Accumulation of potassium by human red cells

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1. A method is described for measuring the accumulation of K at 37°C. by washed human red cells in glucose-containing systems in which the pH is kept constant, the K content of the cells being compared with that of the cells of systems which contain no added glucose but which are otherwise treated similarly. 2. In systems containing added glucose, the accumulation of K begins shortly after the cells have been warmed to 37°C., proceeds to a maximum which is reached after about 10 hours, and then falls exponentially. The maximum rate of accumulation is found during the first 3 hours. In systems which contain no added glucose, the K content of the cells appears to decrease exponentially with time for about 18 to 24 hours; thereafter the K content of the cells may decrease rapidly and the systems may show considerable hemolysis. Sometimes a small accumulation effect is observed during the first 2 to 3 hours; this may be the result of the washed cells not having been completely freed of glucose. 3. The accumulation process proceeds at its maximum rate at pH 7.4 to 7.6, which is also the pH at which the K loss from the red cells is at a minimum in systems containing no added glucose. 4. When red cells are stored at 4°C. for increasing lengths of time, the storage is accompanied by increasing K loss and the maximum rate of accumulation observed when the cells are warmed to 37°C. at first becomes greater. If the storage at 4°C. is continued for more than 3 to 4 days, the rate of the accumulation which occurs at 37°C decreases again, the accumulation mechanism showing progressive deterioration with time even at low temperatures. This deterioration has a counterpart in the progressive deterioration (deduced from the analysis of the curves relating K content and time) of the accumulation mechanism with time at 37°C. 5. The accumulation of K occurs at a maximum rate when the concentration of glucose in the system is between 50 and 200 mg./100 ml. Its temperature coefficient over the range 27–37°C. is 2.4. In the presence of glucose and at pH 7.6, accumulation of K takes place from isotonic mixtures of KCl and LiCl or of KCl and CsCl only a little less actively than from mixtures of KCl and NaCl; i.e., the accumulation of K under optimum conditions seems to be an active process which is at least partly independent of the excretion of Na.

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The province covered by the title assigned to this division of the handbook might reasonably be taken to include an appalling diversity of physiological processes. However, there is no intention here to cover such aspects as the elaboration of special glandular secretions, or the extensive renal and gastrointestinal physiology which might conceivably be included under the heading. A survey of such scope would not only involve a literature of impractically enormous proportions, but would misplace the intended emphasis. The effort here is to cover those lines of investigation which purport to deal more or less directly with the activity of cells in the translocation of substances through the cell surfaces. The transfers concerned will be in general either between the interior and the exterior of the cells, or through layers of cells from one side to the other. Discussion of-the cellular extrusion of special secretory products has been avoided; and details of the operation of the special absorptive and excretory organs are taken up only insofar as the experimental approach has been directed toward analysis of the transport phenomena in the various epithelia involved.
. It is shown that freshlv drawn pigeon blood when placed at body temperature during the first hour will absorb half or more of the plasma potassium. Later the plasma potassium concentration will increase. Lack of oxygen will stop the potassium absorption and instead potassium of the cells is lost. and sodium increased.Addition of isotonic KCl solution will not change the potassium absorption.But if you besides KCl add a hypertonic solution of NaCl a considerable potassium uptake is seen, and it will carry on for more than three hours. In this way the cell potassium concentration can be augmented by more than 20 %.Blood from hen or cock will not absorb potassium unless a hypertonio NaCl solution is added.The hypertonic solution may increase the active potassium absorption or it may tighten the cell membrane so that the loss of potassiiini from the cells is decreased. The second possibility is considered the most probable. The theory is strengthened by an experiment where 1 % ethyl alcohol produced a loss of potassium, whereas simultaneous addition of hypertonic NaCl solution produced a considerable potassium uptake. Simultaneously with the potassium absorption an increase of the haematocrit values is seen.The find solution of the problem may be given by experiments with radioactive potassium.The experiments correspond to similar experiments made with bacillus coli communis (Ørskov 1948).
Studies have been conducted on the movements of sodium and potassium into and out of the Ehrlich ascites tumor cell. Under steady state conditions, at 22 degrees C., in the absence of an exogenous source of glucose, the cell flux for both potassium and sodium averaged 0.8 microM10(7) cells/hr, or 3.0 pM/cm.(2)/sec. The cell can accumulate potassium and extrude sodium against electrochemical gradients for both ions. It is possible under the experimental conditions reported to separate the transport systems for these two ions. Thus, it has been shown that under conditions of low temperature with a diminished metabolism, net fluxes for the two ions are different. Also, following periods of 24 hours at 2 degrees C., an exogenous source of glucose enhances the accumulation of potassium sevenfold while sodium extrusion is uninfluenced by the presence of glucose. Similarly potassium exchange rates are temperature-dependent, with Q(10) values as high as 5, while exchange rates for sodium are temperature-insensitive, with Q(10) values of 1.2 to 1.6. Glycolysis has been eliminated as an energy source for the transport processes since these processes go on in the absence of an exogenous source of glucose. It is estimated that a maximum of 0.3 per cent of the energy derived from the total oxidative metabolism of glucose would be required to support independent transport of potassium and sodium.
