Net Extrusion Research Articles

Further evidence for a potassium‐like action of lithium ions on sodium efflux in frog skeletal muscle

1. The efflux of labelled sodium as well as net sodium and lithium changes were studied in aged high sodium sartorius muscles of the South American frog Leptodactilus ocelatus.2. In the presence of 2.5 mM potassium in the media, the replacement of external sodium with lithium or magnesium resulted in an increase in sodium efflux. The magnitude of such increase was always larger in lithium.3. With the absence of potassium in the media, the response of sodium efflux to replacement of external sodium varied with the cation used as a substitute. In lithium Ringer there was always a noticeable increase, whereas in magnesium there was always a marked reduction. The same results were observed when calcium was substituted for magnesium.4. The replacement of 60 mM external sodium with sucrose did not prevent the stimulating effect of 5 mM potassium on sodium efflux, nor the inhibitory action of 10(-4)M ouabain. This indicates that neither sucrose by itself, nor the lowering of the ionic strength, modified to an appreciable extent the function of the sodium pump.5. Net sodium extrusion took place against an electrochemical gradient in potassium-free - 50 mM sodium - mM lithium Ringer. About 75% of this efflux was ouabain sensitive.6. Muscles made both sodium and lithium rich and incubated in potassium-free - 60 mM sodium - 50 mM lithium Ringer also showed net sodium extrusion against an electrochemical gradient, which was 85% ouabain sensitive. This extrusion took place even under conditions where the changes in free energy favouring lithium entry were always lower than the changes in free energy opposing sodium going out. This indicates that a sodium-lithium exchange by a counter-transport process is unlikely.7. External potassium reduced the ouabain sensitive lithium influx in muscles incubated in lithium Ringer. The values found were 5.90 +/- 0.39 mu-mole/g.hr and 2.66 +/- 0.43 mumole/g.hr in potassium-free and 15 mM potassium respectively. At the same time potassium had no effect on the ouabain-insensitive lithium uptake.8. Muscles incubated in potassium-free-magnesium Ringer had a residual sodium efflux which could not be accounted for by passive movement. About 40% of it was abolished by 10(-4)M ouabain. This ouabain-sensitive part could be a consequence of some stimulation of the sodium pump by potassium leaking out of the cells. If this is correct it should be inhibited by external sodium and should not contribute to the total sodium efflux in potassium-free sodium media.9. Magnesium was used as the reference cation to study the sodium-stimulated sodium efflux under potassium-free conditions. The total sodium efflux amounted to 0.668 hr(-1) (rate constant) and was 71% ouabain sensitive.10. The present experiments demonstrated that lithium ions have a direct stimulating effect on sodium efflux in high sodium skeletal muscle, and strongly support the notion that this effect is produced by an activation of the sodium pump through a potassium-like action.

Relation of intracellular Ca2+ to retention of K+ by liver slices.

CALCIUM ions are necessary for the maintenance of the normal, low permeability of cell plasma membranes1–3: if animal cells are incubated in media free of Ca2+ the passive permeability of the membranes increases, and the cells are unable to maintain their normal concentration gradients of K+ and Na+ (refs. 4, 5). Recent work has suggested, however, that a raised level of intracellular Ca2+ specifically increases the passive permeability of erythrocyte membranes to K+. Liver cells possess an energy dependent mechanism which can bring about the net extrusion of Ca2+ that has accumulated during incubation at 1° C (refs. 7, 8). It was therefore of interest to examine the relationship between the lowering of intracellular Ca2+ thus produced and the accompanying recovery of the K+ and Na+ composition of the cells.

Two modes of Na extrusion in cells from guinea pig kidney cortex slices.

Cells from guinea pig kidney cortex slices, which have been loaded with Na and caused to lose K, by leaching at 0.6° C for 2.5 hours, extrude Na with Cl upon rewarming to 25° C in a medium without K. A subsequent rise in the K concentration in the bath at 25° C induces further net Na extrusion, 1 Na being extruded in exchange for 1 K that is taken up. When the leached tissue is rewarmed to 25°C in the presence of K in the bathing fluid (2 or 16 mM), some Na is extruded accompanied with Cl (by a mechanism that is inhibited by ethacrynic acid) and some Na is extruded maintanining a 1:1 ratio with the K that is taken up, (this system being inhibited by ouabain). Thus two modes of Na extrusion are observed, mode A that is accompanied by net Cl efflux, and that is inhibited by 2 mM ethacrynic acid, but not by 1 or 10 mM ouabain and mode B in which one K is taken up for each Na extruded. Mode B is inhibited by 1 mM ouabain and not by ethacrynic acid. DNP and anoxia inhibit both modes A and B. Insufficient doses of ouabain do not explain the refractoriness of mode A to ouabain. Ouabain and ethacrynic acid are known inhibitors of the Na−K-ATPase at much lower doses. It is concluded that both modes may originate in different Na pumps which may have different energy sources. Pump A should be efficient in the volume regulation of the cell. According to experimental procedure, both modes of Na extrusion appear of comparable magnitude. In the steady-state their relative role may be different.

