Penetration of horseradish peroxidase into the terminal cisternae of frog skeletal muscle fibers and blockade of caffeine contracture by Ca++ depletion

Z. Zellforsch. 124, 57-71 (1972) �9 by Springer-Verlag 1972

Penetration of Horseradish Peroxidase into the Terminal Cisternae of Frog Skeletal Muscle Fibers and Blockade of Caffeine Contracture by Ca ++ Depletion*

RAI~A~L RIZBIO and NICK SPER:ELAKIS Department of Physiology University of Virginia, School of Medicine

Charlottesville, Va., U.S.A.

Received July 26, 1971

Summary. Horseradish peroxidase, an extracellular marker, was given intravenously to frogs, and 40 min later the sartorins muscles were removed. The isolated muscles were exposed for an additional hour to Ringer solution containing peroxidase, then fixed with glutaraldehyde. Peroxidase activity was found in the T tubules, in some of the terminal cisternae (TC) of the SR, and occasionally in the longitudinal tubules of the SlY. In transverse sections, the structures containing tracer formed a pattern of approximately parallel columns reaching to the cell surface; the statistical distribution of their spacing was nearly the same as that of the interdistances between the current-sensitive spots on the Z-line which triggered localized contraction (Huxley and Taylor, 1958). The caffeine contracture of frog sartorius muscles remained unchanged in isotonic Ringer solutions which were Ca++-free or contained Mn ++ or La+++; however, contracture was blocked by prior exposure of the muscles to the same solutions made 2 • hypertonic with sucrose (known to produce swelling of T tubules and TC). Since Mn ++ and La +++ are known to depress Ca ++ influx, these results suggest that washout of Ca +§ from the TC, and penetration of La +++ or Mn ++ into it, occur more rapidly due to the swelling of T tubules and TC associated with hypertonicity. It is concluded that at least some of the terminal cisternae are open to the interstitial fluid via the T tubules. Thus, depolarization of the T tubules could readly depolarize the cisternae and lead to Ca ++ influx into the myoplasm.

Key words: Muscle extracellular compartments - - Hypertonicity effects - - Excitation- contraction coupling - - Terminal cisternae - - Caffeine contracture.

Introduct ion

The lateral cisternae and the longi tudinal tubules of the sarcoplasmic re t iculum (SR) of skeletal muscle are generally considered to be intracel lular compar tments no t cont inuous with the extracellular space (Porter and Palade, 1957; Peachey, 1965 ; Huxley, 1964; Peachey and Schild, 1968; Eisenbcrg and Eisenberg, 1968). However, Luft (1967) found tha t r u then ium red, used as an electron-opaque marker, entered the T tubules and lateral cisternae of mouse diaphragm muscle exposed to the tracer during the period of fixation. Although other studies with other markers demonst ra ted entrance into the I band, the techniques used did no t have sufficient spatial resolution to determine where the marker was located wi thin the I band (Endo, 1966; Hill, 1964). I n addit ion, several physiological observations (see Discussion) suggest t ha t the fluid in the lateral cisternae exchanges relat ively freely with the inters t i t ia l fluid. Previous studies with either

* Supported by grants from the Public Health Service (HE-11155, HE-05815, and HE-10384) and from the American Heart Assocation. The authors are indebted to Mrs. Jan Redick for expert technical assistance.

58 R. Rubio and N. Sperelakis:

horseradish peroxidase or ferri t in (Huxley, 1964; Peachey and Schild, 1968; Eiscnberg and Eisenberg, 1968) have demonst ra ted the presence of the tracer in T tubules bu t no t in the cisternae. However, the rate of entrance of an extracellular marker into a compar tment is a funct ion of its size (Tasker et al., 1959; Bozler, 1961). For example, inul in takes over 1 hr to equil ibrate in frog sartorius compared to a much shorter t ime for sucrose. Thus, it is possible t ha t in the previous studies with peroxidasc, the muscles were not exposed sufficiently long to allow these relatively large molecules to penetra te into the cisternae. There- fore, we decided to reinvestigate this problem in frog sartorius with horseradish peroxidase as an extracellular marker, and using the degree of caffeine contracture as an index of the Ca ++ influx from the cisternae into the cytoplasm. Peroxidase was found in the tubu]es and in the lateral cisternae, and caffeine contracture was abolished by prior exposure of muscles to the following hypertonic solutions (known to produce swelling of the T tubules and SR): (a) Ca++-free, (b) con- t a in ing Mn ++, and (c) containing La +++.

