THE SELECTIVE ABSORPTION OF POTASSIUM BY ANIMAL CELLS : I. CONDITIONS CONTROLLING ABSORPTION AND RETENTION OF POTASSIUM.

1. Individual variations in the potassium content of the fresh muscles of frogs are notable even when computed as percentages of the dry solids. The potassium content averaged higher in freshly collected summer frogs than in winter frogs after a period of captivity. 2. Muscles show a loss of from 8 to 15 per cent of their potassium during perfusion with potassium-free Ringer solution but tenaciously hold the remainder. 3. Muscles, stimulated to contract under conditions that do not produce irreversible stages of fatigue, show losses of potassium no greater than those attributable to the presence of a potassium-free medium. 4. A condition favorable to the taking up of potassium probably occurs in a contracting muscle because rubidium and cesium, substances very similar to potassium in chemical and physiological behavior, are absorbed in retainable form by a contracting muscle but not by a resting one.     

THE SELECTIVE ABSORPTION OF POTASSIUM BY ANIMAL CELLS : III. THE EFFECT OF HYDROGEN ION CONCENTRATION UPON THE RETENTION OF POTASSIUM.

By perfusing frogs for varying periods with potassium-free Ringer solutions having a pH ranging from 6.0 to 8.0, it has been determined that such solutions have little or no effect upon the retention of potassium by muscle cells.     

THE POTASSIUM EQUILIBRIUM IN MUSCLE

1. Analyses were made of the K and HCO₃ content, the irritability, and weight change of isolated frog sartorius muscles after immersion for 5 hours in Ringer''s solutions modified as to pH and potassium content. 2. At each pH a concentration of potassium in the solution was found which was in diffusion equilibrium with the potassium in the muscle. In greater concentrations potassium moved into the muscle against the concentration gradient and vice versa. 3. The greater the alkalinity of the solution the smaller the concentration of the potassium at equilibrium so that the product of the concentrations of OH and K in the solution at equilibrium tends to remain approximately constant. 4. The pH inside the muscle is approximately equal to that outside when first dissected but it tends to change during immersion so as to follow the changes in the pH of the solution. This finding is in direct conflict with the theory according to which the high potassium concentration inside should be accompanied by an equally high hydrogen ion concentration in relation to that outside. 5. The diffusion of potassium into the muscle makes its contents more alkaline but the increase in alkalinity is not always, nor usually, equivalent to the amount of potassium which has diffused and conversely, the pH inside can change in either direction according to the pH outside without there being any diffusion of potassium. Hence potassium is not the only penetrating ion. 6. The irritability of the muscles is at a maximum in concentrations of potassium which are greater than that in normal Ringer''s solution, or about 20 mg. per cent potassium. This optimum does not seem to be a function of pH and is therefore not dependent upon the direction of movement of the potassium but probably on the ratio of potassium outside to that inside. 7. Swelling of the muscles occurs in solutions which injure the muscle so as to permit both cations and anions to enter without permitting the organic protein anions to escape. Anion impermeability is necessary to prevent this same osmotic swelling under normal conditions. 8. An increase in the CO₂ tension in muscle and solution causes a greater increase in acidity in the solution than in the muscle and leads to a loss of potassium. One expects therefore a potassium shift from tissues to blood comparable to the chlorine shift from plasma to corpuscles.     

