Physiological and cytochemical studies of Ca in the primary muscle of the trunk of Sagitta setosa (Chaetognath)

TISSUE & CELL 1986 18 (6) 953-966 0 1986 Longman Group UK Ltd

JEAN-PIERRE

SAVINEAU

and MICHEL DUVERT*

PHYSIOLOGICAL AND CYTOCHEMICAL STUDIES OF Ca IN THE PRIMARY MUSCLE THE TRUNK OF SAG/X.A SETO5A (CHAETOGNATH) Keywords: Contraction,

OF

Ca movement, SR, plasma membrane, Ca localization

ABSTRACT. The movements of Sagirra are conditioned by the presence of CaZ+ in the external medium. When this ion is removed from artificial sea water, animals do not move. They swim again when Ca2+ is present. Among the problems raised by this observation, we have studied the role of CaZ+ in the contraction of the primary musculature. Physiological experiments show the central importance of the extracellular Ca*+ and of its translocation through the membrane during the initiation of the contraction. Cytochemical data correlate these observations. They show that Ca2+ is localized mainly at the level of the plasma membrane, its imaginations and in the poorly developed SR (less than 2% of cell). Like SR, mitochondria accumulate Ca2+ but do not seem to participate in the regulation of these Ca movements except in abnormal situations. Las+ blocks the entry of extracellular Ca*+ and attaches to the membranes; this fixation is not the same on the plasma membrane and in its imaginations. The contractile apparatus of Sagirra primary musculature show remarkable specializations (Duvert and Savineau, 1986). It is composed of ribbon-shaped myofibrils of regular thickness surrounded by external membranes implicated in the fixation and the translocation of a pool of CaZ+ necessary for initiating contraction. The poorly developed SR and the mitochondria probably modulate the functioning of the two types of fibres (A and B).

Introduction

motor nerve endings by the sheet of connective tissue (Duvert and Barets, 1983). This musculature probably acts as a syncytium on an hydroskeleton; it is reminiscent of myocardium for instance (Sommer and Waugh, 1976; Langer et al., 1982). The primary musculature contains two kinds of fibres, A and B. Compared with A fibres, B fibres have irregular myofibrils in cross-section, SR and mitochondria are more abundant (Dress and Duvert, 1983). Both fibres have large and narrow invaginations of the plasma membrane penetrating between the myofibrils and opening regularly in the extracellular space at the M level. Couplings occur mainly on longitudinal tubules of the SR along plasma membrane in A and B fibres, and invaginations in B fibres (Duvert and Salat, 1979, 1980). Preliminary experiments have shown that external calcium is necessary for muscular contraction. Animals placed in artificial sea water (ASW) without Ca*+ do not contract;

The primary musculature of the trunk of Sugitta borders the general cavity. It is separated from the epidermis by a sheet of connective tissue sandwiched between two basal laminae (the basement membrane) (Grassi, 1883; Burfield, 1927). Structural characteristics of this musculature are peculiar and may be summarized as follows. Muscle fibres: (1) are devoid of basal lamina, except against the basement membrane (Duvert and Salat, 1979); (2) are interconnected by cell. junctions which have an obvious mechanical role and by gap junctions (Duvert et al., 1980); (3) are separated from Laboratoire de Physiologic cellulaire, I.B.C.N., CNRS et Universitt de Bordeaux II, 1 Rue Camille Saint-Saens, 33077 Bordeaux Cedex, France. Laboratoire de Cytologic, Universitt de Bordeaux II, U.A. 339 CNRS, Avenue des Facuhes, 33405 Talence Cedex, France. Received 29 May 1986. Revised 1 July 1986. l

953

954

SAVINEAU

when they are put again in normal ASW, or in natural sea water, they contract again. Moreover these data suggest easy passage between the external milieu and intercellular spaces in epidermal, connective and muscular tissue. They raise the problem of the nature and the limits of the internal medium (‘milieu intkrieur’). In fact no structure appear to seal the paracellular pathway; no Zonula occludens was found in muscle and epidermis; there is no cuticle at the surface of the trunk (Duvert etaf., 1984). It seems that, in Sag&a, as in other marine invertebrates, the intercellular space (at least in the trunk wall) may be in equilibrium with the sea water (Meglitsch, 1967) and there is no circulatory or excretory apparatus. Using these exceptional characteristics of permeation we have made both cytological and physiological studies of the cellular localization of Ca2+ and its role in contractile activity. The first results confirm the important role of this ion in the functioning of the primary musculature; they show the central importance of the plasma membrane in the intracellular calcium control of the movements.

