Effect of ouabain on the Ca-dependent increase in K permeability in depleted guinea-pig red cells

1.

1. The hypothesis that the inhibitory action of ouabain on the Ca²⁺-dependent increase in K⁺ permeability observed in depleted human red cells is mediated by changes in the intracellular level of ATP was tested by measuring simultaneously the ouabain sensitive K⁺ loss and the concentration of ATP in depleted guinea-pig red cells in the presence and absence of external Ca²⁺.     

The effect of cellular calcium on Na+/K+ cotransport in human red blood cells

The increase in Ca²⁺ permeability by addition of ionophore A23187 in the presence of external Ca²⁺ did not alter the bumetanide-sensitive Na⁺/K⁺ effluxes in human red blood cells. An inhibition of this pathway by cellular Ca²⁺ could be observed only under conditions in which the cellular ATP content was drastically depleted.     

Calcium-induced potassium pathway in sided erythrocyte membrane vesicles

We have characterized the asymmetric effect of Ca²⁺ on passive K⁺ permeability in erythrocyte membranes, using inside out and right-side out vesicles. Ca²⁺, but not Mg²⁺, can induce an increase in K⁺ uptake in inside out vesicles. The half-maximal concentration of Ca²⁺ required to induce the K⁺ uptake is 0.2 mM, and the permeability increase is not specific for K⁺. Thus, the Ca²⁺-induced permeation process in inside out vesicles is changed from that in the energy-depleted intact cell which requires only micromolar concentrations of Ca²⁺ and is specific for K⁺. Removal of spectrin had no effect on the vesicle permeability increase due to Ca²⁺. Studies with $\text{N-}\text{ethylmaleimide}$ show that the vesicle channel opening is mediated by a protein and passage is controlled by sulfhydryl groups; furthermore, the Ca²⁺-induced vesicle pathway is distinct from the normal channel for passive K⁺ leak in the absence of Ca²⁺. The protein is sensitive to its phospholipid environment since removal of easily accessible phospholipid head groups on the cytoplasmic face of the vesicles inhibits the Ca²⁺-stimulated channel opening.     

ATPase and phosphatase activities from human red cell membranes: I. The effects of N-ethylmaleimide

Summary (i) In human red cell membranes the sensitivity to N-ethylmaleimide of Ca²⁺-dependent ATPase and phosphatase activities is at least ten times larger than the sensitivity to N-ethylmaleimide of (Na⁺+K⁺)-ATPase and K⁺-activated phosphatase activities. All activities are partially protected against N-ethylmaleimide by ATP but not by inorganic phosphate or byp-nitrophenylphosphate. (ii) Protection by ATP of (Na⁺+K⁺)-ATPase is impeded by either Na⁺ or K⁺ whereas only K⁺ impedes protection by ATP of K⁺-activated phosphatase. On the other hand, Na⁺ or K⁺ slightly protects Ca²⁺-dependent activities against N-ethylmaleimide, this effect being independent of ATP. (iii) The sensitivity to N-ethylmaleimide of Ca²⁺-dependent ATPase and phosphatase activities is markedly enhanced by low concentrations of Ca²⁺. This effect is half-maximal at less than 1 m Ca²⁺ and does not require ATP, which suggests that sites with high affinity for Ca²⁺ exist in the Ca²⁺-ATPase in the absence of ATP. (iv) Under all conditions tested the response to N-ethylmaleimide of the ATPase and phosphatase activites stimulated by K⁺ or Na⁺ in the presence of Ca²⁺ parallels that of the Ca²⁺-dependent activities, suggesting that the Ca²⁺-ATPase system possesses sites at which monovalent cations bind to increase its activity.     

Effect of antihistamines and chlorpromazine on the calcium-induced hyperpolarization of the Amphiuma red cell membrane

1. 1. It has previously been demonstrated that an increase in extracellular Ca²⁺ concentration induces a transient increase in K⁺ permeability and associated hyperpolarization of the red cell membrane of the giant salamander, Amphiuma means. This phenomenon is analogous to the Ca²⁺-induced KCl loss observed in ATP-depleted human red cells and red cell ghosts.