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

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

Repolarization of striated muscle fibers by the antifatigue agent,K-Mg-aspartate

Resting membrane potentials of isolated frog sartorius muscles were measured under a variety of conditions using intracellular glass microelectrodes. Muscle cells depolarized by the addition of 5.0 or 10.0 mM KCl to the bathing Ringer solution can be repolarized some 5 to 10 mV by the substitution of an equivalent amount of K-aspartate for KCl in the presence of 2.0 mM Mg⁺⁺. The repolarization produced by this method persists when the muscle is again placed in the initial KCl solution, thus eliminating the possibility that the hyperpolarization is due to the reduction of chloride in the bathing medium. If for some reason the resting membrane potential of the muscle fibers is considerably below (less negative than) the normal level of 92 mV reported for muscles bathed in 2.5 mM Ringer solution, the substitution of 2.5 mM K-aspartate for the 2.5 mM KCl and the addition of 2.0 mM Mg-aspartate to the Ringer solution will, within 15 minutes, repolarize the fiber to the normal level. Magnesium ions alone will not produce the observed repolarization nor can it be attributed to a reduction in the activity of the potassium in the Ringer solution.     

CALCULATIONS OF BIOELECTRIC POTENTIALS : VI. SOME EFFECTS OF GUAIACOL ON NITELLA

Values have been calculated for apparent mobilities and partition coefficients in the outer non-aqueous layer of the protoplasm of Nitella. Among the alkali metals (with the exception of cesium) the order of mobilities resembles that in water and the partition coefficients (except for cesium) follow the rule of Shedlovsky and Uhlig, according to which the partition coefficient increases with the ionic radius. Taking the mobility of the chloride ion as unity, we obtain the following: lithium 2.04, sodium 2.33, potassium 8.76, rubidium 8.76, cesium 1.72, ammonium 4.05, ½ magnesium 20.7, and ½ calcium 7.52. After exposure to guaiacol these values become: lithium 5.83, sodium 7.30, potassium 8.76, rubidium 8,76, cesium 3.38, ammonium 4.91, ½ magnesium 20.7, and ½ calcium 14.46. The partition coefficients of the chlorides are as follows, when that of potassium chloride is taken as unity: lithium 0.0133, sodium 0.0263, rubidium 1.0, cesium 0.0152, ammonium 0.0182, magnesium 0.0017, and calcium 0.02. These are raised by guaiacol to the following: lithium 0.149, sodium 0.426, rubidium 1.0, cesium 0.82, ammonium 0.935, magnesium 0.0263, and calcium 0.323 (that of potassium is not changed). The effect of guaiacol on the mobilities of the sodium and potassium ions resembles that seen in Halicystis but differs from that found in Valonia where guaiacol increases the mobility of the sodium ion but decreases that of the potassium ion.     

THE PENETRATION OF AMMONIA INTO FROG MUSCLE

1. A study was made of the electrolyte changes which occur when frog muscles are immersed in a Ringer solution with 1/5 of the Na replaced by NH₄Cl. Analyses were made in the solution and in the muscles for K and NH₃, and the muscles were also analyzed for Cl, HCO₃, and Na. Control muscles were immersed in normal Ringer''s solution and similarly analyzed. 2. The amount of ammonia taken up was about equal to the K and Na lost. There was also a small increase in chloride content. The bicarbonate content was the same in both experimental and control muscles, indicating no change in the muscle pH due to the NH₃ which penetrated. An increased loss of K due to the penetration of NH₃ was also demonstrated by the use of radioactive K. 3. After 5 hours, the concentration of ammonia per gram of muscle is about the same as the concentration in the solution. After 4 or 5 days, the concentration in the muscle is about 1.5 times that in the solution. The inside to outside NH₃ ratio is about equal to the corresponding H ion ratio, but is much less than the K ratio. 4. The rate of penetration of the NH₃ is increased by a rise of temperature, by stirring the solution, and by decrease in the concentration of Na, K, Ca, or Mg in the solution; it is decreased by increasing the size of the muscles or by killing them with chloroform or boiling. 5. Liver, smooth muscle, skin, and kidney, in a few experiments, behaved much like muscle except that there was a formation of urea in the case of liver. 6. The injection of NH₄Cl into anesthetized cats causes an increase in the level of K in the blood plasma.     

