DANA DEC 7 1979

Canadian Journal of Zoology Published by T H E NATIONAL RESEARCH COUNCIL O F C A N A D A

~ - .

de zoologle Publie pcir LE CONSEIL NATIONAL DE RECHERCHES D U C A N A D A

Volume 57 Number 11 November 1979 Volume 57 numero 11 novembre 1979

The contractile vacuole of Amoebaproteus. 111. Effects of inhibitors

DPparternent des Sciences Biologiques, UnicersitP de MontrPal, MonirPal, (QuP.), Canada H3C 357 Received December 4, 1978

A H M A D , M . 1979. The contractile vacuole ofArnoebuproteus. 111. Effects of inhibitors. Can. J . Zool. 57: 2083-2088.

The effects of cyanide and malonate have been observed on the fine kinetics of the contractile vacuole of Amoebu p ro tc~u~ . Brief exposure to cyanide does not produce any inhibition of vacuolar output rate. Only after keeping the amoeba in cyanide solution for long periods (9 h) and renewing the inhibitor solution frequently was significant (76%) inhibition of output rate ob- served. Owing to this reduction in output rate, the volume of the organism doubles in approxi- mately 9 h. Malonate induces mild (30%) inhibition of the vacuolar output. This inhibition normally is alleviated on return to original Chalkley's medium.

It is concluded that the vacuolar operation in Amo~buproteus is an energy dependent phenom- enon. Such energy can be derived from respiratory or glycolytic processes. However, the congregation of mitochondria around the contractile vacuole suggests that respiratory oxidation may be the chief source of energy supply. The cellular homeostasis is probably maintained through a Na+-K+ pump. operative in the vacuolar membrane.

AHMAD. M . 1979. The contractile vacuole ofArnoebaproteus. 111. Effects of inhibitors. Can. J . 2001.57: 2083-2088.

On trouvera ici la description des effets du cyanure et du malonate sur la cinetique les mouvements de la vacuole contractile chez Amoeba proteus. Un contact de courte duree avec du cyanure ne cause pas d'inhibition du taux d'efflux vacuolaire. L'immersion de I'amibe dans une solution de cyanure pendant une longue periode (9 h), durant laquelle la solution inhibitrice est remplacee frequemment, entraine une inhibition significative (76%) d'efflux vacuolaire. Cette reduction d'efflux cause une augmentation appreciable du volume de l'organisme (deux fois son volume original apres 9 h). Le malonate cause une faible inhibition (30%) d'efflux vacuolaire. L'effet inhibiteur disparait normalement des que I'animal est remis en milieu Chalkley.

Le fonctionnement de la vacuole d'Amoeba proteus semble donc un phenomene a base energetique. L'energie necessaire a la fonctionnement de la vacuole peut venir soit de la respira- tion, soit de la glycolyse. Cependant, le rassemblement des mitochondries autour de la vacuole contractile semble indiquer que c'est I'oxydation respiratoire qui constitue la principale source d'energie. L'homeostasie cellulaire est probablement due a la presence d'une pompe Na-K dans la membrane de la vacuole.

[Traduit par le journal]

Amoeba proteus, a pond-water protozoan, pos- rounded by a single layer of mitochondria. It grows sesses a single, spherical and hyaline contractile in volume, probably due to fusion of microvesicles vacuole. It is a temporary structure which is formed (17). After attaining a critical volume, the main periodically owing to the fusion of many smaller vacuole expels its fluid to the exterior through a vesicles, emerging from all over the cytoplasm (13). temporary pore. It finally disappears in the cyto- Eventuallv. a sinele vacuole results which is sur- ~ l a s m and the vacuolar membrane is transformed

d . u

'Present address: Clinical Biochemistry Department, into tiny (35 nm) vesicles (14). After a brief pause Hotel-Dieu Hospital. 3840 St-urbain Street, Montreal, ( Q U ~ . ) , the cycle starts again. Canada. Osmotic homeostasis is generally believed to be

0008-430 11791 1 12083-06$01.00/0 @ 1979 National Research Council of Canada/Conseil national de recherches du Canada

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2084 CAN. .I. ZOOL. VOL. 57. 1979

one of the prime functions of contractile vacuoles in freshwater protozoa (11). It is shown that the vacuolar output increases or decreases in response to extracellular osmotic pressure and that these changes result in a corresponding swelling or shrinking of the body volume (9, 10). However, the underlying mechanism of vacuolar function is still incompletely understood. Observations have revealed that an accumulation of fluid occurs at the microvesicle level (17), though the origin of these microvesicles remains unclear. It is often suggested that the Golgi apparatus (3) and (or) mitochondria are in some way associated with the segregation of hypoosmotic fluid from distinctly hyperosmotic cytoplasm (20). In a relatively recent work the existence of a Na+-K+ pump has been suggested (19). Nonetheless, the source of energy for operation remains to be evaluated.

