Control of cellular contraction by calcium in Vorticella

The sessile peritrich ciliate Vorticella exhibits contraction of an all-or-nothing type involving shrinkage of the cell body and coiling of the stalk in response to mechanical stimulation. As early as 1958, Hoffmann-Berling found that the stalk of a glycerol-extracted Vorticella coiled when Ca²⁺ was added to the external solution and that it uncoiled when Ca²⁺ was removed (see also Amos et al. 1976). Ochiai et al. (1983) applied Ca²⁺ electrophoretically to a localized portion of the stalk of a glycerol-extracted Vorticella and found that only the portion subjected to Ca²⁺ showed coiling. These results suggest the presence of a mechanism for evoking a regenerative increase in intracellular Ca²⁺ concentration which, in turn, is responsible for an all-or-nothing cellular contraction in living Vorticella.

Allen (1973) reported that external Ca²⁺ was not required for cellular contraction in living specimens of Vorticella. Katoh and Naitoh (1991, 1992a) found that the mechanical threshold for evoking a contraction in Vorticella was not affected by removal of Ca²⁺ from the external solution. These results imply that the Ca²⁺ required for the contraction comes from intracellular Ca²⁺ storage sites and not from the external solution.

In their electron microscopical examinations of vorticellid ciliates, several authors have reported that membranous tubules (tubular endoplasmic reticulum) are present in the spasmoneme, which is responsible for the coiling of the stalk, and in the myoneme, which is responsible for the shrinkage of the cell body, and that endoplasmic reticulum is present around the myoneme (Carasso and Favard, 1966; Allen, 1973; Amos, 1972; Amos et al. 1976). Moreover, Carasso and Favard (1966) demonstrated the presence of calcium in the tubules and the endoplasmic reticulum by cytochemical methods. These membranous systems are, therefore, strong candidates for the Ca²⁺ storage sites.

We recently suggested the possibility that the Ca²⁺-induced Ca²⁺ release mechanism, which was first reported by Endo et al. (1970) (see also Ford and Podolsky, 1970) in the sarcoplasmic reticulum of skeletal muscle fibres, might cause a regenerative increase in the intracellular Ca²⁺ concentration responsible for the all-or-nothing type of contraction in Vorticella (Katoh and Naitoh, 1992a). In the research described in the present paper, we accordingly determined the effects of Ca²⁺ and of drugs that affect the liberation of Ca²⁺ from the sarcoplasmic reticulum of the skeletal muscle fibre (Endo, 1977; Lai et al. 1988) on the contractile activity of both living and Triton-extracted specimens of Vorticella. Some of these results have been presented verbally elsewhere (Katoh and Naitoh, 1991, 1992b).

Specimens of Vorticella sp. were grown at 20 °C on a glass slide in a bacterized saline solution (0.1 mmoll⁻¹ KCl, 0.09 mmoll⁻¹ CaCl₂ and 0.1 mmoll⁻¹ MgSO₄ final concentration) containing 0.1 % (w/w) dehydrated cereal leaves (Sigma). Specimens were washed with a standard saline solution, which contained 1 mmoll⁻¹ KCl, 1 mmoll⁻¹ CaCl₂ and 10 mmoll⁻¹ Tris–maleate buffer (pH 7.0), and then immersed in the same solution for approximately 10 min prior to experimentation.

Detergent-extraction of the specimens of Vorticella was performed according to Naitoh and Kaneko (1972). Washed specimens attached to a glass slide were kept immersed for approximately 1 min in the extraction medium, which consisted of 0.1 % (v/v) octylphenoxypolyethoxyethanol (Triton X-100, Wako Pure Chemicals Co., Tokyo), 20 mmoll⁻¹ KCl, 10 mmoll⁻¹ EDTA and 10 mmoll⁻¹ Tris–maleate buffer (pH 7.0). The extracted specimens were then washed gently three times with a washing medium, which consisted of 50 mmoll⁻¹ KCl, 2 mmoll⁻¹ EGTA and 10 mmoll⁻¹ Tris–maleate buffer (pH 7.0), and kept in this medium for 15 min to remove Triton X-100. Washed extracted specimens were immersed in reactivation media with differing Ca²⁺ concentrations. The reactivation medium consisted of 50 mmoll⁻¹ KCl, 10 mmoll⁻¹ Tris–maleate buffer (pH 7.0) and calcium buffer with 2 mmoll⁻¹ EGTA.