Cation exchange binding of rubidium and cesium by rat liver cell microsomes

The rubidium and cesium binding characteristics of rat liver cell microsomes were studied by an equilibration, centrifugation and washing procedure. Concentration dependence experiments, in which microsomes were equilibrated in media containing 0 to 400 mM rubidium or cesium chloride at pH 6.9, yielded saturation type adsorption isotherms similar to those previously reported for sodium and potassium. Mass law analysis of the data yielded apparent dissociation constants of 21 × 10^?3 eq/liter and 19 × 10^?3 eq/liter for rubidium and cesium binding, respectively. The results indicate that cesium is bound slightly more strongly than rubidium, and that both these cations may be bound more strongly than sodium or potassium. The maximum binding capacity at pH 6.9 was approximately 1.3 meq rubidium or cesium/g nitrogen. Sodium, potassium, magnesium and calcium generally associated with the isolated microsomes decreased concomitantly with increasing bound rubidium or cesium, demonstrating the ion exchange nature of the binding. Results of pH-dependence experiments showed that following equilibration of the microsomes in media containing approximately 96 mM rubidium or cesium at various pH values, bound rubidium or cesium was essentially zero at pH less than five, increased sharply between pH 5 and 7, and tended to level off at higher pH. The present results further characterize the cation binding properties of the microsomal material.     

RADIOACTIVITY AND PHYSIOLOGICAL ACTION OF POTASSIUM

1. The non-radioactive cesium ion can replace the potassium ion almost quantitatively in solutions required for the development of the egg of the sea urchin into swimming blastulæ. 2. Thorium chloride and uranium acetate cannot replace the potassium chloride in the solutions required for the development of the egg. 3. Thorium chloride and uranium acetate do not antagonize the action of the potassium contained in sea water upon the development of eggs.     

STUDIES ON THE FORMATION AND IONIZATION OF THE COMPOUNDS OF CASEIN WITH ALKALI : I. THE TRANSPORT NUMBERS OF ALKALI CASEINATE SOLUTIONS.

1. The deposition of casein on a platinum anode which takes place on the passage of a direct current through solutions of alkali caseinates was quantitatively studied, and it was found that: (a) the amount of casein which is deposited is directly proportional to the current, i.e. it obeys Faraday''s law; (b) the amount of casein deposited is inversely proportional (within the limits studied) to the amount of alkali which is combined with the casein. 2. A method of determining the transport numbers of proteins insoluble at their isoelectric point has been developed. 3. A titration method for determining the amount of alkali in a casein solution is given. 4. Data from the results of transference experiments on sodium caseinate, potassium caseinate, cesium caseinate, and rubidium caseinate solutions are given. It is shown that the data are best explained on the assumption that in these solutions the carriers of the current are alkali metal cations and casein anions. 5. On the basis of our transference results an explanation is given of the results which were obtained by Robertson and by Haas in their migration experiments.     

Untersuchung der im Vergleich zur Kalium-Anreicherung bevorzugten Anreicherung von Cäsium 137 im Säugetierorganismus

Zusammenfassung Es werden Untersuchungen zur Kalium-, Rubidium- und Cäsium- Anreicherung am perfundierten Meerschweinchenherzen durchgeführt. Unter Verwendung der Radionuklide⁴²K,⁸⁶Rb und¹³⁷Cs werden mit Hilfe einer Meßeinrichtung Alkaliionenflüsse zwischen Perfusionslösung und Herzmuskelzelle bestimmt. Es wird die Mehrdeutigkeit der Versuchsergebnisse diskutiert und einexperimentum crucis vorgeschlagen, das entscheiden soll, ob das Ruhepotential als Diffusionsoder als Grenzflächenpotential angesehen werden muß. Im Hinblick auf die unterschiedliche Kalium- und Cäsium-137-Anreicherung im Säugetierorganismus zeigen die Versuche, daß sich Cäsium gegenüber der Muskelzelle qualitativ wie Kalium verhält und daß Cäsium im Herzmuskel nicht bevorzugt angereichert wird.