Material and Methods

Horseradish Peroxidase Tracer Experiments. The experiments were performed on 4 frogs (Rana pipiens) during April and May. Horseradish peroxidase (type II, Sigma Chemical Co.) was injected into the abdominal vein of each frog (70-90 g weight; dose of 10 rag/100 g body weight dissolved in 0.3 ml Ringer solution). The sartorius muscles were removed after 60 min. Thereafter, all of the 8 muscles (mounted on glass rods at their resting length) were soaked in peroxidase-eontaining (0.5 mg/ml) oxygenated frog Ringer solution for an additional hour. In the case of 1 muscle, the peroxidase solution was made 3-fold hypertonic (0.90 osMolar) by addition of excess NaC1. The muscles were fixed with 1.5 % glutaraldehyde in cacodylate buffer (pH 7.4) for 20 min at 23 ~ C. The histochemical localization of peroxidase was carried out according to the protocol of Forssmann (1969), and only the most superficial fibers in each muscle were examined. After alcoholic dehydration, the tissue blocks were embedded in Epon, and sectioned at a thickness of about 700 A. The sections were either stained with lead citrate only or left unstained (see Discussion), and examined with a Zeiss model EM-9A electron microscope.

Ca//eine Contracture Experiments. The effects of previous exposure of muscles to hypertonic Ringer solutions (by sucrose addition) either Ca++-free or containing Mn ++ of La +++ on the caffeine-induced eontracture were studied. The Ringer solution had the following composition (in raM): 111.2 NaC1, 1.88 KC1, 1.08 CaCl 2, 2.38 NaHCO~, and 0.10 NaH2PO4.2H20. The hypertonic Ringer solutions were made two times isotonicity by addition of sucrose (300 m-osM). The Ca++-free Ringer was prepared by omitting the CaC12 . All solutions were gassed with air (pH of 7.4). For the Mn ++ (5 mM MnCl 2) and La +++ (1.5 mY[ LaCI3) experiments, the Ringer composition was kept the same except that the bicarbonate and phosphate were substituted by Tris-HCl to prevent precipitation, and the pI-I was adjusted to 7.4. The sartorius muscles from each frog were paired (26 pairs), one being exposed to hypertonic Ringer solution and the other to hypertonic experimental solutions for periods of 20 or 40 min. The experimental solutions were (a) Ca++-free, (b) Mn++-contaning, and (c) La+++-eontaining. Caffeine (5 10 mM) eontracture was always induced while the muscles were equilibrated in isotonic Ringer at about 23 ~ C. All contractures were recorded isometri- cally with a force transducer and penwriter at the resting muscle length.

Results

Cellular Localization o/Horseradish Peroxidase

Peroxidase ac t iv i ty was observed in most of the T tubules, less f requent ly in the terminal (lateral) cisternae, and occasionally in the longi tudinal tubules of the SR. Longi tudinal sections are i l lustrated in Figs. 1-4. Typically, the peroxi-

Tracer Penetration into Terminal Cisternae 59

Fig. 1. Longitudinal section of frog sartorius muscle fiber showing horseradish peroxidase (HP) activity in T tubules (TT) and in one of the terminal cisternae (TC) (long arrow). Other arrows point to branches of T tubules running longitudinally. A A-band; 1 I-band; Z Z line.

M M line. Section stained with lead. X 18500

dase a c t i v i t y is loca ted : (a) ma in ly in the T tubules (Fig. 1) and (b) in T tubules and m a n y of the t e rmina l cis ternae (TC) of the SR (Fig. 2). F o r example , in Fig. 2, 24 out of the to ta l of 54 cis ternae visible are filled wi th t racer . Fig. 3


Fig. 2 A-C


illustrates, at higher magnification, a T tubule and one of its adjacent terminal cisternae filled with tracer, and shows the dimples characteristic of the junction between T tubules and terminal cisternae (Kelly, 1969). In some fibers from the muscle exposed to hypertonic Ringer, almost every terminal cisterna was filled with the extracellular tracer (Fig. 4). Occasionally, peroxidase was also observed in the longitudinal SR at the level of the A band. Branches of T tubules tha t run axially in the muscle fiber were observed (Figs. 1-3, arrows), as has been described by others (Peachey, 1965; Huxley, 1964; Peachey and Schild, 1968; Eisenberg and Eisenberg, 1968); sometimes these longitudinal branches are very tortuous. Such a high degree of tortuosity is observed only occasionally, as expected in thin tissue sections.

