The effect of monovalent cations on calcium efflux in yeasts

The properties of the calcium efflux system in the yeast Saccharomyces cerevisiae were investigated. After growing the cells overnight in medium containing ⁴⁵Ca, the cells were transferred to medium containing glucose, Hepes buffer (pH 5.2) and monovalent cations. The presence of potassium or sodium in the medium induced efflux of calcium from the cells. The magnitude of the efflux was dependent on the concentration of these cations in the medium. The time course of calcium efflux was analyzed, and two types of exchangeable calcium pools, which turned over at different rates, were detected: ‘Fast turnover’ and ‘slow turnover’. Increase in the concentration of monovalent cations in the medium caused an increase in the fraction of cellular calcium which turned over at a fast rate, and activation of calcium efflux from the ‘slow turnover’ calcium pool. The specific changes in the parameters of calcium efflux induced by monovalent cations were different from those reported previously to be induced by divalent cations. Both processes, i.e. activation of calcium efflux by monovalent and by divalent cations, were found to be additive, indicating that they operate via different mechanisms. Experiments using the respiratory inhibitor Antimycin A, showed that stimulation of calcium efflux by monovalent cations is energy dependent. Lanthanum ions which are known to inhibit calcium influx into yeast cells, inhibitted the activation of calcium efflux by both divalent and monovalent cations. Determination of the cationic composition of the cells indicated that the stimulation of calcium efflux was accompanied by influx of potassium or sodium into the cells.     

Proton-activated rubidium transport catalyzed by the sodium pump

Although the sodium pump normally exchanges three sodium for two potassium ions, experiments with inside-out red cell membrane vesicles show that the stoichiometry is reduced when the cytoplasmic sodium concentration is decreased to less than 1 mM. The present study was designed to gain insight into the question whether other monovalent cations, particularly protons, can act as sodium congeners in effecting pump-mediated potassium transport (ATP-dependent rubidium efflux from inside-out vesicles). The results show that at low cytoplasmic sodium concentration, an increase in proton concentration effects a further reduction in sodium:rubidium stoichiometry, to a value less than the minimal expected (1Na+:3Rb+). Furthermore, when vesicles containing 86RbCl are incubated in nominally sodium-free medium. ATP-dependent net rubidium efflux (normal influx) occurs when the pH is reduced from approximately 7.0 to 6.2 or less. This efflux is inhibited by strophanthidin and vanadate. These experiments support the notion that the sodium pump can operate as an ATP-dependent proton-activated rubidium (potassium) pump without obligatory countertransport of sodium ions.     

Fractionation of Sodium Efflux in Frog Sartorius Muscles by Strophanthidin and Removal of External Sodium

The influence of strophanthidin, ouabain, and the removal of external sodium on the sodium efflux from frog sartorius muscle was measured. In freshly dissected muscles strophanthidin and ouabain in maximally effective concentrations reduced the efflux of sodium by about 50%. Of the sodium efflux which is strophanthidin-insensitive about 75% is inhibited after complete replacement of external sodium by lithium. In the absence of strophanthidin replacement of external sodium by lithium, calcium, or magnesium produces an initial rise in the sodium efflux, followed by a fall in the efflux as the exposure of the muscles to sodium-free media is continued. When the muscles are exposed for prolonged periods in sodium-free media, the fraction of internal sodium lost per minute is higher when returned to normal Ringer fluid than it was initially. The activation of sodium efflux by external sodium after long periods in sodium-free solutions is partly strophanthidin-sensitive and partly strophanthidin-insensitive. The internal sodium concentration is an important factor in these effects. The effects of temperature on the sodium efflux were also measured. Above 7°C the Q₁₀ of both the strophanthidin-sensitive and strophanthidin-insensitive sodium efflux is about 2.0. Below 7°C the strophanthidin-insensitive sodium efflux has a Q₁₀ of about 7.4.     

