Membrane Damage by Hemolytic Viruses, Toxins, Complement ... · Membrane Damage by Hemolytic Viruses, Toxins, Complement, and Other Cytotoxic Agents A COMMON MECHANISM BLOCKED BY

THE J O U R N A L OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Val. 261, No. 20, Issue of July 15, pp. 9300-9308, 1986 Printed in U.S.A.

Membrane Damage by Hemolytic Viruses, Toxins, Complement, and Other Cytotoxic Agents A COMMON MECHANISM BLOCKED BY DIVALENT CATIONS*

(Received for publication, February 26, 1986)

C. Lindsay Bashford$, Glenn M. Alder$, Gianfranco Menestrinas, Kingsley J. Micklemn, John J. Murphy11 , and Charles A. PasternakS From the $Departments of Biochemistry and (1 Immunology, St. George’s Hospital Medical School, Cranmer Terrace, London S W17 ORE, Great Britain, the §Department of Physics, University of Trento, Povo, Italy, and the YNuffield Department of Pathology, John Radcliffe Hospital, Oxford, Great Britain

Hemolytic viruses, bacterial and animal toxins, the components of activated complement, cationic proteins, and detergents induce a sequence of permeability changes at the plasma membrane that are in every case sensitive to changes in ionic strength and to divalent cations. Individually, each agent exhibits positive cooperativity; when two agents are present together, they show synergy. It is concluded that such cytotoxic agents damage membranes by a common mechanism. Hence permeability changes are unlikely to depend on the formation of specific, protein-lined channels, as previously envisaged in the case of activated complement or certain bacterial toxins.

Many clinically important cytotoxic agents that are hemolytic are able to damage other cells without lysis. The basis of membrane damage is the same as in erythrocytes: a breach of the plasma membrane permeability barrier such that ions whose intracellular and extracellular concentrations differ because of ATP-driven pumps leak into or out of cells. An immediate consequence is collapse of the transmembrane potential; a later one is colloid osmotic swelling, which, in the case of cells filled with hemoglobin and devoid of surface membrane elaborations capable of unfolding (l), leads to lysis. For hemolytic viruses it has been shown that, depending on the agent:cell ratio and the capacity of cells to recover (2), progressively more damage leads to the leakage of progressively larger molecules such as phosphorylated metabolites (sugar phosphates, nucleotides, etc.) and eventually cytoplasmic proteins (3).

The permeabilizing action of hemolytic paramyxoviruses (4), melittin (5), Staphylococcus uureus a toxin (6, 7), or the components of activated complement (8) is not due to the possession of lipase or protease activity, and they have therefore been referred to as “pore-formers” or “channel formers” (9-12). Because S. aureus a toxin (13) and the later components (C5b-8,gn) of activated complement (14-16) form struc- tured protein annuli with diameters of 2-10 nm, it has been assumed that the center of the annulus is the pore through which ions, water, and other compounds leak. The evidence

* This work was supported in part by financial assistance from the Medical Research Council, Cell Surface Research Fund, Cancer Re- search Campaign, and the Minister0 di Publica Istruzione and Con- siglio Nationale delle Richerche. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

presented here, however, leads to the conclusion that leakage is likely to be through smaller, rather different distortions or “leaky patches” (17) of plasma membrane structure. It is based on results showing that the action of Triton X-100 (at sublytic concentration) resembles the action of hemolytic viruses, a toxin, activated complement, melittin, and polylysine in four important regards. First, permeability changes are induced sequentially, suggestive of a growing lesion or pore. (We use the word pore to indicate a structural alteration of the plasma membrane such that its permeability is en- hanced; no connotation of a specific channel is implied.) Second, the dose-response curve for each agent is sigmoidal, indicating positive cooperativity; when present in pairs, the agents act synergistically. Third, leakage can be prevented by divalent cations in the order Zn2+ > Ca2+ > M$+. Fourth, the rate of leakage is decreased by lowering the ionic strength of the external medium and can be restored, in a Zn2+- and Ca2+ -sensitive manner, by the addition of salt. Because Zn2+ and Ca2+ are effective at concentrations near those found physiologically, the possibility of affecting the clinical consequences of, for example, staphylococcal infections (la), is opened up. Parts of this work have been briefly reported in a preliminary paper (11) and at two meetings (19,20).

EXPERIMENTAL PROCEDURES

Measurement of Permeability Changes Lettre cells, grown intraperitoneally as an ascitic suspension in

Swiss white mice, were removed from the animals, washed in 150 mM NaCl, 5 mM KCl, 5 mM Hepes’, 1 mM MgS04, pH adjusted to 7.4 at 22 “C with NaOH (Hepes-buffered saline, HBS) and incubated with [3H]choline for 30-45 min at 37 “C in order to label the intracellular phosphorylcholine pool (97). Washed cells were incubated (2-5 X lo6 cells/ml) in Hepes-buffered saline at 37 “C with pore-forming agents +CaC12 and membrane potential (98), leakage of monovalent cations (98), leakage of ph~sphoryl[~H]choline (971, and leakage of lactate dehydrogenase (55) measured.

Pore-forming Agents Used Sendai virus was grown in embryonated eggs for 3 days (97). S.

aureus 01 toxin was isolated (99) from strain Wood 46 (NCTC 7121) and kindly donated by Dr. Joyce de Azavedo, Moyne Institute, Trinity College, Dublin. Melittin was purified (41) and kindly donated by Dr. R. C. Hider, University of Essex. Polylysine (Sigma Type IV, M, 4,000-15,000) and Triton X-100 were commercial samples.