The results obtained in cross section are illustrated in Fig. 5. Peroxidase-filled structures are located at the level of the I band, as indicated by the presence of thin filaments only (Fig. 5A). The structures containing the peroxidase activity tend to surround the myofibrils, and tortuous tubules which are filled with tracer interconnect adjacent wider structures (Fig. 5A, arrows). As demonstrated in the longitudinal sections, some of the structures containing the tracer in these cross sections probably correspond to the cisternae. Other structures having peroxidase activity may correspond to T tubules which lie over or under the cisternae, thereby giving the appearance that the marker is inside the SR; this is probably the case when the tracer incompletely fills a cisterna. Occasionally, peroxidase is also found in the SR at the level of the A band. The sites containing the tracer are aligned in a columnar fashion projecting toward the surface of the cell (Fig. 5 B). This pat tern in the spatial distribution of peroxidase suggests that there is a common channel, possibly the T tubule orifice, for entrance of the marker. That is, one column may be filled by one T tubule. Adjacent columns are approximately parallel to each other. The average distance separating the columns (measured for each pair of adjacent columns a t three different depths in the fiber from the mid-point of one column to the mid-point of its neighbor) is 4.6 ~. The data from 37 fibers (266 values grouped into intervals of 0.6 ~) are plotted in Fig. 6 (dotted line).

Ca//eine Contracture in Muscles Pre-Exposed to Hypertonic Media

The caffeine contracture of skeletal muscle is unaffected by exposing the muscles to isotonic Ca++-free media (see Bianchi, 1968), or to isotonic La +++- containing (see Weiss, 1970) and Mn++-containing Ringer solutions. However, the caffeine contracture of frog sartorius muscles immersed in isotonic Ca++-free Ringer was blocked reversibly by prior exposure of the muscles to hypertonic Ca++-free Ringer (made 2 x isotonic with sucrose) (Fig. 7A-C). Similarly, the

Fig. 2. A: Longitudinal section of frog sartorius fiber showing horseradish peroxidase (HP) activity in T tubules (TT), and in some of the terminal cisternae (TC). Arrows point to branches of the T tubules running longitudinally. B: Pinocytotic vesicles (Ves) at the sarcolemma filled with peroxidase activity. C: Higher magnification of two triads, one with both cisternae filled and the other with only one cistcrna filled. ISF, interstitial fluid; other labels

as in Fig. l. A, x 15400; B, x 25900; C, x 77600

62 R. Rubio and N. Sperelakis : Trucer Peneration into Terminal Cisternae

Fig. 3. Longitudinal section showing horseradish peroxidase (HP) activity in T tubule (TT) and in one of two adjacent terminal cisternae (TC). Arrows point to branches of T tubules

that run axially. SR, sarcoplasmic reticulum. • 53 600

caffeine cont rac ture of muscles immersed in isotonic Mn++ or La+++-containing Ringer was blocked by pr ior exposure of the muscles to the corresponding solut ion made hyper ton ic wi th sucrose (Fig. 7 D, E). I n the case of the Ca++-free exper iments , the magn i tude of the inhibi t ion was a funct ion of the per iod of exposure to hyper ton ic solut ion (Fig. 7B vs C). The inhib i t ion was reversed by add i t ion of Ca ++ (Fig. 7C).

The caffeine cont rac ture of pairs of muscles (protocol followed summar ized a t the b o t t o m of each panel) are i l lus t ra ted in Fig. 7. I n A, bo th the control

Fig. 4. A: Longitudinal section of a fiber from muscle exposed to 3 x hypertonie Ringer (NaCI) showing almost every terminal cisterna filled with peroxidase. B : From same section as in A (boxed-in area upper left) bu t shown a t higher magnification. A, x 4600; B, x 25900


Fig. 5 A and B


20

I0 ,.~

O-

i ~ ~ From: Huxley &Taylor, J. Physlol. 144. ~gU) N--49; Z fibers, O.|~ ~a inter val$.

