Effects of helium and other inert gases on Echinosphaerium nucleofilum (protozoa, heliozoa)

‘ Inert’ gases such as neon, nitrogen, argon, krypton and xenon have been shown to have anaesthetic effects on various organisms at elevated partial pressures (Brauer & Way, 1970; Schreiner, 1968). It has usually been assumed that helium has no such effects, or is at least extremely innocuous even at high pressure. For this reason it has been used in oxygen-helium mixtures for human divers and in experiments showing pressure reversal of anaesthesia in mice (Lever et al. 1971). The mechanism of anaesthesia remains undecided. According to one explanation anaesthetics may act by expanding the cell membranes, whereas hydrostatic pressure might oppose this effect. Other possible explanations of anaesthesia involve the disruption of other cell components, such as microtubules (Allison & Nunn, 1968) or microfilaments (Wicklund & Allison, 1972). Clinical anaesthetics have been shown to disrupt the rather labile microtubules which support the axopods of Heliozoa (Allison et al. 1970).

In this paper we describe the effects of several inert gases on Echinosphaerium (Actinosphaerium) nucleofilum (Heliozoa). This animal has a spherical body and numerous radiating long fine processes known as axopods. The axopods of Echino-sphaerium and of the closely related Actinophrys sol are each supported by a closely and characteristically linked array of microtubules (Macdonald & Kitching, 1967; Roth, Pihlaja & Shigenaka, 1970). Hydrostatic pressure causes a shortening or collapse of the axopods (Kitching, 1957 b) together with disintegration of the supporting systems of microtubules (Tilney, Hiramoto & Marsland, 1966). Breakdown of the microtubules and withdrawal of the axopods is also caused by low temperature (Tilney & Porter, 1967), by colchicine (Tilney, 1968) and by various other treatments. The withdrawal of axopods can clearly be used as an indicator of the disintegration of the supporting system of microtubules. We have used it in this way for the study of elevated pressures of certain gases. This investigation may have some relevance both to diving physiology and to anaesthesia.

Cultures of E. nucleofilum were grown at 21°C in local (Yare or Tiffey) river water which had been heated to 90 °C, allowed to cool, and then filtered and re-aerated ; the pH was 6·8−7·5. They were fed twice weekly on a dense culture of Colpidium with other Protozoa, cultured in the same river water containing boiled wheat grains.

Two pressure vessels were used for this work – the hydrostatic pressure vessel already described by Kitching (1954, 1957a), and a specially designed gas pressure vessel (Fig. 1). The hydrostatic vessel was used for experiments in which the hydro-static pressure was raised without significant change in the gas content of the medium surrounding the animals, and the gas vessel for experiments in which the animals were subjected to elevated partial pressures of one or more gases. In both vessels the animals are suspended in a hanging drop on the underside of the upper window. In the hydrostatic vessel the drop is in direct contact with the medicinal oil (‘liquid paraffin’) used as the hydraulic fluid, and there is no gas phase present; in the gas pressure vessel it hangs over the gas admitted for the experiment.

The basic design of the gas pressure vessel is similar to that of the hydrostatic pressure vessel - a stainless steel box with thick walls, and a pair of glass windows set flush in steel window plates which are held in position by annular holding screws. The central perforation of these screws is sufficiently large to allow adequate scanning movements of the microscope objective and illuminator. PTFE washers are used, and the vessel is designed for a maximum working pressure of 300 atm.

The gas pressure vessel is filled with gas at appropriate pressures through a system of Ermeto in nominal bore high pressure piping, Ermeto shut-off valves, and short lengths of flexible pressure hose connecting it with the gas cylinders (Fig. 2). Gas is supplied to both sides of the pressure vessel at once, so as to reduce the disturbance to the hanging drop during compression or decompression.

The optical system consists of a lamp housing carrying a Zeiss long working dis-tance phase condenser (‘2 stop slider-mounted’) below the vessel, and a microscope head above the vessel fitted with a Zeiss × 10 phase objective or a Union long working distance × 40 phase objective. The complete optical system can be moved as one unit, so that any part of the hanging drop can be brought into the field of view.

The temperature of both vessels was maintained at 21 °C by the circulation of water from a waterbath through channels in the vessel walls. Tests were carried out in the gas pressure vessel in which the temperature in a hanging drop of culture medium, mounted in the usual way, was measured during compression and decompression. For this purpose the leads of a very small copper-nichrome thermocouple were passed through holes in a spare steel window plate (fitted with window) and sealed with Araldite. Measurements showed that there was a rise in the temperature of the water droplet on compression which depended on the rate of compression. For a com-pression to 100 atm extending over 45 s the rise would be approximately 2 °C. This compression rate was never exceeded in our experiments. The heat generated was dispersed very rapidly, and the temperature of the droplet was restored to its original value within 10−20 s of the end of compression. For decompression the fall in temperature was less.

