Cell damage by cytolysin. Spontaneous recovery and reversible inhibition by divalent cations

Cytolysin-induced membrane damage (which requires low Ca2+) has been studied 1) in E by assay of hemolysis, 2) in Lettre cells by measurement of transmembrane potential, intracellular content of K+ and Na+, leakage of phosphoryl[3H]choline or 51Cr from [3H]choline-labeled or 51CrO4(2-)-labeled cells and leakage of lactate dehydrogenase, and 3) in phospholipid bilayers by measurement of electrical conductivity changes. In Lettre cells, damage is restricted and reversible: little lactate dehydrogenase leaks from cells that leak substantial amounts of Na+, K+, and phosphoryl[3H]choline; at low amounts of cytolysin, membrane potential and intracellular content of Na+ and K+ recover within minutes. In E and Lettre cells, membrane damage is inhibited by Zn2+, by high Ca2+, or by low pH. Inhibition is reversible: addition of EGTA to Zn2+-protected E or Lettre cells (incubated in the presence of cytolysin, low Ca2+ and Zn2+) initiates leakage; removal of Zn2+ (and cytolysin and Ca2+) by washing also initiates leakage; such leakage is again sensitive to Zn2+, high Ca2+, or H+. In phospholipid bilayers, channels induced by cytolysin (at low Ca2+) are partially closed by negative voltage; Ca2+, Zn2+, or H+ promote channel closure. Channels are re-opened (only partially in the case of Zn2+) by positive voltage. From all these results it is concluded that the action of cytolysin on membranes is similar to that of other pore-forming agents: damage does not necessarily lead to lysis of nucleated cells, and can be prevented by Ca2+, Zn2+, or H+.     

Protection of cells against membrane damage by haemolytic agents: divalent cations and protons act at the extracellular side of the plasma membrane

The protective effect of Ca2+, Zn2+ and H+ against membrane damage induced by different haemolytic agents has been studied by measuring monovalent cation leakage and haemolysis of erythrocytes, and phosphoryl[3H]choline and adenine nucleotide leakage from Lettre cells prelabelled with [3H]choline. The protective effect of Ca2+ and Zn2+ on erythrocytes damaged by Staphylococcus aureus alpha-toxin, Sendai virus or melittin is unaffected by the addition of A23187, even though this ionophore greatly increases the uptake of 45Ca2+ or 65Zn2+. The same result has been found for the protective effect of Zn2+ on Lettre cells damaged by S. aureus alpha-toxin, Sendai virus, melittin or Triton X-100. Leakage of phosphoryl[3H]choline from prelabelled Lettre cells is inhibited if extracellular pH is lowered; lowering the intracellular pH without affecting the extracellular pH, affords little protection. It is concluded that Ca2+, Zn2+ and H+ protect cells against membrane damage induced by haemolytic agents by an action at the extracellular side of the plasma membrane.     

Cadmium as a tool for studying calcium-dependent cation permeability of the human red blood cell membrane.

Three Ca(2+)-dependent procedures known to increase cation permeability of red blood cell membranes were tested with Cd2+ ions which equal Ca2+ ions both in their charge and the crystal radius, 1. Increase of non-selective permeability for monovalent cations by incubating the red cells in a Ca(2+)-free sucrose medium. Addition of Cd2+ to the suspension of leaky cells failed to restore the initial impermeability of the red cell membrane while a repairing effect of Ca2+ was evident both in the presence and absence of Cd2+. Thus, in low electrolyte medium, Cd2+ could neither mimic Ca2+, nor prevent the latter from interacting with membrane structures which control cation permeability. 2. Increase of the K(+)-selective permeability by propranolol plus Ca2+. Cd2+ added to a Ca(2+)-free Ringer type medium containing propranolol enhanced K+ permeability similar to that obtained with Ca2+. No changes of membrane permeability could be detected in the presence of 0.5 mmol/l Cd2+ in absence of propranolol. The Cd(2+)-stimulated K+ channels were different from those induced by Ca2+. They proved to be insensitive to quinine, exhibited a low K+/Na+ selectivity, and showed no tendency to self-inactivation. 3. Stimulation of K+ permeability by electron donors plus Ca2+. Substitution of Ca2+ by Cd2+ yielded results similar to those obtained with propranolol. The ability of Cd2+ to overtake the role of Ca2+ appears to depend on the system studied. It supplies information allowing to distinguish between the diverse Ca(2+)-dependent systems in cell membranes.     