In a previous study, we have demonstrated that vacuolar output rate in A. proteus changes in ac- cordance with experimental temperatures (2). At low temperatures, the vacuolar output is reduced owing to the appearance of a plateau during which the vacuole remains passive, i.e., vacuolar input is negated by its output.

Following these observations two important questions were raised, (i) What is the cause of the nonasymptotic onset of a plateau which was ob- served invariably during those observations? (ii) What regulates vacuolar operation?

For nonasymptotic initiation of plateau, we analysed our experimental curves at various low temperatures and statistically compared them with computer-generated ones. It was found that errors of less than 0.2 pm3 in our vacuolar diameter mea- surements were sufficient to render undetectable the asymptotic onset of a plateau (4).

The aim of this study is to elucidate the nature of vacuolar operation by using inhibitors on the fine kinetics of vacuolar cycles under controlled ex- perimental conditions.

Materials and Methods This study has been conducted on Amoeba proteus (Leidy)

which was originally obtained from the General Biological Sup- ply House, Chicago. The amoebae were fed with the ciliate Tetruhymenu pyrif)rmis, which was provided by the Carolina Biological Supply House, Burlington, NC.

Culture techniques ofprescott and Carrier (18) and Chalkley's (5) culture medium were employed. The ionic composition of Chalkley's medium was similar to that ofpond water; the osmo- tic concentration varied from 2 to 6 mosmol/kg; and the pH varied between 6.5 and 6.9.

Desired concentrations of sodium cyanide, sodium malonate, sodium succinate, etc., were prepared in double distilled water and the pH was brought to 6.5-6.9 with a 0.1% NaOH solution. The amount of NaOH added was very small and the osmotic concentration of our inhibitor solution was never above 10 mosmol/kg.

Amoebae were starved for 24 h before use. A concentrated sample of amoebae in suspension was taken into a beaker and about 15 mL ofthe inhibitor solution was added. The beaker was placed in the incubator at 20°C for 3 h. Then a drop of amoebae in suspension was placed in the central cavity of our previously prepared microscopic slides (2). About 15 min was allowed to minimize the transfer shock and the excess liquid was pipetted out, leaving just enough to fill the central cavity. A cover slip was applied and the observations were made at 20°C.

For long-term treatment of cyanide, amoebae were housed in a glass tube the bottom of which was closed by a mesh of nylon cloth. The bottom ofthe tube was kept submerged in Chalkley's medium, contained in a deep petri dish. In a funnel, a freshly prepared and pH-adjusted solution of cyanide was placed above the open end of the tube. The cyanide solution flowed down from the funnel along the walls of the tube and thus replaced Chalkley's medium without disturbing the amoebae. The flow was adjusted so that 200 mL of solution lasted almost 3 h after which a fresh solution of cyanide was prepared. Before intro- ducing the fresh solution of cyanide, a sample of amoeba sus- pension was taken out for observations. The procedure was repeated three times and before each change of cyanide a sample of amoeba suspension was withdrawn.

The observations were made under controlled conditions at 20°C with a phase contrast microscope at 12.5 x 40 magnifica- tion. Successive measurements of vacuolar diameter were made with the help of a filar micrometer and volume was calculated with respect to time. Details of this procedure have been pub- lished previously (2).

Results Cy anidc.

(i) Short-term Effects Cyanide if applied at a concentration of lop4 M

for a brief period (15-20 min) produces no effects on the vacuolar output ofA . proteus. A comparison between the average vacuolar cycle in cyanide treated amoeba (A) with that in the control (B) is shown in Fig. 1.

The vacuolar cycles in the cyanide treated amoeba exhibit greater irregularities in their dias- tolic durations and systolic volumes as compared with the control (Fig. 1). It is also observed that these cycles may or may not be terminated by a

too 200 aw 400

Time #n Seconds

FIG. 1 . Short-term effects of NaCN M) on the contractile vacuole of Amoebu proteus. Note the variations in systolic volumes and diastolic durations of the cycles (72 cycles were observed on eight different amoebae).