fixed with an aldehyde mixture (Karnovsky. 1965) or with 3% glutaraldehyde in sea water or in a Na-cacodylate 0. I M buffer. pH 7.5. containing 0.6 M saccharose. for 2 hr at room temperature. Animals were washed in sea water. Some specimens were post-fixed in 1 o/r 0~0, in a 0.1 M Na-cacodylate buffer. pH 7.5. Impregnation with lanthanum hydroxide was performed according to Revel and Karnovsky (1967); ruthenium red was used according to Luft (1971). Cytochemical

studies

No counterstained (unless specified).

1. Light microscopic

preparations

Morphological studies Sugittu were collected in the plankton the Bassin d’Arcachon. The animals

A B bm c @

from were

were done

studies

Animals were frozen in isopentane precooled with liquid nitrogen, cryosections about lo-20pm thick were used. Some cryosections were fixed in formal vapour (Lison 1960). GBHA reaction was made according to Kashiwa and Atkinson (1963) and Kashiwa and House (1964). As described by these authors, controls were done with NaCO,KCN andwith EGTA (5% in water, 15 min). 2. Electron microscopic

Material and Methods

AND DUVERT

studies

(a) The oxalate methods of Costantin er al. (1965) and of Kniprath (1071) were used. In the first, fixation takes place after treatment with potassium and oxalate-chloride; the pre-

A fibre B fibre ‘basement membrane’ coupling general cavity

I m pm

invagination mitochondria plasma membrane “U&US

;R

sarcoplasmlc

reticulum

Fig. 1. Transversal section showing a group of B fibres between two groups ot A fibre,. fibres have more regular myofibrils and few mltchondria. x 16.400.

A

Fig. 2. Fixation in the presence of Ruthenium red. Thea extracellular maker 1s lound in the basement membrane (top of the figure). in the invaginatlons. between contiguous plasma membranes. ~24,200 Fig. 3. Tangential section in mvagmatmns of B fibres showmg numcrow conplmga (cl. Contrary to an early observation (Duvert and Salat. 1980) no fene,trations are seen in the invaginations. X25,600. Fig. 4. Perpendicular

section in a coupling.

note the absence of ‘junctional

granules‘.

x46,X(M)

956

SAVINEAU AND DUVERT

servation of tissues is very poor. The second method combines precipitation of calcium and fixation with osmium. (b) In the pyroantimonate method animals are placed either in sea water or in artificial sea water containing 12.2 mM CaC12 (20 min, 21°C). Pyroantimonate (4% finely crushed in distilled water) and OsOl 4% were mixed (9:l v/v respectively). Animals were rapidly blotted and immersed in the reactive, then washed in bi-distilled water and processed for electron microscopy. (c) Localization of La: animals were kept 20 min in ASW containing various concentrations of LaCh (see below). They were directly fixed in Karnovsky’s fluid only, pH 6.8 (to prevent lanthanum hydroxide formation), without La. Some sections were stained with lead citrate. (d) Microanalysis: unstained sections of pyroantimonate-treated animals were mounted on copper grids and analysed in a Philips electron microscope EM 301, equipped with a goniometer stage, EDAX energy

A

1

20 see

dispersive X-ray analyser model 707 with a specimen holder with Be inserts. The X-ray microanalysis was performed at a tension of 80 kV, the time count was 800 set; the area analysed is indicated on the micrograph. These observations were made by S. D. Van Winjgaarden (Philips, Appl. Lab. E. 0. Eindhoven). Physiological studies

The methods and experimental device are the same as those described in the preceding paper (Duvert and Savineau. 1986). Results Morphological introduction

On a transversal section, one can see that myofibrils are separated by narrow invaginations of the plasma membrane (Fig. 1). In these A fibres, the SR is very reduced and virtually absent against the invaginations. On the contrary, in B fibres the SR is largely developed against the invaginations. Many

2

5vI-

B

Fig. 5. Effects of the calcium external concentration on the twitch of Sagirra. (A) Effect of a (X+-free ASW: 1, ( t ) removal and ( _1) readmission of calcium ions; 2, oscillograph traces of the twitch recorded in ASW (a) and just after the removal of Ca2+ ions (b). Frequency of stimulation: 0.5 c/set. (B) Effect of 18.4 mM Ca*+ (2x [Ca2+],) on the amplitude of the twitch: 1, admission ( t ) and removal ( J ) of the Ca 2+-rich ASW; 2, oscillograph traces of the twitch recorded in ASW (a) and during 30 set after the admission of the Ca2+-rich (18.4 mM) ASW (b). Frequency of stimulation: 0.5 c/set. Note that in each case (a, b), the time to peak of the twitch is not significantly modified.

