Ionic Regulation in the Ciliate Spirostomum Ambiguum

The concentration of sodium, potassium and bromide have been measured in animals grown in medium containing these ions. The rates of entry and loss of these ions have been measured and the results are discussed in the light of current theories of the mechanisms of ionic regulation in other cells.

Spirostomum ambiguum was chosen for the experiments as it is easily cultured, it is relatively large and it survives the experimental conditions. It was cultured in the following medium :

The medium was made up with glass distilled water and the pH was adjusted to 6-3 by addition of the KH₂PO₄-KOH buffer. Boiled wheat grains were added as a source of food for the bacteria on which Spirostomum feeds.

⁴²K was obtained as the ‘Specpure’ carbonate from A.E.R.E., Harwell, and converted to the chloride by addition of hydrochloric acid; the excess acid being removed by evaporation. When the ⁴²K had decayed the concentration of potassium in the experimental solution was confirmed by measurement with a flame photometer. ²²Na was supplied by the Radiochemical Centre, Amersham, as a sodium chloride solution. ⁸²Br was obtained from A.E.R.E., Harwell, as ammonium bromide. The compound was converted to sodium bromide by dissolving it in sodium hydroxide and evaporating the water and ammonia.

In each experiment approximately 50,000 animals were removed from the cultures and concentrated in a small volume of medium by centrifuging at approximately 15 g. for 1 min. The animals were then kept for 3 days in unlabelled medium under experimental conditions to come into a steady state before being transferred to a similar but labelled medium. Several samples of the radioactive medium were taken with an ‘Alga’ pipette for determination of the radioactivity per unit concentration of medium. At suitable times samples of several hundred animals were taken from the labelled solution. These animals were washed free of labelled solution by centrifuging with six changes of unlabelled medium. The time taken for washing was recorded so that correction could be made for the washing of labelled ions out of the animals. From the last wash the animals were transferred to a planchette with a 0·1 ml. pipette operated by a hypodermic syringe. A sample of the last washing medium was also taken with the same pipette to enable correction to be made for any residual radioactivity of the medium on the animals The count of this sample was never more than one or two counts above the background count ; it was used to correct the sample for background count and that of contaminating medium. The animals on the planchette were fixed and stained with a few drops of saturated aqueous picric acid and photographed. Photographing the animals made it possible to carry out the slow process of counting them after all the samples had been assayed for radioactivity. This permitted many more samples to be taken during the useful life of the isotope. The planchettes were dried in an oven at 100° C. before being assayed for radioactivity.

The radioactivity was assayed with a Geiger-Muller end-window counter. The time in half seconds for 1000 counts was recorded for all samples, so that the standard deviation of any count is approximately 3% of the total count. In experiments with ⁴²K and ⁸²Br the counts were corrected for decay to a zero time by calculation using the measured half-lives of the isotopes.

The calculation of the mean volume of a population of animals was based on measurements of photographs of a representative sample of contracted animals. A sample of animals was concentrated into a small volume of medium and transferred to a microscope slide which was held in position with a circle of paraffin wax. When a condenser was discharged through the drop via two electrodes embedded in the wax, the animals contracted. Photomicrographs of the contracted animals were taken. The animal appears as an ellipse on the photograph, and assuming that it is a prolate spheroid the volume may be calculated as ⁠, where a and b are major and minor semi-axes. A micrometer scale was photographed with each sample.

In the experiments to be described the animals were transferred from one medium in which they had reached a steady state to another of the same composition but containing labelled ions. The tracer ion then exchanges with its counterpart inside the animal and eventually the specific activity of the tracer inside the animal becomes the same as that of the medium. There is no evidence to show that tracer ions behave differently from the normal species. With a knowledge of the specific activity of the tracer in the medium, the radioactivity when it has reached a steady state in the animal and the amount of body water, the internal concentration may be calculated. This value does not necessarily correspond to the total concentration of the ion inside the animal ; only material which will exchange with the labelled ion is measured. Morphological or chemical isolation of the ion inside the animal would give a measured value below the true value.

X_in is the transfer constant from out to in and has dimensions of hours^-1. A_out is the transfer constant from in to out and also has the dimensions of hours^-1, t is the time in hours during which the animals are in the labelled solution. C_in is the concentration of the labelled ions in the cell. C_out is the concentration of labelled ions in the medium and may be regarded as constant when the volume of medium is very large compared with that of the animals.

