The non-correlation of bioelectric potentials with ionic gradients

The sartorius muscles of 320 toads have been analyzed for Na⁺ and K⁺. There is a wide variation in the Na⁺ content which when calculated intracellularly varied from 0 m.eq./kg. to 58 m.eq./kg. In particular it was found that the distribution of internal Na⁺ in the intact animal was such that only 17 per cent of the muscles should give from the Nernst equation the observed overshoot of 37 mv. In contrast to this wide variation the K⁺ content is comparatively constant, the range being 71 to 112 m.eq./kg. The mean observed resting potential of 87 mv. agreed well with the potential calculated from the mean intracellular K⁺ by the Nernst equation. Analyses of plasma show that the Na⁺ content is constant at 130 m.eq./liter, and the K⁺ is 3.0 m.eq./liter. The resting and action potentials of 77 muscles have been recorded and then the muscles have been analyzed. The results have shown that there is no correlation between the level of intracellular Na⁺ and the overshoot. Furthermore the apparent correlation between the average K⁺ content and the average resting potential has been shown to be fortuitous, when the correlation in individual muscles is considered. When a muscle is soaked in Ringer solution for several hours there is a gain of Na⁺ and a loss of K⁺. These shifts should result in changes in the respective potentials, but such changes were not found. The above findings have been discussed in the light of the present theories that the resting potential and the action potential are directly related to the ionic ratio across the membrane. Our results very definitely do not support the theory that the overshoot is related to the Na⁺ gradient, and this also applies with respect to the K⁺ gradient and the resting potential.     

Potassium uptake by the dog erythrocyte

The inward transport of potassium by separated dog erythrocytes has been studied at concentrations of potassium in the medium from 2.9 to 25.0 m.eq./liter and at 38.0 and 33.0 degrees C. At the physiological concentration of external potassium (4.06 m.eq./liter medium), the inward potassium flux is 0.11 m.eq./liter cells hour and the glucose consumption is 2.0 mM/liter cells hour. The dependence of potassium influx on extracellular potassium concentration is given by the following equation, K influx (m.eq./liter cells hour) = 0.028 [K](amb.) - 0.003 in which [K](amb.) refers to the potassium concentration in the medium. In a single 93 hour experiment, 94 per cent of the intracellular potassium was exchanged at an apparently uniform rate. The average apparent activation energy for the process is 7,750 calories +/- 2,000 calories/mol and there is some indication that the apparent activation energy of inward K transport decreases with increasing external K concentration.     

The permeability of the human erythrocyte to sodium and potassium

Measurements have been made on the permeability of the human erythrocyte to Na and K in vitro, using radioactive tracers to observe the system in the steady state. The average inward K flux is 1.67 m.eq./liter cells hour, and the apparent activation energy is 12,300 ± 1300 calories/mol. The inward K flux is independent of the external K concentration in the range of concentrations studied (4 to 16 m.eq. K/liter plasma). Rb appears to compete with K for transport into the cell, whereas Na and Li do not. The average inward Na flux is 3.08 ± 0.57 m.eq. Na/liter cells hour, and the apparent activation energies are 20,200 ± 2700 calories/mol for inward transport, and 14,900 ± 3,400 calories/mol for outward transport. The inward Na flux is dependent on the external Na concentration, but not in a linear fashion. Li appears to compete with Na for inward transport, whereas K and Rb do not. An approximate maximum estimate shows that the energy required for cation transport is only 8.8 calories/mol liter cells hour of the 110 calories/mol liter cells hour available from the consumption of glucose. A working hypothesis for the transport of Na and K is presented.     