dis tances

Fig. 6. Histograms showing similar statistical distribution of distances separating adjacent columns filled with peroxidase (broken line) and of distances separating circumferential current-sensitive sites on Z-line (continuous line). Ordinate gives percent of total observations made; abscissa 8ires distances in ~z. Arrows point to average values. Diagram at right repre-

sents single muscle fiber with micropipette placed externally on Z-line (see text)

(trace label led C) and exper imen ta l muscles ( trace label led E) were exposed to hyper ton ic Ringer for 40 rain, b u t the exper imenta l muscle was f irst ba thed in hyper ton ic Ca++-ffee Ringer for 20 rain and then t ransfer red to hyper ton ic Ringer conta ining Ca ++ for 20 min, whereas the control muscle was in hyper ton ic Ringer conta ining Ca ++ for the ent i re 40 min. Fol lowing the per iod of hype r ton ic i ty , bo th muscles were t ransfer red to isotonic Ringer conta in ing Ca ++ for 20 rain, and then caffeine con t rac tu re was induced. The caffe ine-contractures of bo th muscles were very similar, thus ind ica t ing t h a t exposure to hyper ton ic Ca++-free Ringer does no t i r revers ib ly affect the response to caffeine. I n B, the control and exper imen ta l muscles were exposed to hyper ton ic Ringer for 20 min, and then isotonic Ringer for 20 rain; for the exper imenta l muscle, these solut ions d id no t conta in Ca ++. The cont rac tures of the exper imenta l muscles were smal ler t han those of the control muscles. I n C, bo th muscles were exposed to hyper ton ic Ringer for 40 min, and then isotonic Ringer for 20 min. These control muscles were t r e a t e d

Fig. 5. A: Transverse section of fiber illustrating horseradish peroxidase (HP) activity in structures located at level of /-bands. These structures possibly correspond to terminal cisternae (TC) and T tubules above or below the cisternae. Arrows point to tortuous tubules filled with tracer that interconnect adjacent wider structures containing marker. I-bands identified by absence of thick myofilaments. Plane of sectioning passed through Z line of some myofibrils; peroxidase activity is intense in these regions. B: Transverse section of fiber illustrating the approximately parallel columnar pattern of the sites containing peroxidase activity. One of the columns delineated by dotted lines. Peroxidase tracer is usually

found in SR at level of I-bands only. A, • 16900; B, • 4300

5 Z. Zellforsch., Bd. 124


t IOmM.

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t 8mM

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Fig. 7 A-F. Depression of caffeine contracture of sartorius muscles by Ca++-free solutions or by addition of La +++ or Mn ++ facilitated by prior exposure to hypertonic solutions. Traces labelled C and E represent control and experimental muscles, respectively. The protocol followed in each panel is summarized at bottom of that panel: 2 x and 1 x represent 2 x isotonicity and isotonicity; -- or q- represent Ringer solution without or with Ca ++ or addition of La +++ or Mn++; the time of exposure to each solution is given in min, and the sequence of exposure is given from left to right. At first arrow in each panel, caffeine (concentration as indicated) was added. A: Caffeine contracture of the experimental muscle was same as that of control. B: Depressed caffeine contracture; only 20 min exposure to hypertonic solution. C: Caffeine contracture almost completely abolished by longer exposure to hypertonic solution (40 rain) ; addition of Ca ++ (7 raM) at second arrow resulted in contracture. D-E : The caffeine contractures of E muscles were almost completely abolished by addition of 1.5 mM La+++ (D) or 5 mM Mn ++ (E). F: Diagram represents cross-section of skeletal muscle fiber with T tubules (TT) giving rise to tortuous branches opening into the TC. Arrows indicate movement of Ca ++ from TC to myoplasm during membrane activation. Broken line represents muscle fiber during exposure to hypertonic medium; decrease in fiber volume and concomitant swelling of TT and TC should facilitate diffusion and lead to more rapid exchange of TC fluid with ]SF

ident ical ly as those in A ; the exper imenta l muscles were placed in hyper tonic