In the first experiment the level of potassium was measured over a period of 55 hr. in animals kept at room temperature. Results of this experiment are shown in fig. 1, where the count per animal per minute is plotted against time in the radioactive solution. In all experiments each point is the mean activity determined for 50-500 animals, and all samples at a given time were washed together. The scale on the right of the graph indicates the calculated concentration of potassium in the animal on the assumption (to be discussed later) that the animal is 100% water.

In Table 1 the results of several similar experiments are summarized. The level of activity in the animal remains fairly constant during an experiment, but from column 3 of Table 1 it may be observed that the mean volume frequently falls off with time. Assuming that the mean volume alters at a constant rate, calculations of mean volumes have been made to obtain the best estimates at the beginning and end of the period during which the level of activity in the animal was measured. The calculated concentrations based on these volumes are shown in column 5 of Table 1. The means of the levels of the radioactivity in the animals during the period stated are listed in column 4 of Table 1 and they are corrected for loss during the washing period from data obtained in Exp. 4.

It is clear from Table 1 that the potassium concentration inside the animal is at least ten times that in the medium.

To obtain data for plotting according to equations (3) and (4), the rate of uptake of potassium was measured in a population of animals, and when the animals had taken up an appreciable amount of labelled ions they were washed for variable periods in unlabelled medium and the rate of loss measured. The values of were calculated from the uptake data and they are plotted against time in Fig. 2 a. The value of C_{in(t− ∞)} is the mean of all measurements made whilst the animals were in a steady state with the medium. As C_in(t) approaches C_{in(t− ∞)} variation becomes greater, and as Solomon (1952) pointed out only the first few points describe a straight line. Following Treheme (1954) the line drawn through the data is the mean of the slopes calculated for lines adjoining the individual points to the origin. As there is a marked change of slope after 3 hr., only data for this first period are considered. The slope of this line is—K_ont/2·3O3.

The rate of loss of labelled ions from the animals was measured after 5 hr. and after 21 hr. in the labelled solution, that is, prior to and after reaching a steady state with respect to the labelled ion. The data plotted according to equation (4) are shown in Figs. 2 b and c. The slopes of the regression lines through the data are equal to-K_out/2·303.

The slopes of the lines in Figs. 2 a, b and c are practically the same, and so it follows that the values of K_out measured by several methods must be almost identical. This, together with the results of other experiments fisted in Table 2, is considered to be justification for the theoretical treatment and it may be concluded that Spirostomum behaves as a one-compartment system to potassium.

It may be seen from Table 2 that when the concentration of the external medium is altered the values of K_out remain relatively constant, but K_in varies considerably. The significance of this point will be discussed later.

Spirostomum feeds on bacteria which grow in the culture medium when boiled wheat grains are added. To obtain a source of food for these experiments the common bacteria from a thriving Spirostomum culture were plated out and grown on agar slopes.

The uptake of potassium was measured in three groups of animals of common stock. Group i was fed throughout the experiment, whilst group 2 was fed until it was transferred to the labelled solution and group 3 was starved until it was transferred and then fed. After the initial separation of the animals feeding where necessary was carried out every 12 hr. until 6 hr. before placing the animals in the labelled solution. From then onwards it was carried out every 2 hr. in order to maintain the supply of bacteria in the medium.

The results of the experiment are shown in Fig. 3. The animals of group 1 do not reach a steady state with labelled potassium during the experiment ; the value of C_in at 28 hr. is calculated as 9·8 HIM/I. The animals of group 2 attain a maximum activity at 10 hr., i.e. the normal period, and then the level falls. This fall of activity is associated with a similar fall of volume so that the concentration of internal potassium remains fairly constant at 6·7 mM/1. The potassium activity in animals of group 3 continues to rise throughout the experiment at a greater rate than in animals of group 1. The mean volume of these animals does not appear to alter during the experimental period, and the value of the internal potassium at 27 hr. is 12 ·5 mM/1. Obviously the potassium concentration is greatly influenced by feeding, and this probably accounts for much of the variation in the experimental results.

Animals of the same population were acclimatized to culture media containing 0·02, 0 ·08, 0 ·32 and 1·28 mM/1. potassium for 3 days. They were then transferred to similar but labelled media, and after 12 hr. the concentrations of potassium in the animals were measured. The means of the volumes of the animals in the various concentrations were practically the same and the internal potassium concentrations were calculated as 3·2, 4·5, 5·7 and 7·8 mM/I. respectively.