Ca2+-activated K+ channels in human red cells. Comparison of single-channel currents with ion fluxes

Exposure of the inner surface of intact red cells or red cell ghosts to Ca2+ evokes unitary currents that can be measured in cell-attached and cell-free membrane patches. The currents are preferentially carried by K+ (PK/PNa 17) and show rectification. Increasing the Ca2+ concentration from 0 to 5 microM increases the probability of the open state of the channels parallel to the change of K+ permeability as observed in suspensions of red cell ghosts. Prolonged incubation of red cell ghosts in the absence of external K+ prevents the Ca2+ from increasing K+ permeability. Similarly, the probability to find Ca2+-activated unitary currents in membrane patches is drastically reduced. These observations suggest that the Ca2+-induced changes of K+ permeability observed in red cell suspensions are causally related to the appearance of the unitary K+ currents. Attempts to determine the number of K+ channels per cell were made by comparing fluxes measured in suspensions of red cells with the unitary currents in membrane patches as determined under comparable ionic conditions. At 100 mM KCl in the external medium, where no net movements of K+ occur, the time course of equilibration of 86Rb+ does not follow a single exponential. This indicates a heterogeneity of the response to Ca2+ of the cells in the population. The data are compatible with the assumption that 25% of the cells respond with Pk = 33.2 X 10(-14)cm3/s and 75% with Pk = 3.1 X 10(-14)cm3/s. At 100 mM external K+ the zero current permeability of a single channel is 6.1 X 10(-14)cm3/s (corresponding to a conductance of 22 pS).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of hypoxia due to iron-deficiency anemia on the physicochemical properties of biological membranes]

The lipid structure and Ca2+ permeability of red blood cell, hepatocyte and cardiomyocyte membranes were determined while investigating the effect of hypoxia caused by iron deficiency anemia upon the structural and functional state of biological membranes. The lipid composition and barrier characteristics of membranes change under conditions of hypoxia caused by experimental iron deficiency anemia. Quantitative changes in the cell membrane lipids may be considered as an important molecular mechanism of Ca2+ transport disorder in membranes, increase of Ca2+ permeability producing its surplus in the cells and subsequent metabolic homeostatic disturbances.     

Effects of calcium and A23187 on deformability and volume of human red blood cells

Human red blood cells treated in vitro with Ca2+ plus A23187 in low K+ medium exhibited significantly decreased cell volume and deformability, the latter determined by ektacytometry. These effects of Ca2+ plus A23187 were prevented in the presence of high K+ medium. Increased K+ permeability mediated by increased intracellular Ca2+ (Gardos effect) was apparently responsible for decreased cell volume and deformability in low K+ medium. Although it is commonly accepted that Ca2+ accumulation and/or ATP depletion per se cause decreased red blood cell deformability, the present results demonstrate that acutely induced changes in red blood cell volume as promoted by Ca2+ are a more important determinant of red blood cell deformability.     

Is the Ca2+-sensitive K+ channel under metabolic control in human red cells?

It is widely known that a rise in internal Ca2+ leads to an increased K+ permeability of human red blood cells [1,2,3]. Binding of Ca2+ to some membrane receptors is required for the opening of the K+ channel [4]. This requirement, however, seems to alter after "ageing" red cells in vitro in acid-citrate-dextrose solutions. Thus, the free Ca2+ concentration producing half-maximal effect on K+ permeability ([Ca2+]K+-50) of 4-weeks stored cells is approx. 2.10(-4) M (calculated from ref. 3 using 50% free Ca2+ according to Schatzmann [5]); nearly ten times lower than that reported for fresh cells [6]. This observation suggests the possibility that the K+ channel may become more sensitive to Ca2+ on cold storage. The experiments described below support this idea.     