2. 2. Histamine, which enhances the Ca²⁺-induced K⁺ loss from depleted human red cells, is without effect on this Ca²⁺-induced hyperpolarization of Amphiuma red cells.

3. 3. Promethazine (10 μM) and mepyramine (1 mM), which inhibit the Ca²⁺-induced K⁺ loss in depleted human red cells, also block the Ca²⁺-related hyperpolarization of Amphiuma erythrocytes.

4. 4. Chlorpromazine (25 μM), despite being a weak antihistamine, is equally effective in blocking the Ca²⁺-induced hyperpolarization of Amphiuma red cells.

5. 5. Ionophore A23187 causes a large and sustained Ca²⁺/K⁺-dependent hyperpolarization even in the presence of normal (1.8 mM) concentrations of Ca²⁺. This hyperpolarization is relatively insensitive to chlorpromazine and promethazine.

6. 6. The inhibition of the Ca²⁺-induced hyperpolarization of the Amphiuma red cell membrane by chlorpromazine and promethazine may be related to their properties as local anaesthetics.

A calcium-activated potassium channel present in foetal red cells of the sheep but absent from reticulocytes and mature red cells

Red cells of adult sheep, like those of other ruminants, lack the calcium-activated potassium channel which is present in the membrane of human red cells. Since the activities of other transport systems in the sheep red cell are known to decrease during maturation of the cell or during development of the animal it was investigated whether the K⁺ channel is present in red cells from younger animals or in reticulocytes. Using the divalent cation ionophore A23187 to increase the intracellular Ca of intact cells, it was found that the K⁺-selective channel is present in foetal red cells from the foetus or newborn animal but not in reticulocytes. The presence of the channel showed no dependence on the K⁺ genotype of the sheep and was not associated with either “high K⁺”-or “low K⁺”-type Na⁺ pump. No Ca²⁺-dependent change in K⁺ permeability was found in red cells from either newborn or adult donkeys suggesting that its presence in the red cells of the foetus may not be general. The role of the K⁺ channel in the mammalian red cell and the relationship between the K⁺ channel and the Na⁺ pump are discussed.     

The mechanism of vanadium action on selective K+-permeability in human erythrocytes

Low concentrations of chelating agents such as EDTA prevent the air oxidation of vanadyl (VO²⁺, +4 oxidation state) to vanadate (VO₃^?, +5 oxidation state). Under these conditions, the ionophore A23187 mediates the rapid entry of vanadyl into human erythrocytes. In the presence of A23187, vanadyl at concentrations in excess of EDTA gives rise to a dramatic increase in K⁺ permeability, which is very similar to the Gardos Ca²⁺-induced K⁺ permeability increase with respect to ion selectivity, response to inhibitors, effects of pH, and stimulation by external K⁺. In ultrapure media with very low Ca²⁺, however, vanadyl has no effect on K⁺ permeability. These experiments suggest that Ca²⁺ is displaced from EDTA by vanadyl and then enters the cell via A23187 where it triggers the increase in K⁺ permeability. This hypothesis is confirmed by experiments demonstrating that vanadyl does displace Ca²⁺ from EDTA. Vanadate, an inhibitor of Ca²⁺-ATPase, causes a selective increase in K⁺ permeability in metabolically depleted cells, but the increase is abolished by low concentrations of EDTA, indicating that this effect is also due to entry of extracellular Ca²⁺. Earlier observations of effects of vanadyl and vanadate on erythrocyte K⁺ permeability can thus be explained on the basis of inhibition of the Ca²⁺ pump by vanadium, leading to an increase in intracellular Ca²⁺ concentration.     