The relation between sodium ion content and efflux of labelled sodium ions from yeast

1. The activity of the Na(+) pump in an Na(+)-rich yeast was compared with that in an Na(+)-rich frog sartorius muscle, and found to be very similar to it over the first hour if both were immersed in fluid containing 104mm-Na(+) plus 10mm-K(+). 2. The efflux of labelled Na(+) from an Na(+)-rich yeast into an Na(+)-free medium was investigated. In this Na(+)-free medium, Li(+) or choline replaced the Na(+), and the efflux-content curves obtained with either of these ions were very similar. The curves were sigmoid, reaching or approaching a saturation at the higher internal Na(+) concentrations. 3. The curves obtained with yeast resembled those similarly obtained with frog sartorius muscle by Keynes & Swan (1959), Mullins & Frumento (1963), Harris (1965) and Keynes (1965). The slope of the plot of the logarithm of the Na(+) efflux against the logarithm of the Na(+) concentration in the cells reached its highest value at an internal Na(+) concentration of 15m-equiv./kg. (27m-equiv./l. of cell water). 4. The effect of external K(+) concentration on the efflux-content relationship was examined. An increased K(+) concentration was found to increase the Na(+) efflux by raising the saturation value, which is similar to observations made by Harris (1965) with frog muscle. 5. The effect of increasing the external carbon dioxide concentration was investigated. No effect on the slope of the plot of the logarithm of the Na(+) efflux against the logarithm of the Na(+) content was noticed even when the yeast suspension was equilibrated with 100% carbon dioxide. There was, however, a decrease in the amount of Na(+) efflux on equilibrating the solution with carbon dioxide.     

The effect of ACTH and adrenal steroids on K transport in human erythrocytes

The effect of ACTH and adrenal steroids on K transport in human erythrocytes has been studied. A new method of calculation has revealed that in normal human erythrocytes the K transport is not independent of external K concentration as had previously been thought. The equation describing the relationship is, K influx (m.eq./liter cells hour) = [K]_pi/(0.697 + 0.329 [K]_pi) in which [K]_pi refers to the plasma K concentration at the beginning of the experiment. At the physiological plasma K concentration of 4.65 m.eq./liter, K influx is 2.09 m.eq./liter cells hour; K efflux is 1.95 m.eq./liter cells hour and is independent of plasma K concentration. The effect of the infusion of ACTH and adrenal steroids on the K content of the erythrocytes was also studied. Infusions of ACTH or cortisone do not cause the expected loss in erythrocyte K content and may well cause a gain. Infusions of ACTH and cortisone decrease the rate of K influx and efflux slightly at all stages of the infusion, as measured in vitro in blood samples drawn at various times during and following the infusion. However, the erythrocytes incubated in vitro do not exhibit the same changes in K content as are found in vivo. Hydrocortisone added to normal cells in vitro also decreases both influx and efflux of K, without affecting the K content of the cells.     

Rates of efflux and intracellular concentrations of potassium, sodium and chloride ions in isolated fat-cells from the rat

1. The metabolism of K(+), Na(+) and Cl(-) has been investigated in isolated fat-cells prepared from the epididymal adipose tissue of rats. 2. Methods are described for measuring the intracellular water space, the rates of loss of intracellular (42)K(+), (22)Na(+) and (36)Cl(-) and the intracellular concentrations of K(+), Na(+) and Cl(-) in isolated fat-cells. 3. The intracellular water space, measured as the [(3)H]water space minus the [carboxylic acid-(14)C]inulin space, was 3.93+/-0.38mul./100mg. cell dry wt. 4. The first-order rate constants for radioisotope effluxes from isolated fat-cells were 0.029min.(-1) for (42)K(+), 0.245min.(-1) for (22)Na(+) and 0.158min.(-1) for (36)Cl(-). 5. The intracellular concentrations of K(+), Na(+) and Cl(-) were 146m-equiv./l., 18.6+/-2.9m-equiv./l. and 43+/-2.4m-equiv./l. respectively. 6. The total intracellular K(+) content of isolated fat-cells was determined by atomic-absorption spectrophotometry to confirm the value obtained from the radioisotope-efflux data. 7. The ion effluxes from isolated fat-cells were: K(+), 1.5pmoles/cm.(2)/sec., Na(+), 1.6pmoles/cm.(2)/sec., and Cl(-), 2.4pmoles/cm.(2)/sec. 8. The membrane potential of isolated fat-cells calculated from the Cl(-) distribution ratio was -28.7mv.     

