(Received 16 August 1966) frog iliofibularis muscle and the contractile response elicited by applied

Transcription

1 J. Phy8iol. (1967), 188, pp With 6 plates Printed in Great Britain CALCIUM ACTIVATION OF FROG SLOW MUSCLE FIBRES BY L. L. COSTANTIN, R. J. PODOLSKY AND LOIS W. TICE From the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland 20014, U.S.A. (Received 16 August 1966) SUMMARY 1. Skinned muscle fibres were prepared from the tonus bundle of the frog iliofibularis muscle and the contractile response elicited by applied calcium ions was studied. The fibre type was determined by electron microscopy. 2. Fast fibres shortened many times more rapidly than slow fibres, indicating that the slow contraction of slow fibres is an inherent property of the contractile mechanism. 3. The extent of spread of contraction following local calcium application was much greater in slow than in fast fibres, a difference which is consistent with the relative sparsity of the sarcoplasmic reticulum in slow fibres. 4. The ability of the sarcoplasmic reticulum of slow fibres to accumulate calcium was demonstrated by the in situ immobilization of calcium when oxalate solutions were added to the skinned fibre. INTRODUCTION Kuffler & Vaughan Williams (1953) have demonstrated that the 'phasic' and 'tonic' phenomena seen in frog muscles are due to two qualitatively different types of muscle fibre, the fast and the slow fibres. In a study correlating the physiological behaviour of single fibres dissected from the tonus bundle of the frog iliofibularis with their microscopic appearance, Peachey & Huxley (1962) found that fibres of the two types differed in the organization of both the myofibrils and the internal membrane system. In slow fibres the internal membrane system was less extensive than in fast fibres and also appeared to lack both transverse continuity and the triads that had been associated with the inward spread of activation in fast fibres (Huxley & Taylor, 1958), and it was suggested that the slow mechanical response of these fibres was related to these differences in the internal membrane system (Peachey, 1961). Recently, Page (1965) has demonstrated in slow fibres, by bathing

2 262 L. L. COSTANTIN AND OTHERS muscles in ferritin before fixation, the presence of tubules which extend inward from the surface membrane. This observation, coupled with the finding of occasional triads in slow fibres, suggested that these fibres as well as fast fibres were activated by a process faster than diffusion from the surface, and raised the possibility that the slow response originated in the contraction rather than the activation system. To obtain more information on this point, it seemed worth while to study the motion that resulted when myofibrils from fast and slow fibres were activated directly rather than through the normal sequence of events initiated by depolarization of the surface membrane. This was done in the experiments reported here by applying calcium ions to isolated fibres from which the surface membrane had been removed by microdissection. The results provide evidence that the functional difference in the two types of fibre can be accounted for by differences in the contractile mechanism. METHODS All experiments were performed on the Natori (1954) skinned fibre preparation at room temperature (about 230 C). A frog iliofibularis muscle was mounted in a dissecting chamber filled with either a normal Ringer solution (115.5 mm-nacl, 2-5 mm-kcl, 1-8 mm-cacl2, 3 mm sodium orthophosphate, ph 7-0) or sulphate 'Ringer solution (38.75 mm-na2so4, 1-25 mm-k2so4, 8 mm-caso4, 113 mm sucrose, 1-5 mm sodium orthophosphate, ph 7-0, after Hodgkin & Horowicz, 1959). Since slow fibres are known to be concentrated in the tonus bundle of the iliofibularis, single skinned fibres were prepared from small bundles removed from this region of the muscle. The dissection procedure for preparing a skinned fibre has been described in detail elsewhere (Costantin & Podolsky, 1965). The final preparation consisted of a segment of a single muscle fibre in which the sarcolemma and a superficial layer of myofibrils had been dissected away over a length of up to 1 mm. The preparation was covered by paraffin oil on a microscope slide, so that a layer of aqueous fluid only a few microns thick surrounded the isolated fibre. Three criteria were useful in identifying the tonus bundle: (1) the nerve innervating the iliofibularis penetrates the muscle in the region of the tonus bundle (Peachey, 1961); (2) the diameter of both slow and fast fibres within the bundle is about half that of the fibres in the remainder of the iliofibularis (Adrian & Peachey, 1965); (3) the connective tissue in the region of the tonus bundle is more dense than in other portions of the iliofibularis, so that it was more difficult to tease apart fibres removed from this region. Following dissection, the preparation was placed on a microscope stage, permitting simultaneous visual observation and cinephotographic recording. In most experiments, the prep. aration was illuminated by ordinary light with the condenser aperture stopped down until striations were readily visible, and was observed with a 40 x Bausch and Lomb oil-immersion objective (N.A. = 1-00), which made contact with the paraffin oil bathing the preparation. When a shallow depth of field was desirable, the condenser aperture was opened, and the sarcomere pattern visualized with polarization optics. Film records were made at 15 frames/ sec, and the film speed was periodically calibrated by filming a moving stop-watch. Total magnification in the plane of the film was 53 x. Light microscope appearance of fibres from the tonus bundle. Fibres dissected from the frog semitendinosus or from a portion of the iliofibularis outside the tonus bundle usually have a characteristic hyaline appearance when viewed in the dissecting microscope (magnification 40 x ) under dark field illumination. Within the tonus bundle, however, two types of fibre

