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.     

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).     

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.     

pH-dependent glycine uptake in the presence and absence of sodium ions from rat small intestine

Intestinal uptake of glycine in rats was stimulated 15-20% in the presence of 120 mM Na at pH 6.0 and below but around neutral pH, the amino acid uptake was augmented to 60% compared to that in the Na-free medium. Glycine uptake was 30% more at pH 5.5 compared to that at pH 7.3 in the absence of Na. Kinetic analysis revealed a decrease in Kt for glycine uptake (9.62 mM) at pH 5.5 compared to that at pH 7.3 (Kt = 16.67 mM) with no change in maximal velocity (1.51 mumole/10 min/g tissue) in Na-free buffer. Addition of -SH group reacting reagents to the incubation medium produced 36-58% inhibition of glycine uptake in the presence of Na. However, in absence of Na, inhibition of the order of 21-35% and 8-23% was observed at pH 5.5 and 7.0, respectively. These findings suggest that glycine uptake in rat intestine is influenced by pH and -SH groups are implicated in the process(es).     

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.     

Effect of ouabain on amino acid uptake by mouse ascites-tumour cells in the presence of nigericin.

Mouse ascites-tumour cells oxidizing lactate, in a modified Ringer solution, concentrated 2-aminoisobutyrate, L-methionine or 2-(methylamino)isobutyrate about 20-fold from a 0.4 mM solution in the presence of 2-3 micrograms of nigericin/mg cellular dry wt. The ionophore increased cellular [Na+] to almost 100 mM when extracellular [Na+] was about 45 mM. Either valinomycin or the two mitochondrial inhibitors oligomycin and antimycin acting together each markedly lowered the extent to which the tumour cells concentrated amino acid, from the above factor of about 20 to roughly 2-fold. Ouabain (1 mM) had a similar effect, and further raised cellular [Na+]. The sodium pump appeared to be closely involved in amino acid uptake under these conditions.     

Effects of sodium and potassium ions on the uptake of noradrenaline and its precursor tyrosine

The effects of the monovalent cations Na⁺ and K⁺ were studied on the uptake of noradrenaline and tyrosine by a crude synaptosomal fraction in vitro. Sodium ions produced opposite effects on the uptake of noradrenaline and the uptake of tyrosine viz. an increase in noradrenaline uptake and a decrease in the uptake of its precursor tyrosine. A low concentration of K⁺ stimulated the uptake of noradrenaline in the presence of Na⁺, while in the absence of Na⁺ K⁺ had no effect. However, the uptake of tyrosine could be stimulated by low K⁺ in the absence of Na⁺. Besides the increased uptake in the absence of Na⁺, a second uptake was found which was Na⁺, K⁺ activated ATPase dependent. The contribution of this uptake system to the total uptake of tyrosine was about 20%. No evidence was obtained for the involvement of a Na⁺, K⁺ activated ATPase in noradrenaline uptake. It is suggested that another ATPase might be involved in the latter uptake system.     

The effect of calcium ions on the glycolytic activity of Ehrlich ascites-tumour cells

1. Added Ca²⁺ inhibited lactate formation from sugar phosphates by intact Ehrlich ascites-tumour cells. Lactate formation from glucose by these cells was unaffected by added Ca²⁺. 2. The Ca²⁺ inhibition of lactate formation by intact cells occurred in the extracellular medium. 3. Intact ascites-tumour cells did not take up Ca²⁺ in vitro. 4. Glycolysis of sugar phosphates by cell extracts as well as pyruvate formation from 3-phosphoglycerate and phosphoenolpyruvate was inhibited by Ca²⁺. 5. It was concluded that Ca²⁺ inhibited the pyruvate-kinase (EC 2.7.1.40) reaction. Further, Ca²⁺ inhibition of pyruvate kinase could be correlated with the overall inhibition of glycolysis. 6. Concentrations of Ca²⁺ usually present in Krebs–Ringer buffers, inhibited glycolysis and pyruvate-kinase activity by approx. 50%. 7. The inhibition of glycolysis by added Ca²⁺ could be partially reversed by K⁺ and completely reversed by Mg²⁺ or by stoicheiometric amounts of EDTA. 8. The hypothesis is advanced that the inability of tumour cells to take up Ca²⁺ is a factor contributing towards their high rate of glycolysis.     

Acrosome-reacted guinea pig spermatozoa become fusion competent in the presence of extracellular potassium ions

Guinea pig spermatozoa are able to undergo capacitation and the acrosome reaction in a K+-free (-deficient) medium. However, they are unable to fuse with eggs unless they are exposed to a millimolar concentration of extracellular K+ during or after the acrosome reaction. Apparently, the plasma membrane over the equatorial segment gains the ability to fuse with eggs in the presence of K+ during and/or after the acrosome reaction. Once it becomes fusible, the membrane retains its fusibility even in a K+-deficient medium. Rb+ is almost as effective as K+ in rendering the sperm membrane fusible. Li+ and Cs+ are less effective. The molecular mechanism by which K+ renders acrosome-reacted spermatozoa fusion competent is unknown, but it may involve K+-mediated efflux of H+ from the spermatozoa.     

Net uptake of potassium in Neurospora. Exchange for sodium and hydrogen ions

Net uptake of potassium by low K, high Na cells of Neurospora at pH 5.8 is accompanied by net extrusion of sodium and hydrogen ions. The amount of potassium taken up by the cells is matched by the sum of sodium and hydrogen ions lost, under a variety of conditions: prolonged preincubation, partial respiratory inhibition (DNP), and lowered [K]_o. All three fluxes are exponential with time and obey Michaelis kinetics as functions of [K]_o. The V _max for net potassium uptake, 22.7 mmoles/kg cell water/min, is very close to that for K/K exchange reported previously (20 mmoles/kg cell water/min). However, the apparent K_m for net potassium uptake, 11.8 mM [K]_o, is an order of magnitude larger than the value (1 mM) for K/K exchange. It is suggested that a single transport system handles both net K uptake and K/K exchange, but that the affinity of the external site for potassium is influenced by the species of ion being extruded.     

Effects of temperature and dinitrophenol on the uptake of potassium and sodium ions in Ricinus communis roots

Summary Detopped root systems of Ricinus communis plants were used for the study of the effects of temperature and DNP on the uptake of K and Na ions supplied as KNO₃ and NaNO₃.When K and Na ions were offered together in equivalent concentrations, the steady state uptake rates for K⁺ and Na⁺ at 23 to 25° gave a K⁺/Na⁺ ratio of 3. Increasing the Na⁺ concentration relative to K⁺ 3-fold did not alter the preferential uptake of K⁺. The uptake of K⁺ was more sensitive to temperature in the range 10 to 40° and to the application of DNP at 1.5x10^-4 M than was the uptake of Na⁺. When NaNO₃ was the only salt supplied Na⁺ uptake became more sensitive to DNP than when both K⁺ and Na⁺ nitrates were supplied. Prolonged application of DNP led to net K⁺ efflux from the roots, even when no K⁺ was being supplied to the roots. Net Na⁺ efflux under the influence of DNP occurred only in roots previously grown on Na-containing nutrient medium.The different responses of the K⁺ and Na⁺ uptake processes to temperature and DNP suggest the operation of different uptake mechanisms for K⁺ and Na⁺ These results have been considered in relation to the recent concept of dual mechanisms for the absorption of alkali cations by plant tissues.