Intracellular Potassium : A Determinant of the Sodium-Potassium Pump Rate

Normal human red cells which have had their intracellular sodium (Na_c) reduced have a diminished Na-K pump rate, but only if intracellular potassium (K_c) is high. If most of the K_c is replaced by tetramethylammonium or choline, both ouabain-sensitive Na efflux and K influx are significantly increased even with Na_c below normal. Cells with reduced Na_c and high K_c have an unchanged Na efflux if external potassium (K_ext) is removed. In contrast, low-Na, low-K cells have a large ouabain-sensitive Na efflux which shows a normal response to removal of K_ext. Neither low-K nor high-K cells have an altered ouabain-sensitive K efflux. Measurement at constant low Na_c and varying K_c shows the pump Na efflux to be an inverse function of K_c. Thus, in low-Na cells, K_c appears to act as an inhibitor of the pump. Inhibition by high K_c can be seen even when Na_c is normal. The effects attributed to K_c are distinguished experimentally from other variables such as cell volume, adenosine triphosphate concentration, effects of the replacement cations, and the method used to alter intracellular cation concentrations. A role is proposed for K_c, in cooperation with Na_c, in regulating the pump rate of normal human red cells.     

Acute Functional Adaptation to Nephron Loss: Micropuncture Studies

The renal and proximal tubule response to contralateral kidney exclusion was studied in a variety of circumstances. Recollection micropuncture studies were performed to assess the response to contralateral kidney clamping in the normal or a remnant kidney of the dog. Acute clamping of the contralateral kidney for a normal and unilateral remnant kidney resulted in marked reduction in proximal TF/P inulin ratios in the experimental kidney reflecting a 15 percent reduction in fluid reabsorption. Mean fractional excretion of sodium, potassium and water increased significantly in remnant kidney dogs but no significant change was observed in normal dogs except for potassium excretion. The marked reduction in proximal reabsorption occurred as soon as 5-15 minutes after contralateral kidney clamping and was compensated by distal reabsorption. Acute obstruction of the contralateral ureter results in a similar markedly reduced proximal tubular reabsorption. The reduction in proximal reabsorption induced by contralateral clamping occurred in the presence of reduced perfusion pressure and volume expansion and to some extent with renal denervation. When prostaglandin E₂ or acetycholine were infused prior to contralateral kidney clamping, proximal reabsorption remained at control levels and the contralateral clamping response was blocked. Similar blockade occurred after treatment with indomethacin. Acute reduction in nephron mass causes a marked depression of proximal tubular sodium and fluid absorption not obviously accounted for by hemodynamicphysical factors and humoral factors may be involved. The level of distal reabsorption to increased proximal delivery following contralateral clamping, determines the net urinary excretion.     

The Interactions Between Monovalent Cations and Calcium During Their Adsorption on Isolated Cell Walls and Absorption by Intact Barley Roots

The adsorption of sodium, potassium, rubidium and calcium ionsat different concentrations was measured on cell walls frombarley roots (Hordeum vulgare L. cv. Union), prepared by detergenttreatment. It was found that sodium and calcium interacted verystrongly during their simultaneous adsorption, whereas potassiumdid not interfere with calcium. This has led us to concludethat calcium and sodium are adsorbed on identical sites in thecell wall, whereas potassium is adsorbed at another site. Rubidiumseems to be less specific for both sites and interferes onlymoderately with calcium. The adsorption on cell walls of thesecations was compared with their adsorption on intact roots at2 °C, where beside the cell wall, sites may be availableat the outer surface of the membrane, and further measurementswere made of absorption at 25 °C. The fact that sodium interactswith calcium and potassium alters the ratio of K to Na in thecell wall compared to their concentrations in the medium. Thepreferential shift towards potassium when calcium is presentcould be very important for the rates of initial uptake in lowsalt barley roots, since the membrane is in contact with a differentproportion of K to Na in the cell wall from the one suppliedin the medium. Hordeum vulgare L., barley, absorption of cations, adsorption, calcium, sodium, potassium rubidium     

Affinities or Apparent Affinities of the Transport Adenosine Triphosphatase System

The interactions of potassium ions and ATP on transport ATPase activity are discussed, and the interpretation of these interactions is shown to be often ambiguous. Caldwell''s (1968) Physiological Review model is discussed with particular reference to the observed kinetics of sodium: sodium exchange in red cells. Recent experimental work on the properties of the ouabain-sensitive component of potassium efflux from red cells is described. This component of efflux occurs only if either sodium or potassium are present in the external medium, but the effects of external sodium and potassium are not additive. The relation between ouabain-sensitive potassium efflux and the external concentration of sodium (in a potassium-free medium) or of potassium (in low- and high-sodium media) are described. When starved sodium-poor red cells are poisoned with iodoacetamide, loaded with phosphate, and incubated in high-sodium potassium-free media, the ouabain-sensitive efflux of potassium appears to be accompanied by the reversal of the entire ATPase system. About two to three potassium ions leave by the ouabain-sensitive route for each molecule of ATP synthesized. If potassium is present in the external medium, no ouabain-sensitive synthesis of ATP occurs and the ouabain-sensitive efflux of potassium presumably involves the reversal of only the last part of the ATPase system.     

