Acta pharmacol. er roxicol. 1919, 44, 208-21s.
From the Institute of Biology, i(bo Akademi, 20 SO0 Abo SO, Finland
Interactions of Surface-active Alkyltrimethylammonium Salts with the Plasma Membrane of Acanthamoeba castellanii BY Boris Isomaa, Gun Paatero and Christer Lonnqvist (Received August 30, 1978; Accepted October 16, 1978)
Absfracf: The interactions of three surface-active alkyltrimethylammonium salts (C12-Cls) with the plasma membrane of Acanthamoeba cusfellanii were studied. The surfactants caused a release of K’ from the cells at premicellar concentrations. The lytic effectiveness of the surfactants increased with an increase in the length of the alkyl chain with about an order of magnitude for every two carbon atoms added to the alkyl chain. Binding studies with the C Mhomologue revealed that at a concentration corresponding to SO% release of K’ there were about 1.9X 10” molecules bound per cell. At prelytic concentrations the surfactants stimulated phagocytosis and pinocytosis. The mode of action of the surfactants on the plasma membrane ofAcanthamoeba casrellanii is discussed and it is hypothesized that the stimulation of endocytosis is due to a “fluidizing” effect of the surfactants on the lipid bilayer of the plasma membrane. Key-words: Cationic surfactants
- lysis - endocytosis - Acanfhamoeba castellanii.
Because of their importance as antiseptic agents, the actions of surface-active alkyltrimethylammonium salts on cells have been subjected to many studies. Several authors have suggested that the principal point of attack of cationic surfactants is the lipid bilayer of the plasma membrane (Hooghwinkel et al. 1965; Kondo & Tomizawa 1966; Reman et al. 1969; Isomaa et al. 1979). The surfactant molecules are assumed to penetrate into the lipid bilayer which they tend t o break up into localized micellar arrangements (Haydon & Taylor 1963; Seufert 1965). The ability of the membrane to prevent free exchange of ions is abolished and the cell is lysed by secondary osmotic effects (Isomaa 1979). In a previous study with rat erythrocytes it was found that surface-active alkyltrimethylammonium salts, in addition to their lytic effect, stabilize erythrocytes against hypotonic haemolysis at prelytic concentrations (Isomaa 1979). I t was suggested that this stabilizing is due to a
“fluidizing” effect of the surfactants on the lipid bilayer of the plasma membrane. The purpose of this study was to examine the interactions of surface-active alkyltrimethylammonium salts with the plasma membrane of Acanthamoeba castellanii. This small soil amoeba has no surface coat external to the plasma membrane (Bowers & Korn 1968) and the gross chemical composition of its plasma membrane is not very different from the composition of other eucaryotics (Ulsamer et al. 1971). Unlike mammalian erythrocytes this amoeba has a very high rate of endocytosis in laboratory cultures (Bowers 1977) which makes it a very suitable organism when studying the interactions of various agents with membrane-localized processes. Materials and Methods Chemicals. Cetyltrimethylammonium bromide (CTAB,
CATIONIC SURFACTANTS ON PLASMA MEMBRANE c16), 99% pure, was obtained from E. Merck AG, Darmstadt, Germany. Dodecyltrimethylammonium bromide (Cit) and tetradecyltrimethylammonium bromide (CI4) were generously supplied by MoDoKemi AB, Stenungsund, Sweden. These compounds had a chemical purity of about 98%. Trimethyl-(I-"C)cetyltrimethylammonium bromide (%-CTAB) with a specific activity of 6.4 mci/mmol and radiochemical purity of 97%. and 'H-inulin with a specific activity of 900 mci/mmol and a radiochemical purity of 99% were obtained from the Radiochemical Centre, Amersham, Bucks, England. All other chemicals used were of analytical grade. Polystyrene latex beads with a particle diameter of 1.01 pm were obtained as an aqueous suspension from Serva Feinbiochemica, Heidelberg, Germany. To remove emulsifiers and other soluble substances the latex beads were dialyzed against 5 1 distilled water at room temperature for 6 hrs. The latex beads were stored in distilled water and they were sonicated in an ultrasonic bath for about 5 min. prior to their use in the experiments.
Cell culturing. Acanthamoeba castellanii was obtained from the Culture Centre of Algae and Protozoa, Cambridge, England. The cells were cultured axenically in Neffs optimal growth medium (Neff er al. 1964), as modified by Drainvilli & Gagnon (1973). The temperature was maintained at 2 8 f I 0 and the cultures were shaked at 100 r.p.m. on a rotary shaker. The cells were collected from cultures that were in a late log-phase (7-10 days old) by centrifuging at 800Xg for 5 min. The cells were washed three times in a buffer solution of the following composition: 100 mM-NaCI, 10 mM-NaH2P04, 0.5 mM-CaCIz, 0.5 mM-MgSO4, pH 7.0. The cells were then resuspended in the buffer solution and the cell concentration of this stock suspension was adjusted to 4.5X10' cells per ml. Cell counts were made using a haemocytometer. Measuremenr o j ' r release. One ml of the stock amoeba suspension was added to glass test-tubes containing 9 ml buffer solution and various amounts of the surfactants. The contents of the tubes were briefly mixed with a vortex mixer and the tubes were incubated for 1 hr at 28 f0.5"in a shaking thermostat bath. The tubes were centrifuged at 800Xg for 5 min. and washed three times with the buffer solution. The cells were then resuspended in 9 ml distilled water and, to rupture the cells, the suspensions were sonicated in an ultrasonic bath for 5 min. The tubes were centrifuged at 2,OOOXg for 10 min. and the potassium content of the supernatant was determined with an atomic absorption spectrophotometer at 766.5 nm using an air-acetylene flame. The amount of K' released was estimated from standard curves prepared by sonicating known number of cells. The experimental values were corrected for the release of K' occurring in a test-tube without surfactant. Viability counts. Amoeba cells were incubated as described above in a buffer solution containing various
209
amounts of the surfactants. Following incubation the viability of the cells was assessed by adding 1 vol. of tryphan blue (0.16% in buffer solution) to 2 vol. of the amoeba suspensions and counting the number of stained ("dead") and unstained ("living") cells in a haemocytometer. Cell counts were made within 10 min. of mixing the cells with tryphan blue. Adsorption of I4C-CTAB to the amoeba cells. Amoeba cells were incubated as described above in buffer solution with different concentrations (1-70 pM)of '"C-CTABCTAB. The specific activity of the I4C-CTAB-CTAB mixture was 0.35 mci/mmol. Following incubation the suspensions were centrifuged in polyethylene tubes at 9,OOOXg for 10 min. and I ml of the supernatants were taken to determine radioactivity. In someexperiments the cells were washed, following incubation, three times with cold (4') buffer solution by centrifugation at 2,OOOXg for 10 min. and 1 ml of the supernatants were taken to determine radioactivity. finocytic acrivit.p. The pinocytic activity of the amoebae was determined by measuring the uptake of 'H-inulin. This carbohydrate is in the amoeba taken up by pinocytosis (Bowers & Olszewski 1972). Amoeba cells (4.5X 10') were incubated for 1 hr as described above in 10 ml buffer solution containing 10 p i 'H-inulin (56 pg inulin) and various amounts of the surfactants. Pinocytosis was stopped by adding dinitrophenol to a final concentration of 0.2 mM (Wiseman & Korn 1967). The cells were harvested by centrifugation at 2,ooQ)Xgfor 5 min. and washed three times with cold buffer solution. The cell pellets were solubilized in Protosol (New England Nuclear Corp., Boston, Mass., USA) at room temperature and aliquots of the solubilized samples were taken to determine radioactivity. In the control the amount of radioactivity taken up by the cells under these experimental conditions was about 1,500 d.p.m. per 4.5X106 cells. fhagocytic activity. The phagocytic activity was measured by determining microscopically the number of polystyrene latex beads ingested by the amoebae. Amoeba cells were incubated as described above in buffer solution with different concentrations of the surfactants. Following incubation the cells were washed three times with cold (4") buffer solution and resuspended in 2 ml buffer solution containing 43 pg latex beads per ml. The washing of the cells prior to the incubation with latex beads was done in order to minimize the adsorption of unbound surfactant to the latex beads. The test-tubes were incubated at 28f0.5" for 40 to 60 min. The cells were then washed three times with buffer solution. Wet smears were examined with a phase-contrast microscope and the number of ingested beads was assessed by counting the number of beads in each of the cells in fields selected at random. Each sample was examined by two observers who counted a total of about 400 cells.