It is concluded that the animal is capable of regulating its potassium content even when the external concentration is very low.

The potassium activities were measured in groups of animals of the same population kept at different temperatures. It was found that animals kept at 15° C. contain three times the amount of labelled ions found in animals at 25° C. This difference in activity was associated with a difference in volume so that the potassium concentrations were practically the same. Owing to the volume difference, comparable values of transfer constant were not obtained and a Q₁₀ for potassium transport could not be calculated.

A preliminary experiment with labelled sodium indicated that the animals reached a steady state with the ion in a matter of minutes and that the apparent internal concentration was very low. Therefore the rate of loss of sodium was measured from animals which were in a steady state with the labelled ion. The logarithms of the activities (C_in(t)) measured during Exp. 16 are plotted in Fig. 4 against time in unlabelled medium. It is clear from the figure that the washing out process may be divided into two components : a fast component which is calculated to account for 98 % of the sodium in the animal and a slow component which for practical purposes may be disregarded. The animal is considered to behave in the main as a one-compartment system to sodium. The concentration of sodium in the animals shown in Table 3 was calculated from the activity in the animals at zero time which was found from the regression of log C_in against time, only the first 10 min. being considered. A plot of this data according to equation (4) is equivalent to altering the scale of the ordinate so that log C_{in(t− 0)} is zero. The slope of the regression line is equal to—K_out/2·303.

Two further experiments, 17 and 18, were carried out to determine the values of C_in and K_ont for animals kept in media with different sodium concentrations. Fortunately, the volumes of the animals were not significantly different in the two experiments, so that the transfer constants listed in Table 3 may be compared. There is no correlation between the concentration of the medium and the values of K_out, but the values of K_in, calculated from equation (2), decrease with increasing C_out. Allowing for the variability of the material and experimental error it is considered that and C_in are constant when C_out is varied from 1 to 6 mM/1. The significance of constant values for C_in and K_out, but a variable when C_out is altered, will be discussed later.

Before discussing the significance of the concentration of cations in the animals it is necessary to know the concentrations of the anions. Chloride ions are likely to be the most concentrated inorganic anions in the animal, but it was impracticable to use a radioactive isotope of chlorine for their measurement. As an alternative ⁸²Br was used. Preliminary experiments showed that the animals would live in a culture medium where the sodium and potassium chlorides were replaced by bromides.

The uptake of labelled bromide is illustrated in Fig. 5. The scatter of results at the beginning of the experiment is attributed to contamination of the ⁸²Br by ⁸⁰Br. The concentration of bromide in the animal is calculated as 0·34 mM/1., whilst in the medium it was 2·5 mM/1. It is clear that the animals maintain a lower bromide concentration in their bodies than is found in the medium.

Animals were transferred from culture media to similar but labelled culture solutions containing labelled sodium and labelled bromide at three different concentrations. After 12 hr. samples were taken and washed for periods ranging from 5 min. to 4 hr. and they were assayed for radioactivity before and after the bromide had decayed. It is calculated from the regression of the bromide activity against time that animals in a medium containing 3·0 mM/1. bromide lose 4·3% of the labelled ion during the washing period of 5 min.

The calculated concentrations of sodium and bromide in the animals are summarized in Table 4. On the assumption (to be discussed later) that the proportion of bromide to total halide is the same inside as outside the animal, the concentrations of total halide in the animals have been calculated and are shown in the last column of Table 4. Once again there is no correlation between the sodium concentrations in the animal and those in the medium and the values are similar to those of previous experiments.

The mean gross volume has been used in calculating the concentrations of ions in the animals as no accurate method is readily available for measuring the water content of Spirostomum. Grobicka & Wasilewska (1925) estimated that Paramecium caudatum has a water content of 89%, whilst lida (1940) estimated it as 79%. By analogy it is expected that Spirostomum would have a high water content, and this is borne out by a series of histochemical tests which failed to find any appreciable quantities of fat or glycogen present in the experimental animals. It is suggested that the body water is at least 75 % of the body volume, so that the calculated ionic concentrations may be subject to a correction not exceeding 33 % to allow for the non-aqueous content of the animals.

