Double dependence of organic acid active transport in proximal tubules of surviving frog kidney on sodium ions II. Relationship between counter-flows of fluorescein and sodium ion across cell layer

With the aid of a direct microfluorimetric method a dependence of organic onion (fluorescein) transport into proximal tubules of surviving frog kidney on Na⁺-flow in the opposite direction was studied. It was shown that the complete removal of Na⁺ from the tubules lumen resulted in inhibition of fluorescein transport of about 30%. After a specific inhibitor of sodium channels, amiloride (10^-3M) having been introduced into lumen of the tubules, the fluorescein transport was inhibited to the same extent. Amiloride affects only when Na⁺ is present in the tubular lumen. S present in the tubular lumen. Strophantin K (5 · 10^?5 M), a specific inhibitor of (Na⁺, K⁺)-ATPase, reduced fluorescein transport about twice. Substances increasing the 3′,5′-AMP level in cells (theophylline, NaF) and exogenous 3′,5′-AMP inhibited fluorescein transport while substance that decreased the 3′,5′-AMP level intracellularly (carbachol) stimulated it. For realization of these effects Na⁺ should be present in proximal tubules lumen.Thus, the various effects on the Na⁺ flow from lumen of the tubules to medium at the level of both the basal and apical membranes alter the rate of organic acid active transport from medium to lumen as a result of changes in the maximum rate of transport ($\text{V}$) with unchanged $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. It is suggested that the system of Na⁺ extrusion from proximal tubules produces peritubular membrane-side (near the membrane) gradient of Na⁺ concentration which may be higher than the summary Na⁺ gradient between the medium and the cytoplasm. The magnitude of this gradient affects the maximal rate value of Na⁺-dependent organic acid transport. So, there is a double dependence of the active transport system on Na⁺, and the stages where Na⁺ is needed are: (1) the formation of a carrier-substrate-Na⁺ complex and (2) the production of substantial membrane-side Na⁺ gradient at the expense of Na⁺ extrusion from the tubules.     

Localization of Na+, K+-ATPase to the inside of the basolateral cell membranes of epithelial cells of proximal and distal tubules in rabbit kidney

Summary Cysteine-sensitive alkaline phosphatase and/or ouabain-sensitive Na⁺, K⁺-ATPase were studied by ultrastructure cytochemistry in epithelial cells of proximal and distal kidney tubules. Alkaline phosphatase reactivity was confined to the surface of the microvillous luminal cell membrane of proximal tubule cells, whereas distal tubules and collecting ducts were unreactive. The Na⁺, K⁺-ATPase reactivity was localized evenly along the cytoplasmic side of the basolateral cell membrane of cells of proximal and distal tubules and in collecting ducts. In the proximal tubules, where the activity was strongest, the Na⁺, K⁺-ATPase deposits were also found in the 10–50 nm gap between the cell membrane and the cisternae of tubulo-cisternal endoplasmic reticulum (TER) underlying a major part of the basolateral cell membrane. The restriction of Na⁺, K⁺-ATPase sites, which are involved in extrusion of Na⁺ from the cell, to a narrow cytoplasmic compartment located between the cell membrane and the cisternae of TER, is consistent with a transport role for the TER.     

Double dependence of organic acid active transport in proximal tubules of surviving frog kidney on sodium ions I. Influence of sodium ions in bath medium on the uptake and run out of fluorescein and uranin