In order to change cytoplasmic Ca²⁺ concentration of a living specimen, Ca²⁺ buffer solution was injected into the cell body according to the method of Hiramoto (1974). A specimen was first detached from its substratum (a glass slide) and its cell body was fixed at the tip of a suction pipette (about 5 μm in inner diameter) by lowering the hydrostatic pressure inside the pipette. The tip of a micropipette (1–2 μm in inner diameter) for injection was then inserted into the cell body. Ca²⁺ buffer solutions with different Ca²⁺ concentrations were prepared by mixing 100 mmoll⁻¹ EGTA and 100 mmoll⁻¹ calciumbound EGTA in various ratios. The pH of the Ca²⁺ buffers was adjusted to 7.0 with 100 mmoll⁻¹ Pipes–KOH buffer. The volume of Ca²⁺ buffer injected into the cell body was approximately 3.5 pl, which corresponded to about 10 % of the volume of the cell body and was large enough to keep the cytoplasmic free Ca²⁺ concentration at a value identical with that for the Ca²⁺ buffer. The external solution for the injection experiments was a mixture of 1 mmoll⁻¹ KCl, 1 mmoll⁻¹ EGTA and 10 mmoll⁻¹ Tris–maleate buffer (pH 7.0).

Caffeine, ryanodine, procaine, Ruthenium Red (all from Wako Pure Chemicals Co., Tokyo) and Ins(1,4,5)P₃ (D-myo-inositol 1,4,5-trisphosphate; Boehringer GmbH, Mannheim) were dissolved in solutions containing 100 mmoll⁻¹ KCl, 0.02 mmoll⁻¹ EGTA and 5 mmoll⁻¹ Pipes–KOH buffer (pH 7.0), which were used for intracellular injection. The amount of solution injected into the cell body was approximately 3.5 pl. These drugs were dissolved in a solution containing 1 mmoll⁻¹ KCl, 1 mmoll⁻¹ EGTA and 10 mmoll⁻¹ Tris–maleate buffer (pH 7.0) for external application.

A mechanical stimulus was applied to the cell body with a microneedle driven by a piezoelectric transducer according to Katoh and Naitoh (1992a) (see also Naitoh and Eckert, 1969). Magnified images (approximately ×200) of the specimens were recorded on a video tape, and the specimens’ contractile responses were examined on the displayed pictures. All the experiments were performed at room temperature (20–23 °C).

Triton-extracted specimens of Vorticella exhibited a contraction (shrinkage of the cell body and coiling of the stalk) when the free Ca²⁺ concentration in the reactivation medium was raised above a threshold value of approximately 8.9×10⁻⁸ moll⁻¹. Fig. 1 shows a representative series of photographs of a single extracted specimen in five different reactivation media with different free Ca²⁺ concentrations ranging from 8.9×10⁻⁸ to 1.9×10⁻⁶ moll⁻¹.

The relationship between the degree of contraction and the free Ca²⁺ concentration of the reactivation medium is shown in Fig. 2. The degree of shrinkage of the cell body is expressed as the decrease in the length of the longitudinal (oralo-aboral) axis of the cell body relative to the maximum decrease in the length at a Ca²⁺ concentration higher than 1.9×10⁻⁶ moll⁻¹(ΔLc/ΔLc_max; see the inset of Fig. 2). Similarly, the degree of coiling of the stalk is expressed as the decrease in the distance between the ends of the stalk relative to its maximum decrease at a Ca²⁺ concentration higher than 1.9×10⁻⁶ moll⁻¹ (ΔLs/ΔLs_max; see the inset of Fig. 2). Both values increased sigmoidally with a logarithmic increase in the Ca²⁺ concentration, and reached 100 % at approximately 1.9×10⁻⁶ moll⁻¹ Ca²⁺. The Ca²⁺ concentration corresponding to 50 % contraction was approximately 1.9×10⁻⁷ moll⁻¹.