Calcium uptake by smooth endoplasmic reticulum of peeled retinal photoreceptors of the crayfish

Summary The localization and basic properties of Ca²⁺-accumulating sites in crayfish photoreceptors were studied with a novel preparation of peeled retinula cells in suspension. Peeled photoreceptors were obtained by gentle mechanical disruption of the retina, and incubated in media based on a Ca²⁺-EGTA buffer with ATP and oxalate. Electron microscopy of photoreceptors so treated showed the appearance of peculiar dense deposits inside vesicles of smooth endoplasmic reticulum (SER). EGTA-extraction and energy-dispersive X-ray microanalysis identified Ca as a major constituent of such deposits.⁴⁵Ca²⁺ uptake experiments with peeled photoreceptors or with the crude particulate fraction of retinal homogenates revealed a rapid binding of radioactivity over the first 8 min, followed by a slower continued accumulation, which did not occur in the absence of ATP.⁴⁵Ca²⁺ uptake is stimulated by an increase in the concentration of free Ca over the range 4×10^–7 to 5×10^–6 M, and becomes inhibited at higher levels.⁴⁵Ca²⁺ uptake is depressed when K⁺ is replaced by Na⁺ or Li⁺ as the main monovalent cation in the medium, but it is not affected by illumination nor by the presence of caffeine or ruthenium red. These findings attest that the SER has a Na⁺-sensitive capacity for regulating the intracellular concentration of Ca²⁺ in these photoreceptors, and support the hypothesis of its probable role in the control of pigment granule transport and other structural changes involved in light/dark adaptation.     

The membrane potential modulates the ATP-dependent Ca pump of cardiac sarcolemma

The effect of membrane potential on the activity of the ATP-dependent Ca²⁺ pump of isolated canine ventricular sarcolemmal vesicles were investigated. The membrane potential was controlled by the intravesicular and extravesicular concentration of K⁺, and the initial rates of Ca²⁺ uptake both in the presence and the absence of valinomycin were determined. The rate of Ca²⁺ uptake was stimulated by a inside-negative potential induced in the presence of valinomycin. The valinomycin-dependent stimulation was enhanced by the addition of K⁺ channel blocker, tetraethylammonium ion or Ba²⁺. The electrogenicity of cardiac sarcolemmal ATP-dependent Ca²⁺ pump is suggested from the increase of Ca²⁺ uptake by negative potential induced by valinomycin.     

Partial Characterization of K and Ca Uptake Systems in the Halotolerant Alga Dunaliella salina

The uptake of K⁺ and Ca²⁺ in Dunaliella salina is mediated by two distinct carriers: a K⁺ carrier with a high selectivity against Na⁺, Li⁺, and choline⁺ but not towards Rb⁺, K⁺, Cs⁺, or NH₄⁺, and a Ca²⁺ carrier with a high selectivity against Mg²⁺. The latter is specifically blocked by La³⁺ and by Cd²⁺. Apparent K_m values for K⁺ and Ca²⁺ uptake are 2.5 and 0.8 millimolar, respectively, and their maximal calculated fluxes are 22 and 0.8 nanomoles per square meter per second, respectively. Effects of permeable ions and ionophores on K⁺ and Ca²⁺ uptake suggest that the driving force for their uptake is the transmembrane electrical potential. Inhibitors of ATP production, typical inhibitors of plasma membrane H⁺-ATPases and protonionophores inhibit K⁺ and Ca²⁺ uptake and accelerate K⁺ efflux. The results suggest that an H⁺-ATPase in the cell membrane provides the driving force for K⁺ and Ca²⁺ uptake. Efflux measurements from ⁸⁶Rb⁺ and ⁴⁵Ca²⁺ loaded cells suggest that part of the intracellular K⁺ and most of the intracellular Ca²⁺ is nonexchangeable with the extracellular pool. Correlations between phosphate and K⁺ contents and the effect of phosphate on K⁺ efflux suggest intracellular associations between K⁺ and polyphosphates. On the basis of these results, it is suggested that: (a) K⁺ and Ca²⁺ uptake in D. salina is driven by the transmembrane electrical potential which is generated by the action of an H⁺-ATPase of the plasma membrane. (b) Part of the intracellular K⁺ is associated with polyphosphate bodies, while most of the intracellular Ca²⁺ is accumulated in intracellular organelles in the algal cells.     

