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.     

Interactions between potassium ions and glycine transport in the yeast Saccharomyces carlsbergensis

A study has been made of the effects of both varying the pH and extracellular [K(+)] on the initial rate of uptake of glycine (v) by a strain of Saccharomyces carlsbergensis that concentrated the amino acid, with respect to the extracellular phase, by up to 1400 times. When no other substrate than glycine was provided and [glycine] was relatively small (相似文献   

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).     

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.     

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.     

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.     

Alkali grass resists salt stress through high [K+] and an endodermis barrier to Na+

In order to understand the salt-tolerance mechanism of alkali grass (Puccinellia tenuiflora) compared with wheat (Triticum aestivum L.), [K(+)] and [Na(+)] in roots and shoots in response to salt treatments were examined with ion element analysis and X-ray microanalysis. Both the rapid K(+) and Na(+) influx in response to different NaCl and KCl treatments, and the accumulation of K(+) and Na(+) as the plants acclimated to long-term stress were studied in culture- solution experiments. A higher K(+) uptake under normal and saline conditions was evident in alkali grass compared with that in wheat, and electrophysiological analyses indicated that the different uptake probably resulted from the higher K(+)/Na(+) selectivity of the plasma membrane. When external [K(+)] was high, K(+) uptake and transport from roots to shoots were inhibited by exogenous Cs(+), while TEA (tetraethylammonium) only inhibited K(+) transport from the root to the shoot. K(+) uptake was not influenced by Cs(+) when plants were K(+) starved. It was shown by X-ray microanalysis that high [K(+)] and low [Na(+)] existed in the endodermal cells of alkali grass roots, suggesting this to be the tissue where Cs(+) inhibition occurs. These results suggest that the K(+)/Na(+) selectivity of potassium channels and the existence of an apoplastic barrier, the Casparian bands of the endodermis, lead to the lateral gradient of K(+) and Na(+) across root tissue, resulting not only in high levels of [K(+)] in the shoot but also a large [Na(+)] gradient between the root and the shoot.     

An investigation into the effects of inhibitors of fluid production by Locusta Malpighian tubule Type I cells on their secretion and elemental composition

The intracellular elemental concentrations of K, Na, Cl, P, Mg and Ca within Type I cells of the Malpighian tubules of Locusta migratoria have been measured using electron probe X-ray microanalysis. The distribution of Na, K and Cl was not homogeneous within the cells and concentration gradients exist from basal to apical surfaces. The rate of secretion and the cationic composition of the secreted tubule fluid have also been determined. Furosemide (1 mM) inhibited fluid secretion by about 60%, raised the [Na(+)] but did not significantly alter the [K(+)] of the secreted tubule fluid. When Rb(+) replaced K(+) in the saline fluid secretion was also inhibited by about 60%, but no additional inhibition occurred by the simultaneous inclusion of furosemide. Thus, Rb(+) and furosemide probably act at the same transport site, and Rb(+) cannot substitute for K(+) at the basal membrane cotransporter. Bafilomycin (1 μM) dramatically inhibited fluid production by 85%, the [K(+)] of the secreted fluid was reduced by about 30% but the [Na(+)] was almost doubled. Furosemide, in common with other inhibitors of fluid secretion acting at the basal surface (ouabain and Rb(+)), caused a fall in intracellular [K] and a rise in [Na]. Bafilomycin, in common with N-ethyl maleimide, which acts at the apical surface, increased the intracellular [K] but did not affect the [Na].     

