrane marker K⁺-stimulatedp-nitrophenyl phosphatase. In addition²²Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H⁺ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration cosistent with the existence of a single transport system withK _M =8.0mm at 23°C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na⁺/H⁺ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion.     

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).     

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).     

Effect of potassium on cell volume regulation in renal straight proximal tubules

Summary The present study was designed to assess for the influence of extracellular potassium and of inhibitors of potassium transport on cell volume regulatory decrease in isolated perfused straight proximal tubules of the mouse kidney. Volume regulatory decrease is virtually unaffected when bath potassium concentration is elevated from 5 to 20 mmol/liter, and still persists, albeit significantly retarded, in the presence of the potassium channel blocker barium on both sides of the epithelium and during virtually complete dissipation of the transmembrane potassium gradient by increasing extracellular potassium concentration to 40 mmol/liter. As evident from electrophysiologic observations, barium blocks the potassium conductance of the basolateral cell membrane. Reduction of bicarbonate concentration and increase of H⁺ concentration in the bath solution cannot compensate for enhanced potassium concentration and cell volume regulatory decrease is not affected in the presence of the K/H exchange inhibitor omeprazole. Similarly cell volume regulatory decrease is not affected by ouabain. In conclusion, potassium movements through potassium channels in the basolateral cell membrane are important determinants of cell volume and may participate in cell volume regulatory decrease. However, a powerful component of cell volume regulatory decrease in straight proximal tubules of the mouse kidney is apparently independent of potassium conductive pathways, K/H exchange and Na⁺/K⁺-ATPase.     

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.     

Transepithelial Na+ transport and the intracellular fluids: A computer study

Summary Computer simulations of tight epithelia under three experimental conditions have been carried out, using the rheogenic nonlinear model of Lew, Ferreira and Moura (Proc. Roy. Soc. London. B 206:53–83, 1979) based largely on the formulation of Koefoed-Johnsen and Ussing (Acta Physiol. Scand.42:298–308, 1958). First, analysis of the transition between the short-circuited and open-circuited states has indicated that (i) apical Cl^– permeability is a critical parameter requiring experimental definition in order to analyze cell volume regulation, and (ii) contrary to certain experimental reports, intracellular Na⁺ concentration (c _Na ^c ) is expected to be a strong function of transepithelial clamping voltage. Second, analysis of the effects of lowering serosal K⁺ concentration (c _K ^s ) indicates that the basic model cannot simulate several well-documented observations; these defects can be overcome, at least qualitatively, by modifying the model to take account of the negative feedback interaction likely to exist between the apical Na⁺ permeability andc _Na ^c . Third, analysis of the effects induced by lowering mucosal Na⁺ concentration (c _Na ^m ) strongly supports the concept that osmotically induced permeability changes in the apical intercellular junctions play a physiological role in conserving the body's stores of NaCl. The analyses also demonstrate that the importance of Na⁺ entry across the basolateral membrane is strongly dependent upon transepithelial potential,c _Na ^m andc _K ^s ; under certain conditions, net Na⁺ entry could be appreciably greater across the basolateral than across the apical membrane.     

Characterization of ATP-dependent Ca2+ transport in the basolateral membrane vesicles from proximal and distal tubules of the rabbit kidney

Basolateral membrane vesicles were prepared from purified proximal and distal tubules of the rabbit kidney. The properties of the ATP-dependent Ca2+ transport were investigated. In both membranes, there was a high affinity, ATP-dependent Ca2+ transport system (Km = 0.1 microM). The optimal concentration of Mg2+ was 0.5 mM and the optimal concentration of ATP was 1 mM. The nucleotide specificity and pH dependence of the Ca2+ transport in both membranes were similar. In basolateral membrane vesicles, calmodulin had no effect on Ca2+ transport. However, in basolateral membrane vesicles depleted of calmodulin, exogenous calmodulin increased the Ca2+ transport by increasing maximal velocity. There were no major differences in the properties of the ATP-dependent Ca2+ transport system in these two membranes. These findings are discussed in relation to why parathyroid hormone differentially modulates Ca2+ transport in these two segments of the nephron.     