The effects of varying the cellular and extracellular concentrations of sodium and potassium ions on the uptake of glycine by mouse ascites-tumour cells in the presence and absence of sodium cyanide

1. Tumour cells were starved to deplete them of ATP and transferred to 0.9mm-glycine in Ringer solutions containing 2mm-sodium cyanide and various Na(+) and K(+) concentrations. The uptake of glycine then usually reached a peak by about 10min. 2. When cellular [Na(+)] and extracellular [Na(+)] were each about 30m-equiv./l., the maximum amount of glycine absorbed increased between 1.2- and 3.0-fold on lowering extracellular [K(+)] from 128 to 10m-equiv./l. 3. When extracellular [Na(+)] was 150m-equiv./l., the ratio, R, of the cellular to extracellular glycine concentrations increased progressively, from near 1 to about 9, when cellular [Na(+)] was lowered from 120 to 40m-equiv./l. 4. When cellular [Na(+)] was almost constant, either at 45 or 70m-equiv./l., R fell about 14-fold when extracellular [Na(+)] varied from 150 to 16m-equiv./l. 5. Values of R near 0.2 were found when cellular [Na(+)] was about four times as large as extracellular [Na(+)]. 6. R fell about threefold when the cells were put with 12mm- instead of 0.9mm-glycine. 7. The results were taken to imply that, under these conditions, the spontaneous movements of both Na(+) and K(+) across the cell membrane, down their respective concentration gradients, served to concentrate the glycine in the tumour cells (Christensen's hypothesis).     

Furosemide-sensitive Na and K fluxes in human red cells. Net uphill Na extrusion and equilibrium properties

This paper reports experiments designed to find the concentrations of internal and external Na and K at which inward and outward furosemide-sensitive (FS) Na and K fluxes are equal, so that there is no net FS movement of Na and K. The red cell cation content was modified by using the ionophore nystatin, varying cell Na (Nai) from 0 to 34 mM (K substitution, high-K cells) and cell K (Ki) from 0 to 30 mM (Na substitution, high-Na cells). All incubation media contained NaCl (Nao = 130 or 120 nM), and KCl (Ko = 0-30 mM). In high-K cells, incubated in the absence of Ko, there was net extrusion of Na through the FS pathway. The net FS Na extrusion increased when Nai was increased. Low concentrations of Ko (0-6 mM) slightly stimulated, whereas higher concentrations of Ko inhibited, FS Na efflux. Increasing Ko stimulated the FS Na influx (K0.5 = 4 mM). Under conditions similar to those that occur in vivo (Nai = 10, Ki = 130, Nao = 130, Ko = 4 mM, Cli/Clo = 0.7), net extrusion of Na occurs through the FS pathway (180-250 mumol/liter cell X h). The concentration of Ko at which the FS Na influx and efflux and the FS K influx and efflux become equal increased when Nai increased in high-K cells and when Ki was increased in high-Na cells. The net FS Na and K fluxes both approached zero at similar internal and external Na and K concentrations. In high-K cells, under conditions when net Na and K fluxes were near zero, the ratio of FS Na to FS K unidirectional flux was found to be 2:3. In high-K cells, the empirical expression (Nai/Nao)2(Ki/Ko)3 remained at constant value (apparent equilibrium constant, Kappeq +/- SEM = 22 +/- 2) for each set of internal and external cation concentrations at which there was no net Na flux. These results indicate that in the physiological region of concentrations of internal and external Na, K, and Cl, the stoichiometry of the FS Na and K fluxes is 2 Na:3 K. In high-Na cells under conditions when net FS Na and K fluxes were near zero, the ratio of FS Na to FS K unidirectional fluxes was 3:2 (1).(ABSTRACT TRUNCATED AT 400 WORDS)     

Large diameter of palytoxin-induced Na/K pump channels and modulation of palytoxin interaction by Na/K pump ligands