Relationships between Ca2+ release, Ca2+ cycling, and Ca2+-mediated permeability changes in mitochondria

Ruthenium red and/or EGTA prevent cyclic uptake and release of Ca2+ in mitochondria. These compounds inhibit but do not prevent the swelling of liver mitochondria induced by Ca2+ plus t-butyl hydroperoxide or Ca2+ plus N-ethylmaleimide. Ruthenium red and/or EGTA have complex effects on the release rate of Ca2+ and other cations induced by t-butyl hydroperoxide or N-ethylmaleimide. To determine the relationship between permeability changes and Ca2+ release in the absence of Ca2+ cycling, a novel method of data collection and analysis is developed which allows the relative time courses of Ca2+ release and Mg2+ release or swelling to be accurately and quantitatively compared. This method eliminates errors in time course comparisons which arise from the aging of mitochondrial preparations and allows data from different preparations to be directly contrasted. Using the method, it is shown that permeability changes caused by Ca2+-releasing agents are not secondary effects arising from Ca2+ cycling between uptake and release carriers. In the absence of Ca2+-cycling inhibitors, Ca2+ release induced by t-butyl hydroperoxide or N-ethylmaleimide is, in part, carrier-mediated. In the presence of EGTA and ruthenium red, Ca2+ release induced by either agent is mediated solely by the permeability pathway. No differences are apparent in the solute selectivity of the inner membrane permeability defect induced by Ca2+ plus t-butyl hydroperoxide or Ca2+ plus N-ethylmaleimide. A novel type of Ca2+ release from energized liver mitochondria is reported. This release is induced by EGTA, occurs in the absence of other releasing agents or nonspecific permeability changes, and is rapid (greater than or equal to 50 nmol/min/mg protein).     

Divalent cation-sensitive pores formed by natural and synthetic melittin and by Triton X-100

Leakage of ions and low-molecular-weight metabolites from Lettre cells is induced by synthetic melittin, as effectively as by melittin isolated from bee venom; in each case leakage is inhibited by Ca2+, Zn2+ or H+. Inhibition of leakage by divalent cations is reversible in that Lettre cells incubated with melittin (or with Triton X-100) in the presence of inhibitory amounts of Zn2+, when freed of Zn2+ by EGTA or by centrifugation, begin to leak (in Zn2(+)-sensitive manner). Electrorotation of Lettre cells is altered by melittin, compatible with membrane permeabilization; melittin plus Zn2+ does not alter electrorotation until Zn2+ (and unbound melittin) are removed. Melittin or Triton X-100 added to calcein-loaded liposomes induces leakage of calcein; divalent cations inhibit. Energy transfer between liposome-associated melittin and 2-, 7- or 12-(9-anthroyloxy)stearate (AS) is maximal with 12-AS; addition of Zn2+ has little effect. Circular dichroism spectra of melittin plus liposomes are unaffected by Zn2+. These results show that the formation of divalent cation-sensitive pores is not dependent on the presence of endogenous membrane proteins and that the action of divalent cations is not by displacement of melittin (or Triton) from the lipid bilayer.     

