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).     

A sodium ion concentration gradient formed during the absorption of glycine by mouse ascites-tumour cells

1. To deplete them of ATP the tumour cells were starved at 37 degrees in a Ringer solution containing 33m-equiv. of Na(+)/l., 131m-equiv. of Li(+)/l., 2mM-sodium cyanide and 0.1mm-ouabain. The cellular content of K(+) was largely replaced by Li(+), but cellular [Na(+)] remained near 33m-equiv./l. 2. The addition of 12mm-glycine to the system caused cellular [Na(+)] to increase, during the next 4min., by about 4m-equiv./l., so that it slightly exceeded extracellular [Na(+)]. This occurred in parallel with the absorption of glycine. 3. The cellular K(+) content fell by an amount representing about 10% of the amount of Na(+) absorbed. 4. The results provide a clear demonstration that the flow of glycine into the cells is linked to a parallel movement of Na(+); K(+) appears to play a facultative role in the carrier system, whereas Li(+) is almost inert. 5. The effects produced by glycine were not reproduced by l-arabinose.     

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.     

Strophanthidin-sensitive sodium fluxes in metabolically poisoned frog skeletal muscle

Strophanthidin-sensitive and insensitive unidirectional fluxes of Na were measured in fog sartorius muscles whose internal Na levels were elevated by overnight storage in the cold. ATP levels were lowered, and ADP levels raised, by metabolic poisoning with either 2,4-dinitrofluorobenzene or iodoacetamide. Strophanthidin-sensitive Na efflux and influx both increased after poisoning, while strophanthidin-insensitives fluxes did not. The increase in efflux did not require the presence of external K but was greatly attenuated when Li replaced Na as the major external cation. Membrane potential was not markedly altered by 2,4-dinitrofluorobenzene. These observations indicate that the sodium pump of frog skeletal muscle resembles that of squid giant axon and human erythrocyte in its ability to catalyze Na-Na exchange to an extent determined by intracellular ATP/ADP levels.     

Inhibition of sodium for sodium exchange by phlorizin in frog sartorius muscle

The effects of phlorizin (2 X 10(-3) mol X l-1) on the Na transport of frog (Rana esculenta) sartorius muscle were investigated in glucose-free medium. Phlorizin decreased the rate coefficient of 24Na efflux by about 40%. The degree of inhibition was comparable to that caused by ouabain (10(-4) mol X l-1). Phlorizin could evoke a further reduction in the 24Na efflux also in the presence of ouabain. The intracellular Na content of the phlorizin-treated muscles remained unchanged, in contrast to a 60% increase induced by ouabain. 42K uptake was not affected by phlorizin. Data indicate that the ouabain-sensitive Na-K pump was not involved in the action of phlorizin. At the same time, phlorizin failed to alter the residual 24Na efflux measured in Li-Ringer solution containing ouabain. When Na: Na exchange was restored by replacing Na into the washout solution in the presence of ouabain, the increase of 24Na efflux was significantly diminished by phlorizin. Phlorizin reduced the 24Na uptake into a compartment with a half time of 6 min by about 40% without affecting the intracellular compartment. The results suggest that phlorizin inhibits the ouabain-insensitive Na: Na exchange in a superficial Na compartment.     

Responses of frog skin Na(+) and Cl(-) transport to guanylin

A possible role for the peptide hormone guanylin was investigated in frog skin (Rana pipiens) epithelium. Sodium and chloride fluxes in response to this peptide were evaluated in Ussing-type chambers. Net and unidirectional Na(+) fluxes were measured by using (22)Na(+) and atomic absorption analysis of total [Na(+)], whereas net Cl(-) fluxes were measured by using electrometric titration for [Cl(-)]. Mucosal application of guanylin (0.5-2.0 micromol/l) caused marked increases in serosal to mucosal net flux and efflux of Na(+). Serosal application of guanylin over the same dose range caused similar large increases in net serosal to mucosal (S-->M) Na(+) and Cl(-) flux as well as Na(+) efflux. Responses of Na(+) influx were small and inconsistent. When frog skin was bathed on the serosal side with Cl(-)-free Ringer's solution mucosal application of guanylin stimulated large efflux and S-->M net fluxes of Na(+). Serosal treatment yielded large Na(+) effluxes and S-->M Na(+) and Cl(-) net fluxes. When frog skin serosal surfaces were bathed with Na(+)- free Ringer's solution mucosal guanylin treatment had no effect but serosal treatment produced large S-->M Cl(-) net fluxes.     

