Contractile activation in skeletal muscle

Pmg. Biophys. M&c. Biol. 1975. Vol. 29, No. 2. pp. 197-224. Pergamn Press. Printed in Great Britain

CONTRACTILE ACTIVATION IN SKELETAL MUSCLE L. L. C~TAN-I~N Department oj Physiology and Biophysics, Washington University School of Medtcine, St. Louis, 63110, USA

CONTENTS I.

INTR~DU~~ON

199

II. ANAKIMY OF THE I NTERNAL M EMBRANE S YSTEM 1. Comparative Anatomy of the internal Membrane System

III.

R ESTING E LECTRICAL P ROPERTIES 1. Ionic Permeability

OF THE

I NTERNAL M EMBRANE SYSTW

2. Ion Accumulation and Depletion in the T-System (a) Inward-going rectification 3. The EffPectioe Capacity of the Muscle Ceil (a) The equivalent circuit of the frog twitch fibre (b) Conduction velocity of the action potential 4. Properties of the Internal Membrane System in Other Species 5. The Detubulated Muscle

IV.

E XCITATION IN S KELETAL MUSCLE 1. Spread of Depolarization Along the Surjiace Membrane

(a) Twitch fibres (b) Slow jbres

199 200 201 201 201 202 203 203 205 205 205 206 206 206

(c) Invertebrate muscle

V.

I NWARD S PREAD OF A CTIVATION 1. T-System Conductance Changes in Frog

Twitch Fibres

(a) Tubular sodium depletion (b) Tubular potassium accumulation (c) Calculation of the propagated action potential in jog twitch jbres

VI. THE M ECHANISM OF ACTIVATOR RELWSE 1. Magnitude of Calcium Release in a Twitch

VII.

207 208 208 209 210

2. Membrane Potential and Calcium Release (a) Gradation of the calcium release process (b) Inactivation and repriming of the calcium release mechanism (c) The kinetics of contractile activation 3. Electrical Events Associated with Contractile Activation (a) Time course of the charge movement 4. Contractile Activation in Skinned Muscle Fibres (a) Depolarization of the internal membrane system (b) Calcium-induced calcium release (c) Relevance of skinnedfibre results to activation of intact muscle 5. Contactile Activation Without Membrane Depolarization 6. Pharmacological Alterations of Contractile Activation (a) Dantrolene and formaldehyde (b) Twitch potent&ion (c) Caffeine contractures (d) Hypertonic solutions

210 210 211 211 212 213 214 215 216 216 216 217 217 218 218 218 219 219

C ONCLUSIONS

220

R EFERENCES

220

197

CONTRACTILE

ACTWATION

IN SKELETAL

MUSCLE

L. L. COSTANTIN Department of Physiology and Biophysics, Washinotoa University School of Medicine, St. Louis, Missouri 631 I0, USA I. I N T R O D U C T I O N

The role of calcium as the physiological activator of the contractile proteins is now firmly established (Ebashi and Endo, 1968). In the present review, the steps in contractile activation beginning with surface membrane depolarization of the muscle cell and culminating in an increase in the concentration of activator calcium in the myoplasm will be examined. Striated muscle cells usually contain two distinct systems of internal membranes, a surface-connected system of invaginations which form clefts or tubules within the depth of the cell, and a system of intracellular origin, the sarcoplasmic reticulum, or SR, which is derived from the rough-surfaced endoplasmic reticulum. In broad outline, contractile activation may be summarized as follows: In the relaxed muscle cell, activator calcium is stored in the SR. Surface membrane depolarization spreads electrically along the surfaceconnected elements of the internal membrane system, and the depolarization of these elements signals the SR to release calcium. This sequence of events has been most extensively studied in the twitch fibers of frog muscle; thus, it has been convenient in the present review to focus on the properties of these fibers. It should be emphasized, however, that a wide diversity of both structure and physiological properties exists in different types of striated muscle. While it has not been possible to describe this diversity in any detail, an attempt has been made to consider those differences which appear directly related to contractile activation. II. A N A T O M Y

OF THE

INTERNAL

MEMBRANE

SYSTEM

In the frog twitch fiber, and in vertebrate twitch fibers generally, the surface connected elements of the internal membrane system consist of tubules a few hundred Angstroms in diameter which form a predominantly planar network through the entire fiber crosssection. This tubular network is found in a regular position within each sarcomere, at the level of the Z-line in amphibian muscle and at the A-I junction in mammalian skeletal muscle. Electron microscopy of striated muscle from the myotomes of a fish, the Black Mollie, has shown that the lumen of the T-system is in direct continuity with the extraceliular space (Franzini-Armstrong and Porter, 1964). In frog twitch fibers, similar surface connections have only rarely been seen, perhaps because the T-tubule apertures seal during the fixation process (Huxley, 1964; Page, 1964) or because the tubular network is enlarged and irregular immediately beneath the surface membrane (Peachey, 1965; Franzini-Armstrong, 1970). Nevertheless, there is little doubt that continuity between the tubular lumen and the extracellular space is present in vivo, since large molecules which serve as extracellular markers can freely enter the tubule lumen. Fluorescent dyes, ferritin, and peroxidase have been employed for this purpose by various investigators (Huxley, 1964; Page, 1964; Endo, 1966; Eisenberg and Eisenberg, 1968). The contractile proteins or myofilaments of frog twitch fibers are grouped into bundles of myofibrils about l #m in diameter, and each myofibril is surrounded by both tubular and SR elements. The SR consists of flattened sacs which extend from Z-line to Z-line. The configuration of the SR varies along the length of the sarcomere, appearing as terminal cisternae in the region of the I-band and as longitudinal tubules and a fenestrated collar in the A-band region. The lumen of the SR is continuous both along the length of the sarcomere and throughout the fiber cross-section, but does not appear to be continuous with either the extracellular space or the region of the myoplasm which contains the contractile proteins. 199

200

L.L. COS1-ANTIN

The internal membrane system constitutes an appreciable fraction o f the fiber volume, and the surface area of the internal membrane system is considerably greater than that of the external surface membrane; the volumes and surface areas of the SR and T-system, as determined by electron microscopy (Peachey, 1965), are given in Table 1. About 3°,,~; of the T-tubules also extend longitudinally along the sarcomere to run parallel with the elements of the SR (Eisenberg, 1972). The T-system lies in close proximity to the terminal cisternae of the SR, and the grouping of the T-tubule with two laterally disposed terminal cisternae is called a triad. In frog twitch fibers, approximately 80~ of the surface area of the T-system abuts on the terminal cisternae (Peachey, 1965), and anatomically distinctive junctions between tubule and SR membranes are found in the triad. The precise nature of the SR-T junction is still in doubt; this problem will be discussed further when the electrical properties of the internal membrane system are considered.

1. Comparative Anatomy of the Internal Membrane System SR-like elements have been found in all striated muscles which have been intensively studied, although the extent of the SR varies widely in different cell types. In a general way, the degree of development of the SR can be correlated with the mechanical properties of the muscle; where different muscle types from the same animal have been compared (Pellegrino and Franzini, 1963; Page, 1965, 1969), the SR has been found to be more welldeveloped in the more rapidly acting muscles. This results is, of course, consistent with the idea that SR calcium sequestration permits muscle relaxation. There is considerable physiological evidence to indicate that the surface-connected Tsystem serves to transmit surface membrane depolarization to the SR-T junction, and this role of the T-system is reflected in the organization of the T-system in different muscle types. In the rapidly contracting sound-producing muscles of the bat, for example, a branching T-tubule may contact three SR elements, forming pentads at each A-I junction within the sarcomere (Revel, 1962). In more slowly acting muscle, the frequency of SR-T junctional contacts are relatively sparse, and the contacts are mainly in the form of dyads, i.e., junctions between a T-tubule and a single SR element. Certain cardiac muscle cells lack a surface-connected T-system, e.g., the small (about 5 #m in diameter) cells of the frog myocardium (Page and Niedergerke, 1972) and the Purkinje cells of the mammalian ventricle which are specialized for rapid conduction of the action potential (Sommer and Johnson, 1968). In these cases, however, SR elements form junctional contacts directly with the surface membrane, and the structure of these contacts resembles the SR-T junctional complexes seen in skeletal muscle cells. Thus the feature of the internal membrane system which is apparently common to all striated muscle is the presence of the SR in intimate contact with a surface-connected membrane element which can sense surface membrane depolarization. The single exception to the generalization that SR-T or SR-surface junctions are to be found in all striated muscle cells would appear to be the skeletal muscle of Amphioxus. These muscle cells are arranged in broad flat sheets about 1 ~m thick; in osmium-fixed TABLE 1. VOLUMESAND SURFACEAREAS OF SARCOPLASM1CRETICULUMAND TRANSVERSETUBULES

Compartment Transverse tubules Sarcoplasmic reticulum Terminal cisternae Longitudinal tubules, intermediate cisternae, and fenestrated collar Total

Fractional fiber volume

Surface area for a fiber 100 p m in diameter (cm2/cm 2 of outer surface)

0.004

9

0.05

35

0.08

100

0.13

144

(After Peachey, 1965). The values o f T - t u b u l e surface and volume are 3 0 ~ larger than Peachey's original estimates (see Peachey and Schild, 1968).

