1. The intracellular localization and translocation of activator Ca in the longitudinal retractor muscle (LRM) of a sea cucumber Stichopus japonicus were studied by fixing the LRM in a 1 % OsO4 solution containing 2 % K pyroantimonate.

  2. In the resting LRM fibres, electron-opaque pyroantimonate precipitate was mostly localized along the inner surface of the plasma membrane and at the subsarcolemmal vesicles in close apposition to the plasma membrane.

  3. In the LRM fibres fixed during the mechanical response to ACh and high [K]o, the precipitate was diffusely distributed in the myoplasm in the form of numerous particles with corresponding decrease in the amount of the precipitate at the peripheral structures.

  4. Electron probe X-ray microanalysis showed the presence of Ca in the precipitate, indicating that the precipitate provides a valid measure of Ca localization.

  5. These results accord with the view that, in the LRM, the contractile mechanism is activated by the release of Ca from the intracellular structures as well as by the inward movement of extracellular Ca.

Although it has been generally accepted that the mechanical activity in muscle is regulated by the change in free Ca ion concentration in the myoplasm (e.g. Ebashi & Endo, 1968). The sources of Ca ions which activate the contractile mechanism (activator Ca) in the physiological contraction of various kinds of smooth muscle still remain to be determined. Since both vertebrate and invertebrate smooth muscles exhibit considerable functional and structural variations, physiological and structural studies from the viewpoint of comparative physiology may facilitate progress in smooth muscle physiology.

In the preceding paper (Sugi et al. 1982), the mode of activation of the contractile mechanism by acetylcholine (ACh), high [K]o and other factors has been studied on the longitudinal retractor muscle (LRM) of a sea cucumber Stichopus japonicus. The results obtained indicate that the LRM fibres contain intracellularly stored Ca ions available for the activation of the contractile mechanism. The present experiments were undertaken to determine the Ca-accumulating structures in the LRM fibres, and to study the intracellular translocation of activator Ca during the contractionrelaxation cycle, with the pyroantimonate method which has proved useful for studying the intracellular Ca translocation in various types of smooth muscle (Atsumi & Sugi, 1976; Sugi & Daimon, 1977; Suzuki & Sugi, 1978).

The experimental methods used were identical with those previously described (Sugi et al. 1982). The LRM fibre bundle preparation (0·5–1 mm in diameter and 0·8 –1 ·2 cm in length) at rest or during mechanical activity was fixed by replacing the experimental solution with a 1 % OsO4 solution (pH 6 ·0 –6 ·2 by 0 ·01 N acetic acid) containing 2 % K pyroantimonate (K[Sb(OH)6]), which is known to penetrate intact cell membrane in the presence of Os to produce electron-opaque precipitate with intracellular cations (Komnick, 1962; Komnick & Komnick, 1963; Legato & Langer, 1969). Then, the fibres were dehydrated with ethanol, and embedded in Epon 812. Sections with silver colour were cut on a Porter-Blum MT-2 ultramicrotome and examined with a Hitachi HU-12AS electron microscope unstained or stained with uranyl acetate.

For chemical identification of the pyroantimonate precipitate, the sections (unstained, 100 –200 nm in thickness) were mounted on a carbon coated collodium film on copper grid, analysed with an energy dispersive X-ray microanalyser (EDAX 711) attached to a scanning transmission electron microscope (JEOL 100CX). The X-ray microanalysis was performed by focusing the electron beam on a fixed area of the sections (spot analysis; about 20 nm spot diameter) with an accelerating voltage of 20 kV and a sample current of 0·1nA. The X-ray emission was collected over a detecting time of 200 sec. The quantitative analysis of the precipitate was also made by use of a computerized EDIT system together with the X-ray microanalyser (Sugi & Daimon, 1977; Suzuki & Sugi, 1978).

All experiments were made at room temperature (18–25 °C).

Localization of pyroantimonate precipitate in the resting LRM fibres

The intracellular localization of pyroantimonate precipitate was studied by fixing the resting LRM fibres with the pyroantimonate-OsO4 solution and examining the intracellular localization of pyroantimonate precipitate (Atsumi & Sugi, 1976; Sugi & Daimon, 1977; Suzuki & Sugi, 1978).