210
BORIS ISOMAA FT A L .
Concentration of surfactant, M Fig. I . The abscissae show the concentration of surfactants in the incubation buffer. -+--cetyltrimethylammonium bromide (CM); +tetradecyltrimethylammonium bromide ((21,);-A- dodecyltrimethylammonium bromide ( C L ~Cell ) . concentration was 4.5X 10' cells per ml and incubation temperature 28f0.5".Each point represents the mean of three to six separate experiments. Vertical bars indicate S.E.M. a. Effect of surface-active alkyltrimethylammonium salts on pinocytosis in Acanthamoeba casiellunii. The ordinate shows relative pinocytic activity (uptake of 'H-inulin in sample/uptake of 'H-inulin in control) of amoebae incubated in a buffer containing the surfactants. b. Lytic activity of surface-active alkyltrimethylammonium salts in Acanihumoeba castellanii. The ordinate shows release of potassium from amoebae incubated in a buffer containing the surfactants. The dotted curve shows the percentage of cells killed (as judged by tryphan blue staining) by the C M homologue. The curves for the two other homologues has for sake of clarity been omitted from the figure. c. Effect of surface-active alkyltrimethylammonium salts on phagocytosis in Acanthamoeba casiellunii. The ordinate shows relative phagocytic activity (number of cells containing two or more latex beads in sampldnumber of cells containing two or more latex beads in control). In the evaluation of phagocytosis the amoebae were pretreated with the surfactants prior to the determination of phagocytosis.
CATIONIC SURFACTANTS ON PLASMA MEMBRANE
21 1
60 -
-
v)
= a, 0
m
0
I]
50-
-
-
Concentration unbound CTAB, pM Fig. 2. The adsorption of “C-CTAB to Acanthamoeba castellanii. Cell concentration was 4.5X 10’ cells per ml and incubation temperature 28f0.5”. Each point of the adsorption isotherm represents the mean of five separate experiments. Vertical and horizontal bars indicate S.E.M. The inset shows the degree of lysis of amoebae incubated in a buffer containing CTAB.
Determinaiion of radioactivity. Solubilized cells were counted in a toluene-based scintillation fluid and aqueous samples in Aquasol (New England Nuclear Corp.). The samples were counted in a liquid scintillation spectrometer and the values were corrected for quenching by internal standardization using 14C-toluene as internal standard. Treatment of glass ware. Cationic surfactants are readily adsorbed to glass. The adsorbed surfactants do not readily desorbe from the glass surface. This adsorption is trouble-some when working with weak solutions of cationic surfactants because a considerable amount of the surfactant molecules can be adsorbed on to the glass surface. Attempts were made to overcome this problem by soaking the glass ware prior to every experiment in weak solutions of the surfactants and then rinsing them several times with distilled water. This treatment was supposed to leave the glass surface covered by a monolayer of surfactant molecules. The polyethylene tubes used in determining the adsorption of “C-CTAB to the cells were treated in a similar way.
Results
Lyric activity of the alkyltrimethylammonium salts. The alkyltrimethylammonium surfactants caused a release of potassium to the surrounding medium. In fig. 1 b the percentage of potassium released is plotted against the concentration of the surfactants. The C U homologue was the least potent compound. With the cell concentration used (4.5X lo5 cells/ml) this homologue caused a release of 50% of the intracellular potassium at a concentration of about 1.6 mM. When the length of the alkyl chain increased the lytic effectiveness of the surfactants increased with about a factor of ten for every two carbon atoms added to the alkyl chain. This agrees well with observations previously made on rat erythrocytes (Isomaa 1979). Moreover there was a change in the activity versus concentration profile to a steeper curve when the length of the
212
BORIS I S O M A A FT A I . .
alkyl chain increased. When the cells were stained with tryphan blue t o assess the viability of the cells it was found that potassium release preceded cell death (fig. 1b), i.e. the membrane permeability for cations increased prior to penetration of the dye. In some experiments cells incubated with CTAB (&) were examined by transmission electron microscopy. Cells incubated in lytic concentrations of CTAB showed obvious signs of physical disruption indicating that cell death is caused by injuring the plasma membrane. The adsorption of I4C-CTAB to amoeba cells. The adsorption isotherm for the binding of I4C-CTAB to the amoebae is presented in fig. 2. The isotherm corresponds to a L4 type in the classification system of Giles et al. (1960). No saturation of the binding was observed at the concentrations of CTAB used, but there were two distinct inflections in the adsorption isotherm. The first inflection coincides with the beginning of potassium release from the cells and probably represents saturation of “binding sites” at the cell surface. The following increase in binding is probably due to accessibility of internal membrane surfaces in lysed cells. The second inflection could represent saturation of these internal surfaces. The rise in the isotherm following the second inflection is somewhat confusing. It seems as if an increase of the CTAB concentration beyond the second inflection would cause further ruption of the cells which in turn gives rise to additional surfaces on which adsorption can occur. No solubilization of cell components should have occurred at the concentrations of CTAB used. The CMC for CTAB is about 1 mM in distilled water and about 0.1 mM in a buffer solution (Salt & Wiseman 1970). The number of CTAB molecules bound per cell at a concentration corresponding to 50% release of potassium (21 pmol CTABA) was determined from the adsorption data to be 1.9X 10”. Assuming the average surface area of Acanthamoeba castellanii to be 2,200 pm2 (Bowers & Korn 1968) this corresponds to about 8.8X lo6 molecules per pm’ of the cell surface. This number is about ten times higher than that previously determined in rat erythrocytes at a concentration corresponding to 50% haemolysis (Isomaa 1979). The area that could be covered by 1.9 X 10” molecules is about four times the cell
surface assuming the area occupied by a CTAB molecule in a close packed monolayer to be 45 A’. When the amoeba cells were washed three times with cold buffer solution about 20% of the amount of 14C-CTAB initially bound was removed. The effects of alkyltrimethylammonium salts on pinocytosis. The effects of the surfactants on pinocytosis, as determined by the uptake of ’H-inulin, are presented in fig. la. With all the homologues there was an increase in uptake of ’H-inulin at higher concentrations. This increase in uptake coincided with the release of potassium and is apparently due to trapped inulin by lysed cells. At prelytic concentrations the C12 and c14 homologues caused a small increase in the uptake of ’H-inulin. This stimulation of pinocytosis was in the case of the C12 homologue followed by a region with reduced pinocytosis and this reduction occurred at concentrations corresponding to the beginning of potassium release. The C16 homologue did not alter the uptake of ’H-inulin by the amoeba cells, except for the previously mentioned increase at lytic concentrations. The effects of alkyltrimethylammonium salts on phagocytosis. There was a great variation in the number of latex beads taken up by the amoebae. In the control the number usually varied between 0 and 12 beads per cell. If the cells contained more than 10 beads the number of beads could not be accurately counted and the total number of beads ingested by the cells could thus not be quantitatively determined. The evaluation of phagocytosis was instead based on the number of cells containing two or more beads without any other regard to the number of beads in each cell. The assay of phagocytosis is thus to be considered as being only semiquantitative. Lytic concentrations of the surfactants reduced the uptake of latex beads but at prelytic concentrations the uptake was increased (fig. Ic). The stimulating effect appears to increase with an increase in the length of the alkyl chain of the surfactant but due to the great variations this can not be definitely stated. A small increase in phagocytic activity of Tetrahymena pyriformis and Paramecium caudata in the presence of prelytic concen-
CATIONIC SURFACTANTS ON PLASMA MEMBRANE
trations of ionic surfactants has recently been reported (Brutkowska & Mehr 1976). In the present investigation stimulation of phagocytosis occurred also at concentrations causing a slight release of potassium. When comparing the curves expressing phagocytosis to those of potassium release and pinocytosis in fig. 1, however, it should be remembered that the cells were washed three times with buffer solution following preincubation with surfactants in order to minimize adsorption of surfactants to the latex beads. This washing and the following incubation removed some of the surfactant molecules adsorbed to the cells. The amount of surfactant removed should decrease with increasing length of the alkyl chain according to the IipiUwater partition of the compounds. The curves expressing phagocytosis are thus shifted toward higher concentrations and the shift should be smallest for the c 1 6 homologue.