Heavy water inhibition of the transport of alkaline cations across the muscle membrane. I. A comparison of different types of sodium transfer

The sodium efflux from the frog sartorius muscle into the media of different ion composition, prepared with ordinary and heavy water, was measured by radiotracer and flame-emission techniques. About the half of the sodium in muscles was substituted for lithium. The ouabain-sensitive, as well as external potassium- and external sodium-dependent components of the efflux were found to be totally inhibited in D2O, whereas the residual efflux observed in sodium- and potassium-free magnesium medium was diminished in D2O only by one half. A conclusion is made that the decrease in sodium efflux in D2O is due to the inhibition of sodium transfer through the Na, K-ATPase transport system.     

A net gain of sodium ions and a net loss of potassium ions accompanying the uptake of glycine by mouse ascites-tumour cells in the presence of sodium cyanide

1. The tumour cells were starved in a solution lacking Na(+) and then transferred to a Ringer solution containing 2mm-sodium cyanide, 150m-equiv. of Na(+)/l. and 10m-equiv. of K(+)/l. Such cells were depleted of ATP and contained an endogenous pool of various amino acids equivalent to a 26mm solution. 2. At 4min. after the transfer the cellular Na(+) content had increased by about 100% and roughly an equivalent amount of K(+) had left the cells. 3. Under these conditions [(14)C]glycine was absorbed from an 11mm solution and reached the same cellular concentration by about 4min. The pool size increased by approximately the same amount (DeltaGly), so glycine did not simply exchange with the endogenous components. 4. After 4min. with glycine, the cells contained about 20% more Na(+) (DeltaNa(+)) than the control and about 10% less K(+) (DeltaK(+)). The mean values of DeltaNa(+)/DeltaGly and DeltaK(+)/DeltaGly from five experiments were respectively 0.90+/-0.11 and 0.62+/-0.11equiv./mole. 5. A further indication that these two ratios were not equal was that the cells absorbed more water than the movement of glycine itself required. The excess of water was osmotically equivalent to 0.95+/-0.16equiv. of solute/mole of glycine absorbed. 6. The variation of DeltaNa(+)/DeltaGly with the duration of the incubation was consistent with the stimulated uptake of Na(+) being linked to the actual transport of glycine. The same may apply to the movement of K(+), though the time-dependence was not examined in that case. 7. The observations were analysed in terms of a model in which both K(+) and Na(+) moved with a glycine-carrier system without ATP being involved. The analysis supported the idea that the spontaneous movements of the ions through the system might concentrate glycine in the cells significantly by purely physical means (Christensen's hypothesis).     

Effects of lower alcohols on potassium transport and microsomal adenosine-triphosphatase activity of rat cerebral cortex

1. Slices of rat cerebral cortex, incubated anaerobically at 37 degrees , lost K(+) from an initial concentration of 102m-equiv./kg. to a concentration of 57m-equiv./kg. after 10min. On subsequent aerobic incubation they regained K(+) rapidly at a rate that varied with the K(+) concentration of the medium. 2. Lower aliphatic alcohols, present at equal thermodynamic activity, produced approximately equal degrees of inhibition of K(+) uptake during the aerobic incubation. This inhibition was reduced by an increase in K(+) content of the medium. Ethanol did not affect the rate of K(+) loss during anaerobic incubation. 3. Li(+), in concentrations of 1-10mm, also inhibited K(+) uptake by brain-cortex slices, the degree of inhibition varying with the Li(+) concentration. Ouabain also inhibited K(+) uptake. 4. The same series of alcohols, at equal thermodynamic activity, produced comparable degrees of inhibition of Na(+),K(+),Mg(2+)-stimulated adenosine-triphosphatase activity in brain microsomes. 5. It is suggested that inhibition of cation transport is an important, but not a primary, mechanism in the production of central nervous depression by alcohols and other substances.     