Palytoxin binds to Na/K pumps to generate nonselective cation channels whose pore likely comprises at least part of the pump's ion translocation pathway. We systematically analyzed palytoxin's interactions with native human Na/K pumps in outside-out patches from HEK293 cells over a broad range of ionic and nucleotide conditions, and with or without cardiotonic steroids. With 5 mM internal (pipette) [MgATP], palytoxin activated the conductance with an apparent affinity that was highest for Na(+)-containing (K(+)-free) external and internal solutions, lowest for K(+)-containing (Na(+)-free) external and internal solutions, and intermediate for the mixed external Na(+)/internal K(+), and external K(+)/internal Na(+) conditions; with Na(+) solutions and MgATP, the mean dwell time of palytoxin on the Na/K pump was about one day. With Na(+) solutions, the apparent affinity for palytoxin action was low after equilibration of patches with nucleotide-free pipette solution. That apparent affinity was increased in two phases as the equilibrating [MgATP] was raised over the submicromolar, and submillimolar, ranges, but was increased by pipette MgAMPPNP in a single phase, over the submillimolar range; the apparent affinity at saturating [MgAMPPNP] remained approximately 30-fold lower than at saturating [MgATP]. After palytoxin washout, the conductance decay that reflects palytoxin unbinding was accelerated by cardiotonic steroid. When Na/K pumps were preincubated with cardiotonic steroid, subsequent activation of palytoxin-induced conductance was greatly slowed, even after washout of the cardiotonic steroid, but activation could still be accelerated by increasing palytoxin concentration. These results indicate that palytoxin and a cardiotonic steroid can simultaneously occupy the same Na/K pump, each destabilizing the other. The palytoxin-induced channels were permeable to several large organic cations, including N-methyl-d-glucamine(+), suggesting that the narrowest section of the pore must be approximately 7.5 A wide. Enhanced understanding of palytoxin action now allows its use for examining the structures and mechanisms of the gates that occlude/deocclude transported ions during the normal Na/K pump cycle.     

Potassium transport in human erythrocytes: evidence for a three compartment system

Whole human blood is incubated for periods of ½ to 3 hours with K⁴² at 37°C. At the close of this period, called pre-incubation, the plasma is removed from the cells and the cells, now become radioactive, are again incubated in a mixture of plasma and buffer for periods of up to 10 additional hours. The time course of the K⁴² activity of the incubating medium is followed. Characteristically, after 2 hours of pre-incubation, the activity in the medium rises to a peak about 1 and ½ hours after resuspension, and then falls slowly until at 10 hours it is very close to its initial value at the beginning of the resuspension interval. This transient rise in K⁴² activity in the medium is taken to indicate that the red cell does not consist of a single uniform K compartment, but contains at least two compartments. Thus one cellular compartment contains a reservoir of high specific activity K which provides the specific activity gradient necessary to drive the K⁴² content of the medium to its transient peak. Experiments with Na indicate that its behavior in this respect is unlike that of K. The experimental data are matched to a simple model system which is capable of theoretical analysis with the aid of an analogue computer. The model system, whose characteristics agree fairly well with those observed experimentally on red cell suspensions, comprises two intracellular compartments, one containing 2.35 m.eq. K/liter blood, and the other 44.1 m.eq. K/liter blood. The plasma K content is 2.64 m.eq./liter blood. The flux between plasma and the smaller intracellular compartment is 0.65 m.eq. K/liter blood hour; that between the smaller and the larger intracellular compartment, 1.77 m.eq. K/liter blood hour; and that between the larger intracellular compartment and the plasma is 0.34 m.eq. K/liter blood hour.     

The transport of sodium into human erythrocytes in vivo

The relative Na²⁴ specific activity of red cells and plasma was measured at periods up to 30 hours following a single intravenous injection of Na²⁴ in normal healthy young adults. The average specific activity of the red cells relative to that of the plasma at 24 hours and beyond was found to average 0.83 ± 0.05 in a series of five normal individuals, significantly different from 1.0. This indicates that all the intracellular Na is not exchangeable in 24 hours, and confirms earlier in vitro results. The red cell Na concentration in man was shown to be 12.1 ± 1.1 m.eq. Na/liter red cell, as measured in a series of nineteen normal healthy young adults. A theoretical analysis of the data on exchangeable cell Na suggests that the red cell Na (5.3 m.eq. Na/liter blood) is divided into a fast compartment comprising 4.25 m.eq. Na/liter blood, and a slow compartment comprising 1.07 m.eq. Na/liter blood. If these compartments are arranged in parallel, the flux between plasma and fast compartment is 1.32 m.eq. Na/liter blood hour, and that between plasma and slow compartment is 0.016 m.eq. Na/liter blood hour. Results of experiments on two patients with congenital hemolytic jaundice suggest that the fraction of slowly exchanging Na may increase with the age of the red cell.     

