THE SARCOPLASMIC RETICULUM AND TRANSVERSE TUBULES OF THE FROG''S SARTORIUS

The sarcoplasmic reticulum of the frog's sartorius muscle was examined by electron microscopy following sequential fixation in glutaraldehyde and osmium tetroxide and embedding in Epon. The earlier results of Porter and Palade on Ambystoma muscle were confirmed in the sartorius. In addition, the transverse tubules were observed to be continuous across the width of the fiber, a set of flat intermediate cisternae was seen to connect the terminal cisternae to the longitudinal tubules in the A band, and the continuous reticulum collar at the center of the A band was found to be perforated by circular and elongated pores (the fenestrated collar). The transverse tubules have a volume about 0.3 per cent of the fiber volume, and a surface area about 7 times the outer cylindrical surface area for a fiber 100 µ in diameter. The terminal cisternae, the intermediate cisternae, and the longitudinal tubules together with the fenestrated collar each have a volume of 4 to 5 per cent of the fiber volume and a surface area 40 to 50 times the outer surface area of a fiber 100 µ in diameter. Some evidence for continuity of the transverse tubules with the fiber surface is presented, but this is thought to be not so convincing as evidence presented by others. The results are discussed in terms of a possible mechanism for a role of the transverse tubules and sarcoplasmic reticulum in excitation-contraction coupling, as suggested by their morphology and a variety of physiological studies. In this scheme, the transverse tubules are thought to be electrically coupled to the terminal cisternae, so that depolarization of the fiber surface spreads inward along the transverse tubules and to the terminal cisternae, initiating the release of a contraction-activating substance.     

Scanning electron-microscopic studies on the three-dimensional structure of sarcoplasmic reticulum in the mammalian red,white and intermediate muscle fibers

Summary The three-dimensional structure of the sarcoplasmic reticulum (SR) in the red, white and intermediate striated muscle fibers of the extensor digitorum longus muscle of the rat was examined under a field-emission type scanning electron microscope after removal of cytoplasmic matrices by the osmium-DMSO-osmium procedure.In all three types of fibers, the terminal cisternae and transverse tubules form triads at the level of the A-I junction. Numerous slender sarcotubules, originating from the A-band side terminal cisternae, extend obliquely or longitudinally and form oval or irregular shaped networks of various sizes in front of the A-band, then become continuous with the tiny mesh (fenestrated collar) in front of the H-band. The A-and H-band SR appears as a single sheet of anastomotic tubules. Numerous sarcotubules, originating from the I-band side terminal cisternae, extend in threedimensional directions and form a multilayered network over the I-band and Z-line regions. At the I-band level, paired transversely oriented mitochondria partly embrace the myofibril. The I-band SR network is poorly developed in the narrow space between the paired mitochondria, but is well developed in places devoid of these mitochondria.The three-dimensional structure of the SR is basically the same in all three muscle fiber-types. However, the SR is sparse on the surface of mitochondria, so the mitochondria-rich red fiber has a much smaller total volume of SR than the mitochondria-poor white fiber. Moreover, the volume of SR of the intermediate fiber is intermediate between the two.     

High -resolution scanning electron-microscopic studies on the three-dimensional structure of mitochondria and sarcoplasmic reticulum in the different twitch muscle fibers of the frog

Summary The three-dimensional structure of the mitochondria and sarcoplasmic reticulum (SR) in the three types of twitch fibers, i.e., the red, white and intermediate skeletal muscle fibers, of the vastus lateralis muscle of the Japanese meadow frog (Rana nigromaculata nigromaculata Hallowell) was examined by high resolution scanning electron microscopy, after removal of the cytoplasmic matrices.The small red fibers have numerous mitochondrial columns of large diameter, while the large white fibers have a small number of mitochondrial columns of small diameter. In the medium-size intermediate fibers, the number and diameter of the mitochondrial columns are intermediate between those of the red and white fibers.In all three types of fibers, the terminal cisternae and transverse tubules form triads at the level of each Z-line. The thick terminal cisternae continue into much thinner flat intermediate cisternae, through a transitional part where a row of tiny indentations can be observed. Numerous slender longitudinal tubules originating from the intermediate cisternae, extend longitudinally or obliquely and form elongated oval networks of various sizes in front of the A-band, then fuse to form the H-band collar (fenestrated collar) around the myofibrils. On the surface of the H-band collar, small fenestrations as well as tiny hollows are seen. The three-dimensional structure of SR is basically the same in all three muscle fiber-types. However, the SR is sparse on the surface of mitochondria, so the mitochondria-rich red fiber has a smaller total volume of SR than the mitochondria-poor white fiber. The volume of SR of the intermediate fiber is intermediate between other the two.     