An accurate method for measurement of aldosterone production

A method is described for isolation of the acid-hydrolysable metabolite of aldosterone in sufficient purity to measure accurately the daily production rate. Values obtained with six hospital patients were 84-131mug./day on a daily intake of 100m-equiv. of Na(+) and 227-464mug./day on a daily intake of 10m-equiv. of Na(+). Corresponding values for aldosterone excretion were also recorded, but these are a poor index of production rate since they represent from 1.6 to 9.8% of the total daily output of aldosterone.     

The effects of sodium ions and potassium ions of glycine uptake by mouse ascites-tumour cells in the presence and absence of selected metabolic inhibitors

1. The initial rate, v, of glycine uptake by ascites-tumour cells respiring their endogenous nutrient reserves was studied as a function of the respective extracellular concentrations of glycine, Na(+) and K(+). With the extracellular concentration of Na(+)+K(+) constant at 158m-equiv./l. and that of glycine either 4 or 12mm, v tended to zero as the extracellular concentration of Na(+) approached zero. Glycine appeared to enter the cells as a ternary complex with a carrier and Na(+). K(+) competed with Na(+) for one of the carrier sites, whereas glycine was bound at a second site. The values of the five relevant binding constants showed that the two sites interacted. 2. The glycine uptake rate at various extracellular concentrations of glycine and Na(+) was scarcely affected by starving the cells for 30min. in the presence of 2mm-sodium cyanide provided that cellular Na(+) and K(+) contents were kept at the normal values. When the cells took up Na(+), however, v decreased approximately threefold. 3. When their Na(+) content was relatively small and the extracellular concentration of Na(+) was large, the starved cells accumulated glycine in the presence of cyanide for about 15min. Glycine then tended to leave the cells. An average of about 5mumoles of glycine/ml. of cell water was taken up from a 1mm solution, representing about 20% of the accumulation observed during respiration. Studies with fluoride, 2,4-dinitrophenol and other metabolic inhibitors supported the view that ATP and similar compounds were not implicated. The relation between the transient accumulation of glycine that occurred in these circumstances and the normal mode of active transport was not established.     

Further observations on the inhibitory effect of extracellular potassium ions on glycine uptake by mouse ascites-tumour cells

1. The initial rate of uptake of glycine by the tumour cells was measured as a function of the Na(+) and K(+) concentrations in the solution in which the cells were suspended. When [Gly] was 1mm or 12mm, the rate in the absence of Na(+) was independent of [K(+)] and about 3% or 10% respectively of the rate when [Na(+)] was 150m-equiv./l. 2. The Na(+)-dependent glycine entry rate, v, at a given value of [Na(+)] was successively lowered when [K(+)] was increased from 8 to 47 to 96m-equiv./l. A kinetic analysis indicated that K(+) competitively inhibited the action of Na(+). The results were in fair agreement with previous determinations of the kinetic parameters. 3. The presence of 2mm-sodium cyanide and 10mm-2-deoxyglucose lowered the cellular ATP content to less than 3% of the value in the respiring cells. Although v was then about 50% smaller, the relative effects of K(+) and Na(+) on the system were similar to those observed during respiration. 4. A theoretical analysis indicated that the variation of v with [K(+)] is not a reliable guide to the extent to which the K(+) gradient between the cells and their environment may contribute to the net transport of glycine.     

Stimulation-enhanced 3-O-methylglucose efflux from the frog sartorius: kinetics and properties of the system.

The characteristics of the process by which contraction enhances glucose transport in the frog sartorius were studied. Electrical stimulation increased the permeability of muscles to 3-O-methylglucose (3-O-MeGlc), a nonmetabolizable glucose analogue, increasing efflux as well as uptake. Enhanced efflux was due to an increase in Vmax of the efflux process. A lactacidosis had no effect on basal 3-O-MeGlc efflux, and replacement of media Na+ with Li+ did not affect stimulation-induced uptake. Also, basal and stimulated uptake was not affected by 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator. Lastly, N-carbobenzoxy-glycyl-L-phenylalaninamide, which inhibits insulin-enhanced, but not basal, glucose uptake in adipocytes, inhibited both basal and stimulated 3-O-MeGlc fluxes in the frog sartorius. From these findings, we conclude: (1) contraction and exercise enhance glucose transport in muscle by increasing the number of transporters in the plasma membrane, or their turnover, by an unknown process; and (2) basal glucose transport of muscle, unlike that of adipocytes, can not be distinguished from stimulated transport on the basis of its insensitivity to N-carbobenzoxyglycyl-L-phenylalaninamide.     

