Selective inhibition of calcium-stimulated cation-induced pinocytosis by starvation and inhibitors of protein synthesis in Amoeba proteus

Experimental Cell Research 154 (1984) 367-375

Selective Inhibition of Calcium-stimulated Cation-induced Pinocytosis by Starvation and Inhibitors of Protein Synthesis in Amoeba proteus P. JOHANSSON and J.-O. JOSEFSSON* Department of Pharmacology, University of Lund, S-22362 Lund, Sweden

The capacity of Amoeba proteus to form pinocytotic channels after pretreatment with either puromycin, cycloheximide, emetine or a long period of starvation was studied. The effect on pinocytosis of the three inhibitors of protein synthesis was similar. They preferentially affected pinocytosis induced by Na+ with little effect on Kf-induced pinocytosis. In Ca’+-deficient media, Nat-induced pinocytosis was inhibited, while the addition of Ca*+ restored channel formation. The degree of inhibition of Na+-induced pinocytosis was influenced by the concentration of Ca2+ in the inducing solution. Selective Ca’+reversible inhibition of Na+-induced pinocytosis also occurred after starvation or treatment with a proteolytic enzyme, subtilisin. The membrane potential in starved or emetinetreated cells in culture medium was normal and their depolarising response to inducers was not diminished in solutions containing NaC. The resting input resistance of these cells was higher than in normal amoebae, but no significant difference in electrical parameters was observed after pinocytosis was induced. It is suggested that starvation, inhibition of protein synthesis, and enzyme digestion deplete the membrane of structures which are necessary for normal Ca2+ functions during induction of pinocytosis by Na+-like inducers.

Experimentally induced pinocytosis in Amoeba proteus has been widely used to study the mechanism of invagination of the cell membrane. Many ionic compounds with a positive net charge are potent inducers of pinocytosis in the amoeba [l]. With La3+ present during pinocytosis, it is possible to differentiate between two classes of cationic inducers represented by Na+ and K+ [2]. Pinocytosis induced by Na+ is inhibited by La3+, while K+-induced pinocytosis is stimulated by this ion. The former type of pinocytosis is, in addition, susceptible to inhibition by EGTA and local anesthetic drugs and may therefore depend upon the movement of Ca*+ from the medium into the cell [3]. Sanders & Bell [4] reported that puromycin inhibits pinocytosis in Amoeba proteus by reducing the area of cell surface available for invagination. In their study pinocytosis was induced exclusively by Na+. Here we examine whether inhibition of protein synthesis affects different types of pinocytosis similarly, and how the effect of Ca*+ on pinocytosis is altered in these cells. * To whom offprint requests should be sent. Copyright @ 1984 by Academic Ress, Inc. All rights of reproduction in any form reserved 0014-4827/84 $03.00

368 Johansson and Josef’son The pinocytotic activity induced by various cations was studied in amoebae, subjected to a long period of starvation or treated with puromycin, cycloheximide or emetine. It was found that pinocytosis induced with Na+ was the first to be affected by starvation or inhibition of protein synthesis. Addition of Ca*+ to the inducing NaCl solution increased the number of pinocytotic channels and so reversed inhibition of pinocytosis. It is suggested that inhibition of protein synthesis primarily reduces the availability of binding sites or pathways for Ca*+ in the cell surface. Inducers like Na+, which make use of Ca*+ in the cell surface, will therefore become inefficient, while cations like K+, which are less dependent on extracellular Ca*+, are effective in spite of diminished protein synthesis. METHODS Amoeba proteus Bristol was cultured in modified Pringsheim’s solution and fed daily with TetrahyThe cells were routinely starved 3 days before they were assayed for pinocytosis. During starvation the medium was changed once a day. Immediately before incubation with drugs or enzymes, the cells were washed in the culture medium (Pringsheim’s) and transferred to clean culture dishes. In some experiments Pringsheim’s solution was substituted with Chalkley’s medium 12 h before incubation with antibiotics. The composition of Pringsheim’s solution in mM is: Na+, 0.22; K+, 0.35; Ca’+, 0.85; Mg*+, 0.08; HPG* + H2PO;, 0.11; Cl-, 0.35; NO;, 1.70; SO;‘, 0.08, at pH 7.0. Chalkley’s medium (pH 6.9) contained in mM: Na+, 1.43; K+, 0.027; Ca*+, 0.007; HPO;* + H,PO;, 0.014; Cl-, 1.40; HCO;, 0.047. The assay of pinocytosis was carried out at 23-25°C according to the technique described earlier [5, menu pyriformis.