We examined the contractile responses of living specimens of Vorticella to injection of Ca²⁺ buffer solution into the cell body. Fig. 3 shows three representative pairs of photographs of three different specimens into which were injected Ca²⁺ buffer solutions with different free Ca²⁺ concentrations.

The relationships between the degree of contraction and free Ca²⁺ concentration of injected buffer solution are shown in Fig. 4. Neither coiling of the stalk nor shrinkage of the cell body was evoked when the free Ca²⁺ concentration was lower than 5.1× 10⁻⁸ moll⁻¹ (corresponding to Fig. 3Aii). When the free Ca²⁺ concentration was slightly higher than 5.1×10⁻⁸ moll⁻¹ the stalk showed more than 79 % of the maximum coiling, whereas the cell body remained relaxed (corresponding to Fig. 3Bii). When the free Ca²⁺ concentration was further increased the cell body started to show shrinkage. The degree of shrinkage increased with increasing Ca²⁺ concentration, reaching its maximum value (100 %) at 2.0×10⁻⁷ moll⁻¹. The degree of coiling of the stalk was also 100 % at this Ca²⁺ concentration (corresponding to Fig. 3Cii). The Ca²⁺ concentrations that corresponded to 50 % contraction were approximately 5.1×10⁻⁸ moll⁻¹ for coiling of the stalk and 1.4×10⁻⁷ moll⁻¹ for shrinkage of the cell body.

In this series of experiments, we examined the effects of drugs that affect Ca²⁺ release from the sarcoplasmic reticulum in skeletal muscle fibres on the contractile activities of both living and Triton-extracted specimens of Vorticella.

Living specimens of Vorticella exhibited spontaneous contractions at very low frequency (0.1±0.09 min⁻¹) in the standard saline solution (the reference solution). The frequency of contraction, which was determined for successive 1 min periods, increased suddenly when caffeine was introduced into the standard saline solution at a final concentration of 25 mmoll⁻¹ (Fig. 5). The frequency decreased gradually with time, reaching a steady value that was significantly (P<0.05) higher than that before application of caffeine in about 7 min. In contrast, the duration of contraction, which was defined as the period from the beginning of contraction to the time when the stalk had uncoiled by 80 % of the maximum degree of coiling, increased immediately after application of caffeine and remained unchanged for more than 10 min. The increase in the duration was mostly due to prolongation of the relaxation phase of the contraction.

To examine the effects of caffeine concentration more precisely, both the frequency and the duration of contraction were determined by averaging the values for 3 min after administration of caffeine at different concentrations ranging from 2.5 to 50 mmoll⁻¹. As shown in Fig. 6, both values increased with increasing caffeine concentration above respective threshold values (approximately 2.5 mmoll⁻¹ for frequency and 5 mmoll⁻¹ for duration). The frequency decreased when caffeine concentration was more than 50 mmoll⁻¹.

An injection of a saline solution containing 50 mmoll⁻¹ caffeine into the cell body of a living specimen caused a sudden increase in the frequency of contraction to 3.0±0.9 min⁻¹ (N=7); a value significantly (P<0.025) higher than that measured immediately after an injection of the caffeine-free reference solution (0.57±0.18 min⁻¹, N=7). In contrast, the duration of contraction was relatively unaffected by the injection of caffeine (control, 0.85±0.15 s, N=4; caffeine-injected, 0.90±0.12 s, N=5). In contrast to living specimens, administration of caffeine into reactivation medium did not produce contraction in specimens extracted in Triton X-100.