The acute action of ammonia on rat brain metabolism in vivo

1. Acute NH(4) (+) toxicity was studied by using a new apparatus that removes and freezes the brains of conscious rats within 1s. 2. Brains were removed and frozen 5min after intraperitoneal injection of ammonium acetate (2-3min before the onset of convulsions). Arterial [NH(4) (+)] rose from less than 0.01 to 1.74mm at 4-5min. The concentrations of all glycolytic intermediates measured, except glucose 6-phosphate, were increased by the indicated percentage above the control value as follows: glucose (by 41%), fructose 1,6-diphosphate (by 133%), dihydroxyacetone phosphate (by 164%), alpha-glycerophosphate (by 45%), phosphoenolpyruvate (by 67%) and pyruvate (by 26%). 4. Citrate and alpha-oxoglutarate concentrations were unchanged and that of malate was increased (by 17%). 5. Adenine nucleotides and P(i) concentrations were unchanged but the concentration of creatine phosphate decreased slightly (by 6%). 6. Brain [NH(4) (+)] increased from 0.2 to 1.53mm. Net glutamine synthesis occurred at an average rate of 0.33mumol/min per g. 7. The rate of brain glucose utilization was measured in vivo as 0.62mumol/min per g in controls and 0.81mumol/min per g after NH(4) (+) injection. 8. The arteriovenous difference of glucose and O(2) increased by 35%. 9. No significant arteriovenous differences of glutamate or glutamine were detected. Thus, although much NH(4) (+) was incorporated into glutamine the latter was not rapidly released from the brain to the circulation. 10. Plasma [K(+)] increased from 3.3 to 5.4mm. 11. The results indicate that NH(4) (+) stimulates oxidative metabolism but does not interfere with brain energy balance. The increased rate of oxidative metabolism could not be accounted for only on the basis of glutamine synthesis. We suggest that increased extracellular [NH(4) (+)] and [K(+)] decreased the resting transmembrane potential and stimulated Na(+),K(+)-stimulated adenosine triphosphatase activity thus accounting for the increased metabolic rate.     

Dopamine-induced epithelial K(+) and Na(+) movements in the salivary ducts of Periplaneta americana

K(+)- and Na(+)-selective double-barrelled microelectrodes were used for intracellular and luminal measurements in salivary ducts of Periplaneta americana. The salivary ducts were stimulated with dopamine (10(-6) mol l(-1)). Dopamine decreased intracellular [K(+)] from 112+/-17 mmol l(-1) to 40+/-13 mmol l(-1) (n=6) and increased intracellular [Na(+)] from 22+/-19 mmol l(-1) to 92+/-4 mmol l(-1) (n=6). Luminal [K(+)] was 15+/-3 mmol l(-1) in the unstimulated salivary ducts and increased to 26+/-11 mmol l(-1) upon stimulation with dopamine (n=10). Luminal [Na(+)] was insignificantly increased from 105+/-25 mmol l(-1) to 116+/-22 mmol l(-1) (n=12) by stimulation with dopamine. The potential difference across the basolateral membrane (PD(b)) was depolarized from -65+/-6 mV to -31+/-13 mV (n=12) and the transepithelial potential difference (PD(t)) was hyperpolarized from -13+/-6 mV to -22+/-7 mV (n=22, lumen negative) upon stimulation with dopamine. The re-establishment of prestimulus values of intracellular [K(+)] and [Na(+)] and PD(b) was inhibited by basolateral addition of ouabain (10(-4) mol l(-1)). Furosemide (10(-4) mol l(-1)) in the bath inhibited the dopamine-induced increase in intracellular [Na(+)], the decrease in intracellular [K(+)] and the depolarization of PD(b). We propose a model for dopamine-stimulated ion transport in the salivary ducts involving basolateral Na(+)-K(+)-2Cl(-) cotransport and active extrusion of K(+) via the apical membrane.     