3 CALCIUM ACTIVATION OF SLOW FIBRES 263 can be identified, one with the typical hyaline appearance of other muscles and the other with a much more granular cytoplasm. When the response of a typical fibre from each group to calcium application was examined (see Results), to our surprise the granular-appearing fibres were almost invariably found to be fast fibres while the more hyaline ones were usually slow fibres. It should be emphasized, however, that the light microscope appearance was not an adequate criterion for distinguishing between slow and fast fibres, since half or more of the fibres within the tonus bundle presented an appearance intermediate between these two types. When the granular- and hyaline-appearing fibres from the tonus bundle were compared in the electron microscope, the granularity was correlated with lipid droplets associated with the mitochondria. Application of solutions to the skinned fibre. Small volumes of solution were applied to the surface of the skinned fibre by means of a micropipette whose tip was inserted into the oil layer above the fibre. The preparation and filling of the micropipettes is described elsewhere (Costantin & Podolsky, 1966). Controlled application of pressure to the lumen of the micropipette formed a droplet which could be lowered to the skinned fibre; when the pipette tip was applied to the fibre surface, a small region of the preparation could be perfused directly. Potassium propionate was added to the 1-0, 3 0 and 30-0 mm calcium chloride solutions used to study the contractile response of the skinned fibre so that the total cation concentration was 140 mm. Sodium citrate (80 mm) was added to the 10 mm calcium chloride solution. The ph of all these solutions was 7-2. Preparation of agar-filled pipettes. Agar was prepared by heating 1 g of agarose (Mann Research Laboratories) in 100 ml. of a 0-14 M-K propionate solution. Agar prepared in this manner contained 0-25 mm of calcium by direct analysis (500 mg of the dry agarose was ashed, resuspended in 1 M-HCI, and assayed for calcium on a flame photometer). When a higher calcium concentration was desired, 0-5, 1-0 or 2 0 mm-cacl2 was added to the K propionate solution used to prepare the agar. Conventional micropipettes about 6 cm long were ground on a motor-driven diamond wheel until the tip outer diameter was about 75 ji. The debris introduced into the pipette lumen by this procedure was easily removed by flushing with absolute alcohol. The pipette was fixed with Araldite (Ciba (A.R.C.) Ltd.) to the hub of a hypodermic needle, and mounted vertically with the tip down under a heating lamp. The needle hub was then filled with warm agar, and the lamp promptly removed. As the pipette cooled, the agar flowed down the pipette shaft to within a few millimetres of the tip before it gelled. The agar-filled pipette was mounted via a three-way stopcock to a gas-tight mercury. filled 1 ml. syringe equipped with a threaded plunger (Hamilton Company). This system was nearly incompressible, so that the position of the agar column in the pipette was readily controlled by a slight turn of the plunger. The entire apparatus was mounted on a sliding micromanipulator (Zeiss-Jena), and the pipette tip was introduced into the oil layer surrounding the preparation. When the agar was driven by pressure into the tapering portion of the pipette, it broke into columns mm in length separated by liquid extruded from the gel. Pressure was applied until /i of agar protruded from the pipette tip, the stopcock was opened to the air, and the excess fluid was blotted by touching the agar to the slide. The agar was then ready for application to the preparation, which was suspended in oil by means of two jewellers' forceps arranged as stage micromanipulators. When a new preparation was to be studied, the agar column was expelled and a slight negative pressure applied, filling the pipette tip with a small amount of paraffin oil. The new preparation was mounted on the microscope stage, the pipette was reintroduced, and the procedure for forming a short length of agar protruding from the pipette tip was repeated. In this manner, drying of the agar in air was avoided, and one pipette could be used for many studies. Filled pipettes could be stored for weeks before use, with the pipette tip under paraffin oil. Preparation for electron microscopy. Fibres to be studied by electron microscopy were fixed by covering the entire preparation with a large drop of buffered glutaraldehyde (6-5 % glutaraldehyde and 200 mm sodium cacodylate at ph 7-2). Most of the paraffin oil