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On the preferred accumulation of cesium 137 in mammalian organism in comparison with the accumulation of potassiumI. Accumulation of potassium, rubidium and cesium in the perfused guinea-pig heart

Summary Experiments concerning the accumulation of potassium, rubidium and cesium in the perfused guinea-pig heart were performed. Using the radionuclides⁴²K,⁸⁶Rb,¹³⁷Cs and a scintillation counter, the alkali ion fluxes between the perfusion solution and the heart muscle cells are evaluated. The ambiguity of the results is discussed and anexperimentum crucis is proposed which shall decide wether the resting potential has the character of a diffusion or of a phase-boundary potential. As to the different accumulations of potassium and cesium in mammalian organism, our experiments demonstrate that the transport mechanism into the cell is similar for potassium and cesium and that cesium is not accumulated at a higher degree than potassium in the heart muscle cell.

     

Sodium channel selectivity. Dependence on internal permeant ion concentration

The selectivity of sodium channels in squid axon membranes was investigated with widely varying concentrations of internal ions. The selectivity ratio, PNa/PK, determined from reversal potentials decreases from 12.8 to 5.7 to 3.5 as the concentration of internal potassium is reduced from 530 to 180 to 50 mM, respectively. The internal KF perfusion medium can be diluted by tetramethylammonium (TMA), Tris, or sucrose solutions with the same decrease in PNa/PK. The changes in the selectivity ratio depend upon internal permeant ion concentration rather than ionic strength, membrane potential, or chloride permeability. Lowering the internal concentration of cesium, rubidium, guanidnium, or ammonium also reduces PNa/Pion. The selective sequence of the sodium channel is: Na greater than guanidinium greater than ammonium greater than K greater than Rb greater than Cs.     

Rubidium and cesium fluxes in muscle as related to the membrane potential

The reduction of membrane potential in frog sartorius muscle produced by rubidium and cesium ions has been studied over a wide concentration range and compared with depolarization occasioned by potassium ions. The constant field theory of passive flux has been used to predict the potential changes observed. The potential data suggest certain permeability coefficient ratios and these are compared with ratios obtained from flux data using radioactive tracers. The agreement of the flux with the potential data is good if account is taken of the inhibition of potassium flux which occurs in the presence of rubidium and cesium ions. A high temperature dependence has been observed for cesium influx (Q₁₀ = 2.5) which is correlated with the observation that cesium ions depolarize very little at low temperatures. The observations suggest that cesium ions behave more like sodium ions at low temperatures and more like potassium ions at room temperature with respect to their effect on the muscle cell resting potential. The constant field theory of passive ion flux appears to be in general agreement with the experimental results observed if account is taken of the dependence of permeability coefficients on the concentrations of ions used and of possible interactions between the permeabilities of ions.     

INFLUENCE OF EXTERNAL POTASSIUM ON THE SYNTHESIS AND DEPOSITION OF MATRIX COMPONENTS BY CHONDROCYTES IN VITRO

The effect of a high external potassium concentration on the synthesis and deposition of matrix components by chondrocytes in cell culture was determined. There is a twofold increase in the amount of chondroitin 4- and 6-sulfate accumulated by chondrocytes grown in medium containing a high potassium concentration. There is also a comparable increase in the production of other sulfated glycosaminoglycans (GAG) including heparan sulfate and uncharacterized glycoprotein components. The twofold greater accumulation of GAG in the high potassium medium is primarily the result of a decrease in their rate of degradation. In spite of this increased accumulation of GAG, the cells in high potassium fail to elaborate appreciable quantities of visible matrix, although they do retain the typical chondrocytic polygonal morphology. Although most of the products are secreted into the culture medium in the high potassium environment, the cell layer retains the same amount of glycosaminoglycan as the control cultures. The inability of chondrocytes grown in high potassium to elaborate the typical hyaline cartilage matrix is not a consequence of an impairment in collagen synthesis, since there is no difference in the total amount of collagen synthesized by high potassium or control cultures. There is, however, a slight increase in the proportion of collagen that is secreted into the medium by chondrocytes in high potassium. Synthesis of the predominant cartilage matrix molecules is not sufficient in itself to ensure that these molecules will be assembled into a hyaline matrix.     

