Acidity and solubility of gramicidin S in water

The acid-base transformations of the gramicidin S molecule in water were studied. The protonization constants of the antibiotic amino group were calculated by the data of the potentiometric titration and the antibiotic distribution in the system of chloroform-water: K1 1.55 X 10(10), K2 1.38 X 10(6), the logarithm of the distribution coefficient of gramicidin S in the system of chloroform-water (1:1) lg alpha G 4.10. By the same data the constants of water solubility of gramicidin S base (1.02 X 10(7) mol/l), gramicidin S monohydrate (1.06 X 10(-4) mol/l) and gramicidin S dihydrochloride (2.08 X 10(-4) mol/l) were calculated.     

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

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

Heavy water inhibition of alkali cation transport across the muscle membrane. II. A comparison of the action of D20 and ouabain on the sodium efflux and rubidium influx in magnesium media

The action of heavy water and ouabain on sodium effluxes and rubidium influxes has been measured and compared in frog muscles (m. sartorius, R. temporaria). Approximately half of muscle sodium was substituted by lithium by preliminary incubation in mixed sodium-lithium media. The ratio of the ouabain-sensitive parts of rubidium influx and sodium efflux is 7.3:10.5, and that of D2O-sensitive parts of corresponding parameters is 7.5:11.3. A conclusion is made that D2O-effect on the Na, K-ATPase system of muscles under investigation resembles ouabain-effect on sodium effluxes as well as on rubidium influxes.     

Dependence of ion transport across the plasma membrane on cell culture density. II. Active and passive cation transport during the growth of L cell cultures

Rubidium and lithium influxes as well as intracellular potassium and sodium contents were investigated in L cells during the culture growth. In sparse culture over the cell densities 0.5-3 X 10(4) cells/cm2 ouabain-sensitive rubidium influx is small and ouabain-resistant lithium influx in high. With the increase in culture density up to 4-5 X 10(4) cells/cm2 the active rubidium influx, mediated by ouabain-sensitive component, is enhanced, and ion "leakage" tested by lithium influx is diminished. Simultaneously with the exponential growth of culture the intracellular potassium content is increased and the intracellular sodium content is decreased resulting in the higher K/Na ratio in cell. During the further transition to dense culture and in stationary state (10-17 X 10(4) cells/cm2) the sodium content and lithium influx do not change significantly, but the potassium content is decreased. The decrease in intracellular potassium is correlated with that in the portion of cells in S-phase from 27-30 to 12%. Thus, in transformed cells the density-dependent alterations in membrane cation transport are observed.     

Transmembrane effects in the sodium pump system. I. The effect of external potassium and rubidium on the dependence of sodium efflux on sodium concentration in the frog muscle

The dependence of sodium efflux on intracellular sodium content with various potassium and rubidium concentration in the external medium has been studied on frog sartorious muscle. In potassium-sodium-free magnesium medium ouabain-sensitive sodium efflux was shown to be proportional to internal sodium concentration. In the presence of external ribidium (0.5--5.0 mM) the efflux concentration relations are non-linear, being closely described by assuming that 3 Na+ are transported per pump cycle. In sodium loaded muscles the efflux concentration curve was found to be dependent on the external rubidium concentration, becoming linear instead of S-shaped with the decrease in internal rubidium concentration from 5.0--2.5 to 1.0--0.5 mM. The apparent affinity constant for the internal sodium pump site increased with increasing the external rubidium (potassium) concentration. The data obtained may contribute to the kinetic evaluation of the type of Na-K pump mechanism, being more consistent with simultaneous model of pump operation.     

Furosemide-sensitive cation transport in frog skeletal muscle fibers

Furosemide-inhibitable components in unidirectional cation fluxes have been identified in frog skeletal muscle. In sodium loaded muscles, placed in sodium-free rubidium lithium media, furosemide (1 mM) inhibits partially rubidium and lithium influxes as well as potassium and sodium outfluxes. The furosemide-inhibitable components were found to depend on the presence of ouabain. They were greatly diminished in sodium-free magnesium media and were present in chloride-free nitrate containing media. The dependence of furosemide-inhibitable sodium efflux on internal sodium content was also described.     