1.1. Net movements of sodium and potassium in liver slices prepared from rats at different stages of foetal and post-natal growth have been studied in vitro.2.2. During incubation at 1° slices prepared from rats of most of the ages studied lost potassium and gained sodium and water. These changes could be accounted for by a 1:1 exchange of Na+ from the medium for K+ of the cells and by the further entry of sodium as a solution having the same composition as the medium.3.3. In liver slices from the youngest rats studied (17–18 days gestation) the extent of the changes during incubation at 1° was much less than in slices prepared from older animals. There was no significant increase in the water content and the net gain of sodium was equivalent to the loss of potassium.4.4. During subsequent incubation at 38° under aerobic conditions the slices from rats of all ages regained potassium and lost sodium.5.5. The net uptake of potassium during the incubation at 38° of liver slices prepared from foetuses at 17–20 days gestation was completely inhibited by cyanide. At 21–22 days gestation about 40% of the potassium uptake persisted in the presence of cyanide; after birth about 10% of the potassium uptake was resistant to cyanide. The cyanide-resistant potassium uptake was inhibited by the further addition of iodoacetate to the incubation medium.6.6. Liver slices prepared from foetuses at 17–20 days gestation showed a net loss of sodium during incubation at 38° in the presence of cyanide.7.7. The relation of the sodium and potassium movements at 38° to the respiratory and anaerobic glycolytic activity of the rat liver during the growth of the animal is discussed.
1.1. The effects of oligomycin on the net accumulation of potassium and net loss of sodium by whole mammalian cells have been studied. Three types of cell system, differing in the relationship of the cation movements to the energy-providing metabolism, were used.2.2. In liver slices prepared from adult rats oligomycin inhibited sodium and potassium movements by a maximum of 50%. Half-maximal inhibition of potassium uptake was given by oligomycin at a concentration of approx. 3 μg/ml (0.29 μg/mg liver-slice protein). The 50% of the potassium uptake which persisted in the presence of oligomycin was largely inhibited by the further addition of cyanide or 2,4-dinitrophenol.3.3. Oligomycin inhibited respiration of the adult tissue by a maximum of 20%, half-maximal inhibition being given by 3 μg/ml oligomycin (0.29 μg/mg protein). The inhibition was released by the further addition of dinitrophenol.4.4. Slices of liver prepared from rat foetuses during the last 2 days of gestation can accumulate a considerable amount of potassium when incubated in the presence of cyanide. Oligomycin gave approx. 50% inhibition of this cyanide-resistant accumulation of potassium, the half-maximal effect being given by an oligomycin concentration of about 3 μg/ml (0.41 μg/mg liver-slice protein). In the absence of oligomycin, dinitrophenol almost completely inhibited cyanide-resistant potassium accumulation. In the presence of oligomycin, part of the potassium accumulation remained uninhibited by dinitrophenol.5.5. Oligomycin did not inhibit anaerobic glycolysis in the slices of foetal liver.6.6. Oligomycin, at concentrations of 10 and 20 μg/ml, had no effect upon net sodium movements in human erythrocytes. In contrast, strophanthin G inhibited the sodium movements.7.7. Consideration of these results in relation to the known effects of oligomycin on enzyme activities in subcellular particles suggests that oligomycin probably inhibited cation movements in liver slices by virtue of its inhibitory effect upon oxidative phosphorylation, rather than by inhibition of a reaction directly involved in cation transport. If this conclusion is correct, it follows that part of the energy required for cation transport by liver cells can be derived directly from an energy-rich intermediate of oxidative phosphorylation.
1. The kinetics of the movements of glucose in both directions across the surface of the human red cell were studied by optical recording (Ørskov method) of resultant cell volume changes. 2. A wide experimental variety was arranged in the relations between the several quantitative factors contributing to the glucose gradient and the volume changes expected, in order to provide a maximum variety of systematic relations between those factors and the rate of glucose transfer. 3. The kinetics were shown to follow the patterns predicted on the basis of a simple carrier system, involving formation of a highly undissociated complex between the sugar and some factor in the cell surface, provided the glucose concentrations used did not exceed about 3/4 isosmotic. Certain simple properties of this system are derived from the data. 4. At very high glucose concentrations, this system apparently gradually fails to operate; this failure is reversible upon lowering of the excessive glucose concentration. 5. An empirical correction was derived for a previously known but uncalibrated optical disturbance complicating the use of the Ørskov method with media containing appreciable concentrations of non-electrolytes.