Na+ and K+ ion imbalances in Alzheimer's disease

Alzheimer's disease (AD) is associated with impaired glutamate clearance and depressed Na(+)/K(+) ATPase levels in AD brain that might lead to a cellular ion imbalance. To test this hypothesis, [Na(+)] and [K(+)] were analyzed in postmortem brain samples of 12 normal and 16 AD individuals, and in cerebrospinal fluid (CSF) from AD patients and matched controls. Statistically significant increases in [Na(+)] in frontal (25%) and parietal cortex (20%) and in cerebellar [K(+)] (15%) were observed in AD samples compared to controls. CSF from AD patients and matched controls exhibited no differences, suggesting that tissue ion imbalances reflected changes in the intracellular compartment. Differences in cation concentrations between normal and AD brain samples were modeled by a 2-fold increase in intracellular [Na(+)] and an 8-15% increase in intracellular [K(+)]. Since amyloid beta peptide (Aβ) is an important contributor to AD brain pathology, we assessed how Aβ affects ion homeostasis in primary murine astrocytes, the most abundant cells in brain tissue. We demonstrate that treatment of astrocytes with the Aβ 25-35 peptide increases intracellular levels of Na(+) (~2-3-fold) and K(+) (~1.5-fold), which were associated with reduced levels of Na(+)/K(+) ATPase and the Na(+)-dependent glutamate transporters, GLAST and GLT-1. Similar increases in astrocytic Na(+) and K(+) levels were also caused by Aβ 1-40, but not by Aβ 1-42 treatment. Our study suggests a previously unrecognized impairment in AD brain cell ion homeostasis that might be triggered by Aβ and could significantly affect electrophysiological activity of brain cells, contributing to the pathophysiology of AD.     

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.     

Transport of monosaccharides in kidney-cortex cells

1. The aerobic transport of d-glucose and d-galactose in rabbit kidney tissue at 25 degrees was studied. 2. In slices forming glucose from added substrates an accumulation of glucose against its concentration gradient was found. The apparent ratio of intracellular ([S](i)) and extracellular ([S](o)) glucose concentrations was increased by 0.4mm-phlorrhizin and 0.3mm-ouabain. 3. Slices and isolated renal tubules actively accumulated glucose from the saline; the apparent [S](i)/[S](o) fell below 1.0 only at [S](o) higher than 0.5mm. 4. The rate of glucose oxidation by slices was characterized by the following parameters: K(m) 1.16mm; V(max.) 4.5mumoles/g. wet wt./hr. 5. The active accumulation of glucose from the saline was decreased by 0.1mm-2,4-dinitrophenol, 0.4mm-phlorrhizin and by the absence of external Na(+). 6. The kinetic parameters of galactose entry into the cells were: K(m) 1.5mm; V(max) 10mumoles/g. wet wt./hr. 7. The efflux kinetics from slices indicated two intracellular compartments for d-galactose. The galactose efflux was greatly diminished at 0 degrees , was inhibited by 0.4mm-phlorrhizin, but was insensitive to ouabain. 8. The following mechanism of glucose and galactose transport in renal tubular cells is suggested: (a) at the tubular membrane, these sugars are actively transported into the cells by a metabolically- and Na(+)-dependent phlorrhizin-sensitive mechanism; (b) at the basal cell membrane, these sugars are transported in accordance with their concentration gradient by a phlorrhizin-sensitive Na(+)-independent facilitated diffusion. The steady-state intracellular sugar concentration is determined by the kinetic parameters of active entry, passive outflow and intracellular utilization.     

Quantitative analysis of serum sodium concentration after prolonged running in the heat.