Contractile activation in skeletal muscle

201

preparations, numerous subsarcolemmal vesicles, which may represent SR elements, have been seen, but no T-system and no SR-surface junctional complexes have been found (Flood, 1968). Fixation with glutaraldehyde, which has been shown to result in better preservation of the internal membrane system than osmium (Franzini-Armstrong and Porter, 1964), has confirmed this result (Hagiwara et al., 1971). III. R E S T I N G

ELECTRICAL

PROPERTIES

MEMBRANE

OF THE

INTERNAL

SYSTEM

I. Ionic Permeability Although electron microscopic studies clearly indicate that the T-system is open to the extracellular space and that the SR-T junction serves as a barrier to the passage of large molecules such as ferritin or peroxidase, the fine structure of the SR-T junction has not been firmly established. It has been said by some investigators to resemble the low resistance cell-to-cell junctions found in other cell types (Fahrenbach, 1965; Peachey, 1965). In other studies (Walker and Schrodt, 1965; Franzini-Armstrong, 1970, 1971) the tubule and SR membranes appear to be separated by a gap of about 12 nm which is bridged by periodic projections from the SR membrane called SR feet; this wide separation between the two membranes would preclude the ready passage of solutes directly between the SR and the T-tubule lumen. Freeze-fracture studies of the dyads of spider muscle also suggest that the SR feet do not form continuous structures passing through the SR or T-tubule membranes (Franzini-Armstrong, 1974). There is, however, some evidence that the SR-T junction might be permeable to small ions or molecules. The osmotic behavior of the SR of frog twitch fibers in response to altered osmolarity of the bathing medium is. consistent with the idea that the SR-T junction is permeable to extracellular ions or small non-electrolytes; the addition of NaCI or sucrose to the bathing medium causes the muscle fiber to shrink and the SR to swell, presumably because Solute movement into the SR results in water movement from the myoplasm to the SR (Birks and Davey, 1969, 1972). The presence of a relatively large extracellular compartment within the muscle fiber has also been suggested by measurements of muscle volume changes in hypertonic solutions (Dydynska and Wilkie, 1963; Blinks, 1965), by the kinetics of ion movement in muscle and by the distribution of muscle chloride and sodium (Harris, 1963; Rogus and Zierler, 1973), and by comparison of the extracellular space accessible to small and large extracellular markers (Tasker et al., 1959; Page, 1962; Bozler, 1967); the volume of this compartment is consistent with the volume of the SR. It should be noted, however, that these various techniques may reflect only a relatively slow permeation of extracellular solutes into the SR. An important question with regard to the mechanism of SR calcium release is whether the ionic permeability of the SR-T junction is high enough so that sufficient ionic current can flow across the SR membrane when the surface membrane is depolarized. The evidence on this point appears to be quite definite: a variety of electrophysiological studies have failed to demonstrate that the SR membranes provide a pathway for current flow from the sarcoplasm to the extracellular space. These studies are of two general types, (1) studies of ion accumulation and depletion within the internal membrane system and (2) studies of the effective membrane capacity of the muscle cell. 2. Ion Accumulation and Depletion in the T-System Hodgkin and Horowicz (1960a), working with isolated single fibers from the frog semitendinosus muscle, were able to examine the time course of membrane potential changes following a sudden alteration of the extracellalar ionic concentration, and they showed that repolarization of the fiber, following a sudden decrease in potassium concentration, required many seconds, much longer than would be expected from the time constant of the muscle membrane. The slow time course of repolarization was consistent with the slow diffusion of potassium from a restricted space such as the internal membrane system. The repolarization following a decrease in potassium was two to three times more rapid in the ~,.,~

242

~,

202

L.L. COSTANTIN

presence of a permeable anion such as chloride, presumably because under these conditions, the potassium concentration in the restricted space was reduced primarily by a KC1 influx into the fiber. By calculation of the chloride influx during repolarization, Hodgkin and Horowicz were able to estimate the volume of the space which would contain this quantity of potassium. This volume was about 0.2 to 0.5~o of the fiber volume, in good agreement with the electron microscopic estimates of the volume of the T-system and considerably less than the volume of the SR (see Table 1). Thus, under the conditions of these experiments, only the T-system, and not the SR, appeared to have rapid access to the extracellular space. Although the resting chloride conductance of the frog twitch fiber is about twice the potassium conductance (Hurter and Noble, 1960; Adrian and Freygang, 1962), Hodgkin and Horowicz found little slow component of membrane potential change following alteration of the extracellular chloride ion concentration. This result implies that the chloride permeability is largely restricted to the surface membrane so that, in the resting muscle cell, the major carrier of ionic current across the T-tubule membrane is the potassium ion. It should be possible, therefore, to alter the potassium ion concentration of the T-system by prolonged current flow, and this expectation was confirmed by Adrian and Freygang (1962). Application of prolonged inward current pulses to a fibre in normal Ringer solution containing 2.5 mR potassium resulted in a hyperpolarization which developed with the membrane time constant and was followed by a slow "creep" in potential over about 1 sec to a more hyperpolarized value. The "creep" was not seen in potassium-free or in high potassium (100 m~) solutions. Adrian and Freygang proposed that this "creep" was due to a depletion of potassium in the tubule lumen and a resultant decrease in the potassium conductance of the T-tubule membrane. Estimates of the space in which potassium depletion occurred were consistent with the volume of the T-system. With sufficiently large hyperpolarizing voltages, the fall in membrane conductance arises, not only from a current-dependent depletion of tubular potassium, but also from a voltage-dependent decrease in potassium permeability (Adrian et al., 1970b). Almers (1972a, b) has shown that these two processes can be clearly dissociated by studies at different temperatures, since the Q~o for recovery from tubular potassium depletion is about 1.3, a value consistent with a diffusion process, and the Q ~o for the conductance change is 2.8. Almers' estimate of the volume of the space which can be depleted of potassium by prolonged inward current was also consistent with the volume of the T-system; again no indication of significant current flow through the SR membranes was found (see also Barry and Adrian, 1973). (a) Inward-going rectification The resting potassium conductance in the frog twitch muscle fiber is markedly nonlinear; the conductance is much greater when measured with inward than with outward currents. This behavior, which has been termed anomalous or inward-going rectification, has been reported in a variety of nerve and muscle cells; inward-going rectification in muscle has been reviewed by Adrian (1969). It has not been established whether the high conductance pathway for inward current is confined to the T-tubule membranes or is a property of both tubule and surface membranes. A consequence of this inward-rectifying characteristic of the muscle membrane is that the success of the experiments described above on potassium ion depletion and accumulation in the T-system is dependent on the direction of the gradient for potassium ion movement across the T-system. Because outward potassium currents are extremely small, at least in the range where delayed rectification is not activated (see below), prolonged depolarizing steps can provide no clear evidence of potassium accumulation in the T-system (Almers, 1972a, b; Barry and Adrian, 1973). Similarly a sudden increase in extracellular potassium in the Hodgkin and Horowicz (1960a) experiments did not reveal a slow component of depolarization, presumably because under these conditions, the driving force for potassium current across the tubular membranes was outward and the surface membrane dominated the recorded transmembrane potential (see Nakajima et al., 1973).

Contractile activation in skeletal muscle

203

3. The Effective Capacity of the Muscle Cell The second line of evidence which indicates that the SR membranes do not provide a significant pathway for current flow from the sareoplasm to the extracellular space is provided by capacity measurements of the muscle fiber, Since the capacity of biological membranes appears to be about 1/~F/cm2 of membrane, the.effective membrane area through which current passes can be estimated by the size of the effective capacity of the cell. The initial measurements in frog twitch fibers (Katz, 1948; Fatt and Katz, 1951) showed that the effective capacity which was charged by application of steady currents was about 57 #F/cm 2 of fiber surface area. Although this large value might conceivably have arisen from extensive folding of the surface membrane (Katz, 1948), it was realized quite soon afterward by A. L. Hodgkin (see Huxley, 1971) that the membrane capacity was strongly frequency-dependent. The estimate of capacity obtained with a high frequency signal, the exponential foot of the propagated action potential, was considerably lower (about 2.6/zF/ cm 2 of fiber surface). This frequency-dependenceof the effective capacity of the muscle cell cannot be explained by a simple folding of the surface membrane but is quite consistent with the idea that some fraction of the internal membrane system provides a pathway for current flow in parallel with the surface membrane. Since the internal membrane system penetrates into the depths of the fiber, its membrane capacity might be expected to be in series with the resistance of the T-tubule lumen so that only a fraction of the total membrane capacity will be charged when rapid potential changes are applied to the surface membrane. It is clear from the anatomical data in Table 1 that this additional capacity of 3-5 #F/cm 2 of fiber surface which is seen with low frequency signals must be attributed to the T-system. As is the case with the ionic depletion and accumulation studies, there is no indication of current flow through the large membrane area of the SR. On the contrary, the correlation between the total measured capacity and the combined area of Surface and T-tubular membranes is quite good. Since the T-system is distributed through the fiber volume, the total area of tubule and surface membrane, referred to the fiber surface, increases linearly with the fiber diameter, and Hodgkin and Nakajima (1972a) have reported that the effective membrane capacity for low frequency signals shows a similar dependence on fiber diameter.

