Ouabain-sensitive Potassium Influx Research Articles

Potassium influx in human neonatal red blood cells. Partition into its major components

The potassium influx in human neonatal red blood cells (nRBC) shows an approximately 25% lower value compared to the total potassium influx in adult red blood cells (aRBC). The ouabain-sensitive potassium influx component represents approximately 70-75% of the total potassium influx for both types of cells but with an absolute value significantly lower in nRBC. In nRBC, the half maximum inhibitory effect for ouabain was obtained at a 10(-9) M concentration. The ouabain-insensitive nRBC potassium influx fractions showed two components: (i) a bumetanide-sensitive component, significantly lower than that of aRBC, (ii) a ouabain-bumetanide-insensitive (leak) component with a similar value in both cell types. The sum of the ouabain-sensitive and furosemide-sensitive components amounted in nRBC to a greater value than the total potassium influx. This behaviour could be interpreted as a superposition of the action of the inhibitors on the components affected.

The effects of alterations in the external sodium concentration on human leucocyte sodium and potassium transport in vitro.

Human leucocytes incubated in tissue culture fluid of low-sodium concentration (2 mM; iso-osmolarity maintained with choline chloride) reached a new equilibrium within 1 hour and lost approximately 25% of intracellular potassium and 70% of intracellular sodium. The rate constant for ouabain-sensitive sodium efflux fell by more than 50% and the ouabain-insensitive rate constant increased nearly threefold in the low-sodium medium. Total sodium efflux fell in proportion to internal sodium whereas ouabain-insensitive sodium efflux remained unchanged. A reduction in external sodium from 140 to 2 mM was associated with a 75% fall in sodium influx. In the low-sodium medium ouabain-sensitive potassium influx exceeded ouabain-sensitive sodium efflux and no ouabain-sensitive potassium efflux could be demonstrated. Ouabain-insensitive potassium influx and that portion of potassium efflux which is dependent on external potassium fell in parallel in low-sodium cells, suggesting reduced activity of a ouabain-insensitive K:K exchange system.

Effect of extracellular potassium on the transport of sodium and potassium in rat thymocytes.

1. The effect of extracellular potassium on the transport of sodium and potassium in rat thymocytes has been studied in vitro. 2. A significant increase in the rate constant for total and ouabain-sensitive sodium efflux was demonstrated at an extracellular potassium concentration of 1 mmol/l as compared with that at either 0 or 2 mmol/l. 3. At potassium concentrations below 3 mmol/l ouabain-sensitive sodium influx was observed suggesting sodium-sodium exchange catalysed by the sodium pump. 4. Both total and ouabain-insensitive potassium efflux rose with external potassium. A small ouabain-sensitive potassium efflux was observed at all levels of external potassium studied. 5. Total and ouabain-insensitive potassium influx increased with external potassium, but did not appear to saturate. Ouabain-sensitive potassium influx reached a maximum at an external potassium concentration of 2 mmol/l then decreased with increasing external potassium.

Serum stimulation of potassium fluxes, ouabain binding, and sodium fluxes in quiescent chicken embryo fibroblasts.

Growth-contingent alterations in potassium and sodium fluxes, ouabain binding, and potassium ion content were examined following serum stimulation of quiescent, density-inhibited chicken embryo fibroblasts. Serum stimulation resulted in very rapid 1.5- to 1.8-fold increases in ouabain-sensitive potassium influx and lesser 1.4- to 1.5-fold increases in potassium efflux and sodium influx. Potassium influx stimulation was maximal after addition of 5-20% calf serum and was unaffected by cycloheximide inhibition of protein synthesis. Reflecting the slightly greater stimulation of potassium influx versus potassium efflux, potassium ion levels were 10-15% higher in serum-stimulated compared to unstimulated cells. Specific ouabain binding levels in stimulated and unstimulated control cells were initially similar, however, by four hours after stimulation a 40-50% increase in specific ouabain binding was observed. Incubation with ouabain was found also to inhibit later serum-stimulated hexose uptake and thymidine incorporation; this blockage may be consequence of subnormal potassium levels rather than ouabain inhibition of the serum-stimulated potassium influx.

Lithium and erythrocyte membrane cation carrier studies in normal and manic depressive subjects.