With the aid of direct microfluorimetric determination of marker organic anions (fluorescein and uranin) accumulated in the proximal tubules the influence of Na⁺ in the bath medium on the active transport of these anions was studied. Kinetic analysis of the rate dependence of organic acid active transport into tubules on their concentration in the bath medium with a constant Na⁺ concentration permitted to define values of apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and $\text{V}$ for uranin and fluorescein transport in the medium with different Na⁺ content. It was shown that a decrease of Na⁺ concentration in the medium increases $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and lowers the $\text{V/K}{}_{\mathrm{<mtext>m</mtext>}}$ ratio with uncharged $\text{V}$. By varying the Na⁺ concentration in the medium with a constant concentration of the marker anion the and values for fluorescein and uranin transport were determined. A value for fluorescein in twice as much that for uranin. The $\text{1/K}{}_{\mathrm{<mtext>m</mtext>}}$ value for uranin transport is a linear function of Na⁺ concentration, while for fluorescein transport it is a quadratic one. Therefore it is concluded that two Na⁺ from the medium participate in active transfer of one fluorescein anion whereas only one Na⁺ from the medium is required for active transfer of one uranin anion. The run out of fluorescein from tubules preloaded with this acid is sharply reinforced by the Na⁺ omission from the medium. Thus, active transport of organic acids in proximal tubules of frog kidney is Na⁺-dependent, and Na⁺ from the medium is likely to participate directly in formation of a transport complex. When Na⁺ is absent in the medium a carrier fulfils a facilitated diffusion only.     

Effect of acetate on transport of organic acid (fluorescein) in renal proximal tubules of frog

The effect of acetate on active fluorescein transport in intact proximal tubules of surviving frog kidney was studied. When the kidneys were incubated in a 120 mM Na⁺ medium, 10 mM acetate stimulated fluorescein uptake in the tubules. The stimulation was more pronounced if the kidneys had been previously preincubated for 3 h in the substrate-free solution. Lowering of the Na⁺ concentration in the bathing medium to 10 mM resulted in the disappearance of the acetate effect. Preincubation of the kidneys with acetate at 2–4°C gave rise to stimulation of the fluorescein transport only in the 120 mM Na⁺ acetate-free medium. The acetate effect on the fluorescein uptake was partially prevented by ouabain. The stimulation of the uptake by acetate in the 120 mM Na⁺ medium correlated with an increase in the extent of reduction of pyridine nucleotides in the tubules. The pyridine nucleotides were reduced more markedly after incubation of the kidneys in the 10 mM Na⁺ medium, when acetate had no effect on the fluorescein transport. In both the 120 mM and the 10 mM Na⁺ media, the cold preincubation of the kidneys with 2.5 mM ADP or 2.5 mM ATP resulted in only slight stimulation of the fluorescein uptake. But in both media the uptake was significantly enhanced after cold preincubation of the kidneys with 2 mM NADH. After the cold precincubation with ADP, stimulation of the fluorescein transport by acetate was observed in the case of the 10 mM Na⁺ medium also. The absence of any stimulatory effect of acetate on the organic acid transport in the 10 mM Na⁺ medium is explained as the result of the transformation of mitochondria in the tubular cells into the inactive state 4 due to a decrease in the intracellular ADP level. Reducing equivalents are supposed to take part in energization and/or regulation of transport processes in plasma membranes of the renal proximal tubules.     

On the active transport of organic acids (fluorescein) in the choroid plexus of the rabbit

The kinetics of active transport of an organic acid (fluorescein) through the membranes of the choroid plexus from the lateral ventricules of the brain of rabbit was studied both morphologically and functionally. It was shown that fluorescein is actively translocated through the apical and basal membrane of the epithelium and is accumulated in blood capillaries at a concentration exceeding one order of magnitude that in the incubation medium. The kinetic curves displaying saturation and the demonstration of inhibition by other acids shows that a specific carrier is involved in the transfer across the membrane. The active transport of fluorescein at 20°C was found to be sodium independent. Total exclusion of sodium from the incubation medium does not change the Michaelis constant ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}$) and maximal velocity ($\text{V}$). The active transport depends on the operation of (Na⁺ + K⁺)-ATPase as energy source but obviously no specific complexes with the participation of sodium are involved.     