The LRM fibres, which had been kept in the standard solution (artificial sea water, Sugi et al. 1982), however, developed considerable tension in response to the application of the pyroantimonate-OsO4 solution. In order to fix the LRM fibres in a completely relaxed state, La ions (10–20 mM) were found to be effective in eliminating the mechanical response to the pyroantimonate-OsO4 solution (Fig. 1 A). This effect of La ions may be explained as being due to their strong block of Ca-flux across the fibre membrane (e.g. van Breemen, 1969), since 10–20 mM La caused an almost complete inhibition of the mechanical response of the LRM to ACh, high [K]o and other agents (Suzuki & Sugi, unpublished).

Fig. 1.

Tension changes in the LRM fibres during the course of fixation with the pyroantimonate-OsO4 solution. (A) The fibres were first soaked in the standard solution containing 20 mM-La (La) and then fixed in the pyroantimonate-OsO4 solution with no tension development. (B) The fibres were made to contract with 10−3 M-ACh (ACh), and the pyroantimonateOsO4 solution (PAOs) was applied at the peak of contracture tension to fix the fibres in the contracted state. (C) The fibres were made to contract with 200 min-K (High-K), and the pyroantimonate-OsO4 solution (PAOs) was applied at the peak of contracture tension. Note additional tension development in response to the pyroantimonate-OsO4 solution in B.

Fig. 1.

Tension changes in the LRM fibres during the course of fixation with the pyroantimonate-OsO4 solution. (A) The fibres were first soaked in the standard solution containing 20 mM-La (La) and then fixed in the pyroantimonate-OsO4 solution with no tension development. (B) The fibres were made to contract with 10−3 M-ACh (ACh), and the pyroantimonateOsO4 solution (PAOs) was applied at the peak of contracture tension to fix the fibres in the contracted state. (C) The fibres were made to contract with 200 min-K (High-K), and the pyroantimonate-OsO4 solution (PAOs) was applied at the peak of contracture tension. Note additional tension development in response to the pyroantimonate-OsO4 solution in B.

Fig. 2 shows the cross-section of the resting fibres fixed in the pyroantimonate-OsO4 solution. A continuous line of electron-opaque pyroantimonate precipitate was always observed along the plasma membrane of the LRM fibres (Fig. 2 A). The precipitate was also seen in close apposition to the plasma membrane.

Fig. 2.

Intracellular localization of electron-opaque pyroantimonate precipitate in the resting LRM fibres fixed in a 1 % OsO4 solution containing 2 % K pyroantimonate. (A) Cross-section showing the localization of the precipitate at the peripheral part of the fibres. Lightly stained with uranyl acetate, × 14000. (B) High-magnification view around the plasma membrane, illustrating the localization of the precipitate along the inner surface of the plasma membrane and at the subsarcolemmal vesicles in close apposition to the plasma membrane. Lightly stained with uranyl acetate, × 51800.

Fig. 2.

Intracellular localization of electron-opaque pyroantimonate precipitate in the resting LRM fibres fixed in a 1 % OsO4 solution containing 2 % K pyroantimonate. (A) Cross-section showing the localization of the precipitate at the peripheral part of the fibres. Lightly stained with uranyl acetate, × 14000. (B) High-magnification view around the plasma membrane, illustrating the localization of the precipitate along the inner surface of the plasma membrane and at the subsarcolemmal vesicles in close apposition to the plasma membrane. Lightly stained with uranyl acetate, × 51800.

Closer examination of the plasma membrane with higher magnifications indicated that the precipitate was localized along the inner surface but not along the outer surface of the plasma membrane (Fig. 2B), while the precipitate in close apposition to the plasma membrane was observed to be located at the flattened subsarcolemmal vesicles (Sugi et al. 1982). Minute particles of the precipitate were also seen in the myoplasm and the nucleus.