Complement-mediated permeability changes were initiated by

The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-pipera- zineethanesulfonic acid HBS, Hepes-buffered saline; PC, phosphatidylcholine; PS, phosphatidylserine; Mes, 4-morpholineethanesul- fonic acid; HAU, hemagglutination unit.

9300

Prevention of Membrane L treating Lettre cells with (a ) an antibody preparation and ( b ) a complement source. Antibody against Lettre cells was raised in a rabbit by intramuscular injection of an approximately 1:l mixture of Lettre cells and whole mouse brain in Freund's adjuvant. The rabbit was bled and serum obtained either prior to injection (preimmune serum) or at monthly intervals thereafter (a Lettre serum). The antiserum was either heated at 55 "C for 30 min to inactivate its constituent complement components or was passed through a protein A-Sepharose column and an IgG fraction (2.3 mg of protein/ml) isolated. The source of complement was either whole human serum which had been pre-adsorbed with Lettre cells or a preparation kindly donated by Dr. H. J. Muller-Eberhard of human serum depleted of C9 by immunoabsorption on an anti-C9 Sepharose column. In the latter instance a lesser amount of whole human serum (preadsorbed with Lettre cells) was added as source of C9, so that maximum leakage is dependent on the presence of both C9-depleted and whole serum. The procedures used were as follows.

Method A-Cells were incubated at 37 'C with preimmune or a Lettre serum (final concentration 3 or 4%) and whole human serum (final concentration 1 or 4%).

Method B-Cells were incubated at 37 "C with preimmune or a Lettre serum (final concentration 2%) and C9-depleted serum (final concentration 2%) for 20 min. Cells were spun, the pellets resuspended, and incubation at 37 "C continued in the presence of whole human serum (final concentration 0.5 or 1%).

Method C-Cells were incubated at 4 "C with a Lettre IgG (final concentration 46 pg of total protein/ml) for 20 min, followed by incubation at 37 "C with whole human serum (final concentration 1%). In each case, the incubation medium contained 0.1% bovine serum albumin.

Fluorescent Assay of C9 Binding to Cells

Concomitant with measurement of complement-induced permeability changes, samples of cell suspensions were spun, resuspended in ice-cold HBS containing 0.1% albumin and either analyzed immedi- ately or fixed with paraformaldehyde (1% in HBS) and analyzed after storing at 4 "C for up to 1 week. Washed cell pellets (approximately 2 X IO6 cells) were treated at 4 "C with either 0.05 ml of a goat anti- C9 serum (diluted X 1000), or with 0.05 ml of a mouse monoclonal anti-neo C9 serum (diluted X 1000), each kindly donated by Dr. H. J. Muller-Eberhard, for a minimum of 45 min, followed by two washes in HBS containing 0.1% albumin. The cell pellets were then treated at 4 "C with either 0.05 ml of fluorescein isothiocyanate-conjugated rabbit anti-goat IgG (Sigma; diluted X 1000) or with 0.05 ml of fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG (Sigma; diluted x 1000) for a minimum of 45 min, followed by two washes in HBS containing 0.1% albumin.

Cell pellets were diluted in Isoton and analyzed in a Coulter EPICS V Flow Cytometer by two-parameter analysis, namely brightness (LIGFL) uersus size (FALS). Two populations were discernible: one population which consisted of small, bright cells and one population which consisted of variable-sized, not so bright cells (Fig. 1). The proportion of small, bright cells was calculated as indicated in Fig. 1.

Cell Size - (FALSI

FIG. 1. Fluorescent assay of C9 binding to cells. Lettre cells were incubated, according to Method A, with preimmune serum (left panel) or a Lettre serum (right panel) (each at 4%), plus human serum (4%), for 16 min and analyzed with the monoclonal a neo-C9 preparation as described under "Experimental Procedures." For all fluorescent assays, percentage bright cells refers to the number in the top left quadrant compared with the total number. Three levels of

B). frequency are indicated dots, 1; stripes, 4; filled, 38 (in A) or 33 (in

lamage by Divalent Cations 9301

Measurement of Conductivity Changes in Lipid Bilayer Planar phospholipid bilayers were prepared at room temperature

by the apposition of two monolayers (59) of either reduced egg phosphatidylcholine (PC; PL Biochemicals) or a 1:l mixture of PC and phosphatidylserine (PS; Calbiochem-Behring). Monolayers were spread from a 5 mg/ml solution of lipid in hexane and, after evapo- ration of hexane, membranes were formed across a 200-rm hole punched through a 15-pm-thick Teflon sept pretreated with hexa- decane. To the compartments on either side of the sept was added 4 ml of bathing solution containing KC1 (various concentrations), 1 mM EDTA (Merck), buffered at pH 7.0 with Tris-C1 (Calbiochem- Behring). The conductivity of each bathing solution was measured with a Philips PW 9509 digital conductimeter equipped with a PW 9514 cell (cell constant 1 cm". The membrane potential was clamped through Ag-AgC1 electrodes at specified voltages and the current monitored by means of a virtual grounded operational amplifier (Burt-Brown DPA 104C) with feed-back resistors ranging from lo7 to 10" ohms. During the experiment divalent cations were added to one or both of the bathing solutions: concentrations quoted have been corrected for the presence of EDTA. S. aurells a toxin, kindly donated by Dr. K. D. Hungerer, Behringwerke, Marburg, Germany, was added (final concentration 5-30 pg/ml) to one of the bathing solutions (cis compartment) after the membrane was completely formed and sta- bilized. The cis compartment was connected to the virtual ground and voltage signs are referred to it.