Changes in the erythrocyte membrane cation carrier following lithium ingestion in normal human subjects were studied; ouabain sensitive potassium influx fell significantly during the lithium treated phase. Lithium was fed to rats and no change in erythrocyte Na-K ATPase was shown. These findings contrast with studies of lithium in manic depressive psychosis. The fluctuations in the erythrocyte membrane cation carrier were studied in 5 normal subjects over 12 weeks and the correlations between the parameters calculated. The erythrocyte sodium concentration correlated positively with the ouabain sensitive potassium influx. This too contrasts with findings in manic depressive psychosis.

Erythrocyte membrane cation carrier in mania.

Erythrocyte sodium and potassium concentrations, erythrocyte membrane ATPase (Na-K specific and non-specific) and the rate of potassium influx into erythrocytes (ouabain-sensitive and insensitive) were estimated in a group of female patients suffering from mania and repeated on about two thirds of them when they had recovered. With recovery there was a statistically significant increase in the erythrocyte ouabain-sensitive potassium influx. The other parameters showed no significant overall change with recovery but the initial severity correlated significantly and negatively with the change in erythrocyte Na-K ATPase with recovery. The changes that occurred in the erythorcyte sodium concentration and Na-K ATPase activity were not random since they correlated significantly with changes in the active potassium influx.

A biochemical study of short-cycle manic-depressive psychosis in mental defectives.

Four mentally defective patients who were suffering from short-cycle manic-depressive psychosis were each studied under metabolic ward conditions for about six months. Weekly estimations of erythrocyte Na-K ATPase activity, ouabain sensitive potassium influx, sodium concentration, whole blood cell ATP concentration and blood haematocrit were made. The findings in each patient differed, but the erythrocyte Na-K ATPase activity, sodium and ATP concentration appeared to show cyclic changes related to, though out of phase with, the clinical state.

The effect of external potassium concentration on leucocyte cation transport in vitro.

1. Sodium and potassium transport rates in human leucocytes were measured in vitro at different external potassium concentrations. 2. At nominally zero external potassium concentrations, the ouabain-sensitive sodium efflux was reduced to less than 20% of its maximum value. There was evidence that under these conditions a ouabain-sensitive sodium-sodium exchange occurs. 3. Both total and ouabain-insensitive potassium influx increased with increasing external potassium concentration. The ouabain-sensitive potassium influx showed saturation. 4. Ouabain-insensitive potassium efflux was also stimulated by increasing the external potassium concentration, suggesting significant potassium-potassium exchange at physiological external potassium concentrations.

Erythrocyte membrane cation carrier in depressive illness.

SynopsisErythrocyte sodium concentration, potassium concentration, Na-K ATPase activity, and ouabain sensitive potassium influx were determined in female inpatients suffering from a depressive illness. In most patients the biochemical values were also determined shortly before the patient's discharge from hospital. The Na-K ATPase and the ouabain sensitive potassium influx increased significantly with improvement in the depressive mood rating.

Ouabain binding and cation transport in human erythrocytes.

In the present studies we have explored the relation between ouabain binding and the inhibition of potassium influx in intact human erythrocytes. The rate at which bound ouabain molecules dissociate from the erythrocyte membrane is not altered by complete replacement of choline with sodium or by partial replacement with potassium. These findings indicate that the effects of these cations on ouabain binding reflect alterations in the rate of association of ouabain molecules with the erythrocyte membrane. Variations in the cation composition of the incubation solution did not alter the relation between the fraction of the glycosidebinding sites occupied by ouabain or the fraction of ouabain-sensitive potassium influx which was inhibited. That is, irrespective of the affinity of the erythrocyte membrane for ouabain molecules and irrespective of the magnitude of glycoside-sensitive potassium influx, occupation of a given fraction of the glycoside-binding sites by ouabain results in the inhibition of an equal fraction of the ouabain-sensitive potassium transport sites.

