POTASSIUM EFFECTS ON ION TRANSPORT IN BRAIN SLICES

—(1) Fluxes of sodium, potassium, chloride and glutamate ions were studied in brain slices by aid of radio-isotopes. Desaturation curves showed the efflux to occur from at least two compartments with widely different kinetics. (2) The slowly exchanging component comprises from about 10 (sodium, potassium, chloride) to about 30 (glutamate) per cent of the radioactivity in the tissue. An energy-requiring uptake of potassium and extrusion of sodium seems to occur in this compartment, which probably includes the nerve cells. (3) A rather slow efflux of especially potassium ions from the rapidly exchanging fraction indicates that this component may not be purely extracellular, but also seems to include cells, which possibly are neuroglial. The hypothesis of a cellular origin is supported by the demonstration of an increase in the rate constant of the potassium efflux evoked in the presence of oxygen by high concentrations of potassium. (4) Evidence is presented that the increase in the rate constant of the potassium efflux is due to a potassium-induced stimulation of active transport. No coupling seems to occur between the stimulated potassium transport and movements of sodium, but potassium ions may be accompanied by glutamate ions.     

Temporal relationship between neurotransmitter release and ion flux during spreading depression and anoxia

Brain ion homeostasis is severely perturbed during spreading depression of Leao and during anoxia. The ionic composition of the extracellular space changes abruptly and approaches the intracellular concentrations owing to an increase in cell permeability. In spreading depression, synchronous transmitter efflux caused by a depolarization of the presynaptic terminals has been implicated as a possible mechanism that would explain the concomitant movement of ions. Anoxia, having many features in common with spreading depression, may follow the same mechanism. We have measured the concentrations of extracellular potassium with ion-selective microelectrodes and dopamine by in vivo voltammetry with carbon fiber microelectrodes during spreading depression and anoxia to compare the temporal relationship between the release of dopamine and ion movements in the striatum. There is a pronounced release of dopamine during both spreading depression and anoxia. In spreading depression, the sharp increase of potassium concentration that follows an initial smaller and slower increase of potassium is accompanied by the release of dopamine. In anoxia, the dopamine release clearly precedes the fast rise of extracellular potassium concentration. We conclude that in striatum, there is a pronounced dopamine release during spreading depression and anoxia, but that the relationships between ionic changes and transmitter release for these two phenomena are different and probably reflect different mechanisms.     

Phenothiazine inhibition of calmodulin stimulates calcium-dependent potassium efflux in human red blood cells

Elevation of red blood cell calcium increases the efflux of potassium. The active extrusion of calcium from the red cell is regulated by calmodulin. Phenothiazines bind to calmodulin in a calcium-dependent manner preventing the calmodulin from activating a wide variety of cellular processes. The present study shows that phenothiazines increase the efflux of potassium from red cells incubated with the calcium ionophore A23187. The dose dependent effect of trifluoperazine on potassium efflux correlates with its inhibition of Ca-ATPase activity. The phenothiazine effects are dependent upon ATP in that increases in potassium efflux are not observed in energy depleted cells. In calcium buffered ghosts no direct effect of calmodulin or an antibody to calmodulin can be shown. These data suggest that phenothiazines stimulate calcium-dependent potassium loss indirectly by a drug-induced blockage of the calmodulin-activated Ca-ATPase.     

Decreased membrane permeability to potassium is responsible for the cell volume increase that drives lens fiber cell elongation

Differentiation of lens epithelial cells into fiber cells involves an increase in cell volume which previously was proposed to be the direct cause of the extensive cell elongation which accompanies fiber formation. In this study we have continued to investigate the mechanism underlying cell elongation by defining the minimum nutrient and ion requirements of elongating cells, measuring potassium and sodium fluxes in stimulated and unstimulated lens epithelia, and determining the effects of several pharmacological agents on elongation and ion transport. We have shown that elongation will occur in a basic salt/glucose solution with Insulin-like growth factor I/somatomedin-C stimulation. Neither sodium nor any metabolite appears to be the osmotically active species which drives the increase in cell volume. However, potassium efflux rate coefficient was 47% lower in differentiating cells than in unstimulated cells, whereas potassium uptake rates and ouabain effects were similar. Cells did not elongate in potassium-free medium nor in the presence of several drugs which prevent the accumulation of intracellular potassium or hinder osmotic water flux. Unstimulated cells elongated, however, with the application of quinidine, a drug known to block potassium channels. We propose that stimulation of lens epithelial cells with an insulin-like growth factor signals the closure of a certain population of potassium channels. As a result, potassium efflux from the differentiating cells slows while active potassium uptake continues at a constant rate. Potassium then accumulates within the cell causing water influx, an increase in cell volume, and cell elongation.     