The calmodulin-stimulated (Ca2+ + Mg2+)-ATPase in hemoglobin S erythrocyte membranes: effects of sickling and oxidative agents

A decrease in the reactivity of erythrocyte membrane (Ca2+ + Mg2+)-ATPase to calmodulin stimulation has been observed in aging red cells and in various types of hemolytic anemias, particularly in sickle red cell membranes. Unlike the aging process, the defect in the (Ca2+ + Mg2+)-ATPase from SS red blood cells is not secondary to a decrease in calmodulin activity and is already present in the least dense SS red blood cells separated on a discontinuous density gradient. Deoxygenated AS red cells were forced to sickle by lowering the pH, raising the osmolarity of the buffer (sickling pulse). Under these conditions an inhibition of the calmodulin-stimulated enzyme was observed only if several cycles of oxygenation/deoxygenation were applied. No alteration of the enzyme could be detected after submitting AS red blood cells to other conditions or in AA red blood cells submitted to the same treatments. This suggests that oxidative processes are involved in the alterations of the (Ca2+ + Mg2+)-ATPase activity. Treatment of membranes from AA erythrocytes by thiol group reagents and malondialdehyde, a by-product of auto-oxidation of membrane unsaturated lipids and a cross-linking agent of cytoskeletal proteins, led to a partial inhibition of the calmodulin-stimulated (Ca2+ + Mg2+)-ATPase. We postulate that the hyperproduction of free radicals described in the SS red blood cells and involved in the destabilization of the membrane may be also responsible for the (Ca2+ + Mg2+)-ATPase failure.     

The role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes.

To elucidate the role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes, we examined the interaction of erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion of human erythrocytes was monitored by light microscopy as well as spectrophotometrically by the octadecylrhodamine dequenching assay. Phospholipid translocation and distribution between the inner and the outer leaflet of intact red blood cells were determined with spin-labeled phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Significant fusion of lipid-asymmetric red blood cells where PS and PE are predominantly oriented to the inner leaflet was only observed at Ca2+ concentrations greater than or equal to 10 mM (in the presence of 10 mM phosphate buffer) while fusion of lipid-symmetric erythrocyte membranes was established at greater than or equal to 1.5 mM Ca2+. The Ca2+ threshold of fusion of lipid-asymmetric red blood cells was significantly reduced (i) after exposure of PS to the outer layer but not after redistribution of PE alone, and (ii) upon incorporation of spin-labeled PS into the outer leaflet of red blood cells. Spin-labeled PE or PC did not affect fusion, suggesting that the serine headgroup is an important factor in calcium-phosphate-induced fusion.     

The use of ionophores of rapid loading of human red cells with radioactive cations for cation-pump studies.

Techniques are described for the rapid loading of intact human red cells with radioactive isotopes of alkali cations or Ca2+ by using ionophorous compounds (nigericin, gramicidin D and A 23187). Loading was rapid and efficient if the membrane potential of the cells was rendered more negative inside. After cation loading the ionophores could be bound to albumin and removed by repeated washings. The ATP and 2,3-DPG contents of the cells were practically unaltered by this treatment. Passive membrane permeability to Na+ and Ca2+ returned to normal. Loaded erythrocytes pumped out Na+ in a ouabain-sensitive and Ca2+ in a lanthanum-sensitive way. Ca2+ -loaded red cells were microspherocytes and exhibited a rapid K+ -efflux. Parallel with the extrusion of Ca2+ cells regained their biconcave shape and normal passive permeability to K+.     

Sendai virus membrane fusion: time course and effect of temperature, pH, calcium, and receptor concentration

The conditions that optimize Sendai virus membrane fusion with liposomes have been studied. No fusion occurs in the absence of ganglioside receptors. Maximum fusion occurs when the molar ratio of ganglioside GD1a to phospholipid is 0.02 or greater. The amount of fusion at 37 degrees C increases with time up to at least 6.5 h. The rate of fusion increases from the lowest temperature tested, 10 degrees C, to 40 degrees C. Above 43 degrees C the amount of fusion decreases because of thermal inactivation of the viral proteins. There is a broad pH maximum between pH 7.5 and pH 9.0. At both ends of the pH range the amount of fusion increases and exceeds that found in the physiologic pH range. Neither ethylenediaminetetraacetic acid nor Ca2+ changes the amount of membrane fusion. The optimal conditions for membrane fusion of Sendai virus membranes with liposomes are the same as the optimal conditions for fusion with host cells and with red blood cells. Since the liposomes contain no proteins, the optimal conditions for Sendai virus membrane fusion must be determined by the viral proteins and be mostly independent of the nature or presence of the host proteins.     