An Analysis of the Leakages of Sodium Ions into and Potassium Ions out of Striated Muscle Cells

Net sodium influx under K-free conditions was independent of the intracellular sodium ion concentration, [Na]_i, and was increased by ouabain. Unidirectional sodium influx was the sum of a component independent of [Na]_i and a component that increased linearly with increasing [Na]_i. Net influx of sodium ions in K-free solutions varied with the external sodium ion concentration, [Na]_o, and a steady-state balance of the sodium ion fluxes occurred at [Na]_o = 40 mM. When solutions were K-free and contained 10^-4 M ouabain, net sodium influx varied linearly with [Na]_o and a steady state for the intracellular sodium was observed at [Na]_o = 13 mM. The steady state observed in the presence of ouabain was the result of a pump-leak balance as the external sodium ion concentration with which the muscle sodium would be in equilibrium, under these conditions, was 0.11 mM. The rate constant for total potassium loss to K-free Ringer solution was independent of [Na]_i but dependent on [Na]_o. Replacing external NaCl with MgCl₂ brought about reductions in net potassium efflux. Ouabain was without effect on net potassium efflux in K-free Ringer solution with [Na]_o = 120 mM, but increased potassium efflux in a medium with NaCl replaced by MgCl₂. When muscles were enriched with sodium ions, potassium efflux into K-free, Mg⁺⁺-substituted Ringer solution fell to around 0.1 pmol/cm²·s and was increased 14-fold by addition of ouabain.     

The influence of monovalent cations upon influx and efflux of Ca2+ in barley roots

The effects of monovalent cations, inhibitors of metabolism dinitrophenol (DNP), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and KCN and temperature variations upon Ca²⁺ fluxes in intact roots of barley (Hordeum vulgare L. cv. Fergus and Herta) seedlings were investigated. ⁴⁵Ca²⁺ influx was depressed in CaSO₄-grown (low-salt) plants by the presence of NH₄⁺, K⁺, or Na⁺ in the uptake medium. In contrast Ca²⁺ influx was slightly increased by Li⁺. In low-salt roots pretreated with KCN and in roots preloaded with K⁺ (high-K⁺ plants), the presence of K⁺ in the medium had no significant effect on Ca²⁺ influx, while in roots preloaded with Na⁺, the presence of K⁺ in the medium depressed Ca²⁺ influx. In absolute terms, Ca²⁺ influx was significantly greater in high-salt (both K⁺ or Na⁺ preloaded) than in low-salt roots.Patterns of ⁴⁵Ca²⁺ efflux in the absence and in the presence of K⁺, NH₄⁺, or Li⁺ in the external medium showed that these monovalent cations caused stimulation of ⁴⁵Ca²⁺ efflux both from the cytoplasmic and vacuolar phases.It was noted that these modifications of Ca²⁺ fluxes by monovalent cations are transient and characteristic of a transitional stage of cation uptake by low-salt roots. We conclude that, together with stimulated active H⁺ efflux (another characteristic of this transitional stage), modifications of Ca²⁺ fluxes during monovalent cation uptake by low-salt roots is a response directed towards the maintenance of electrical neutrality.Determination of net fluxes revealed that the plants were close to Ca²⁺ flux equilibrium in the growth medium (0.5 mM CaSO₄). Transfer of these plants to 0.5 mM CaSO₄ + 0.25 mM K₂SO₄ caused a net release of CA²⁺ into the external medium.     

