Changes induced in the permeability barrier of the yeast plasma membrane by cupric ion.

A specific effect of Cu2+ eliciting selective changes in the permeability of intact Saccharomyces cerevisiae cells is described. When 100 microM CuCl2 was added to a cell suspension in a buffer of low ionic strength, the permeability barrier of the plasma membranes of the cells was lost within 2 min at 25 degrees C. The release of amino acids was partial, and the composition of the amino acids released was different from that of those retained in the cells. Mostly glutamate was released, but arginine was mainly retained in the cells. Cellular K+ was released rapidly after CuCl2 addition, but 30% of the total K+ was retained in the cells. These and other observations suggested that Cu2+ caused selective lesions of the permeability barrier of the plasma membrane but did not affect the permeability of the vacuolar membrane. These selective changes were not induced by the other divalent cations tested. A novel and simple method for differential extraction of vacuolar and cytosolic amino acid pools by Cu2+ treatment was established. When Ca2+ was added to Cu2+-treated cells, a large amount of Ca2+ was sequestered into vacuoles, with formation of an inclusion of a Ca2+-polyphosphate complex in the vacuoles. Cu2+-treated cells also showed enhanced uptake of basic amino acids and S-adenosylmethionine. The transport of these substrates showed saturable kinetics with low affinities, reflecting the vacuolar transport process in situ. With Cu2+ treatment, selective leakage of K+ from the cytosolic compartment appears to create a large concentration gradient of K+ across the vacuolar membrane and generates an inside-negative membrane potential, which may provide a driving force of uptake of positively charged substances into vacuoles. Cu2+ treatment provides a useful in situ method for investigating the mechanisms of differential solute pool formation and specific transport phenomena across the vacuolar membrane.     

Potassium release from erythrocytes of different age rats in 9 simulated lactic acidosis

The methods are described for determination of potassium content in erythrocytes by means of a K+ selective electrode, for evaluation of changes in potassium permeability through their membranes and for detection of the Na+, K+-ATPase contribution to spontaneous and induced cation release from cells. Preincubation of erythrocytes with 5 mM lactate has no effect on the potassium release rate and content in cells of young and adult rats. Under the effect of 25 mM lactate the rate of K+ release from erythrocytes of adult animals lowers and sensitivity to ouabaine of this process stimulated by valinomycin increases. The latter effect is less pronounced in erythrocytes of young rats and is accompanied by a rise in potassium total content in cells.     

A kinetics study of pig erythrocyte hemolysis induced by polyene antibiotics

The kinetics of the hemolysis induced by filipin is of the damage type, indicating the formation of large nonselective perforations of erythrocyte membranes. The process is relatively independent of the ionic composition of the incubation medium, and the differences between the hemolysis induced by filipin in pig and human erythrocytes are not significant. In a sucrose medium, filipin-induced hemolysis is inhibited in humans, whereas it is stimulated in pig erythrocytes. It is suggested that low ionic strength is the reason for the different modifications of complexation of filipin in pig and human erythrocyte membranes in a sucrose medium. The kinetics of the hemolysis induced in pig erythrocytes by amphotericin B and nystatin is of the permeability type, indicating the formation of selective channels in erythrocyte membranes and colloid osmotic hemolysis. The rate of the hemolysis, which is high in a KCl medium, is decreased in all the other media tested (CaCl2, MgCl2, potassium phosphate buffer, K2SO4, sucrose), although there are no changes in the kinetics of hemolysis. The results are interpreted as the formation of highly selective channels at a low concentration of the antibiotics. At increasing concentrations, channels of decreasing selectivity occur. The resistances of pig erythrocytes to amphotericin B and nystatin are lower than those of human erythrocytes.     