It has been shown that potassium can be replaced in both dog and human erythrocytes against a concentration gradient when cells are incubated with acetylcholine and cholinesterase activity is maintained, provided the cell has previously lost a part of its normal complement of potassium. This process is inhibited by physostigmine.Replacement of potassium in human cells during metabolism of glucose is similarly blocked by physostigmine, which is considered to be a specific inhibitor of cholinesterase activity.These results point to a single mechanism for potassium replacement regardless of whether the substrate added is glucose or acetylcholine, and that mechanism depends on an active cholinesterase.
In writing this chapter for “Protoplasmatologia,” I have tried to confine the material to a consideration of the problems of the red cell and its breakdown as they appear at the moment. The problems of 1955 are very different from those which presented themselves tea years ago, as will he realized by anybody who compares this chapter with my monograph. “Hemolysis and Related Phenomena.” 1948. In these ten years. the situation has changed because of six new departures: the observation of fine structure. made with the electron microscope, the realization that there are many varieties of ghosts which have properties of their own, the increasing amount of evidence that some of the simplifying hypotheses regarding the osmotic behaviour of the red cell have broken down, the observation of the hitherto negleeted fragmentation phenomena, the realization that many Iytic reactions cannot be described by the equations for simple chemical reactions. and. finally. the appreciation of the fact that the mammalian red cell has a complex metabolism and that this metabolism is concerned with processes such as active ion transport.
More than thirty years have passed since the appearance of Höber’s treatise1 on the alkali metal ions in this handbook. Since then the literature on the biological functions and effects of these ions has encreased enormously. The mere bulk of material has forced the authors to abandon the vain attempt to cover all the papers with a bearing on the field. Instead they have tried to choose from the abundance primarily what they feel can be organized into a coherent picture of the biological role of the alkali metal ions.
This chapter describes some recent developments in the field of alkali cation transport. It focuses on problems of sodium and potassium movements. Dean stated that the cell membrane is permeable for sodium as well as for potassium and that the leakage of sodium into the cells is compensated by a sodium pump located in the membrane. For those cell types in which the distribution of not only sodium but also potassium is far from equilibrium, the leak and pump concept possibly has to be extended to potassium, assuming the existence of a potassium pump. On the basis of various evidence, the conclusion was drawn that the leak and pump are spatially separated—leakage occurs by simple diffusion, possibly through aqueous channels, while the pump operates across a lipoid layer. The leak and pump system is utilized for specific cell functions, such as the excitation and recovery cycle in excitable tissues and the ion transports across epithelium cells in the frog skin and in the kidney.
1. In Analogie zum Erythrocyten wurde gezeigt, daß der Mechanismus der Gewichtszunahme der Linse spontan und unter Einwirken von Äthylurethan kolloidosmotischer Natur ist. Die Gewichtszunahme läßt sich durch Kompensation mit Saccharose verhindern, vorausgesetzt, daß dabei für einen optimalen Elektrolytgehalt des Milieus gesorgt ist. 2. Glutathion kann die durch Äthylurethan bedingte Gewichtszunahme zum Teil deutlich vermindern. — Die Bedeutung des Stoffwechsels für die physiologischen Durchlässigkeitseigenschaften der Linsenkapsel wird diskutiert.