A drop of the culture fluid containing several (usually 4−5) animals was transferred with a pipette to the upper window of the gas pressure vessel, where it measured about 1·5−2·5 mm across. After the window had been fitted in place and the holding screw had been tightened, a small amount of humidified air containing 0·5 % CO² was passed through the vessel, and the animals were allowed to recover from the handling for 1 h. By this time the axopods were fully extended.

Estimates of the diameter of the cell body, and of the lengths of axopods from cell surface to tip, were made for each animal by means of a graticule in the eyepiece of the microscope. The axopods were measured in the plane of focus in which the body size was greatest, and the average length of the axopods was taken as the length over which the majority extended. This measurement was made in four directions at right angles, and the mean was recorded, so that slight variations sometimes produced by the lighting, and the position of the animals in the water droplet, were eliminated. All measurements in any one series of experiments were made by the same observer. The mean axopod length at any time is expressed as a percentage of the original length at the beginning of the experiment. Great care was taken to ensure consistency of method.

After the initial measurements had been made, some of the air-CO² mixture was passed through the vessel, the taps were closed, and the appropriate gas or gases were admitted to the required pressure. As the gas was let in directly from cylinders filled to not more than 130 atm, this was the maximum pressure used in these experiments. Before the second gas of a mixture was admitted to the vessel, the remainder of the first was released from the supply tubing, and the connexion between pressure vessel and delivery pipes was closed as soon as the pressure in the vessel and pipes had equalized. Measurements of axopod lengths were made at 5, 10, 15, 20, 30, 45 and 60 min after compression, and a record was also kept of the condition of the animals, of the extent of any cell damage, and of the time at which this occurred.

After 60 min of treatment, the pressure in the vessel was checked and then released. If the animals were to be observed afterwards, the decompression was very slow (taking 5 min or longer), and some of the air-CO² mixture was then passed through.

For each group of observations, the mean and standard deviation of the axopod lengths for all the animals present (expressed as a percentage of the original axopod lengths) was calculated. The standard deviation was normally between 5 % and 15 % of the original axopod length, although it was sometimes less and occasionally more.

There was some considerable variation in the susceptibility of animals from different cultures, and of animals taken from the same culture over a period of time. Accordingly most of the experiments were carried out in short series of 4−8 done within 2−3 days on animals from a single culture. In some, but not all, of these series a hydrostatic control experiment was included. Experiments from different cultures or times could only be considered together after a large number had been carried out.

In four experiments animals were mounted and equilibrated in the usual way and were observed as in a normal experiment but at atmospheric pressure in the air-CO² mixture. The animals remained in good condition, with no shortening of the axopods.

Thirty experiments (on 112 animals) were carried out with the hydrostatic vessel, with pressures ranging from 40 to 130 atm. These comprised four series in which the effects of a range of hydrostatic pressures were compared, and also a number of individual experiments carried out simultaneously with experiments in which a gas was used at the same pressure. No change was seen in the cell body of the animal at any pressure up to 130 atm, nor was there any leakage of the cell contents. Up to about 80 atm the axopods remained long and fine; above 80 atm they shortened over the first 10−20 min after compression, but then began to lengthen again, even though the pressure was maintained. The amount and duration of shortening was greater at higher pressures. Recovery was sometimes complete within an hour of compression at 80−100 atm, but not so at higher pressures.

The results for a typical series of experiments are shown in Fig. 3. The area en-closed between the curve for any one pressure and the horizontal at 100 % (representing no change in axopod length) can be used as a measure of the intensity of the response, since it reflects both the degree of shortening and the period over which it persists. The mean values for all the experiments at each pressure are given in Fig. 4. The higher the pressure, the more severe is the effect on the axopods. These results are consistent with those of Tilney & Porter (1967) for this species and of Kitching (1957) for A. sol but are more comprehensive for the low range of pressures under consideration.

The effects of helium have been investigated in 70 full experiments on 336 animals at 10−130 atm. General observations were made in 25 other experiments on 128 animals, without measurement of axopod lengths. At between 60 and 90 atm, helium caused some of the animals to leak, and a few of those which leaked severely cytolysed.

The maximum incidence of leakage was about 12 %, at 80 atm (Fig. 5). No leakage or death occurred below 60 atm or above 90 atm, but changes in the appearance of the cell body were sometimes observed ; the endoplasm appeared denser, the cortex paler and more reticulate, and the cell surface more fuzzy.