4 264 L. L. COSTANTIN AND OTHERS was drawn off by blotting, and the preparation was placed in a vial of the same glutaraldehyde solution at 00 C for 1 hr. It was then washed in cacodylate buffer overnight, postfixed in 1 % OS04 for 1 hr, dehydrated in graded alcohols, and cleared in propylene oxide. The preparation was then embedded in a thin layer of Maraglas so that it could be examined in the light microscope before sectioning; this initial embedding made it possible to reexamine the preparation and to identify the fibre which had been studied physiologically. The desired portion of the preparation was then re-embedded in Maraglas in an appropriate orientation for sectioning. Calcium-accumulating structures were localized by a slight modification of the procedure described by Costantin, Franzini-Armstrong & Podolsky (1965); 10 mm sodium oxalate was present in all aqueous solutions used in fixation and the concentration of OS04 was reduced to 1 %. Sections for microscopy were collected on either distilled water or 1 0 mm- Na oxalate solution. When distilled water was used, many deposits were dissolved away, leaving clearly recognizable holes in the section; this effect was considerably reduced in sections collected on the oxalate solution, but the latter procedure produced extraneous precipitates randomly distributed on the section. Studies of oxalate localization were done either on sections cut into distilled water or in those regions of sections collected on oxalate solution which showed minimal extraneous precipitate. RESULTS Characterization of the response to calcium application. When solutions containing 1-10 mm calcium were applied to the surface of skinned fibres prepared from the tonus bundle of the iliofibularis, two distinct types of responses were readily distinguishable; repeated testing of different sites along the skinned region demonstrated that a given preparation gave exclusively one or the other type of response. The first, which seemed identical with the response of skinned fibres prepared from the frog semitendinosus muscle (Podolsky & Costantin, 1964), was characterized by a rapid twitch-like contraction localized to the immediate site of calcium application. The duration of contraction was dependent on the amount of added calcium; with 1 mm-ca droplets less than 50,Ca in diameter, the entire contraction-relaxation cycle was completed within about 5 sec. In general, these contractions were fully reversible; perfusion with 10 mm calcium, however, which produced very strong local contractions, frequently resulted in a slight residual distortion of the sarcomere pattern at the site of application. The second type of response, which is shown in P1. 1, was characterized by a much slower contraction which spread extensively into the bulk of the fibre, even when the applied volume of calcium solution was confined to a small region of the fibre surface. When relatively small droplets of 1 mm calcium solution were applied, relaxation began within about 3 sec (P1. 1E); only partial relaxation occurred, however, and some residual shortening persisted for many minutes. Little or no relaxation was seen when larger amounts of calcium were applied. Correlation of response to calcium application with fibre structure. It appeared probable that the two different types of response to calcium