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Time in seconds

FIG. 2. Long-term effects of NaCN (3 x M). The cyanide solution was renewed every 3 h with afreshly prepared one (47 cycles were recorded on 9 different amoebae).

systole as is the case in normal cycles (control). In line of the nucleus is well defined. The vacuole is such cases, the vacuolar growth proceeds normally very clear and it moves slowly with the plasmasol. up to a given diameter then the vacuole suddenly In the early stages of its growth, the vacuole mig- stops growing and remains at a constant volume for rates to the extremity of the pseudopodium and it some time before being terminated by a complete systole. This period of vacuolar inactivity is termed plateau. Out of 72 cycles observed on eight differ- ent amoebae, 14 cycles did not end in a systole; rather, they continued as plateaus while the rest achieved rapid and complete systole. The total du- ration of these occasional and irregular plateaus varied between 43 and 78s. They also show significant variations in their inceptions and termi- nations. Because of inconsistency of vacuolar cy- cles in general and plateaus in particular, only the active phases of all cycles have been taken into consideration and no attempt has been made to

remains there for some time; then it returns to the posterior region where it remains almost stationary without any change in volume (plateau) for a de- finite period of time until systole.

Fig. 2 and Table 1 show that the vacuolar cycle in the cyanide treated amoeba is clearly divisible into four phases: the coalescence period, the con- tinuous growth phase, the plateau, and the systole. Following 9 h of contact with 3 x lop4 M cyanide solution, the vacuolar output falls from 62 pm3/s in the control to 15 pm3/ s. There is a net inhibition of 76%.

However, if the cyanide solution is not replaced analyse the plateau at this stage. periodically by a fresh1 y prepared one, aberrations

in vacuolar rhythm occur, with highly irregular ( i i ) Long-term Effects cycles, both in systolic volume and total duration. At slightly higher concentration, 3 x M, and Plateau may or may not appear and if it appears its

renewed periodically by freshly prepared solution, inception and termination is variable. cyanide significantly inhibits vacuolar activity in A . proteus. The results are summarized in Fig. 2 and Effects qf Mulonute . .

Table 1. under phase contrast microscopy, amoebae Morphologically, after being in contact with the treated with 5 x M sodium malonate for 3 h

cyanide solution for 3 h, the majority of amoebae are show viscous cytoplasm and slow movement. They monopodial. Their locomotion increases consid- become round and several peripheral projections erably, the cytoplasm becomes dense, and the nu- appear. However, the mitochondria do not seem to cleus is almost invisible. The contractile vacuole is be distorted in their morphology. No visible in- also difficult to observe. After 6 h the amoebae crease in the volume of the amoebae was observed. round up and their movement slows down. Many A comparison between the average vacuolar peripheral projections are produced. The cyto- cycle in malonate treated amoebae and the control plasm is less dense and there is a visible increase in is shown in Fig. 3 and Table 2. In amoebae treated amoeba volume. The nucleus and the vacuole are with malonate the average diastolic duration of the easily distinguished. After 9 h (three changes) the cycle is almost the same as the average diastolic cytoplasm becomes more transparent, the volume duration of the control (272 s versus 301 s in the of the amoeba increases appreciably, and the out- control). However, the systolic volume is consid-

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2086 CAN. J. ZOOL. VOL. 57, 1979

TABLE 1. Long-term effects of NaCN on the contractile vacuole of A. proteus

Active phase, s Rate of efflux, pm3/s

Period of Continuous Plateau, Total time, Active Total % coalescence growth phase s s phase cycle inhibition

Control 82k 7 225+ 17 307+ 7 62 .5k7 62.5+7

Renewal of cyanide a t : 3 h 83+ 14 288k 36 1 3 4 + 4 7 5 0 5 k 2 3 37.0+8 27.0+6 57 6 h 86+ 36 326+ 59 1 6 1 _ + 2 5 5 7 3 k 2 8 37.0+9 24 .0k8 62 9 h 146+23 672+ 200 542+140 1364k348 24.0+4 15.0+2 76