Effect of dihydropyridines on cation transport across the skeletal muscle membrane in the frog

2,6-Dimethyl-3,5-dimethoxycarbonyl-4-(o-difluromethoxyphenyl)- 1,4-dihydropyridine (ryodipine) blocks Ca-channels in phasic muscle fibres from ileofibularis and semitendinosus muscle of Rana esculenta. Ryodipine and some other newly synthesized dihydropyridines (10(-7)-10(-4) M) exerted a slight, if any, effect on the steady-state of potassium chord conductance in isotonic K+-sulfate solution. The effluxes of potassium and rubidium from the sartorius muscle of Rana temporaria also remained unchanged after addition of 2 x 10(-4) M ryodipine. Thus, the nonspecific dihydropyridine effect on ion transport seems to be poorly expressed.     

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.     

Effect of triamterene on leucocyte sodium and potassium levels in heart disease.

Sodium and potassium levels in plasma and leucocytes and the sodium efflux rate constants of leucocytes were measured in patients with congenital heart disease not on treatment, patients with valvular heart disease being treated with digoxin and conventional diuretics, and patients with valvular heart disease receiving digoxin and either conventional diuretics or triamterene or both. The group being treated with digoxin and conventional diuretics showed low cellular potassium levels, low sodium efflux rate constants, and a rise in cellular sodium levels. Patients given triamterene showed a rise in potassium levels in plasma and cells and in the sodium efflux rate constant.     

Axoplasmic free magnesium levels and magnesium extrusion from squid giant axons

The free magnesium concentration in the axoplasm of the giant axon of the squid, Loligo pealei, was estimated by exploting the known sensitivity of the sodium pump to intracellular Mg2+ levels. The Mg- citrate buffer which, when injected into the axon, resulted in no change in sodium efflux was in equilibrium with a Mg2+ level of about 3- -4 mM. Optimal [Mg2+] for the sodium pump is somewhat higher. Total magnesium content of axoplasm was 6.7 mmol/kg, and that of hemolymph was 44 mM. The rate coefficient for 28Mg efflux was about 2 X 10(-3) min-u for a 500-mum axon at 22-25degreesC, with a very high temperature coefficient (Q10=4-5). This efflux is inhibited 95% by injection of apyrase and 75% by removal of external sodium, and seems unaffected by membrane potential or potassium ions. Increased intracellular ADP levels do not affect Mg efflux nor its requirement for Na+/o, but extracellularl magnesium ions do. Activation of 28Mg efflux by Na+/o follows hyperbolic kinetics, with Mg2+/o reducing the affinity of the system for Na+/o. Lanthanum and D600 reversibly inhibit Mg efflux. In the absence of both Na+ and Mg2+, but not in their presence, removal of Ca2+ from the seawater vastly increased 28Mg efflux; this efflux was also strongly inhibited by lanthanum. A small (10(-14) mol cm-2) extra Mg efflux accompanies the conduction of an action potential.     

Sodium binding sites of gramicidin A: sodium-23 nuclear magnetic resonance study.

In ethanol-water mixtures (90:10), the gramicidin dimer binds Na+ cations at well-defined sites, with a binding constant K = 4 M-1. Partial desolvation of Na+ occurs upon binding, as judged from the magnitude of the quadrupolar coupling constant (1.7 MHz) for bound sodium. The binding sites are identified with the outer sites flanking the channel entrances. The rate constants for binding and release are k+ less than or equal to 2.2 X 10(9) M-1 s-1 and k- less than or equal to 5.5 X 10(8) s-1, respectively.     

Racial differences in bumetanide-sensitive cotransport and N-ethylmaleimide-stimulated potassium efflux

Racial differences in erythrocyte potassium effluxes mediated by two loop-diuretic sensitive modes of cotransport were compared. In red cells loaded to contain approximately equimolar amounts of sodium and potassium, black subjects had lower bumetanide-sensitive sodium-dependent net potassium effluxes as compared to whites. In fresh, washed erythrocytes pretreated with N-ethylmaleimide (NEM), maximal net potassium efflux was greater in blacks than in whites. NEM-stimulated potassium efflux was partially inhibited by bumetanide but only at very high concentrations. The quantitative differences in these two modes of potassium efflux suggest that NEM-stimulated potassium efflux is not an altered mode of sodium-dependent potassium efflux.     