61.

Electrical Measurements For electrical measurements the cells were in a 10 ml perspex bath at room temperature (22-26°C). They were impaled when adhering to the bottom surface using a piezoelectric driver element (type P172, Physik-Instrumente, Waldbronn, FRG) mounted on a Leitz micromanipulator [7]. To preserve the speed of advancement the protection resistance at the output of the high voltage supply was limited to 10’ Q. Microelectrodes were made from borosilicate glass capillaries with inner filament (Clark Electromedical Instruments, Pangboume, Reading, Berks., England) and filled manually with 3 M KCI. The microelectrodes (resistance 330 MB in Pringsheim solution, 220 mQ in 0.1 M NaCI, tip potentials < 10 mV) were connected via agar-salt bridges and Ag-AgC1 electrodes to an electrometer with 2~ 10” P input impedance. The input resistance of the cell was generally determined with a single electrode for current injection and potential measurements. Hyperpolarizing current pulses (0.06-6x lO-9 A, 200-l 500 msec from a Grass S4SIU) were injected via the high impedance (>2~ 10” Q) current generator of the electrometer. The electrotonic potentials, displayed on a Tektronix 502 A oscilloscope and recorded with an inkwriter (Elema mingograph), were used for calculation of the resistance. A fast component of this potential representing the electrode and tip resistance was subtracted to give the input resistance of the cell, cf [8]. Measurements were taken 60 set after impalement, when potential and resistance of the membrane had reached stable values. When the time constant of the cell membrane was low as it is in 100 mM NaCl, the membrane resistance had to be measured by the use of two electrodes. In these experiments stable values were not obtained until 5 min after impalement.

Measurements of Protein Synthesis Incorporation of [i4C]L-leucine (270 mCi/mmole, The Radiochemical Centre, Amersham, Bucks., England) was studied in cells incubated in conical polystyrene tubes. The cells were sampled from a culture agitated with a magnetic bar (200 rpm). Each tube contained 3 000 cells and 0.25-0.5 uCi Lleucine in 0.5-l ml. At the end of the incubation period, 1 h, the cells were centrifuged (1000 g, 5 min) and the cell pellet suspended in 7% trichloroacetic acid (TCA) containing 100 mM leucine, sonicated Exp Cell Res 154 (1984)

Inhibition

of calcium-dependent

pinocytosis

369

and hydrolysed at 90°C for 15 min. After rinsing three times in leucine-TCA and once in 90 % ethanol the precipitate was lyophilized and dissolved together with the tip of the tube in 1 ml Soluene (Packard Instrument Comp., Downers Grove, Ill.) in a counting vial. Radioactivity was measured in toluene containing 5 g PPO and 0.1 g dimethyl-POPOP per liter.

Statistics The Mann-Whitney U-test was used for statistical analysis [9]

Materials The experimental salt solutions were made from stock solutions stored at 4°C. Glass bidistilled water and chemicals of analytical reagent grade were used throughout this study. Cycloheximide, puromycin and puromycin aminonucleoside were obtained from Sigma, St. Louis, MO. and Calbiothem, San Diego, Calif. Subtilisin from Bacillus subtilis (1500 PUN units/mg) was from BDH, Poole, Dorset , England.