Effects of temperature on water diffusion in human erythrocytes and ghosts--nuclear magnetic resonance studies

The temperature-dependence of water diffusion across human erythrocyte membrane was studied on isolated erythrocytes and resealed ghosts by a doping nuclear magnetic resonance technique. The conclusions are the following: (1) The storage of suspended erythrocytes at 2 degrees C up to 24 h or at 37 degrees C for 30 min did not change the water exchange time significantly, even if Mn2+ was present in the medium. This indicates that no significant penetration of Mn2+ is taking place under such conditions. (2) In case of cells previously incubated at 37 degrees C for longer than 30 min with concentrations of p-chloromercuribenzene sulfonate (PCMBS) greater than 0.5 mM, the water-exchange time gradually decreased if the cells were stored in the presence of Mn2+ for more than 10 min at 37 degrees C. (3) When the Arrhenius plot of the water-exchange time was calculated on the basis of measurements performed in such a way as to avoid a prolonged exposure of erythrocytes to Mn2+ no discontinuity occurred, regardless of the treatment with PCMBS. (4) No significant differences between erythrocytes and resealed ghosts regarding their permeability and the activation energy of water diffusion (Ea,d) were noticed. The mean value of Ea,d obtained on erythrocytes from 35 donors was 24.5 kJ/mol. (5) The value of Ea,d increased after treatment with PCMBS, in parallel with the percentage inhibition of water diffusion. A mean value of 41.3 kJ/mol was obtained for Ea,d of erythrocytes incubated with 1 mM PCMBS for 60 min at 37 degrees C and 28.3 kJ/mol for ghosts incubated with 0.1 mM PCMBS for 15 min, the values of inhibition being 46% and 21% respectively.     

Interaction of dichlone with human erythrocytes. I. Changes in cell permeability

The uptake of the fungicide dichlone (2,3-dichloro-1,4-naphthoquinone) by human erythrocytes was extremely rapid, reaching a maximum within 5 min of treatment. Most of the dichlone taken up was present in the interior of the cell; only a small fraction of the pesticide (less than 5%) was bound to the cell membrane. Dichlone (3 · 10^?5M-10^?4M) induced a rapid loss of intracellular potassium from the erythrocytes; the leakage of K⁺ varied with the fungicide concentration as well as with cell concentration. Pretreatment of the cells with glutathione was able to reduce potassium loss. Cells exposed to dichlone showed increased osmotic fragility. Dichlone also inhibited Na⁺-K⁺ ATPase, which is associated with active ion transport. However, the leakage of potassium in dichlone-treated cells does not appear to be related to the interference with active ion transport. An extensive loss of potassium within a relatively short time after treatment suggests that dichlone produces its effect by increasing passive cation permeability, probably as a result of direct action on the membrane structure. Dichlone was able to induce hemolysis, but only at concentrations higher than those which resulted in K⁺ loss. The loss of hemoglobin appeared to be mainly due to osmotic swelling of the treated cells. Exposure of red cells to dichlone also resulted in a rapid and extensive formation of methemoglobin as well as a denaturation of hemoglobin. Thus, dichlone not only may be capable of lowering the capacity of erythrocytes to transport oxygen but also alters their permeability.     

Effects of divalent cation ionophore A23187 on potassium permeability of rat erythrocytes.

A23187 transports calcium rapidly into rat erythrocytes, apparently by an electroneutral exchange for intracellular magnesium and protons. When red cells are incubated in the absence of any added divalent cations, A23187 transports internal magnesium out of the cells, in exchange for extracellular protons. Magnesium uptake into erythrocytes is produced by A23187, providing the extracellular concentration of this cation exceeds intracellular levels, and the ionophore also transports strontium, but not barium, into red cells. A23187 produces a rapid and extensive loss of intracellular potassium from erythrocytes during uptake of calcium or strontium, but not magnesium. When red cells are incubated in the absence of any exogenous divalent cations, A23187 still produces a potassium efflux and this is inhibited completely by small amounts of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and restored by the addition of calcium in excess of the chelator. Although EDTA enhances the extent of magnesium release from erythrocytes incubated with A23187, it prevents the potassium efflux. Dipyridamole and 4-acetamid-4'-isothiocyano-stilbene-2,5'-disulfonic acid, which decrease chloride premeability of erythrocytes, inhibit the A23187-induced potassium loss from red cells. Rutamycin, peliomycin, venturicidin, and A23668B also inhibit potassium efflux from intact cells incubated with A23187, but this effect is not correlated with their abilities to inhibit various ATPases in red cell membrane preparations. It is concluded that A23187 does not transport potassium directly across the erythrocyte plasma membrane, but permits small amounts of endogenous calcium to interact with some membrane component to enhance potassium permeability of the cell.     