Will man ihn erkennen, dann rut man gut, den Zustand des Zelltodes zum Vergleich heranzuziehen. Man denke sich die Bereitstellung yon Energie durch eine pl6tzliche Einstellung aller Stoffwechselprozesse angehalten. Keinesfalls wiirde dann die Ordnung der Zelle unver~indert bleiben, vielmehr wtirden sich Konzentrationsunterschiede, Potentiale usw. ausgleichen. Die vorhandenen klein- und grogmolekularen organischen Stoffe wtirden in Gegenwart der Fermente bald das Gleichgewicht erreichen, welches ihnen auch im Reagenzglas ohne den Ablauf energieliefernder Vorg~inge zuktime. Es stellte sich nach einem kurzen reversiblen Stadium der Dauerzustand des thermodynamischen Gleichgewichtes ein, in dem sich keine feststellbaren Vorg~inge mehr vollziehen. Die Anderung der freien Energie (A F) bzw. der freien Enthalpie (A G) und die Anderung der Entropie (A S) sind gleich null gewordenl). Die freien Energien haben dann ihr Minimum und die zugeharigen Entropien aller abgelaufenen Entspannungsprozesse ihr Maximum erreicht. Der Zelltod kann so thermodynamisch nicht anders als durch Abnahme der Ordnung bzw. Zunahme der Zustandswahrscheinlichkeit gekennzeiehnet werden. W~hrend des Lebens wird also ein haherer Ordnungszustand durch Energiezuftihrung aufrechterhalten. So wird der spontan angestrebten Entropiezunahme entgegengearbeitet, d.h. sie selbst auf einem niedrigen Weft gehalten: die Teilmechanismen werden an der Erreichung ihrer Gleichgewichte gehindert. Sie bleiben so zur Leistung yon Arbeit I~ihig, die sie unter Verlust ihrer Ordnung bzw. Zunahme ihrer Entropie bei Ann~herung an das Gleichgewicht abgeben. Sinn des Energiestoffwechsels ist also Niedrighaltung der *) Vortrag beim I. Mosbacher Colloquium am 28. September
Die Wirkungen von Calcium und Digitalis auf die aktiven und passiven Kaliumverschiebungen durch die Erythrocytenmembran wurden verglichen. Die Beeinflussung der Kalium-Natriumpumpe wurde mit der von Harris angegebenen Methodik untersucht. Die passiven Kaliumverschiebungen wurden nach Depolarisation der Zellmembran mittels Inkubation der Zellen in Gemischen von isotonischer Salzlösung und isotonischer Saccharoselösung bei niedriger Elektrolytkonzentration verfolgt. Die Kalium-Natriumpumpe wurde von Digitalis blockiert, von Calcium nicht beeinflußt. Die passiven Kaliumverschiebungen wurden von Calcium gehemmt, wogegen Herzglykosid ohne Wirkung war. Die Bedeutung der Befunde für die Interpretation des Wirkungsmechanismus der Herzglykoside wird diskutiert.
Es wurden die Beziehungen zwischen dem aktiven Kationentransport und der Methmoglobinrckbildung in roten Blutkrperchen untersucht. Daraus ergaben sich folgende Feststellungen: 1. Glucose unterhlt beide Vorgnge, die Rckbildung hat in normalem osmotischem Milieu den Vorrang. — Verlieren die Erythrocyten die Fhigkeit, Methmoglobin zurckzubilden, ist auch die Erhaltung des Kationenbestandes der Zellen nicht mehr gesichert. 2. Lactat unterhlt nur die Methmoglobinrckbildung. 3. Gleichzeitige Zugabe von Glucose und Lactat vermag sowohl die Hb(3)-Rckbildung, als auch den aktiven Kationentransport zu unterhalten. 4. Die Methmoglobinrckbildung in hypertonem Medium ist deutlich verlangsamt, die Ursachen dafr sind noch nicht geklrt. 5. Es bestehen Unterschiede im osmotischen Verhalten normaler und methmoglobinhaltiger Erythrocyten; sie haben ihre Ursache in der Konkurrenz der Methmoglobinrckbildung und des aktiven Kationentransports um gleiche Stoffwechselsysteme und fhren dazu, da die Methmoglobinrckbildung mit einem Absinken der osmotischen Resistenz verbunden sein kann.
Digitoxin, ouabain, digoxin and their derivatives dihydrodigitoxin, dihydro-ouabain, and dihydrodigoxin were investigated in regard to their effects on the Na+ and K+ transport in incubated cold stored guinea-pig erythrocytes. The ion transport is inhibited by 50% in the following concentrations of the different glycosides: digitoxin 3.0 × 10−7 M, ouabain 1.0 × 10−6 M, digoxin 1.2 × 10−6 M. The saturation of the lactone ring decreases the potency of all three glycosides by the same order of magnitude. The potencies of the dihydro-derivatives in relation to their parent compounds are: dihydro-digitoxin 1/22, dihydro-ouabain 1/34, and dihydro-digoxin 1/25.
1.(1) An outline is given of some of the biologically important properties of inorganic ions and of the part played by different ion species in the metabolism of cells.2.(2) The composition of about 100 physiological salines for use with fish, amphibia, mammals, birds, annelids, crustacea, insects and molluscs is given together with analyses of the blood composition of some of the species of animals most frequently used in physiological experiments.