Cloning, functional expression and primary characterization of Vibrio parahaemolyticus K+/H+ antiporter genes in Escherichia coli

The regulation of internal Na(+) and K(+) concentrations is important for bacterial cells, which, in the absence of Na(+) extrusion systems, cannot grow in the presence of high external Na(+). Likewise, bacteria require K(+) uptake systems when the external K(+) concentration becomes too low to support growth. At present, we have little knowledge of K(+) toxicity and bacterial outward-directed K(+) transport systems. We report here that high external concentrations of K(+) at alkaline pH are toxic and that bacteria require K(+) efflux and/or extrusion systems to avoid excessive K(+) accumulation. We have identified the first example of a bacterial K(+)(specific)/H(+) antiporter, Vp-NhaP2, from Vibrio parahaemolyticus. This protein, a member of the cation : proton antiporter-1 (CPA1) family, was able to mediate K(+) extrusion from the cell to provide tolerance to high concentrations of external KCl at alkaline pH. We also report the discovery of two V. parahaemolyticus Na(+)/H(+) antiporters, Vp-NhaA and Vp-NhaB, which also exhibit a novel ion specificity toward K(+), implying that they work as Na(+)(K(+))/H(+) exchangers. Furthermore, under specific conditions, Escherichia coli was able to mediate K(+) extrusion against a K(+) chemical gradient, indicating that E. coli also possesses an unidentified K(+) extrusion system(s).     

Permeability properties and intracellular ion concentrations of epithelial cells in rat duodenum.

Effects of the K+ concentration in the bathing fluid ([K+]l) on the intracellular K+, Na+ and Cl- concentrations ([K+]i [Na+]i and [Cl-]i) as well as on the electrical potential were studied in rat duodenum. Changes in the mucosal K+ concentration ([K+]m), bringing the sum of Na+ and K+ concentrations to 147.2 mM constant, had little effect on the transmural potential difference (PDt), but did induce marked changes in the mucosal membrane potential (Vm). As [K+]m increased, Vm was depolarized gradually and obeyed the Nernst equation for a potassium electrode in the range of [K+]m greater than approx. 60 mM. Experiments of ion analyses were carried out on strips of duodenum to determine the effect of changing the external K+ concentrations on [K+] i, [Na+]i and [Cl-]i. An increase in [K+]o resulted in increases in [K+]i and [Cl-]i and a decrease in [Na+]i, [K+]i approaching its maximum at [K+]o greater than 70 mM. Such changes in [K+]i and [Na+]i seem to correlate quantitatively with the changes in [K+]o and [Na+]o. The values of the ratio of permeability coefficients, Pna+/PK+ were estimated using the Vm values and intracellular ion concentrations measured in these experiments. The results suggested that there appeared a rather abrupt increase in the PNa+/PK+ ratio from 0 to approx. 0.1, as [K+]m decreased.     

Active and Passive Transport of the Major Nutrient Ions across the Root of Ricinus communis

Mesurements have been made of the electrical potential differncebetween the exuding sap of detopped castor-oil plants and anutrient solution bathing the roots. The initial concentrationsof the major nutrient ions in the root medium were (m.equiv./I.):K 0.7, Ca 0.8, Mg 0.4, NO₂ 1.4, So₄ 0.4, and phosphate 0.2,when present, Na was at a level of 0.3 m.equiv./I. and Cl at0.1 m.equiv./I. The concentration of each of these ions in thesap was of the order of 10 times that in the external solution,was —50 mV. Comparison of the measured potential differencewith the Nernst potential calculated for each ion incicatesthat, under the experimental conditions used here, the influxof K, Na, Ca, and Mg is a passive process while the transportof No₃, Cl, SO₄, H₂PO₄ and HPO₄ is in each case an active processagainst the electrochemical potential gradient. There is no evidence that a K—Na pump is working in theseroots although, in the absence of estimates for flux ratios,an outward ‘pump’ is not ruled out for Na, or Caand Mg. K is the only ion which appears to be at equilibriumconcentration in the sap.     