Injury to nerves and the initiation of amphibian limb regeneration

The force produced within skeletal muscle fibers is transmitted to the bone via a myotendinous junction. This junctional region was examined by light and electron microscopy in the sartorius muscles of three Rana temporaria. The muscle fibers tapered and inserted at an angle of about 25 degrees with the connective tissue fascia near the bone. The composition of the structures within the last 100 microns of the fiber was analyzed morphometrically. The T-system, terminal cisternae, and caveolae were the same as in the central region of the muscle fiber. However, the mitochondrial content was higher and the volume of longitudinal sarcoplasmic reticulum was lower than elsewhere in the fiber. The membrane at the end of the fiber had extensive villiform processes interdigitating with the tendon. The surface area of the membrane around the villiform processes was estimated with point-counting techniques and calculated from the stereological equations appropriate for partially anisotropic structures. The extra membrane involved in the myotendinous junction was about 32 times that of the cross-sectional area of the fiber. Part of this additional membrane contained specialized adherens junctions through which the contractile proteins of the muscle are anchored to collagen. The increased area at the myotendinous junction presumably provides greater mechanical strength than a flat termination. The high values of membrane capacitance and specific resistance measured electrophysiologically at the end of the fiber also can be attributed to the characteristics of the terminal membrane structure.     

Ultrastructure of the radula protractor of Busycon canaliculatum

Summary The distribution of the sarcoplasmic reticulum and sarcolemmic tubules in the radula protractor muscle of the whelk, Busycon canaliculatum, has been investigated. The sarcoplasmic reticulum consists of an interconnected system of cisternae and tubular channels. The cisternae are closely associated with the sarcolemma. The tubular channels project from the cisternae into the interior of the cell and run parallel to the long axis of the myofilaments. Parallel tubular channels are interconnected with one another by short branches. This finding of an elaborate sarcoplasmic reticulum supports previous physiological work on this smooth muscle which indicated the presence of an intracellular compartmentalization of calcium ions. There is also an extensive system of tubular invaginations of the sarcolemma which we have termed sarcolemmic tubules. These tubules are 600 Å in diameter and about 0.5 microns in length. There is a substructure associated with the leaflet of the tubular membrane bordering the extracellular space. The sarcolemmic tubules penetrate only half a micron from the surface of the cell and interdigitate with the sarcoplasmic reticulum associated with the sarcolemma. Calculations have shown that the surface area of this smooth muscle cell is more than doubled by the presence of sarcolemmic tubules.     

Is a guanine nucleotide-binding protein involved in excitation-contraction coupling in skeletal muscle?

Plasma membrane depolarization causes skeletal muscle contraction by triggering Ca2+ release from an intracellular membrane network, the sarcoplasmic reticulum. A specialized portion of the sarcoplasmic reticulum, the terminal cisternae, is junctionally associated with sarcolemmal invaginations called the transverse tubules, but the mechanism by which the action potential at the level of the transverse tubules is coupled to Ca2+ release from the terminal cisternae is still mysterious. Here we show that: (i) GTP gamma S, a non-hydrolyzable analog of GTP, elicits isometric force development in skinned muscle fibre; (ii) GTP gamma S is unable to release CA2+ from isolated sarcoplasmic reticulum fractions; (iii) the threshold for tension development is shifted to higher GTP gamma S concentrations by pre-incubation with pertussis toxin. These results suggest that a GTP-binding protein is involved in coupling the action potential of transverse tubules to Ca2+ release from the terminal cisternae.     