Strophanthidin-sensitive components of potassium and sodium movements in skeletal muscle as influenced by the internal sodium concentration

"Low sodium" muscles were prepared which contained around 5 mmoles/kg fiber of intracellular sodium. "High sodium" muscles containing between 15 and 30 mmoles/kg fiber of intracellular sodium were also prepared. In low sodium muscles application of 10^-5 M strophanthidin reduced potassium influx by about 5%. Potassium efflux was unaffected by strophanthidin under these conditions. In high sodium muscles, 10^-5 M strophanthidin reduced potassium influx by 45% and increased potassium efflux by 70%, on the average. In low sodium muscles sodium efflux was reduced by 25% during application of 10^-5 M strophanthidin while in high sodium muscles similarly treated, sodium efflux was reduced by about 60%. Low sodium muscles showed a large reduction in sodium efflux when sodium ions in the Ringer solution were replaced by lithium ions. The average reduction in sodium efflux was 4.5-fold. Of the amount of sodium efflux remaining in lithium. Ringer''s solution, 40% could be inhibited by application of 10^-5 M strophanthidin. The total sodium efflux from low sodium muscles exposed to Ringer''s solution in which lithium had been substituted for sodium ions for a period of 1 hr can be fractionated as 78% Na-for-Na interchange, 10% strophanthidin-sensitive sodium pump, and 12% residual sodium efflux. It is concluded that large strophanthidin-sensitive components of sodium and potassium flux can be expected only at elevated sodium concentrations within the muscle cells.     

Ouabain-insensitive salt and water movements in duck red cells. I. Kinetics of cation transport under hypertonic conditions

Duck red cells in hypertonic media experience rapid osmotic shrinkage followed by gradual reswelling back toward their original volume. This uptake of salt and water is self limiting and demands a specific ionic composition of the external solution. Although ouabain (10(-4)M) alters the pattern of cation accumulation from predominantly potassium to sodium, it does not affect the rate of the reaction, or the total amount of salt or water taken up. To study the response without the complications of active Na-K transport, ouabain was added to most incubations. All water accumulated by the cells can be accounted for by net salt uptake. Specific external cation requirements for reswelling include: sufficient sodium (more than 23 mM), and elevated potassium (more than 7 mM). In the absence of external potassium cells lose potassium without gaining sodium and continue to shrink instead of reswelling. Adding rubidium to the potassium- free solution promotes an even greater loss of cell potassium, yet causes swelling due to a net uptake of sodium and rubidium followed by chloride. The diuretic furosemide (10(-3)M) inhibits net sodium uptake which depends on potassium (or rubidium), as well as inhibits net sodium uptake which depends on sodium. As a result, cell volume is stabilized in the presence of this drug by inhibition of shrinkage, at low, and of swelling at high external potassium. The response has a high apparent energy of activation (15-20 kcal/mol). We propose that net salt and water movements in hypertonic solutions containing ouabain are mediated by direct coupling or cis-interaction, between sodium and potassium so that the uphill movement of one is driven by the downhill movement of the other in the same direction.     