Coupled transepithelial sodium and potassium transport across isolated frog skin: Effect of ouabain,amiloride and the polyene antibiotic filipin

Summary Addition of the polyene antibiotic filipin (50 m) to the outside bathing solution (OBS) of the isolated frog skin resulted in a highly significant active outward transport of K⁺ because filipinper se increases the nonspecific Na⁺ and K⁺ permeability of the outward facing membrane. The K⁺ transport was calculated from the chemically determined changes in K⁺ concentrations in the solution bathing the two sides of the skin. The active transepithelial K⁺ transport required the presence of Na⁺ in the OBS, but not in the inside bathing solution (IBS), and it was inhibited by the Na⁺, K⁺-ATPase inhibitor ouabain. The addition of Ba⁺⁺ to the IBS in the presence of filipin in the OBS resulted in an activation of the transepithelial K⁺ transport and in an inhibition of the active Na⁺ transport. This is in agreement with the notion that Ba⁺⁺ decreases the passive K⁺ permeability of the inward facing membrane. In the presence of amiloride (which blocks the specific Na permeability of the outward facing membrane) and Ba⁺⁺ there was a good correlation between the active Na⁺ and K⁺ transport. It is concluded that the active transepithelial K⁺ transport is carried out by a coupled electrogenic Na–K pump, and it is suggested that the pump ratio (Na/K) is 1.5.     

Phenylalanine uptake in isolated renal brush border vesicles

The uptake of l-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques.Brush border microvilli but not basolateral plasma membrane vesicles take up l-phenylalanine by an Na⁺-dependent, saturable transport system. The apparent affinity of the transport system for l-phenylalanine is 6.1 mM at 100 mM Na⁺ and for Na⁺ 13 mM at 1 mM l-phenylalanine. Reduction of the Na⁺ concentration reduces the apparent affinity of the transport system for l-phenylalanine but does not alter the maximum velocity.In the presence of an electrochemical potential difference for Na⁺ across the membrane (η_Na0 >η_Na1) the brush border microvilli accumulate transiently l-phenylalanine over the concentration in the incubation medium (overshoot phenomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K⁺ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient.These results indicate that the entry of l-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na⁺ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na⁺ and on the electrical potential difference. The exit of l-phenylalanine across the basolateral plasma membranes is Na⁺-independent and probably involves facilitated diffusion.     

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.     

Silver ion (Ag+)-Induced increases in cell membrane K+ and Na+ permeability in the renal proximal tubule: Reversal by thiol reagents

Summary The initial mechanisms of injury to the proximal tubule following exposure to nephrotoxic heavy metals are not well established. We studied the immediate effects of silver (Ag⁺) on K⁺ transport and respiration with extracellular K⁺ and O₂ electrodes in suspensions of renal cortical tubules. Addition of silver nitrate (AgNO₃) to tubules suspended in bicarbonate Ringer's solution caused a rapid, dose-dependent net K⁺ efflux (K _m =10^–4 m,V _max=379 nmol K⁺/min/mg protein) which was not inhibited by furosemide, barium chloride, quinine, tetraethylammonium, or tolbutamide. An increase in the ouabain-sensitive oxygen consumption rate (QO₂) (13.9±1.1 to 25.7±4.4 nmol O₂/min/mg,P<0.001), was observed 19 sec after the K⁺ efflux induced by AgNO₃ (10^–4 m), suggesting a delayed increase in Na⁺ entry into the cell. Ouabain-insensitive QO₂, nystatin-stimulated QO₂, and CCCP-uncoupled QO₂ were not significantly affected, indicating preserved function of the Na⁺, K⁺-ATPase and mitochondria. External addition of the thiol reagents dithiothreitol (1mm) and reduced glutathione (1mm) prevented and/or immediately reversed the effects on K⁺ transport and QO₂. We conclude that Ag⁺ causes early changes in the permeability of the cell membrane to K⁺ and then to Na⁺ at concentrations that do not limit Na⁺, K⁺-ATPase activity or mitochondrial function. These alterations are likely the result of a reversible interaction of Ag⁺ with sulfhydryl groups of cell membrane proteins and may represent initial cytotoxic effects common to other sulfhydryl-reactive heavy metals on the proximal tubule.     

Solute Transport Process in Intestinal Epithelial Cells

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na⁺ and K⁺ concentrations, significant gains of Na⁺ and K⁺ occurred with glucose transport. The quantitative relationships among net gains of Na⁺, K⁺ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na⁺ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na⁺-efflux and K⁺-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.     