STEREOLOGICAL ANALYSIS OF MAMMALIAN SKELETAL MUSCLE : I. Soleus Muscle of the Adult Guinea Pig

A quantitative analysis of the volumes, surface areas, and dimensions of the ultrastructural components in the soleus muscle fibers of the guinea pig was made by using point counting methods of stereology. Muscle fibers have structural orientation (anisotropy) and have spatial gradients of the structures within the fiber; therefore the standard stereological methods were modified where necessary. The entire analysis was repeated at two section orientations to test the modifications and identical results obtained from both. The volume of lipid droplets was 0.20 ± 0.06% (mean ± standard error, n = 5 animals) and the nuclei volume was 0.86 ± 0.20% of the fiber volume. The total mitochondrial volume was 4.85 ± 0.66% of the fiber volume with about one-third being found in an annulus within 1 µm of the sarcolemma. The mitochondrial volume in the remaining core of the fiber was 3.6 ± 0.4%. The T system has a volume of 0.14 ± 0.01% and a surface area of 0.064 ± 0.005 µm²/µm³ of the fiber volume. The surface area of the sarcolemma is 0.116 ± 0.013 µm²/µm³ which is twice the T system surface area. The volume of the entire sarcoplasmic reticulum is 3.52 ± 0.33% and the surface area is 0.97 ± 0.09 µm²/µm³. The sarcoplasmic reticulum is composed of the terminal cisternae whose volume is 1.04 ± 0.19% and surface area is 0.24 ± 0.05 µm²/µm³. The tubules of the sarcoplasmic reticulum in the I band and A band have volumes of 1.97 ± 0.24% and 0.51 ± 0.08%, and the surface areas of the I and A band reticulum are 0.56 ± 0.07 µm²/µm³ and 0.16 ± 0.04 µm²/µm³, respectively. The Z line width, myofibril and fiber diameters were measured.     

Antibodies to junctional sarcoplasmic reticulum proteins: probes for the Ca2+-release channel.

The junctional face membrane plays a key role in excitation-contraction coupling in skeletal muscle. A protein of 350 kDa, tentatively identified as a component of the junctional feet, connects transverse tubules to terminal cisternae of sarcoplasmic reticulum [Kawamoto, Brunschwig, Kim & Caswell (1986) J. Cell Biol. 103, 1405-1414]. The membrane topology and protein composition of sarcoplasmic reticulum Ca2+-release channels of rabbit skeletal muscle were investigated using an immunological approach, with anti-(junctional face membrane) and anti-(350 kDa protein) polyclonal antibodies. Upon preincubation of the terminal cisternae with anti-(junctional face membrane) antibodies, Ca2+-ATPase and Ca2+-loading activities were not affected, whereas anti-(350 kDa protein) antibodies stimulated Ca2+-ATPase activity by 25% and inhibited Ca2+-loading activity by 50% (at an antibody/terminal cisternae protein ratio of 1:1). Specific photolabelling of terminal cisternae proteins with [14C]doxorubicin was prevented by both anti-(junctional face membrane) and anti-(350 kDa protein) antibodies. Stimulation of Ca2+ release by doxorubicin was prevented by both anti-(junctional face membrane) and anti-(350 kDa protein) antibodies. Half-maximal inhibition was obtained at an antibody/terminal cisternae protein ratio of 1:1. Kinetic measurements of Ca2+ release indicated that anti-(350 kDa protein) antibodies prevented Ca2+-induced Ca2+ release, whereas the ATP-stimulation and the inhibition by Mg2+ were not affected. These results suggest that: (i) Ca2+- and doxorubicin-induced Ca2+ release is mediated by Ca2+ channels which are selectively localized in the junctional face membrane; (ii) the 350 kDa protein is a component of the Ca2+-release channel in native terminal cisternae vesicles; and (iii) the Ca2+-activating site of the channel is separate from other allosteric sites.     

Morphology of isolated triads

The triad is the junctional association of transverse tubule with sarcoplasmic reticulum terminal cisternae. A procedure for the isolation of highly enriched triads from skeletal muscle has been described in the previous paper. In the present study, the structural features of isolated triads have been examined by thin-section, negative-staining, and freeze-fracture electron microscopy. In isolated triads, key features of the structure observed in situ have been retained, including the osmiophilic "feet," junctional structures between the transverse tubule and terminal cisternae. New insight into triad structure is obtained by negative staining, which also enables visualization of feet at the junctional face of the terminal cisternae, whereas smaller surface particles, characteristic of calcium pump protein, are not visualized there. Therefore, the junctional face is different from the remainder of the sarcoplasmic reticulum membrane. Junctional feet as viewed by thin section or negative staining have similar periodicity and extend approximately 100 A from the surface of the membrane. Freeze-fracture of isolated triads reveals blocklike structures associated with the membrane of the terminal cisternae at the junctional face, interjunctional connections between the terminal cisternae and t-tubule, and intragap particles. The intragap particles can be observed to be closely associated with the t-tubule. The structure of isolated triads is susceptible to osmotic and salt perturbation, and examples are given regarding differential effects on transverse tubules and terminal cisternae. Conditions that adversely affect morphology must be considered in experimentation with triads as well as in their preparation and handling.     