Dramatic magnesium efflux induced by high potassium in rat thymocytes

When incubated in 150 mM KCl, rat thymocytes exhibited a very important magnesium efflux (11.4 +/- 0.7 mmoles/liter cells/20 min, n = 29), about 90 times higher than the physiological magnesium efflux catalyzed by the Na-Mg exchanger (0.126 +/- 0.093 mmoles/liter cells/20 min). Cells remained viable (trypan blue test) and membrane integrity was shown by the absence of an increase in sodium permeability. K(+)-induced magnesium efflux exhibited the following properties: (i) it required the presence of external chloride; (ii) it was fully blocked by DIOA, a selective KCl-cotransporter inhibitor (IC(50) = 35 microm); and (iii) it was associated to a progressive increase in cell volume via the DIOA-sensitive K-Cl cotransporter. Such cell swelling seems to play a causal role, because (i) hypertonic media (+400 mM sucrose) abolished K(+)-induced magnesium efflux and (ii) hypotonic Ringer media (205 mOsm) increased both cell volume and magnesium efflux (from a basal value of 0.35 +/- 0.03 mmoles/liter cells/20 min up to 1.44 +/- 0.24 mmoles/liter cells/20 min), even in the presence of DIOA. In conclusion, high potassium induced a dramatic release of intracellular magnesium from rat thymocytes. Such a phenomenon was, at least in part, caused by cell swelling via the DIOA-sensitive K-Cl cotransporter. The nature of the magnesium transport mechanism and its role in the transduction signal of K-Cl cotransporter activation by cell swelling deserve further investigation.     

Purification and biochemical characterization of tellurite-reducing activities from Thermus thermophilus HB8.

Cell-free extracts of Thermus thermophilus HB8 catalyze the in vitro, NADH-dependent reduction of potassium tellurite (K2TeO3). Three different protein fractions with tellurite-reducing activities were identified. Two exhibited high molecular weight and were composed of at least two different polypeptides. The protein in the third fraction was purified to homogeneity and had a single polypeptide chain of 53 to 54 kilodaltons, with an isoelectric point of 8.1. Each enzyme was thermostable, the temperature optimum was 75 degrees C, and 30 mM NaCl, 1.5 M urea, or 0.004% sodium dodecyl sulfate caused 50% inhibition of the enzymes. However, 2% Triton X-100 did not have an inhibitory effect. The enzymes were also able to catalyze the reduction of sodium selenite and sodium sulfite in vitro. NADH was replaceable by NADPH. Divalent cations, such as Ca2+ and Ba2+, had no effect on the activity, while similar concentrations of Zn2+, Ni2+, and Cu2+ abolished the activity. This reductase activity could enable these bacteria both to reduce K2TeO3 and to increase their tolerance toward this salt.     