Discussion In the present study it was found that surface-active alkyltrimethylammonium salts lysed amoeba cells at premicellar concentrations. The lytic effectiveness of the surfactants increased with about an order of magnitude for every two carbon atoms added to the alkyl chain. This is in accordance with observations previously made on rat erythrocytes (Isomaa 1979) and indicates a hydrophobic nature of the interaction between the alkyltrimethylammonium surfactants and the amoeba plasma membrane. When comparing the lytic activity of the surfactants on rat erythrocytes and amoeba cells, however, they turn out to be more effective on rat erythrocytes. The amount of CTAB bound (calculated as number of CTAB molecules bound per surface unit of the plasma membrane) at concentrations giving 50% release of potassium is about ten times higher in the amoeba. This can hardly be explained by differences in the chemical composition between the erythrocyte and amoeba plasma membrane. A more probable explanation for the lower effectiveness on amoebae seems to be the very rapid surface turnover in this organism. A turnover rate of 2-10 times per hour due to pinocytosis has been reported for Acanthamoeba castellarrii and it has been suggested that the membrane deletion of the cell surface is in part replaced by rle
213
n o w synthesis and in part by recirculation ofmem-
brane components (Bowers &Olszewski 1972). I t is obvious that such a rapid turnover of the membrane interferes with the events leading to lysis of the cell. The rapid turnover of the plasma membrane has apparently also affected the shape of the adsorption isotherm. Firstly, the continuous renewal of cell surface creates fresh surfaces on which adsorption can occur. Secondly, at lethal concentrations of CTAB the renewal of cell surface should cease and no new cell surface is produced. Because of the continuous renewal of cell surface and because of the change in rate of renewal due to experimental conditions the situation is far from a true adsorption. A change in the turnover rate should affect the adsorption of CTAB to the amoeba cells and consequently the shape of the adsorption isotherm. The interpretation of the adsorption isotherm presented on p. 212 thus may suffer from much uncertainty. At prelytic concentrations the surface-active alkyltrimethylammonium salts were found to stimulate endocytosis. Phagocytosis was effected to a greater extent than pinocytosis and with the CI6 homologue there was no stimulation of pinocytosis at prelytic concentrations. The lack of effect of the c 1 6 homologue on pinocytosis is somewhat confusing. It has been shown, however, by Bowers (1977) that an increase in phagocytosis decreases the rate of pinocytosis in Acanthamoeba castellanii. In the present study the c16 homologue had a more pronounced effect on phagocytosis than the two other homologues. It is possible that the lack of effect of the c16 homologue on pinocytosis is due to this relationship between phagocytosis and pinocytosis. One possible explanation for the change in phagocytic activity is that the surfactants interfere with the attachment of the latex beads to the cell surface. Several authors have emphasized the importance of interactions between the cell surface and the surface of ingested particles in phagocytosis (Rabinovitch 1967; Nagura et a/. 1973; Griffin et al. 1975; Deierkauf et a/. 1977). Polystyrene latex beads may be negatively charged due to negative end groups of the polymer chain (Van den Hul & Vanderhoff 1968). Apart from the negatively charged groups the latex beads have
214
BORIS ISOMAA ET AI.
