J. Cell Sci. 8, 601-609 (1971) 601

Printed in Great Britain

CAVEOLAE INTRACELLULARES AND

SARCOPLASMIC RETICULUM IN

SMOOTH MUSCLE

G.GABELLA

Department of Anatomy, University College London, England

SUMMARY

In the smooth-muscle cells from guinea-pig ileum a highly developed sarcoplasmic reticulumhas been observed immediately underneath the plasma membrane in close relationship to thecaveolae intracellulares. It is suggested that sarcoplasmic reticulum in smooth muscle may playa similar role as in skeletal muscle, constituting the main intracellular store for calcium.

INTRODUCTION

In the last 15 years several electron-microscope studies of visceral and vascularsmooth muscle of vertebrates have been published (see reviews by Dewey & Barr,1968, and Burnstock, 1970). The presence of bottle-shaped invaginations of thesarcolemmal membrane (micropinocvtotic vesicles, caveolae intracellulares) (Caesar,Edwards & Ruska, 1957) is well known. However, it is generally accepted that sarco-plasmic reticulum is poorly developed in smooth muscle, especially when comparedwith striated muscle (Mark, 1956; Caesar et al. 1957; Prosser, Burnstock & Kahn,i960; Dewey & Barr, 1968). In this paper the occurrence of well developed sarco-plasmic reticulum in the intestinal smooth muscle of guinea-pig is reported.

MATERIALS AND METHODS

The guinea-pigs used were 6-10 months old.Under ether anaesthesia a length of approximately 6 cm of the ileum at 15-20 cm from the

ileo-colonic junction was gently injected intraluminarly with the fixative at room temperature.Both ends of the injected loop were tied in order to maintain the distension, the mesentery wasthen cut at 1 cm from the attachment to the intestinal wall, and the loop was isolated and im-mersed in the fixative. After 1 h the length of gut was cut into small rings, which were immersedin fresh fixative.

The primary fixative was 5 % glutaraldehyde in 0 1 M sodium cacodylate buffer at pH 7 4 ;aldehyde fixation lasted 2 h. Specimens were then washed overnight in several changes ofsodium cacodylate buffer, 0125 M, with 5 % sucrose. After postfixation either in 133 % osmiumtetroxide in collidine or in unbuffered 2 % osmium tetroxide the specimens were dehydratedin graded ethanol and infiltrated in Araldite. Sections were cut with a Reichert ultramicrotome,stained with uranyl acetate (1 % in 70% ethanol) and lead citrate, and observed in a Siemens1 A electron microscope.

OBSERVATIONS

At the surface of smooth-muscle cells the plasma membrane shows numerous oval-or bottle-shaped invaginations about 70 x 150 nm (caveolae intracellulares) (Fig. 1),

6o2 G. Gabella

with circular cross-section (Fig. 2). The caveolar membrane is trilaminar (Fig. 5) andstructurally similar to the plasma membrane, with which it is in physical continuity.The content of the caveolae is relatively homogeneous, with the same electron densityas the extracellular substance. In the longitudinal muscle coat, groups of caveolae donot always open directly at the cell surface but occasionally into a larger invaginationconnected with the extracellular space (Figs. 3, 4). Larger and more open invaginationsof the plasma membrane are sometimes observed, coated on the cytoplasmic side withdense granular material (Fig. 5). Whether this coat is composed of the hexagons andpentagons described by Kanaseki & Kadota (1969) remains to be seen.

Smooth-muscle cells are surrounded by an uninterrupted basement lamina exceptwhere nexuses and desmosome-like attachments between adjacent cells occur. Nointerruption of basement lamina is observed at the level of caveolae or where neuro-muscular transmission presumably takes place.

Tubules, sacs and cisternae of smooth endoplasmic reticulum (sarcoplasmic reti-culum) are commonly observed in proximity to the cell surface and in close relationshipwith the caveolae. Sarcoplasmic reticulum may lie immediately beneath the sarcolemma(Fig. 1) or beneath the caveolae (Fig. 2). Often the sarcoplasmic reticulum constitutesa network spread among the caveolae, which is clearly shown in sections tangential tothe cell surface (Fig. 6), or long tubules running parallel to the longitudinal axis ofthe cell. Only rarely has a conspicuous sarcoplasmic reticulum extending from beneaththe plasma membrane deep into the cell towards the nuclear poles been observed.

The distance between sarcoplasmic reticulum and caveolae or plasma membraneis variable and can be only 10 nm. No clear picture has been obtained either of openingof sarcoplasmic reticulum at the surface or of direct communication between sarco-plasmic reticulum and caveolae or fusion of their membranes.

Numerous glycogen-like granules are found, as well as ribosomes either free orassociated with membranes. In smooth muscle mitochondria are mainly situated at thenuclear poles or beneath the surface of the cell (Fig. 1).