5 CALCIUM ACTIVATION OF SLOW FIBRES 265 application reflected the two discrete fibre types known to be present in the tonus bundle of the iliofibularis muscle (see Introduction). This possibility was examined directly by electron microscopic identification of a series of skinned fibres whose response to calcium had been characterized before fixation. Fourteen skinned fibres from four different frogs were studied. Five gave the first type of response (a twitch-like contraction) and nine the second. Electron microscopic identification of the fibres was carried out by one of us (L.W.T.) without knowledge of the physiological results. The criteria for identification of slow fibres were (1) the absence of an M-line, and (2) the relative scarcity of the sarcoplasmic reticulum (Peachey & Huxley, 1962; Page, 1965). In all cases, the fibres which gave a twitch-like response to calcium were found to be fast fibres and those which gave the slow response were slow fibres. Velocity of contraction of slow fibres. This was measured by applying a relatively large droplet (about 50,t in diameter) of a calcium solution to the surface of the skinned fibre and recording the resulting contraction on motion-picture film at 15 frames/sec. In an attempt to obtain full activation over an extensive region of the fibre, a calcium concentration of 3 mm was generally used; this is at least 10 times the lowest source concentration that will activate slow fibres (see below). The preparation was observed in polarized light with a large condenser aperture. The depth of field with this arrangement was about 5,u, and, by focusing very near the surface of the preparation, an optical section of the site of calcium application could be obtained. If shortening were rapid in comparison with the time required for diffusion into the bulk of the fibre, activation by surface calcium application would result in a dispersion of sarcomere lengths through the depth of the fibre as contraction was occurring. This effect is, in fact, observed with fast fibres, and when a fast fibre is studied by ordinary light microcopys, the superimposition of sarcomeres at different depths of the fibre precludes an accurate estimate of the rate of shortening. The shallow depth of field obtainable with a large numerical aperture in polarization optics permits the visualization of a small thickness of the preparation without interference from underlying sarcomeres. Plate 2 is a record of one of the experiments on a fibre whose response to calcium application characterized it as a slow fibre. As can be seen, the striation spacing in the contracting region appears relatively uniform along the fibre axis in each panel; this was a consistent finding in slow fibres and permitted an estimate of individual sarcomere length as the average of ten successive sarcomeres. Seven applications of calcium droplets were studied in three slow fibre preparations, all with initial sarcomere lengths greater than 3 g. The mean velocity of shortening was 1,/zsec. sarcomere with a range of 0-8,t-1-5 i/sec. sarcomere, so that shortening to a sarcomere length of 2,u, where the striation pattern is virtually obscured in polarization optics, required at least 1 sec in all fibres examined. The contraction