Potassium Transport and the Relationship Between Intracellular Potassium Concentration and Amino Acid Uptake by Cells of a Marine Pseudomonad

Transport of K(+) by K(+)-depleted cells of marine pseudomonad B-16 (ATCC 19855) exhibited saturation kinetics. Rb(+) inhibited both K(+) transport and the K(+)-dependent transport of alpha-aminoisobutyric acid (AIB) into K(+)-depleted cells of the organism in proportion to the concentration of Rb(+) in the suspending medium. Inhibition of the K(+)-dependent uptake of AIB into K(+)-depleted cells by Rb(+) could be overcome by increasing the concentration of K(+) in the medium. When AIB and K(+) were added simultaneously to a suspension of K(+)-depleted cells, the uptake of K(+) occurred immediately and rapidly, whereas the accumulation of AIB occurred only after a lag. The initial uptake rate of AIB was directly proportional to the intracellular K(+) concentration. The intracellular concentration of K(+) and AIB at their steady-state levels increased to a maximum as the Na(+) concentration in the suspending medium was increased. At Na(+) concentrations between 0.2 and 0.3 M, the molar ratio of K(+) to AIB at their intracellular steady-state concentrations was constant at 1.6. At external Na(+) concentrations less than 0.2 M, the cells maintained a relatively higher K(+) intracellular steady-state level than AIB.     

The influence of external sodium ions on the sodium pump in erythrocytes

1. A study has been made of the interaction between Na(+) and K(+) on the adenosine triphosphatase activity of erythrocyte ;ghosts', and on the K(+) influx and Na(+) efflux of intact erythrocytes. The adenosine triphosphatase activity and the ion movements were greater at a low external K(+) concentration in the absence of Na(+) than they were in the presence of 150mm-Na(+). The inhibition by external Na(+) of K(+) influx had an inhibitory constant of 5-10mm. 2. Activation by K(+) of kidney microsomal adenosine triphosphatase was retarded by Na(+), and activation by Na(+) was retarded by K(+). Fragmented erythrocyte membranes behaved similarly. 3. These observations suggest that there is competition between Na(+) and K(+) at the K(+)-sensitive site of the membrane.     

Ultracytochemical location of Na(+)/K(+)-atpase activity and effect of high salinity acclimation in gill and renal epithelia of the freshwater shrimp Macrobrachium olfersii (Crustacea, Decapoda).

Accumulation sites of lead phosphate reaction product consequent to Na(+)/K(+)-ATPase activity in gill and renal epithelia of the freshwater shrimp Macrobrachium olfersii were located ultracytochemically by para-nitrophenyl-phosphate hydrolysis and lead precipitation, and quantified per unit membrane area and cytoplasmic volume. In shrimps in freshwater (<0.5 per thousand S, 20 mOsm/kg H(2)O, 0.7 mEq Na(+)/liter), numerous sites of electron-dense, Na(+)/K(+)-ATPase reaction product accumulation were demonstrated in the membrane invaginations of the mitochondria-rich, intralamellar septal cells (12.5 +/- 1.7 sites/microm(2) membrane, 179 +/- 22 sites/microm(3) cytoplasm, mean+/- SEM, N 相似文献   

Sertraline inhibits pre-synaptic Na⁺ channel-mediated responses in hippocampus-isolated nerve endings