957

Ca IN PRIMARY MUSCLE OF THE TRUNK

couplings are seen at this level (Figs 3,4) and below the plasma membrane. When animals are placed in ASW containing an electronopaque tracer such as horseradish peroxidase, or when they are fixed with a mixture containing ruthenium red or lanthanum hydroxide, the tracer penetrates the intercellular space, between the muscle cells and in the invaginations (Fig. 2). Physiological data

(a) Effects of external calcium ions When calcium ions are removed from the ASW, the electrically induced contractions of muscular strips are rapidly diminished (75% reduction after 10 set) and are completely abolished after 1 min. Readmission of calcium ions (9.2 mM) into ASW produces a fast (~8 set) and full recovery of twitches (Fig. 5A). On the other hand, the amplitude of twitches is increased by about 100% when the calcium concentration of ASW is twice (18.4 mM). concentration the normal Electrically induced contraction goes back

progressively to its original value with the removal of excess calcium (Fig. SB). (b) Effects of multivalent cations (a) La3+ ions. When La3+ ions (0.1-l mM) are added to ASW, electrically induced contractions of Sagitta are inhibited in a dose and time dependent manner. Fig. 6A shows a typical experiment with 1 mM La3+. A short application (30 set) of this La”+ concentration is sufficient to abolish the contraction but the reversibility of the mechanical response is slow; 50% of the maximal electrically induced contraction is recovered after 6 min and 100% after 10 min of washing the preparation in L$+-free ASW. (b) Mn2+ ions. Manganese ions (1.5-5 mM) also produce an inhibition of the twitch. The decrease of the amplitude is more progressive but the recovery is more rapid than those observed with lanthanum (full inhibition and recovery are respectively in 90 and 40 set-Fig 6B).

A

B

2

1

svln 2oscc

Pmscc

_

Fig. 6. Effect of multivalent cations on the amplitude of the twitch. [A) effect of the La’+ ions: 1, a brief application of 1 mM La’+ ( t ) fully inhibits the twitch which very progressively reappears after the removal ( _1) of the cation; 2, oscillograph traces of the twitch recorded in ASW (a) and 10 set after the addition of 1mM La’ (b). (B) Effects of Mn” ions: 1, progressive abolition of the twitch in the presence ( t J ) of 2.5 mM Mn2+ in the ASW; 2, superimposed oscillograph traces during the first 40 set of perfusion with manganese ions. Frequency of stimulation: 0.5 c/set.

SAVINEAU

Similar ions.

results

Hi&chemical

are observed

data

Fig.

Localizatton

7. The

Animals treated first with oxalate solution and then fixed. were poorly preserved. In the best preserved areas, deposits were found in the SR: they are to be seen in serial sections (Figs 10. 11). Deposits were enclosed in the SR (Fig. 12) and in some mitochondria. but these organelles were largely destroyed. Animals fixed in 0~0, oxalate appear to be

of C;I wtth

reaction

DLJVEK?

The control of specificity of the reaction with NaCO, and KCN shows that only the calcium chelates remain in the section. Treatment with EGTA 5%. 15 min. suppressed the reaction.

with cobalt

GBHA A positive reaction was seen along the plasma membranes (Figs 7, 8) and perhaps in the invaginations (Fig. 7). Mitochondria were seen in oblique section (Fig. 9). This method often allowed a clear distinction between A and B fibres: many preparations show a strong reaction in A fibres and no reaction in B fibres, but in other preparations (Fig. 8) B fibres contain more calcium than A fibres.

Figs 7-Y.

AND

is positive

the CiBHA

method

along the pkma

membrane

and prohabl!,

m invagmatwns

x IXOO. Fig. 8. Owing

to the clear-cut

Fig.