Effects of gramicidin and tryptophan-N-formylated gramicidin on the sodium and potassium content of human erythrocytes

Summary

In order to get a better understanding in the mechanism by which tryptophan-N-formylated gramicidin (NFG) and gramicidin kill the malaria parasite Plasmodium falciparum in vitro, we studied the capacity of these peptides to change the potassium, as well as the sodium, composition of normal human erythrocytes, and their ability to cause cell lysis. It is shown that both peptides are able to induce potassium leakage from, and sodium flux into, erythrocytes in such a manner that it is most likely that they are able to form cation channels in the membrane of these cells. For both peptides, potassium efflux proceeds at a faster rate than sodium influx, but this difference is greater for NFG than for gramicidin. This explains the observation that gramicidin is more lytic than NFG is, even when comparing concentrations that show the same antimalarial activity. The finding that gramicidin is approximately 10 times more active than NFG in causing potassium efflux from normal erythrocytes, as well as in killing the malaria parasite, supports the hypothesis that peptideinduced parasite death is related to their capacity to induce potassium leakage from infected erythrocytes. Finally, the observation that erythrocytes are able to restore their normal ion contents after losing more than 50% of their potassium content by incubation with NFG or gramicidin, suggests that, in vivo, and upon treatment with drug concentrations that cause full inhibition of parasite growth, these cells would not be irreversibly damaged by action of the drugs.     

The influence of potassium- and sodium-free solutions on sodium efflux from squid giant axons

The sensitivity of sodium efflux to the removal of potassium ions from the external solution and the change in sodium efflux occurring when sodium ions are also removed were observed to be related. When Tris was used to replace external sodium ions, increases in sodium efflux were always observed whether the sensitivity of sodium efflux to external potassium ions was weak or strong. Greater percentage increases in sodium efflux occurred, however, the greater the sensitivity of sodium efflux to external potassium ions. When lithium ions were used to replace external sodium ions, increases in sodium efflux occurred if the sensitivity of efflux to external potassium ions was strong whereas decreases in sodium efflux took place if the sensitivity of efflux to external potassium ions was weak. Intermediate sensitivities of efflux to external potassium resulted in no change in efflux upon substitution of lithium ions for external sodium ions. In the presence of 10^-5 M ouabain, substitution of Tris for external sodium ions always resulted in a small decrease in sodium efflux. The data can be described in terms of a model which assumes the presence of efflux stimulation sites that are about 98% selective to potassium ions and about 2% selective to sodium or lithium ions.     

An Na+-stimulated Mg2+-transport system in human red blood cells

The initial rate of net Mg2+ efflux was measured in human red blood cells by atomic absorption. In fresh erythrocytes incubated in Na+,K+-Ringer's medium this rate was 7.3 +/- 2.8 mumol/l cells per h (mean +/- S.D. of 14 subjects) with an energy of activation of 13 200 cal/mol. Cells with total Mg2+ contents ([ Mg]i) ranging from 1.8 to 24 mmol/l cells were prepared by using a modified p-chloromercuribenzenesulphonate method. Mg2+ efflux was strongly stimulated by increases in [Mg]i and in external Na+ concentrations ([ Na]o). A kinetic analysis of Mg2+ efflux as a function of [Mg]i and [Na]o revealed the existence of two components: an Na+-stimulated Mg2+ efflux, which exhibited a Michaelian-like dependence of free internal Mg2+ content (apparent dissociation constant = 2.6 +/- 1.4 mmol/l cells; mean +/- S.D. of six subjects) and on external Na+ concentration (apparent dissociation constant = 20.5 +/- 1.9 mM; mean +/- S.D. of four subjects) and a variable maximal rate ranging from 35 to 370 mumol/l cells per h, and an Na+-independent Mg2+ efflux, which showed a linear dependence on internal Mg2+ content with a rate constant of (6.6 +/- 0.7) X 10(-3) h-1. Fluxes catalyzed by the Na+-stimulated Mg2+ carrier were partially dependent on the ATP content of the cells and completely inhibited by quinidine (IC50 = 50 microM) and by Mn2+ (IC50 = 0.5-1.0 mM).     