Factors of the shape change of human erythrocytes induced with lidocaine

We studied the molecular mechanism of the shape change of erythrocytes with a local anesthetic, lidocaine. The shape of human erythrocytes changed from discocytes to stomatocytes in the presence of lidocaine when ATP was present. But, the shape of resealed cells which were prepared with 10 mM Tris-HCl buffer (pH 7.4) containing 2 mM ATP-MgCl2 and various substances was not changed from discocytes to stomatocytes with lidocaine. When intact cells and resealed cells which were prepared with various concentrations of Tris-HCl buffer (pH 7.4) were incubated with various concentrations of lidocaine and their membrane proteins were analyzed by SDS-PAGE, the densities of bands 62K, 28K and 22K depended on lidocaine concentration: in particular, that of band 28K changed remarkably. These membranous 62K-, 28K- and 22K-proteins agreed with cytoplasmic 62K-, 28K- and 22K-proteins in molecular weight. We propose that not only ATP but also the 62K-, 28K- and 22K-proteins in the cytoplasm are concerned with the shape change of human erythrocytes induced with lidocaine.     

Inhibition by isoproterenol of the passive potassium efflux from pigeon erythrocytes

Pigeon erythrocytes were carefully washed in an isotonic neutral buffer, devoid of potassium, and the rate of passive unidirectional efflux of potassium from the cells into a K+-free medium was measured after 20 min, at 40 degrees C. Isoproterenol inhibits K+-efflux by 35-45%, at a cell concentration of 1%; the isoproterenol effect is mediated by beta-adrenergic receptors. Cyclic AMP mimics the effect of isoproterenol, but at 4-5 orders of magnitude higher concentrations. Cyclic AMP increases 20-fold the phosphorylation of purified cell membranes by [gamma 32P]ATP.     

Activation of potassium channels in erythrocytes of marine teleost Scorpaena porcus

To assess the possibility of stimulating Ca2+-activated K+ channels, marine fish erythrocytes were incubated at 20-22 degrees C in saline containing a Ca2+-ATPase inhibitor (orthovanadate), a Ca2+ ionophore (A23187), propranolol or Pb2+. Incubation of the cells for up to 2 h under control conditions or in the presence of 5 mM NH4VO3 and 1 mM Ca2+ did not affect the intracellular K+ and Na+ concentrations. About 50% cellular K+ was lost from erythrocytes incubated in the presence of 0.01 mM A23187, 1 mM EGTA and 0.4-1.0 mM Ca2+. There was a significant loss of cellular K+ after the addition of 0.05-0.2 mM propranolol to the incubation medium. The stimulatory effect of propranolol on the K+ efflux was independent of external Ca2+. Blockers of Ca2+ transport, verapamil and Co2+, caused only a small decrease in the K+ loss induced by propranolol. The treatment of erythrocytes with 1-2 microM Pb2+ led to a minor K+ loss, but at a Pb2+ concentration of 20-50 microM, about 70% cellular K+ was lost. The K+ efflux induced by propranolol or Pb2+ was completely blocked by 1 mM quinine. The induced K+ loss from the erythrocytes was accompanied by a slight increase in the intracellular Na+ concentration. These data indicate the possibility of inducing Ca2+- and Pb2+-activated potassium channels in erythrocytes of S. porcus. A distinctive feature of the cells is a high sensitivity to propranolol, which activates K+ channels in the absence of external Ca2+.     

Extracellular ATP increases cation fluxes in human erythrocytes by activation of the P2X7 receptor

Canine erythrocytes are known to undergo a reversible increase in cation permeability when incubated with extracellular ATP. We have examined the expression and function of P2X receptors on human erythrocytes using confocal microscopy and a panel of anti-P2X(1-7) antibodies and have measured monovalent cation fluxes in the presence of various nucleotide agonists. Human erythrocytes expressed P2X7 receptors on all cells examined from eight of eight subjects, as well as P2X2 at a far lower staining intensity in six of eight subjects. ATP stimulated the efflux of 86Rb+ (K+) from human erythrocytes in a dose-dependent fashion with an EC50 of approximately 95 microM. Other nucleotides also induced an efflux of 86Rb+ from erythrocytes with an order of agonist potency of 2'- and 3'-O(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP (2MeSATP) > adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), whereas ADP or UTP had no effect. ATP-induced efflux of 86Rb+ from erythrocytes was inhibited by extracellular Na+ and oxidized ATP, as well as by KN-62, an antagonist specific for the human P2X7 receptor. When erythrocytes were incubated in isotonic KCl medium, the addition of ATP stimulated an 86Rb+ influx approximately equal in magnitude to ATP-stimulated 86Rb+ efflux from the same cells. BzATP also stimulated the influx of 22Na+ into erythrocytes incubated in isotonic NaCl medium. Both ATP-induced efflux and influx of 86Rb+ and 22Na+ were impaired in erythrocytes from subjects who had inherited loss-of-function polymorphisms in the P2X7 receptor. These results suggest that the reversible permeabilization of erythrocytes by extracellular ATP is mediated by the P2X7 receptor.     