Doxorubicin induces calcium release from terminal cisternae of skeletal muscle. A study on isolated sarcoplasmic reticulum and chemically skinned fibers

In this study, we investigated the effect of the anticancer drug doxorubicin on Ca2+ fluxes of isolated highly purified sarcoplasmic reticulum fractions (longitudinal tubules and terminal cisternae (Saito, A., Seiler, S., Chu, A., and Fleischer, S. (1984) J. Cell Biol. 99, 875-885] and of chemically skinned skeletal muscle fibers of the rabbit. In terminal cisternae, doxorubicin inhibits Ca2+ uptake (IC50 at 0.5 microM) and increases 2.6-fold Ca2+-dependent ATPase rate (half-maximal activation at 3 microM) and unidirectional Ca2+ efflux (8-fold stimulation at 25 microM). On the contrary, doxorubicin is without effect on longitudinal tubules. In skinned muscle fibers, doxorubicin induces rapid and transient Ca2+ release, as measured by tension development (half-maximal stimulation at 6 microM), which is completely and reversibly inhibited by ruthenium red, a known inhibitor of Ca2+ release from isolated terminal cisternae. Doxorubicin has no effect on the sarcoplasmic reticulum Ca2+ pump and on the contractile apparatus of skinned muscle fibers. It is concluded that doxorubicin activates Ca2+ release from sarcoplasmic reticulum and opens a Ca2+ efflux pathway (Ca2+ channel) selectively localized in terminal cisternae. Doxorubicin might interact with Ca2+ channels involved in physiological Ca2+ release.     

Ultrastructure and morphometry of the stomach muscle of Amphiuma tridactylum

An ultrastructural and stereological examination was performed on stomach smooth muscle of the salamander Amphiuma. This tissue has very large cells, ranging up to 12 X 1500 micron when relaxed. The extracellular space is 31% of the tissue volume, and the tissue contains 84.6% water. These values are similar to those of other amphibian and mammalian gastrointestinal smooth muscle. The cells possess the usual smooth muscle organelles. Thick, thin and intermediate filaments are present, along with membrane-associated and cytoplasmic dense regions. There is a well-developed sarcoplasmic reticulum and many microtubules. Caveolae are found in rows along the cellular surface; the caveolae increase the cellular surface area by about 70%. The ratio mean volume: surface area of the cells is 1.26 micron. This tissue appears to be typical of gastrointestinal smooth muscle, with the exception of the very large size of the cells.     

On the unity of cytomembrane system in the skeletal muscle

In situ cytochemical evidence for specific Ca-binding sites in the cytomembrane system of skeletal muscle fibers is reported. High Ca accumulation was found at the junctions between different types of cytomembranes. Such junctions might represent "gate-locks' for intracellular Ca movements. Openings of sarcoplasmic reticulum (SR) in frog muscle fibers and of T-tubules in rat muscle fibers are described. Coated and noncoated caveolae were found in rat muscle fibers. The same positive reaction for TPP-ase was found in trans-Golgi zone, terminal cisternae and subsarcolemmal cisternae. These results suggest the membrane continuity and ontogenetic relationships in the cytomembrane system of skeletal muscle fibers.     