Bioelectrochemical throttle of metabolism

Electrochemical principles indicate how living membranes may regulate the rate of a bioelectrochemical reaction. If this reaction is the rate limiting biochemical link in an electrogenic metabolic pathway, to regulate its rate is to regulate the pathway. This metabolic regulation is called the Bioelectrochemical throttle of metabolism. The throttle concept is employed to interpret propagated action potentials, excitation-contraction coupling in muscle, and very briefly, other instances of metabolic regulation. For the propagated action potential, there is feedback in an electrogenic metabolic pathway, between the rates of a bioelectrochemical and a non-bioelectrochemical reaction. For excitation-contraction coupling, there is the change in state of constituents of this pathway as its rate changes, constituents which regulate tonus. And there are other instances to which the throttle applies, in which the bioelectrochemical reaction may occur in different biological membranes, and in different electrogenic metabolic pathways. Section 1. If a metabolic reaction proceeds as an electric current, regulating this current could regulate not only the rate of this reaction itself, but other, rate-linked metabolic reactions. Selected electrochemical principles indicate how living membranes may regulate metabolism by regulating bioelectric currents. Section 2. By applying the bioelectrochemical throttle to feedback between rate changes of a non-bioelectrochemical reaction in an electrogenic metabolic pathway, and of the bioelectrochemical reaction, I have developed a metabolic interpretation of electric excitation: o (1) the rate of all reactions of an electrogenic metabolic pathway is slow in the resting, steady-state membrane; (2) an action potential is initiated when the rate of a non-bioelectrochemical link in this pathway increases to a threshold value, in a given area of the membrane; (3) this alters the state of its reaction constituents in this area. Since these are also membrane constituents, it alters the state of the membrane; (4) bioelectric current flows between the altered area of the membrane and unaltered area, or in other words, the rate of the bioelectrochemical link in the metabolic pathway increases; (5) the bioelectrochemical reaction rate increase is coupled to further rate increase of the non-bioelectrochemical reaction, etc., until neither reaction is rate limiting; in the excited membrane; (6) the pathway subsequently slows to the resting rate, and the pathway constituents return to the pre-excitation steady state; (7) metabolic potassium extrusion is suggested as the bioelectrochemical reaction, and metabolic calcium release from its carrier as the non-bioelectrochemical one. Section 3. biochemical reaction rate changes of a propagated action potential alter the states of biochemical reaction constituents in a muscle fiber, and these constituents regulate tonus. It is pointed out that to avoid negative electrostatic charge build up in the myoplasm when there is net potassium extrusion, net electron transfer from myoplasmic oxidation-reduction couples to membrane couples would be necessary. The potassium extrusion rate increase, part and parcel of electric excitation, therefore shifts the oxidation-reduction potential of myoplasmic couples toward the oxidizing direction, and contraction occurs as a result. Experimental evidence is presented according to which oxidized muscle does in fact contract; reduced muscle relaxes. Section 4. The throttle concept is applicable for metabolic regulations in which all-or-none bioelectric changes do not occur.

Seawater teleosts: evidence for a sodium-potassium exchange in the branchial sodium-excreting pump.

The net sodium extrusion rate by the gill of the seawater-adapted euryhaline flounder is identical to the potassium influx. The excretion of sodium is blocked in K(+)-free seawater solutions. The instantaneous sodium outflux readjustment pattern of flounders transferred from seawater to solutions of various sodium chloride or potassium chloride concentrations is consistent with the hypothesis of a linkage between Na(+) outflux and K(+) influx through a common exchange carrier. External Na(+) and K(+) compete for this comnmonz carrier. It is suggested that the exchange diffusion mechanism (linkage of sodium influx and outflux) and the high internal sodium turnover rate which characterizes all seawater teleosts are the results of this competitive process. The sodium-potassium dependent adenosine triphosphatase system occurring in the gill of the seawater teleosts may play a central role in this sodium-potassium exchange pump.