Cr(VI) Induces Lipid Peroxidation, Protein Oxidation and Alters the Activities of Antioxidant Enzymes in Human Erythrocytes

The effect of potassium dichromate (K(2)Cr(2)O(7)), a hexavalent chromium compound, on human erythrocytes was studied under in vitro conditions. Incubation of erythrocytes with different concentrations of K(2)Cr(2)O(7) resulted in marked hemolysis in a concentration-dependent manner. K(2)Cr(2)O(7) treatment also caused significant increase in protein oxidation, lipid peroxidation and decrease in total sulfhydryl content, indicating that it causes oxidative stress in human erythrocytes. However, there was no concomitant nitrosative stress as the nitric oxide levels in hemolysates from K(2)Cr(2)O(7)-treated erythrocytes were lower than in control. Exposure of erythrocytes to K(2)Cr(2)O(7) decreased the activities of catalase, glutathione peroxidase, thioredoxin reductase, glucose-6-phosphate dehydrogenase, and glutathione reductase, whereas the activities of Cu-Zn superoxide dismutase and glutathione S-transferase were increased. These results show that K(2)Cr(2)O(7) induces oxidative stress and alters the antioxidant defense mechanism of human erythrocytes.     

Selective inhibition by bis(2-chloroethyl)methylamine (nitrogen mustard) of the Na+/K+/Cl- cotransporter of murine L1210 leukemia cells

Incubation of L1210 murine leukemia cells in vitro with 10 microM of the bifunctional alkylating agent bis(2-chloroethyl)methylamine (nitrogen mustard, HN2) for 10 min brought about a fall of more than 99.9% in their ability to form colonies when the cells were suspended in 0.5% nutrient agar. Incubation with HN2 also inhibited the influx of the potassium congener 86Rb+ to exponentially proliferating L1210 cells in a concentration-dependent manner. This inhibition was specific and was accounted for by a reduction of a diuretic-sensitive component of 86Rb+ influx, identified in the preceding paper (Wilcock, C. and Hickman, J.A. (1988) Biochim. Biophys. Acta 946, 359-367) as being mediated by a Na+/K+/Cl- cotransporter. Inhibition by 10 microM HN2 was complete after a 3-h incubation. There was no inhibition at this time of the ouabain-sensitive component of 86Rb+ influx, mediated by Na+/K+-ATPase. After 3 h of incubation with 10 microM HN2 there was also no change in the membrane potential of the treated cells as measured by the distribution of the [3H]TPMP+, no decrease in cellular ATP concentration and no change in intracellular pH, and the ability of the cells to exclude the vital dye Trypan blue was not significantly different from control values. These effects of HN2, therefore, appeared to follow lethal damage, but precede cell death. In the stationary phase of L1210 cell growth, the component of HN2 and diuretic-sensitive K+ influx to L1210 cells was reduced, whilst the component constituting the HN2-insensitive ouabain-sensitive sodium pump was increased. The monofunctional alkylating agent MeHN1 (2-chloroethyldimethylamine) which cannot cross-link cellular targets and has no antitumor activity, did not inhibit 86Rb+ influx to L1210 cells when incubated at equimolar or equitoxic concentrations to HN2. Intracellular potassium concentration was maintained close to control values of 138 +/- 10 mM in HN2-treated cells because of an approx. 35% fall in cell volume. The results suggest that the Na+/K+/Cl- cotransporter is a selectively inhibitable target for HN2, and the lesion is discussed with reference to the cytotoxic effects of this agent.     