Ringer containing Ca § for 20 rain, hyper tonic Ca++-free Ringer for 20 min, and

then isotonic Ca++-free Ringer for 20 min. The caffeine contracture of the experi- menta l muscles was a lmost completely blocked. However , addi t ion of Ca ~+ pro-

duced contracturc. Thus, a l though the exper imenta l muscles of both B and C

were exposed to a Ca++-frec medium for 40 rain, the muscles in C were preceded by a 20 min period in hyper tonic Ringer containing Ca ++ whereas those in B

were not. Therefore, the effect of Ca++-free solution was increased by prolonging


the period of exposure to hypertonicity. Since muscles exposed to isotonic Ca ++- free Ringer for 1 hr gave caffeine contractures similar to those of the control muscles (not illustrated), a period of exposure to hypertonicity is necessary to demonstrate an effect of Ca++-free solution on caffeine contracture. Some effects of hypertonicity on the caffeine contracture of single muscle fibers (Caputo, 1966) and on the contracture of whole muscle exposed to Ca++-free solution for prolonged periods (Gebert, 1968) have been described.

Measurements of the Ca ++ content (by atomic absorption spectrophotometry of ashed muscles) of four pairs of muscles treated as those in Fig. 7 but not given caffeine gave a mean values of 3.2 ~moles/g tissue (range of 4.8 to 2.4) for the controls and a mean of 2.0 ~moles/g (range of 2.6 to 1.5) for the experimental muscles. The Ca ++ content of the experimental muscles averaged 64 % of that of the control muscles. In all cases, the Ca ++ content of the experimental muscle was less than that of its control muscle. Therefore, exposure to a Ca++-free solution which is also hypertonic partially depletes the muscle of its Ca++ content.

Blockade of the caffeine contracture of muscles tha t were previously exposed to hypertonic Ringer solutions containing La +++ or Mn++is illustrated in Fig. 7 D-E. The control and experimental muscles were exposed to hypertonic Ringer without La +++ or Mn ++ for 20 rain. Then the control muscle was transferred to isotonic Ringer for 10 min and then to isotonic Ringer containing La +++ (D) or Mn ++ (E) for 50 min; the experimental muscle was transferred to hypertonic Ringer containing La +++ or Mn ++ for 25 min and then placed in isotonic Ringer containing La +++ (D) or Mn++ (E) for 25 rain. Thus, both the experimental and control muscles were exposed to La +++ or Mn ++ for 50 min, but the experimental muscles were preceded by a period in hypertonic Ringer containing La +++ or Mn ++. The caffeine contracture of the experimental muscles was almost completely blocked. Since this difference between the caffeine contracture of the control and experimental muscles was observed even when the final period in isotonic Ringer containing La +++ or Mn++ lasted 2.5 hr, a period of exposure to hypertonicity is necessary to demonstrate the blocking effect of La +++ and Mn ++ on the caffeine contracture.

Discussion

The presence of horseradish peroxidase, an extracellular tracer, in some of the compartments that morphologically are defined as the lateral cisternae, suggests tha t a t least a fraction of the cisternae communicates freely with the interstitial fluid, presumably via the T tubules. The complex T-tubular branches tha t run axially to the muscle fiber may open into the cisternae, thus constituting the communicating channels with the cisternae. This communication occurs even under normal isotonic conditions, although many of the connections may be opened only in hypertonic solutions. That is, under normal physiological conditions, some of the connections between cisternae and T tubules may be closed par t of the time, particularly since prolonged exposures to isotonic Ca++-free, Mn ++, or La +++- Ringer do not abolish the caffeine contracture.

The present results appear to be in disagreement with previous studies in which marker was found in the T tubules but not in the cisternae (Huxley, 1964; Peachey and Schild, 1968 ; Eisenberg and Eisenberg, 1968). However, our protocol

5*

68 R. l%ubio and N. 8perelakis:

was different in several ways: (1) A "f luffy" coat of a gel substance may form and surround fibers in isolated muscles and depress diffusion of tracer substances ; therefore, the muscles were exposed to peroxidase both in situ and after isolation for a total period of 2 hr. (2) The usefulness of peroxidase as a tracer depends on its enzyme activity which is impaired by glutaraldehyde (Forssmann, 1969), and any reduction of enzymatic activity at regions where the enzyme is in low concentration may yield negative results; therefore, both the period of exposure to the fixative and the concentration used were minimized. (3) Opaque material indicating the presence of peroxidase was removed during staining with uranyl acetate, particularly at sites where it was not heavily concentrated; therefore, our sections were either left unstained or stained with lead citrate only.