STUDIES CONCERNING THE NATURE OF THE SECRETORY ACTIVITY OF THE ISOLATED RINGER-PERFUSED FROG LIVER : II. THE INHIBITORY AND THE PROMOTING INFLUENCE OF ORGANIC ELECTROLYTES AND NON-ELECTROLYTES UPON THE SECRETION OF DYESTUFFS

The ability of the isolated Ringer-perfused frog liver, to concentrate dyestuffs in its secretion several hundred times, can be abolished entirely and reversibly by replacing in the Ringer solution about 1/8 of the NaCl by the isosmotic amount of a surface-inactive non-electrolyte (disaccharide, hexose, pentose, polyhydric alcohol, amino acid, acid amide) or electrolyte (salts of lower fatty acids, hydroxyl carboxylic, and dicarboxylic acids). This effect is not dependent upon changes in the perfusion rate. The opposite effect, promotion of secretory activity, can be brought about by polar-non-polar electrolytes (salts of higher fatty acids, bile acids, and other aromatic carboxylic acids, aromatic sulfonic acids) and surface-active non-electrolytes (anesthetics, alkaloids, digitonin). However, reversibility of this effect cannot be regularly observed, since cytolysis is frequently the end result. Suitable concentrations of inhibitory and promoting substances, simultaneously applied, counteract each other. Inhibitory and promoting substances, in general, exhibit opposite effects upon the dispersion of colloids (starch, lecithin, gelatin). The correlation between the physicochemical and the physiological action of the organic compounds is discussed.     

The independence of membrane potential and potassium activation of the sodium pump in muscle.

The membrane potential (Em) of sartorius muscle fibers was made insensitive to [K+] by equilibration in a 95 mM K+, 120 mM Na+ Ringer solution. Under these conditions a potassium-activated, ouabain-sensitive sodium efflux was observed which had characteristics similar to those seen in muscles with Em sensitive to [K+]. In addition, in the presence of 10 mM K+, these muscles were able to produce a net sodium extrusion against an electrochemical gradient which was also inhibited by 10- minus 4 M oubain. This suggests that the membrane potential does not play a major role in the potassium activation of the sodium pump in muscles.     

THE EFFECT OF CALCIUM AND OTHER IONS ON THE AUTOCATALYTIC FORMATION OF TRYPSIN FROM TRYPSINOGEN

Crystalline trypsinogen is completely transformed into trypsin by means of trypsin in the presence of calcium salts. The process follows the course of a pure autocatalytic unimolecular reaction. In the absence of calcium salts, the autocatalytic formation of trypsin from trypsinogen is complicated by the transformation of part of the trypsinogen into an inert protein which cannot be changed into trypsin by any known means. Salts increase or decrease the rate of both reactions so that the ultimate amount of trypsin formed varies with the nature and concentration of the salt used. With equivalent concentrations of salt the percentage of trypsinogen changed into trypsin is greatest in the presence of calcium ion followed in order by strontium; magnesium and sodium; rubidium, ammonium, lithium, and potassium; caesium and barium. With the anions the largest percentage of trypsinogen transformed into trypsin was found with the acetate, sulfate, oxalate, citrate, tartrate, fluoride, and chloride ions followed in order by bromide, nitrate, and iodide. The formation of inert protein is completely suppressed by concentrations of calcium ion greater than 0.02 M.     

Effects of external cesium and rubidium on outward potassium currents in squid axons

We have studied the effects of external cesium and rubidium on potassium conductance of voltage clamped squid axons over a broad range of concentrations of these ions relative to the external potassium concentration. Our primary novel finding concerning cesium is that relatively large concentrations of this ion are able to block a small, but statistically significant fraction of outward potassium current for potentials less than approximately 50 mV positive to reversal potential. This effect is relieved at more positive potentials. We have also found that external rubidium blocks outward current with a qualitatively similar voltage dependence. This effect is more readily apparent than the cesium blockade, occurring even for concentrations less than that of external potassium. Rubidium also has a blocking effect on inward current, which is relieved for potentials more than 20-40 mV negative to reversal, thereby allowing both potassium and rubidium ions to cross the membrane. We have described these results with a single-file diffusion model of ion permeation through potassium channels. The model analysis suggests that both rubidium and cesium ions exert their blocking effects at the innermost site of a two-site channel, and that rubidium competes with potassium ions for entry into the channel more effectively than does cesium under comparable conditions.