Transbilayer movement of cholesterol in the human erythrocyte membrane

The rate of transbilayer movement of cholesterol was measured in intact human erythrocytes. Suspended erythrocytes were incubated briefly with [3H]cholesterol in ethanol at 4 degrees C, or with liposomes containing [3H]cholesterol over 6 hr at 4 degrees C to incorporate the tracer into the outer leaflet of erythrocyte plasma membranes. The erythrocytes were then incubated at 37 degrees C to allow diffusion of cholesterol across the membrane bilayer. Cells were treated briefly with cholesterol oxidase to convert a portion of the outer leaflet cholesterol to cholestenone, and the specific radioactivity of cholestenone was determined over the time of tracer equilibration. The decrease in specific radioactivity of cholestenone reflected transbilayer movement of [3H]cholesterol. The transbilayer movement of cholesterol had a mean half-time of 50 min at 37 degrees C in cells labeled with [3H]cholesterol in ethanol, and 130 min at 37 degrees C in cells labeled with [3H]cholesterol exchanged from liposomes. The cells were shown, by the absence of hemolysis, to remain intact throughout the assay. The presence of 1 mM Mg2+ in the assay buffer was essential to prevent hemolysis of cells treated with cholesterol oxidase perturbed the cells, resulting in an accelerated rate of apparent transbilayer movement. Our data are also consistent with an asymmetric distribution of cholesterol in erythrocyte membranes, with the majority of cholesterol in the inner leaflet.     

Peroxide-induced membrane damage in human erythrocytes

Erythrocytes exposed to H2O2 or t-butyl hydroperoxide (tBHP) exhibited lipid peroxidation and increased passive cation permeability. In the case of tBHP a virtually complete inhibition of both processes was caused by butylated hydroxytoluene (BHT), whereas pretreatment of the cells with CO increased both lipid peroxidation and K+ leakage. In the experiments with H2O2, on the other hand, both BHT and CO strongly inhibited lipid peroxidation, without affecting the increased passive cation permeability. These observations indicate different mechanisms of oxidative damage, induced by H2O2 and tBHP, respectively. The SH-reagent diamide strongly inhibited H2O2-induced K+ leakage, indicating the involvement of SH oxidation in this process. With tBHP, on the contrary, K+ leakage was not significantly influenced by diamide. Thiourea inhibited tBHP-induced K+ leakage, without affecting lipid peroxidation. Together with other experimental evidence this contradicts a rigorous interdependence of tBHP-induced lipid peroxidation and K+ leakage.     

Permeabilization of rat hepatocytes with Staphylococcus aureus alpha- toxin

Pathogenic staphylococci secrete a number of exotoxins, including alpha-toxin. alpha-Toxin induces lysis of erythrocytes and liposomes when its 3S protein monomers associate with the lipid bilayer and form a hexomeric transmembrane channel 3 nm in diameter. We have used alpha-toxin to render rat hepatocytes 93-100% permeable to trypan blue with a lactate dehydrogenase leakage less than or equal to 22%. Treatment conditions included incubation for 5-10 min at 37 degrees C and pH 7.0 with an alpha-toxin concentration of 4-35 human hemolytic U/ml and a cell concentration of 13-21 mg dry wt/ml. Scanning electron microscopy revealed signs of swelling in the treated hepatocytes, but there were no large lesions or gross damage to the cell surface. Transmission electron microscopy indicated that the nucleus, mitochondria, and cytoplasm were similar in control and treated cells and both had large regions of well-defined lamellar rough endoplasmic reticulum. Comparisons of the mannose-6-phosphatase and glucose-6-phosphatase activities demonstrated that 5-10 U/ml alpha-toxin rendered cells freely permeable to glucose-6-phosphate, while substantially preserving the selective permeability of the membranes of the endoplasmic reticulum and the functionality of the glucose-6-phosphatase system. Thus, alpha-toxin appears to have significant potential as a means to induce selective permeability to small ions. It should make possible the study of a variety of cellular functions in situ.     