Basolateral membrane potassium conductance is independent of sodium pump activity and membrane voltage in canine tracheal epithelium

Summary When secretagogues stimulate Cl secretion in canine tracheal epithelium, apical membrane Cl conductance (G _a ^Cl ) increases, and then basolateral membrane K conductance (G _b ^K ) increases. Conversely, inhibition ofG _a ^Cl results in a secondary decrease inG _b ^K . The coordination of the two membrane conductances and regulation ofG _b ^K is critical for maintaining constant intracellular ion concentrations and transepithelial Cl secretion. The purpose of this study was to test two hypotheses about the regulation ofG _b ^K . First, we asked whetherG _b ^K is directly linked to the activity of the Na,K-ATPase. We found that pump activity could be dissociated from K conductance. Inhibition of the Na pump with ouabain, in nonsecreting tissues led to an increase inG _b . Elevation of the bathing solution K concentration produced a similar effect. Addition of ouabain to secreting tissues did not appear to alterG _b . These results indicate thatG _b ^K does not directly parallel Na pump activity. Second, we asked whether changes inG _b ^K are voltage dependent. We prevented secretagogue-induced depolarization of the electrical potential difference across the basolateral membrane _b by clamping _b at its resting value during stimulation of Cl secretion with epinephrine. Despite maintaining _b constant, the typical changes in transepithelial resistance and the ratio of membrane resistances persisted. This observation indicates that depolarization is not required for the secretagogue-induced increase inG _b ^K . In addition we examined the effect of depolarizing and hyperpolarizing _b by passing transepithelial current in secreting and nonsecreting epithelia. Despite depolarizing and hyperpolarizing _b within the physiologic range, we observed no significant changes in transepithelial resistance or the ratio of membrane resistance that would suggest a change inG _b ^K . This observation indicates that changes in _b are not sufficient to alterG _b ^K . Thus,G _b ^K appears to be regulated by factors other than membrane voltage, or direct coupling to the Na pump.     

Na+ and K+ transport at basolateral membranes of epithelial cells. III. Voltage independence of basolateral membrane Na+ efflux

Na+ efflux across basolateral membranes of isolated epithelia of frog skin was tested for voltage sensitivity. The intracellular Na+ transport pool was loaded with 24Na from the apical solution and the rate of isotope appearance in the basolateral solution (JNa23) was measured at timed intervals of 30 s. Basolateral membrane voltage was depolarized by either 50 mM K+, 5 mM Ba++, or 80 mM NH+4. Whereas within 30 s ouabain caused inhibition of JNa23, depolarization of Vb by 30-60 mV caused no significant change of JNa23. Thus, both pump-mediated and leak Na+ effluxes were voltage independent. Although the pumps are electrogenic, pump-mediated Na+ efflux is voltage independent, perhaps because of a nonlinear relationship between pump current and transmembrane voltage. Voltage independence of the leak Na+ efflux confirms a previous suggestion (Cox and Helman, 1983. American Journal of Physiology. 245:F312-F321) that basolateral membrane Na+ leak fluxes are electroneutral.     

ATP-dependent calcium transport in rat parotid basolateral membrane vesicles is modulated by membrane potential

Summary The ATP-dependent Ca²⁺ transport activity (T. Takuma, B.L. Kuyatt and B.J. Baum,Biochem. J. 227:239–245, 1985) exhibited by inverted basolateral membrane vesicles isolated from rat parotid gland was further characterized. The activity was dependent on Mg²⁺. Phosphate (5mm), but not oxalate (5mm), increased maximum Ca²⁺ accumulation by 50%. Half-maximal Ca²⁺ transport was achieved at 70nm Ca²⁺ in EGTA-buffered medium while maximal activity required >1 m Ca²⁺ (V _max=54 nmol/mg protein/min). Optimal rates of Ca²⁺ transport were obtained in the presence of KCl, while in a KCl-free medium (mannitol or sucrose) 40% of the total activity was achieved, which could not be stimulated by FCCP. The initial rate of Ca²⁺ transport could be significantly altered by preimposed membrane potentials generated by K⁺ gradients in the presence of valinomycin. Compared to the transport rate in the absence of membrane potential, a negative (interior) potential stimulated uptake by 30%, while a positive (interior) potential inhibited uptake. Initial rates of Ca²⁺ uptake could also be altered by imposing pH gradients, in the absence of KCl. When compared to the initial rate of Ca²⁺ transport in the absence of a pH gradient, pH _i =7.5/pH _o =7.5; the activity was 60% higher in the presence of an outwardly directed pH gradient, pH _i =7.5/pH _o =8.5; while it was 80% lower when an inwardly directed pH gradient was imposed, pH _i =7.5/pH _o =6.2. The data show that the ATP-dependent Ca²⁺ transport in BLMV can be modulated by the membrane potential, suggesting therefore that there is a transfer of charge into the vesicle during Ca²⁺ uptake, which could be compensated by other ion movements.     

Reversal of Na+-dependent glycine transport in sheep reticulocyte membrane vesicles

Inside-out membrane vesicles have been prepared from sheep reticulocytes. With these vesicles, Na⁺-dependent glycine uptake and net accumulation have been demonstrated to occur in reverse, i.e., from extravesicular (normal cytoplasmic) to intravesicular (normal extravesicular) surface. Uptake and accumulation are inhibited by energization of the sodium pump by ATP whereby the Na⁺ electrochemical gradient is dissipated. Glycine-dependent Na⁺ uptake was also observed, providing evidence that Na⁺-dependent glycine influx into these vesicles, equivalent to normal efflux, is characterized by Na⁺-glycine co-transport.     