RESULTS

The Onset of Increased Permeability Is Sequential-Fig. 2 shows that agents as diverse as Sendai virus (like influenza virus at pH 5 (21, 22)), the later components (C5b-8,9n) of immune-activated complement, i.e. the "membrane attack complex" (23), the a toxin of S. aureus, the bee venom protein melittin, polylysine, or Triton X-100 each induces an increased permeability of the plasma membrane of Lettre cells that is reflected by the following sequence of events: (i) collapse of membrane potential, (ii) an increase in intracellular Na' relative to K', (iii) a loss of phosphorylated metabolites and, a t high agent:cell ratio, (iv) a loss of cytoplasmic proteins. In every case the changes can be prevented by the addition of Ca2+ to the extracellular medium. For technical reasons it has proved difficult to measure membrane potential in the presence of complement, polylysine or Triton.

Agents Exhibit Cooperativity and, in Pairs, Show Synergy- The extent of leakage induced by different concentrations of each agent is illustrated in Fig. 3. In no case is a simple, hyperbolic dependency of leakage on agent concentration observed; all the agents exhibit positive cooperativity with Hill coefficients lying in the range 1.3 (for Sendai virus) to 6.4 (for complement).

If similar membrane lesions are formed by different pore- forming agents, it might be anticipated that when two agents are added at concentrations at which each is near its threshold value (i.e. little leakage), extensive leakage is induced. This is illustrated for combinations of the pore-forming agents under study in Fig. 4. Whereas panels e-g, in each of which a toxin is one of the agents, show no more than additivity, panels a and h, involving a toxin with Triton or complement, respec- tively, do indicate some synergy, making it likely that a toxin can act in a synergistic manner. In any case, even additivity of response can be indicative of a similarity of action, since extent of leakage (measured for the whole cell population), at least for Sendai virus (20) and complement (24, 25), is dependent on the number of cells sufficiently damaged to allow leakage of a particular metabolite ( i e . past threshold), and results such as those illustrated are incompatible with the notion that different populations of cells are sensitive to different agents. The fact that synergy is clearly seen with most agents, is in agreement with the observation that each agent acts in a "synergistic" manner with itself, i.e. exhibits

9302 Prevention of Membrane Damage by Divalent Cations

nu 0 5 IO 0 5 IO

Time (minutes)

' 50- b 9 .5 J O 3 0 4.

30 60 0 15 30 0 10 20

0 0 20 40 0 15 X) 0 30 60

Time (minutes) FIG. 2. Sequential onset of permeability changes induced

by pore-forming agents. Membrane potential, shown as a contin-

depolarization). Leakage of monovalent cations, (calculated as Na+/ uous trace, is expressed as A630-690 (an increase of which indicates

Na+ + K+ in cells and shown in circles), ph~sphoryl[~H]choline (squares) and lactate dehydrogenase (triangles) is expressed as follows: 0% leakage is that before addition of agent and 100% leakage is that that would occur if cells were completely permeabilized. The lower curve (filled-in symbols) in each case refers to changes induced in the presence of CaCL Upper panel, Sendai virus, 128 HAU/ml; a toxin, 1 pg/mb melittin, 0.5 pM. CaC12, when present, was at 2.5 mM. Lower panel, Sendai virus, 450 HAU/ml; a toxin, 4 pg/ml; melittin, 1 p ~ ; complement, Method A, 3% a Lettre serum, 1% whole human serum; polylysine, 14 pg/ml; Triton, 0.005%. CaCI2, when present, was at 10 mM.

positive co-operativity (Fig. 3). Such a result has also been obtained from the induction of conductivity in lipid bilayers by melittin (9) and a toxin (26). Synergy between Sendai virus and various membrane-active drugs has previously been reported (27). In contrast, as demonstrated for Sendai virus (2) and noted for complement,2 the concentration of divalent cation required to inhibit leakage increases as the agent:cell ratio increases (in the absence of any effect of divalent cation on binding of agent to cells).

Stimulation of Respiration-One consequence of an increased Na+ content of cells is likely to be a stimulation of the Na+ pump. Since increase in pump activity leads to an increased hydrolysis of ATP to ADP-a known stimulator of respiration (28)-one might expect cells affected by "permeabilizing" agents to show increased oxygen consumption. That this indeed occurs is documented in Table I, and confirms that cells affected by low concentrations of Sendai virus, complement, or melittin are damaged but not lysed. The

C. A. Pasternak, unpublished observations.

Cwnplemant (%raurnl Polylyrin (pglrnll Triton X-IO0 (K FIG. 3. Dependency of permeability changes on the concen-

tration of pore-forming agents. Lettre cells (2.4 x 10' cells/ml in HBS) prelabeled with [3H]choline were treated with Sendai virus (30 rnin), S. aureus a toxin (30 min), melittin (16 min), complement (Method C: % whole serum as indicated) (30 min), polylysine (30 rnin), or Triton X-100 (30 min) a t the concentrations indicated. Intracellular cations (calculated as Na+/(Na+ + K+) and shown as circles) and ph~sphoryl[~H]choline (squares) were measured as described under "Experimental Procedures." The line drawn in each case is the best fit (least squares) to the Hill equation. The Hill coefficients used were as follows: Sendai virus, 1.32; S. aureus a toxin, 3.28; mellitin, 2.56; complement, 6.41; polylysine, 1.66; Triton X-100, 2.13.