CALCIUM UPTAKE INTO RAT PHEOCHROMOCYTOMA CELLS1

Abstract– The uptake of ⁴⁵Ca²⁺ into cell suspensions prepared from a transplantable rat pheochromocytoma was measured. The uptake of Ca²⁺ into these cells is biphasic; there is a rapid, initial uptake of Ca⁺, followed by a slower uptake that proceeds at a linear rate for at least 10min at 37°C. The uptake of Ca²⁺ is a linear function of the external Ca²⁺ concentration over the range of 0.13-2.5 mm -Ca²⁺ Incubation of the cells in a medium containing 56mm -K⁺ results in a 2-3 fold increase in the uptake of Ca²⁺ into the cells; 56mm -K⁺ increases both phases of Ca²⁺ uptake. The cells apparently lack a mechanism to inactivate this 56 mm -K⁺-induced increase-in Ca²⁺ permeability. Two inhibitors of K⁺ stimulated catecholamine secretion, diphenylhydantoin and verapamil, both inhibit K ⁺-stimulated Ca²⁺ uptake. These results provide a direct demonstration of the stimulus-coupled uptake of Ca²⁺ into chromaffin cells, and provide additional evidence for the correlation of Ca²⁺ uptake with catecholamine secretion by these cells.     

The role of calcium in transferrin and iron uptake by reticulocytes

The possible role of calcium in the uptake of transferrin and iron by rabbit reticulocytes was investigated by altering cellular calcium levels through the use of the chelating agents EDTA and $\text{ethyleneglycol-bis}\text{-(3-}\text{aminoethylether}\text{)-N,N′-}\text{tetraacetic}$ acid (EGTA) and the ionophores, A23187 and X537A. Incubation of reticuloyctes with EDTA or EGTA at 4°C had no effect on transferrin and iron uptake but incubation at 37°C resulted in an irreversible inhibition associated with decreased adsorption of transferrin to the cells and evidence of inactivation or loss of the transferrin receptors. Transferrin and iron uptake were also inhibited when the cells were incubated with A23187 or X537A. In the case of A23187 the action was primarily exerted on the temperature-sensitive stage of transferrin uptake and was associated with loss of cellular K⁺ and decrease in cell size. The effect was greater when Ca²⁺ was added to the incubation medium than its absence. X537A produced relatively greater inhibition of iron uptake than of transferrin uptake, associated with a reduction in cellular ATP concentratio. The action of X537A was unaffected by the presence of Ca²⁺ in the incubation medium.The results obtained with EDTA and EGTA indicate that cell membrane Ca²⁺ is required for the integrity or binding of transferrin receptors to the reticulocyte membrane. No evidence was obtained from the experiments with ionophores that an increase of cellular Ca²⁺ affects transferrin and iron uptake directly. The inhibition caused by A23187 was mainly due to a reduction in cell size resulting from increased membrane permeability to K⁺ and that caused by X537A appeared to result from an inhibition of energy metabolism and ATP production.     

Characteristics and regulation of active calcium transport in inside-out red cell membrane vesicles

Sealed, inside-out human red cell membrane vesicles, prepared by a modified method of Steck (Steck T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed.), Vol 2, pp. 245–281, Plenum Press, New York), accomplish an ATP and Mg²⁺-dependent uphill calcium uptake with a reproducible maximum rate of 12–15 nmol/mg vesicle protein per min under physiological conditions. This maximum rate is increased by about 60–70% in the presence of a heatstable cytoplasmic activator protein (calmodulin) obtained from red cells. Calcium efflux from inside-out vesicles is smaller than 0.01 nmol/mg vesicle protein per min at intravesicular calcium concentrations between 0.1 and 20.0 mM.In the presence of Mg²⁺, active calcium uptake is supported by ATP, ITP, or UTP, but not by ADP, AMP, or p-nitrophenyl phosphate. The optimum pH for the process is 7.4–7.6, and the activation energy is 19–20 kcal/mol, irrespective of the presence or absence of calmodulin. Calcium uptake in inside-out vesicles is unaffected by ouabain or oligomycin, but blocked by low concentrations of lanthanum, ruthenium red, quercetin and phloretin. K⁺ and Na⁺, when compared to choline⁺ or Li⁺, significantly increase active calcium uptake. This stimulation by K⁺ and Na⁺ is independent of that by calmodulin.Concentrated red cell cytoplasm activates calcium uptake at low soluble protein:membrane protein ratios, while a ‘deactivation’ of the transport occurs at high cytoplasm: membrane protein ratios. A heat-labile cytoplasmic protein fraction antagonizing calmodulin activation, can be separated by DEAE-Sephadex chromatography. Based on these findings the regulation of active calcium transport in human red cells is discussed.     