hydrophobic regions (Oss & Singer 1966). Thus there is the possibility of an adsorption of the alkyltrimethylammonium surfactants to the latex beads by electrostatic or/and hydrophobic forces. This would alter the charge or charge density of the latex beads and consequently affect phagocytosis. Although unadsorbed surfactant was removed by washing the cells prior to the evaluation of phagocytic activity there remains the possibility that surfactant molecules, desorbed from the cells during the experiment, adsorbed to the latex beads to such extent that the surface properties of the latex beads were significantly altered. Moreover, if the adsorption of surfactants to the amoeba plasma membrane is by electrostatic forces this should alter the charge density at the cell surface and probably also the phagocytic activity. It has been proposed by Brewer & Bell (1969) that surfaceactive alkyltrimethylammonium salts are bound to negatively charged groups of mucopolysaccarides of the surface coat of Amoeba proteus. There are, however, many studies indicating that cationic surfactants are adsorbed into the lipid bilayer of the plasma membrane (Hooghwinkel e t a / . 1965; Kondo & Tomizawa 1966; Reman e f al. 1969; Isomaa et al. 1979; Isomaa 1979). In Acanthamoeba, which lacks a surface coat, adsorbed alkyltrimethylammonium salts are probably buried with their alkyl chain between the lipid molecules of the lipid bilayer of the membrane and with their polar heads at the lipid-water interface. This would, of course, introduce positive charges into the plasma membrane, but these charges are probably not relevant in phagocytosis. In the present investigation both pinocytosis and phagocytosis were stimulated at about the same concentrations of surfactants. According to Bowers & Olszewski (1972) pinocytosis in Acanfhamoeba is a continuous process which does not depend on surface binding of molecules and which is not enhanced by molecules that induce pinocytosis in Chaos chaos and Amoeba proteus. Thus the fact that both pinocytosis and phagocytosis were stimulated by the surfactants does not support the idea that the increased phagocytic activity is due to alterations of surface charges of the plasma membrane or/and of the latex beads. In trying to explain the stimulating effect of the alkyltrimethylammonium surfactants at prelytic
concentrations there are several processes, apart from surface interactions, which call for attention. These include for example microtubule-dependent membrane alterations, permeability properties of the plasma membrane, binding of Ca” to phospholipids of the lipid bilayer and the “fluidity” of the plasma membrane. Although present data d o not allow any elucidation of the mechanism underlying the stimulation of endocytosis by the alkyltrimethylammonium surfactants we would like to draw the attention to the possibility that this effect could be produced by an increase in the “fluidity” of the plasma membrane. In a previous work (Isornaa 1979) it was found that surface-active alkyltrimethylammonium salts at prelytic concentrations protect erythrocytes from hypotonic haemolysis. Protection against hypotonic haemolysis is a n effect shown by many lipid-soluble anaesthetics and tranquilizers (Seeman 1972). As far as these agents are concerned the protection from haemolysis is associated with an expansion of the plasma membrane (Seeman et al. 1969; Roth & Seeman 1972), and it is assumed that the expansion of the membrane stems primarily from a “fluidizing” effect of the anaesthetics on the lipid bilayer (Seeman 1972; Lee 1976). Surface-active alkylttimethylammonium salts have been found to initially lower the transition temperature of phospholipid model membranes (Eliasz et al. 1976). Thus it appears that the protection against hypotonic haemolysis in the case of surface-active alkyltrimethylammonium salts also could be attributed a “fluidizing” effect of the surfactants on the lipid bilayer. An increase in the “fluidity” of the bilayer of the plasma membrane could possibly increase the rate of endocytosis. References Bowers, B.: Comparison of pinocytosis and phagocytosis in Acanthamoeba castellanii. Exp. Cell. Res. 1977, 110. 409-417. Bowers, B. & E. D. Korn: The fine structure ofdcanthamoeba castellanii. I. The trophozoite.J. Cell. Biol. 1968, 39, 95-1 1 1 . Bowers, B. & T. E. Olszewski: Pinocytosis in Acanthamoeba castellanii. Kinetics and morphology. J. Cell. Biol. 1972, 53, 681-694. Brewer, J. E. & L. G. E. Bell: Reaction of detergents with Amoeba proteus. Nature (London) 1969,222,891-892. Brutkowska, M. & K. Mehr: Effects of ionic detergents on
CATIONIC SURFACTANTS ON PLASMA MEMBRANE phagocytic activity of Tetrahymena pyriformis G L and Paramecium caudatum. Acta protozool. 1976,15,66-76. Dierkauf, F. A., H. Beukers, M. Deierkauf & J. C. Riemersma: Phagocytosis by rabbit polymorphonuclear leukocytes: The effect of albumin and polyamino acids on latex uptake. J. Cell. Physiol. 1977, 92, 169176. Drainville, G. & A. Gagnon: Osmoregulation in Acanthamoeba castellanii. I. Variations of the concentrations of free intracellular amino acids and of the water content. Comp. Biochem. Physiol. 1973, 45A, 379-388. Eliasz, A. W., D. Chapman & D. F. Ewing: Phospholipid phase transitions. Effects of n-alcohols, n-monocarboxylicacids, phenylalkyl alcohols and quaternary ammonium compounds. Biochim. Biophys. Acta 1976, 448, 220-230. Giles, C. H., T. H. MacEwan, S. N. Nakhwa & D. Smith: Studies in adsorption. Part XI. A system of classification of solution adsorption isotherms, and its use in diagnosis of adsorption mechanisms and in measurement of specific surface areas of solids. J. Chem. Soc. 1960, 3973-3993. Griffin, F. M., Jr., J. A. Griffin, J. E. Leider & S. C. Silverstein: Studies on the mechanism of phagocytosis. J. Exp. Med. 1975, 142, 1263-1282. Haydon, D. A. & J. Taylor: The stability and properties of bimolecular lipid leaflets in aqueous solutions. J. Theoret. Biol. 1963,4, 281-296. Hooghwinkel, G. J. M., R. E. De Rooij & H. R. Dankmeijer: The mechanism of hemolysis by various types of surfactants. A d a Physiol. Neerl. 1965, 13, 303-316. Hul, H. J. van den & J. W. Vanderhoff: Well-characterized monodisperse latexes. J. Coll. Interface Sci. 1968, 28, 336-337. Isomaa, B.: Interactions of surface-active alkyltrimethylammonium salts with the erythrocyte membrane. Biochem. Pharmacol. 1979, 27, in press. Isomaa, B., H. Bergman & P. Sandberg: The binding of CTAB, a cationic surfactant, to the rat erythrocyte membrane. Actapharmarol. et ioxicol. 1979,44,36-42. Kondo, T. & M. Tomizawa: Hemolytic action of surfaceactive electrolytes. J. Coll. Interface Sci. 1966, 21, 224-228.
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Lee, A. G.: Interactions between anaesthetics and lipid mixtures amines. Biochim. Biophys. Acta 1976, 448, 34-44. Nagura, H., J . Asai, Y. Katsumata & K. Kojima: Role of electric surface charge of cell membrane in phagocytosis. Acta Path. Jap. 1973, 23, 279-290. Neff, R. J., S. A. Ray, W. F. Benton & M. Wilborn: Induction of synchronous encystment (differentiation) in Acanthamoeba sp. In: Methods in cellphysiology. Volume I. Ed.: D. M. Prescott. Academic Press,NewYork and London, 1964, pp. 55-83. Oss, C. J. van & J. M. Singer: The binding of immune globulins and other proteins by polystyrene latex particles. J. Reticuloendothel. Soc. 1966, 3, 29-40. Rabinovitch, M.: The dissociation of the attachment and ingestion phases of phagocytosis by macrophages. Exp. Cell. Res. 1967, 46, 19-28. Reman, F. C., R. A. Demel, J. De Gier, L. L. M. Van Deenen, H. Eibl& 0. Westphal: Studies on the lysis of red cells and bimolecular lipid leaflets by synthetic lysolecithins, lecithins and structural analogs. Chem. Phys. Lipids 1969, 3, 221-233. Roth, S. & P. Seeman: Anesthetics expand erythrocyte membranes without causing loss of K'. Biochim. Biophys. Acta 1972, 255, 190-198. Salt, W. G . & D. Wiseman: The effect of magnesium ions and Tris buffer on the uptake of cetyl trimethyl ammonium bromide by Escherichia coli. J. Pharm. Pharmacol. 1970, 22, 767-773. Seeman, P.: The membrane actions of anesthetics and tranquilizers. Pharmacol. Rev. 1972, 24, 583-655. Seeman, P., W. 0. Kwant, T. S a u k s t W. Argent: Membrane expansion of intact erythrocytes by anesthetics. Biochim. Biophys. Acta 1969, 183, 490-498. Seufert, W. D.: Induced permeability changes in reconstituted cell membrane structure. Nature (London) 1965, 207, 174-176. Ulsamer, A. G., P. L. Wright, M. G . Wetzel & E. D. Korn: Plasma and phagosome membranes of Acanthamoeba castellanii. J. Cell Biol. 1971, 51, 193-215. Wiseman, R. A. & E. D. Korn: Phagocytosis of latex beads by Acanthamoeba castellanii. I. Biochemical properties. Biochemistry 1967, 6, 485-497.