Translocation of pyroantimonate precipitate during mechanical activity

The translocation of the precipitate during the mechanical activity of the LRM fibres was examined by fixing them during the mechanical response to acetylcholine (ACh,10−4 to 10−3 M), and high (K]o (200–400 mM) (Sugi et al. 1982). The application of the pyroantimonate-OsO4 solution at the peak of ACh- or K-induced contractures produced further development of isometric tension, which decayed slowly until the completion of fixation (Fig. 1 B, C). Consequently, the isometric tension of the LRM fibres at the completion of fixation amounted to 70–80% of the peak contracture tension.

Fig. 3 A shows the cross-section of the LRM fibres fixed during ACh-contractures. It can be seen that the pyroantimonate precipitate is diffusely distributed in the myoplasm in the form of a number of particles, while the precipitate at the peripheral part of the fibres was markedly decreased (Fig. 3 B).

Fig. 3.

Intracellular translocation of the precipitate during the mechanical activity produced by ACh or high [K]o. (A) Cross-section of the LRM fibres fixed during the mechanical response to 10−3 M-ACh. Note the diffuse distribution of the precipitate in the myoplasm in the form of numerous particles. Lightly stained with uranyl acetate, x 13600. (B) High-magnification view around the plasma membrane of the fibres fixed during the mechanical response to 10−3 M-ACh. Note that the amount of the precipitate along the inner surface of the plasma membrane and at the subsarcolemmal vesicles is markedly reduced as compared with that shown in Fig. 2B. Lightly stained with uranyl acetate, × 68200. (C) Cross-section of the fibres fixed during the mechanical response to 200 mM-K. Note the diffuse distribution of the precipitate in the myoplasm as in A. Lightly stained with uranyl acetate, × 22000.

Fig. 3.

Intracellular translocation of the precipitate during the mechanical activity produced by ACh or high [K]o. (A) Cross-section of the LRM fibres fixed during the mechanical response to 10−3 M-ACh. Note the diffuse distribution of the precipitate in the myoplasm in the form of numerous particles. Lightly stained with uranyl acetate, x 13600. (B) High-magnification view around the plasma membrane of the fibres fixed during the mechanical response to 10−3 M-ACh. Note that the amount of the precipitate along the inner surface of the plasma membrane and at the subsarcolemmal vesicles is markedly reduced as compared with that shown in Fig. 2B. Lightly stained with uranyl acetate, × 68200. (C) Cross-section of the fibres fixed during the mechanical response to 200 mM-K. Note the diffuse distribution of the precipitate in the myoplasm as in A. Lightly stained with uranyl acetate, × 22000.

Similar results were obtained on the fibres fixed during K-contractures (Fig. 3 C), except that the amount of precipitate remaining at the peripheral part of the fibres appeared to be larger in K-contractures than in ACh-contractures.

Though the LRM can also be made to contract by various factors other than ACh and high [K]o (Sugi et al. 1982), it was not possible to examine the intracellular Ca translocation during such mechanical responses, since the tension developed by the application of the pyroantimonate-OsO4 solution was much larger than those mechanical responses to mask the Ca translocation during the preceding mechanical response.

Electron probe X-ray microanalysis of the precipitate

Since pyroantimonate is known to produce electron-opaque precipitate not only with Ca but also with other cations, the presence of Ca in the pyroantimonate precipitate within the LRM fibres was examined by means of the electron probe X-ray microanalysis to ascertain whether the precipitate served as a valid measure of intracellular Ca localization.

Fig. 4 shows typical examples of the spot analysis of the precipitate at the plasma membrane and the subsarcolemmal vesicles of the resting fibres (A, B) and the precipitate distributed in the myoplasm of the contracted fibres (C). In both cases, the X-ray spectra of the precipitate always exhibited the most distinct peak at 3620 eV, which has been shown to result from a combination of the Sb-La emission (at 3600 eV) and the Ca-Kα emission (at 36906V) (Mizuhira, 1976; Suzuki & Sugi, 1978). The presence of the peak at 3620 eV is therefore taken to indicate that the pyroantimonate precipitate contains Ca, though the peak for Ca-Kαemission at 3690 eV is not directly observable in the X-ray spectrum of the precipitate.