The T tubules, cisternae, and SR swell when the muscle is exposed to hypertonic media (Birks and Davey, 1961; Sperelakis and Schneider, 1968; Huxley et aI., 1963). The diagram in Fig, 7F represents a hypothetical skeletal muscle fiber in cross section with T tubules giving rise to tortuous branches opening into the eisternae. If a muscle is exposed to hypertonic solution, the cell shrinks and the cisternae and T tubules swell. Thus, if the cisternae are continuous with the ISF, diffusion would be facilitated and depletion of their Ca ++ content or entrance of Mn +++ and La +§ into them should occur more rapidly. Caffeine, which is thought to induce muscle contraction by releasing Ca ++ from the cisternae (Bianchi, 1968), has a negligible effect under conditions when Ca ++ movement down its electrochemical gradient is prevented by Ca ++ depletion or by presence of La+++ or Mn +§ (Weiss, 1970). Thus, pre-exposure to hypertonic media either Ca++-free or containing La +++ or Mn++, followed by their corresponding isotonic media, should reduce the influx of Car+ from the eisternae into the cytoplasm, thereby causing a decrease in the caffeine eontraeture (Fig. 7). The im- portance of hypertonicity is emphasized by the fact that the caffeine contracture is not decreased in muscles exposed to isotonic Ca++-free Ringer for 60 min or in Mn ++ or La+++-Ringer for 2.5 hr. The lower Ca ++ content of the muscles exposed to hypertonic Ca++-free Ringer, is consistent with the assumption of Ca ++ depletion of the eisternae. When the total exposure period to hypertonic medium was prolonged, a more complete washout of Ca ++ from the cisternae may occur and therefore a smaller caffeine eontracture (Fig. 7 B vs C) because the T tubules and cisternae are already dilated when exposed to the Ca++-free medium. Depending on the relationship between tension and [Ca++]TC, washout of Ca ++ from the cisternae may need to be nearly complete in order to diminish contraction significantly; similarly, the return of caffeine contracture upon re-introduction of Ca ++ would be rapid if low [Ca++]T c were sufficient to give nearly maximal contraction. In agreement with our view, hypertonicity causes a faster onset and rate of tension development of the caffeine contraeture of single fibers (Caputo, 1966), suggesting that caffeine has a more rapid access to its site of action.

Since no tracer was found in the cytoplasm (Huxley, 1964; Peachey and Schild, 1968; Eisenberg and Eisenberg, 1968), the T tubules are the preferential sites of entrance. Depolarizing current pulses applied through a micropipette in contact with the Z-line caused a localized contraction of the two adjacent half- sareomeres in frog muscle fibers (Huxley and Taylor, 1958). Only some spots around the circumference of the fiber at the Z line were sensitive to current, and


the solid line in Fig. 6 gives the statistical distribution of the distances (grouped into intervals of 0.55 ~z) which separated the sensitive spots from the work of these authors. The average separating distance was 4.8 ~z. Comparison of both histograms shows a striking congruence, and suggests that when localized depolarizing current is applied, this current flows through the membranes of the structures comprising the columns. That is, the current-sensitive spots may represent the opening of the columns to the outside.

The mechanism by which the electrical signal is transmitted from the T tubules to the lateral cisternae, causing the release of calcium into the myoplasm, has been a matter of speculation (Walker and Schrodt, 1968). For example, it has been suggested that the evaginations of the lateral cisternal wall toward the T-tubular membrane may represent low-resistance pathways for current flow (Peachey, 1965). However, it is doubtful whether there is a close junctional relationship between the two apposing membranes (Kelly, 1969); in addition, the measured area of contact between the T-tubular and TC membranes is thought to be too small to have functional implications (Franzini-Armstrong, 1970). If there is continuity of the T tubular membrane with the cisternal membrane, then in the normal process of excitation, the action potential may be transmitted inward by the T tubules (Huxley and Taylor, 1958) and the electrical signal directly delivered to the cisternal membrane. Depolarization of the cisternal membrane, if it produced an increase in Ca++ permeability, would lead to Ca ++ influx into the cytoplasm and thereby trigger contraction. I t is interesting that Natori (1965) observed an electrical potential change in a bundle of myofibrils during propagated contractions; this electrical signal may represent the activation of the T tukule- cisternal system. SR is implicated in lowering myoplasmic Ca ++ (Ebashi and Lipmann, 1962; Costantin et al., 1965; Winegrad, 1968), and these structures may equilibrate their Ca ++ content with the interstitial space.