From the Institute of Biology, i(bo Akademi, 20 SO0 Abo SO, Finland
Interactions of Surface-active Alkyltrimethylammonium Salts with the Plasma Membrane of Acanthamoeba castellanii BY Boris Isomaa, Gun Paatero and Christer Lonnqvist (Received August 30, 1978; Accepted October 16, 1978)
Absfracf: The interactions of three surface-active alkyltrimethylammonium salts (C12-Cls) with the plasma membrane of Acanthamoeba cusfellanii were studied. The surfactants caused a release of K’ from the cells at premicellar concentrations. The lytic effectiveness of the surfactants increased with an increase in the length of the alkyl chain with about an order of magnitude for every two carbon atoms added to the alkyl chain. Binding studies with the C Mhomologue revealed that at a concentration corresponding to SO% release of K’ there were about 1.9X 10” molecules bound per cell. At prelytic concentrations the surfactants stimulated phagocytosis and pinocytosis. The mode of action of the surfactants on the plasma membrane ofAcanthamoeba casrellanii is discussed and it is hypothesized that the stimulation of endocytosis is due to a “fluidizing” effect of the surfactants on the lipid bilayer of the plasma membrane. Key-words: Cationic surfactants
- lysis - endocytosis - Acanfhamoeba castellanii.
Because of their importance as antiseptic agents, the actions of surface-active alkyltrimethylammonium salts on cells have been subjected to many studies. Several authors have suggested that the principal point of attack of cationic surfactants is the lipid bilayer of the plasma membrane (Hooghwinkel et al. 1965; Kondo & Tomizawa 1966; Reman et al. 1969; Isomaa et al. 1979). The surfactant molecules are assumed to penetrate into the lipid bilayer which they tend t o break up into localized micellar arrangements (Haydon & Taylor 1963; Seufert 1965). The ability of the membrane to prevent free exchange of ions is abolished and the cell is lysed by secondary osmotic effects (Isomaa 1979). In a previous study with rat erythrocytes it was found that surface-active alkyltrimethylammonium salts, in addition to their lytic effect, stabilize erythrocytes against hypotonic haemolysis at prelytic concentrations (Isomaa 1979). I t was suggested that this stabilizing is due to a
“fluidizing” effect of the surfactants on the lipid bilayer of the plasma membrane. The purpose of this study was to examine the interactions of surface-active alkyltrimethylammonium salts with the plasma membrane of Acanthamoeba castellanii. This small soil amoeba has no surface coat external to the plasma membrane (Bowers & Korn 1968) and the gross chemical composition of its plasma membrane is not very different from the composition of other eucaryotics (Ulsamer et al. 1971). Unlike mammalian erythrocytes this amoeba has a very high rate of endocytosis in laboratory cultures (Bowers 1977) which makes it a very suitable organism when studying the interactions of various agents with membrane-localized processes. Materials and Methods Chemicals. Cetyltrimethylammonium bromide (CTAB,
CATIONIC SURFACTANTS ON PLASMA MEMBRANE c16), 99% pure, was obtained from E. Merck AG, Darmstadt, Germany. Dodecyltrimethylammonium bromide (Cit) and tetradecyltrimethylammonium bromide (CI4) were generously supplied by MoDoKemi AB, Stenungsund, Sweden. These compounds had a chemical purity of about 98%. Trimethyl-(I-"C)cetyltrimethylammonium bromide (%-CTAB) with a specific activity of 6.4 mci/mmol and radiochemical purity of 97%. and 'H-inulin with a specific activity of 900 mci/mmol and a radiochemical purity of 99% were obtained from the Radiochemical Centre, Amersham, Bucks, England. All other chemicals used were of analytical grade. Polystyrene latex beads with a particle diameter of 1.01 pm were obtained as an aqueous suspension from Serva Feinbiochemica, Heidelberg, Germany. To remove emulsifiers and other soluble substances the latex beads were dialyzed against 5 1 distilled water at room temperature for 6 hrs. The latex beads were stored in distilled water and they were sonicated in an ultrasonic bath for about 5 min. prior to their use in the experiments.
Cell culturing. Acanthamoeba castellanii was obtained from the Culture Centre of Algae and Protozoa, Cambridge, England. The cells were cultured axenically in Neffs optimal growth medium (Neff er al. 1964), as modified by Drainvilli & Gagnon (1973). The temperature was maintained at 2 8 f I 0 and the cultures were shaked at 100 r.p.m. on a rotary shaker. The cells were collected from cultures that were in a late log-phase (7-10 days old) by centrifuging at 800Xg for 5 min. The cells were washed three times in a buffer solution of the following composition: 100 mM-NaCI, 10 mM-NaH2P04, 0.5 mM-CaCIz, 0.5 mM-MgSO4, pH 7.0. The cells were then resuspended in the buffer solution and the cell concentration of this stock suspension was adjusted to 4.5X10' cells per ml. Cell counts were made using a haemocytometer. Measuremenr o j ' r release. One ml of the stock amoeba suspension was added to glass test-tubes containing 9 ml buffer solution and various amounts of the surfactants. The contents of the tubes were briefly mixed with a vortex mixer and the tubes were incubated for 1 hr at 28 f0.5"in a shaking thermostat bath. The tubes were centrifuged at 800Xg for 5 min. and washed three times with the buffer solution. The cells were then resuspended in 9 ml distilled water and, to rupture the cells, the suspensions were sonicated in an ultrasonic bath for 5 min. The tubes were centrifuged at 2,OOOXg for 10 min. and the potassium content of the supernatant was determined with an atomic absorption spectrophotometer at 766.5 nm using an air-acetylene flame. The amount of K' released was estimated from standard curves prepared by sonicating known number of cells. The experimental values were corrected for the release of K' occurring in a test-tube without surfactant. Viability counts. Amoeba cells were incubated as described above in a buffer solution containing various
209
amounts of the surfactants. Following incubation the viability of the cells was assessed by adding 1 vol. of tryphan blue (0.16% in buffer solution) to 2 vol. of the amoeba suspensions and counting the number of stained ("dead") and unstained ("living") cells in a haemocytometer. Cell counts were made within 10 min. of mixing the cells with tryphan blue. Adsorption of I4C-CTAB to the amoeba cells. Amoeba cells were incubated as described above in buffer solution with different concentrations (1-70 pM)of '"C-CTABCTAB. The specific activity of the I4C-CTAB-CTAB mixture was 0.35 mci/mmol. Following incubation the suspensions were centrifuged in polyethylene tubes at 9,OOOXg for 10 min. and I ml of the supernatants were taken to determine radioactivity. In someexperiments the cells were washed, following incubation, three times with cold (4') buffer solution by centrifugation at 2,OOOXg for 10 min. and 1 ml of the supernatants were taken to determine radioactivity. finocytic acrivit.p. The pinocytic activity of the amoebae was determined by measuring the uptake of 'H-inulin. This carbohydrate is in the amoeba taken up by pinocytosis (Bowers & Olszewski 1972). Amoeba cells (4.5X 10') were incubated for 1 hr as described above in 10 ml buffer solution containing 10 p i 'H-inulin (56 pg inulin) and various amounts of the surfactants. Pinocytosis was stopped by adding dinitrophenol to a final concentration of 0.2 mM (Wiseman & Korn 1967). The cells were harvested by centrifugation at 2,ooQ)Xgfor 5 min. and washed three times with cold buffer solution. The cell pellets were solubilized in Protosol (New England Nuclear Corp., Boston, Mass., USA) at room temperature and aliquots of the solubilized samples were taken to determine radioactivity. In the control the amount of radioactivity taken up by the cells under these experimental conditions was about 1,500 d.p.m. per 4.5X106 cells. fhagocytic activity. The phagocytic activity was measured by determining microscopically the number of polystyrene latex beads ingested by the amoebae. Amoeba cells were incubated as described above in buffer solution with different concentrations of the surfactants. Following incubation the cells were washed three times with cold (4") buffer solution and resuspended in 2 ml buffer solution containing 43 pg latex beads per ml. The washing of the cells prior to the incubation with latex beads was done in order to minimize the adsorption of unbound surfactant to the latex beads. The test-tubes were incubated at 28f0.5" for 40 to 60 min. The cells were then washed three times with buffer solution. Wet smears were examined with a phase-contrast microscope and the number of ingested beads was assessed by counting the number of beads in each of the cells in fields selected at random. Each sample was examined by two observers who counted a total of about 400 cells.