THE ORGANIZATION OF FLIGHT MUSCLE FIBERS IN THE ODONATA

The cytological organization of flight muscle fibers of Odonata has been investigated. These fibers, in representatives of the Zygoptera and Anisoptera, have been compared and found to be similar, except that, in the former, pairs of lamellar fibrils, rather than single fibrils, alternate with the mitochondria. In each instance, in these synchronous muscles, the actin filaments of the myofibrils are found to lie opposite to and midway between pairs of myosin filaments—a configuration previously reported in asynchronous flight muscle fibers. The disposition of the T system and sarcoplasmic reticulum membranes in glutaraldehyde-fixed anisopteran muscle is described in detail: the T system tubules are shown to be radially continuous across the fiber, and are derived as openmouthed invaginations from the surface cell-membrane. The detailed organization of the dyad junctions between these tubules and the adjoining cisternae of the sarcoplasmic reticulum is described. The accessibility of the T system interior to diffusion exchange with the general extracellular milieu has been investigated by studies on the penetration of ferritin into the fiber: molecules of this marker have been found to diffuse solely along the T system tubules, and their presence in the tubule extremities adjoining the centrally placed nuclei confirms the morphological evidence suggesting that these tubules provide open diffusion channels extending across the radius of the fiber. The possible physiological role of these membrane components and their distribution in synchronous muscles of insects and vertebrates and in asynchronous insect flight muscle are discussed.     

The relation between excitation-contraction coupling and fine structure of a molluscan muscle,the radular retractor of the whelk,Buccinum undatum

The Buccinum radula is of the rachiglossate type with two outer rows of fierce hook-like attack teeth and a medial row of straight sharp-pointed shredding teeth. Individual cells of the radular retractor muscle are 10–12 m in diameter and separated at the closest by gaps of only 40 nm, providing areas of potential electrical contact. The cell membranes are heavily invested with long finger-like invaginations, associated with sarcoplasmic reticular cisternae, and surface caveolae; the latter are associated with the numerous dense body membrane attachment plaques found in this muscle. The radular retractor muscle possesses a significant sarcoplasmic reticulum of peripheral cisternae and deeper vesicles associated with mitochondria. The surface caveolae may result from myofilament force exerted via attachment plaques at the cell membrane, while deeper invaginations may constitute a rudimentary transverse tubular system to relay surface depolarization to associated sarcoplasmic reticular cisternae inducing calcium release to effect excitation-contraction coupling. The radular retractor muscle possesses the usual thick paramyosin and thin actin myofilaments, the latter associated with dense bodies and attachment plaques presumably to transduce force to the cell membrane. The mitochondria are unusually large and packed into dense central clusters surrounded by large deposits of glycogen granules. The nerve endings on the radular retractor muscle fibres show four different types of transmitter vesicle, presumably related to the four kinds of agonist action in this muscle, cholinergic, serotonergic, peptidergic and purinergic. All nerve endings have mixed vesicle populations, clear evidence of co-transmission. In this muscle we see a modification of usual smooth muscle structure to effect fast sustained contractions, an ultrastructural configuration functionally designed for the muscle's central role in the feeding cycle.Abbreviations ABRM anterior byssus retractor muscle - EC coupling excitation-contraction coupling - RP radular protractor muscle - RR radular retractor muscle - SR sarcoplasmic reticulum - T-system transverse tubular system     

THE ORGANIZATION OF FLIGHT MUSCLE IN AN APHID, MEGOURA VICIAE (HOMOPTERA) : With a Discussion on the Structure of Synchronous and Asynchronous Striated Muscle Fibers

The organization of the indirect flight muscle of an aphid (Hemiptera-Homoptera) is described. The fibers of this muscle contain an extensive though irregularly disposed complement of T system tubules, derived as open invaginations from the cell surface and from the plasma membrane sheaths accompanying the tracheoles within the fiber. The sarcoplasmic reticulum is reduced to small vesicles applied to the T system surfaces, the intermembrane gap being traversed by blocks of electron-opaque material resembling that of septate desmosomes. The form and distribution of the T system and sarcoplasmic reticulum membranes in flight muscles of representatives of the major insect orders is described, and the extreme reduction of the reticulum cisternae in all asynchronous fibers (to which group the aphid flight muscle probably belongs), and the high degree of their development in synchronous fibers is documented and discussed in terms of the contraction physiology of these muscle cells.     

Sequential expression during postnatal development of specific markers of junctional and free sarcoplasmic reticulum in chicken pectoralis muscle.