The Dual Effect of Lithium Ions on Sodium Efflux in Skeletal Muscle

Sartorius muscle cells from the frog were stored in a K-free Ringer solution at 3 degrees C until their average sodium contents rose to around 23 mM/kg fiber (about 40 mM/liter fiber water). Such muscles, when placed in Ringer's solution containing 60 mM LiCl and 50 mM NaCl at 20 degrees C, extruded 9.8 mM/kg of sodium and gained an equivalent quantity of lithium in a 2 hr period. The presence of 10(-5)M strophanthidin in the 60 mM LiCl/50 mM NaCl Ringer solution prevented the net extrusion of sodium from the muscles. Lithium ions were found to enter muscles with a lowered internal sodium concentration at a rate about half that for entry into sodium-enriched muscles. When sodium-enriched muscles labeled with radioactive sodium ions were transferred from Ringer's solution to a sodium-free lithium-substituted Ringer solution, an increase in the rate of tracer sodium output was observed. When the lithium-substituted Ringer solution contained 10(-5)M strophanthidin, a large decrease in the rate of tracer sodium output was observed upon transferring labeled sodium-enriched muscles from Ringer's solution to the sodium-free medium. It is concluded that lithium ions have a direct stimulating action on the sodium pump in skeletal muscle cells and that a significantly large external sodium-dependent component of sodium efflux is present in muscles with an elevated sodium content. In the sodium-rich muscles, about 23% of the total sodium efflux was due to strophanthidin-insensitive Na-for-Na interchange, about 67% being due to strophanthidin-sensitive sodium pumping.

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

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

The role of ion transport in the regulation of respiration in the Ehrlich mouse ascites-tumor cell

1. 1. When suspensions of Ehrlich mouse ascites-tumor cells are chilled to 2° in media free of K +, the cells gain large amounts of Na + and lose K +. 2. 2. When re-warmed to 23° the net active extrusion of Na + may be totally uncoupled from the net active accumulation of K +. 3. 3. Cell suspensions transitioned from low temperature to 23° in K +-free medium respire at rates only one-sixth the rate of cells normally maintained at 23° with 6 mM K +. Addition of K + to the external medium stimulates respiration. 4. 4. The relation between respiration rate and external K + follows Michaelis-Menten kinetics. The K m was 0.43 mM and v max was 1.0 μmole O 2 10 7 cells·h. 5. 5. Ouabain inhibited K +-stimulated respiration at a concentration effective against the active transport of Na + and K +. 6. 6. External K + and 2,4-dinitrophenol affect a common source of respiratory intermediate but act at different sites. 7. 7. The hypothesis is proposed that components of oxidative phosphorylation participate in the transport of Na + and K + and that intermediates may communicate between cell membrane and mitochondria.

Energy barriers to sodium extrusion from sodium-rich kidney cortex slices.

1. Rat and guinea-pig kidney cortex slices were made Na-rich by leaching in cold NaCl and then allowed to recover in Ringer solution at 30 or 25 degrees C. Net Na extrusion was systematically decreased by the use of re-immersion media with constant Na and diminishing K, or with constant K and increasing Na, and the energy requirements for Na extrusion under these conditions were calculated.2. The critical energy barrier at which no net Na loss can be achieved was somewhat lower in these tissues than found by other authors for frog sartorius muscle.3. Inclusion of pyruvate or insulin and lactate in the recovery fluid had no effect.4. Methylsulphate apparently enters renal cells during incubation and is therefore unsuitable for use with this tissue as a source of impermeant anion.

CATION TRANSPORT IN ESCHERICHIA COLI. IV. KINETICS OF NET K UPTAKE.

The resuspension of K-poor, Na-rich stationary phase E. coli in fresh medium at pH 7.0 results in a rapid uptake of K and extrusion of Na by the cells. In all experiments net K uptake exceeded net Na extrusion. An investigation of the uptake of glucose, PO(4), and Mg and the secretion of H by these cells indicates that the excess K uptake is not balanced by the simultaneous uptake of anions but must be accompanied by the extrusion of cations from the cell. The kinetics of net K uptake are consistent with the existence of two parallel influx processes. The first is rapid, of brief duration, and accounts for approximately 60 per cent of the total net K uptake. This process is a function of the extracellular K concentration, is inhibited in acid media, and appears to be a 1 for 1 exchange of extracellular K for intracellular H. The second influx process has a half-time of approximately 12 minutes, and is not affected by acid media. This process is a function of the intracellular Na concentration, is dependent upon the presence of K in the medium, and may be ascribed to a 1 for 1 exchange of extracellular K for intracellular Na.

Influence of pilocarpine on transport by salivary gland slices.