Interrelation between Ca2+ and K+ ions in the flocculation of two Brewers yeast strains

Summary Flocculation in two strains of Saccharomyces uvarum appears to be governed by the ratio of K⁺ and Ca²⁺ concentrations ([K⁺]/[Ca²⁺]). When the ratio is diminished either by an increased [Ca²⁺] or by a decreased [K⁺], the intensity and rate of flocculation are increased.K⁺ seems to be an antagonist of Ca²⁺ in the flocculation mechanism since enhancement of [K⁺] in the medium decreases uptake of Ca²⁺. Conversely an increase in [Ca²⁺] decreases the uptake of K⁺.     

Effects of calcium on potassium and water transport in human erythrocyte ghosts

Bulk water transport in reconstituted ghosts is statistically comparable to that in the parent red cells, and is unaffected by incorporation of Ca²⁺ over the range of 0.01 to 1 mM. Brief exposure of ghosts to p-chloromercuribenzene sulfonate results in a supression of osmotic water flow but leaves K⁺ permeability unchanged. Incorporation of p-chloromercuribenzene sulfonate provokes extremely rapid K⁺ loss which can be counteracted by simultaneous inclusion of Ca²⁺.Erythrocyte ghosts, when prepared with a small amount of Ca²⁺, demonstrate recovery of normal impermeability to choline, sucrose, Na⁺ and inulin and have an improved K⁺ retention over Ca²⁺-free preparations.The rate of passive transport of K⁺ from unwashed erythrocyte ghosts was measured during the initial few minutes of efflux. The initial rates vary in a bimodal fashion with the concentration of Ca²⁺ incorporated at the time of hemolysis. In low concentrations (0.01–0.1 mM), Ca²⁺ protects the K⁺ barrier while at higher concentrations (0.1–1.0 mM) it provokes a K⁺ leakage ranging from 7 to 50 times the normal rate of passive K⁺ loss. The Ca²⁺-induced K⁺ leak is thus a graded response rather than a discrete membrane transport state. The transition from a Ca²⁺-protected to a Ca²⁺-damaged membrane occurs upon an increase in Ca²⁺ concentration of less than 50 μmoles/l.     

A possible role of the phosphorylation of synaptic membrane proteins in the control of calcium ion permeability

Incubation of synaptosomes under conditions which result in complete phosphorylation of membrane bound accepter proteins does not affect the permeability to Na⁺ or K⁺ as measured by a spectrophotometric method. This technique was not, however, sensitive enough to determine permeability to Ca²⁺ which was thus estimated using ⁴⁵Ca²⁺. It was found that although phosphorylation did not affect the equilibrium binding of ⁴⁵Ca it did lower the rate of both Ca²⁺ uptake and efflux. The most likely interpretation of these results is that phosphorylation of proteins in the synaptic membrane lowers the permeability of the membrane to Ca²⁺. This could have a role in the regulation of synaptic transmission.     

Mechanism of Ca2+-dependent selective rapid K+-transport induced by propranolol in red cells

Summary Passive Ca²⁺ influx is gradually enhanced by 0.5 to 5mm propranolol in fresh and phosphate ester-depleted human red cells. In fresh cells the active Ca²⁺ efflux tends to counteract Ca²⁺ uptake. Membrane hyperpolarization, induced by the K⁺ transport that accompanies Ca²⁺ uptake, further enhances the rate of Ca² uptake. The dissociated, positively charged form of propranolol seems to be crucial in the increase of passive Ca²⁺ influx caused by the drug. The effect can be attributed to the release of structural Ca²⁺ from the membrane (lipids).The release of structural Ca²⁺ promotes the formation of the selectively K⁺-permeable membrane structure as well. The transitions of lipid structure responsible for the opening of the passive Ca²⁺ and K⁺ pathways, however, are not identical. The opening of the K⁺ pathways is prevented by certain highly lipid-soluble substances (chlorobutanol, heptanol, oligomycin, etc.), whereas the formation of the Ca²⁺ pathways is unaffected. Passive K⁺ transport is inhibited by high propranolol concentrations (more intensively at alkaline pH), whereas Ca²⁺ transport is promoted. A further difference between the passive K⁺ and Ca²⁺ pathways is that SH-proteins also seem to be involved in the formation of the K⁺ pathways, whereas they do not play a specific role in the opening of the passive Ca²⁺ channels. The additional Ca²⁺ binding that triggers the formation of the K⁺ pathways also seems to occur in the protein area of the inner membrane surface.     