This study compared measured serum [Na(+)] (S([Na+]); brackets denote concentration) with that predicted by the Nguyen-Kurtz equation after manipulating ingested [Na(+)] and changes in body mass (DeltaBM) during prolonged running in the heat. Athletes (4 men, 4 women; 22-36 yr) ran for 2 h, followed by a run to exhaustion and 1-h recovery. During exercise and recovery, subjects drank a 6% carbohydrate solution without Na(+) (Na(+)0), 6% carbohydrate solution with 18 mmol/l Na(+) (Na(+)18), or 6% carbohydrate solution with 30 mmol/l Na(+) (Na(+)30) to maintain BM (0%DeltaBM), increase BM by 2%, or decrease BM by 2% or 4% in 12 separate trials. Net fluid, Na(+), and K(+) balance were measured to calculate the Nguyen-Kurtz predicted S([Na+]) for each trial. For all beverages, predicted and measured S([Na+]) were not significantly different during the 0%, -2%, and -4%DeltaBM trials (-0.2 +/- 0.2 mmol/l) but were significantly different during the +2%DeltaBM trials (-2.6 +/- 0.5 mmol/l). Overall, Na(+) consumption attenuated the decline in S([Na+]) (-2.0 +/- 0.5, -0.9 +/- 0.5, -0.5 +/- 0.5 mmol/l from pre- to postexperiment of the 0%DeltaBM trials for Na(+)30, Na(+)18, and Na(+)0, respectively) but the differences among beverages were not statistically significant. Beverage [Na(+)] did not affect performance; however, time to exhaustion was significantly shorter during the -4% (8 +/- 3 min) and -2% (14 +/- 3 min) vs. 0% (22 +/- 5 min) and +2% (26 +/- 6 min) DeltaBM trials. In conclusion, when athletes maintain or lose BM, changes in S([Na+]) can be accurately predicted by changes in the mass balance of fluid, Na(+), and K(+) during prolonged running in the heat.     

AMPK activation with AICAR provokes an acute fall in plasma [K+

AMP-activated protein kinase (AMPK), activated by an increase in intracellular AMP-to-ATP ratio, stimulates pathways that can restore ATP levels. We tested the hypothesis that AMPK activation influences extracellular fluid (ECF) K(+) homeostasis. In conscious rats, AMPK was activated with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) infusion: 38.4 mg x kg bolus then 4 mg x kg(-1) x min(-1) infusion. Plasma [K(+)] and [glucose] both dropped at 1 h of AICAR infusion and [K(+)] dropped to 3.3 +/- 0.04 mM by 3 h, linearly related to the increase in muscle AMPK phosphorylation. AICAR treatment did not increase urinary K(+) excretion. AICAR lowered [K(+)] whether plasma [K(+)] was chronically elevated or lowered. The K(+) infusion rate needed to maintain baseline plasma [K(+)] reached 15.7 +/- 1.3 micromol K(+) x kg(-1) x min(-1) between 120 and 180 min AICAR infusion. In mice expressing a dominant inhibitory form of AMPK in the muscle (Tg-KD1), baseline [K(+)] was not different from controls (4.2 +/- 0.1 mM), but the fall in plasma [K(+)] in response to AICAR (0.25 g/kg) was blunted: [K(+)] fell to 3.6 +/- 0.1 in controls and to 3.9 +/- 0.1 mM in Tg-KD1, suggesting that ECF K(+) redistributes, at least in part, to muscle ICF. In summary, these findings illustrate that activation of AMPK activity with AICAR provokes a significant fall in plasma [K(+)] and suggest a novel mechanism for redistributing K(+) from ECF to ICF.     

Sodium and ATP affinities of the cardiac (Na,K)-ATPase in relation to nitric oxide synthesis in spontaneously hypertensive rats

The (Na,K)-ATPase is hypothesized to be involved in systemic vascular hypertension through its effects on smooth muscle reactivity and cardiac contractility. Investigating the kinetic properties of the above enzyme we tried to assess the molecular basis of alterations in transmembraneous efflux of Na(+) from cardiac cells in spontaneously hypertensive rats (SHR) with increased synthesis of nitric oxide (NO). In the investigated group of SHR the systolic blood pressure was increased by 64% and the synthesis of NO was increased by 60% in the heart. When activating the cardiac (Na,K)-ATPase with substrate, its activity was higher in SHR in the whole concentration range of ATP. Evaluation of kinetic parameters revealed an increase of the V(max) (by 37%) probably due to increased affinity of the ATP-binding site as indicated by the lowered K(m) value (by 38%) in SHR. During activation with Na(+), we observed no change in the enzyme activity below 10 mmol/l of NaCl whereas in the presence of higher concentrations of NaCl the (Na,K)-ATPase was stimulated. The value of V(max) increased (by 64%), however the K(Na) increased (by 106%), indicating an adaptation of the Na(+)-binding site of the enzyme to increased [Na(+)](i). Thus the (Na,K)-ATPase in our SHR group is able to extrude the excessive Na(+) from myocardial cells more effectively also at higher [Na(+)](i), while the enzyme from controls is unable to increase its activity further. This improvement of the (Na,K)-ATPase function is supported also by increased affinity of its ATP-binding site probably due to enhanced NO-synthesis.     

