The Physiology of Contractile Vacuoles: X. Effects of High Hydrostatic Pressure on the Contractile Vacuole of A Suctorian

The effects of high pressure on living material are little understood and probably complicated. In a number of cases the tendency of cytoplasmic inclusions to become stratified by centrifuging is increased, and this is attributed to a decrease in protoplasmic viscosity (Marsland, 1942). High pressure is therefore considered to solate protoplasmic gels. In addition, high pressure affects contractile structures ; at ordinary temperatures it increases the tension of muscle during contraction (Brown, 1934, 1936) and it temporarily increases the frequency of beat of certain cilia (Pease & Kitching, 1939). The effect of pressure on the rate of beat of tadpole heart tissue depends upon temperature; increased pressure causes a temporary increase in frequency at temperatures above 16°C., but a decrease below 14°C. (Landau & Marsland, 1952). It appears that several reactions or processes are differentially affected. It seems likely that all the effects mentioned, including the solation of protoplasmic gels, have a common basis in the folding of protein molecules. This might be caused either directly or perhaps indirectly, through some contraction-causing reaction in which ATP might be concerned.

It has been suggested by Taylor (1923) that sol-gel changes are involved in the cycle of activity of contractile vacuoles, and cyclic changes occur in the viscosity of the neighbouring protoplasm, as judged by Brownian movement, in the Ophryo-scolecidae (MacLennan, 1933). MacLennan attributed systole to a solation of the pore plug. It seems likely that the configuration of proteins in the vacuolar wall also plays an important part in systole and is perhaps responsible for the contraction (Kitching, 19546). Moreover, according to Goldacre (1952), the contraction of protein molecules also offers a possible mechanism for secretion. It therefore seemed of interest to study the effects of high hydrostatic pressure upon vacuolar activity.

The suctorian Discophrya piriformisGuilcher (1947) was chosen for this work because it is extremely convenient material. It has rotational symmetry, it remains completely still, and its protoplasm is clear except soon after a meal. Pressures of 2000 Ib./sq.in. (136 atm.) or more cause a wrinkling of the body surface, which is associated with an expansion of the pellicle and sometimes with a decrease in protoplasmic volume (Kitching, 1954a). After release of pressure the normal body shape is slowly restored over a period of many hours.

Discophrya piriformis was cultured in Bristol tap water and fed on Paramecium spp. and Colpidium sp. No food was given for several days before an experiment.

The pressure vessel and associated equipment have already been described (Kitching, 1954 a). The glass windows are set in steel disks which can be removed. The vessel was filled with medicinal paraffin (‘mineral oil’). A small drop of the culture medium containing Discophrya was placed on the inner side of one of the windows, and the window was mounted in position in contact with the medicinal paraffin. Pressure was applied from a hydraulic pump in from 1 to 3 sec., and was released in 1 sec. or less.

The technique of observation was as described in previous papers of this series. Normally it was possible to choose a Discophrya with only one contractile vacuole, the experiment illustrated in Fig. 2 being an exception. Observations were carried out before treatment, at one or more high pressures, and after release of pressure. In several cases the organism was re-examined the following day, and was apparently healthy. The room temperature was between 15·7 and 20·0°C., and varied very little in any one experiment.

The drawings shown with the graphs (Figs. 1–3) were reconstructed from rough sketches and measurements made hastily during the course of the experiments. They are intended to show the general condition of the animal, and may not be accurate in detail.

Eleven experiments were carried through successfully, and are summarized in Table 1. Some others had to be abandoned because the contractile vacuole was obscured by the creasing of the body surface, which always occurs at pressures of 2000 Ib./sq.in. or more. Three experiments are illustrated in Figs. 1–3.

The rate of vacuolar output was depressed at all pressures from 1000 Ib./sq.in. (68 atm.) upwards, but the effect was slight in the lowest part of the range (Fig. 1). No vacuolar activity could be detected at 7000 Ib./sq.in. (476 atm.) or over. On release of pressures of 1000–5000 Ib./sq.in. (68–340 atm.) the rate of output recovered almost immediately to about normal (Figs. 1, 2). After 10,000–14,000 lb./ sq.in. (680–952 atm.) there was some delay before the contractile vacuole started working again (Fig. 3), and when that delay was considerable the protoplasm appeared to swell within the wrinkled pellicle and to fill it out. There was no recovery after 16 min. at 15,000 Ib./sq.in. (1020 atm.) in experiment 210452a (Table 1); in other observations a small proportion of individuals showed some recovery of vacuolar activity after 7 min. at this pressure (Kitching, 1954a). This treatment is therefore close to the limit.

The vacuolar frequency was rather variable at pressures below 2000 Ib./sq.in. (136 atm.), and no particular significance can be attached to the changes observed. At 2000–3000 Ib./sq.in. (136–204 atm.) the frequency increases markedly, and the increase was maintained until the pressure was released (Fig. 1). There appears to have been no correlation with creasing of the pellicle, as in experiment 020352 b (Table 1) the increased frequency was maintained throughout the period of exposure to 2000 Ib./sq.in. even though the pellicle completely lost its wrinkles during the later part of this treatment. This conclusion is also supported in Fig. 1. Moreover, the frequency decreased to about its original level on release of pressure in the second part of experiment 020352b, even though the pellicle remained creased. At pressures of 5000–6000 Ib./sq.in. (340–408 atm.) the frequency decreased considerably, but after release of pressure it rose sharply to a peak before becoming steady. After release from a pressure of 7000 Ib./sq.in. (476 atm.) or more, when the vacuole resumed activity the frequency rose to a level considerably above the original.

It has been suggested that systole is associated with a folding up of protein molecules, ultimately to globular form, this process providing not only for the opening of the pore but also for the contraction of the vacuolar wall and its ultimate solation and disappearance (Kitching, 19546). The nature of the hypothetical protein structure in the vacuolar wall is unknown. The increase of vacuolar frequency at 2000–3000 Ib./sq.in. accords well with this suggestion, as it might reduce the stability of the pore plug, promote contraction and solation of the vacuolar wall, or influence the chemical processes controlling the rhythm of contraction. At higher pressure the rate of secretion is considerably depressed, and with it the vacuolar frequency. The linkage between the two processes has already been noted and discussed (Kitching, 1954b).

Marsland (1950) found that cortical gelation in the eggs of Arbacia decreases with pressure but increases with temperature, these two influences acting in opposition. On the other hand, they both act together in accelerating the vacuolar rhythm of Discophrya. In the latter case we are probably concerned with a rhythmic change in protein configuration instead of a steady state of solation and gelation. The frequency (and the amplitude) of the isolated frog’s heart is increased at moderately high pressure (60 atm.) (Edwards & Cattell, 1928).

It is too soon to discuss the various other observations recorded in this paper.