(a) The equivalent circuit of the frog twitch fiber Numerous attempts have been made to derive an equivalent electrical circuit which would describe the impedance between myoplasm and extracellular space. Detailed impedance measurements initially carried out by Falk and Fatt (1964) and extended by a number of workers have demonstrated that two distinct pathways exist for current flow between the myoplasm and the extracellular space. One pathway, consisting of a resistor and capacitor in parallel, corresponds to the surface membrane of the muscle cell. The second pathway corresponds to the T-system, and thus its electrical properties should provide information on the passive electrical properties of the T-system. The impedance data obtained on frog twitch fibers in normal Ringer solution can be fitted by a "T-system" pathway consisting of a single resistor and capacitor in series (Falk and Fatt, 1964; Freygang et al., 1967; Schneider, 1970; Valdiosera et al., 1974). In this "lumped" circuit model, the surface membrane capacity is about 2/~F/cm 2 of fiber surface (1.6/~F/cm2: Schneider, 1970; ---2.6 #F/cm2: Falk and Fatt, 1964; Valdiosera et al., 1974), while the tubular capacity is about twice this value. Since there is some uncertainty in the estimate of fiber surface area arising both from the possibility of membrane folding and from the presence of small invaginations of the surface membrane or caveolae (Dulhunty and Franzini-Armstrong, 1974), this distribution of surface and tubular capacity is not unreasonable, and the physiological validity of this model must be decided on other grounds. An important implication of this model is that there exists a "lumped" resistive element in series with the T-tubule membrane capacity which is large in comparison with the distributed resistance of the fluid in the T-system lumen, so that radial potential gradients do not develop along the T-system. Such a lumped resistance might arise at the mouths of

204

L.L. COSTANTIN

the T-tubules or perhaps within the SR itself(Falk and Fatt, 1964). Since the initial work of Falk and Fatt, however, experimental evidence has been obtained for the presence of radial potential gradients within the T-system. Contractile activation spreads radially with a measurable delay (Gonzalez-Serratos, 1966, 1971) and the radial spread of activation is decremental in the absence of an action potential (Adrian et al., 1969b). Although these studies involved relatively large depolarizations (beyond the contraction threshold), recent experiments (Adrian and Almers, 1974) suggest that potential gradients can be established along the T-system even with small displacements of the surface membrane potential. An alternative representation of the T-system current pathway is as a distributed electrical circuit with the membrane capacitance of the more axially located T-tubules in series with a larger luminal resistance than the membranes of the more superficial T-tubules (Falk and Fatt, 1964; Adrian et al., 1969a). This "distributed" model does predict the development of radial potential gradients along the T-system with surface membrane depolarization. When the parameters of this model derived from impedance data are related to the anatomical measurements of the T-system, the predicted value of capacity of both tubular and surface membrane is about 1 #F/cm 2 of membrane, and the conductivity of the fluid in the tubular lumen appears to be about ½ to ½ of the conductivity of the bathing solution (Schneider, 1970). In view of the uncertainties in the anatomical measurements (see above), this close correspondence between the value of membrane capacity in the "distributed" model and the generally accepted value for biological membranes should probably be regarded as no better (or no worse) than the fit obtained with the "lumped" model. However, Valdiosera et al. (1974) have shown that alterations in the conductivity of the bathing medium produce qualitatively appropriate alterations in the lumen conductivity predicted by the "distributed" model; this result supports the idea that the resistance in series with the T-tubule membranes is located in the tubular lumen. A satisfactory explanation for the apparently reduced conductivity of the tubular lumen has not been established. It seems unlikely that the cross-sectional area of the T-tubules is over-estimated by electron microscopic techniques, and in fact, the electrophysiological estimates ofT-system volume are somewhat larger than the values given in Table 1 (Hodgkin and Horowicz, 1960a). One possible explanation is that ionic mobilities are reduced by the presence of macromolecules within the tubular lumen; there is some evidence that the diffusion coefficients of sodium (Nakajima and Nakajima, 1974) and of potassium (Barry and Adrian, 1973) are reduced within the T-system. In mammalian cardiac muscle, the protein-polysaccharide coating of the surface membrane clearly extends into the Tsystem (Fawcett and McNutt, 1969), but this has not been seen in frog skeletal muscle. Another possibility is that the branching within the tubular network is sufficiently irregular to increase the path length for ion movement within the T-system by a greater amount than the value derived for a regular tubular network (Adrian et al., 1969a). Electron microscopic studies have shown that the tubules develop extensive tortuosities immediately beneath the fiber surface. Huxley and Taylor (1958) suggested that the tubular mouths are rather widely separated around the circumference of the fiber, and Adrian and Peachey (1973) have proposed that these limited openings of the tubules should be represented electrically as an access resistance to the T-system, i.e. as a "lumped" resistance in series with the distributed tubular network. Valdiosera et al. (1974) have attempted to fit the impedance data on muscle with the T-system represented as a distributed network in series with an access resistance (a hybrid model); a best fit analysis of the data required a relatively small access resistance which has little effect on the calculated properties of the T-system. This result, however, does not rule out the presence of a significant access resistance in the muscle fiber. As noted previously, an equivalent circuit of the muscle cell in which the surface pathway has a capacity of about 2 ltF/cm 2 of fiber surface and the T-system pathway has a single lumped resistance and capacity yields impedance data which is essentially similar to a circuit with a surface capacity of 1 #F/cm 2 and a distributed T-system; presumably, a "hybrid" model with an appreciable lumped access resistance and a surface capacity between 1 and 2 ~F/ c m 2 would also fit the impedance data.

Contractileactivationin skeletalmuscle

205

(b) Conduction velocity of the action potential The passive electrical properties of the muscle fiber are an important determinant of the velocity of propagation of the action potential, since the initial phase of depolarization, the exponential foot of the action potential, is brought about by passive electrotonic current flow between excited and unexcited regions of the fiber. In frog twitch fibers, the foot of the action potential is quite rapid, and thus the membrane currents in the unexcited region of the fiber are largely capacitive. Hodgkin and Nakajima (1972b) have shown that the extent to which the T-tubule capacity is discharged during the foot of the action potential is dependent on the time constant (rl) of the foot. At room temperature, the effective capacity of the fiber during the foot of the action potential (zI = 127 #sec) was 2.6 gF/cm 2, while at 3.5°C, with zl = 823/~sec, the effective capacity increased to 3.9/~F/cm 2. Assuming a distributed model of the T-system, these values imply that the space constant of the Tsystem during the foot of the action potential is 6.5 pm at room temperature and 13.3 #m at 3.5°C. Thus, only the outer zones of the tubular network are discharged during the foot of the action potential, and, as shown by Hodgkin and Nakajima (1972b), the effective capacity during the foot is nearly independent of fiber diameter between 50 and 150/~m. The velocity of conduction of the action potential is much slower in frog muscle than in the squid axon, even if allowances are made for differences in cell diameter and internal conductivity. Hodgkin and Nakajima (1972b) have suggested that this difference in membrane properties between these two cell types can be attributed to the added capacity contributed by the T-system rather than to a difference in the magnitude of active ionic currents. They showed that the conduction velocity was considerably increased in detubulated muscle fibers (see below) in which the effective capacity during the foot of the action potential was 0.9/~F/cm 2, and that these detubulated fibers were quite comparable to the squid axon in membrane properties.

4. Properties of the Internal Membrane System in Other Species Although the internal membrane system of other muscles has not been as extensively studied as that of the frog twitch fiber, sufficient data exists in certain arthropod muscles to indicate that here too the T-system provides a pathway for current flow between the myoplasm and the extracellular space. Thus in crab muscle, the low frequency capacity is 40--50 #F/cm 2 of fiber surface, a value consistent with the extensive surface-connected internal membrane system in these cells (Fatt and Katz, 1953; Eisenberg, 1967; Selverston, 1967), and impedance studies have revealed a pathway for current flow in parallel with the surface membrane (Eisenberg, 1967). In crab (Selverston, 1967) and in crayfish (Girardier et al., 1963) muscle, the T-tubule membrane appears to be selectively permeable to chloride rather than potassium. 5. The Detubulated Muscle Exposure of a frog twitch fiber to a Ringer solution plus 400 mM glycerol for 1 hr followed by a return to normal Ringer solution has been found to interrupt the continuity between the tubule lumen and the extracellular space (Eisenberg and Eisenberg, 1968; Howell, 1969; Krolenko, 1969). This interruption can occur with little or no decrease in the resting membrane potential (Howell, 1969; Eisenberg et al., 1971), and action potentials can be elicited in these detubulated fibers by electrical stimulation (Gage and Eisenberg, 1969b). The action potential, however, does not result in a muscle twitch. Since contractile activation can be produced in these muscles by caffeine, a drug which apparently acts directly on the SR to cause an increase in myoplasmic calcium (Axelsson and Thesleff, 1958; Weber, 1968), the disappearance of the twitch provides strong support for the essential role of the T-system in transmitting the signal for calcium release to the SR. Electrophysiological studies of glycerol-treated muscle fibers have generally supported the description of the T-system obtained from studies in the intact cell. Thus the chloride conductance was essentially unchanged while the potassium conductance was significantly reduced in detubulated fibers. Furthermore, the slow hyperpolarization, or "creep" during application of prolonged inward current, which has been attributed to depletion of tubular

206

L.L. COSTANTIN

potassium, was not found in these fibers (Eisenberg and Gage, 1969). These results provide striking confirmation for the proposal that the T-system is selectively permeable to potassium. Similarly the assymetry in the response to an increase and a decrease in extracellular potassium which has been attributed to retention of potassium in the T-system was abolished in the detubulated fiber (Nakajima et al., 1973). The total capacity of detubulated fibers is also strikingly reduced to about 2 #F/cm 2 (Gage and Eisenberg, 1969a; Hodgkin and Nakajima, 1972b). The precise mechanism by which glycerol treatment affects the internal membrane system has not been established. Glycerol penetrates the cell membrane rather slowly, so that, following immersion in a Ringer solution plus glycerol, the cell shrinks transiently and then returns to its normal volume as glycerol enters the cell (Krolenko and Adamyan, 1967; Miyamoto and Hubbard, 1972). Return of the cell to a glycerol-free Ringer solution results in transient swelling of the cell and swelling and vesiculation of the T-system (Eisenberg and Eisenberg, 1968; Howell, 1969; Krolenko, 1969). The swollen vesicles of the Tsystem range up to a few micrometers in diameter, and the muscle fiber takes on a characteristic light microscopic appearance of vacuolation; these vacuoles usually persist for prolonged periods of time in normal Ringer solution (Krolenko et al., 1967). In some instances, however, both the inaccessibility of the tubules to ferritin (Krolenko, 1968) and the decrease in effective capacity (Nakajima and Bastian, 1974) are partially reversed with prolonged immersion in Ringer solution, and this reversal is accompanied by a return of twitch tension. Although it was initially thought that separation of the tubules from the surface membrane was the predominant effect of glycerol treatment, these signs of reversibility suggest that the primary effect may be simply a narrowing or obliteration of the tubule lumen (Krolenko and Fedorov, 1972; Nakajima et al., 1973). The possibility that some disturbance of the SR-T junction also occurs during glycerol treatment cannot be excluded (Dulhunty and Gage, 1973), but direct evidence for this mechanism, which should presumably result in a fiber with a relatively normal total capacity and no mechanical response to an action potential, has not yet been produced. IV. E X C I T A T I O N