751 e I f 1 9 I h

Time (mid

FIG. 4. Synergistic action of pore-forming agents. Leakage of ph~sphoryl[~H]choline from prelabeled Lettre cells was measured as described under "Experimental Procedures." Lettre cells (2-4 X 10' cells/ml) were treated with S. aureus a toxin (1 pg/ml), melittin (0.34 p ~ ) , polylysine (5 pglml), Sendai virus (50 HAU/ml), complement (method B: 1% whole serum), or Triton X-100 (0.0025%). For

was added after the initial incubation with a Lettre serum and C9- experiments involving complement, the second pore-forming agent

depleted serum, i.e. together with the whole serum. Open symbols indicate each pore-forming agent added separately; closed symbols indicate pore-forming agent added in pairs. Panel a, a toxin (sqwres) and Triton (Circles); panel b, melittin (triangles) and Triton (circles); panel c, polylysine (triangles) and Triton (circles); panel d, Sendai ViNS (triangles) and Triton (circles); panel e, a toxin (squares) and melittin (triangles); panel f, a toxin (squares) and polylysine (triangles); panel g, a toxin (squares) and Sendai virus (triangles); panel h, a toxin (squares) and complement (triangles); panel i, melittin (upward-facing triangles) and Sendai virus (right-side-facing triangles); panel j , melittin (upward-facing triangles) and polylysine (downward- facing triangles); panel k, Sendai virus (right-side-facing triangles) and polylysine (downward-facing triangles); panel 1, Sendai virus (right-side-facing triangles) and complement (left-side-facing triangles).

increase in respiration is inhibited by Ca2+ or Zn", which prevent the increased influx of Na' (see below). That the stimulated respiration is due to an increased Na' pump activity is shown by the fact that it is inhibited by ouabain.

Prevention of Membrane Damage by Divalent Cations 9303

TABLE I Respiration of Lettre cells in the presence of pore-forming agents 10’ Lettre cells/ml in 150 mM NaCI, 5 mM KCl, 5 mM glucose, 5

mM Hepes, 1 mM MgS04, pH adjusted to 7.4 with NaOH (HBS) at 37 “C. ResDiration rates are ng atom 02 consumed/rnin/lO’ cells.

Medium 47 HAU/ml pM 4%

No agent melittin complement virus

HBS 35.0 f 4.9 47.1 48.3 52.7 HBS + 2.5 mM 35.5 f 4.1 39.1 39.6 41.9

CaC1,

ZnCl,

ouabain

HBS + 0.25 mM 31.9 2 1.0 ND“ 28.2 33.5

HBS + 2.5 mM 30.1 f 3.6 30.3 29.9 30.1

ND, not determined.

IO-^ IO-^ IO-^ IO-^ IO-^ IO-^ IO-^ IO-^ ~ g ~ + ( * ) , Ca2*(.),or Zn2’(A) M

FIG. 5. Inhibition of permeability changes by divalent cations. Leakage of ph~sphoryl[~H]choline from prelabeled Lettre cells was measured as described under “Experimental Procedures.” Sendai virus, 128 HAU/ml; a toxin, 3 pg/ml; melittin, 0.5 pM; complement, method C; polylysine, 10 pg/ml; Triton, 0.005%. Divalent cations (MgCI,, CaC12, or ZnS04) were present as indicated.

Increased Permeability Is Inhibited by Divalent Cations- The permeabilizing action of each agent is inhibited by Ca2+ and other divalent cations such as Zn2+ and M$+. Although the efficacy of Ca2+ varies from agent to agent (Fig. 5), as it does with different cell types (see below), it is remarkable that the relative potency of the divalent cations is the same, namely Zn2+ > Ca2+ > M F , in every case (Fig. 5); Mn2+ has the same potency as Ca2+ for Sendai virus-induced leakage (29). Note that above approximately 0.5 mM, Zn2+ itself permeabilizes Lettre and other cells.

The fact that all the agents so far tested show a similarity of response to extracellular (19) divalent cations supports the notion that the membrane lesions induced by the various agents share some common features and suggests that the action of divalent cations is, at least in part, on the structure of those lesions, rather than on the mechanism of their formation. In the case of complement, for which the induction mechanism is the most complicated, divalent cations are required for the formation of the membrane attack complex C5b-9 (30), whether triggered by the “classical” (Cl-C3) or by the “alternative” pathway. However, concentrations of Ca2+ or Zn2+ that inhibit leakage from sensitized Lettre cells (Fig. 5), Daudi cells ( l l ) , or tonsil lymphocytes (19) do not affect the steps leading to the formation of the membrane attack complex (19). Indeed Zn2+, at a concentration that inhibits leakage, has no effect on subsequent leakage when cells are resuspended in Zn2+-free medium (Table 11). A similar observation has been made in regard to the inhibition of complement-induced hemolysis (31). Furthermore, the binding and polymerization of C9 to Lettre cells, assessed by immunoflu- orescence with a C9 and CY neo-c9 antibody, is relatively

TABLE I1 Action of complement on Lettre cells

Lettre cells prelabeled with [3H]choline were incubated (9 X lo6 cells/ml) according to Method B (0.5% whole human serum). ZnSO4 was added at the same time as whole human serum. 10 min later, cells were spun, the 3H content of the supernatant determined, and the cell pellet chilled and assayed for C9 binding. A portion of cells incubated in the presence of Zn2+ was spun after only 3 min, the pellet resuspended in Zn2+-free HBS (containing 0.1% albumin and 10 mM EDTA), and incubation at 37 “C continued for a further 10 min.