Taurine transport by brush border membrane vesicles isolated from the flounder kidney

Summary Taurine transport was investigated in brush border membrane vesicles isolated from renal tubules of the winter flounder (Pseudopleuronectes americanus). Taurine uptake by the vesicles was greater in the presence of NaCl as compared to uptake in KCl. The Na⁺-dependent taurine transport was electrogenic and demonstrated tracer replacement and inhibition by -alanine and HgCl₂, indicating the presence of Na⁺-dependent, carrier-mediated taurine transport. In contrast to Na⁺-dependent taurine transport across the basolateral membrane, there was not a specific Cl^– dependency for transport in the brush border membrane. No evidence was obtained for Na⁺-independent carrier-mediated taurine transport. The possible involvement of the brush border Na⁺-dependent transport system in the net secretion of taurine from blood to tubular lumen in vivo (Schrock et al. 1982) is discussed.     

Neutral amino acid transport in surface membrane vesicles isolated from mouse fibroblasts: Intrinsic and extrinsic models of regulation

Membrane transport carrier function, its regulation and coupling to metabolism, can be selectively investigated dissociated from metabolism and in the presence of a defined electrochemical ion gradient driving force, using the single internal compartment system provided by vesiculated surface membranes. Vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated transport of several neutral amino acids into an osmotically-sensitive intravesicular space without detectable metabolic conversion of substrate. When a Na⁺ gradient, external Na⁺ > internal Na⁺, was artifically imposed across vesicle membranes, accumulation of several neutral amino acids achieved apparent intravesicular concentrations 6- to 9-fold above their external concentrations. Na⁺-stimulated alanine transport activity accompanied plasma membrane material during subcellular fractionation procedures. Competitive interactions among several neutral amino acids for Na⁺-stimulated transport into vesicles and inactivation studies indicated that at least 3 separate transport systems with specificity properties previously defined for neutral amino acid transport in Ehrlich ascites cells were functional in vesicles from mouse fibroblasts: the A system, the L system and a glycine transport system. The pH profiles and apparent K_m values for alanine and 2-aminoisobutyric acid transport into vesicles were those expected of components of the corresponding cellular uptake system. Several observations indicated that both a Na⁺ chemical concentration gradient and an electrical membrane potential contribute to the total driving force for active amino acid transport via the A system and the glycine system. Both the initial rate and quasi-steady-state of accumulation were stimulated as a function of increasing concentrations of Na⁺ applied as a gradient (external > internal) across the membrane. This stimulation was independent of endogenous Na⁺, K⁺-ATPase activity in vesicles and was diminished by monensin or by preincubation of vesicles with Na⁺. The apparent K_m for transport of alanine and 2-aminoisobutyric acid was decreased as a function of Na⁺ concentration. Similarly, in the presence of a standard initial Na⁺ gradient, quasi-steady-state alanine accumulation in vesicles increased as a function of increasing magnitudes of interior-negative membrane potential imposed across the membrane by means of K⁺ diffusion potentials (internal > external) in the presence of valinomycin; the magnitude of this electrical component was estimated by the apparent distributions of the freely permeant lipophilic cation triphenylme thylphosphonium ion. Alanine transport stimulation by charge asymmetry required Na⁺ and was blocked by the further addition of either nigericin or external K⁺. As a corollary, Na⁺-stimulated alanine transport was associated with an apparent depolarization, detectable as an increased labeled thiocyanate accumulation. Permeant anions stimulated Na⁺-coupled active transport of these amino acids but did not affect Na⁺-independent transport. Translocation of K⁺, H⁺, or anions did not appear to be directly involved in this transport mechanism. These characteristics support an electrogenic mechanism in which amino acid translocation is coupled t o an electrochemical Na⁺ gradient by formation of a positively charged complex, stoichiometry unspecified, of Na⁺, amino acid, and membrane component. Functional changes expressed in isolated membranes were observed t o accompany a change in cellular proliferative state or viral transformation. Vesicles from Simian virus 40-transformed cells exhibited an increased Vmax of Na⁺-stimulated 2-aminoisobutyric acid transport, as well as an increased capacity for steady-state accumulation of amino acids in response t o a standard Na⁺ gradient, relative t o vesicles from nontransformed cells. Density-inhibition of nontransformed cells was associated with a marked decrease in these parameters assayed in vesicles. Several possibilities for regulatory interactions involving gradient-coupled transport systems are discussed.     