Effects on sodium efflux of treating frog sartorius muscles with hypertonic glycerol solutions

Summary Efflux of sodium from frog sartorius muscles was measured during and after exposure to Ringer's fluid made hypertonic by addition of 400mm glycerol. Effects of strophanthidin, removal of external Na, and variation of external K were determined. During exposure to glycerol-containing solutions, Na efflux increased. Upon return to Ringer's fluid, Na efflux at first increased further. After the initial increase, Na efflux gradually declined; for the first two hours the efflux of Na from treated muscles was higher than that from untreated muscles. In the second hour, the strophanthidin-sensitive fractions of Na efflux were slightly increased while the strophanthidin-insensitive fractions were slightly decreased when compared with untreated muscles. The responses of Na efflux to removal of external sodium and to varying external K were comparable in both treated and untreated muscles. This shows that, at first, the membranes which remained after glycerol treatment exhibited the normal characteristics of Na extrusion. For at least eight hours after glycerol withdrawal the Na efflux from treated muscles declined relative to that of untreated muscles. The decline was largely due to reduction in strophanthidinsensitive fractions of efflux. Six to eight hours after glycerol withdrawal the Na efflux in treated muscles was less responsive to alterations in external K and Na than it was in untreated muscles. This indicates that aged glycerol-treated sartorii lost a substantial part of their capacity to actively transport sodium.     

Kinetic investigation of K+ efflux during glycerol treatment of muscle

The kinetics of K+ efflux was investigated in the membranes of frog sartorius muscle after disintegration by glycerol treatment. Data of the K+ concentration in the muscle as a function of time of the glycerol treatment fitted well the sum of two exponential fractions (with the correlation coefficient of more than 0.98). The half-lives of the two fractions of the K+ efflux were 1 and 75 hours respectively. On the basis of the value of its half life the efflux of the faster fraction was suggested to correspond to the free diffusion. At low temperature the magnitude of the faster fraction increased in a Na+-containing milieu. This could be due to K+-Na+ ion exchange. From the rate of loss of the slower fraction of K+ one finds that movement of K+ in cells without membranes is significantly slower than free diffusion. Presumably, part of the bound potassium exists in intra- or intermolecular "ion-bridges" of muscle proteins.     

Heavy water inhibition of alkali cation transport across the muscle membrane. II. A comparison of the action of D20 and ouabain on the sodium efflux and rubidium influx in magnesium media

The action of heavy water and ouabain on sodium effluxes and rubidium influxes has been measured and compared in frog muscles (m. sartorius, R. temporaria). Approximately half of muscle sodium was substituted by lithium by preliminary incubation in mixed sodium-lithium media. The ratio of the ouabain-sensitive parts of rubidium influx and sodium efflux is 7.3:10.5, and that of D2O-sensitive parts of corresponding parameters is 7.5:11.3. A conclusion is made that D2O-effect on the Na, K-ATPase system of muscles under investigation resembles ouabain-effect on sodium effluxes as well as on rubidium influxes.     

Effect of acetylcholine on ion transport in the frog skeletal muscle

1. The effect of acetylcholine (ACh) on the ion transport of frog (Rana esculenta) sartorius muscles was studied. ACh was applied in bathing solution, Na influx and K efflux were measured using 24Na and 42K isotopes. 2. Na influx of sartorius muscles was increased by 1 mmol/1 ACh 2-10 fold depending on the experimental arrangement. The increase was greater if Na influx was measured at the beginning of ACh depolarization. During ACh treatment the Na influx took about the same time course as the depolarization recorded extracellularly. This type of recording approximately reflects the depolarization proceeding on the sartorius muscle fibres. 3. The presence of 31 nmol/l tetrodotoxin (TTX) did not modify the degree of increase of Na influx. 4. Rate coefficients for K efflux were increased 2-5 fold by ACh. The maximum rate coefficients were obtained in the first minute of ACh treatment. 5. Increase in K loss evolves also in the presence of 31 nmol/l TTX. The increase in rate coefficients was found to be about 30% less than without TTX in the first minute of ACh action. 6. The results indicate that in the presence of ACh the observed increase in Na influx and K efflux is brought about mainly by changes in Na and K conductance induced by ACh at the end-plates rather than by the action potentials accompanying ACh depolarization.     