IN

SKELETAL

MUSCLE

1. Spread of Depolarization Alony the Surface Membrane (a) Twitch fibers Depolarization of the muscle fiber is initiated at the neuromuscular junction and spreads along the fiber to produce a more-or-less uniform surface membrane depolarization. In vertebrate twitch fibers this spread of depolarization is produced by a propagated action potential. The ionic conductance changes which give rise to the action potential are essentially similar to those of the squid axon; depolarization results from a voltage and time-dependent increase in sodium conductance (Nastuk and Hodgkin, 1950) and repolarization from a delayed voltage and time-dependent increase in potassium conductance, or delayed rectification (Heistracher and Hunt, 19.69a; Adrian et al., 1970a). A significant difference between delayed rectification in nerve and in muscle is that, in muscle, the potassium conductance is inactivated when depolarization is maintained for several hundred milliseconds (Nakajima et al., 1962; Heistracher and Hunt, 1969a, b; Adrian et al., 1970a, b). (b) Slow fibers In amphibia (Kuffler and Vaughan-Williams, 1953a, b), fish (Bone, 1964) and in certain mammalian (Hess and Pilar, 1963; Pilar, 1967; Peachey, 1968; Hess, 1970) muscles, a second distinct motor system, the slow fiber system, has been described. The muscle fibers of this system appear to be employed for the production of more-or-less steady tension. In those species of fish where a dual motor system is found, the slow fiber system is active during quiet maintained swimming and the fast system is utilized for bursts of quick swimming (Bone, 1966), while in frogs the slow fiber system appears to be involved in the maintenance of postural tone (Kuffler and Vaughan-Williams, 1953a, b). Slow muscle fibers of the frog have been most extensively studied. They show no regenerative increase in sodium

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conductance with depolarization and thus cannot propagate an action potential. Instead a. fairly uniform spatial distribution of depolarization is achieved by passive electrotonic spread of depolarization from multiple neuromuscular junctions distributed along the length of the fiber. Delayed rectification is present in the slow muscle fiber membrane (Burke and Ginsborg, 1956; Stefani and Steinbach, 1969). Surprisingly, chronic denervation results in the appearance of sodium-dependent action potentials which are blocked by TTX (Miledi et al., 1971). (c) Invertebrate muscle The striated muscle fibers of invertebrates resemble the fibers of the vertebrate slow muscle system in that they are multiply-innervated and do not propagate a sodium-dependent action potential. In a number of arthropods, however, an inward calcium current, which is sufficiently large to lead to a regenerative depolarization of the cell, can be produced by membrane depolarization. The magnitude of the regenerative response shows considerable variability even in different fibers from the same muscle, and can range from local graded responses to all-or-none propagated action potentials. The subject of calcium currents in excitable membranes has been reviewed by Reuter (1973).

V. I N W A R D

SPREAD

OF ACTIVATION

Following surface membrane depolarization, the next essential step in contractile activation appears to be the spread of depolarization within the T-system. Both the anatomical organization of the T-system and the disappearance of the twitch in detubulated muscle support this view, but the most direct evidence has come from a series of experiments by A. F. Huxley and his co-workers [summarized by Huxley (1971)]. When highly localized depolarizations were produced by application of a small extracellular pipette to the surface membrane of a muscle fiber, local contractions could be elicited only when the pipette was positioned at specific sites along the sarcomere. The location of these sites in muscles from different species could be correlated with the location within the sarcomere of the T-tubule element which formed a specialized junction with the SR. Thus, in frog twitch fibers, where SR-T tubule triadic junctions are located at theZ-line, depolarizations were effective only when the lumen of the pipette overlapped the Z-line while, in lizard and crab muscle, where dyadic junctions are found at the boundary between A- and I-bands, the sensitive spots were localized to the A-I boundary. In frog muscle, contraction involved both halves of the I-band; with increasing depolarizatiorL the depth to which the contraction spread inward was increased, but spread to adjacent I-bands was not seen. In crab and lizard muscle, on the other hand, contractions of a single half-sarcomere could be obtained. Although a second set of tubules opens to the fiber surface at the Z-line in crab muscle, these tubules apparently do not form specialized junctions with the SR, and depolarizations localized to the Z-line level were not effective in eliciting contraction. Since the direct measurement of transmembrane potential within the T-system has not been possible because of the small diameter of the T-tubules, the spread of mechanical activation from superficial to more axially located myofibrfls has been employed as an index of the spread of depolarization along the T-system. One problem with this approach is that the change in striation spacing and I-band width along a myofibril are the same with both active and passive shortening, at least until contraction bands are formed with shortening below 2/lm (Huxley and Gordon, 1962). This difficulty was overcome by GonzalezSerratos (1966, 1971)who found that longitudinal compression of a resting frog muscle fiber set in gelatin caused the myofibrils to be thrown into folds at striation spacings below about 2 pm and that, following a single action potential, active shortening of the myofibrils resulted in straightening of these folds throughout the fiber cross-section. Thus the entire cross-section became activated in the course of a single twitch. Straightening was found to begin earlier in superficial than in more axially located fibrils. This delay in the radial spread of activation was about 0.6 msec at 20°C for a fiber 100 pm in diameter and increased with decreasing temperature with a Q lo of about 2.0.

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The radial spread of mechanical activation has also been examined in voltage clamped frog twitch fibers. In fibers in which the action potential was blocked by TTX, a just-threshold depolarizing step applied to the surface membrane produced contraction of only the most superficial myofibrils, presumably because the surface depolarization decayed electrotonically along the tubular network to a sub-threshold value in the most axial Ttubules. An increment in depolarization of a few millivolts was required to produce extensive shortening of the entire cross-section (Adrian et al., 1969b). In fibers not exposed to TTX, the radial spread of activation was much more effective, and in some fibers, the radial gradient of activation was actually reversed, with a just-threshold depolarization eliciting contraction of only the most axial myofibrils (Costantin, 1970). This reversal of the gradient of activation in TTX-free fibers implies a reversal of the gradient of depolarization along the T-system, and therefore implies the presence of a net inward current in the Tsystem. Since the radial spread of activation in a low sodium Ringer was similar to that seen in TTX-treated fibers, this net inward current appeared to be carried by sodium ions. In the normal muscle cell, this inward sodium current would result in a propagated action potential along the T-system. Keynes et al. (1973) have proposed that a propagated action potential is present in the cleft and T-system membranes of the barnacle, Megabalanus psittacus (Darwin); the ionic mechanism of the action potential in this muscle appears to be an increase in calcium permeability. The question of whether a tubular action potential is required for the effective spread of depolarization within the T-system of amphibian muscle cells has been investigated by Bastian and Nakajima (1974). Working with single fibers of the African clawed toad (Xenopus laevis) in a double sucrose gap voltage clamp, they demonstrated that, at 23°C, the twitch produced by an action potential in a normal fiber was about three times larger than that produced by application of an action potential waveform to the surface membrane of TTX-treated fibers. At 10°C, where the duration of the normal action potential is prolonged so that the passive spread of depolarization along the T-system is more effective, the twitch produced by a simulated action potential in a TTX-treated fiber increased to about 85Voof that elicited by a normal action potential.

1. T-System Conductance Changes in Fro9 Twitch Fibers Although the experiments described above clearly demonstrate that a tubular action potential is involved in the radial spread of the activation signal, they provide no estimate of the magnitude Of the tubular ionic currents. Since outward current through the inward rectifier potassium channel is very small with depolarizing signals, the increase in sodium conductance required to produce a net inward current in the T-system could also be quite small, and T-tubule depolarization might result largely from local circuit currents flowing between the T-system and the surface membrane. Alternatively, if the tubular sodium current was appreciable, the tubule membrane would be depolarized by a sodium influx and, if delayed rectification also occurred in the T-system, the tubule would be repolarized by a potassium efflux. Hodgkin and Horowicz (1959) found that in a frog twitch fiber, the net sodium entry per impulse is about 15.6 pu/cm 2 of fiber surface and the net potassium loss is about 9.6 pM/cm 2. If these ion fluxes are distributed equally over the surface and the tubular membranes, appreciable concentration changes would occur in the tubular lumen during rapid repetitive stimulation. For a fiber 100 #m in diameter in which the tubular surface area is about nine times that of the surface membrane (see Table 1), the net flux per cm 2 of tubule membrane would be 1.56 pM for sodium and 0.96 pM for potassium with each action potential. If the tubule surface-volume ratio is taken a s 106 cm- 1 (Peachey, 1965), the tubular sodium concentration would decrease by 1.56mM and the tubular potassium concentration would increase by 0.96 mM with each potential. In the absence of delayed rectification, the expected tubular potassium flux and concentration increase would be at least twenty-five-fold smaller (Hodgkin and Horowicz~ 1959). (a) Tubular sodium depletion In an attempt to demonstrate tubular sodium depletion during rapid repetitive stimulation, Bezanilla et al. (1972) have examined the time course of tetanic tension in frog