% 3H in supernatant % bright cells

Complete system 90 83 50 Plus 0.1 mM Zn2+ 31 76 48 Plus 0.1 mM Zn2+ followed by its 82 88 70

Preimmune serum instead of a 11 8 8

Minus whole human serum (comple- 17 31 23

removal

Lettre serum

ment source)

unaffected by the presence of Zn2+ (Table 11). This shows that, under the conditions of the experiment, Zn2+ does not inhibit leakage by, for example, preventing the binding of C9 to cells through polymerization to inactive polymer (32, 33). The effect of Ca2+ is essentially the same as that of Zn2+.3

Increased Permeability Is Inhibited by Lowering Ionic Strength-Divalent cations are known to affect the surface potential of cells (34, and it is therefore possible that leakage induced by pore-forming agents is affected by surface potential. In media of low ionic strength, where 150 mM NaCl is replaced by 300 mM mannitol, leakage induced by all the agents so far tested is reduced, even though cationic proteins such as melittin bind better in mannitol medium than in saline. Leakage in mannitol is stimulated by the addition of salt (Fig. 6) under conditions where excess agent has been removed by centrifugation. Salt-stimulated leakage is inde- pendent of the nature of either cation or anion used (Fig. 7), and is prevented by Ca2+ or Zn2+ (Fig. 6). The concentration dependence of salt-stimulated leakage is illustrated, for the case of Sendai virus, in Fig. 7, panel A . Panel B shows the effect of pH on leakage in mannitol medium; this is to be contrasted with an insensitivity to pH in 150 mM NaCl (22).

Increased Leakage through Pure Lipid Bilayers Is Sensitive to Divalent Cations-The similarity of response to divalent cations by cells permeabilized by different agents (Figs. 2 and 5) some of which, like melittin (35), polylysine, or Triton, lack credible binding sites for positively charged molecules, suggests a binding site for cations that is the cell membrane itself. That this may involve phospholipids is indicated by the following observation. S. aureus CY toxin, which, like melittin (9) or complement (8, 36, 37), is able to induce leakage in pure phospholipid bilayers (38), does so in a divalent cation- sensitive manner. Fig. 8, panel A, shows a toxin-induced ion conductivity through a planar phosphatidylcholine (PC) bilayer: addition of 60 mM Ca2+ causes a rapid decay of conductivity (which is voltage-dependent). With mixed, negatively charged phosphatidylserine (PS):PC bilayers the concentration of Ca2+ required to show this effect is much reduced, inactivation being achieved at 1-3 mM Ca2+ (Fig. 8, panel B ) . This implies binding by PS, or at least the attraction of Ca2+

C. L. Bashford, G. M. Alder, K. J. Micklem, J. J. Murphy, and C. A. Pasternak, unpublished observations.

9304 Prevention of Membrane Damage by Divalent Cations Sendai virus Complement

c

Triton X- 100 Controls

Time (minutes)

FIG. 6. Salt-stimulated leakage from cells at low ionic strength. Lettre cells (2.6 X IO6 cells/ml) prelabeled with [3H] choline, were incubated either in mannitol medium (0.3 M mannitol, 0.005 M Hepes pH adjusted to 7.4 with NaOH) with 26 HAU/ml Sendai virus for 10 min or with 0.00625% Triton X-100 (w/w) for 3.5 min at 37 'C or in HBS with complement (method A: 0.9% whole serum) at 37 "C for 2 min. Cells were then pelleted and resuspended in mannitol medium in the presence of 40 mM NaCl (squares) or 15 mM MgCl, (triangles) for the times indicated. Experiments were conducted in the absence (open symbols) or presence (closed symbols) of 2.5 mM CaCI, (Sendai virus and Triton X-100) or 0.15 mM ZnSOI (complement); control cells were treated exactly as described above except that the pore-forming agent was, in each case, omitted.