DISCUSSION

The present observations show that a prominent feature of the surface of smooth-muscle cells of guinea-pig ileum is the occurrence of a tubular or labyrinthine systemof smooth endoplasmic reticulum (sarcoplasmic reticulum) spread like a net aroundthe caveolae or immediately beneath the plasma membrane. This arrangement is pos-sibly related to the mechanisms of excitation-contraction coupling and to the problemof compartments for the ions active in these processes.

On a purely morphological basis, 4 compartments can be described in smoothmuscle tissue: (1) an extracellular compartment, (2) a surface intracellular compart-ment (caveolae intracellulares) directly (i.e. without intervening membranes) communi-cating with the extracellular space, (3) an intracellular compartment delimited by thesarcoplasmic reticulum, and (4) an intracellular sarcoplasmic compartment. Of interestis the fact that 4 similar compartments are present in striated muscle: (1) extracellular,(2) surface intracellular (T-system), (3) intracellular-sarcoplasmic reticulum, and

Caveolae and sarcoplasmic reticulum 603

(4) intracellular sarcoplasmic. In smooth muscle the second compartment is less welldeveloped than in skeletal muscle, and moreover the sarcoplasmic reticulum can be indirect relationship with the plasma membrane. The difference, however, is less strikingif it is taken into account that smooth muscle attains a diameter of only 6-10 /im (thatis, -J—>/jjthe diameter of skeletal muscle fibres). It is not known whether the caveolaecommunicate freely with the extracellular space, as is the case with T-system tubules(Franzini-Armstrong & Porter, 1964; Huxley, 1964). This seems highly likely, sinceJ. L. S. Cobb & T. Bennett (personal communication) observed penetration oflanthanum nitrate into caveolae. Lanthanum nitrate is considered as an extracellulartracer, which permeates only those structures which are continuous with the extra-cellular space (Revel & Karnovsky, 1967; Brightman & Reese, 1969).

The notion of'compartments' (Goodford, 1970) or 'sites' (Daniel, 1965; Hurwitz,Joiner & Von Hagen, 1967) is currently used by smooth-muscle physiologists, inrelation to ion exchange and ion sequestration, calcium being the main ion takeninto account. In striated muscle the calcium is stored in sacs of the sarcoplasmicreticulum, in proximity to the tubules of the T-system. On excitation the calciumleaves the sacs and activates the filaments, while in relaxation the calcium is pickedup again by the sarcoplasmic reticulum (see Sandow, 1965). Since the biochemicalconstituents of muscle contractile machinery in smooth muscle are similar to thosefound in striated muscle, the assumption that calcium ions are also involved in thecontraction of smooth muscle is widely justified and supported by much experimentalevidence (see Biilbring, Brading, Jones & Tomita, 1970).

In the intestinal muscle there is evidence that the calcium reaches the sarcoplasmby 2 different pathways: part enters the cytoplasm through an intracellular store(which can serve to sustain muscle tone when the tissue is put in a calcium-freeenvironment (Bozler, 1969)), and part enters by-passing this store (Daniel, 1965;Hurwitz et al. 1967). The existence of an intracellular store of calcium is shown by thefact that a fraction of calcium is kept by smooth muscle even when the tissue is putin a Ca-free solution (Schatzmann, 1961; Lullmann & Siegfriedt, 1968), notwith-standing that all the cellular calcium is relatively quickly exchanged with extracellularcalcium when the tissue is put in a solution containing ^Ca (Lullmann, 1970). Severalauthors have supposed that sarcolemmal membrane can store and release calcium andact like the sarcoplasmic reticulum of skeletal muscle (Daniel, 1965; Goodford, 1965;Lullmann, 1970). Bozler (1969) has argued strongly against this assumption.

On the basis of the rather peculiar development of sarcoplasmic reticulum at thesurface of smooth-muscle cells, the hypothesis is here put forward that sarcoplasmicreticulum constitutes a major store of intracellular calcium. This adds a furthersimilarity in excitation-contraction mechanisms between smooth and striated muscle.

As a working hypothesis it is suggested that in smooth-muscle cells of adult guinea-pig ileum, sarcoplasmic reticulum plays a similar role as in the striated muscle: it isclosely related to the plasma membrane or to the caveolae (surface intracellular com-partment), and can free calcium into the sarcoplasm, activating contractile machinery,and reabsorb calcium, sequestrating it from the cytoplasm.

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I wish to thank Professors J. Z. Young, F.R.S., and E. G. Gray for their support and en-couragement. A fellowship from the Wellcome Trust is gratefully acknowledged.

REFERENCES

BOZLER, E. (1969). Role of calcium in initiation of activity of smooth muscle. Am. J. Pliysiol.216, 671—674.