The kinetics of sodium extrusion in striated muscle as functions of the external sodium and potassium ion concentrations

After a 20 min initial washout, the rate of loss of radioactively labeled sodium ions from sodium-enriched muscle cells is sensitive to the external sodium and potassium ion concentrations. In the absence of external potassium ions, the presence of external sodium ions increases the sodium efflux. In the presence of external potassium ions, the presence of external sodium ions decreases the sodium efflux. In the absence of external potassium ions about one-third of the Na⁺ efflux that depends upon the external sodium ion concentration can be abolished by 10^-5 M glycoside. The glycoside-insensitive but external sodium-dependent Na⁺ efflux is uninfluenced by external potassium ions. In the absence of both external sodium and potassium ions the sodium efflux is relatively insensitive to the presence of 10^-5 M glycoside. The maximal external sodium-dependent sodium efflux in the absence of external potassium ions is about 20% of the magnitude of the maximal potassium-dependent sodium efflux. The magnitude of the glycoside-sensitive sodium efflux in K-free Ringer solution is less than 10% of that observed when sodium efflux is maximally activated by potassium ions. The inhibition of the potassium-activated sodium efflux by external sodium ions is of the competitive type. Reducing the external sodium ion concentration displaces the plots of sodium extrusion rate vs. [K]_o to the left and upwards.     

The effects of alterations in the external sodium concentration on human leucocyte sodium and potassium transport in vitro

Human leucocytes incubated in tissue culture fluid of low-sodium concentration (2 mM; iso-osmolarity maintained with choline chloride) reached a new equlibrium within 1 hour and lost approximately 25% of intracellular potassium and 70% of intracellular sodium. The rate constant for ouabainsensitive sodium efflux fell by more than 50% and the ouabain-insensitive rate constant increased nearly threefold in the low-sodium medium. Total sodium efflux fell in proportion to internal sodium whereas ouabain-insensitive sodium efflux remained unchanged. A reduction in external sodium from 140 to 2 mM was associated with a 75% fall in sodium influx. In the low-sodium medium ouabainsensitive potassium influx exceeded ouabain-sensitive sodium efflux and no ouabain-sensitive potassium efflux could be demonstrated. Ouabain-insensitive potassium influx and that portion of potassium efflux which is dependent on external potassium fell in parallel in low-sodium cells, suggesting reduced activity of a ouabain-insensitive K:K exchange system.     

Effect of Low pH on Glutamate Uptake and Release in Isolated Presynaptic Endings from Rat Brain

The effect of acidification of the incubation medium on the membrane potential and glutamate uptake and release was studied in isolated presynaptic neuronal endings (synaptosomes) from rat brain. Using the fluorescent probe diS-C₃-(5), a rapid depolarization of plasma membrane was detected at pH 6.0, most probably as a result of the inhibition of the sodium pump and potassium channel blockade. The membrane potential decrease did not result in increase of basal efflux of glutamate. Glutamate release following K⁺-induced depolarization was decreased upon lowering pH to 6.0. Acidosis inhibited mainly calcium-dependent (vesicular) release of glutamate and did not significantly reduce [¹⁴C]glutamate uptake. This inhibition of glutamate release but not of glutamate uptake may be a mechanism of the protective effect of acidosis during brain ischemia.     

Effects of Low Root Temperature on Ion Uptake and Ion Translocation in Wheat

Roots of wheat seedlings (Triticum aestivum L. cv. Weibulls Starke) were cooled (+1°C) for 24 h while the shoots were kept at 25°C. The treatment induced an increased water deficit in the leaves. Fresh weight, dry weight, and the uptake and distribution of potassium and calcium were measured before and after cooling. Growth, measured both as fresh weight and dry weight increase, was reduced during the cold treatment. Afterwards (at 20°C), growth recovered to nearly pre-stress rates. Analysis of the potassium fluxes in and out of the roots by ⁸⁶Rb techniques showed that influx, and to a lesser extent efflux, were inhibited at low temperature. The result was a net potassium uptake rate of one-third that of unstressed plants. After the cooling period the potassium influx increased to the rate of control plants. The potassium efflux increased to one and one-half times the rate of unstressed wheat so that net uptake was negative. The increase in potassium efflux was explained by a higher permeability of the root cell membranes after cooling. The net uptake of calcium was reduced to one-third by root cooling. Contrary to potassium uptake, calcium uptake increased under post-stress conditions, partly due to a low efflux rate. During root cooling there was a redistribution of dry matter from the leaves down towards the lower part of the shoot. Afterwards the original distribution of dry matter was reestablished. The net flow of potassium and calcium followed a similar pattern as dry matter, suggesting a growth-regulated flow.     