I Na Succinate: A

Control: B

Na Malonate: C

", 20 000 Z

- 4 >

10 DUD -

100 200 300

Time in Seconds

FIG. 3. Comparison of sodium malonate and sodium succinate (5 x IW4 M and 3 h preconditioning) on the contractile vacuole (38 cycles on six different amoebae were observed in the case of malonate treatment while only 29 cycles on four amoebae were noted for succinate).

erably lower ( 1 1 580 pm3 compared with 16 980 pm3). Therefore, the rate of vacuolar output de- creases from 60 pm3/s in the control to 42 pm3/s, representing a 30% inhibition of vacuolar output due to malonate. This inhibition is mild and once the malonate treated amoebae are returned to Chalkley's medium, the vacuolar activity gradually returns to normal. No plateau has been observed in amoebae treated with malonate.

Effects of Succinate We also treated amoebae with sodium succinate

(5 x lop4 M) for 3 h. After this treatment the aver- age systolic volume of 26 vacuolar cycles is 16 320 pm3, obtained in 238 s. Therefore, the output rate is 68 pm3/s which means a 12% increase in output rate due to succinate (Fig. 3 and Table 2). It must be added that no plateau occured in succinate treated amoebae.

Discussion The results we report here demonstrate that brief

exposure to cyanide does not alter the overall vac-

uolar output rate in A. proteus. Such treatment only induces aberrations in the cyclic pattern of vacuo- lar function. However, prolonged immersion in the medium containing this inhibitor, causes significant depression of vacuolar output rate. This discrep- ancy may be due to the fact that the amoeba cell is surrounded by a layer of mucopolysaccharide or that the salts of cyanide are less penetrant in the ionic form than as the weak acid (HCN) (8). It may also be possible that amoebae have no short-term dependence on the ATP-generating oxidative mechanism.

It has previously been reported that cyanide strongly inhibits vacuolar function in peritrich ciliates and the decrease in vacuolar function re- sults in an increase in body volume. When these cyanide treated organisms were returned to fresh- water, the inhibition was alleviated and the body volume decreased to its original level (9, 10). Con- trary to these observations, we have observed neither an immediate depression of vacuolar output rate nor an increase in body volume of A. proteus. We will refer to this point later on.

Under proper conditions and concentrations, cyanide directly affects the physiological state of the amoeba's cytoplasm since the contractile vac- uole, which is normally situated posterior to the nucleus, migrates to the pseudopodial extremity. It has been mentioned that A. protrus has a typical network type of plasmagel. Under normal condi- tions, the vacuole is retained in the posterior posi- tion by this thick plasmagel (7). It is reasonable to assume that cyanide action damages this network type of plasmagel structure. Such an action might cause the formation of broader channels through which the contractile vacuole would move to the pseudopodial apex. In the past, such liquefaction of the plasmagel network has been observed by micro- injection of heparin or by heating the amoeba to 40°C (7).

\ ,

Effects of cyanide on the vacuolar cycle are more discrete: the coalescence period increases, the continuous growth phase lengthens, and plateau

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TABLE 2. Effects of sodium malonate and sodium succinate on the contractile vacuole

Active phase

Inhibitor and Period of Continuous Total time, Total volume, Rate of efflux, % of activator coalescence growth phase s urn3 um3/s inhibition

Control 72+ 12 229+17 3 0 1 k 6 17980+992 6 0 . 0 k 6

Na malonate (5 x M ) 60+ 14 212+ 52 272k53 11 580_+4228 42.6k 5 30%

Na succinate (5 x M ) 90f 19 150f 62 240+63 1632223073 68.0+10 12%

invariably appears, (Fig. 2 and Table 1). If one takes plateau into consideration, the vacuolar out- put decreases from 62 pm3/s in the control to 27 pm3/s after 3 h of cyanide treatment; after 6 h the vacuolar output decreases to 24 pm3/s; after 9 h it decreases to 15 pm3/s signifying 57, 61, and 76% inhibition of output rate'after 3 ,6 , and 9 h, respec- tively.

The decrease in output rate is reflected by an increase in amoeba volume which is shown by a corresponding increase in cytoplasmic transpa- rency. However, the above mentioned decrease in vacuolar output rate does not bring about any im- mediate increase in amoeba volume. Only after 9 h does increased volume become visually detectable. This may be explained as follows: actual volume of an average amoeba (measured as a cylinder) is 1.5 x lo6 pm3. After being in cyanide solution for 3 h the deficit in output is 35.8 pm3/s; therefore, an average amoeba will require 11.4 h to double its volume (if the output rate remains constant at 27 pm3/s). It will take 10.8 h if the deficit in output rate is 38.5 pm3/s (6 h contact) and 8.8 h if such reduc- tion is 47.5 pm3/s (9 h contact). This explains why we observed no increase in amoeba volume when amoebae are treated with cyanide for 15-20 min.