In the present study, a possible sertraline action on cerebral pre-synaptic Na(+) channels was investigated. For this purpose, the effect of sertraline on responses induced by the Na(+) channel opener, veratridine, namely the increase in Na(+) and in neurotransmitter release in hippocampus-isolated nerve endings was investigated. Results show that sertraline in the low μM range (1.5-25?μM) progressively inhibits the rise in Na(+) and the release of pre-loaded [(3) H]Glu as well as the release of endogenous 5-HT, Glu and GABA (detected by HPLC) induced by veratridine depolarization either under external Ca(2+) -free conditions or in the presence of external Ca(2+) . In addition, under non-depolarized conditions, sertraline (25 μM) increased the external concentration of 5-HT at expense of its internal concentration, and unchanged the external and internal concentrations of the amino acid neurotransmitters and of the 5-HT main metabolite, 5-HIAA. This result is consistent with the sertraline inhibitory action of the serotonin transporter. However, sertraline is unlikely to inhibit pre-synaptic Na(+) channels permeability by increasing external 5-HT. Because 5-HT in a wide concentration range (1-1000 μM) did not change the veratridine-induced increase in Na(+) . In summary, present findings demonstrate that besides the inhibition of 5-HT reuptake, sertraline is an effective inhibitor of pre-synaptic Na(+) channels controlling neurotransmitter release.     

Kinetic properties of Na(+) -H(+) antiport in Escherichia coli membrane vesicles: Effects of imposed electrical potential, proton gradient, and internal pH

Modifications of the kinetic properties of the Escherichia coli (RA11) Na(+) - H(+) antiport system by imposed pH gradients (deltapH, interior alkaline) and membrane potential(delta(psi), interior negative) were studied by looking at the accelerating effects of deltapH and delta on downhill Na(+) efflux from membrane vesicles incubated at different external pHs. First,variations of the Na(+) efflux rate ( VNa) as a function of imposed delta pH appear to be strongly dependent on the external pH value.The individual VN, vs. deltapH relationships observed between pH 5.5 and pH 6.6 are all nonlinear and indicate the existence of a threshold deltapH above which V(Na) increases steeply as the deltapH magnitude increases; threshold deltapH values progressively decrease as the pH is raised from 5.5 to 6.6. In contrast, at or above neutrality, V(Na) acceleration is linearly related to deltapH amplitude. Strikingly, it is shown that the deltapH-dependent variations in the Na(+) efflux rate measured in vesicles incubated at different external pHs can be accounted for by variations of internal pH; the observed relationship suggests that a high internal H(+) concentration inhibits the Na(+) -H(+) antiport activity.This inhibition results from a drastic increase in the apparent K(m), of the Na(+) efflux reaction as the internal H(+) concentration increases. On the other hand, imposed Δ increases the Na(+) efflux rate linearly by a selective modification of the V(max) value of the Na(+) efflux. Together, these data indicate that the internal H(+) concentration controls the Na(+)-H(+) antiport activity and that the chemical and electrical proton gradients affect two different kinetic steps of the Na(+)-H(+) exchange reaction.     

Iodoacetate action on endocytic uptake of different fluid-phase markers by OK renal epithelial cells

When grown in monolayer culture, OK cells display endocytic uptake of soluble fluid-phase markers such as lucifer yellow (LY) and horseradish peroxidase (HRP). The response of this process to metabolic inhibitors was characterized in the present study. Inhibition of cell metabolism by cyanide produced a decrease in cell ATP content which was accompanied by a decrease in uptake of both LY and HRP, confirming the energy-dependence of fluid-phase endocytosis in OK cells. Use of iodoacetate also decreased cell ATP content but its action on endocytosis was unexpected. Cell uptake of HRP was decreased by iodoacetate, similar to the effect of cyanide, but there was a marked increase in LY uptake. Additional studies showed that cyanide did not change intracellular Na+ or intracellular K+ and did not interfere with the Na(+)-dependency of Pi uptake. In contrast, iodoacetate produced a marked increase in Na+, a decrease in K+, and abolished the Na(+)-dependency of Pi transport. The latter was due primarily to a 10-fold increase in Na(+)-independent uptake of Pi. These findings suggest, indirectly, that plasma membrane permeability to Na+, K+, Pi, and small molecules such as LY, may be increased by iodoacetate, possibly through its action as an alkylating agent. This mechanism may allow increased cell uptake of LY through a non-endocytic pathway, and may mask the inhibitory action of iodoacetate on endocytic uptake of LY. These additional effects complicate the use of iodoacetate to interrupt endocytosis.