The modifications of the final stages of the complement reaction by alkali metal cations.

We studied the effects of alkali metal cations on the terminal stages of complement lysis of human and sheep HK erythrocytes. Sensitized erythrocytes (EA) were reacted with limited amounts of complement for 1 hr at 37 degrees C in buffer containing 147 mM NaCl (Na buffer), which resulted in 10-40% lysis. The unlysed cells were washed with Na buffer at 0-2 degrees C and incubated for 1 hr at 37 degrees C in buffers containing 147 mM of the various alkali metal cations. Although additional lysis (25 to 65%) occurred with K, Rb, or Cs buffer, only minor degrees developed with Na or Li buffer, only minor degrees developed with Na or Li buffer. Intermediate levels occurred with 100 mM of the divalent alkali cations. Halogen ions and SCN-(147 MM), Ca++ (0.15mM), and Mg++ (0.5 mM) did not alter the effect of the alkali metal cations. Lysis occurring in K+, Rb+ or Cs+ proceeded without lag, was temperature dependent with an optimum of 43 degrees C, and had a pH optimum of 6.5. Lysis in K and Na buffers was unaffected by 10(-3) to 10(-5) M ouabain. Experiments with mixtures of cations indicated that Na+ had a mild inhibitory effect that could be totally overcome by K+, partially by Rb+, and not at all by Cs+. Li+ had a strong inhibitory effect, 6 X 10(-5) M causing 50% inhibition in buffers containing 147 mM K+, Rb+, or Cs+. By using intermediate complexes of EA and purified complement components we demonstrated that K+ enhances the lytic action of C8 on EAC1-7 as well as that of C9 on EAC1-8. It was known that Li+ facilitates lysis when acting on the entire complement reaction. We found that Li+ enhanced the lytic action of C8 on EAC1-7, with a kinetic that differed from that of the K+ effect. In addition, Li+ inhibited the enhancing effect of K+ upon lysis of EAC1-8 by C9. This occurred at concentration of Li+ similar to those which inhibited the additional lysis by K+, Rb+, and Cs+ of cells that were pretreated in Na buffer with the entire complement sequence. We propose that the major effects of alkali metal cations on complement lysis are due to their interaction with C8 and/or membrane constitutes.     

Extracellular calcium is required for the maintenance of plasma membrane integrity in nucleated cells

In contrast to rat and human erythrocytes, nucleated erythrocytes from two fish species (Cyprinus carpio and Salmo trutta) underwent almost complete haemolysis in 20 min of EDTA addition. Using Ca2+/Mg2+ EGTA-citrate buffer, we observed that half-maximal haemolysis of fish erythrocytes occurs at [Ca2+]o approximately 10 microM independently of extracellular Mg2+ concentration. Attenuation of [Ca2+]o with EGTA also decreased stability of the plasma membrane of vascular smooth muscle cells (VSMC) and HeLa cells, indicated by a three- to five-fold elevation of lactate dehydrogenase release and passive permeability of plasma membrane for Na+. In VSMC, EGTA lowered [Ca2+]i by approximately 20%. This effect was absent in VSMC-loaded with the intracellular Ca2+ chelator BAPTA. In contrast to EGTA, BAPTA did not affect haemoglobin release from fish erythrocytes and passive permeability for Na+ in VSMC. Viewed collectively, our data show that in nucleated cells, extracellular Ca2+ plays a crucial role in the maintenance of plasma membrane integrity.