210
BORIS ISOMAA FT A L .
Concentration of surfactant, M Fig. I . The abscissae show the concentration of surfactants in the incubation buffer. -+--cetyltrimethylammonium bromide (CM); +tetradecyltrimethylammonium bromide ((21,);-A- dodecyltrimethylammonium bromide ( C L ~Cell ) . concentration was 4.5X 10' cells per ml and incubation temperature 28f0.5".Each point represents the mean of three to six separate experiments. Vertical bars indicate S.E.M. a. Effect of surface-active alkyltrimethylammonium salts on pinocytosis in Acanthamoeba casiellunii. The ordinate shows relative pinocytic activity (uptake of 'H-inulin in sample/uptake of 'H-inulin in control) of amoebae incubated in a buffer containing the surfactants. b. Lytic activity of surface-active alkyltrimethylammonium salts in Acanihumoeba castellanii. The ordinate shows release of potassium from amoebae incubated in a buffer containing the surfactants. The dotted curve shows the percentage of cells killed (as judged by tryphan blue staining) by the C M homologue. The curves for the two other homologues has for sake of clarity been omitted from the figure. c. Effect of surface-active alkyltrimethylammonium salts on phagocytosis in Acanthamoeba casiellunii. The ordinate shows relative phagocytic activity (number of cells containing two or more latex beads in sampldnumber of cells containing two or more latex beads in control). In the evaluation of phagocytosis the amoebae were pretreated with the surfactants prior to the determination of phagocytosis.
CATIONIC SURFACTANTS ON PLASMA MEMBRANE
21 1
60 -
-
v)
= a, 0
m
0
I]
50-
-
-
Concentration unbound CTAB, pM Fig. 2. The adsorption of “C-CTAB to Acanthamoeba castellanii. Cell concentration was 4.5X 10’ cells per ml and incubation temperature 28f0.5”. Each point of the adsorption isotherm represents the mean of five separate experiments. Vertical and horizontal bars indicate S.E.M. The inset shows the degree of lysis of amoebae incubated in a buffer containing CTAB.
Determinaiion of radioactivity. Solubilized cells were counted in a toluene-based scintillation fluid and aqueous samples in Aquasol (New England Nuclear Corp.). The samples were counted in a liquid scintillation spectrometer and the values were corrected for quenching by internal standardization using 14C-toluene as internal standard. Treatment of glass ware. Cationic surfactants are readily adsorbed to glass. The adsorbed surfactants do not readily desorbe from the glass surface. This adsorption is trouble-some when working with weak solutions of cationic surfactants because a considerable amount of the surfactant molecules can be adsorbed on to the glass surface. Attempts were made to overcome this problem by soaking the glass ware prior to every experiment in weak solutions of the surfactants and then rinsing them several times with distilled water. This treatment was supposed to leave the glass surface covered by a monolayer of surfactant molecules. The polyethylene tubes used in determining the adsorption of “C-CTAB to the cells were treated in a similar way.
Results
Lyric activity of the alkyltrimethylammonium salts. The alkyltrimethylammonium surfactants caused a release of potassium to the surrounding medium. In fig. 1 b the percentage of potassium released is plotted against the concentration of the surfactants. The C U homologue was the least potent compound. With the cell concentration used (4.5X lo5 cells/ml) this homologue caused a release of 50% of the intracellular potassium at a concentration of about 1.6 mM. When the length of the alkyl chain increased the lytic effectiveness of the surfactants increased with about a factor of ten for every two carbon atoms added to the alkyl chain. This agrees well with observations previously made on rat erythrocytes (Isomaa 1979). Moreover there was a change in the activity versus concentration profile to a steeper curve when the length of the
212
BORIS I S O M A A FT A I . .
alkyl chain increased. When the cells were stained with tryphan blue t o assess the viability of the cells it was found that potassium release preceded cell death (fig. 1b), i.e. the membrane permeability for cations increased prior to penetration of the dye. In some experiments cells incubated with CTAB (&) were examined by transmission electron microscopy. Cells incubated in lytic concentrations of CTAB showed obvious signs of physical disruption indicating that cell death is caused by injuring the plasma membrane. The adsorption of I4C-CTAB to amoeba cells. The adsorption isotherm for the binding of I4C-CTAB to the amoebae is presented in fig. 2. The isotherm corresponds to a L4 type in the classification system of Giles et al. (1960). No saturation of the binding was observed at the concentrations of CTAB used, but there were two distinct inflections in the adsorption isotherm. The first inflection coincides with the beginning of potassium release from the cells and probably represents saturation of “binding sites” at the cell surface. The following increase in binding is probably due to accessibility of internal membrane surfaces in lysed cells. The second inflection could represent saturation of these internal surfaces. The rise in the isotherm following the second inflection is somewhat confusing. It seems as if an increase of the CTAB concentration beyond the second inflection would cause further ruption of the cells which in turn gives rise to additional surfaces on which adsorption can occur. No solubilization of cell components should have occurred at the concentrations of CTAB used. The CMC for CTAB is about 1 mM in distilled water and about 0.1 mM in a buffer solution (Salt & Wiseman 1970). The number of CTAB molecules bound per cell at a concentration corresponding to 50% release of potassium (21 pmol CTABA) was determined from the adsorption data to be 1.9X 10”. Assuming the average surface area of Acanthamoeba castellanii to be 2,200 pm2 (Bowers & Korn 1968) this corresponds to about 8.8X lo6 molecules per pm’ of the cell surface. This number is about ten times higher than that previously determined in rat erythrocytes at a concentration corresponding to 50% haemolysis (Isomaa 1979). The area that could be covered by 1.9 X 10” molecules is about four times the cell
surface assuming the area occupied by a CTAB molecule in a close packed monolayer to be 45 A’. When the amoeba cells were washed three times with cold buffer solution about 20% of the amount of 14C-CTAB initially bound was removed. The effects of alkyltrimethylammonium salts on pinocytosis. The effects of the surfactants on pinocytosis, as determined by the uptake of ’H-inulin, are presented in fig. la. With all the homologues there was an increase in uptake of ’H-inulin at higher concentrations. This increase in uptake coincided with the release of potassium and is apparently due to trapped inulin by lysed cells. At prelytic concentrations the C12 and c14 homologues caused a small increase in the uptake of ’H-inulin. This stimulation of pinocytosis was in the case of the C12 homologue followed by a region with reduced pinocytosis and this reduction occurred at concentrations corresponding to the beginning of potassium release. The C16 homologue did not alter the uptake of ’H-inulin by the amoeba cells, except for the previously mentioned increase at lytic concentrations. The effects of alkyltrimethylammonium salts on phagocytosis. There was a great variation in the number of latex beads taken up by the amoebae. In the control the number usually varied between 0 and 12 beads per cell. If the cells contained more than 10 beads the number of beads could not be accurately counted and the total number of beads ingested by the cells could thus not be quantitatively determined. The evaluation of phagocytosis was instead based on the number of cells containing two or more beads without any other regard to the number of beads in each cell. The assay of phagocytosis is thus to be considered as being only semiquantitative. Lytic concentrations of the surfactants reduced the uptake of latex beads but at prelytic concentrations the uptake was increased (fig. Ic). The stimulating effect appears to increase with an increase in the length of the alkyl chain of the surfactant but due to the great variations this can not be definitely stated. A small increase in phagocytic activity of Tetrahymena pyriformis and Paramecium caudata in the presence of prelytic concen-
CATIONIC SURFACTANTS ON PLASMA MEMBRANE
trations of ionic surfactants has recently been reported (Brutkowska & Mehr 1976). In the present investigation stimulation of phagocytosis occurred also at concentrations causing a slight release of potassium. When comparing the curves expressing phagocytosis to those of potassium release and pinocytosis in fig. 1, however, it should be remembered that the cells were washed three times with buffer solution following preincubation with surfactants in order to minimize adsorption of surfactants to the latex beads. This washing and the following incubation removed some of the surfactant molecules adsorbed to the cells. The amount of surfactant removed should decrease with increasing length of the alkyl chain according to the IipiUwater partition of the compounds. The curves expressing phagocytosis are thus shifted toward higher concentrations and the shift should be smallest for the c 1 6 homologue.