A New Variant of Hereditary Hemolytic Anemia With Stomatocytosis and Erythrocyte Cation Abnormality

Abstract A new variant of congenital hemolytic anemia associated with stomatocytosis, reticulocytosis, decreased osmotic fragility, type I autohemolysis and shortened erythrocyte survival without specific splenic sequestration was discovered in three siblings of Swiss-German ancestry. Increased intracellular sodium (two to three times normal) and slightly decreased intracellular potassium were detected. Total sodium efflux was eight-fold greater than normal but total potassium influx was normal and ouabain-sensitive potassium influx was decreased. The ouabain-sensitive sodium efflux: potassium influx ratio was 26:1 rather than the 3:2 ratio noted in normal cells. The consanguineous parents, four other siblings, and 44 other family members had mild stomatocytosis, reticulocytosis, and, when studied, decreased osmotic fragility, increased autohemolysis, intermediate abnormalities of cation content, cation flux, and moderate shortening of erythrocyte survival. Autosomal dominant inheritance was suggested. No abnormalities of RBC enzymes, hemoglobin or lipids were observed. No abnormalities of membrane protein were detected on acrylamide gel. Substrate depletion of these hypermetabolic cells resulted in intracellular dehydration with potassium loss in excess of sodium gain and decreased deformability. Although the exact nature of the defect responsible for hemolysis is unknown, this syndrome differs from other hereditary hemolytic anemias associated with stomatocytosis.

The influence of the chloride gradient across red cell membranes on sodium and potassium movements.

1. A study has been made to see whether active and passive movements of sodium and potassium in human red blood cells are influenced by changing the chloride gradient and hence the potential difference across the cell membrane.2. Chloride distribution was measured between red cells and isotonic solutions with a range of concentrations of chloride and non-penetrating anions (EDTA, citrate, gluconate). The cell chloride concentration was greater than that outside with low external chloride, suggesting that the sign of the membrane potential was reversed. The chloride ratio (internal/external) was approximately equal to the inverse of the hydrogen ion ratio at normal and low external chloride, and inversely proportional to external pH. These results show that chloride is passively distributed, making it valid to calculate the membrane potential from the chloride ratio.3. Ouabain-sensitive (pump) potassium influx and sodium efflux were decreased by not more than 20 and 40% respectively on reversing the chloride gradient, corresponding to a change in membrane potential from -9 to +30 mV. In contrast, passive (ouabain-insensitive) movements were reversibly altered - potassium influx was decreased about 60% and potassium efflux was increased some tenfold. Sodium influx was unaffected by the nature of the anion and depended only on the external sodium concentration, whereas ouabain-insensitive sodium efflux was increased about threefold. When external sodium was replaced by potassium there was a decrease in ouabain-insensitive sodium efflux with normal chloride, but an increase in low-chloride medium.4. Net movements of sodium and potassium were roughly in accord with the unidirectional fluxes.5. The results suggest that reversing the chloride gradient and, therefore, the sign of the membrane potential, had little effect on the sodium pump, but caused a marked increase in passive outward movements of both sodium and potassium ions.

The effect of 2,4,6-trinitro-m-cresol on cation and anion transport in sheep red blood cells.

2,4,6-Trinitro-3-methyl-phenol (trinitrocresol, H(+)TNC(-)) was found to inhibit anion and stimulate cation movements across the membranes of both high potassium (HK) and low potassium (LK) sheep red blood cells. The concentration of TNC(-) required to inhibit SO(4) (-) and Cl(-) efflux (10(-5)-10(-3)M) was less than that required to increase Na(+) and K(+) leakage (10(-3)-10(-2)M). Both the inhibition of anion and stimulation of cation permeation were reversed if TNC(-) was washed from the red cells. The cation leak caused by TNC(-) was much greater at 0 degrees and 37 degrees C than at room temperature (23 degrees C). In sheep red cells, TNC(-) was found to be about 20 times more effective than salicylate and about 40 times more effective than thiocyanate in increasing cation leak. TNC(-) also inhibited the ouabain-sensitive potassium influx.

Synthesis of adenosine triphosphate at the expense of downhill cation movements in intact human red cells.

1. When red cells that have been starved for about 6 hr are loaded with inorganic phosphate and incubated in high-sodium potassium-free media, the ouabain-sensitive efflux of potassium from the cells is accompanied by a ouabain-sensitive incorporation of inorganic phosphate into ATP.2. The magnitude of the incorporation varies roughly linearly with the concentration of sodium in the medium. The ratio (ouabain-sensitive potassium efflux)/(ouabain-sensitive ATP synthesis) is probably not much less than 2 nor much greater than 3.3. Potassium in the medium inhibits the ouabain-sensitive incorporation of phosphate. The concentration of potassium necessary for half-maximal inhibition is about the same as the concentration at which, under similar conditions, ouabain-sensitive potassium influx and the stimulation of ouabain-sensitive potassium efflux are both half-maximal.4. These observations suggest that the ouabain-sensitive efflux of potassium from red cells incubated in high-sodium potassium-free media is associated with a reversal of the entire pump cycle. In media containing sufficient potassium to saturate the pump, the efflux appears to involve the reversal of only part of the cycle.