Freezing of isolated thylakoid membranes in complex media: I. The effect of potassium and sodium chloride,nitrate, and sulfate

Thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were subjected to a freeze-thaw cycle in the presence of a buffered medium containing sorbitol as a cryoprotectant and various combinations of potassium and sodium chloride, nitrate, and sulfate. Above a certain total salt concentration, an increase in the concentration of a single electrolyte, or of potassium plus sodium salts with identical anions, always led to a decrease in photophosphorylation activity. A similar effect was obtained with combinations of nitrate plus chloride with identical cations and of KNO₃ plus NaCl. By contrast, in the presence of suitable combinations of NaNO₃ plus KCl, NaNO₃ plus sulfates, and chlorides plus sulfates, inactivation of photophosphorylation was diminished, sometimes dramatically, at initial molarities of nitrate or chloride which alone caused partial or complete membrane damage. When NaNO₃, KCl, and potassium or sodium sulfate were simultaneously present during freezing, thylakoids were affected very little over a wide range of concentration. Diminution or prevention of inactivation of photophosphorylation by suitable combinations of two or more cryotoxic inorganic salts can be explained by postulating that the different solutes act on different sites and that each reduces the concentration of the others by colligative action, together with specific effects of the various electrolytes on individual membrane sites.     

Determination of Unidirectional Sodium Fluxes in Roots of Intact Sunflower Seedlings

The method of compartmental analysis was applied to study sodiumfluxes in roots of intact seedlings of Helianthus annuus L.By measuring sodium uptake and transport to the shoots of theseedlings in parallel experiments, transport of tracer sodiumto shoots and net accumulation of Na⁺ in the roots during theflux measurements was accounted for. The steady-state sodiumfluxes in the intact sunflower roots were similar in size tothose in excised roots but in general they were somewhat higher.This indicates more metabolic activity in the intact tissues.Using whole plants it is possible to study the response of ionfluxes in roots to ecophysiological stimuli received by theshoots, and in the present experiments the effect of continuouslight versus long-day growth conditions was investigated. Potassium,when continually present, depressed all fluxes and the cytoplasmiccontent of sodium but tended to increase the vacuolar sodiumcontent, in particular when this was related to the cytoplasmiccontent. When added to sodium-loaded roots, potassium stimulatedthe plasmalemma sodium efflux but slightly, suggesting a lowefficiency of K⁺-Na⁺ exchange across the plasmalemma in intactas well as excised sunflower roots. Subsequently, however, potassiuminduced a transient decrease in the ²²Na efflux that was followedby oscillations in tracer efflux. These changes were attributedto potassium-induced transfer of sodium to vacuoles. Moreover,the oscillations seem to indicate the operation of negativefeedback control of sodium fluxes.     

Changes in the Membrane Permeability of Frog's Sartorius Muscle Fibers in Ca-Free EDTA Solution

The changes in the membrane permeability to sodium, potassium, and chloride ions as well as the changes in the intracellular concentration of these ions were studied on frog sartorius muscles in Ca-free EDTA solution. It was found that the rate constants for potassium and chloride efflux became almost constant within 10 minutes in the absence of external calcium ions, that for potassium increasing to 1.5 to 2 times normal and that for chloride decreasing about one-half. The sodium influx in Ca-free EDTA solution, between 30 and 40 minutes, was about 4 times that in Ringer's solution. The intracellular sodium and potassium contents did not change appreciably but the intracellular chloride content had increased to about 4 times normal after 40 minutes. By applying the constant field theory to these results, it was concluded that (a) P_Cl did not change appreciably whereas P_K decreased to a level that, in the interval between 10 and 40 minutes, was about one-half normal, (b) P_Na increased until between 30 and 40 minutes it was about 8 times normal. The low value of the membrane potential between 30 and 40 minutes was explained in terms of the changes in the membrane permeability and the intracellular ion concentrations. The mechanism for membrane depolarization in this solution was briefly discussed.     