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twitch fibers in solutions of varying sodium concentrations. In a normal Ringer solution, tetanic tension was well maintained during I sec of stimulation at 60 shocks/sec, but when the sodium concentration of the bathing solution was reduced to 50% of normal, the tension response showed an initial peak and a subsequent fall, with a half-time of about 260 msec, to a lower steady plateau of tension. They attributed this fall in tension to tubular sodium depletion with repetitive stimulation and a resultant failure of activation of axial myofibrils. Although the height of the recorded surface action potential also decreased with rapid stimulation, presumably because of some sodium inactivation with repetitive action potentials, Bezanilla et al. argued that this decrease in action potential height did not significantly affect contractile activation. This does not appear to be the case, however. In 50~ sodium Ringer, the action potential overshoot declined to + 13 mV after 290 msec of stimulation at 50 shocks/sec, while a similar decline in normal Ringer required 2.5 sec. The time course of tetanic tension in normal Ringer during rapid repetitive stimulation of this duration was apparently not examined by Bezanilla et al. (1972). A comparable decrease in the height of the action potential can be produced by lowering the extracellular sodium to one-third of normal (Nastuk and Hodgkin, 1950); since at this sodium concentration the fibers studied by Bezanilla et al. showed a marked reduction in both twitch and tetanic tension, the progressive fall in tetanic tension seen in 50~o sodium Ringer may have resulted from the progressive fall in action potential amplitude with rapid repetitive stimulation. (b) Tubular potassium accumulation An appreciable increase in tubular potassium concentration with repetitive stimulation was proposed by Freygang et al. (1964) to explain the slow time course of repolarization in frog twitch fibers following a train of impulses. Following a single impulse, the rapid repolarization phase of the action potential ends at a membrane potential of about 70 mV and the potential returns to the resting value of - 90 mV over several milliseconds. With repetitive stimulation, this negative after-potential (Nastuk and Hodgkin, 1950), or early after-potential (Freygang et al., 1964) is followed by a slower repolarization phase with a half-time of about 350 msec which has been termed the late after-potential. A late after-potential is not found in detubulated muscle, a result which implies that it is associated in some way with the T-system (Gage and Eisenberg, 1969b). In intact fibers, Freygang et al. (1964) found that the peak amplitude of the late after-potential after ten impulses spaced 10 msec apart was 10.6 mV, and they estimated that an increase in tubular potassium concentration of at least 5 mM was required to account for a depolarization of this amplitude. If the late after-potential does arise from tubular potassium accumulation, these results would seem to require that delayed rectification is comparable in magnitude in tubular and surface membranes. The voltage clamp experiments of Adrian et al. (1970a, b), however, have not supported the idea that an appreciable increase in tubular potassium conductance occurs during depolarization. In these experiments, large depolarizing pulses which resulted in appreciable outward current did not produce the shift in equilibrium potential which would be expected if a significant fraction of the outward current were carried by potassium ions moving into the tubular lumen. A slow increase in membrane conductance, distinct from delayed rectification, was found with prolonged depolarization, and it was proposed that the slow deactivation of this conductance increase, and not an accumulation of potassium in the T-system, might give rise to the late after-potential. A detailed analysis of the kinetics of this late conductance increase has not been done, however, and it has not been established that this slow conductance change can quantitatively account for the late afterpotential. The possibility should also be considered that the failure of voltage-clamp experiments to detect a shift in equilibrium potential with large depolarizing currents was due to the combined effect of several experimental factors. Since the equilibrium potential was measured immediately following a large depolarization which increased the potassium conductance of the surface membrane, surface membrane currents could have overshadowed the contribution of tubular ionic currents. Adrian and Peachey (1973) have calculated voltage -

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clamp records for a model fiber with a distributed T-system and access resistance and have shown that tubular ionic currents have little effect on the apparent equilibrium potential estimated by the transient "tail" currents following a depolarizing step. It is also possible that, even if delayed rectification is present in the T-system, little potassium current passes the more axially located T-tubule membranes during large surface depolarizations. Adrian et al. (1969b) have shown that the radial spread of contraction in TTX-treated fibers is rather less effective than is predicted from the apparent resting length constant of the Tsystem, and have suggested that an increase in tubular potassium conductance resulted in an attenuation along the T-system of an applied surface depolarization. Recent studies by Valdiosera et al. (1974) indicate that the tubular lumen resistance increases eight-fold in a hypertonic sucrose medium similar to that employed by Adrian et al. (1970a, b) in their voltage-clamp studies; an increase in lumen resistance of this magnitude could effectively isolate the more deeply situated tubules from surface potential changes under conditions where the tubular membrane conductance is high. (c) Calculation of the propagated action potential in frog twitch fibers Adrian et al. (1970a) have attempted to compute the muscle action potential from voltage clamp data in terms of the parameters of the Hodgkin-Huxley model of the squid axon. Since the voltage clamp data provided no evidence for delayed rectification in the tubular system, they assumed that ionic conductance changes were confined to the surface membrane. The T-system was represented as a "lumped" circuit element with a single resistor and capacitor in series. One difference between the recorded and computed action potentials was that the rate of rise and conduction velocity of the computed action potential were smaller than the experimentally observed values, and Adrian et al. noted that the omission of a tubular sodium current in their model might account for this discrepancy. Analysis of early inward currents obtained by the sucrose-gap voltage-clamp technique also suggests that a regenerative sodium current within the T-system can contribute to the recorded ionic current (Ildefonse and Roy, 1972). A second difference between the recorded and computed action potentials was seen in the early after-potential. The recorded early after-potential consists of two phases, a late phase during which the membrane resistance is at its resting value and repolarization proceeds exponentially with the time constant of the resting membrane, and an initial phase during which the membrane resistance is low (Persson, 1963). The time course of repolarization during the initial phase varies considerably in different fibers; occasionally a secondary hump of depolarization is seen. In the computed action potential, however, the early after-potential falls smoothly to the resting potential, and no hump is seen. In the model proposed by Adrian et al., the potential at the beginning of the early after-potential corresponds to the equilibrium potential for delayed rectification, and the monotonic decay of the initial phase of the early after-potential is largely due to a slow deactivation of delayed rectification. More recently, Adrian and Peachey (1973) have extended this analysis with a model in which the admittance of the T-system is represented as a distributed cable network with an access resistance to the network at the fiber surface and have suggested a rather different mechanism for the early after-potential. This distributed model introduces a transmission delay within the T-system, so that depolarization of the surface membrane occurs before depolarization of the most axially located T-tubule membranes. Because of this delay, the surface membrane repolarizes while the T-system is still depolarized, and the interaction between these two membranes serves to slow the terminal phase of repolarization of the surface membrane. In large fibers, the predicted delay in the T-system is sufficiently long so that a secondary hump, similar to the recorded early after-potential, is produced by the model. The introduction of active tubular sodium current in the Adrian and Peachey calculations increased the size of the secondary hump. VI. T H E

MECHANISM

OF

ACTIVATOR

RELEASE

1. Magnitude of Calcium Release in a Twitch In a variety of isolated muscle model systems, the free calcium concentration required for just-threshold activation is about 10-6 M, and full activation is produced by 10-5M

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calcium. However, since calcium is bound to the contractile proteins during activation, the increase in total myoplasmic calcium concentration in the intact cell is considerably greater. In vertebrate muscle, activation results from calcium binding to troponin, a protein component of the thin filament (Ebashi and Endo, 1968). From the relation between free calcium concentration and calcium binding to troponin (Bremel and Weber, 1972) and the troponin concentration of intact vertebrate muscle (about 33 #M, Ebashi and Endo, 1968), the total myoplasmic calcium concentration required for full activation can be estimated as about 10- 4 M. Although any attempt to relate the properties of isolated protein molecules to their behavior in the intact cell involves considerable uncertainty, this calculation does serve to emphasize that an appreciable increase in the total myoplasmic calcium is probably required for the development of full tetanic tension. The influx of extracellular calcium during a single action potential in frog muscle is about 0.2 pM/cm 2 of cell surface (Bianchi and Shanes, 1959). For a muscle cell 100 micrometers in diameter, this represents an increase of about 8 x 10-aM/i. for each action potential. Since a single action potential results in a twitch which can be as large as 90~ of full tetanic tension (Jewell and Wilkie, 1958), there is a large discrepancy between extracellular calcium influx and the estimated increase in myoplasmic calcium during activation. The only obvious intracellular source of calcium in vertebrate striated muscle is the sarcoplasmic reticulum. Although an appreciably larger influx of extracellular calcium occurs with depolarization in certain invertebrate striated muscles, similar quantitative considerations suggest that in these muscles also, the SR serves as an intracellular calcium source in normal activation (Edwards et al., 1964). Not only is the extracellular calcium influx during a twitch clearly inadequate to produce contractile activation but the total inward current associated with the action potential is also less than the calcium flux required for activation (Freygang, 1965). Thus even if the action potential currents were in some way channeled through the internal membrane system, some form of current amplifier would be required to account for the quantity of activator calcium released in a twitch. 2. Membrane Potential and Calcium Release

(a) Gradation of the calcium release process If the action potential of vertebrate twitch fibers is blocked by TI'X or by the replacement of extracellular sodium with choline, contractile activation can be produced by passive depolarization of the muscle cell membrane. Under these conditions, contractile force appears to be a continuously graded function of membrane depolarization. Hodgkin and Horowicz (1960b), found that depolarization of frog twitch fibers by elevation of extracellular potassium produced a prolonged mechanical response, or contracture. Both the rate of tension development and the final level of tension were dependent on membrane potential over a range of potential from about - 5 0 to - 3 0 m V . Similar results have been obtained in frog tonic fibers (Nasledov et al., 1966; Lannergren, 1967a, b) and in arthropod fibers (Zachar and Zacharova, 1966; Gainer, 1968). Since these potassium-induced contractures are promptly and completely reversed by a decrease in extracellular potassium to normal levels, and since the maximal tension developed with large depolarizations is comparable to full tetanic tension, activation initiated by potassium depolarization appears to be quite similar to the normal process by which the action potential initiates a contractile response. The time course of depolarization following an elevation of extracellular potassium is, of course, considerably slower than the time course of the action potential, but subsequent studies have utilized voltage-clamp techniques to obtain more rapid changes in membrane potential. In these studies, short fibers from the scale muscles of garter snakes (Heistracher and Hunt, 1969a, b) or from the lumbricalis muscle of the frog (Bezanilla et al., 1971) were employed to minimize the decremental spread of depolarization along the length of the fiber. Again, gradation of contractile force was obtained as the amplitude of depolarization was varied. Since the contractile force developed by a muscle fiber presumably reflects the myoplasmic calcium concentration, the implication of these studies is that the quantity