The experiments concerned with axopod lengths included 6 series, each on uniform material as already described. The results of a typical series are shown in Fig. 6. Many other experiments with helium were carried out as controls for others involving mixtures of helium and argon (see below). The mean values of axopod lengths for each pressure at each time yield curves similar to those shown in Fig. 6; the areas enclosed are plotted against pressure in Fig. 7, which shows a maximum effect of helium at 80 atm. Thus the pressure of helium most effective for shortening axopods is the same as that which causes the highest incidence of leakage or death.

In eight experiments the helium was released slowly after the usual 1 h of treatment, and there was a further period (60 min) of observation in the air-CO² mixture.

The axopods, which had already begun to recover in length while still in helium, continued to do so after a short pause which was probably due to the physical effects of decompression.

Nitrogen was used in 24 full experiments on 102 animals at 10−102 atm. At pressures of 60 atm upwards, nitrogen caused leakage in some animals and sometimes death. Damage occurred more quickly, and was more severe, the higher the pressure (Fig. 8). These effects are fully confirmed in 45 earlier experiments which are not included in Figs. 7 and 8 because the period of observations varied. Some changes in appearance, similar to those already described for helium, were recorded at pressures below 60 atm.

For observation of axopod lengths, the 24 full experiments were performed in four series. In general, the higher the pressure of nitrogen, the greater and the faster was the shortening ; and it was followed by less recovery within the period of treatment. However, the results were rather variable. A typical series is shown in Fig. 9. The areas enclosed by the curves for the combined results of all 24 experiments (Fig. 10) show an irregular drift upward with increasing pressure.

Argon was used in 60 experiments on 250 animals at 10−130 atm. (Single animals were used in eight of the earliest of these.) It caused leakage and death, the effect being both more rapid and more severe at higher pressures (Fig. 11). Death often took place by sudden bursting and disintegration. The lengths of axopods could only be studied at pressures up to 40 atm, because of the rapid cell damage at higher pressures. There was no significant shortening, and even after bursting at higher pressures isolated axopods with a small globule of cell material at the base remained long and fine for periods of up to 15 min.

Krypton was used in 39 experiments on 193 animals at 5−40 atm. It damaged the cells severely, even at low pressures (Fig. 12). Some cells burst suddenly, as with argon. Damage was more severe, took place more quickly, and affected more animals, with increasing pressure. In all these respects krypton was more effective than argon at corresponding pressures. The length of axopods could be measured only at pressures up to 20 atm, because of the rapid onset of cell damage at higher pressures. No significant shortening of axopods could be detected. As with argon, isolated axopods urvived for up to 15 min after the cell had burst.

In two preliminary series of experiments, different amounts of helium were added to fixed amounts (60 and 80 atm) of argon. (At this time the pressure vessel lacked the facility for releasing residual gas from the supply tubing, so that the argon content of the vessel could have been raised when the helium was added.) In both series it was found that in appropriate mixtures the cell damage normally found with these pressures of argon was abolished, and the axopod shortening normally found with these pressures of helium was reduced.

After modification of the gas supply system for more precise mixing, experiments were carried out in groups, each group comprising one experiment with a known gas mixture, one experiment with argon at the partial pressure of argon used in the mixture, one experiment with helium at the partial pressure of helium used in the mixture, and one experiment with helium at the total gas pressure used in the mixture. The results for one typical group of experiments are shown in Fig. 13. Again it was established that addition of argon greatly reduced the axopod shortening found other-wise with helium, while the presence of sufficient helium prevented the cell damage found otherwise with argon. The results for the 8 groups of experiments on a total of 157 animals are summarized in Table 1. It appears that 40−60 atm of helium and 40 atm of argon balance each other in respect of both cell damage and shortening of axopods. Cell damage occurred with too little helium (He 20 atm + A 40 atm), and axopod shortening with too much (He 80 atm + A 40 atm). Because of the natural variability of the material and because the treatments were less severe, it is not clear whether a similar balance exists at lower pressures.

In a further series of six experiments, different pressures of argon were added to a fixed pressure of 60 atm of helium. Argon was increasingly effective (up to 40 atm) in reducing the amount of axopod shortening (Fig. 14).

In these experiments we have shown that helium causes a shortening of the axopods of Echinosphaerium at pressures below those at which hydrostatic pressure causes shortening. It also causes leakage in some animals, and death in a few, within a time of treatment and range of pressures at which hydrostatic pressure has no such effects

All these effects of helium reach a maximum at 80 atm, and decline at still higher pressures.