Efflux of chloride from lactating rat mammary tissue slices

1. The efflux of chloride (using 36Cl) from lactating rat mammary tissue slices has been investigated. 2. Chloride efflux was found to be temperature dependent; lowering the temperature of the incubation medium reduced the fractional efflux. 3. The stilbene derivatives DIDS was without effect on the fractional release of Cl when studied at 20 degrees C. However, DIDS was found to attenuate the increase in efflux found upon transferring the tissue from a medium maintained at 4 degrees C to one at 20 degrees C. 4. The loop-diuretic furosemide, also reduced the temperature-sensitive portion of Cl efflux. 5. Chloride efflux was transiently increased when tissue slices were transferred from a medium containing gluconate as the principal anion to one containing Cl. 6. The results appear to confirm that mammary Cl transport is mediated via anion exchange and via (Na + K + Cl) cotransport.     

Transmembrane effects in the sodium pump system. II. The ratio of the sodium efflux to the sodium concentration in frog muscle in media with various sodium substitutes

The dependence of sodium efflux on the internal sodium concentration on sodium-free magnesium, Tris, coline and lithium media was investigated on frog striated muscle. In all the sodium-substituted media, the efflux concentration curve was found to be dependent on the external rubidium concentration, being S-shaped at the saturating external rubidium (potassium) concentration and becoming close to linear at the low external rubidium concentration (0.5-1.0 microM). The maximal sodium efflux at saturating levels of internal sodium concentrations remains unchanged with various sodium substitutes in the medium, whereas the affinity constant of internal sodium sites is dependent on the external cations.     

Effect of lysophosphatidylcholine on salt permeability through the erythrocyte membrane under haemolytic conditions

Human erythrocytes were incubated in haemolytic salt or sucrose media and the amount of potassium and haemoglobin released were monitored. In hypotonic NaCl and KCl solutions potassium release and haemolysis increased with time showing that the cell membrane had been injured and became permeable to intra- and extracellular cations which, due to intracellular haemoglobin, causes water influx and continuous haemolysis. Both potassium release and haemolysis remained, however, at their 2-minute level in the presence of LPC. Thus, LPC could reseal the membrane and prevent continuous salt fluxes. It protected erythrocytes from hypotonic haemolysis and the protection was more efficient in NaCl than in sucrose media. This suggests that the increase in the critical volume of erythrocytes caused by LPC occurs both in electrolyte and sucrose media, and the additional protection observed in electrolyte media is due to the resealing of the injured cell membrane by LPC. The repairing mechanism was mediated via the membrane lipids or integral proteins, since the time-course of haemolysis of erythrocytes swollen in NaCl media at the spectrin-denaturing temperature of 49.5 degrees C was similar to that at room temperature with and without LPC. LPC did not protect erythrocytes from colloid osmotic haemolysis caused by ammonia influx in an isotonic NH4Cl medium, but protected the cells from colloid osmotic haemolysis caused by sodium influx through nystatin-channels in NaCl media without any area or volume increase. Hence, LPC could not prevent ammonia influx through the lipid bilayer, but suppressed sodium influx through nystatin-channels presumably via LPC interference with cholesterol.     

Uptake and distribution of sodium and potassium by corn seedlings : I. Role of the mesocotyl in ;sodium exclusion'

The distribution of sodium and potassium throughout corn (Zea mays L. [A632 × Crows 3640] × Oh 43) plants is not simply a matter of uptake by cortical cells and irreversible delivery to the xylem for upward transport. We show that sodium, but not potassium, accumulates in the mesocotyl of corn seedlings grown on NaCl medium. Upon transfer to NaCl-free medium, total sodium is reduced by export through the roots but remains at high levels within the mesocotyl. We report experiments which consider uptake from the xylem.

Shoots excised at the seed were allowed to transpire solutions containing ²²Na and ⁴²K. Potassium uptake within the mesocotyl was very sensitive to concentration, increasing 27-fold between 1 and 10 millimolar. Sodium uptake was dependent upon the square root of the concentration suggesting active accumulation. At sodium concentrations below 1 millimolar, more than 80% of the sodium in the plant was retained in the mesocotyl. Both the uptake by and retention within the mesocotyl were dependent upon transpiration rate as well as concentration. We discuss the limitations of measuring uptake from a finite, depletable medium. The mesocotyl is a modified root with a cuticularized epidermis. We discuss the feasibility of using this `plastic-coated root' as a model for root transport studies.