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of calcium released from the SR is controlled by membrane depolarization and that membrane depolarization to some critical threshold does not trigger an all-or-none calcium release from the SR. Working with large muscle fibers of the barnacle which had been injected with the protein aequorin which emits visible light in the presence of ionized calcium, Ashley and Ridgway (1970) have succeeded in directly demonstrating the release of calcium in response to membrane depolarization. Depolarization beyond the contraction threshold resulted in an increase in light emission, and the magnitude of the light response was dependent on the amplitude of the depolarizing stimulus. A possible objection to these studies is that the applied surface depolarization attenuates along the T-system so that the observed increase in contractile force or light emission with increasing depolarization might arise from a progressive recruitment of more axially located T-tubules with an all-or-none threshold response occurring at individual SR-T junctions. The recruitment of axial myofibrils does occur with increasing depolarization in voltage-clamped frog twitch fibers (Adrian et al., 1969b), but Costantin and Taylor (1973) have reported that, even with depolarizations large enough to activate the entire fiber cross-section, graded contractile responses can apparently be elicited over a membrane potential range of at least 6-9 mV. (b) Inactivation and repriming of the calcium release mechanism If membrane depolarization beyond the contraction threshold is maintained in frog twitch fibers, the steady level of contracture tension is followed by a slow spontaneous relaxation, and tension finally returns to its resting value (Hodgkin and Horowicz, 1960b). The duration of contracture is reduced by an increase in the amplitude of depolarization. In frog twitch fibers at room temperature, the contracture plateau is decreased from about 7 sec at a membrane potential of - 40 mV to 2 sec at + 2 mV. The ability of the fiber to produce a maximal contracture is restored by repolarization to the resting potential for about 1 min, and the degree of restoration is dependent on the level of repolarization. Hodgkin and Horowicz (1960b) proposed that the spontaneous relaxation seen with maintained depolarization is caused by depletion of a supply of activator and that the abbreviated time course of the contracture with progressively larger depolarizations is due to a more rapid release and consequent depletion of activator. Repriming of the contractile mechanism by repolarization then results from a slow restoration of activator to a state where it can again be rapidly released by depolarization. Both the contracture duration and the time course of the repriming process are greatly increased at low temperatures (Caputo, 1972); at 3°C the contracture duration is 35 sec at a membrane potential of + 2 mV, and complete repriming with repolarization to the resting potential requires about 5 min. Some support for the proposal that restoration of activator to the site of release may require some time after a contracture has come from studies of calcium localization within muscle by autoradiography (Winegrad, 1970). Prolonged contractile activation results in a displacement of calcium-45 from its location in the I-band region of resting muscle (presumably in the terminal cisternae of the SR) to the A-band region; thus, it would appear that activator calcium released by depolarization is taken up by the longitudinal elements of the SR and only slowly returned to the terminal cisternae. If the calcium in the longitudinal elements were in an inactive form, this calcium movement would provide a physical counterpart to the depletion and restoration of activator discussed above. One difficulty with this idea, however, is that the return of calcium to the terminal cisternae appears to proceed even during a sustained depolarization produced by high external potassium, so that contractile inactivation in a depolarized muscle presumably involves some factor other than the site within the SR where calcium is located. An alternative explanation for the relaxation seen with sustained depolarization and the restoration of the ability of the fibers to contract with repolarization is that the calcium release mechanism itself is inactivated with sustained depolarization and reprimed during repolarization (Hodgkin and Horowicz, 1960b; Luttgau, 1963), Chandler and his coworkers have described a voltage-dependent charge movement in frog twitch fibers which appears to be a step in contractile activation (see Section VI.3); the time course of inactiva-

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213

tion and repriming of this charge movement is quite similar to the time course of inactivation and repriming of contractile activation (Chandler et al., 1975). (c) The kinetics o f contractile activation Adrian et al. (1969a) have examined the relation between membrane depolarization and contractile activation in frog twitch fibers under conditions where the amplitude and duration of a locally applied depolarization could be controlled by a voltage-clamp technique. At 4°C and with long depolarizing pulses (100 msec in duration), the contraction threshold was about - 5 2 mV, quite similar to the threshold for potassium contracture; progressively larger depolarizations were required as the pulse duration was decreased below 10 msec so that, with 2 msec pulses, the contraction threshold was + 40 inV. In this study, the contraction threshold was determined by visual observation with a relatively low-power microscope so that it was not possible to evaluate the extent of the fiber which was activated at threshold. Recently, however, quite similar results have been obtained with higher resolution microscopy which permitted detection of just-threshold contraction of only a few surface myofibrils (Costantin, 1974). The strength-duration relation for just-threshold mechanical activation by brief ( < 6 msec)depolarizations can be fitted by a simple hyperbolic curve; the amplitude of depolarization beyond a membrane potential of - 3 0 mV times the pulse duration, that is, the area of the pulse beyond - 30 mV, is constant and equal to about 120 mV x msec. This empirical "constant quantity relationship" has been employed to evaluate the temporal summation of two sub-threshold pulses. Thus, following a brief pulse whose area was about 90% of threshold, excitability fell to 50% of its initial value within about 4 msec at 4°C (Adrian et al., 1969); a second slower component of the decay of excitability proceeds with a half-time of 20 msec (Costantin, 1974). The time course of decay of mechanical excitability following brief depolarizations is dependent on the amplitude of the initial subthreshold pulse; with weak brief pulses (about 40% of threshold), the slow component of decay is much less prominent. Costantin (1974) has suggested that the initial rapid component of decay reflects deactivation of a voltage-dependent membrane process which regulates calcium release while the second slow component reflects uptake by the SR of the calcium released by the sub-threshold pulse. The half-time of this slow component is five to ten times faster than the half-time for twitch relaxation at a comparable temperature (Jewell and Wilkie, 1958), and it is of interest that the decay of the calcium transient following tetanic stimulation in aequorin-injected amphibian muscle is also considerably faster than the decay of muscle tension (Taylor, 1974). Adrian et al. (1969a) found that just sub-threshold long (100 msec) depolarizations produce a relatively small increase in mechanical excitability, since a brief pulse applied immediately afterwards must be at least 50% of threshold to elicit a visible contraction. Since a long pulse 1-2 mV larger is by itself able to elicit a contraction, this result implies that the increment of only 1-2 mV has a disproportionately large effect on activation, perhaps by triggering a regenerative release of activator calcium. An alternative possibility suggested by Adrian et al. is that SR calcium uptake might be inhibited by an increase in myoplasmic calcium over a critical concentration range. Thus a 1-2 mV increment in the amplitude of a long depolarizing pulse could produce only a slight increase in the rate of calcium release, and a disproportionate increase in myoplasmic calcium to a threshold level would result from a decrease in calcium uptake. However, the rate of calcium uptake both by isolated SR and by skinned fibers appears to increase monotonically with increasing calcium over a concentration range which is sub-threshold for contractile activation (Nakajima and Endo, 1973). At present a satisfactory explanation for this phenomenon is not available. It is possible, however, that this effect arises from the use of brief test pulses to define excitability. If membrane depolarization initiates a voltage and time dependent process which regulates calcium release (see the experiments of Chandler and his coworkers discussed below), the effects of a brief sub-threshold depolarization and of a long sub-threshold depolarization on the time course of the myoplasmic calcium concentration should be quite different. The brief depolarization might be expected to produce a transient increase in the calcium release rate so that the increase in myoplasmic calcium should

214

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continue for a few milliseconds following the pulse; with a long depolarization, on the other hand, the calcium release rate during the pulse should reach a steady level which just balances SR calcium uptake. A small test depolarization immediately following a brief pulse should transiently increase the myoplasmic calcium concentration by slowing the deactivation of the calcium release process; Costantin (1974) has found that such an interaction appears to occur between two brief pulses, since a 5 msec post-pulse to a membrane potential of - 70 mV decreased the magnitude of a brief pulse required to elicit a threshold contraction. 3. Electrical Events Associated with Contractile Activation

In frog twitch fibers, membrane depolarization to a potential range where the contractile mechanism is activated results in three distinct voltage-dependent conductance changes: (1) a decrease in the resting potassium conductance (inward rectification), (2) a transient increase in sodium conductance which is responsible for the upstroke of the action potential, and (3) a delayed increase in potassium conductance (delayed rectification) which is responsible for action potential repolarization. None of these three processes, however, appears to be directly related to the mechanism by which contractile activity is initiated. Thus, inward rectification can be seen in muscles in which the normal calcium release mechanism has been inactivated by prolonged depolarization (Nakajima et al., 1962), and the transient increase in sodium conductance can be abolished by TTX without inhibition of contractile activation in response to depolarization. A number of similarities between delayed rectification and contractile activation has been reported. The time course of inactivation and repriming of these two processes is quite similar (Heistracher and Hunt, 1969a, b). Furthermore, just-threshold activation of contraction and of delayed rectification both occur at a membrane potential of about - 50 mV, and a variety of anions and divalent cations which are thought to act by altering the membrane surface charge produce similar shifts in both thresholds (Costantin, 1968; Kao and Stanfield, 1968, 1970). Nevertheless, it is clear that contractile activation is not simply related to an outward potassium current during depolarization. Thus, contractile activation can be produced in fibers in which 90% of the potassium current through the delayed rectifier has been blocked by TEA (Stanfield, 1970). Moreover, in potassium-depolarized fibers in which contractile activation and delayed rectification have been reprimed by hyperpolarization, depolarization produces a mechanical response and an inward potassium current through the delayed rectifier channel (Heistracher and Hunt, 1969b). In an attempt to detect an electrical signal uniquely related to contractile activation, Schneider and Chandler (1973) have examined the ionic currents in voltage-clamped frog twitch fibers exposed to a bathing medium which was designed to eliminate the known voltage dependent conductance changes. Inward rectification was blocked by 5 mM RbC1 (Adrian, 1964), and TTX and 117.5 mM TEA + were employed to block the active sodium and potassium conductances. Muscle movement due to contraction was suppressed by making the solution hypertonic by addition of sucrose. Step depolarizations were applied to the muscle fiber by a three microelectrode voltage-clamp technique, and the currents produced by two depolarizations of equal amplitude, one from the resting potential into the range where contraction is normally initiated and the other from a hyperpolarized membrane potential into the range of the resting potential, were compared. Although there was no sign of a large conductance increase following the depolarization into the range for contractile activation, such as might be expected if a decrease in the SR-T junction resistance were associated with activation, small differences between the two currents were seen. The depolarizing step from the resting potential produced a small transient outward current over and above that seen following the depolarizing step from a hyperpolarized potential level, and repolarization resulted in a small transient inward current. When the duration of the depolarizing pulse was increased, the outward current decayed to a small steady value, while the amplitude of the inward current tail on repolarization increased to a constant value. These results are not readily accounted for by a time-dependent change in membrane conductance which allows the flow of ionic current across the membrane. Thus, if the transient outward current were due to an early increase and subsequent