6 266 L. L. COSTANTIN AND OTHERS velocity was not noticeably changed when the calcium concentration in the applied droplet was increased from 3 to 30 mm. When the speed of shortening of fast fibres was examined in a similar fashion, a few sarcomeres at the site of application contracted vigorously before the applied droplet could spread widely over the fibre surface (P1. 3). Because of this, the mean sarcomere length in the contracting region was calculated, in these preparations, from measurement of only five successive sarcomeres. Seven applications of calcium were studied in four different fast fibre preparations with initial sarcomere lengths ranging from 2-8 to 3-6,u. In contrast to the results with slow fibres the mean sarcomere length in these preparations decreased by at least 1 #t within two motion-picture frames after droplet application (about 130 msec), and the velocity of shortening ranged from 6 to 12,u/sec. sarcomere. The failure of the droplet to spread over the fibre surface during the rapid contraction of fast fibres introduced an uncertainty in these values for shortening velocity. Since the droplet itself invariably obscured a portion of the contracting region (see arrow in P1. 3B), it was possible that the sarcomeres which were measurable in the film record were not those which shortened most rapidly. With this in mind, the value of 6-12 /t/sec. sarcomere can be regarded only as a lower limit; nevertheless, it is obvious that this is many times faster than the shortening velocity of slow fibres under similar conditions. Spread of contraction. As noted previously, the local application of a calcium droplet to a slow fibre results in an extensive contraction, in contrast to the local response seen in a fast fibre; this distinction, however, is to some extent obscured by the variable distribution of the applied volume of calcium-containing solution over the fibre surface. A more unequivocal demonstration of the difference between the two fibre types was obtained when a calcium-containing agar gel was applied to the lateral border of a preparation suspended in an oil bath. In this situation, there was no extensive bulk flow of liquid over the fibre surface, and the diffusion path of calcium from the applied source to the fibre was well defined. The 0-25 mm calcium normally present in the 1% agar gel produced a contraction in a slow fibre which, in comparison with a fast fibre, spread extensively from the site of application (P1. 4A and C). When 0*5 mm calcium was added to the agar, the contraction spread further in both slow and fast fibres, but again the depth of penetration of the contraction was much greater in the slow fibre preparation (P1. 4B and D). If the agar pipette remained in contact with the surface of a fast fibre for more than 3-4 sec, at least partial relaxation was seen, presumably because of a local decrease in the calcium concentration at the tip of the agar column. In slow fibres, however, more variable results were obtained. In some fibres, the contraction appeared quite stable for a few seconds, while, in others, although the radial extent of the contraction appeared stable, sarcomeres lateral to the site of agar application continued to contract until the pipette was removed. This slow increase in the extent of contraction was more marked when agar containing 0 5 mm added CaCl2 was applied and, on occasion, led to contraction of the entire skinned region.

7 CALCIUM ACTIVATION OF SLOW FIBRES.267 Relaxation in slow fibres. The extent of relaxation in slow fibres following calcium application was much more variable than in fast fibres. This was most readily observed in those experiments where the calcium was applied locally by agar pipettes. In fast fibres, even when as much as 2 mm-cacl2 had been added to the agar, prompt relaxation occurred when the agar was removed, leaving at most a slight distortion of the superficial 2-3,C of the fibre. In slow fibres, with only 0 5 mm added calcium, many preparations showed little or no relaxation and, on occasion, progressive shortening of the fibre continued even after removal of the agar. It seemed possible that this failure of the slow fibre to relax following calcium application was due to the persistence of calciuhl in the myofilament space. In an attempt to test this directly, slow fibres which had been made to contract by a brief application of agar with 0 5 or 1-0 mm CaCl2 added were perfused locally with a 30 mm EGTA solution (ethyleneglycol bis(aminoethylether) tetra-acetic acid titrated to ph 7-3 with KOH). In one of five fibres examined, EGTA elicited a brisk relaxation, while in the remaining four preparations little or no relaxation was seen. Since perfusion with EGTA alone might have lowered the ATP and Mg concentration of the sarcoplasm below levels required for relaxation (Bozler, 1954; Weber & Herz, 1963), similar experiments were performed on three additional preparations, using a solution known to act as a relaxing medium for fast fibres (3 mm EGTA, 5 mm ATP, 1 mm-mgcl2, 10 mm imidazole, 140 mm-kcl, ph 7 0 (Hellam & Podolsky, 1966)). Perfusion with this medium following a calcium-induced contraction resulted in relaxation in all three preparations. Localization of calcium-accumulating structures within slow fibres. The localization of calcium oxalate within the sarcoplasmic reticulum following the addition of sodium oxalate to skinned fast fibres has been taken as evidence that the reticulum serves as an intracellular calcium sink and thus controls relaxation (Costantin et al. 1965). In an attempt to see whether such a mechanism was present in slow fibres, similar experiments were carried out with fibres from the tonus bundle (see Methods). Electronopaque deposits confined to the lumen of the internal membrane system were readily demonstrated (P1. 6B). Since both the sarcoplasmic reticulum and the transversely oriented tubules which correspond to the T-tubules of fast fibres are composed of slender tubules in slow fibres (Page, 1965), it was not possible to decide within which of these elements the precipitate was located. Four preparations in which oxalate deposits were abundant were studied with respect to the relative localization of the deposits within the sarcomere. In one preparation, the deposits were predominantly in the I band region (P1. 5), as was the case in fast fibres, while, in two others, the deposits were more equally distributed between A and I band levels (P1. 6A).