Sodium gradient- and sodium plus potassium gradient-dependentl-glutamate uptake in renal basolateral membrane vesicles

Summary A membrane preparation enriched in the basolateral segment of the plasma membrane was isolated from the rat renal cortex by a procedure that included separation of particulates on a self-generating Percoll gradient. The uptake ofl-glutamate by the basolateral membrane vesicles was studied. A Na⁺ gradient ([Na⁺] _o >[Na⁺] _i ) stimulated the uptake ofl-glutamate and provided the driving force for the uphill transport of the acidic amino acid, suggesting a Na⁺-l-glutamate cotransport system in the basolateral membrane. A K⁺ gradient ([K⁺] _i >[K⁺] _o ) increased the uptake additionally. This effect was specific for K⁺ (Rb⁺). The action of the K⁺ gradient in enhancing the uptake ofl-glutamate had an absolute requirement for Na⁺. In the presence of Na⁺, but in the absence of a Na⁺ gradient. i.e., [Na⁺] _o =[Na⁺] _i , the K⁺ gradient also energized the concentrative uptake ofl-glutamate. This effect of the K⁺ gradient was not attributable to an alteration in membrane potential. The finding of a concentrative uptake system forl-glutamate energized by both Na⁺ ([Na⁺] _o >[Na⁺] _i and K⁺ ([K⁺] _i >[K⁺] _o ) gradients in the basolateral membrane, combined with previous reports of an ion gradient-dependent uphill transport system for this amino acid in the brush border membrane, suggests a mechanism by whichl-glutamate is accumulated intracellularly in the renal proximal tubule to extraordinarily high concentrations.     

Sodium and potassium fluxes and compartmentation in roots of atriplex and oat

K⁺ and Na⁺ fluxes and ion content have been studied in roots of Atriplex nummularia Lindl. and Avena sativa L. cv Goodfield grown in 3 millimolar K⁺ with or without 3 or 50 millimolar NaCl. Compartmental analysis was carried out with entire root systems under steady-state conditions.

Increasing ambient Na⁺ concentrations from 0 to 50 millimolar altered K⁺, in Atriplex, as follows: slightly decreased the cytoplasmic content (Q_c), the vacuolar content (Q_v), and the plasma membrane influx and efflux. Xylem transport for K⁺ decreased by 63% in Atriplex. For oat roots, similar increases in Na⁺ altered K⁺ parameters as follows: plasma membrane influx and efflux decreased by about 80%. Q_c decreased by 65%, and xylem transport decreased by 91%. No change, however, was observed in Q_v for K⁺. Increasing ambient Na⁺ resulted in higher (3 to 5-fold) Na⁺ fluxes across the plasma membrane and in Q_c of both species. In Atriplex, Na⁺ fluxes across the tonoplast and Q_v increased as external Na⁺ was increased. In oat, however, no significant change was observed in Na⁺ flux across the tonoplast or in Q_v as external Na⁺ was increased. In oat roots, Na⁺ reduced K⁺ uptake markedly; in Atriplex, this was not as pronounced. However, even at high Na⁺ levels, the influx transport system at the plasma membrane of both species preferred K⁺ over Na⁺.

Based upon the Ussing-Teorell equation, it was concluded that active inward transport of K⁺ occurred across the plasma membrane, and passive movement of K⁺ occurred across the tonoplast in both species. Na⁺, in oat roots, was actively pumped out of the cytoplasm to the exterior, whereas, in Atriplex, Na⁺ was passively distributed between the free space, cytoplasm, and vacuole.