Yarrowia lipolytica possesses two plasma membrane alkali metal cation/H+ antiporters with different functions in cell physiology

The family of Nha antiporters mediating the efflux of alkali metal cations in exchange for protons across the plasma membrane is conserved in all yeast species. Yarrowia lipolytica is a dimorphic yeast, phylogenetically very distant from the model yeast Saccharomyces cerevisiae. A search in its sequenced genome revealed two genes (designated as YlNHA1 and YlNHA2) with homology to the S. cerevisiae NHA1 gene, which encodes a plasma membrane alkali metal cation/H+ antiporter. Upon heterologous expression of both YlNHA genes in S. cerevisiae, we showed that Y. lipolytica antiporters differ not only in length and sequence, but also in their affinity for individual substrates. While the YlNha1 protein mainly increased cell tolerance to potassium, YlNha2p displayed a remarkable transport capacity for sodium. Thus, Y. lipolytica is the first example of a yeast species with two plasma membrane alkali metal cation/H+ antiporters differing in their putative functions in cell physiology; cell detoxification vs. the maintenance of stable intracellular pH, potassium content and cell volume.     

Plasma-membrane hyperpolarization diminishes the cation efflux via Nha1 antiporter and Ena ATPase under potassium-limiting conditions

Saccharomyces cerevisiae extrudes K(+) cations even when potassium is only present in scarce amounts in the environment. Lost potassium is taken up by the Trk1 and Trk2 uptake systems. If the Trk transporters are absent or nonfunctional, the efflux of potassium is significantly diminished. A series of experiments with strains lacking various combinations of potassium efflux and uptake systems revealed that all three potassium-exporting systems the Nha1 antiporter, Ena ATPase and Tok1 channel contribute to potassium homeostasis and are active upon potassium limitation in wild-type cells. In trk1Δ trk2Δ mutants, the potassium efflux via potassium exporters Nha1 and Ena1 is diminished and can be restored either by the expression of TRK1 or deletion of TOK1. In both cases, the relative hyperpolarization of trk1Δ trk2Δ cells is decreased. Thus, it is the plasma-membrane potential which serves as the common mechanism regulating the activity of K(+) exporting systems. There is a continuous uptake and efflux of potassium in yeast cells to regulate their membrane potential and thereby other physiological parameters, and the cells are able to quickly and efficiently compensate for a malfunction of potassium transport in one direction by diminishing the transport in the other direction.     

Action of steroidal diamines on active transport and permeability properties of Escherichia coli

The steroidal diamine irehdiamine A (IDA) is a potent inhibitor of bacteriophage growth and macromolecular synthesis in Escherichia coli. By using radioactive (42)K and (14)C-thiomethylgalactoside (TMG), rapid effects of IDA and related steroids, both on the influx of potassium and TMG via their respective transport systems and on the efflux (leakage) of radioactivity from the treated cells, have been measured. IDA affects both the influx and efflux of (42)K at concentrations of steroid as low as 2 x 10(-5)m. Because of the increased leakage, it is not possible to tell whether there is a direct effect reducing the rate of active transport of potassium. The primary diamine, IDA, and its bis-secondary, bis-tertiary, and bis-quaternary diamine analogues are decreasingly effective in altering cell permeability properties in the order 1 degrees > 2 degrees > 3 degrees > 4 degrees . The effects of IDA on potassium transport are mirrored by similar effects on the transport of TMG. Therefore, the action of IDA is on the cell membrane and not directly on one or another transport system. The effects of IDA on cell permeability can reasonably explain the inhibitory actions of the drugs on bacteriophage growth and cellular metabolism.     

Transport of sugars or amino acids increases potassium efflux from isolated enterocytes

A technique to isolate epithelial cells from rabbit jejunum using hyaluronidase is described. The cells obtained retained their abilities to accumulate sugars and potassium (86Rb) against concentration gradients. Potassium efflux was monitored using cells preloaded with 86Rb and the rate constant of efflux was seen to increase when actively transported sugars or amino acids are added to the bathing medium. The increase is related to the transport of the non-electrolyte, but not to volume regulatory events.     