Onset of plateau, under these conditions, is once again nonasymptotic. However, we now under- stand that this nonasymptotic situation, indeed, re- presents a small error of about 0.2 pm3 in our read- ings of vacuolar diameter (4). In fact, the transfer from active growth phase to plateau is gradual. The occasional appearance of a plateau in amoebae treated with M cyanide solution for a short period of time and its regular appearance in amoebae treated for longer duration, confirms our hypothesis concerning the onset of plateau at low temperatures (2). It is obvious that cyanide, as does low temperature, slows down the active processes responsible for vacuolar filling whereas diffusion of fluid from the vacuole to the surrounding cytoplasm remains unchanged. When active processes are

equalized by passive ones, a steady state equilib- rium is established at 20°C.

The toxicity of cyanide is due to the fact that it combines readily with the oxidized form of cyto- chrome oxidase and forms a stable complex, thus preventing the reduction of the cytochrome a + a 3 fraction of the respiratory terminal chain (22). Be- sides, cyanide affects a number of respiratory en- zymatic systems which require metallic ions as prosthetic groups such as Fe2+, Zn2+, Cu2+, etc. (6, 12). Our results indicate that at appropriate con- centrations and under proper conditions, cyanide effectively suppresses vacuolar activity in A . pr-o- teus. However, it is difficult to identify the energy source of vacuolar function. Respiration seems to be one of the processes which may furnish this energy. In fact, the congregation of mitochondria around the vacuole supports such an assumption, though one must assume that it may not be the only one. Nonetheless, the mechanism through which the energy is made available for vacuolar function, is not yet clearly understood. Riddick (19) has sug- gested the existence of a Na+-K+ pump in the vacuolar membrane. According to him, in the initial stages the vacuolar fluid is isoosmotic to cyto- plasm. Then the salts are excreted out actively, leaving the vacuolar fluid hypoosmotic with respect to cytoplasm. In our view, the energy for this pro- cess is probably derived from respiratory oxidation which is sensitive to inhibitors like cyanide.

The role of malonate on amoebae has not been previously investigated. However, it is known from the studies on plants (21) and animals (15) that it is a competitive inhibitor of succinic dehydrogenase of Kreb's tricarboxylic acid cycle. It is also known that at higher concentrations it effectively inhibits water uptake by barley root hairs, whereas at low concentrations it acts as a metabolite and enhances water absorption (1).

At high concentrations, malonate is extremely toxic for amoebae, i.e., M malonate kills the entire population, whereas at low concentration

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2088 CAN. J . ZOOL. VOL. 57. 1979

(lop6 M ) malonate does not enhance vacuolar ac- Numerical analysis of the steady state hypothesis. J . Pro-

tivity. The former suggests that the energy supply from the respiratory source is inhibited, while the latter indicates that unlike microorganisms and plants, amoebae may be incapable of using malo- nate as a metabolite. Even if amoebae metabolize malonate, this supplementary energy is not access- ible to the vacuolar system. Indeed, it is logical to assume that if such extra energy were used by the vacuole, it would have evacuated cellular fluid more rapidly and thus the amoeba would have de- hydrated itself. To verify this assumption, we used succinate as an exogenous energy source. Succi- nate is a natural metabolite of the living cells and penetrates freely into the mitochondria. Our results are consistent with this assumption as we observed only 12% increase in vacuolar output rate owing to the presence of succinate in the amoeba medium. In a similar study, but on a different animal, Paramecium mulrimicronucleatum, Organ et al. (16) noted that an application of 3 x M ATP increases the vacuolar frequency by 88%. At this concentration the organism starts dehydrating it- self and in only 6min it loses 27% of its body volume. Organ et al. (16) concluded that fluid segregation is an active process which accelerates in the presence of energy-rich compounds such as ATP. However, our results with succinate suggest that the exogenous source of energy is either not directly available to the mechanism responsible for vacuolar function or if it is available the system does not use it spontaneously, since this would perhaps result in a self-dehydration process.

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