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.     

Calcium transport across the basolateral membrane of isolated Malpighian tubules: a survey of several insect orders

The Malpighian tubules play a major role in haemolymph calcium homeostasis in insects by sequestering excess Ca²⁺ within the biomineralized granules that often accumulate in the tubule cells and/or lumen. Using the scanning ion‐selective microelectrode technique, measurements of basolateral Ca²⁺ transport are determined at several sites along the length of the Malpighian tubules isolated from the eight insects representing seven orders: Drosophila melanogaster (Diptera), Aedes aegypti (Diptera), Tenebrio molitor (Coleoptera), Acheta domesticus (Orthoptera), Trichoplusia ni (Lepidoptera), Periplaneta americana (Blattodea), Halyomorpha halys (Hemiptera) and Pogonomyrmex occidentalis (Hymenoptera). Ca²⁺ transport is specific to tubule segments containing Ca‐rich granules in D. melanogaster and A. aegypti, whereas Ca²⁺ transport is relatively uniform along the length of whole tubules in the remaining species. Generally, manipulation of second messenger pathways using cAMP and thapsigargin has little effect on rates of basolateral Ca²⁺ transport, suggesting that previous effects observed across midtubules of A. domesticus are unique to this species. In addition, the present study is the first to provide measurements of basolateral Ca²⁺ across single principal and secondary tubule cells, where Ca²⁺ uptake occurs only across principal cells. Estimated times for all tubules to eliminate the entire haemolymph Ca²⁺ content in each insect range from 6 min (D. melanogaster) to 19 h (H. halys) or more, indicating that rates of Ca²⁺ uptake by the Malpighian tubules are not always rapid. The results of the present study suggest that the principal cells of the Malpighian tubules contribute to haemolymph calcium homeostasis by sequestering excess Ca²⁺, often within specific tubule segments.     

Na+-dependent transport of alpha-aminoisobutyrate in isolated basolateral membrane vesicles from rat parotid glands

Basolateral plasma membranes were prepared from rat parotid gland after centrifugation in a self-orienting Percoll gradient. K+-dependent phosphatase [Na+ + K+)-ATPase), a marker enzyme for basolateral membranes, was enriched 10-fold from tissue homogenates. Using this preparation, the transport of alpha-aminoisobutyrate was studied. The uptake of alpha-aminoisobutyrate was Na+-dependent, osmotically sensitive, and temperature-dependent. In the presence of a Na+ gradient between the extra- and intravesicular solutions, vesicles showed an 'overshoot' accumulation of alpha-aminoisobutyrate. Sodium-dependent alpha-aminoisobutyrate uptake was saturable, exhibiting an apparent Km of 1.28 +/- 0.35 mM and Vmax of 780 +/- 170 pmol/min per mg protein. alpha-Aminoisobutyrate transport was inhibited considerably by monensin, but incubating with ouabain was without effect. These results suggest that basolateral membrane vesicles, which possess an active amino acid transport system (system A), can be prepared from the rat parotid gland.     

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.     

Active and passive Na+ fluxes across the basolateral membrane of rabbit urinary bladder

Summary The apical membrane of rabbit urinary bladder can be functionally removed by application of nystatin at high concentration if the mucosal surface of the tissue is bathed in a saline which mimics intracellular ion concentrations. Under these conditions, the tissue is as far as the movement of univalent ions no more than a sheet of basolateral membrane with some tight junctional membrane in parallel. In this manner the Na⁺ concentration at the inner surface of the basolateral membrane can be varied by altering the concentration in the mucosal bulk solution. When this was done both mucosal-to-serosal²²Na flux and net change in basolateral current were measured. The flux and the current could be further divided into the components of each that were either blocked by ouabain or insensitive to ouabain. Ouabain-insensitive mucosal-to-serosal Na⁺ flux was a linear function of mucosal Na⁺ concentration. Ouabain-sensitive Na⁺ flux and ouabain-sensitive, Na⁺-induced current both display a saturating relationship which cannot be accounted for by the presence of unstirred layers. If the interaction of Na⁺ with the basolateral transport process is assumed to involve the interaction of some number of Na⁺ ions,n, with a maximal flux,M _max, then the data can be fit by assuming 3.2 equivalent sites for interaction and a value forM _max of 287.8pm cm^–2 sec^–1 with an intracellular Na concentration of 2.0mm Na⁺ at half-maximal saturation. By comparing these values with the ouabain-sensitive, Na⁺-induced current, we calculate a Na⁺ to K⁺ coupling ratio of 1.40±0.07 for the transport process.