Effects of pilocarpine on net movements of water and electrolytes in gland cells were investigated in vitro, using slices from submaxillary gland of rat. Slices were depleted of K, and loaded with Na, Cl, and water, by incubation in Krebs-Ringer phosphate with nitrogen atmosphere. After this, the slices were transferred to Krebs-Ringer phosphate with oxygen atmosphere. During this period with O2, pilocarpine caused apparent loss of water from cells, since tissue total water decreased and inulin space remained almost unchanged. Without pilocarpine during this time, water in cells increased. Electrolyte movements were also affected by pilocarpine. Specifically, there occurred reduction in net accumulation of K in total tissue and cells. Reduction in net extrusion of Na was suggested. Since, in vivo, an early effect of stimulation involves depletion of gland K, it appears that the current observations have relevance to normal secretion to the extent, at least, that in both circumstances stimulating agents reduce the ability of the cells to maintain stores of K.

ELECTROLYTE METABOLISM IN HELA CELLS.

Methods have been developed to study cellular Na, K, and Cl concentrations in HeLa cells. Cell [Na] and [K] are functions of the age of the culture. As the culture grows [K], expressed in mmols/liter cell H(2)O, rises from an initial value of 121 to a peak of 206 at about 4 days, and thereafter falls until it has almost returned to the initial value by the 9th day. [Na] falls as [K] rises, but there is no fixed relationship between the cellular concentrations of the two cations. There is, however, a correlation between generation time and cellular [K]. Measurements of net K uptake and net Na extrusion were carried out during 1 hour incubation at 37 degrees C of low K cells. Both net K uptake and net Na extrusion took place against chemical concentration gradients, so that at least one transport system must be active; if the Cl distribution is passive both net K uptake and net Na extrusion are active. Studies with inhibitors of respiration and glycolysis lead to the conclusion that respiration is not required for these net transports, which appear to derive their energy from glycolytic sources.

Potassium accumulation and sodium efflux by Porphyra perforata tissues in lithium and magnesium sea water.

Cells of Porphyra perforata, a red marine alga, accumulate K in the absence of concomitant Na or Li extrusion while immersed in Li- or Mg-sea waters lacking Na. This suggests that the coupling observed between K and Na transport is facultative. No evidence is obtained for net extrusion of Li. Na efflux, with the concentration gradient, is facilitated by K and is proportional to the cellular Na content. Either Na efflux does not involve an ion carrier or the number of Na sites is large. Because K accumulation has been observed in the absence of Na extrusion, but not vice versa, it seems that K uptake is the primary secretory event, with Na extrusion a secondary process dependent upon K accumulation.

Studies on the sodium and potassium transport in rabbit polymorphonuclear leukocytes.

Rabbit polymorphonuclear leukocytes obtained from peritoneal exudates, incubated at 37 degrees C. following exposure to 4 degrees C., actively reaccumulate potassium while little or no net extrusion of sodium takes place. Preventing the utilization of oxidative metabolism with potassium cyanide, 2,4-dinitrophenol, or a nitrogen atmosphere does not inhibit the recovery process. Inhibitors blocking anaerobic glycolysis (sodium iodoacetate and sodium fluoride in low concentrations) completely abolish the capacity to reaccumulate potassium and cause a further dissipation of the sodium and potassium gradients. Water movements have been shown to be secondary to cation shifts. It is postulated that separate transport mechanisms exist for sodium and potassium and that the process of potassium reaccumulation relies on anaerobic glycolysis as a source of energy.

POTASSIUM-DEPENDENT SODIUM EXTRUSION BY CELLS OF PORPHYRA PERFORATA, A RED MARINE ALGA

Potassium-free artificial sea water causes a loss of cell potassium and a gain of cell sodium in Porphyra perforata, which is not attributable to an inhibition of respiration. On adding KCl or RbCl to such low potassium, high sodium tissues, net accumulation of potassium or rubidium takes place, accompanied by net extrusion of sodium. Rates of potassium or rubidium accumulation and sodium extrusion are proportional to the amount of KCl or RbCl added only at low concentrations. Saturation of rates is evident at KCl or RbCl concentrations above 20-30 mM, suggesting the role of an ion carrier mechanism of transport. Evidence for and against mutually dependent sodium extrusion and potassium or rubidium accumulation is discussed.

Action of protamine on electrolyte transport in kidney slices

Conditions are described under which K accumulation by K-deficient slices of the renal cortex of the rabbit continues in the absence of simultaneous net Na extrusion. The independence of K movement from Na movement confirms the presence in kidney of a K-transporting system. This is specifically inhibited by protamine and certain other basic compounds. Protamine has little or no effect on Na extrusion and respiration. The mechanism of the protamine inhibition is discussed.