Non-reciprocal interactions between K+ and Na+ ions in barley (Hordeum vulgare L.)

The interaction of sodium and potassium ions in the context of the primary entry of Na(+) into plant cells, and the subsequent development of sodium toxicity, has been the subject of much recent attention. In the present study, the technique of compartmental analysis with the radiotracers (42)K(+) and (24)Na(+) was applied in intact seedlings of barley (Hordeum vulgare L.) to test the hypothesis that elevated levels of K(+) in the growth medium will reduce both rapid, futile Na(+) cycling at the plasma membrane, and Na(+) build-up in the cytosol of root cells, under saline conditions (100 mM NaCl). We reject this hypothesis, showing that, over a wide (400-fold) range of K(+) supply, K(+) neither reduces the primary fluxes of Na(+) at the root plasma membrane nor suppresses Na(+) accumulation in the cytosol. By contrast, 100 mM NaCl suppressed the cytosolic K(+) pool by 47-73%, and also substantially decreased low-affinity K(+) transport across the plasma membrane. We confirm that the cytosolic [K(+)]:[Na(+)] ratio is a poor predictor of growth performance under saline conditions, while a good correlation is seen between growth and the tissue ratios of the two ions. The data provide insight into the mechanisms that mediate the toxic influx of sodium across the root plasma membrane under salinity stress, demonstrating that, in the glycophyte barley, K(+) and Na(+) are unlikely to share a common low-affinity pathway for entry into the plant cell.     

Influence of permeant ions on voltage sensor function in the Kv2.1 potassium channel

We previously demonstrated that the outer vestibule of activated Kv2.1 potassium channels can be in one of two conformations, and that K(+) occupancy of a specific selectivity filter site determines which conformation the outer vestibule is in. These different outer vestibule conformations result in different sensitivities to internal and external TEA, different inactivation rates, and different macroscopic conductances. The [K(+)]-dependent switch in outer vestibule conformation is also associated with a change in rate of channel activation. In this paper, we examined the mechanism by which changes in [K(+)] modulate the rate of channel activation. Elevation of symmetrical [K(+)] or [Rb(+)] from 0 to 3 mM doubled the rate of on-gating charge movement (Q(on)), measured at 0 mV. Cs(+) produced an identical effect, but required 40-fold higher concentrations. All three permeant ions occupied the selectivity filter over the 0.03-3 mM range, so simple occupancy of the selectivity filter was not sufficient to produce the change in Q(on). However, for each of these permeant ions, the speeding of Q(on) occurred with the same concentration dependence as the switch between outer vestibule conformations. Neutralization of an amino acid (K356) in the outer vestibule, which abolishes the modulation of channel pharmacology and ionic currents by the K(+)-dependent reorientation of the outer vestibule, also abolished the K(+)-dependence of Q(on). Together, the data indicate that the K(+)-dependent reorientation in the outer vestibule was responsible for the change in Q(on). Moreover, similar [K(+)]-dependence and effects of mutagenesis indicate that the K(+)-dependent change in rate of Q(on) can account for the modulation of ionic current activation rate. Simple kinetic analysis suggested that K(+) reduced an energy barrier for voltage sensor movement. These results provide strong evidence for a direct functional interaction, which is modulated by permeant ions acting at the selectivity filter, between the outer vestibule of the Kv2.1 potassium channel and the voltage sensor.