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decrease in conductance with time, the current tails on repolarization would be expected to decrease with prolongation of the depolarizing pulse. On the other hand, if the slow decrease in net outward current during depolarization were due to a slow increase in inward current, the current tails on repolarization would be expected to increase in a regular manner as the outward current decayed with prolongation of the depolarizing pulses. Such a relation between the outward transient and the inward tail was not observed. With depolarizations sufficiently brief so that no decline in outward current was seen, an appreciable tail current was still recorded. When the amplitude and duration of the depolarizing step were varied, the area of the "on" transient and of the "off" transient were equal, that is, depolarization and repolarization resulted in the movement of a constant quantity of charge across or within the cell membrane. A charge movement with qualitatively similar characteristics but a much more rapid time course has been detected in voltage-clamped squid axons (Armstrong and Bezanilla, 1973); since this charge movement appears to be associated with the opening and closing of the activation "gates" for the sodium channels, the term "gating current" has been applied to this phenomenon. Schneider and Chandler found that the total charge movement with prolonged depolarizations in frog twitch fibers was related to membrane potential by a sigmoid curve rather similar to the relation between contracture tension and membrane potential reported by Hodgkin and Horowicz (1960b), and on this basis they proposed that this gating current reflected the displacement within the tubule membrane of a charged molecule which controls the release of calcium from the SR. One attractive aspect of this model is that it suggests that calcium release by the SR is localized to the SR-T junction and thus could account for the Huxley and Taylor (1958) observation that contractile activation following T-tubule depolarization is restricted to the half-sarcomere immediately adjacent to the SR-T junction. Subsequent studies by Chandler and his co-workers (Chandler et al., 1975) have provided additional evidence relating these gating currents to contractile activation. Thus the gating current was inactivated with maintained depolarization, and the time required for inactivation was similar to the time course of potassium contracture with maintained depolarization. Repriming of the gating current by membrane repolarization resulted in a just detectable mechanical response when about 8-20Vo of the maximal available charge movement was restored; repriming of the gating current at 1.5°C proceeded exponentially with a time constant of 21-53 sec. It is, of course, not surprising that sufficiently sensitive recording methods can detect the movement of charged particles within complex biological membranes, and it should be emphasized that at present the idea that these intra-membrane charge movements represent a gating step in contractile activation rests largely on the parallel behavior of these two phenomena. An alternative possibility is that this charge movement regulates, not calcium permeability, but potassium permeability in the muscle membrane. As noted previously, the time course of inactivation and repriming of delayed rectification and contractile activation are quite similar. Moreover, Chandler and his co-workers (1974) have found that the charge movement and the activation variable for delayed rectification show an identical steady-state dependence on membrane potential. The sole difference between these two processes which has thus far been detected is that the rate constant for the decay of the charge movement appears to be one and two-thirds to four times faster than the rate constant for potassium activation. (a) Time course of the charge movement Although the charge movement appears to decay exponentially with a voltage-dependent rate constant, the charge movement transient frequently shows a distinct rising phase, especially with only slightly supra-threshold depolarizations. While it is possible that this rising phase is an inherent property of the charge movement, an alternative explanation is that it reflects the time required for depolarization to spread down the T-system. In hypertonic solutions in which, as noted earlier (see Valdiosera et al., 1974), a large increase in T-tubule lumen resistance develops, the time-to-peak of the charge movement can be as long as 10 msec. This time delay poses a significant difficulty in any attempt at a quantitative analysis of the kinetics of the charge movement. A much shorter time-to-peak is

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consistently found in isotonic solutions, but the full range of depolarization cannot be explored in these solutions because of muscle movement. 4. Contractile Activation in Skinned Muscle Fibers (a) Depolarization of the internal membrane system Although it might be anticipated that disruption of the surface membrane of a muscle fiber would lead to a sustained depolarization of the T-tubule membranes and thus to inactivation of the calcium release mechanism, this does not seem to be the case. Costantin and Podolsky (1967)have shown that depolarization of the internal membrane system, induced either by changes in the ionic concentration of the myoplasm or by application of longitudinal current to the skinned fiber, results in contractile activation. The contractile response to electrical stimulation was abolished when the skinned fiber was prepared from a muscle exposed to a cardiac glycoside, and it was proposed that the T-tubules resealed following disruption of the surface membrane and that active sodium-potassium exchange across the internal membranes restored electrical excitability by reestablishing a potential gradient across the internal membrane system. Although the element of the internal membrane system which was responsive to depolarization could not be determined in these studies, the magnitude of the applied longitudinal currents were suffienciently small so that the depolarization produced in the small transversely-oriented T-system was estimated to be less than 1 mV. Significant changes in membrane potential could be expected only if the responsive element were continuous over many sarcomeres, and it was proposed that appreciable longitudinal continuity of the internal membrane system was present in skinned fibers, either between adjacent terminal cisternae of the SR itself or perhaps through low resistance junctions between each of the two cisternae and the central Ttubule at the triad. Either of these alternatives implies that the element of the internal membrane system which was responsive to depolarization was the SR itself. The subsequent work of Eisenberg (1972), however, has shown that longitudinally disposed elements of the T-system connecting the transverse tubules of adjacent sarcomeres are not uncommon and could produce the longitudinal continuity required for appreciable depolarization of the T-system in response to applied current. Recently, Nakajima and Endo (1973) have produced contractile activation by a depolarizing stimulus in partially skinned muscle fibers in which the continuity of the T-system with a portion of the surface membrane is believed to be intact. Since the T-tubule membrane in these partially skinned fibers was presumably depolarized and thus refractory to depolarizing stimuli, Nakajima and Endo suggested that calcium release was due to a direct depolarization of the SR. The depolarizing stimulus employed by Nakajima and Endo (1973) was an alteration in the ionic concentration of the myoplasm (an increase in myoplasmic chloride or a decrease in myoplasmic potassium), and the contractile response to this stimulus was not altered by treatment with cardiac glycosides. The possibility does exist that alterations in myoplasmic ion concentrations act, not by membrane depolarization but rather by altering the capacity of the SR for calcium. Although Nakajima and Endo (1973) have demonstrated some inhibition of SR calcium uptake in the presence of chloride, the effect appears to be too small to explain the apparently large increase in myoplasmic calcium elicited by elevation of myoplasmic chloride. Moreover, an inhibition of SR calcium uptake, by itself, would not account for the phasic nature of the chlorideinduced contracture. (b) Calcium-induced calcium release Under appropriate experimental conditions, calcium release from the SR of skinned muscle fibers can also be elicited by an increase in myoplasmic calcium (Endo et al., 1970; Ford and Podolsky, 1970). A skinned segment from a frog twitch fiber which is exposed to a bathing medium containing 10-4M calcium develops force relatively slowly; the increase in force is preceded by a latent period of many seconds which has been attributed, at least in part, to uptake of calcium by the SR (Ford and Podolsky, 1972a) and possibly also to a slow rate of activation at low calcium concentrations (Endo, 1973). If the fiber