Ryanodine did not affect the contraction of living specimens of Vorticella when it was applied externally (1–10 μmoll ^{− 1}), but its injection into the cell body (2 μmoll ^{− 1}) caused a sudden increase in the frequency of contraction to 0.56±0.06 min⁻¹ (N=8), a value significantly (P<0.001) greater than that obtained immediately after an injection of the ryanodine-free reference solution (0.19±0.06 min⁻¹, N=7). In contrast, administration of ryanodine into the reactivation medium (1–10 μmoll⁻¹) did not produce contraction in Triton-extracted specimens.

Neither external application (100 μmoll⁻¹) nor injection (100 μmoll⁻¹) of InsP₃ affected the frequency or duration of contraction. InsP₃ (2–100 μmoll⁻¹) did not affect the Ca²⁺-induced contraction of Triton-extracted specimens.

We examined the effects of procaine on the caffeine-induced contraction. As shown in Fig. 7, external application of procaine (1–50 mmoll⁻¹) suppressed the increase in the frequency of contraction expected to be evoked by 10 mmoll⁻¹ caffeine in living specimens of Vorticella. Procaine slightly affected the duration of contraction.

A mechanical stimulus given to the cell body of living specimens failed to evoke a cellular contraction in the presence of 50 mmoll⁻¹ procaine. The inhibitory effect of procaine disappeared 10–20 min after removal of the drug from the external solution.

Injection of a solution containing 5 mmoll⁻¹ procaine into the cell body of living specimens of Vorticella suppressed an increase in the frequency of contraction evoked by external application of 10 mmoll⁻¹ caffeine. Specimens injected with procaine did not contract in response to mechanical stimulation. In contrast to living specimens, procaine had no effects on Ca²⁺-evoked contraction in Triton-extracted specimens.

External application of Ruthenium Red (1–500 μmoll⁻¹) did not affect the caffeine-evoked increase in contraction frequency in living specimens. Living specimens contracted in response to a mechanical stimulus applied to their cell body in the presence of Ruthenium Red in the external solution (1–500 μmoll⁻¹). In contrast, intracellular injection of a solution containing 1 μmoll⁻¹ Ruthenium Red suppressed the caffeineevoked increase in the frequency of contraction in living specimens. Specimens injected with Ruthenium Red failed to show a contraction in response to mechanical stimulation.

In the next series of experiments, a solution containing 1 μmoll⁻¹ Ruthenium Red was first injected, and a Ca²⁺ buffer solution was subsequently injected, into the cell body of a living specimen of Vorticella. When a Ca²⁺ buffer solution with a free Ca²⁺ concentration of 8.9×10⁻⁸ moll⁻¹ was injected within 3 min of Ruthenium Red injection, the stalk showed maximum coiling (98.3±3.8 %, N=3), while the cell body shrank by only 13.8±6.3 % (N=3). When the same buffer solution was injected more than 5 min after Ruthenium Red injection, the degree of coiling of the stalk was as low as that of the cell body (14.5±5.1 %, N=6, for the stalk; 20.9±3.9 %, N=6, for the cell body). Fig. 8 shows three representative pairs of photographs of three different specimens of Vorticella into which Ca²⁺ buffer solutions with different free Ca²⁺ concentrations (A, 1.4×10⁻⁷ moll⁻¹; B, 2.1×10⁻⁷ moll⁻¹; C, 4.8×10⁻⁷ moll⁻¹) had been injected 5 min after preinjection of Ruthenium Red.

The relationship between the degree of contraction and free Ca²⁺ concentration of the injected Ca²⁺ buffer solution is shown in Fig. 9. The degree of shrinkage of the cell body and of coiling of the stalk increased sigmoidally with logarithmic increases in free Ca²⁺ concentration. The Ca²⁺ concentration that corresponded to 50 % contraction was approximately 1.4×10⁻⁷ moll⁻¹. In contrast to living specimens, administration of 1–10 μmoll⁻¹ Ruthenium Red into the reactivation medium had no effect on Ca²⁺-evoked contraction in Triton-extracted specimens. Similar results were obtained when Ca²⁺ buffer solutions were injected into procaine-injected specimens (data not shown).