Skeletal muscle sarcoplasmic reticulum comprises two distinct membrane domains, i.e., the Ca(2+)-pump membrane, corresponding mainly to longitudinal tubules, and the junctional membrane of the terminal cisternae containing the ryanodine receptor/Ca(2+)-release channel. Additional minor proteins previously shown in rabbit fast-twitch skeletal muscle to fractionate selectively to each membrane domain comprise 160- and 53-kDa glycoproteins and 170-kDa low-density lipoprotein (LDL)-binding protein, respectively (Damiani and Margreth, 1991, Biochem. J. 277, 825-832). We report evidence in chicken pectoralis, a predominantly fast muscle, on two closely immunologically related glycoproteins, a minor component of 130-kDa and a major 53-kDa protein. In contrast to the seemingly highly conserved structure of this protein, our results show marked differences in mobilities for chicken 125I-LDL that were detected as a 130- to 116-kDa protein doublet after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, although being otherwise indistinguishable from rabbit 170-kDa protein in LDL-binding characteristics, as well as for preferential association to junctional terminal cisternae. Chicken Ca(2+)-ATPase, although being extensively homologous to rabbit Ca(2+)-ATPase, is shown to be less active and to differ slightly in electrophoretic properties. We have investigated the time course of expression of the specific protein components of longitudinal and of junctional sarcoplasmic reticulum in chick pectoralis muscle from late embryonic development up to 2 months after hatching. Coincident with the posthatching increase in membrane density of high-affinity [3H]ryanodine-binding sites in muscle, both calsequestrin and the species-specific LDL-binding protein(s) are detected in increasing amounts, using ligand blot techniques. In contrast, the appearance and steady accumulation in muscle of Ca(2+)-ATPase, like the time-correlated increase of sarcoplasmic reticulum glycoproteins, are relatively delayed, the most striking changes occurring from 1 week after hatching onward. The sequential expression in chick developing muscle of proteins selectively associated with the junctional terminal cisternae and with longitudinal sarcoplasmic reticulum, respectively, argues for a similar morphogenetic program in avian and mammalian species and, to account for that, for the existence of common epigenetic differentiating influences on the expression of sarcoplasmic reticulum protein genes.     

Quantitation of 'junctional feet' content in two types of muscle fiber from hind limb muscles of the rat

1. Transverse tubules in fibers from rat soleus and extensor digitorum longus (EDL) muscles of the rat were infiltrated with silver dichromate (black reaction of Golgi). This provides a faithful, high-contrast outline of the tubules, which allows distinction between segments involved in junction formation with the sarcoplasmic reticulum and segments that are free. 2. Electron micrographs of semithin transverse sections were used to quantitate T tubule parameters and to measure cross-sectional area and perimeter of individual fibers. Thin sections and data from the literature were used to obtain the contribution of caveolae to external surface area and the frequency of junctional feet along the junctional T tubule membrane. 3. From the above data we calculate the ratio of number of feet to total external surface area for a given fiber segment. The ratio is compared with data in the literature on the total amount of 'charge movement' (in nC/uF of total external surface area). 4. The average feet/surface area ratio is twice as large in EDL than in soleus fibers, while the charge movement is up to five-fold larger. Probably some of the total charge movement is not directly associated with events related to the turning on of the SR permeability to calcium.     

Specialized contacts between sarcolemma and sarcoplasmic reticulum at the ends of muscle fibers in the diaphragm of the rat

Summary An electron-microscopic study of the myotendinous portion of the diaphragm in the Wistar rat has shown that at the ends of muscle fibers, longitudinally oriented invaginations and peripheral furrows of the sarcolemma establish specialized contacts with individual sacs of the sarcoplasmic reticulum. The construction of these terminal contacts is similar to that of contacts between sarcolemmic T-tubules and terminal cisternae of the sarcoplasmic reticulum, characterized by formation of triads. The contact zones of the sac membrane are undulated and bound to the adjoining sarcolemma via electron-dense profiles of varying forms. Frequently, the terminal contacts and triads are located at the same level within the muscle fiber, at the borderline between A- and I-bands of the sarcomeres. At the ends of muscle fibers combined contacts between peripheral furrows of the sarcolemma, terminal cisternae of the sarcoplasmic reticulum, and T-tubules of the triads are also disclosed. The implications of the terminal contacts for muscle contraction are discussed.     