Time (mid pH

FIG. 7. Effect of ionic strength on permeability changes induced by Sendai virus. Leakage of ph~sphoryl[~H]choline from prelabeled Lettre cells was measured as described under "Experimen- tal Procedures." In panel A (i) , labeled cells in HBS (10' cells/ml) were treated with Sendai vius (5000 HAU/mI) a t 37 "C. At the time indicated by the arrow, cells were diluted 100-fold into HBS (closed circles) or into low ionic strength medium (open circles) containing 0.3 M mannitol, 5 mM Hepes, 1 mM MgS04, pH adjusted to 7.4 with NaOH (mannitol medium) and incubation at 37 "C continued. In panel A ( i i ) , labeled cells in mannitol medium (5 X lo6 cells/ml) were treated with Sendai virus (26 HAU/ml) at 37 "C. At the time indicated by the arrow, NaCl was added to give a final concentration of 60 mM (downward-facing triangles), 40 mM (upward-facing triangles), or 20 mM (diamonds) NaCl; cells to which no NaCl was added are shown as open circles. In panel A (i i i) , stimulation of leakage by various salts, carried out as described in (ii), is shown plotted against concentration of salt. NaCI, closed left-facing triangles; Na2S04, closed circles; sodium phosphate, closed squares; KCl, open left-fucing triangles; K,SO,, open circle; potassium phosphate, open square; K citrate, open right-facing triangle. In panel B (i), labeled cells in mannitol medium containing 5 mM Mes, pH 7.5, were treated with Sendai virus (25 HAU/ml) at 37 "C. At the time indicated by the arrow the cells were spun and resuspended at 37 "C in the same medium containing 50 mM NaCl at pH 7.5 (open circles), 7.0 (open squares), 6.5 (open upward-facing triangles), 6.0 (open downward-facing triangles), or 5.5 (open right-facing triangles). In (ii), stimulation of leakage, carried out as described in (i), is shown plotted against pH.

a

2 m p s l P a)

3osec

I I I J IO-^ lo-* IO"

[CO''] M FIG. 8. S. aweus (I toxin-induced ion channels in lipid bi-

layers. Panel A, trace a, current steps following the addition of a toxin (20 pg/ml) to one side of a PC planar bilayer in 0.2 M KC1, clamped at +40 mV: each step is due to the opening of a new ionic channel. Three changes in the current scale are shown. Vertical bars, 200 picosiemens in each case; horizontal bar, 30 s. The base line indicates zero current. Traces b and c, current response (lower trace in each case) to the application of voltage pulses (upper truce in each case) to a PC membrane, in 0.1 M KC1, containing many a toxin channels (added at 25 pglml). b, no added CaCI,; c, in the presence of 60 mM CaCl,. In the absence of CaCL ( b ) , the current remains constant after each voltage step (apart from a continuous slight increase due to the insertion of new channels). In the presence of CaC12 ( e ) , a potential-dependent relaxation of current after each voltage step is seen. Panel B, channel inactivation measured by the ratio of steady state current (I,) to instantaneous current ( Io ) , as a function of CaC1, concentration, for three different applied voltages. An ZJIo value of 0 corresponds to complete inactivation, The applied voltages were -60 mV (circles), -80 mV (squares), and -100 mV (triangles). Closed symbols refer to membranes composed of PC only; open symbols refer to membranes composed of PC and PS (molar ratio 1:l). The bathing solution was 0.1 in KC1 in each case.

by PS so as to increase the effective concentration near the cy toxin at the surface of the bilayer. When other cations were tested in this system, the order of efficacy was Znz+ > Ca2+ > M$+, exactly as found for inhibition of leakage from cells (Fig. 5).

DISCUSSION

The general similarity of action of different agents is surprising, in view of the fact that the agents interact with cells in quite distinct ways. Thus, hemolytic viruses induce permeability changes through the fusion of an inherently leaky viral envelope with the plasma membrane (39); S. aureus LY toxin and the membrane attack complex of complement form oli- gomers with hydrophobic regions capable of insertion into the plasma membrane (12,40); cationic proteins such as melittin


and polylysine may act partly by cross-linking intrinsic membrane proteins (41), partly by insertion and distortion of the plasma membrane (42), and partly by activation of endogenous phospholipases (43); detergents, at concentrations below their critical micellar concentration, act by insertion and distortion (44,45). Nevertheless, the induction of an increased permeability is quite specific: nonhemolytic Sendai virus (46), for example, fuses with cells as effectively as hemolytic virus but causes no permeability change (39), and C9-induced leakage from cells occurs only in the presence of the relevant antibody and the earlier proteins of the complement cascade (Table II).3

Irrespective of the specific mechanism of induction, then, the pattern of damage inflicted on the plasma membrane is common to all the agents tested in the following regards: 1) the onset of damage follows a characteristic sequence, 2) the dose-dependence of damage shows a strong positive cooperativity and combinations of the agents act synergistically, 3) the extent of damage is diminished by divalent cations in the order Zn2+ > Ca2+ > M P , 4) the extent of damage for a given amount of cell-associated agent is diminished in low ionic strengths and can be restored by the addition of salt in a divalent cation-sensitive manner.

That the sequential onset of permeability changes reflects a graded derangement of membrane structure and not merely an increase in the number of cells lysed is indicated by the following observations. First, membrane potential can collapse almost completely (implying that most of the cells have been affected) before significant leakage of phosphorylated metabolites or cytoplasmic proteins takes place (Fig. 2, panek; ii and iii and previous results with Sendai virus (3, 47, 48)). Second, the effect of some of the agents is reversible, in that cells can spontaneously recover their original permeability characteristics, sometimes even without removal of the permeabilizing agent. This has been most clearly demonstrated in the case of Sendai virus (49). Third, permeability changes may result in an increase in the metabolic potentiality of cells, as in the co-mitogenic role of melittin in 3T3 cells (50), the induction of insulin release from pancreatic islet cells by melittin (51), the production of prostacyclin from S. aureus a toxin-treated endothelial cells (52), or the stimulation of oxygen consumption by eosinophil cationic protein in heart cells (53), or by Sendai virus, complement, or melittin in Lettre cells (Table I).