Active transport of potassium by insect midgut

Midguts isolated from fifth-instar larvae of the insert Hyalophora cecropia actively transport potassium in the hemolymph to lumen direction. No specific co- or counter-ion is required and other alkali ions are actively transported in the same direction as potassium. No specific inhibitor of K+ active transport has been found although most metabolic inhibitors reduce the net K+ flux, potential difference, and short-circuit current to zero. The site of the epithelial active transport of potassium has been identified by microelectrode measurements of intracellular resistance as the goblet cell, one of the two major cell types in the single-layered midgut. Under certain external conditions, the neighboring columnar cells are added to the goblet cell transport route through intercellular electrical coupling that occurs after application of external depolarizing current. Tracer influx kinetics were used to establish that the fraction of exchangeable K involved in the transport route under open-circuit conditions is small, corresponding to a goblet cell pathway. Under depolarizing current conditions, virtually all of the exchangeable midgut K is involved in the transport route, corresponding to a goblet and columnar cell pathway. These results and others are used to construct a model for rheogenic active transport of potassium in insect midgut.     

Influence of Glucose on the Transmembrane Action Potential of Anoxic Papillary Muscle

The response of the cat papillary muscle to anoxia has been found to alter depending on the glucose concentration in the medium. At a glucose concentration of 5 mM anoxia caused a marked reduction in force of contraction and action potential duration within 20 minutes. At a glucose concentration of 50 mM anoxia induced similar changes in the force of contraction but little or no change in action potential duration. Elevation of glucose concentration during an anoxic interval reversed the anoxia-induced changes in action potential but had little effect on force of contraction. This effect of glucose could be partially duplicated by xylose and 2-deoxyglucose and in addition, 2-deoxyglucose has been found to prevent the effect of subsequently added glucose. These sugars appear to be transported by a system responsible for glucose transport but are not metabolized to any extent. It would appear therefore that transport of glucose is in some way related to transport of potassium as increased potassium permeability is thought by many to be responsible for anoxia-induced changes in action potential duration.     

Early and late changes in potassium transport during the start of proliferation in CHO cell cultures. The action of serum, isoproterenol, propranolol and theophylline

The stimulation of DNA synthesis by serum is accompanied by early (30 minutes) and late (2-8 hours) increase in ouabain-sensitive rubidium (potassium) influx and the elevation of intracellular potassium content from 0.5-0.6 to 0.7-0.8 mmole per gram protein in CHO-K1 cells. Isoproterenol alone induces the transient increase both in potassium influx via Na,K-ATPase and in potassium efflux without any effect on intracellular potassium content and cell proliferation. Isoproterenol acts synergistically with serum in eliciting the early and late changes in potassium transport and in stimulating G1----S transition. The combination of serum and theophylline produces a rapid increase in potassium influx, however, it does not stimulate DNA synthesis and does not induce any later increase in intracellular potassium content. It is concluded that early and late activation of Na,K-ATPase by mitogens can be dissociated; the Na,K-ATPase activation is involved in mitogenic response when producing the sustained potassium influx and the elevation of intracellular potassium content during G1----S transition.     

The mechanism of energy-dependent ion transport in mitochondria

Summary The transport of potassium, sodium and various anions in rat-liver mitochondria was studied mainly by analysis of ion content and water compartmentation of the mitochondrial pellet. A comparison of spontaneous transport with valinomycin- or gramicidin-stimulated transport is made. The rate or extent of uptake, the internal concentrations and the concentration ratio (C_in/C_out) are calculated and compared to test existing models for ion transport in mitochondria.Several models of ion transport in mitochondria are based on a cation-pump which is directed inward. This hypothesis is rejected because of the following findings: (1) Valinomycin stimulates the rate of potassium uptake but does not increase the potassium concentration ratio that can be actively maintained in a steady state (in which there is no potassium flow). (2) Valinomycin greatly stimulates the efflux of⁴²K from mitochondria during the process of potassium accumulation. When potassium accumulation is stimulated the flux ratio, i.e. influx/efflux, decreases; in the presence of valinomycin this ratio approaches 1. (3) In the presence of gramicidin, the concentration ratios of potassium and sodium are about the same under a variety of conditions. These findings indicate that potassium and sodium transport are passive processes of relaxation towards electro-chemical equilibrium (of the potassium and sodium). In high external potassium concentrations the extent of potassium uptake is limited by the permeation of anions; of the permeating anions multivalent acids support a higher extent than monovalent acids. It was found that succinate, acetate and oxalate which are transported together with potassium are distributed in accordance with the pH and without any relation to the potassium concentration ratio. These findings are compatible with the hypothesis that an outward-directed proton pump creates an electrical potential gradient, which shifts the equilibrium state for the cations and drives sodium and potassium inward, and also creates proton gradient that is the driving force for anion transport.     