     

In Vivo Study of Transepithelial Potential Difference (TEPD) in Proximal Convoluted Tubules of Rat Kidney by Synchronization Modulation Electric Field

Synchronization modulation (SM) electric field has been shown to effectively activate function of Na⁺/K⁺ pumps in various cells and tissues, including skeletal muscle cells, cardiomyocyte, monolayer of cultured cell line, and peripheral blood vessels. We are now reporting the in vivo studies in application of the SM electric field to kidney of living rats. The field-induced changes in the transepithelial potential difference (TEPD) or the lumen potential from the proximal convoluted tubules were monitored. The results showed that a short time (20 s) application of the SM electric field can significantly increase the magnitude of TEPD from 1–2 mV to about 20 mV. The TEPD is an active potential representing the transport current of the Na/K pumps in epithelial wall of renal tubules. This study showed that SM electric field can increase TEPD by activation of the pump molecules. Considering renal tubules, many active transporters are driven by the Na⁺ concentration gradient built by the Na⁺/K⁺ pumps, activation of the pump functions and increase in the magnitude of TEPD imply that the SM electric field may improve reabsorption functions of the renal tubules.     

Effect of amiloride,ouabain and Ba++ on the nonsteady-state Na−K pump flux and short-circuit current in isolated frog skin epithelia

Summary Effect of amiloride, ouabain, and Ba⁺⁺ on the nonsteady-state Na–K pump flux and short-circuit current in isolated frog skin epithelia.The active Na⁺ transport across isolated frog skin occurs in two steps: passive diffusion across the apical membrane of the cells followed by an active extrusion from the cells via the Na⁺–K⁺ pump at the basolateral membrane. In isolated epithelia with a very small Na⁺ efflux, the appearing Na⁺-flux in the basolateral solution is equal to the rate of the pump, whereas the short-circuit current (SCC) is equal to the active transepithelial Na⁺ transport. It was found that blocking the passive diffusion of Na⁺ across the apical membrane (addition of amiloride) resulted in an instantaneous inhibition of the SCC (the transepithelial Na⁺ transport, whereas the appearing flux (the rate of the Na⁺–K⁺ pump) decreased with a halftime of 1.9 min. Addition of the Na⁺–K⁺ pump inhibitor ouabain (0.1mm) resulted in a faster and bigger inhibition of the appearing flux than of the SCC. Thus, by simultaneous measurement of the SCC and the appearing Na⁺ flux one can elucidate whether an inhibitor exerts its effect by inhibiting the pump or by decreasing the passive permeability. Addition of the K⁺ channel inhibitor Ba⁺⁺, in a concentration which gave maximum inhibition of the SCC, had no effect on the appearing flux (the rate of the Na–K pump) in the first 2 min, although the inhibition of the SCC was already at its maximum.It is argued that in the short period, where the Ba⁺⁺-induced inhibition of SCC is at its maximum and the appearing flux in unchanged, the decrease in the SCC (SCC) is equal to the net K⁺ flux via the Na⁺–K⁺ pump, and the coupling ratio () of the Na⁺–K⁺ pump can be calculated from the following equation =SCC _t=0/SCC where SCC _t=0 is the steady-state SCC before the addition of Ba⁺⁺.     

The effects of potassium and membrane potential on sodium-dependent glutamic acid uptake

The uptake of l-glutamic acid into brush-border membrane vesicles isolated from rat renal proximal tubules is Na⁺-dependent. In contrast to Na⁺-dependent uptake of d-glucose, pre-equilibration of the vesicles with K⁺ stimulates l-glutamic acid uptake. Imposition of a K⁺ gradient ($\text{[}\text{K}{}_{\mathrm{i}}{}^{\mathrm{+}}\text{] > [}\text{K}{}_{\mathrm{o}}{}^{\mathrm{+}}\text{]}$) further enhances Na⁺-dependent l-glutamic acid uptake, but leaves K⁺-dependent glucose transport unchanged. If K⁺ is present only at the outside of the vesicles, transport is inhibited. Intravesicular Rb⁺ and, to a lesser extent, Cs⁺ can replace intravesicular K⁺ to stimulate l-glutamic acid uptake. Changes in membrane potential incurred by the imposition of an H⁺-diffusion potential or anion replacement markedly affect Na⁺-dependent glutamic acid uptake only in the presence of K⁺. Experiments with a potential-sensitive cyanine dye also indicate that, in the presence of intravesicular K⁺ a charge movement is involved in Na⁺-dependent transport of l-glutamic acid.The data indicate that Na⁺-dependent l-glutamic acid transport can be additionally energized by a K⁺ gradient. Furthermore, intravesicular K⁺ renders Na⁺-dependent l-glutamic acid transport sensitive to changes in the transmembrane electrical potential difference.     