Each agent shows a concentration dependency that is indicative of a cooperative mechanism of action (Fig. 3). The degree of cooperativity may reflect the number of molecules of agent required to create a particular lesion, as has been argued for the action of melittin in phospholipid bilayers (9). One might anticipate, then, that leakage of pho~phoryl[~H] choline would show a higher Hill coefficient than leakage of ions or membrane depolarization, but this does not appear to be the case. Instead it is possible that the physical basis of the cooperative effect depends on factors in the plasma membrane itself, particularly in the case of viruses where the number of virions/cell causing damage is rather low. Certainly the synergy of diverse agents suggests cooperativity via common, membrane-associated molecules and provides the strongest evidence for a common mechanism of action. Never- theless, differences in degree of synergy between agents suggests that there are some differences of action too.

The ability of divalent cations to reduce the damage to the membrane caused by cytotoxic agents has elements common to all agents and elements specific to each agent. In all cases the relative efficacy is Znz+ > Ca2+ > M P . However, the exact concentration of divalent cation required depends also

on the nature of the agent (a toxin is, in general, less sensitive to inhibition by Ca2+ than other agents) and on the agentxell ratio. The latter observation suggests that binding sites both on the agent and on the membrane may contribute to the effect. What is clear is that binding of agent to cells is not the primary action of divalent cations, i.e. divalent cations block leakage at a stage of lesion formation subsequent to this (see, e.g. Fig, 6). This has previously been shown for Sendai virus (39, 54) and melittin (20). In the experiment illustrated in Fig. 8, the action of Ca2+ cannot be to prevent binding of S. aureu a toxin to the lipid bilayer, since it acts to close already formed pores. These observations clearly indicate that, irrespective of whether divalent cations in certain situations affect the induction of membrane lesions, their role in preventing permeability changes cannot be ascribed merely to such an effect. The sensitivity to divalent cations of pore formation is not restricted to Lettre cells. Although Sendai virus-induced, calcium-sensitive, permeability changes have been detected in many different cell types (48, 55-58),4 the M2+-sensitive nature of permeability changes induced by other agents has, apart from erythrocytes (60-67), not previously been recorded. Erythrocytes have been most studied simply because hemolysis is a straightforward assay of cell permeabilization. However, the concentration of Ca2+ required to prevent hemolysis by, for example, Sendai virus (11, 5 4 , S. aureus a toxin, complement (II), or melittin? namely 20-40 mM, is more than 10 times higher than that required to prevent permeabilization of most other cell types by the same amount of agent; as little as 10 p~ Ca2+ is, for example, sufficient to prevent permeabilization of mast cells by Sendai virus (57). Hence the physiological significance of the potential role of divalent cations in protecting cells against pore- forming agents has not previously been appreciated. More- over, non-erythroid cells possess mechanisms for membrane repair of virus-induced (2) or complement-induced (68) damage not available to red cells, and Ca2+ itself promotes repair (2).

In a similar vein, the reduced ability of cytotoxic agents to permeabilize cells at low ionic strength strongly suggests an effect at the cell membrane itself, as the agents themselves are negatively charged (virus, a toxin (12)), positively charged (melittin, polylysine), or neutral (Triton X-100). Again the action must be subsequent to binding of the agent because the stimulation of leakage by salt occurs in the absence of excess agent (Fig. 6) in a manner suggestive of the “opening” of a preformed pore.

Our results, then, lead to two new conclusions regarding the action of cytotoxic agents. The first is that hemolytic agents of widely differing type have a similar, “detergent-like” effect on membrane permeability, although the precise nature of lesions formed, ( i e . whether lipid-lipid, lipid-protein, or protein-protein interactions are involved) cannot be decided at present. The lesions clearly differ from endogenous features such as K+, Na+ (69, 70), H+ (711, or anion (72) channels or communicating junctions (73) (although these, like the Na’ channel (74), can be blocked by Ca2+ (73)), from the lesions created by compounds such as gramicidin which have a de- fined internal structure (75) through which specific ions, but not phosphorylated metabolites, leak in a Ca2+-insensitive manner (20) and from lesions like the Na+ pump-related channel created by palytoxin (76). The pores under discussion have more in common with the membrane changes reported to occur when amphipathic molecules, at sublytic concentration, interact with membranes (77). In short, leakage is less

‘ M. M. Barrowman, S. Cockcroft, and B. D. Gomperts (1986) Nature 319, 504-507.

9306 Prevention of Membrane Damage by Divalent Cations

likely to be through the entire 2-10-nm pores at the center of the annular protein assemblies created by S. aureus (Y toxin (12, 13) or by the membrane attack complex at high concentrations of C9 (16), than through distortions at the internal or external edges of these assemblies. Such a view is compat- ible with other types of evidence regarding the action of complement (17, 78, 79); the fact that the lesions are sometimes crescent-shaped, not closed (12), is in accord with the central portion being covered by a lipid bilayer. Since hemolysis results from membrane rupture due to colloid osmotic swelling and does not occur when albumin is present in the medium, rather than from the creation of pores large enough to accommodate hemoglobin, it is clear that the creation of relatively narrow pores, sufficient in number to overcome the volume-regulating ability of the Na+ pump (80), is adequate to induce hemolysis.