Blockade of NMDA-receptors or calcium-channels attenuates the ischaemia-evoked efflux of glutamate and phosphoethanolamine and depression of neuronal activity in rat organotypic hippocampal slice cultures

We have investigated the effects of various insults on extracellular glutamate and phosphoethanolamine levels as well as electrical activity alterations in the early period following these insults in organotypic hippocampal slice cultures. Cultures prepared from 7-day-old rats were maintained in vitro for 7-14 days and then metabolic inhibition was induced: cultures were briefly exposed to potassium cyanide to induce chemical anoxia, 2-deoxyglucose with glucose removal to produce hypoglycaemia, or a combination of both to simulate ischaemia. Chemical anoxia induced a small increase in glutamate and a reversible decrease in evoked field potentials and these were greatly potentiated following simulated ischaemia: high, biphasic glutamate efflux and irreversible field potential abolition as well as increase in phosphoethanolamine levels were observed. We have characterised the effects of treatments using NMDA-receptor antagonists and the L-type calcium channel blocker diltiazem. Anoxia-induced glutamate accumulation was prevented by MK-801 and diltiazem D-AP5. Following simulated ischaemia, diltiazem totally prevented glutamate and phosphoethanolamine accumulations, whereas MK-801 did not block the first phase of glutamate accumulation and D-AP5 prevented none. We demonstrated that glutamate and phosphoethanolamine ischaemic-evoked efflux as well as the recovery of electrical activity in organotypic hippocampal slice cultures are sensitive to both NMDA-receptor and calcium-channel blockade. This model thus represents a useful in vitro system for the study of ischaemic neurodegeneration paralleling results reported using in vivo models.     

Voltage-current relation and K+ transport in tobacco hornworm (Manduca sexta) midgut

Summary Voltage-current curves for the isolated midgut of the tobacco hornworm were determined by transient and steady voltage clamping over the range of 200 to –200 mV. Over this range the transient method yields a linear relation while the steady method usually yields a curve consisting of two lines of differing slope which intersect at zero voltage. The difference between the results of the methods is due to a slow decline in total conductance which accompanies steady voltage clamping.Holding the midgut at short circuit increases the total conductance of the tissue in a manner consistent with increasing shunt conductance; this effect was seen in both diet-reared and leaf-reared animals.When potassium transport is inhibited by substitution of choline or sodium for potassium in bathing solution the total conductance decreases and the voltage-current curve intersects the normal curve in the hyperpolarizing region. Applying a simple equivalent circuit analysis to the results from partial or total potassium replacement suggests that the electromotive force of the potassium transport system is of the order of 140–190 mV. The conductance decrease during inhibition of potassium transport by transient anoxia is of similar magnitude, suggesting that a major effect of metabolic inhibition is to decrease the active conductance of the potassium transport pathway.     

Ouabain-dependent potassium-potassium exchange in the toad bladder

Summary Recent results from this laboratory have indicated the existence of two potassium compartments in the isolated toad bladder. Only one of these, containing less than 10% of total intracellular potassium, appears to be related to the sodium transport system, since potassium influx at the serosal border of this compartment is coupled to the sodium efflux which occurs there. Ouabain, which specifically inhibits serosal sodium exit, has no effect on potassium fluxes and compartment sizes in bladders mounted in normal (2.5mm K) Ringer's solution. However, in the presence of this inhibitor, removal of serosal potassium results in a significant decrease in the rate coefficient for potassium efflux into the serosal medium, while an increase in serosal potassium results in a significant rise in this parameter, which appears to saturate at approximately 5mm K. This sensitivity to serosal potassium is seen neither in the absence of ouabain nor when the sodium pump is inactivated by removal of sodium from the mucosal medium. Furosemide, which also inhibits the sodium transport system, both inhibits potassium transport parameters in normal Ringer's and abolishes the potassium-sensitive potassium efflux seen in the presence of ouabain. Thus, the Na–K pump appears to operate as a K–K exchanger when the sodium system is inhibited by ouabain; this K–K exchange mechanism is inhibited by furosemide. One explanation for these results is that ouabain effects an alteration in the affinities of the transport system for sodium and potassium.     