BRIGHTMAN, M. W. & REESE, T. S. (1969). Junctions between intimately apposed cell membranesin the vertebrate brain. J. Cell Biol. 40, 648-677.

BOLBRING, E., BRADING, A. F., JONES, A. W. & TOMITA, T. (1970). Smooth Muscle. London:Arnold.

BURNSTOCK, G. (1970). Structure of smooth muscle and its innervation. In Smooth Muscle (ed.E. Biilbring, A. F. Brading, A. W. Jones & T. Tomita), pp. 1-69. London: Arnold.

CAESAR, R., EDWARDS, G. A. & RUSKA, H. (1957)- Architecture and nerve supply of mammaliansmooth muscle tissue. J. biophys. biochem. Cytol. 3, 867-878.

DANIEL, E. E. (1965). Attempted synthesis of data regarding divalent ions in muscle function.In Muscle (ed. W. M. Paul, E. E. Daniel, C. M. Kay & G. Monckton), pp. 293-313. London:Pergamon.

DEWEY, M . M . & B A R R , L.(i968). Structure of vertebrate intestinal smooth muscle. In Handbookof Physiology, section 6, Alimentary Canal, vol. 4, Motility (ed. C. F. Code), pp. 1629-1654.Washington: American Physiological Society.

FRANZINI-ARMSTRONG, C. & PORTER, K. R. (1964). Sarcolemmal imaginations and the T-system in fish skeletal muscle. Nature, Land. 202, 355—357.

GOODFORD, P. J. (1965). The distribution of calcium in intestinal smooth muscle. In Muscle(ed. W. M. Paul, E. E. Daniel, C. M. Kay & G. Monckton), pp. 219-227. London: Pergamon.

GOODFORD, P. J. (1970). Ionic interactions in smooth muscle. In Smootli Muscle (ed. E. Biilbring,A. F. Brading, A. W. Jones & T. Tomita), pp. 100—121. London: Arnold.

HURWITZ, L., JOINER, P. D. & VON HAGEN, S. (1967). Calcium pools utilized for contraction insmooth muscle. Am. J. Physiol. 213, 1299-1304.

HUXLEY, H. E. (1964). Evidence for continuity between the central elements of the triads andextracellular space in frog sartorius muscle. Nature, Lond. 202, 1067—1071.

KANASEKI, T. & KADOTA, K. (1969). The 'vesicle in a basket'. J. Cell Biol. 42, 202-220.LOLLMANN, H. (1970). Calcium fluxes and caJcium distribution in smooth muscle. In Smooth

Muscle (ed. E. Biilbring, A. F. Brading, A. W. Jones & T. Tomita), pp. 151-165. London:Arnold.

LOLLMANN, H. & SIEGFRIEDT, A. (1968). Ober den Calcium-Gehalt und den 45Calcium-Austausch in Langsmuskulatur der Meerschweinchendiindarms. Pfliigers Arch. ges. Physiol.300, 108-119.

MARK, J. T. S. (1956). An electron microscope study of uterine smooth muscle. Anat. Rec. 125,473-493-

PROSSER, C. L., BURNSTOCK, G. & KAHN, J. (i960). Conduction in smooth muscle: comparativestructural properties. Am. J. Physiol. 199, 545-552.

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(Received 13 November 1970)

Fig. 1. Two adjacent smooth-muscle cells. A number of caveolae are clearly apparent.Sacs of sarcoplasmic reticulum are underneath the caveolae or directly underneath theplasma membrane. In the cell to the left a large mitochondrion is present. Circularmuscle coat of guinea-pig ileum. x 81 000.

Caveolae. and sarcoplasmic reticulum

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Fig. 2. Section tangential to the surface of a smooth-muscle cell. Rows of caveolae cross-sectioned, and among them long tubules of sarcoplasmic reticulum are observed.Circular muscle coat of guinea-pig ileum. x 120000.Figs. 3, 4. Groups of caveolae which do not open directly at the cell surface but intolarger invaginations of the plasma membrane. Longitudinal muscle coat of guinea-pigileum. x 107000.Fig. 5. Caveolae and sarcoplasmic reticulum in 2 muscle cells. In the cell to the righta larger and more open invagination of the plasma membrane, coated on the sarco-plasmic side with dense granular material, is observed. Circular muscle coat of guinea-pig ileum. x 106000.

Caveolae and sarcoplasmic reticulum 607

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Fig. 6. Section tangential to the surface of a smooth muscle cell. Caveolae are cross-sectioned and a labyrinthine system of sarcoplasmic reticulum is spread around them.Circular muscle coat of guinea-pig ileum. x 125000.

Caveolae and sarcoplasmic reticulum