Even allowing for this correction it is clear that the concentration of bromide is much lower in the cell than in the medium. Experiments with other animals suggest that cells do not discriminate between bromide and chloride ions (e.g. Koch (1938), Krogh (1938), Frey (1937)). Therefore it is assumed that when Spirostomum is in a steady state with the labelled ion the concentration of bromide may be used to calculate the total halide content of the cells, but the rate of uptake of bromide is not a measure of the rate of entry of halide.

In table 5 the concentrations of inorganic ions inside the animal are compared with the corresponding concentrations in the media. It may be seen that anions other than halide must be present inside the cells to balance the charges of cations. Conway (1945) suggested that an anion deficit in frog’s muscle may be made up by low molecular weight compounds sue has creatine phosphate, carnosine and adenosine triphosphate. If similar compounds make up the deficit in Spirostomum then it becomes clear that the animals are hypertonic to the medium.

It appears that a Donnan equilibrium might account for the high concentration of potassium and low concentration of halide in the cell if due allowance is made for experimental error and the variability of the animals.

Without a knowledge of the electrical potential gradient across the cell membrane of Spirostomum it cannot be conclusively proved that active transport of any ion is taking place, since the only certain criterion, defined by Rosenberg (1948), is that the ion in question is transported against the electrochemical gradient. There is no direct evidence to show that sodium is actively transported, but it does seem likely since otherwise it would be difficult to account for the low content of the cell when the external concentration is high. The fact that the exchange of sodium is much more rapid than that of potassium points to sodium passing through the cell membrane as an undissociated complex, since in the ionic state sodium diffuses more slowly than potassium.

The concentration of sodium in the cell is in some circumstances the same as the medium. Therefore osmotic equality between cell and medium cannot be obtained by opposing concentration gradients of sodium and potassium. However, osmotic influx of water into Spirostomum could be counterbalanced by the contractile vacuole pumping out water. Where the concentration of sodium in the medium is high there are opposing concentration gradients so that the osmotic entry of water into the animal will be slowed down.

The values of K_out and C_in for potassium ions (Table 2) remain relatively constant when C_out varies greatly, and as an approximation it may be said that varies inversely with C_out. K_in is a measure of the proportion of the external potassium concentration entering the animal in unit time, so it appears at first sight that an important part of the regulation of potassium is the result of the animals taking it up at the constant rate in spite of changes of the concentration in the medium. Similarly, in the case of sodium ions, C_in and K_out remain relatively constant when the concentration in the medium is altered (Table 3). The apparent movement of these ions independent of their concentration gradients could readily be explained by a system of exchange diffusion similar to that proposed by Ussing (1947) to account for the rapid sodium exchange in frog muscle. However, exchange diffusion cannot bring about changes of the total amount of any ion in the cell, and since the animals grow and multiply they must take up sodium and potassium. It is unlikely that the concentration of ions could be maintained constant indefinitely without the intervention of some mechanism other than exchange diffusion.

1. The concentrations of potassium, sodium and bromide in the ciliate Spirostomum ambiguum have been measured by equilibration with radioactive media.

2. The potassium concentration in the animal is at least ten times greater than the concentration in the experimental media.

3. At the higher concentrations of the experimental media, sodium is kept out of the animal against the concentration gradient. The rate of uptake and loss of sodium is largely independent of the external concentration.

4. The time of half exchange of cell sodium is approximately 3 min., whereas for potassium it is between 2 and 3 hr.

5. After equilibration of the animals in culture medium containing bromide the cell concentration of bromide is found to be lower than the external concentration.

6. The effects of temperature changes on the uptake of potassium have been investigated. The size of the animal decreases with increased temperature but the internal potassium concentration remains fairly constant.

7. Feeding the animals increases the rate of uptake and the internal concentration of potassium.

8. It is concluded that the cytoplasm of Spirostomum is hypertonic to the medium.

9. The experimental results for cation exchange might largely be explained by exchange diffusion, but actively regulating processes seem necessary to account for the maintenance of constant internal concentrations when the animals grow and multiply.

10. It is suggested that potassium is concentrated in the cell in accordance with a Donnan equilibrium arising from the presence of indiffusible anions in the cell and an active process which keeps out sodium.

I would like to thank Dr J. A. Kitching, under whose supervision this work was carried out, for his advice and encouragement, and Prof. J. E. Harris for his active interest in the problem.

The isotopes were purchased from a grant to Dr J. A. Kitching by the Royal Society, and the work was carried out during the tenure of a grant from the Department of Scientific and Industrial Research.