Alkali Cation/Sucrose Co-transport in the Root Sink of Sugar Beet

The mechanism of sucrose transport into the vacuole of root parenchyma cells of sugar beet was investigated using discs of intact tissue. Active sucrose uptake was evident only at the tonoplast. Sucrose caused a transient 8.3 millivolts depolarization of the membrane potential, suggesting an ion co-transport mechanism. Sucrose also stimulated net proton efflux. Active (net) uptake of sucrose was strongly affected by factors that influence the alkali cation and proton gradients across biological membranes. Alkali cations (Na⁺ and K⁺) at 95 millimolar activity stimulated active uptake of sucrose 2.1- to 4-fold, whereas membrane-permeating anions inhibited active sucrose uptake. The pH optima for uptake was between 6.5 and 7.0, pH values slightly higher than those of the vacuole. The ionophores valinomycin, gramicidin D, and carbonyl cyanide m-chlorophenylhydrazone at 10 micromolar concentrations strongly inhibited active sucrose uptake. These data are consistent with the hypothesis that an alkali cation influx/proton efflux reaction is coupled to the active uptake of sucrose into the vacuole of parenchyma cells in the root sink of sugar beets.     

Uphill transport of monosaccharides inCandida beverwijkii

The yeastCandida beverwijkii was found to transport several monosaccharides against a concentration gradient. The process is mediated by a mobile carrier and shows a pronounced pH dependence. It is tightly coupled with metabolism and only potassium sorbate uncoupled the equilibrating from the active transport component. Kinetic analysis of uptake and efflux at high monosaccharide concentrations indicates an active transport operating either into or out of cells. T. Deák carried out the work in Prague while supported by the Hungarian Academy of Sciences.     

Inhibition of 2,4-dinitrophenol-induced potassium efflux by adenine nucleotides in mitochondria

The influence of nucleotides on 2,4-dinitrophenol (DNP)-induced K+ efflux from intact rat liver mitochondria has been studied. ATP and ADP at micromolar concentrations were found to inhibit mitochondrial potassium transport, whereas GTP, GDP, CTP, and UTP did not show tha same effect. The values of half-maximal inhibition (IC50) were approximately 20 microM for ATP and approximately 60 microM for ADP. It is suggested that adenine nucleotides exert their inhibitory action at the matrix side of the inner mitochondrial membrane since the inhibitor of adenine nucleotide translocase atractyloside at concentration of 1 microM completely removed the inhibitory effect of ATP and ADP. The mitochondrial ATPase inhibitor oligomycin (2 microg/ml) was found to reduce slightly the rate of DNP-induced K+ efflux and had no effect on inhibition by adenine nucleotides; the latter was insensitive to Mg2+ and the changes in pH. It seems likely that the regulation of potassium transport is not due to phosphorylation of the channel-forming protein but to binding of the nucleotides in specific regulatory sites. The possibility of potassium efflux from mitochondria in the presence of uncoupler via the ATP-dependent potassium channel is discussed.     

Effects of External Potassium and Strophanthidin on Sodium Fluxes in Frog Striated Muscle

Unidirectional Na fluxes in isolated fibers from the frog''s semitendinosus muscle were measured in the presence of strophanthidin and increased external potassium ion concentrations. Strophanthidin at a concentration of 10^-5 M inhibited about 80 per cent of the resting Na efflux without having any detectable effect on the resting Na influx. From this it is concluded that the major portion of the resting Na efflux is caused by active transport processes. External potassium concentrations from 2.5 to 7.5 mM had little effect on resting Na efflux. Above 7.5 mM and up to 15 mM external K, the Na efflux was markedly stimulated; with 15 mM K the Na influx was 250 to 300 per cent greater than normal. On the other hand, Na influx was unchanged with 15 mM K. The stimulated Na efflux with the higher concentrations was not appreciably reduced when choline or Li was substituted for external Na, but was completely inhibited by 10^-5 M strophanthidin. From these findings it is concluded that the active transport of Na is stimulated by the higher concentrations of K. It is postulated that this effect on the Na "pump" is produced as a result of the depolarization of the muscle membranes and is related to the increased metabolism and heat production found under conditions of high external K.