Triton-extracted specimens of Vorticella exhibited shrinkage of the cell body and coiling of the stalk when the free Ca²⁺ concentration of the reactivation medium was raised above approximately 8.9×10⁻⁸ moll⁻¹. Shrinkage of the cell body is caused by Ca²⁺-mediated contraction of the myoneme and coiling of the stalk is caused by Ca²⁺-mediated contraction of the spasmoneme (Allen, 1973; Amos et al. 1976). The degree of contraction increased with increasing free Ca²⁺ concentration (Figs 1, 2). The relationship between free Ca²⁺ concentration and the degree of shrinkage of the cell body was essentially identical to that between free Ca²⁺ concentration and the degree of coiling of the stalk (Fig. 2). This indicates that the Ca²⁺ sensitivity of the myoneme is almost identical with that of the spasmoneme.

In contrast to Triton-extracted Vorticella, living Vorticella showed spontaneous or mechanically evoked all-or-nothing contractions. We therefore assumed that a regenerative increase in the intracellular Ca²⁺ concentration is involved in the contraction of living Vorticella. Moreton and Amos (1979) proposed that an influx of Ca²⁺ into the cell from the external solution, accompanied by a regenerative Ca²⁺ action potential, was responsible for the regenerative increase in the intracellular Ca²⁺ concentration in Zoothamnium geniculatum. This hypothesis is, however, not applicable to Vorticella, for contraction of Vorticella occurs in the absence of external Ca²⁺ (Allen, 1973; Katoh and Naitoh, 1992a). Katoh and Naitoh (1992a) therefore suggested that a Ca²⁺-induced Ca²⁺ release mechanism, first observed in the sarcoplasmic reticulum of skeletal muscle fibres by Endo et al. (1970) (see also Ford and Podolsky, 1970) was involved in the regenerative increase in the intracellular Ca²⁺ concentration in Vorticella.

As shown in Figs 3 and 4, injection of Ca²⁺ buffer solution with a free Ca²⁺ concentration of approximately 5.1×10 ^{− 8} moll ^{− 1} [a value lower than the threshold for evoking contraction in Triton-extracted specimens (approximately 8.3×10 ^{− 8} moll ^{− 1}, see Fig. 2)] into the cell body of a living Vorticella produced nearly maximal coiling of the stalk, but it did not produce shrinkage of the cell body. It is therefore assumed that a mechanism that causes a regenerative increase in the free Ca²⁺ concentration in the stalk is activated by injection of Ca²⁺ buffer into the cell body. It is interesting to note that, when the cell body was stimulated in a living Carchecium polypinum, a relative of Vorticella, coiling of the stalk started from the junction between the cell body and the stalk and propagated down the stalk (Sugi, 1960). The Ca²⁺ required for coiling of the stalk must be supplied from some Ca²⁺ storage sites in the stalk, because the external solution for the injection experiments contains 1 mmoll⁻¹ EGTA, so its free Ca²⁺ concentration is less than 10 ^{− 9} moll ^{− 1}.

A similar mechanism that causes a regenerative rise in the Ca²⁺ concentration in the cell body is assumed to be activated by injection of the Ca²⁺ buffer. However, the buffer action is strong enough to keep the Ca²⁺ concentration in the cell body below the threshold for shrinkage. Ca²⁺ buffer injected into the cell body is assumed to diffuse only slightly into the stalk, because the stalk is thin (approximately 3.4 μm in inner diameter) and long (more than 100 μm). Therefore, a regenerative increase in Ca²⁺ concentration can take place in the stalk. The degree of coiling evoked by injection of Ca²⁺ buffer into the cell body was always slightly smaller than its maximum value (Fig. 4). This must be attributable to a slight diffusion of Ca²⁺ buffer into the stalk from the cell body.