Increased permeability of mitochondria during Ca2+ release induced by t-butyl hydroperoxide or oxalacetate. the effect of ruthenium red

Ca2+ release from mitochondria induced by oxalacetate or t-butyl hydroperoxide is accompanied by loss of endogenous Mg2+ and K+, swelling, loss of membrane potential, and other alterations which indicate that Ca2+ release is a result of increased inner membrane permeability. When ruthenium red is added after Ca2+ uptake, but before the releasing agent, the extent of Ca2+ release is diminished as is the extent of Mg2+ and K+ depletion and the extent of swelling. Under these conditions, the membrane potential appears to remain at a high value. When Ca2+ release is induced by oxalacetate or t-butyl hydroperoxide and ruthenium red is added subsequently, an apparent regeneration of membrane potential is observed providing that the associated swelling and Mg2+ loss had not been completed at the time ruthenium red was added. Under these conditions subsequent swelling and Mg2+ loss are inhibited.l Ultrastructural observations show the mitochondria become permeable in response to Ca2+ plus oxalacetate or Ca2+ plus t-butyl hydroperoxide in a heterogeneous manner. Conditions which appear to separate Ca2+ release from a decline in membrane potential or to produce an apparent recovery of membrane potential following partial collapse are shown to prevent a subpopulation of the mitochondria from becoming permeable. It is shown that membrane potential probes will not indicate a decline in potential or the presence of a permeable fraction under these conditions. It is concluded that the presence of Ca2+ accumulation inhibitors does not separate Ca2+ release from the development of increased inner membrane permeability.     

Permeability changes resulting from virus-cell fusion: temperature-dependence of the contributing processes

Summary A new assay for membrane fusion, using the fluorescent probe pyrene-sulphonyl-phosphatidyl ethanolamine, has been developed. Fusion between the envelope of Sendai virus and human erythrocytes or Lettre cells has a Q₁₀ of 4 at 37° C, increasing to 7 at 7 ° C; there is no lag to onset of fusion. Viral neuraminidase has a Q₁₀ of 2.3 between 37° C and 4° C. Its action limits the extent of fusion by causing the elution of virus; this effect is particularly marked at low temperature because of the difference in Q₁₀ for fusion and neuraminidase. The temperature-dependence of the initiation of permeability changes following the removal of inhibitory amounts of Ca²⁺ is 2; thus membrane fusion is the principal temperature-sensitive step during the permeabilization of cells by Sendai virus. A recovery process, by which cells become insensitive to the removal of Ca²⁺ and which therefore limits the extent of permeabilization, has a Q₁₀ of 7.4 between 37° C and 21° C. It is concluded that the lag to onset of permeability changes is not due to a lag in virus-cell membrane fusion, but to the gradual acquisition of a threshold level of membrane damage; the extent of permeabilization depends on the rate of fusion relative to the rates of neuraminidase and recovery.     

The plasma membrane calcium pump: regulation by a soluble Ca2+ binding protein

The Ca2+ pump of the plasma membrane of human red blood cells is associated with the activity of a (Ca2+ + Mg2+)-ATPase. Both the ATPase and the pump are stimulated above basal activities by calmodulin, an ubiquitous Ca2+-binding protein. Calmodulin isolated from human red blood cells was shown to be equipotent and equieffective with that isolated from beef brain. Half-maximal activation of ATPase (isolated red blood cell membranes, 37 C) and transport (inside-out red blood cell membrane vesicles, 25 C) were obtained with 2.5 and 4.4 nM calmodulin, respectively. Ca2+ dependence of Ca2+ transport was measured in the absence and in the presence of 50 nM calmodulin. At all Ca2+ concentrations above 2 X 10(-7) M Ca2+, the rate of transport was greater in the presence of calmodulin. The results implicate calmodulin in the regulation of the plasma membrane Ca2+ pump, but the mechanism(s) remain to be elucidated.     