This may be a manifestation of a ‘pressure reversal’ effect, comparable with the pressure reversal of anaesthesia in vertebrates (Lever et al. 1971). In accordance with this interpretation, we might suppose that the severity of the helium effect would increase with increasing concentration of helium, but is in fact counteracted by the associated increase in hydrostatic pressure. The latter would eventually become pre-dominant, thus giving rise to the peak in the graph of axopod shortening (or cell damage) against pressure of helium. In the case of vertebrate anaesthesia it has been suggested that pressure counteracts an expansion of the cell membrane produced by the anaesthetic, and a similar explanation might apply in the case of helium. The fact that both the actions of helium, on axopod length and on cell stability, have their maxima at the same pressure might suggest that these actions are perhaps associated with the same site, but it is difficult to reconcile this with the results for argon and krypton.

The inert gases are effective in causing leakage and cell disintegration in the order helium < nitrogen < argon < krypton. This is the usual order of potency, in physiological tests on a variety of material ranging from protoplasmic streaming to narcosis in mammals (Schreiner, 1968). On the other hand these gases are effective in the reverse order for the shortening of axopods: krypton and argon had no visible effect, and helium was the most effective. This suggests that there may be two separate sites of reactivity. Speculatively, these could be the microtubules (for shortening of axopods) and the cell surface or cortical structure (for cell damage). In either case the gas might act directly or indirectly on these structures. Alternatively, it might be supposed that shortening is a reaction to a moderate stimulus and cell damage to a strong stimulus acting at the same site. It would be necessary to assume that cell damage could completely override the preliminary shortening reaction if the timulus were sufficiently intense ; this seems unlikely.

The microtubules of Heliozoa are labile and easily caused to aggregate or disaggregate. It is not known to what extent clinical anaesthetics act directly upon them, or indirectly through some other controlling mechanism. The disaggregating effects of colchicine (Tilney, 1968) and of urea (Shigenaka, Roth & Pihlaja, 1971) have been interpreted as direct. A possible interpretation of our results is that gases of low molecular weight have a great affinity for the microtubular proteins, while those of higher molecular weight preferentially affect other cell components associated with cell stability. The order of effectiveness of these gases in causing cell damage is the same as that found by other workers using different animals, and corresponds with their oil-water (Seeman, 1972) or oil-gas (Miller, 1972, Fig. 6) partition coefficient.

It is as yet difficult to discuss the counteracting effects of argon and helium. One possibility is that solubility of the two gases is altered by the higher total pressure of the mixture. Enns et al. (1965) have shown that the equilibrium pressure of a fixed quantity of gas dissolved in water is increased if the hydrostatic pressure is raised, thus demonstrating that hydrostatic pressure as such renders a gas less soluble ; and Schröder (1969) has shown that the solubility coefficient −the quantity of gas dissolved in water per atmosphere of partial pressure − decreases with increased pressure of that gas. From Schröder’s data we should expect a reduction in the quantity of argon dissolved at 20 °C, and 40 atm partial pressure, of about × 0·9 by the addition of 60 atm of helium. This seems hardly enough to account for the whole of the effect of the opposing argon, although it could contribute. Also, we are uncertain whether Schröder’s results are due solely to hydrostatic pressure. We do not know whether the solubility of a single gas (e.g. argon) is modified by additional pressure of that gas specifically, or whether the addition of a second gas (e.g. helium) would alter the solubility coefficient of the first (e.g. argon) in the same way as would the addition of more argon.

A second possible explanation arises from the suggestion that the effect of helium may be counteracted by hydrostatic pressure. The addition of argon to a fixed quantity of helium in the pressure vessel would raise the hydrostatic pressure without changing the partial pressure of helium. This might explain the fact that the addition of argon can prevent the shortening of axopods which would otherwise be caused by the helium. Although our results do not give any indication either way, it is possible that the effect of argon in causing cell damage might also be counteracted by hydro-static pressure, and thus by the addition of helium.

A third possibility is that there is a simple competition between argon and helium for the supposed two sites of action. This implies that argon can occupy the site at which helium is active in causing axopod shortening, but that it is ineffective in this respect while at this site. Similarly, helium might occupy the argon site, preventing argon from doing so and from causing cell damage. There is no evidence as yet to support this third possible explanation. The use of the term ‘site’ in this discussion need not necessarily imply a specific structural point of action, such as the cell membrane. The cytolytic effect of the inert gases might possibly be interpreted in terms of an interference with metabolic processes, which in turn might lead to cortical break-down. Nevertheless the rapidity of cytolysis in krypton suggests a direct action.

All our comments are speculative. At present a combination of the first and second explanation offers the simplest interpretation. In any case the observations reported may suggest that in experiments on animals, or in deep-diving by man, helium should not be regarded as entirely neutral.

Our thanks go to Mr. G. R. Bevin for constructing the gas pressure vessel. We are also grateful to the Science Research Council for generous support.