0. In this oblique

Figs

10.

mainly

B lihrcscont,un

mitochondria

arc to hc Van.

sectton.

1I. Sendscctmns

in the longitudinal

Ftg.

typcsofrcactwn.

m oxalatc-trcatcd

tubules

12. The

Ca oxalate

13. 0~0,

fixation

of the SK

deposit

n~u\cIc flhrc\

more C‘a than A fihrc\

x I200

x 1701). hclorc

tv.ation

I he dcpouts

arc

x7200.

is enclowd

tn a membrane-bound

compartment

(arrow)

x70.200. Fig.

in presence of oxalate.

Deposits

are mainly

found in all the SR (arrow\).

x 9200. Figs

1617.

Fig.

14. Fixation

membranes Fig.

Pyroantimonate

0~0,

wtth pyroantimonate-OsO,;

and invaginations.

15. After

precipitates.

incubation

the deposits

arc largely found

along the plasma

x7200. in ASW

enriched

with

Ca. all the mitochondrln

arc filled

with

x2000.

Fig.

16. Microanalysis

Fig.

17. Detail

Figs

1X-20.

Fig.

1X. After

data.

of a mitochondrie

Locahration incubation

showmg

the ~onc’ \tudlcd

of lomc lanthanum

and lanthanum

m ASW

5 mM LaCl,.

containing

hy X-ray

microanalysi\.

hydroxldc. ammals are fixed m aldehyde

onI>.

the pH IS mamtained <7; unstained preparation. Depostts are found along the mcmbrancs facing the extracellular space. The deposits are irregular along the external fibre membranes: they arc regular Fig.

in the invaginations. 10. Same preparation

Fig. 20. Lanthanum

tracer is uniformly

x10.500. hut the aectwn was contrasted

hydroxrde

was wed in a double

found in all the extracellular

with

lcad citrate

fixation procedure

space. x?ll,200.

x4Y.200

(aldehyde-WO,)

The

SAVINEAU

better preserved. Deposits were found in some longitudinal tubules of the SR and in cisternae at the I-band level (Fig. 13). Pyroantimonate method

Deposits were always found in the extracellular spaces (Fig. 14); mitochondria and SR were not convincingly marked. If animals were kept in ASW containing 12.2 mM CaC12, instead of 9.2 mM, mitochondria were regularly filled with electron-dense deposits (Fig. 15). The nature of the mitochondrial deposits was studied with X-ray microprobe an energy-dispersive analysis (Figs 16, 17). They show the presence of Ca and Sb; Fig. 16 shows, as well as Os, Mg (I&), Cl (&I. B)>K (K, a)> Sb (Lo.B,) and Ca (K, B). The Cu radiations came from the grid and Fe radiations from the beryllium holder. Lanthanum

When animals were placed in ASW containing 5 mM LaCl,, pH 6.8 (we choose a pH lower than that of sea water to avoid or minimize lanthanum colloidal formation) and only fixed in glutaraldehyde in cacodylate buffer (pH 6.8, also), deposits were found along the plasma membranes and the invaginations (Fig. 18). The deposits were discontinuous along the plasma membrane and continuous along the invaginations. Exhaustive washing gave the same pattern. The deposits became faint with LaC13 4 mM and hardly visible at lower concentration. After staining with lead citrate, the plasma membrane facing the deposits were more visible than elsewhere where the lanthanum was not bound (Fig. 19). Three controls were performed: When animals were kept in artificial sea water containing 5-6 mM LaCl, pH 7, and fixed in an aldehyde mixture pH 7.8, irregular deposits were found in all the extracellular space according to gradient (abundant near the basement membrane, they were rare near the general cavity). After fixation in Karnovsky fluid only, when sections are stained with lead citrate only, the leaflets of the membranes could never be seen, as in zones where the La is not deposited after ASW-La incubation. If the fixative contains lanthanum hydroxide, the tracer is homogeneously present in all the extracellular space (Fig. 20).