Fig. 4.

X-ray spectra of the pyroantimonate precipitate in the LRM fibres. (A) X-ray spectrum of the precipitate at the plasma membrane of the resting LRM fibres. Note the peaks for Na, K and Mg as well as the combined peak for Sb-Ca. (B) Enlarged X-ray spectrum around the peak for Sb-Ca shown in A. Note the combined peak for Sb-Ca at 3620 eV. Vertical white line indicates the position of Sb-Lα emission at 3600 eV. (C) X-ray spectrum of the precipitate diffusely distributed in the myoplasm of the contracted fibres. Note the peak for Sb-Ca at 3620 eV as in B. In all cases, X-ray emission was collected for 200 s.

Fig. 4.

X-ray spectra of the pyroantimonate precipitate in the LRM fibres. (A) X-ray spectrum of the precipitate at the plasma membrane of the resting LRM fibres. Note the peaks for Na, K and Mg as well as the combined peak for Sb-Ca. (B) Enlarged X-ray spectrum around the peak for Sb-Ca shown in A. Note the combined peak for Sb-Ca at 3620 eV. Vertical white line indicates the position of Sb-Lα emission at 3600 eV. (C) X-ray spectrum of the precipitate diffusely distributed in the myoplasm of the contracted fibres. Note the peak for Sb-Ca at 3620 eV as in B. In all cases, X-ray emission was collected for 200 s.

The X-ray spectra of the precipitate also exhibited high peaks of Si-Kα and Cl-Kα emissions and low peaks of K-Kα, Mg-Kα and Na-Kα emissions (Fig. 4A). The large peak of Si-Kα emission was observed even when pure carbon was analysed, indicating that the Si peak originates from the recording system per se. The epoxy resin, on the other hand, exhibited a high Cl-Kα emission, indicating that the Cl peak mainly comes from the epoxy resin (Atsumi & Sugi, 1976). It has been known that pyroantimonate binds with Ca most readily in physiological conditions, though it also binds with Na and Mg. In ethanol, pyroantimonate also binds with K. Thus, it may be that, when the pyroantimonate-OsO4 solution is applied to the LRM fibres, the formation of Ca-pyroantimonate first take place reflecting the intracellular Ca distribution: other cations may further precipitatete around the Ca-pyroantimonate precipitate already present (Suzuki & Sugi, 1978).

To obtain more direct evidence for the presence of Ca in the precipitate, the concentration ratio of Ca relative to the concentration of Sb was computed from the data of the X-ray microanalysis (Russ, 1974). The concentration ratio of Ca in the precipitate was 0·22 + 0·05 (mean + s.D., n = 20) in the resting fibres, and 0·25±0·10 (mean + s.D., n = 20) in the contracted fibres. Since the amount of Ca ions involved in the activation of the contractile mechanism is far smaller than those of K, Na and Mg ions in the myoplasm (Ebashi & Endo, 1968), these values of concentration ratio of Ca in the precipitate may be taken to indicate that the precipitate serves as a valid measure of Ca localization within the LRM fibres.

Intracellular translocation of Ca during mechanical activity

The results described in the preceding paper (Sugi et al. 1982) indicate that the LRM fibres contain intracellularly stored Ca which is available for the activation of the contractile mechanism. The present experiments show that, in the resting fibres, the Ca contained in the electron-opaque pyroantimonate precipitate is mostly localized along the plasma membrane (probably along its inner surface) and at the subsarcolemmal vesicles in close apposition to the plasma membrane (Fig. 2). Since the contraction-relaxation cycle in living muscle fibres is primarily controlled by the potential or permeability changes across the plasma membrane, the above localization of intracellular Ca in the peripheral structures strongly suggests that these structures serve as the sources of activator Ca in addition to the inward moving extracellular Ca.