The stoicheiometry of the sodium pump.

1. When resealed ghosts containing adenosine triphosphate (ATP), magnesium and sodium were incubated in a medium containing potassium, ATP was hydrolysed vigorously by a ouabain-sensitive mechanism. If the ghosts contained potassium instead of or in addition to sodium, and the external solution contained sodium but no potassium, there was little ouabain-sensitive hydrolysis of ATP. As it is known that the ouabain-sensitive ATPase in fragmented ghosts requires both sodium and potassium ions, these results show that the ATPase is activated by potassium externally and by sodium internally, and suggest that the ions activating the ATPase are the ions that are transported.2. Resealed ghosts containing ATP, magnesium and sodium were incubated in sodium-free media containing potassium, with and without ouabain, and the rate of loss of sodium and rate of hydrolysis of ATP were measured. The hydrolysis of 1 molecule of ATP by the ouabain-sensitive mechanism was accompanied by the ouabain-sensitive loss of about 3 sodium ions.3. (24)Na and (42)K were used to measure sodium efflux and potassium influx in identical batches of fresh red cells under the same conditions and at the same time. Each flux was measured in the presence and absence of ouabain. The ratio (ouabain-sensitive sodium efflux)/(ouabain-sensitive potassium influx) was significantly greater than 1 (1.20 +/- 0.01 and 1.35 +/- 0.01 in two experiments). If a small fraction of the potassium influx represented a ouabain-sensitive potassium: potassium exchange, the ratio of the numbers of ions moved in the sodium: potassium exchange catalysed by the pump must have been even further from unity.4. Resealed ghosts containing [gamma-(32)P]ATP, magnesium, (24)Na and orthophosphate were incubated in balanced salt solutions with and without potassium and with and without ouabain. A comparison of sodium efflux, estimated from (24)Na loss, with ATP hydrolysis, estimated from the formation of [(32)P]orthophosphate, showed that the sodium:sodium exchange in a potassium-free medium was accompanied by little or no ouabain-sensitive hydrolysis of ATP.5. Experiments on intact red cells loaded with (24)Na showed that both sodium:sodium exchange in a potassium-free medium, and sodium:potassium exchange in a medium containing potassium, were partially inhibited by oligomycin (1-10 mug/ml.). Inhibition of the sodium:potassium exchange was not affected by raising the external potassium concentration.

Facftors affecting the relative magnitudes of the sodium:potassium and sodium:sodium exchanges catalysed by the sodium pump.

1. The effects of external potassium on sodium: potassium exchange and sodium: sodium exchange in human red cells have been estimated from measurements of ouabain-sensitive potassium influx and ouabain-sensitive sodium influx in media containing different concentrations of potassium.2. As the external potassium concentration is increased from zero to 5 mM, sodium:sodium exchange-as judged by ouabain-sensitive sodium influx-is progressively suppressed, and sodium:potassium exchange-as judged by ouabain-sensitive potassium influx-is progressively increased. Both exchanges are half-maximal between 1 and 2 mM-K, and at 5 mM-K sodium: sodium exchange becomes very small as sodium: potassium exchange approaches a maximum.3. Experiments have been carried out, mainly on resealed ghosts, to determine what factors affect the magnitude of the sodium:sodium exchange in potassium-free solutions.4. Sodium:sodium exchange does not occur in the absence of adenosine triphosphate (ATP).5. Ghosts containing high concentrations of sodium, no potassium and high concentrations of ATP show no ouabain-sensitive loss of sodium into potassium-free solutions. The ability to carry out sodium:sodium exchange can be restored by replacing most of the internal sodium with potassium or by preparing the cells so that they contain much more orthophosphate (P(i)) than ATP.6. Ghosts containing sodium in low concentration, potassium in high concentration and with a low [ATP]/([ADP].[P(i)]) ratio show a greater ouabain-sensitive loss of sodium into potassium-free media than into media containing potassium; i.e. external potassium reduces ouabain-sensitive sodium efflux.7. The effect of P(i) is not the result of competitive inhibition of the transport ATPase since P(i) at the concentrations used does not inhibit ATPase activity in fragmented ghosts.