RESULTS Effect of puromycin

on cation-induced

pinocytosis

Puromycin, 20-25 t&ml, was a weak inhibitor of pinocytosis when applied to cells in Pringsheim’s solution. However, when the cells were transferred to Chalkley’s medium, which means a lOO-fold reduction of extracellular Ca*+, pretreatment with the antibiotic reduced the intensity of pinocytosis and decreased the slope of the dose-response curve of Naf-induced pinocytosis. Thus, within 5 h after addition of 15 ug/ml puromycin, the pinocytotic activity in 125 mM NaCl was reduced to below 20%. Cells treated for 15 h with the inactive puromycin aminonucleoside were, however, not affected. The early effect of puromycin was not a general reduction of the capacity of the amoeba but a diminution of its pinocytotic response to some inducers (fig. 1). Thus, after 5 h of treatment with puromycin, pinocytosis induced by Naf, Tris+ or spermine, was strongly inhibited, while that induced by K+ or U02*+ remained intense for another 10-20 h. Previous experiments have shown that the puromycin-sensitive inducers (Na+, Tris+, spermine) unlike those resistant to the antibiotic (K+, UOz2’) are inhibited by La 3+ [2]. This similarity between La3+, which competes with Ca*+ at the cell surface, and puromycin may indicate that inhibition of protein synthesis interferes with the binding and movement of calcium in the cell membrane. Accordingly, in the course of treatment with puromycin, it was possible to maintain a high pinocytotic activity by increasing the Ca*+ concentration of the inducing solution. Fig. 2 shows the results of an experiment in which the capacity of puromycin-treated cells to form channels was examined at intervals by using 100 mM NaCl solutions with different concentrations (7-44 PM) of Ca*+. As the effect of puromycin developed, the Ca*+ sensitivity of pinocytosis decreased, so that , after 24 h of treatment when the pinocytotic response to the solution with 7 uM Ca’+ was inhibited, the inducer containing 44 uM Ca*+ became effective and elicited formation of a greater number of pinocytotic channels than before application of puromycin. Exp CellRes 154(1984)

370 Johansson and Josefsson

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Fig. 1. Time course of the pinocytotic activity induced by various cations in cells treated from time zero with 15 &ml puromycin in Chalkley’s medium. The following cationic inducers of pinocytosis were used; 0, 125 mM NaCl pH 6.0; Cl, 1 mM spermine 4 HCl pH 5.5; A, 125 mM Tris-chloride pH 7.0; 0, 25 mM KCl, pH 5.8; W, 1 mM U02(N03)2 pH 3.5. Fig. 2. (A) Time course of puromycin block of Na+-inducecd pinocytosis. The cells were treated with 15 @ml puromycin in Chalkley’s medium from time zero. The capacity of the cells to form pinocytotic channels was assayed with 100 mM NaCl at pH 6.0 containing different Ca*+ concentrations. The curves represent, from left to right, pinocytosis induced in the presence of 7, 16, 34 and 44 pM CaC12. A, Pinocytosis in the presence of 7 uM CaC& after treatment with 50 @ml of puromycinaminonucleoside. (B) Effect of Ca’+ on pinocytosis induced with 100 mM NaCl in cells treated with emetine for 3,4 or 6 h. The corresponding curves are indicated by stars, open squares and circles. An aliquot of cells in Pringsheim without emetine was used as controls (0).

Effect of Emetine Emetine, which inhibits protein synthesis in the amoeba [lo], diminished Nafinduced pinocytosis. Treatment with 200 ug/ml emetine in Pringsheim for 3 h reduced pinocytosis in 100 mM NaCl to 25% of normal, while K+-induced pinocytosis remained normal. Addition of Ca2+ to the inducing solution did, however, restore a normal pinocytotic cycle (fig. 2B). In the course of emetine treatment the Ca2+ requirement for intense pinocytosis increased continuously until after about 12 h when the pinocytotic response was near zero irrespective of type of inducer or concentration of Ca2+. Effect of Cycloheximide (CHX) High concentrations of CHX caused a parallel reduction of leucin incorporation and Na+-induced pinocytosis in the amoeba (fig. 3A) with little effect on pinocytosis induced with KCl. To obtain inhibition with lower concentrations of CHX the cells had to be treated for about 3 days. It took a further 5 h in normal culture medium to restore pinocytosis. A period of enhanced capacity for pinocytosis preceded inhibition and was also observed during recovery from CHX (fig. 3B). Exp Cell Res I54 (1984)

Inhibition of calcium-dependent pinocytosis

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Fig. 3. (A) 0, Pinocytotjc activity and 0, rate of incorporation of [‘4C]t.-leucine into TCA-insoluble material of cells during treatment with 1 mg/ml cycloheximide (CHX). Pinocytosis was induced by 100 mM NaCl at the times indicated. 0, TCA-precipitated radioactivity in control cells. All values are percentage of the readings taken in control cells at time zero. (B) Effect of cycloheximide on Na’induced pinocytosis. G-0, The pinocytotic activity of cells from Pringsheim’s medium or O-O, this medium containing 100 @ml CHX were measured. The inducer was 200 mM NaCl at pH 6.0. After 73 h when the CHX-treated cells were inhibited, the medium was replaced by one containing no antibiotic.