8 268 L. L. COSTANTIN AND OTHERS In the fourth preparation, deposits were seen mainly at the I band level in one portion of the fibre, and no such localization was seen in micrographs from another portion of the fibre. Thus, although localization of oxalate deposits within the I band region was seen in slow fibres, this was not a consistent finding, as it is in the case of fast fibres. DISCUSSION Shortening velocity of intact and skinned muscle fibres. A valid comparison of the shortening velocity of intact and skinned fibres requires a knowledge of the load against which the fibre must shorten. In the present study, a small segment of fibre (5-15 sarcomeres in length) was activated, presumably to a maximal extent, by the application of a calcium concentration at least 10 times threshold to its surface. This active region in shortening must elongate the relaxed sarcomeres in series with it and, at least in the case of a fast fibre where the contraction did not spread throughout the fibre diameter, must also shorten, or distort, those segments of the relaxed myofibrils in parallel with the active region. Since the active region is but a small proportion of the entire length of the fibre, the passively stretching sarcomeres could be expected to represent a relatively light load. Similarly, the lateral coupling between myofibrils in both slow and fast fibres seems quite flexible, as can be seen by the sharp angulation of the striation pattern between contracted and relaxed regions in P1. 4, so that it seems likely that a locally activated skinned fibre actually contracts against a very light load. This conclusion is supported by the velocity of shortening obtained for skinned fast fibres. Buchthal & Kaiser (1951, p. 175) have reported that, for a single frog fast fibre at 260 C, shortening velocity with fractional loads of 0-2Po or less was 5-9LO/sec, where Lo0 measured with a load of 0-005PO would represent a sarcomere length close to 2,u. This speed of shortening, then, would be equivalent to about It/sec. sarcomere, a value close to the 6-12,i/sec. sarcomere found in fast fibres (at 23 C) in the present experiments. Complete data on the force-velocity relation in slow fibres from the frog iliofibularis are not available, but the experiments of Peachey & Huxley (1960) and Huxley & Taylor (1958) on local activation of slow and fast fibres by local membrane depolarization do offer a direct comparison of shortening velocity in these two fibre types. As in the present study, the load on the active region was presumably very small, since only one to three sarcomeres were actually activated. Measurements from films of these experiments show that the time required to obliterate one I band was about 7 times longer in slow as compared to fast fibres (L. D. Peachey, personal communication), a value which is quite similar to the results of the present study. It would appear, then, that the inherent velocity of shortening of slow fibres is much less than of fast fibres, whether the contractile