Caffeine can produce a contracture of skeletal muscle fibres. The contracture is attributable to a sustained increase in the sarcoplasmic Ca²⁺ concentration, caused by caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum (Endo et al. 1970). External application of caffeine caused an increase in the frequency and the duration of contraction in living Vorticella (Fig. 6). When the final concentration was more than 50 mmoll ^{− 1}, it even caused a sustained contraction, which corresponds to the contracture of skeletal muscle fibres. An intracellular injection of caffeine also produced an increase in the frequency of contraction. All the caffeine experiments were performed in Ca²⁺-free solution containing EGTA. The Ca²⁺ required to evoke contraction is, therefore, presumed to be supplied from some intracellular Ca²⁺ storage sites. In contrast to living Vorticella, Triton-extracted Vorticella did not show a contraction following administration of caffeine into their reactivation medium.

Procaine and Ruthenium Red are known to inhibit the Ca²⁺-induced Ca²⁺ release from sarcoplasmic reticulum (Endo, 1977; Volpe et al. 1986). Living Vorticella immersed or injected with a solution containing procaine showed neither an increase in the frequency of contraction in response to caffeine nor a contraction in response to mechanical stimulation.

Living Vorticella injected with Ruthenium Red showed neither an increase in the frequency of contraction in response to caffeine nor a contraction in response to mechanical stimulation. However, living Vorticella injected with Ruthenium Red showed contraction upon injection of a Ca²⁺ buffer solution with a free Ca²⁺ concentration higher than the threshold for evoking contraction in Triton-extracted Vorticella. The contraction was not of an all-or-nothing type, but graded. The degree of contraction was dependent on the Ca²⁺ concentration of the injected buffer. The relationship between the free Ca²⁺ concentration and the degree of contraction was identical with that for Triton-extracted specimens (compare Fig. 9 with Fig. 2). This indicates that the contractile activities of the spasmoneme and the myoneme are not affected by Ruthenium Red. This was also shown by the observation that the presence of Ruthenium Red in the reactivation medium did not affect the Ca²⁺-activated contraction in the Triton-extracted specimens.

The present findings strongly support the idea that the regenerative increase in the intracellular free Ca²⁺ concentration responsible for the all-or-nothing contractions of living Vorticella is mediated by Ca²⁺-induced Ca²⁺ release from some membrane-bound system that is similar in its characteristics to the Ca²⁺-induced Ca²⁺ release reported in the sarcoplasmic reticulum of skeletal and cardiac muscle (Endo, 1977) and the endoplasmic reticulum of nerve cells (Neering and McBurney, 1984). Recently, InsP₃-mediated Ca²⁺-induced Ca²⁺ release mechanisms have been reported in sarcoplasmic reticulum of smooth muscle fibres, in the endoplasmic reticulum of some egg cells and in endoplasmic reticulum vesicles from cerebellum incorporated into planar bilayers (Iino and Endo, 1992; Miyazaki et al. 1992; Bezprozvanny et al. 1991). Vorticella did not show contraction in response to an injection of InsP₃.

Several authors have reported that membranous tubules (tubular endoplasmic reticulum) are present in the spasmoneme and the myoneme of Vorticella and that endoplasmic reticulum is present around the myoneme (Carasso and Favard, 1966; Allen, 1973). The Ca²⁺-induced Ca²⁺ release mechanism probably resides in these reticular systems in living Vorticella.

We found that a protozoan, Vorticella, utilizes its endoplasmic reticulum for the control of its Ca²⁺-dependent contractile activity in a way similar to the skeletal muscle fibre, although its contractile mechanism is very different from that of the skeletal muscle fibre. This finding is important for understanding the evolution and differentiation of mechanisms of cellular contractile activity in the animal kingdom.

This work was supported by a Grant in Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and a grant from the Mitsubishi Foundation to Y.N. We are grateful to Dr D. Macer for reading the manuscript critically and to Dr H. Horigami of Hosei University for giving us specimens of Vorticella.