The localization of intracellular Ca at the plasma membrane and other structures in the vicinity of the plasma membrane has also been demonstrated in various vertebrate and invertebrate smooth muscles by use of K oxalate (Jonas & Zelck, 1974; Popescu & Diculescu, 1975; Popescu et al. 1974). 45Ca (Jonas & Zelck, 1974) and K pyroantimonate (Debbas et al. 1975; Atsumi & Sugi, 1976; Sugi & Daimon, 1977; Suzuki & Sugi, 1978).

When the LRM fibres were fixed with the pyroantimonate-osmium solution during the mechanical response to ACh and high [K]o, the pyroantimonate precipitate containing Ca was observed to diffusely distribute in the myoplasm in the form of numerous particles with a corresponding decrease in the amount of precipitate at the peripheral part of the fibres (Fig. 3). This result accords with the view that, during ACh-and K-induced contractures, the contractile mechanism may be activated not only by the inward moving extracellular Ca but also by the Ca release from the plasma membrane and the subsarcolemmal vesicles. Similar intracellular Ca translocation during the mechanical activity has already been observed in various vertebrate and invertebrate smooth muscles by means of the pyroantimonate method (Atsumi & Sugi, 1976; Sugi & Daimon, 1977; Suzuki & Sugi, 1978), suggesting that the mode of intracellular Ca translocation is essentially the same in various kinds of smooth muscle. The mechanism of Ca release from the plasma membrane and other intracellular structures remains to be investigated to clarify excitation-contraction coupling mechanism in smooth muscles.

Comparison between the LRM and other smooth muscles

Physiological experiments on the LRM (Sugi et al. 1982) indicate that the amount of activator Ca in the intracellular Ca-accumulating structures may not be sufficient to fully activate the contractile mechanism. This contrasts with molluscan somatic smooth muscles, such as the anterior byssal retractor muscle (ABRM) of Mytilus edulis and the longitudinal body wall muscle (LBWM) of Dollabella auricularia, which contain enough intracellularly stored Ca to activate fully the contractile mechanism (Sugi & Yamaguchi, 1976; Sugi & Suzuki, 1978). Accordingly, the intracellular membranous structures in close apposition to the plasma membrane in the LRM are poorly developed as compared to those in the ABRM and LBWM, suggesting that these structures are on the intracellular sources of activator Ca.

On the other hand, intracellular Ca has also been shown to be localized along the inner surface of the plasma membrane in various smooth muscles, from which it is released into the myoplasm (Atsumi & Sugi, 1976; Sugi & Daimon, 1977; Suzuki & Sugi, 1978). The amount of activator Ca present at the inner surface of the plasma membrane can not, however, be estimated by the pyroantimonate method, since the precipitate contains not only Ca but also other cations (Fig. 4A). It seems possible that the amount of activator Ca at the inner surface of the plasma membrane is smaller in the LRM than in the ABRM and the LBWM. The X-ray microanalysis of intracellular Ca on the cryo sections of various smooth muscles may be useful for the quantitative studies on intracellular Ca translocation. Quantitative estimation of intracellular ionic concentrations on the cryo sections has already been made on a vertebrate vascular smooth muscle (Somlyo et al. 1977, 1979), though the identification of Ca-accumulating structures on the cryo sections is difficult.

The LRM fibres showed a marked mechanical response to OsO4, which was inhibited by La ions (Fig. 1). In this connection, the LRM resembles vertebrate visceral smooth muscles which also exhibit marked OsO4-induced contraction which is inhibited by Mn ions (Sugi & Daimon, 1977). Meanwhile, molluscan smooth muscles are insensitive to OsO4, and can be fixed with no marked tension development (Atsumi & Sugi, 1976; Suzuki & Sugi, 1978). On the other hand, the LRM fibres can be made to contract by hypertonic solutions (Sugi et al. 1982), while molluscan smooth muscles do not develop tension in hypertonic conditions (Sugi, Yamaguchi & Tanaka, 1977). The mechanical response to hypertonic solutions can also be seen in vertebrate vascular and visceral smooth muscles (Nakayama, Sugi & Suzuki, unpublished). The above similarities in physiological properties between the LRM and vertebrate smooth muscles seem to suggest a close phylogenetic relation between the vertebrates and the echinoderms.

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