The slow inhibition by CHX gave opportunity for a detailed study of the Ca2+ sensitivity of pinocytosis. Cells incubated with 50, 100 and 200 yglml CHX for 70 to 75 hours were investigated (fig. 4A). The cells treated with the lowest concentration developed Na+-induced pinocytosis in the presence of 35 uM CaC12, while treatment with higher concentrations of CHX increased the requirement for Ca2+. Sensitivity to Ca2+ reappeared gradually, however, when the antibiotic

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Fig. 4. (A) Effect of Ca2+ on pinocytosis in cycloheximide-treated

cells. Cells were incubated for 70-74 h in Pringsheim’s medium containing cycloheximide of the following concentrations: 0, 50; W, 100; 0, 200 @ml. Pinocytosis was induced with 100 mM NaCl with Ca2+ added as indicated on the abscissa. Inset: Recovery of Ca2+ sensitivity after incubation for 72 h in CHX 100 ug/ml. At time zero the medium was exchanged for Pringsheim’s medium with no antibiotic. Pinocytosis was induced by 100 mM NaCl containing 27 pM CaCl,. Arrows indicate corresponding experimental points in figure and inset. (II) The effect of Ca*+ on Na+-induced pinocytosis in cells starved for A, 2; 0, 7; n , IO; and q , 12 days respectively. To induce pinocytosis the Pringsheim’s medium was replaced by a solution of 100 mM NaCl at pH 6.0 containing various concentrations of Ca’+. Exp Cell Res 154 (1984)

372 Johansson

and Jose&on

Fig. 5. (A) Effect of subtilisin on Na+- and K+-induced

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pinocytosis. Pinocytosis was measured in cells treated with subtilisin (0.1-100 ug/ml) for 1 h at 23°C. The enzyme was dissolved in Pringsheim’s medium, pH 6.9. Pinocytosis was induced by 0, 20 mM KC1 pH 5.8; q i, 100 mM NaCl pH 6.0. The horizontal lines give the pinocytotic activity in aliquots of cells treated with a solution of boiled enzyme (100 ).&ml). (B) Time course of recovery in Pringsheim’s medium after treatment with 20 &ml subtilisin for 30 min. Pinocytosis was induced by 100 mM NaCl pH 6.0. (C) Effect of Cazf on Na+-induced pinocytosis in O-0, normal amoebae; O---O, in cells treated 30 min with 20 ug/ml subtilisin. Abscissa: Ca’+ concentration of the inducer (100 mM NaCl at pH 6.0).

was removed. This is shown in fig. 4A, inset. Aliquots of cells inhibited by 100 ug/ml CHX were thereafter transferred to Pringsheim’s solution, and repeatedly tested for pinocytotic response to the inducer (100 mM NaCl) containing a concentration of Ca*+ (27 uM Ca*+, in fig. 4A, arrow) which was initially too low to stimulate pinocytosis. As the cells recovered in the culture medium, however, their capacity for pinocytosis increased to reach a maximum after 90 min and thereafter decline to the pinocytotic level typical for normal amoebae. Effect of Starvation

During the course of starvation the capacity to develop pinocytotic channels has been reported to diminish progressively in the amoebae [l]. We found that the effect of starvation on pinocytosis was most obvious in cells cultured in low calcium media like Chalkley’s. It is therefore possible that a starving amoeba, like an amoeba with reduced protein synthesis, first loses the Ca*+-dependent Na+induced pinocytotic activity before the motile system is strongly affected and the capacity to invaginate the cell membrane is reduced. Experiments with K+- and Naf-induced pinocytosis in starving amoebae support this view (table 1). In the course of starvation (from 3 to 10 days) channel formation in 100 mM NaCl Table 1. Effect of starvation on pinocytosis induced by Na+ or K+ Inducer

Starved 3 days

Starved 10-12 days

100 mM NaCl 20 mM KC1

10.0+0.39 (50) 9.4kO.68 (18)

6.0f0.19 (22)

2.4kO.17 (42)

The number of cell cultures used for the study is given within parentheses. Pinocytotic activity + SE refers to the mean number of channels formed per amoeba Exp Cell Res 154 (1984)

Inhibition of calcium-dependent pinocytosis

373

Table 2. Membrane potentials and input resistance of amoebae in different stages of starvation or after treatment with emetine 0.4 mglml in Pringsheim’s solution for 3.5 h Pretreatment of cells