Further characterization of light and heavy sarcoplasmic reticulum vesicles. Identification of the ‘sarcoplasmic reticulum feet’ associated with heavy sarcoplasmic reticulum vesicles

Light and heavy sarcoplasmic reticulum vesicles were isolated from rabbit leg muscle using a combination of differential centrifugation and isophycnic zonal ultracentrifugation. Light sarcoplasmic reticulum vesicles obtained from the 30–32.5% and heavy sarcoplasmic reticulum vesicles obtained from the 38.5–42% sucrose regions of the linear sucrose gradient were determined to be free of surface and mitochondrial membrane contamination by marker enzyme analysis and electron microscopy. Thin sections of the light vesicles revealed empty vesicles of various sizes and shapes. Freeze-fracture replicas of the light vesicles showed an asymmetric distribution of intramembranous particles with the same orientation and distribution as the longitudinal sarcoplasmic reticulum in vivo. Heavy vesicles appeared as rounded vesicles of uniform size filled with electron dense material, similar to that seen in the terminal cisternae of the sarcoplasmic reticulum. The cytoplasmic surface of the membrane was decorated by membrane projections, closely resembling the ‘feet’ which join the sarcoplasmic reticulum to the transverse tubules in the intact muscle fiber. Freeze-fracture replicas of the heavy vesicles revealed an asymmetric distribution of particles which in some areas of the vesicle's surface are larger and less densely aggregated than those of the light vesicles. In the best quality replicas, some regions of the luminal leaflet were not smooth but showed evidence of pits. These structural details are characteristic of the area of sarcoplasmic reticulum membrane which is covered by the ‘feet’ in the intact muscle.Heavy vesicles contained greater than six times the calcium content of light vesicles, 54 vs. 9 nmol Ca²⁺/μl of water space. After KCl washing both contained less than 4 nmol Ca²⁺/μl of water space. Although they transported at the same rate and the same total amount of calcium, the rate of passive Ca²⁺ efflux from the heavy vesicles was double that of light vesicles. The higher rate of calcium efflux from the heavy vesicles was inhibited by dantrolene, an inhibitor of Ca²⁺ release. High resolution sodium dodecyl sulfate gel electrophoresis showed that the light vesicles contained predominantly Ca²⁺-ATPase along with several approx. 55 000-dalton proteins and a 5000-dalton proteolipid, while the heavy vesicles contained Ca²⁺-ATPase and calsequestrin along with several approx. 55 000-dalton proteins, extrinsic 34 000- and 38 000-dalton proteins, intrinsic 30 000- and 33 000-dalton proteins and two proteolipids of 5000 and 9000 daltons. KCl washing of the heavy vesicles removed both the approx. 34 000- and 38 000-dalton proteins, and the ‘sarcoplasmic reticulum feet’ were no longer seen on the heavy vesicles. The KCl supernatant was enriched in the 34 000- and 38 000-dalton proteins, indicating that these proteins are possible components of the sarcoplasmic reticulum feet. The biochemical and morphological data strongly support the view that the light vesicles are derived from the longitudinal sarcoplasmic reticulum and that the heavy vesicles are derived from the terminal cisternae containing junctional sarcoplasmic reticulum membrane with the intact ‘sarcoplasmic reticulum feet’.     

The membrane systems in different fibre types of the triceps surae muscle of cat

Summary The volume and surface area of mitochondria and sarcoplasmic reticulum in fast and slow twitch fibres of the cat triceps surae muscle were determined from thin sections. The width of the Z-line and the array of glycogen granules identified fast and slow twitch fibres.The relative volume occupied by mitochondria was largest in slow twitch gastrocnemius fibres. Fast twitch fibres showed the greatest scatter of mitochondrial content. This corresponds with the fact that motor units of the fast twitch type differ most with respect to resistance to fatigue.The relative volume of the sarcoplasmic reticulum was twice as large in fast as in slow twitch fibres. The volume fraction occupied by longitudinal tubules of the reticulum was the same in fast and slow twitch gastrocnemius fibres but was only half as large in the slow twitch soleus fibres. This difference may be related to post-tetanic potentiation: this property is present in all gastrocnemius fibres but is absent in soleus fibres.The specific tetanic force is 3 to 5 times smaller in slow twitch gastrocnemius than in slow twitch soleus fibres or fast gastrocnemius fibres. There was, however, no detectable morphological difference that might be related to this difference in force.Freeze fractures demonstrated directly that, in soleus fibres, terminal cisternae and longitudinal tubules of the reticulum were scarce as compared to gastrocnemius fibres. The plasma membranes of some gastrocnemius fibres displayed square arrays of 60-nm particles; these arrays were absent in other gastrocnemius fibres and in all soleus fibres. They probably characterize plasma membranes of fast twitch fibres.This study was supported by grants from the Danish Medical Research Council. I wish to thank Mrs. M. Bjærg for valuable technical help