Assessment of lipid peroxidation and plasma membrane permeability for Ca2+ in the red blood cells of cattle after the long term grazing on radioactively contaminated territory

Ten years after the Chernobyl accident a physiological condition of cows was examined on radioactivy contaminated territory of the Novozibkov district of the Bryansk region. The long grazing of cattle on radioactivly contaminated territory revealed the increase in permeability of plasmatic membrane of the red blood cells to Ca2+ and the activation of process of lipid peroxidation. The sensitivity of the red blood cells of cows to incubations in hypertonic conditions was demonstrated.     

Lead-induced activation and inhibition of potassium-selective channels in the human red blood cell

The selective increase of net K+ permeability in human red cells brought about by either Ca2+ or lead was studied using a light scattering technique to measure net K+ fluxes in cell suspensions and the patch-clamp technique to study K+ transport in individual K+-selective channels of the red cell membrane. Using ultrapure solutions it was demonstrated that the effect of lead is neither the indirect consequence of a lead-induced increase of the accessibility of the receptor sites of the K+-selective channels to traces of Ca2+ that are present as contamination in analytical grade reagents nor to the release of Ca2+ from intracellular Ca2+ stores. It is further shown that in cell-free membrane patches low concentrations of lead (10 microM) in Suprapur solutions evoke the same single-channel events as added Ca2+ and that this activity can be inhibited by high concentrations of lead (100 microM), similar to the net KCl efflux measured by means of the light scattering technique. It is concluded, therefore, that both Ca2+ and lead independently activate the same K+-selective channels in the red cell membrane.     

Increased potassium permeability in erythrocytes from patients with hyperparathyroidism

Parathyroid hormone (PTH) has been shown to modify Ca2+ and Na+ transport in several epithelia. The molecular mechanisms of these effects are poorly understood. We investigated here whether PTH may modify Na+ and K+ transport across the human red blood cell membrane in vitro and ex vivo. Fourteen patients with severe primary or secondary hyperparathyroidism and hypercalcemia were studied before and 5-7 days after surgical parathyroidectomy. Erythrocyte ouabain-sensitive as well as furosemide-sensitive Na+ efflux rates of the patients were comparable to that of healthy volunteers and remained unchanged after parathyroidectomy. Moreover, erythrocyte Na+ fluxes of control subjects remained unchanged when red blood cells were incubated in the presence of 1.0 IU/ml of bovine PTH (1-85). However, erythrocytes from hyperparathyroid patients showed a significant increase in passive K+ permeability when compared to that of healthy controls (p less than 0.05). This abnormality could be corrected in vivo after parathyroidectomy and in vitro using quinine, respectively. It is concluded that hyperparathyroidism induces a moderate increase in Ca2+ dependent K+ permeability of erythrocytes ("Gardos effect") which is reversible after parathyroidectomy.     

Parathyroid hormone secretion in the absence of extracellular free Ca2+ and transmembrane Ca2+ influx

The involvement of extracellular Ca2+ and Ca2+ influx across the plasma membrane in parathyroid hormone (PTH) secretion was investigated in vitro using a new preparation of bovine parathyroid cells. Incubation of these cells in the presence of 25 microM or 2.5 microM free ambient Ca2+ induced a maximal rate of PTH secretion. Low free Ca2+ secretion is not associated with changes in membrane permeability, requires metabolic energy, and is reversible. The Ca2+ channel blocker D600 had no effect on either 45Ca-influx or PTH secretion in these cells. These results, showing that extracellular Ca2+ and Ca2+ influx across the plasma membrane are not required for PTH secretion by parathyroid cells, emphasize the differences in the cellular mechanisms underlying the secretion of PTH vs that of other secretory cells.