Extracellular medium

Membrane potential (mV)

Input resistance (MQ)

Controls Emetine Subtilisin Starvation (8 days) Controls Emetine Starvation (8 days)

Pringsheim Pringsheim Pringsheim Pringsheim 100 mM NaCl pH 6.0 100 mM NaCl pH 6.0 100 mM NaCl pH 6.0

-97.3 (n= 10) -94.3 (n=lO) -83.7 (n=lO)” -91.9 (n=7) -7.8 (n=7) - 10.7 (n=6) -8.0 (n=7)

91.6 (n=7) 233.4 (n=S) 153.6 (n=l2) NS 165.5 (n=6)’ 1.6 (n=8) 3.4 (n=6) NS 1.3 (n=5) NS

NS NS NS NS

NS, Non-significant, pCO.05. 0 pQO.05; b ~0.01. Measurements were made within 30 min after transfer of the cells to fresh Pringsheim or to 100 mM NaCl at pH 6.0.

decreased more than in 20 mM KCl. Addition of Cazf to the NaCl solution increased Na+-induced pinocytosis to normal intensity (fig. 4B). The optimal Ca2+ concentration for pinocytosis remained almost constant (16-25 FM) during the course of starvation. So, in this respect starved cells were different from cells treated with inhibitors of protein synthesis. Effect of Treatment with Subtilisin Since starvation and treatment with inhibitors of protein synthesis selectively decreased Na+-induced pinocytosis, it was of interest to examine whether hydrolysis of proteins in the membrane would produce similar effects. We therefore applied subtilisin (0.1-100 ug) for 25 min to normal amoebae in Pringsheim’s medium. Treatment with 20 pg/ml subtilisin decreased the capacity for Na+induced pinocytosis while a five-fold concentration of the enzyme was required to inhibit channel formation induced by K+ (fig. 5A). Normal capacity for pinocytosis was restored within 4 h after transfer of the cells to enzyme-free culture medium (fig. 5B). The enzyme did not itself induce pinocytosis, nor did it affect cation-induced pinocytosis after boiling for 5 min. The effect of Ca2+ on Na+-induced pinocytosis in subtilisin-treated cells was similar to that described above for starving cells. Low concentrations of Ca2+ stimulated pinocytosis with the maximum activity occurring in solutions of 100 mM NaCl containing 20 uM CaC12(fig. 5 C). Electrical Properties of Starved or Emetine-treated Cells Electrical depolarisation of the plasma membrane is an early event during induction of pinocytosis in Amoeba proteus followed within minutes by channel formation [ 11, 121.The correlation between depolarizing and inducing potency of 25-848340

Exp Cell Res 154 (1984)

374 Johansson and Josefsson the alkali metal ions suggests a cause-effect relationship between interaction of the inducer with the plasma membrane and channel formation. It was therefore important to know whether starvation and inhibition of protein synthesis affects electrophysiological parameters of the cell. As is clear from table 2 the membrane potential did not change, whereas the input resistance increased during starvation of the amoeba and after treatment with emetine. The depolarising effect of the inducing Na+ ions and their effect on the ionic conductance was not reduced in these cells. Thus, membrane potentials measured 2-30 min after the exchange of Pringsheim’s solution for 100 mM NaCl did not differ between normal, emetinetreated or starved cells (table 2). DISCUSSION We have shown that puromycin, emetine and CHX selectively inhibit Ca*+dependent types of pinocytosis. These drugs are inhibitors of protein synthesis in the amoeba, although emetine and CHX (fig. 3) were effective only when present in high concentrations [IO]. Similar effects on pinocytosis, not reported here, were observed after treatment with actinomycin D, which indirectly blocks protein synthesis in the amoeba [13]. Taken together, the data indicate that the effects of these drugs on pinocytosis are the result of inhibition of protein synthesis. Inhibition of protein synthesis in Amoeba proteus cultured in calcium-poor medium (Chalkley’s) is accompanied by inhibition of pinocytosis and locomotion [4]. Starvation decreases the ability to form pinocytotic channels [l] as does the preceding cell division [4] or pinocytotic cycle [14]. These are motile processes in which consumption of membrane is apparent. The main cause of the low intensity of pinocytosis has therefore been ascribed to limited availability of cell membrane [15]. Inhibition of exocytosis, as well as endocytosis, has also been observed in T. pyriformis after treatment with CHX and puromycin [16]. The findings presented here are compatible with this hypothesis. Our study indicates that puromycin, emetine and cycloheximide, starvation and treatment with a proteolytic enzyme primarily inhibit pinocytosis induced by Na+-like inducers which require for their inducing effect a certain minimum of Ca*+ in the cell surface or the extracellular medium [6]. Accordingly, blockage by the drugs was reversed by adding Ca*’ to the inducer, and recovery of Na+induced pinocytosis in cycloheximide-treated cells included a graded return of normal Ca*+ sensitivity. These drugs also reduced the inhibitory effect of higher Ca*+ concentrations on pinocytosis (fig. 2A, B). This may indicate that the competition between the inducing cation and Ca*+, i.e. the cation exchanging properties of the membrane, is altered. Excitation of the membrane by the inducer appeared, however, not to be affected by starvation or inhibition of protein synthesis. The membrane potentials of emetine-treated cells and cells starved for up to 10 days were similar to those Exp Cell Res 154 (1984)