H+- and K+-Dependence of Ca2+ Uptake in Lung Lamellar Bodies

Lung lamellar bodies maintain an acidic interior by an energy-dependent process. The acidic pH may affect the packaging of surfactant phospholipids, processing of surfactant proteins, or surfactant protein A-dependent lipid aggregation. The electron-probe microanalysis of lamellar body elemental composition has previously suggested that lamellar bodies contain high levels of calcium some of which may be in ionic form. In this study, we investigated the Ca²⁺ uptake characteristics in isolated lung lamellar bodies. The uptake of Ca²⁺ was measured by monitoring changes in the fluorescence of Fluo-3, a Ca²⁺ indicator dye. The uptake of Ca²⁺ in lamellar bodies was ATP-dependent and increased with increasing concentrations of Ca²⁺. At 100 nm Ca²⁺, the uptake was almost completely inhibited by bafilomycin A1, a selective inhibitor of vacuolar type H⁺-ATPase, or by NH₄Cl, which raises the lamellar body pH, suggesting that the pH gradient regulates the uptake. The uptake of Ca²⁺ increased as the Ca²⁺ concentration was increased, but the relative contribution of bafilomycin A1-sensitive uptake decreased. At 700 nm, it comprised only 20% of the total uptake. These results suggest the presence of additional mechanism(s) for uptake at higher Ca²⁺ concentrations. At 700 nm Ca²⁺, the rate and extent of uptake were lower in the absence of K⁺ than in the presence of K⁺. The inhibitors of Ca²⁺-activated K⁺-channels, tetraethylammonium, Penitrem A, and 4-aminopyridine, also inhibited the K⁺-dependent Ca²⁺ uptake at 700 nm Ca²⁺. Thus the uptake of Ca²⁺ in isolated lung lamellar bodies appears to be regulated by two mechanisms, (i) the H⁺-gradient and (ii) the K⁺ transport across the lamellar body membrane. We speculate that lamellar bodies accumulate Ca²⁺ and contribute to regulation of cytosolic Ca²⁺ in type II cells under resting and stimulated conditions. Received: 18 August 1999/Revised: 9 November 1999     

The role of calcium ions in the permeability changes produced by external ATP in transformed 3T3 cells

External ATP causes a rapid increase in passive permeability to nucleotides and phosphate esters in transformed cell lines, such as 3T6 mouse fibroblasts. However, untransformed lines, such as 3T3, do not show a similar sensitivity to external ATP. Ca²⁺ inhibits permeabilization, but only at concentrations approaching those of external ATP. In contrast, La³⁺ and Tb³⁺ inhibit ATP-dependent permeabilization at one-fifth the concentration of external ATP. Considering reports that lanthanides can substitute for calcium ion in many enzymatic reactions, often with a higher affinity, it would appear that Ca²⁺ plays a specific role in the maintenance of a passive membrane permeability barrier and in opposing the effects of external ATP.Other data suggest a regulatory role for the Ca²⁺-calmodulin complex in the permeabilization process. Trifluoperazine, chlorpromazine and W-7, compounds which inhibit cellular functions dependent on the Ca²⁺-calmodulin complex, are able to enhance the effect of external ATP. Thus, a dramatic stimulation of nucleotide permeability occurs with concentrations of external ATP and inhibitor that are ineffective when added alone. Calmodulin antagonists and low concentrations of external ATP increased membrane permeability to Na⁺ and K⁺ as was previously shown for permeabilization with ATP alone. Earlier studies have shown that energy inhibitors which reduce intracellular ATP levels greatly increase the sensitivity of transformed cells to external ATP. However, the Ca²⁺-calmodulin antagonists used in the present study exert their effects at concentrations which do not alter intracellular ATP levels.