9 CALCIUM ACTIVATION OF SLOW FIBRES 269 mechanism is activated indirectly by membrane depolarization or directly by calcium application. It should be noted that the present results, while implicating the contractile mechanism as the cause of the slow contraction of slow fibres, give no information about the way the effect is produced. The most likely possibility is that in slow fibres the contractile mechanism simply generates motion more slowly than in fast fibres, either because the myofilaments interact at a slower rate or because the average displacement generated by each interaction is smaller. However, Page (1965) has reported that the thin filaments of slow fibres lack a regular array in the I band and that the hexagonal array of the thick filaments is occasionally found to be distorted; this disorder in the filament array could conceivably give rise to a viscous resistance to shortening which is not present in fast fibres, and thus account for a slow velocity of contraction. Spread of contraction following local calcium application. Although the difference in extent of contraction following local calcium application to slow and fast fibres is quite striking, the physiological basis for this difference is by no means clear. The effectiveness of locally applied calcium in inducing contraction will be determined by many factors, including the mobility of calcium in the fibre, the rate of calcium uptake and release by the myofilaments, the calcium threshold for contraction, and the rate of calcium removal by the sarcoplasmic reticulum, and no evidence is available concerning possible differences in most of these factors between slow and fast fibres. The relative sparseness of the sarcoplasmic reticulum in electron micrographs of slow fibres (Peachey & Huxley, 1962; Page, 1965), however, suggests that the rate of calcium removal by the reticulum should be much less in slow fibres. With this in mind, it seems possible that the extensive spread of contraction in slow fibres, as demonstrated in the present experiments, is in large part a reflexion of a less effective calcium pump. The authors are grateful to Dr L. D. Peachey for valuable discussions during the course of this work. REFERENCES ADRIAN, R. H. & PEACHEY, L. D. (1965). The membrane capacity of frog twitch and slow muscle fibres. J. Phy8iol. 181, BOZLER, E. (1954). Relaxation in extracted muscle fibers. J. gen. Physiol. 38, BUCHTHAL, F. & KAiSER, E. (1951). The rheology of the cross striated muscle fibre. Dan. Biol. Medd. 21, no. 7, COSTANTIN, L. L., FRANZINI-ARMSTRONG, C. & PODOLsxY, R. J. (1965). Localization of calcium-accumulating structures in striated muscle fibers. Science, N.Y. 147, COSTANTIN, L. L. & PODOLsKY, R. J. (1965). Calcium localization and the activation of striated muscle fibers. Fedn Proc. 24, COSTANTIN, L. L. & PODOLSKY, R. J. (1966). Depolarization of the internal membrane system in the activation of frog skeletal muscle. J. gen. Physiol. jin the Press.) HELTAM, D. C. & PODOLSKY, R. J. (1966). The relation between calcium concentration and isometric force in skinned frog muscle fibers. Fedn. Proc. 25, 466. HODGKiN, A. L. & HOROWICZ, P. (1959). The influence of potassium and chloride ions on the membrane potential of single muscle fibres. J. Phy8iol. 148, HUxLEY, A. F. & TAYLOR, R. E. (1958). Local activation of striated muscle fibres. J. Physiol. 144, i8 Physiol. i88

10 270 L. L. COSTANTIN AND OTHERS KUJFFLER, S. W. & VAUGHAN WILLIAMS, E. M. (1953). Properties of the 'slow' skeletal muscle fibres of the frog. J. Physiol. 121, NATORi, R. (1954). The property and contraction process of isolated myofibrils. Jikei Med. J. 1, PAGE, S. G. (1965). A comparison of the fine structures of frog slow and twitch muscle fibres. J. cell Biol. 26, PEAcHEY, L. D. (1961). Structure and function of slow striated muscle. In Biophysic8 of Phy8iological and Pharmacological Actions, ed. SHANEs, A. M. Washington: American Association for the Advancement of Science. PEACHEY, L. D. & HuxLrEY, A. F. (1960). Local activation and structure of slow striated muscle fibers of the frog. Fedn Proc. 19, 257. PEACHEY, L. D. & HuxLEY, A. F. (1962). Structural identification oftwitch and slow striated muscle fibers of the frog. J. cell Biol. 13, PODOLSKY, R. J. & CosTANTIN, L. L. (1964). Regulation by calcium of the contraction and relaxation of muscle fibers. Fedn Proc. 23, WEBER, A. & HERZ, R. (1963). The binding of calciium to actomyosin systems in relation to their biological activity. J. biol. Chem. 238, EXPLANATION OF PLATES PLATE 1 Calcium activation of skinned slow fibre. A. Portion of a skinned slow fibre before calcium application. B. About 70 msec following application of a 1 mm calcium droplet 35 It in diameter to fibre surface. C-F. 1, 2, 3 and 8 sec after calcium application. Maximum contraction occurred 2 sec after calcium application (panel D). Relaxation was still incomplete after 8 sec (panel F). The horizontal line in each panel marks five successive sarcomeres. It can be seen that shortening occurs throughout the fibre diameter in the region of calcium application. Ordinary light. Grid spacing = 10 l,. PLATE 2 Velocity of shortening of a skinned slow fibre. A. Portion of a skinned slow fibre before calcium application. Average sarcomere length = B. 05 sec after application of a 3 mm calcium droplet 55 It in diameter to fibre surface. Sarcomere length in contracted region = 3 0,u. C. 1-0 sec after calcium application. Sarcomere length = 2-4,u. D. 1-5 sec after calcium application. Sarcomere length = 2-2 Pt. E. 2-0 sec after calcium application. Sarcomere length = 2-0 gz. F. 2-5 sec after calcium application. Sarcomere length = 1 9 p. The horizontal line in each panel marks five successive sarcomeres. Polarized light. Grid spacing = 10,t. PLATE 3 Velocity of shortening of a skinned twitch fibre. A. Portion of a skinned twitch fibre before application of calcium. Average sarcomere length = 3-4 /%. B. About 130 msec after application of a 3 mm calcium droplet 60It in diameter to fibre surface. Sarcomere length in contracted region = 2-2 It. The arrow indicates the applied droplet, which is still visible on the fibre surface and which partially obscures the underlying sarcomeres. The horizontal line in each panel marks five successive sarcomeres. Polarized light. Grid spacing = 10 /t.