1.1. Sodium and potassium have been measured in the plasma and erythrocytes of inbred strains of mice and of anemic mutants.2.2. In Mus musculus, and also in M. molossinus and M. poschiavinus, the plasma Na and K are in the range 150 and 5 m-equiv/l. respectively. The erythrocyte Na and K are about 13 and 118 m-equiv/l. respectively.3.3. Among ten hereditary anemias, plasma Na and K are within normal limits.4.4. Five of the anemias (mk/mk, nb/nb, ja/ja, ha/ha and sph/sph) have from two to five times the normal cell Na concentrations. On a per cell basis, the Na levels range from less than 1·2 times (mk/mk) to twelve times (sph/sph) normal.5.5. Cell K is normal in two of the anemias with high cell Na (nb/nb and ja /ja), and reduced in ha/ha and sph/sph.6.6. In one anemia (an/an) with normal cell Na, the cell K level is slightly elevated.
Sodium-loaded human erythrocyte ghosts, incubated for 24 h in medium containing low external potassium and high external sodium, catalyzed net movements of sodium and potassium against their respective concentration gradients, resulting in partial restoration of cation gradients.
As compared to that of active animals, blood from P. fallax torpid at an ambient temperature (TA) of 15°C (hibernation) shows a decrease in red cell K and increases in cell Na and plasma K, and analyses of blood from animals hypothermic at TA = 25°C (aestivation) suggest increases in cell Na and plasma K. Blood electrolyte metabolism of this hibernating rodent shows no special resistance to hypothermia.
Measurements have been made on the permeability of the human erythrocyte to Na and K in vitro, using radioactive tracers to observe the system in the steady state. The average inward K flux is 1.67 m.eq./liter cells hour, and the apparent activation energy is 12,300 ± 1300 calories/mol. The inward K flux is independent of the external K concentration in the range of concentrations studied (4 to 16 m.eq. K/liter plasma). Rb appears to compete with K for transport into the cell, whereas Na and Li do not. The average inward Na flux is 3.08 ± 0.57 m.eq. Na/liter cells hour, and the apparent activation energies are 20,200 ± 2700 calories/mol for inward transport, and 14,900 ± 3,400 calories/mol for outward transport. The inward Na flux is dependent on the external Na concentration, but not in a linear fashion. Li appears to compete with Na for inward transport, whereas K and Rb do not. An approximate maximum estimate shows that the energy required for cation transport is only 8.8 calories/mol liter cells hour of the 110 calories/mol liter cells hour available from the consumption of glucose. A working hypothesis for the transport of Na and K is presented.
Curves describing the loss of K from human red cells as a function of time can be interpreted in terms of an equation which treats the K content of the cell (varphi) as the result of an accumulation process occurring at a rate P and an outward diffusion process regulated by a constant a. The equation is useful for describing the observations and for exploring the mechanisms which may be responsible for the K losses, although it cannot be used for analyzing the experimental data in a strict sense in the absence of independent metabolic data because P and a may both be functions of time. The applicability of the equation is illustrated by its use in connection with experimental curves showing K loss as a function of time at 4 degrees , 25 degrees , and 37 degrees C. for systems containing human red cells in isotonic NaCl or NaCl-buffer. At 4 degrees C., the K loss follows an exponential curve approaching an asymptote in the neighborhood of varphi = 0.50 +/- 0.15. The corresponding value of P implies that the cells are able to accumulate about 0.6 per cent of their initial K per hour under these conditions. At 25 degrees C., the K loss starts exponentially but becomes roughly linear with time after 24 to 48 hours. The change of form is probably due to the appearance of autolysins in the system. Curves of a similar mixed or intermediate form may be obtained even at 4 degrees C. if the observations are sufficiently extended and if spontaneous hemolysis becomes appreciable. At 37 degrees C., the K loss is exponential for the first 24 to 36 hours, the curves approaching asymptotes which, translated into terms of P, indicate that the cells can accumulate about 7 +/- 3 per cent of their initial K per hour. After this time autolysis begins to affect the shape of the curves, the rate of K loss increasing rapidly. The effect of adding fluoride or iodoacetate is to lower the position of the asymptote to which the curves proceed; i.e., to decrease the accumulation rate P, to increase the diffusion constant a, or both. Cyanide has almost no effect. Hypotonicity has little effect on the rate of K loss at 37 degrees C.; at 4 degrees C., the rate of loss is somewhat less in hypotonic NaCl. The observation that the K loss in systems at 4 degrees C. and containing as much as 0.086 M NaF does not become complete, but proceeds exponentially towards an asymptote between varphi = 0.2 and 0.4, suggests that 20 to 40 per cent of the cell K is much less diffusible than the remainder at low temperatures and in the absence of lytic substances. A similar conclusion is suggested by the form of the curve for K loss into saline at 4 degrees C., an accumulation rate of 0.6 m. eq./litre of cells/hour at the end of 100 hours or more being improbably great for a system at such a low temperature and containing no added glucose.
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