Discussion In the present study it was found that surface-active alkyltrimethylammonium salts lysed amoeba cells at premicellar concentrations. The lytic effectiveness of the surfactants increased with about an order of magnitude for every two carbon atoms added to the alkyl chain. This is in accordance with observations previously made on rat erythrocytes (Isomaa 1979) and indicates a hydrophobic nature of the interaction between the alkyltrimethylammonium surfactants and the amoeba plasma membrane. When comparing the lytic activity of the surfactants on rat erythrocytes and amoeba cells, however, they turn out to be more effective on rat erythrocytes. The amount of CTAB bound (calculated as number of CTAB molecules bound per surface unit of the plasma membrane) at concentrations giving 50% release of potassium is about ten times higher in the amoeba. This can hardly be explained by differences in the chemical composition between the erythrocyte and amoeba plasma membrane. A more probable explanation for the lower effectiveness on amoebae seems to be the very rapid surface turnover in this organism. A turnover rate of 2-10 times per hour due to pinocytosis has been reported for Acanthamoeba castellarrii and it has been suggested that the membrane deletion of the cell surface is in part replaced by rle
213
n o w synthesis and in part by recirculation ofmem-
brane components (Bowers &Olszewski 1972). I t is obvious that such a rapid turnover of the membrane interferes with the events leading to lysis of the cell. The rapid turnover of the plasma membrane has apparently also affected the shape of the adsorption isotherm. Firstly, the continuous renewal of cell surface creates fresh surfaces on which adsorption can occur. Secondly, at lethal concentrations of CTAB the renewal of cell surface should cease and no new cell surface is produced. Because of the continuous renewal of cell surface and because of the change in rate of renewal due to experimental conditions the situation is far from a true adsorption. A change in the turnover rate should affect the adsorption of CTAB to the amoeba cells and consequently the shape of the adsorption isotherm. The interpretation of the adsorption isotherm presented on p. 212 thus may suffer from much uncertainty. At prelytic concentrations the surface-active alkyltrimethylammonium salts were found to stimulate endocytosis. Phagocytosis was effected to a greater extent than pinocytosis and with the CI6 homologue there was no stimulation of pinocytosis at prelytic concentrations. The lack of effect of the c 1 6 homologue on pinocytosis is somewhat confusing. It has been shown, however, by Bowers (1977) that an increase in phagocytosis decreases the rate of pinocytosis in Acanthamoeba castellanii. In the present study the c16 homologue had a more pronounced effect on phagocytosis than the two other homologues. It is possible that the lack of effect of the c16 homologue on pinocytosis is due to this relationship between phagocytosis and pinocytosis. One possible explanation for the change in phagocytic activity is that the surfactants interfere with the attachment of the latex beads to the cell surface. Several authors have emphasized the importance of interactions between the cell surface and the surface of ingested particles in phagocytosis (Rabinovitch 1967; Nagura et a/. 1973; Griffin et al. 1975; Deierkauf et a/. 1977). Polystyrene latex beads may be negatively charged due to negative end groups of the polymer chain (Van den Hul & Vanderhoff 1968). Apart from the negatively charged groups the latex beads have
214
BORIS ISOMAA ET AI.