Intracellular pH recovery from lactic acidosis of single skeletal muscle fibers

Intracellular pH (pHi), measured with H+-selective microelectrodes, in quiescent frog sartorius muscle fibres was 7.29 +/- 0.09 (n = 13). Frog muscle fibres were superfused with a modified Ringer solution containing 30 mM HEPES buffer, at extracellular pH (pHo) 7.35. Intracellular pH decreased to 6.45 +/- 0.14 (n = 13) following replacement of 30 mM NaCl with sodium lactate (30 mM MES, pHo 6.20). Intracellular pH recovery, upon removal of external lactic acid, depended on the buffer concentration of the modified Ringer solution. The measured values of the pHi recovery rates was 0.06 +/- 0.01 delta pHi/min (n = 5) in 3 mM HEPES and was 0.18 +/- 0.06 delta pHi/min (n = 13) in 30 mM HEPES, pHo 7.35. The Na+-H+ exchange inhibitor amiloride (2 mM) slightly reduced pHi recovery rate. The results indicate that the net proton efflux from lactic acidotic frog skeletal muscle is mainly by lactic acid efflux and is limited by the transmembrane pH gradient which, in turn, depends on the extracellular buffer capacity in the diffusion limited space around the muscle fibres.     

THE EFFECT OF POTASSIUM ON THE ACID METABOLISM OF SURVIVING SKELETAL AND CARDIAC MUSCLES OF THE FROG

The potassium contraction of skeletal muscle and relaxation of cardiac muscle have been correlated with the carbon dioxide and total acid production of these tissues. 1. The immersion of surviving sartorius muscles of the frog in isotonic potassium chloride solution causes a marked increase in the rate of acid production. 2. It is probable that carbon dioxide is the principal acid involved in the above effect. 3. The immersion of surviving cardiac muscle of the frog in isotonic potassium chloride solution causes a pronounced depression in the rate of survival acid production. 4. Reasons are given for believing that these changes in metabolism may be independent of the stimulation and inhibition of contraction which potassium simultaneously produces in these tissues.     

The effect of changes of environment on the electrical and ionic pattern of muscle

The resting and action potentials of sartorius muscles of the toad, Bufo marinus, have been measured under varying conditions of external environment. At the same time, analyses for Na(+) and K(+) content were carried out. There was a slight elevation of 2 mv. when the measurements were made in phosphate-Ringer instead of in bicarbonate-Ringer. The R.P. was independent of the hydrogen ion concentration between pH 6.5 and 8.5, although at these pH's there was marked alteration in the level of Na(+) and K(+) in the muscle. Alteration of the external K(+) level between 0 and 50 m.eq./liter has little influence on the internal K(+) concentration. When the log of the external K(+) concentration is plotted against the R.P. there is not a linear relationship until the external K(+) is raised above 12 m.eq./liter, at which point the cell is unexcitable. Above this value a straight line with a slope of 58 mv. per ten-fold change in concentration is obtained, but the absolute values at any point are about 35 per cent higher than those which would be given by the Nernst equation. Alteration of the external Na(+) level within a range of 45 to 650 m.eq./liter resulted in marked changes in the internal Na(+) content, without, however, having any effect on the ratio Na(+) (out)/Na(+) (in). This ratio has remained at about 3 in spite of marked fluctuations in the absolute value of the internal and external Na(+) levels. When the Na(+) level is lowered there is a decrease in the height of the action potential although there is no alteration in the ratio Na(+) (out)/Na(+) (in). As the Na(+) level is raised the height of the action potential is not affected even in the presence of a fivefold increase in Na(+) in the Ringer. The results do not support the conclusion that the bioelectric potentials can be calculated from the ionic ratios by means of simple physical chemical hypotheses such as the Nernst or Goldman equations. The maintenance of the normal K(+) content of the cell cannot be accounted for by a Donnan mechanism. No definite evidence has been produced to explain the mechanism of a Na(+) "pump." In other words, the concept of a Na(+) pump requires that there shall be a physico- or organochemical mechanism which will distinguish between Na(+) and K(+) (or other) ions. There is evidence that Na(+) can be extruded against a concentration gradient. On the other hand the cell is able to maintain a constant ratio of external to internal Na(+) even when the cell has been severely damaged by very high external Na(+) levels.