AND DUVERT

Discussion

Our experiments clearly show the role of external Ca in the contraction of the primary musculature. Three compartments contain Ca; among these, two seem to have a central physiological importance: (1) the plasma membrane and the invaginations surrounding the myofibrils; (2) the SR which is more abundant in B fibres, where it surrounds the contractile apparatus and has couplings facing the plasma membrane and the invaginations. I. Physiological

data

Electrically induced contractions are rapidly abolished in the absence of calcium ions in ASW and, on the contrary, are strongly increased in Ca-rich ASW (18.4 mM). These effects are fast and completely reversible within few seconds. It is well known that calcium ions play a key role in the initiation of muscular contraction. Whereas in vertebrate skeletal muscle the activator Ca2+ of myofilaments is released from and reaccumulate into the SR (Ebashi and Endo, 1968), in vertebrate cardiac and smooth muscles (Chapman, 1979; Kuruyama, 1981) as well as in many invertebrate striated muscles (Atwater et al., 1974; Zacharova and Zachar, 1967) an influx of extracellular calcium ions is required to trigger the contractile process. Our results clearly suggest that in Sagitta an influx of calcium ions from the extracellular medium into the muscle cells is necessary to induce the mechanical response. In mammalian cardiac muscle (Fabiato and Fabiato, 1975) and in some smooth muscles (Cheung, 1976; Huddart and Price, 1976; Itoh et al., 1981) the influx of calcium during each action potential is not sufficient by itself to trigger the contraction; a release of stored calcium mainly from the SR occurs (e.g. Ca2+-induced Ca’+ release). But in amphibian cardiac muscle (Vassort and Rougier, 1972), in some smooth muscles (Mironneau, 1973) and in some invertebrate striated muscles (Goblet and Mounier, 1982; Gilly and Scheurer, 1984) the calcium current during the action potential is able to directly induce the contractile response. From our physiological studies it is not possible to discriminate between these two possibilities for Sagitta contraction. Moreover, cytochemical data show cellular Ca stored in SR, sometimes in mitochondria,

Ca IN PRIMARY

MUSCLE OF THE TRUNK

and probably linked to the surface of the cell membrane. Further experiments performed in Ca-free ASW and in the presence of substances known to displace intracellular stored calcium (e.g. caffeine) will be considered in order to characterize the possible role of intracellular calcium. Twitch contraction of Sagitta is rapidly and fully inhibited by multivalent cations such as Mn2+ Co*+ or La3+. Mn2+ and Co2+ block select:vely the slow inward calcium current in cardiac muscle (Rougier et al., 1969) and 1974; in smooth muscle (Mironneau, Muraumatsu et al., 1978), while La3+ interacts with all calcium movements (influx and efflux) by competition with calcium for the anionic sites of the plasma membrane (Weiss, 1974). This latter mechanism could account for the very slow recovery of the contraction after La3+ blockage. More information about the process by which calcium ions penetrate the muscle cells of Sugittu could be obtained from electrophysiological studies. Except for the first recordings by Bone and Pulsford (1984) no data are available in the litterature, about the electrical activity of the muscles of Sugitta and thus about a possible electromechanical coupling. But it is possible to external calcium that ions suggest cytochemically localized in the extracellular space, cross the plasma membrane through calcium channels, particularly at the level of the invaginations (as the La experiments suggest). Thus, it is reasonable to think that electrical activity in trunk muscles of Sugitta is mainly dependent on extracellular calcium as in many other muscles of invertebrates (Hagiwara et al., 1964). Nevertheless, a small sodium component cannot be entirely excluded (Duvert and Savineau, 1986). II. Cytochemical data We have presented evidence showing that (1) calcium is stored in SR, mitochondria and at the level of the plasma membrane invaginations; (2) the ionic intercellular medium bathing the fibres is in equilibrium with the sea water; (3) the contraction of the primary musculature depends on extracellular calcium and this may be related to the poor development of the SR and to the large surface of contact between the plasma membrane and invaginations with the contractile apparatus correlated to the very regular