Spontaneous induction of superoxide release and degranulation of neutrophils in isotonic potassium medium: the role of intracellular calcium

When guinea pig peritoneal neutrophils were suspended in the isotonic medium of potassium, rubidium, and cesium ions at 37 degrees C, the cells released superoxide, while low activity was observed in the isotonic medium of sodium and lithium ions. The activity induced in the potassium medium was enhanced by potassium-ionophores, valinomycin, and gramicidin, and decreased by a potassium channel blocker, 4-aminopyridine. The superoxide-releasing activity was not affected by the presence or absence of extracellular calcium but was inhibited by an intracellular calcium antagonist-8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate(TMB-8) with the half-inhibition concentration of 50 microM. The release of granular enzymes, lysozyme and beta-glucuronidase, was also induced in the isotonic potassium medium in the absence of extracellular calcium and inhibited by TMB-8. A remarkable elevation of the intracellular free calcium concentration in neutrophils, which was monitored by quin-2 fluorescence, was found when the cells were added to the potassium medium without calcium. The elevation was inhibited by the addition of TMB-8. These observations suggest that calcium mobilization from intracellular storage sites, not an influx of calcium from the extracellular medium, causes the release of superoxide and the granular enzymes in isotonic potassium medium.     

Sodium,potassium, and rate constants for sodium efflux in leucocytes from hypertensive Jamaicans.

Leucocyte sodium and potassium content and concentrations were measured along with ouabain-sensitive and ouabain-insensitive rate constants for sodium efflux in 14 controls and 20 black patients with essential hypertension. Leucocyte sodium content was significantly increased in the patients (mean 101.1 +/- 7.8 mmol/kg dry solids v 74.5 +/- 7.6 mmol/kg dry solids; p less than 0.05), whereas the rate constants for sodium efflux were not significantly reduced. There was no difference between the two groups in cell potassium values. The increase in leucocyte sodium content in the presence of normal rate constants for sodium efflux suggests an increase in membrane permeability to sodium, which might be important in the pathogenesis of essential hypertension.     

Proton-activated rubidium transport catalyzed by the sodium pump

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

Cation transport and content in serum-stimulated CHO-773 cells. I. Rapid changes in rubidium and lithium influxes and intracellular sodium content

The cultures of Chinese hamster ovary cells (CHO-K1 clone 773) can be brought to the stationary state with most of cellular populations in G1 phase by growing continuously for 4 days up to the cultural density (10-12) X 10(4) cells/cm2. Upon introduction of fresh Eagle medium with 10% calf serum the cells progress from G1 to S phase for 7-9 hours. It is shown that within the first minutes of serum addition ouabain-sensitive rubidium influx increases, however, lithium influx, which serves a test for passive sodium pathways in the membrane, increases or does not change. No correlation was found between the rubidium influx and intracellular sodium changes, induced by serum. From comparative studies of ouabain-sensitive rubidium influx, lithium influx and intracellular sodium content it is concluded that the increase in intracellular sodium is not responsible for serum-induced Na,K-ATPase activation.     

Salt excretion by the perfused head of trout adapted to sea water and its inhibition by adrenaline

Summary Isolated heads of trout (Salmo gairdneri) were used to study the unidirectional flux of sodium and chloride across the gills in salt water.Two perfusion techniques were employed. Under constant pressure perfusion, the addition of adrenaline during the perfusion causes an increase in the flow-rate. Under constant flow-rate conditions, adrenaline provokes a decrease in pressure. A comparison of influx determination made with these two techniques of perfusion shows that variations in flow-rate of perfusion do not affect the assessment of these fluxes.A net efflux of sodium, but not of chloride, is demonstrated in sea water. The effluxes of sodium and chloride observed in sea water are decreasedd about 50% during a rapid transfer to fresh water. The addition of potassium to this medium stimulates the effluxes of sodium and chloride, suggesting a Na/K exchange participating in the chloride excretion.Adrenaline causes an inhibition of sodium and chloride efflux in sea water which persists after transfer to fresh water and the addition of potassium. Only the influx of chloride is inhibited at a concentration of 10^–5 M whereas the sodium influx is unaffected. The presence of adrenaline results in a net influx of both sodium and chloride.The differential action of adrenaline on the influxes of sodium and chloride suggests that the hemodynamic modifications provoked by this catecholamine occur independently of its aforementioned ion exchange effects.