11 l l l l l C~~~ The Journal of Physiology, Vol. 188, No. 2 Plate 1 W~min. dwdlffiwft".4...w L. L. COSTANTIN, R. J. PODOLSKY AND LOIS W. TICE (Facing p. 270)

12 ~~~~~~C The Journal of Physiology, Vol. 188, Nto. 2 A ;, 1w1T7W1F1r.jrT: k 4.. :~~~~~~~A. _Sli_Dl S_.ili *St i W r w B E r..1 _ i Plate 2 D U F L. L. COSTAN-TIN, R. J. PODOLSKY AND LOIS W. TICE

13 The Journal of Physiology, Vol. 188, No. 2 Plate 3 L. L. COSTANTIN, R. J. PODOLSKY AN-D LOIS W. TICE

14 The Journal of Physiology, Vol. 188, NYo. 2 Plate 4 IL NA L. L. COSTANTIN, R. J. PODOLSKY AND LOIS W. TICE

15 The Journal of Physiology, Vol. 188, No. 2 Plate 5 <34 ' - E tt'.: 01: L. L. COSTANTIN, R. J. PODOLSKY AND LOIS W. TICE

16 The Journal of Physiology, Vol. 188, No. 2 Plate 6 L. L. COSTANTIN, R. J. PODOLSKY AND LOIS W. TICE

17 CALCIUM ACTIVATION OF SLOW FIBRES 271 PLATE 4 Extent of contraction following local calcium application. A. Skinned slow fibre 2 sec after application of agar containing 0-25 mm calcium. B. Another skinned slow fibre 3 sec after application of agar containing 0-75 mm calcium. C. Skinned fast fibre 1 sec after application of agar containing 0-25 mm calcium. D. Another skinned fast fibre 2 sec after application of agar containing 0 75 mmr calcium. In each panel the maximum depth of penetration of the calcium-elicited contraction is shown; the black spots mark the apparent extent of the actively contracting region. Ordinary light. Grid spacing = 10 ps. PLATE 5 Electron micrograph of a skinned slow fibre doubly fixed in glutaraldehyde and osmium after treatment with 10 mm calcium solution followed by 10 mm-na oxalate. Sections cut into 1 mm Na oxalate. Electron-dense deposits of Ca oxalate are found predominantly in the I band region. PLATE 6 Slow fibre prepared in the same manner as in P1. 5. A. Precipitates are found both at the level of A and I bands. In a few locations some extraction of precipitate from the section has occurred (arrows). B. In this preparation the internal membrane system was distended; however, many precipitates of Ca oxalate can be seen lying within or in close relation to the inner surface of the membranes. I8-2