A model that accommodates the present findings is shown in Fig. 9. Its key feature is the proposal that ionizable groups at the mouth of each pore affect the movement of compounds through it: when the groups are ionized, as is the case in normal saline, leakage is maximal; when the groups are un- ionized, as is the case (a) in mannitol medium, in which the concentration of H+ relative to Na' or K+ is high enough to protonate weak anionic groups, or ( b ) when liganded to divalent cations, leakage is minimal. Whether such changes affect the function of pores by altering their shape, or affect merely the rate of leakage as a result of an electrostatic effect at the mouth of the pore (81-82)"or both-requires further experimentation. The observation that decreasing ionic strength increases the surface potential of cells through a reduction of ion screening (83, 84) is not incompatible with our model. Decreased screening by Na' or K' would certainly promote the ability of H' to bind to ionizable groups such as phosphate or carboxyl. The extent to which divalent cations

Leaky Membrane Non-leaky Membrane

+ -i EGTA etc

FIG. 9. A model for the control of permeability changes caused by pore-forming agents. A number of ionizable groups (most likely carboxyl, but could also be sulfate) at the outer leaflet of the membrane are indicated. The groups could be on phospholipid, glycolipid, or protein. In order to inhibit leakiness (illustrated for an

phorylated metabolites) through a membrane defect that is induced outward movement of normally impermeant molecules such as phos-

by pore-forming agent, protonation or chelation with divalent metal is required. The affected groups may need to be at, or merely near, the mouth of the pore. This model stresses the effect of protonation and chelation on surface charge, rather than on the geometry of the membrane defect.

bind to such groups, as opposed to a screening effect (85), depends on the particular type of agent and cell. At the high concentrations of calcium ions required to prevent hemolysis (11, 54, 63, 66, 67), at which there is little discrimination between Ca2+ and M e , a screening effect may predominate. At the lower concentrations of Zn2+ and Ca2+ required to prevent leakage from Lettre (Fig. 5) and other cells (11, 48, 55-58, 64-67), specific binding is likely to predominate; this is indicated also by the observation that concentrations of divalent cation having similar effects on leakage, have differing effects on electrophoretic mobility? Moreover, the concentration of divalent cation at which leakage from Lettre cells is approximately 50% inhibited, namely 10-5-10-4 M Zn2+, 10-4-10-3 M Ca", and 5 X 10-3-5 X lo-' M M e (Fig. 5 and Refs. 19 and 20), bears remarkable similarity to the stability constants (log K) for the binding of these cations by a model compound, phosphonomethyliminodiacetate (86): that is, 5.4 for Zn2+, 3.5 for Ca", and 2.4 for Mg2+ (as calculated for pH 3 . 6 Since the binding sites on the compound are phosphonate and carboxyl, the analogy with a phospholipid such as PS (Fig. 9, panel B) is obvious. But binding to other molecules, including proteins, is not ruled out.

The presence of divalent cations such as Ca2+ and Zn2+, and the absence of monovalent cations such as Na+, might protect cell membranes as a consequence of their actions on cell metabolism. For example, entry of Ca2+ through a lesion might trigger intracellular signaling systems which subse- quently affect the membrane (87). However, exposure of cells to the ionophore A23187, which substantially alters the dis- tribution of cellular Ca2+, neither activates nor antagonizes the protective effect of Ca2+ (19). Furthermore, it would be surprising if Zn2+, which is much more potent in preventing membrane damage than is Ca2+, were acting indirectly through intracellular Ca2+-regulated systems. Removal of extracellular Na' certainly reduces intracellular Na', which leads to an inhibition of the Na' pump. But this potentiates, not reduces, Sendai virus-induced leakage from cells sus- pended in conventional media (3). In Sendai virus-treated cells in the absence of ouabain the Na' pump is unaffected, or actually stimulated somewhat (55 and Table I). Hence it is likely that the ionic effects discussed in relation to the model illustrated in Fig. 9 are indeed manifest at the cell surface.

Insofar as the model depicted in Fig. 9 implies the existence of similar membrane lesions induced by quite different means, it may extend to cells that are merely in physiologically unfavorable conditions, such as low temperature, anoxia, physical trauma (insertion of microelectrodes), and so forth (88). Although the protective role of divalent cations is often forgotten in discussion of their biological f'unctions (89), Ca2+ (90) and Zn2+ (91) have long been regarded as membrane protective agents, and divalent cations are known to be required for the effective reconstitution of solubilized membranes (92).

The second conclusion to be drawn from our results is that pathophysiological changes resulting from the action of pore formers in uiuo may be subject to fluctuations in the extracellular concentration of divalent cations such as Ca2' or Zn2+. Conversely, it may be possible to boost the lytic capacity of complement where desirable by the administration of other agents or drugs such as calmodulin inhibitors (calmidazolium and trifluoperazine) and Ca" "antagonists" (verapamil and prenylamine) that have been found to act synergistically with Sendal virus (27). The observation that Zn2' decreases the lytic activity of NK cells (93), coupled with the suspicion that

C. L. Bashford and J. M. Graham, unpublished observations. R. J. P. Williams, personal communication.


T cell cytotoxicity involves membrane lesions similar to those induced by complement (94-96)) implies that cellular as well as humoral immunity may be modulated by an effect of divalent cations at the plasma membrane.

Acknowledgments-We are grateful to Drs. P. J. Lachmann and R. J. P. Williams for helpful discussion of this article and to V. Marvel1 and B. Bashford for preparation of the typescript.

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