Characterization of glutamate transport system in hydrophobic protein (H protein) of Bacillus subtilis

Hydrophobic protein (H protein) was isolated from membrane fractions of Bacillus subtilis and constituted into artificial membrane vesicles with lipid of B. substilis. Glutamate was accumulated into the vesicle when a Na⁺ gradient across the membrane was imposed. The maximum effect of Na⁺ on the transport was achieved at a concentration of about 40 mM, while the apparent K_m for Na⁺ was approximately 8 mM. On the other hand, K_m for glutamate in the presence of 50 mM Na⁺ was about 8 μM. Increasing the concentration of Na⁺ resulted in a decrease in K_m for glutamate, maximum velocity was not affected. The transport was sensitive to monensin (Na⁺ ionophore).Glutamate was also accumulated when pH gradient (interior alkaline) across the membrane was imposed or a membrane potential was induced with K⁺-diffusion potential. The pH gradient-driven glutamate transport was sensitive to carbonylcyanide m-chlorophenylhydrazone and the apparent K_m for glutamate was approximately 25 μM.These results indicate that two kinds of glutamate transport system were present in H protein: one is Na⁺ dependent and the other is H⁺ dependent.     

A mathematical model for membrane transport of amino acid and Na+ in vesicles

Summary A model with a carrier having sites for both amino acid and Na⁺ can account for AIB (-aminoisobutyric acid) transport kinetics observed in membrane vesicles from SV3T3 (simian virus 40-tranformed Balb/c3T3 cells) and 3T3 (the parent cell line). The main feature of this cotransport model is that Na⁺ binding to carrier decreases the effectiveK _m for AIB transport, Na⁺ transport kinetics observed in both vesicle systems can be described by passive (possibly facilitated) diffusion. The lag of Na⁺ transport across the membrane compared to that for AIB, coupled to the Na⁺-dependent decrease in theK _m for AIB, accounts for the overshoot in intravesicular AIB observed for SV3T3 in the presence of an initial Na⁺ gradient. Extra-vesicular Na⁺ maintains a derease in theK _m for AIB influx before intra-vesicular Na⁺ has accumulated to balance it with a comparable decrease in theK _m for AIB efflux. 3T3 vesicles display little overshoot, and this finding can be explained mostly by a lower carrier affinity for Na⁺.     

Accumulation of sodium and potassium ions in oocytes of the river lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis> at the prespawning period

Accumulation of Na⁺ and K⁺ ions in oocytes of the river lamprey Lampetra fluviatilis and their transport across the plasma membrane is realized by two main mechanisms-Na,K-pump and Na,K,Cl-cotransport. At the prespawning period from December to May, the intracellular Na⁺ concentration was observed to increase from 10 to 25 mM and the K⁺ concentration-from 28 to 45 mM. Results obtained on isolated oocytes with aid of radioactive labels ²²Na and ²⁰⁴Tl have shown that contributions of the Na,K-pump and Na,K,Cl-cotransport to potassium accumulations were close until March. In spring, the total K⁺ inflow almost doubled owing to activation of the Na,K-pump, whereas contribution of Na,K,Cl-cotransport did not change. It seems that an increase of the intracellular content of the main inorganic cations in oocytes resulted in parallel activation of the Na,K-pump and probably of Na/H-exchange. The biological significance of activation of these mechanisms of ion transport at the prespawning period might be due to a necessity of accumulation of Na⁺ and K⁺ ions in concentrations optimal for subsequent embryonic development.