The following observations are also consistent with and support the conclusion concerning the free diffusion of ions and larger molecules between the cisternae and T tubules. (1) The capacitance of skeletal muscle fibers, the largest portion of which is attributed to the T tubular system (Fatt, 1964; Falk and Fatt , 1964; Eisenberg and Gage, 1967), is sensitive to changes in tonicity of the surrounding fluid (Fatt, 1964). (2) In voltage-clamped frog skeletal muscle fibers immersed in hypertonie media, the variation of the K + equilibrium potential (EK) with the estimated amount of outward K + current suggests that K+ accumulates in a space of 1/3 to 1/6 of the fiber volume (Adrian et al., 1970). If K+ were to accu- mulate in the T tubules only (1/300 of the cell volume), then much greater variation in EK would be expected. The volume of the SR in hypertonic solution (3.5 • isotonicity) is about 1/6 of the fiber volume (Birks and Davey, 1969). (3) There is a linear relationship between estimated muscle cell volnme and the reciprocal of the osmotic pressure of the surrounding fluid; however, cell volume changes calculated from optical measurements of diameter (Blinks, 1965) give a different slope from that calculated from weight and sucrose space determina- tions (Dydynska and Wilkie, 1963). At isotonicity, the estimated aqueous portion of the cell from the optical method yields a value of 67% compared to 80% from the other method, a difference of 13% (Blinks, 1965). The difference between these two curves is a straight line, having a small negative slope, which may


represent the volume of the SR because i t super imposes on the inverse re la t ionship between the volume of the SR and the reciprocal of the ton ic i ty (Birks and Davey , 1969); this was in t e rp re t ed as ind ica t ing t h a t the SR is accessible to sucrose and therefore is an ext race l lu lar space. (4) To expla in the a p p a r e n t dis- crepancies in the d i s t r ibu t ion and kinet ics of Cl-, Na +, and K + in skele ta l muscle (Shaw et al., 1957; Simon et al., 1957), a t w o - c o m p a r t m e n t cellular model was proposed b y Har r i s (1963). One c o m p a r t m e n t admi t s the three ions as well as HCO~ and me thy l sulphate , contains 15% of the ceil water , has a low Q10 for C1- loss, and is insensi t ive to the ex te rna l concent ra t ion of C1- and o ther anions; the second c o m p a r t m e n t contains K + and CI-, b u t N a § is excluded. I t has also been proposed t h a t N a + is conta ined in two in t race l lu lar c o m p a r t m e n t s (Conway, 1957; Rogus and Zierler, 1970). One c o m p a r t m e n t exchanges slowly, is sensit ive to t e m p e r a t u r e and ouabain, and contains the smaller f rac t ion of the to ta l cellular Na+. The o ther c o m p a r t m e n t exchanges r ap id ly wi th the I S F via the T tubules , is insensi t ive to ouabain , has a N a + concent ra t ion close to t h a t of the I S F , and has a f rac t ional cell volume of 13% which decreases l inear ly with the reciprocal of osmotic s t rength ; i t was suggested t h a t th is c o m p a r t m e n t represents the SR (Rogus and Zierler, 1970, and personal communicat ion) .

References

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muscle fibres. J. Physiol. (Lond.) 177, 42-57 (1965). Bozler, E. : Distribution of non-electrolytes in muscle. Amer. J. Physiol. 200, 651-655 (1961). Caputo, C. : Caffeine- and potassium-induced contractures of frog striated muscle fibers in

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ions in skeletal muscle. Physiol. Rev. 37, 84-132 (1957). Costantin, L. L., Franzini-Armstrong, C., Podolsky, R. J.: Localization of calcium-accumu-

lating structures in striated muscle fibers. Science 147, 158-159 (1965). Dydynska, M., Wilkie, D. R. : The osmotic properties of striated muscle fibers in hypertonic

solutions. J. Physiol. (Lond.) 169, 312-329 (1963). Ebashi, S., Lipmann, F. A. : Adenosine triphosphate-linked concentration of calcium ions

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Dr. Rafael Rubio Department of Physiology School of Medicine University of Virginia Charlottesville, Virginia 22903 U.S.A.

Documents

Penetration of horseradish peroxidase into the terminal cisternae of frog skeletal muscle fibers and blockade of caffeine contracture by Ca++ depletion