hydrophobic regions (Oss & Singer 1966). Thus there is the possibility of an adsorption of the alkyltrimethylammonium surfactants to the latex beads by electrostatic or/and hydrophobic forces. This would alter the charge or charge density of the latex beads and consequently affect phagocytosis. Although unadsorbed surfactant was removed by washing the cells prior to the evaluation of phagocytic activity there remains the possibility that surfactant molecules, desorbed from the cells during the experiment, adsorbed to the latex beads to such extent that the surface properties of the latex beads were significantly altered. Moreover, if the adsorption of surfactants to the amoeba plasma membrane is by electrostatic forces this should alter the charge density at the cell surface and probably also the phagocytic activity. It has been proposed by Brewer & Bell (1969) that surfaceactive alkyltrimethylammonium salts are bound to negatively charged groups of mucopolysaccarides of the surface coat of Amoeba proteus. There are, however, many studies indicating that cationic surfactants are adsorbed into the lipid bilayer of the plasma membrane (Hooghwinkel e t a / . 1965; Kondo & Tomizawa 1966; Reman e f al. 1969; Isomaa et al. 1979; Isomaa 1979). In Acanthamoeba, which lacks a surface coat, adsorbed alkyltrimethylammonium salts are probably buried with their alkyl chain between the lipid molecules of the lipid bilayer of the membrane and with their polar heads at the lipid-water interface. This would, of course, introduce positive charges into the plasma membrane, but these charges are probably not relevant in phagocytosis. In the present investigation both pinocytosis and phagocytosis were stimulated at about the same concentrations of surfactants. According to Bowers & Olszewski (1972) pinocytosis in Acanfhamoeba is a continuous process which does not depend on surface binding of molecules and which is not enhanced by molecules that induce pinocytosis in Chaos chaos and Amoeba proteus. Thus the fact that both pinocytosis and phagocytosis were stimulated by the surfactants does not support the idea that the increased phagocytic activity is due to alterations of surface charges of the plasma membrane or/and of the latex beads. In trying to explain the stimulating effect of the alkyltrimethylammonium surfactants at prelytic
concentrations there are several processes, apart from surface interactions, which call for attention. These include for example microtubule-dependent membrane alterations, permeability properties of the plasma membrane, binding of Ca” to phospholipids of the lipid bilayer and the “fluidity” of the plasma membrane. Although present data d o not allow any elucidation of the mechanism underlying the stimulation of endocytosis by the alkyltrimethylammonium surfactants we would like to draw the attention to the possibility that this effect could be produced by an increase in the “fluidity” of the plasma membrane. In a previous work (Isornaa 1979) it was found that surface-active alkyltrimethylammonium salts at prelytic concentrations protect erythrocytes from hypotonic haemolysis. Protection against hypotonic haemolysis is a n effect shown by many lipid-soluble anaesthetics and tranquilizers (Seeman 1972). As far as these agents are concerned the protection from haemolysis is associated with an expansion of the plasma membrane (Seeman et al. 1969; Roth & Seeman 1972), and it is assumed that the expansion of the membrane stems primarily from a “fluidizing” effect of the anaesthetics on the lipid bilayer (Seeman 1972; Lee 1976). Surface-active alkylttimethylammonium salts have been found to initially lower the transition temperature of phospholipid model membranes (Eliasz et al. 1976). Thus it appears that the protection against hypotonic haemolysis in the case of surface-active alkyltrimethylammonium salts also could be attributed a “fluidizing” effect of the surfactants on the lipid bilayer. An increase in the “fluidity” of the bilayer of the plasma membrane could possibly increase the rate of endocytosis. References Bowers, B.: Comparison of pinocytosis and phagocytosis in Acanthamoeba castellanii. Exp. Cell. Res. 1977, 110. 409-417. Bowers, B. & E. D. Korn: The fine structure ofdcanthamoeba castellanii. I. The trophozoite.J. Cell. Biol. 1968, 39, 95-1 1 1 . Bowers, B. & T. E. Olszewski: Pinocytosis in Acanthamoeba castellanii. Kinetics and morphology. J. Cell. Biol. 1972, 53, 681-694. Brewer, J. E. & L. G. E. Bell: Reaction of detergents with Amoeba proteus. Nature (London) 1969,222,891-892. Brutkowska, M. & K. Mehr: Effects of ionic detergents on
CATIONIC SURFACTANTS ON PLASMA MEMBRANE phagocytic activity of Tetrahymena pyriformis G L and Paramecium caudatum. Acta protozool. 1976,15,66-76. Dierkauf, F. A., H. Beukers, M. Deierkauf & J. C. Riemersma: Phagocytosis by rabbit polymorphonuclear leukocytes: The effect of albumin and polyamino acids on latex uptake. J. Cell. Physiol. 1977, 92, 169176. Drainville, G. & A. Gagnon: Osmoregulation in Acanthamoeba castellanii. I. Variations of the concentrations of free intracellular amino acids and of the water content. Comp. Biochem. Physiol. 1973, 45A, 379-388. Eliasz, A. W., D. Chapman & D. F. Ewing: Phospholipid phase transitions. Effects of n-alcohols, n-monocarboxylicacids, phenylalkyl alcohols and quaternary ammonium compounds. Biochim. Biophys. Acta 1976, 448, 220-230. Giles, C. H., T. H. MacEwan, S. N. Nakhwa & D. Smith: Studies in adsorption. Part XI. A system of classification of solution adsorption isotherms, and its use in diagnosis of adsorption mechanisms and in measurement of specific surface areas of solids. J. Chem. Soc. 1960, 3973-3993. Griffin, F. M., Jr., J. A. Griffin, J. E. Leider & S. C. Silverstein: Studies on the mechanism of phagocytosis. J. Exp. Med. 1975, 142, 1263-1282. Haydon, D. A. & J. Taylor: The stability and properties of bimolecular lipid leaflets in aqueous solutions. J. Theoret. Biol. 1963,4, 281-296. Hooghwinkel, G. J. M., R. E. De Rooij & H. R. Dankmeijer: The mechanism of hemolysis by various types of surfactants. A d a Physiol. Neerl. 1965, 13, 303-316. Hul, H. J. van den & J. W. Vanderhoff: Well-characterized monodisperse latexes. J. Coll. Interface Sci. 1968, 28, 336-337. Isomaa, B.: Interactions of surface-active alkyltrimethylammonium salts with the erythrocyte membrane. Biochem. Pharmacol. 1979, 27, in press. Isomaa, B., H. Bergman & P. Sandberg: The binding of CTAB, a cationic surfactant, to the rat erythrocyte membrane. Actapharmarol. et ioxicol. 1979,44,36-42. Kondo, T. & M. Tomizawa: Hemolytic action of surfaceactive electrolytes. J. Coll. Interface Sci. 1966, 21, 224-228.
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Lee, A. G.: Interactions between anaesthetics and lipid mixtures amines. Biochim. Biophys. Acta 1976, 448, 34-44. Nagura, H., J . Asai, Y. Katsumata & K. Kojima: Role of electric surface charge of cell membrane in phagocytosis. Acta Path. Jap. 1973, 23, 279-290. Neff, R. J., S. A. Ray, W. F. Benton & M. Wilborn: Induction of synchronous encystment (differentiation) in Acanthamoeba sp. In: Methods in cellphysiology. Volume I. Ed.: D. M. Prescott. Academic Press,NewYork and London, 1964, pp. 55-83. Oss, C. J. van & J. M. Singer: The binding of immune globulins and other proteins by polystyrene latex particles. J. Reticuloendothel. Soc. 1966, 3, 29-40. Rabinovitch, M.: The dissociation of the attachment and ingestion phases of phagocytosis by macrophages. Exp. Cell. Res. 1967, 46, 19-28. Reman, F. C., R. A. Demel, J. De Gier, L. L. M. Van Deenen, H. Eibl& 0. Westphal: Studies on the lysis of red cells and bimolecular lipid leaflets by synthetic lysolecithins, lecithins and structural analogs. Chem. Phys. Lipids 1969, 3, 221-233. Roth, S. & P. Seeman: Anesthetics expand erythrocyte membranes without causing loss of K'. Biochim. Biophys. Acta 1972, 255, 190-198. Salt, W. G . & D. Wiseman: The effect of magnesium ions and Tris buffer on the uptake of cetyl trimethyl ammonium bromide by Escherichia coli. J. Pharm. Pharmacol. 1970, 22, 767-773. Seeman, P.: The membrane actions of anesthetics and tranquilizers. Pharmacol. Rev. 1972, 24, 583-655. Seeman, P., W. 0. Kwant, T. S a u k s t W. Argent: Membrane expansion of intact erythrocytes by anesthetics. Biochim. Biophys. Acta 1969, 183, 490-498. Seufert, W. D.: Induced permeability changes in reconstituted cell membrane structure. Nature (London) 1965, 207, 174-176. Ulsamer, A. G., P. L. Wright, M. G . Wetzel & E. D. Korn: Plasma and phagosome membranes of Acanthamoeba castellanii. J. Cell Biol. 1971, 51, 193-215. Wiseman, R. A. & E. D. Korn: Phagocytosis of latex beads by Acanthamoeba castellanii. I. Biochemical properties. Biochemistry 1967, 6, 485-497.