963

thickness of the myofibrils sandwiched between the invaginations (Duvert and Salat, 1980). GBHA and some of its derivatives are highly selective and sensitive to calcium (Milligan and Lindstrom, 1972). The Kashiwa et al. protocols (1964, 1966) allow clear visualization of calcium, in some compartments, at the light microscope level only. Ca was localized at the plasma membrane and probably at the level of the invaginations, and in mitochondria. The SR cannot be seen in the light microscope owing to its poor development and the low diameter of the tubules and saccules. The sensitivity of the oxalate method is lower than that of the GBHA but it gives electron-dense deposits. Oxalate precipitates some of the calcium which is not firmly bound, so that Ca-oxalate electron-opaque deposits may occur (Diculescu et al., 1971). With the Kniprath’s method, the tissues were correctly preserved but gave a low yield method in our experience. As shown by Costantin et al. (1985) in the skeletal muscle fibres, the deposits are found in the lumen of the SR. Contrary to these observations and others reported in heart muscle (Diculescu etal., 1971) the Ca-oxalate deposits in Sagitta are found throughout the SR. Only some poorly preserved mitochondria show deposits and no reaction is seen on the membranes (plasma membrane and invaginations) contrary to observations made with GBHA. These conflicting data suggest that the oxalate methods we used, allow only a visualization of Ca in some limited membrane compartments. In combination with OsO,, pyroantimonate penetrates the plasma membrane and produces electron-opaque deposits with a large variety of substances (Clark and Ackerman, 1971). Deposits also appear during ethanol dehydration (Hayat, 1981). Owing to the numerous large precipitates observed, this method gives a rough estimate on the presence of Ca or other substances. Moreover, the microanalysis method we used was not of fine definition and gives a broad evaluation of the localization of the Ca. Deposits are not convincingly observed at the SR level in A or B fibres and they are not abundant in the contractile apparatus contrary to the obvservations reported in vertebrate skeletal and cardiac fibres for exam-

964

ple (Legato and Langer, 1969; Yarom and Meiri, 1973). Ca was seen in mitochondria but only in an artificial situation, in Ca-rich sea water where animals swim. It seems that this ion is not usually accumulated in mitochondria. As in other muscles (Somlyo, 1984) these organelles do not seem to play an important role in Ca movements in Sagitfa muscles. physiological situations mitochondria can store Ca and act as a cytoplasmic Ca-buffer. With these three cytochemical methods, probably the best ones for the cytochemistry of Ca, apart from cryo-ultramicrotomy coupled to elemental analysis, we have seen that this ion is localized in SR, mitochondria and at the plasma membrane level. More precise data cannot be achieved at present. La is localized at the extracellular surface of the membranes; it allows a distinction between the superficial plasma membrane with discontinuous deposits, and the internal invaginations with continuous deposits. These data may reflect the heterogeneity of the membrane. They do not seem to be very useful in the study of the localization of Ca: La reacts somewhat with the membranes since the part of this organelle which is only covered with La can be selectively contrasted with lead citrate; this element can directly act on phospholipid vesicles (Lussan and Faucon, 1974). This heterogeneous reaction is not found if lanthanum is used as a colloidal extracellular marker. Our observations suggest that La is largely bound to the surface of the membrane and no intracellular penetration can be detected in the sections. Our results differ from those reported in vertebrate hearts (Langer and Frank, 1972; Martinez Palomo et al., 1975). These discrepancies can be explained by the complex action of this element. La can displace Ca from its fixation sites. block the Ca transmembrane movements and stabilize

SAVINEAU AND DUVERT

the membranes (Weiss. 1974). Apart from these various effects, binding differences exist between La and Ca (Silber, 1974); affinities of La change according to various anionic groups and its effects are dose and time dependent (Weiss, 1974). This suggests that La may be bound to various membrane compounds and more particularly to anionic groups. Some correlations can be made between structural and physiological data, more particularly with respect to the role of Ca and the development of Ca-containing organelles. They make it possible to understand the architecture of the muscles fibres of the trunk primary musculature; particularly interesting is the architecture of A and B fibres with the ribbon-shaped myofibrils of regular thickness (400-600 nm) surrounded by membranes facing the extracellular space. They also make it possible to understand the importance of the extracellular calcium and the very rapid initiation of the contraction of the trunk musculature (Duvert and Savineau. 1986). Furthermore, the great regularity of the architecture of the fibres and the high specialization of the contractile apparatus renders difficult any research for primitive features and thus complicates any conclusions concerning the phylogenesis of chaetognaths on the sole basis of their peculiar muscular structure. Acknowledgements We are grateful to Dr J. Mironneau for helpful discussions and to Dr A.-L. Barets for discussions and critical reading of the manuscript. We thank Mrs C. Salat for technical assistance and Mrs C. Fargues for help in preparing the manuscript. We also thank Dr C. Cazaux and the sailors who provide us with chaetognaths, and Mrs Mendes-France for correcting the English text.

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Ca IN PRIMARY

MUSCLE

OF THE TRUNK

965

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