Inhibition of calcium-dependent pinocytosis

375

of normal cells in both Pringsheim’s and in 100 mM NaCI. Their input resistances in Pringsheim’s solution were, however, higher than normal, which may indicate that the area of the cell membrane or its ionic permeability had decreased. There are several potential mechanisms for these effects on the pinocytotic activity of the cell and its input resistance. Depressed synthesis or increased degradation of proteins may affect binding and mobility of CaZf in the membrane and so decrease the intracellular calcium concentration. The exocytotic growth and renewal of the cell membrane may be retarded because of low cytoplasmic free Ca2+ [17]. This could reduce the Kf permeability [18], which dominates membrane conductance during culture conditions [12], and so raise the input resistance of the cell. Because of shortage of membrane area [15], or inactivation of a Ca2+-dependent contractile machinery at the cell cortex [6, 191, the formation of pinocytotic channels would cease unless extra CaZf is added to the cell. Consequently inducers, such as Na+ and K+, may differ because they require different levels of Ca*+ for invagination or exocytosis. Our present results do not distinguish between these alternative mechanisms, but indicate that starvation and inhibition of protein synthesis are useful techniques for the study of Ca*+ regulation of pinocytosis and for characterizing various types of inducers. This work was supported by grants from the Swedish MRC and the Medical Faculty of the University of Lund.

REFERENCES 1. Chapman-Andresen, C, C r lab Carlsberg 33 (1962) 73. 2. Josefsson, J-O & Hansson, S E, Acta physiol stand 96 (1976) 443. 3. Josefsson, J-O, Johansson, G & Hansson, S E, Acta physiol stand 95 (1975) 270. 4. Sanders, E J & Bell, L G E, Exp cell res 63 (1970) 379. 5. Josefsson, J-O, Acta physiol stand 73 (1968) 481. 6. - Ibid, suppl. 432 (1975). 7. Fromm, M, Westkamp, P & Hegel, U, Pfltigers arch 384 (1980) 69. 8. Peskoff, A & Eisenberg, R S, Ann rev biophys bioeng 2 (1973) 65. 9. Siegel, S, Nonparametric statistics for the behavioral sciences. McGraw-Hill, New York (1956). 10. Maruta, H & Goldstein, L, J cell bio165 (1975) 631. 11. Josefsson, J-O, Acta physiol stand 66 (1%6) 395. 12. Josefsson, J-O, Holmer, N-G & Hansson, S E, Acta physiol stand 94 (1975) 278. 13. Hawkins, S E, The biology of the amoeba (ed R W Jeon) p. 371. Academic Press, New York (1973). 14. Chapman-Andresen, C, Proc 1st intern conf protozool Prague 1961 (ed J Ludvik, J Lom Br J Vavra) p. 267. Academic Press, New York (1963). 15. Chapman-Andresen, C, Ann rev microbial 15 (1971) 27. 16. Ricketts, T R & Rappitt A F, Arch microbial 102 (1975) 1. 17. Douglas, W W, Calcium transport in contraction and secretion (ed E Carafoli, F Clementi, W Drabikowski L A Margreth) p. 167. North-Holland Publ. Co., Amsterdam (1975). 18. Meech, R W, Ann rev biophys bioeng 7 (1978) 1. 19. Klein, H & Stockem, W, Cell & tiss res 197 (1979) 263. Received January 16, 1984

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