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segment is preloaded with calcium in a Ca-EGTA buffered medium with a free Ca + + of 1 0 - 6 ~ exposure to a free Ca ++ of 10-'*M initiates a rapid and transient contraction, followed by a secondary slow rise of force to a fina ! steady level; the initial response can be interrupted by high concentrations of EGTA and thus appears to result from an increase in myoplasmic calcium. The rapid onset of the initial response suggested that it might be due to a regenerative release of calcium, presumably from the SR, and subsequent tracer flux studies in similarly treated preparations have confirmed that calcium is released from fiber stores during the brief contraction (Ford and Podolsky, 1972b). The final level of force developed in 10-4 M calcium is well-maintained for prolonged periods, and there is no obvious explanation for the transient relaxation following the initial contraction in calcium-loaded skinned fibers. (c) Relevance of skinned fiber results to activation of intact muscle Although isolated SR vesicles are capable of actively sequestering calcium it has not been possible to demonstrate a release of calcium in such preparations by stimuli which might play a role in the normal activation process. Since the physiological properties of the internal membrane system should be more nearly normal in skinned fibers than in isolated SR vesicles, the finding that the SR of such preparations appears capable of a depolarization-induced or a calcium-induced calcium release is of considerable interest. It is by no means clear, however, that these phenomena are involved in the normal activation process. Thus while SR depolarization may produce calcium release in skinned fibers, there is considerable electrophysiological evidence to indicate that, in intact cells, current flow through the SR-Tjunction is much too small to produce a significant depolarization of the SR membranes (Hodgkin and Horowicz, 1960a; Falk and Fatt, 1964) even with surface membrane depolarizations which produce contractile activation (Chandler et al.. 1975). Similarly, the phenomenon of calcium-induced calcium release has no obvious counterpart in intact muscle cells. Thus the micro-injection of calcium into intact cells results in a highly localized contraction with no evidence of a propagated response (Niedergerke, 1955). Furthermore, the small calcium influx associated with the action potential in frog twitch fibers (Bianchi and Shanes, 1959) is not required for contractile activation, since a mechanical response can be elicited even after prolonged immersion in a virtually calcium-free solution (Costantin, 1971; Armstrong et al., 1972). The gradation of contractile activation with graded increases in membrane depolarization (Hodgkin and Horowicz, 1960b; Costantin and Taylor, 1973) is also inconsistent with a regenerative mechanism of SR calcium release. One possibility which should not be overlooked is that calcium release in skinned fibers results from alterations in the properties of the internal membrane system. In freshly skinned frog twitch fibers, the organization of the internal membrane system appears to be well-preserved, including the SR-T junction which presumably serves as a critical link in the normal activation process (Franzini-Armstrong, 1971). In such fibers, electrical responsiveness, which probably results from T-tubule depolarization, is abolished by cardiac glycoside treatment, and a regenerative calcium-induced calcium release is not seen (Podolsky and Costantin, 1964). Following immersion of a skinned fiber in an aqueous relaxing solution, however, both a glycoside-insensitive depolarization-induced calcium release and a regenerative calcium-induced calcium release can be elicited. Electron microscopy of such preparations reveals that in many triads the T-tubules and the lateral sacs of the SR have become separated (see Discussion in Costantin and Taylor, 1973). 5. Contractile Activation without Membrane Depolarization Although the normal stimulus to activation is a depolarization of the muscle membrane, spontaneous contractions in both vertebrate and crustacean muscles have been found under a variety of conditions. Such contractions involve small segments of the fiber ranging from a few sarcomeres to a few hundred micrometers in extent and may sometimes propagate along the length of the fiber. Propagation is extremely slow (a few millimeters per second) so that it appears unlikely that these contractions are elicited by the spread of depolarization along the surface membrane. Spontaneous slow contraction waves were I J'll.

~-~,.'2

H

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reported as early as 1840 by Bowman in skate muscle; they have been seen in crustacean muscle (Fatt and Ginsborg, 1958), and in frog twitch fibers during recovery from hypertonic glycerol treatment (Krolenko et al., 1967). In the latter case microelectrode recordings have confirmed that such waves are not associated with a change in membrane potential (Zachar et al., 1972). As discussed earlier, a small increase in myoplasmic calcium can, under certain conditions, trigger calcium release from the SR, but it has not been possible to initiate these slow waves by the iontophoretic injection of calcium (Niedergerke, 1955; Zachar et al., 1972). Similar contractions have been induced in frog twitch fibers by the application of caffeine or of quaternary ammonium compounds (Marco and Nastuk. 1968), and in this instance also the absence of a change in transmembrane potential associated with activation has been confirmed. Slow contraction waves have also been reported in skinned frog muscle fibers (Natori, 1965; Costantin and Podolsky, 1967). It is not clear that all of these slow contraction waves share a common mechanism. They are of interest, however, in indicating that some signal for activation can be transmitted, presumably within the internal membrane system, without depolarization of the surface membrane. 6. Pharmacological Alterations of Contractile Activation Many pharmacological agents have been shown to affect the relation between the membrane potential and mechanical activation in vertebrate twitch fibers, and considerable attention has been focused on these agents in the hope that they might provide information concerning the basic mechanism of calcium release. For the most part, however, these efforts have been unsuccessful; the observed alterations in the mechanical response can usually be explained by the effect of these agents on a known step in the activation process. For example, the disappearance of the twitch following exposure to a hypertonic glycerol solution is readily explained by disruption of the T-system, while the "uncoupling" effect of the complete removal of extracellular divalent cations, at least in vertebrate twitch fibers, can be accounted for by membrane depolarization and a resultant inactivation of the calcium release process. (a) Dantrolene and formaldehyde Two agents which may act directly on contractile activation are dantrolene sodium and formaldehyde. Dantrolene, which markedly depresses the muscle twitch without affecting the action potential (Ellis and Bryant, 1972), has been shown to change the shape of the strength-duration relation for contractile activation in TTX-treated frog twitch fibers; the threshold for brief depolarizations is shifted to more positive values of membrane potential while the threshold for long depolarizations is essentially unchanged (Gilly and Costantin, 1974). Complete block of contractile activation has not been achieved, presumably because dantrolene is only sparingly soluble in aqueous media. Formaldehyde, in 10 mM concentrations, abolishes the contractile response to membrane depolarization in frog twitch fibers without eliminating delayed rectification; this contractile "uncoupling" is apparently not due to disruption of T-tubule continuity with the surface membrane (Argibay and Hutter, 1973). It has not yet been established whether formaldehyde blocks calcium release or myofilament interaction in the presence of calcium. (b) Twitch potentiation The tension output of the twitch in frog skeletal muscle can be considerably increased by a wide variety of pharmacological agents. These agents have been divided into two general types (Sandow et al., 1965): (1) type A potentiators, such as NO3, SCN-, and other foreign anions which shift the contractile threshold toward the resting potential, presumably by altering the surface charge on the muscle membrane (Hodgkin and Horowicz, 1960c; Kao and Stanfield, 1968), and (2) type B potentiators, such as TEA +, Zn + +, and UO~- + which prolong the duration of the action potential by blocking delayed rectification (Kao and Stanfield, 1970). The effect of both A and B type potentiators is thus to increase the time during which the action potential depolarizes the cell beyond the contractile threshold, and it is thought that this effect, by increasing the amount of activator calcium

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released by the action potential, is the basis of their pharmacological action on the muscle twitch. This idea is supported by the finding that these agents enhance the rate of tension development within the first few milliseconds of the twitch (Taylor et al., 1969, 1972), while the rapid onset and reversal of potentiation with alterations of the extracellular medium (Hodgkin and Horowicz, 1960c; Sandow and Isaacson, 1966) also suggests that their mode of action involves an effect on the surface and T-tubule membranes rather than, for example, the presumably less accessible SR membranes. A direct effect of both type A and B potentiators in inhibiting calcium uptake by SR vesicles has been reported, however (Carvalho, 1968), and it is possible that a prolonged duration of calcium action during the twitch is also involved in the potentiating effect of these agents. (c) Caffeine contractures Reversible contractures can be induced in both vertebrate (Axelsson and Thesleff, 1968; Luttgau and Oetliker, 1968)and crustacean (Caldwell and Walster, 1963) skeletal muscle by millimolar concentrations of caffeine and the related methylxanthines, theophylline and theobromine. In normal muscle, these contractures are not accompanied by a change in membrane potential, and caffeine contractures can be elicited both in detubulated muscle fibers (Howell, 1969) and in fibers in which the normal calcium release mechanism has been inactivated by the prolonged depolarization. Thus, it appears that caffeine does not act on the link between membrane depolarization and calcium release but instead produces activation by release of calcium from intracellular stores. This idea has been supported by the finding of a direct effect of caffeine on the SR (Weber, 1968; Weber and Herz, 1968). In SR vesicle preparations which had accumulated appreciable amounts of calcium, treatment with caffeine released up to 40Yoof the accumulated calcium; caffeine was without effect in less heavily loaded vesicles. Caffeine is thought to act by inhibiting SR calcium uptake, and the observed release has been attributed to a transient imbalance between passive efflux and active uptake with the subsequent establishment of a lower steady level of intravesicular calcium. In subcontracture concentrations, caffeine also affects the properties of the surface and tubular membranes. Thus caffeine shifts the contraction threshold in frog twitch fibers toward the resting potential, acting as a type A potentiator (Taylor et al., 1969), and in crustacean muscle, caffeine induces calcium action potentials (Chiarandini et al., 1970). It is not known whether these effects are related to the action of caffeine on the SR. (d) H ypertonic solutions Exposure of frog twitch fibers to solutions made two and a half to three times isotonic with non-penetrating solutes results in a disappearance of the muscle twitch with little apparent change in the muscle action potential (Hodgkin and Horowicz, 1957). Tetanic tension in these solutions is reduced but not abolished, and the disappearance of the twitch response has been attributed to a marked decrease in the velocity of shortening in hypertonic solutions (Howarth, 1958; Podolsky and Sugi, 1967). The decrease in contractile response arises primarily from a direct effect of the increase in intracellular ionic strength on the contractile mechanism, and, at least with moderate hypertonicity, the activation process appears to be little altered. At tonicities two to two and a half times normal, the maximal contracture tension elicited by caffeine and the maximal tetanic tension are equally reduced, and a disproportionate fall in tetanic tension is seen only at three times normal tonicity (Gordon and Godt, 1970). Recent measurements of heat production in frog semitendinosus muscle have shown that, in muscles progressively stretched to decrease filament overlap and thus the ATP-splitting associated with actin-myosin interaction, a fixed quantity of activation heat is released following each action potential (Homsher et al., 1972; Smith, 1972); at 0°C, this activation heat, which presumably arises from the cyclic release and uptake of calcium, is essentially unchanged at tonicities up to two and a half times normal(Smith, 1972). At three times normal tonicity, a decrease in activation heat is seen, so that these results also suggest that, with extreme hypertonicity, a direct effect on SR calcium release is produced.

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The effect of hypertonic solutions on the contractile response is of considerable physiological interest. Since the absence of movement is a requirement for maintained impalement of the cell by microelectrodes, hypertonic solutions have been employed in studies of the electrical properties of muscle cells over the potential range in which a vigorous contractile response is normally elicited (Adrian et al., 1970a; Schneider and Chandler, 1973). The results cited above indicate that, at least with moderate hypertonicity, the mechanical response can be virtually eliminated with little qualitative alteration in the electrophysiological behavior of the muscle cell. VII. C O N C L U S I O N S

Many of the steps in contractile activation of skeletal muscle are now well-established. Muscle contraction and relaxation are mediated by an increase and a subsequent decrease in myoplasmic calcium ion concentration; this calcium is released from an intracellular store, the sarcoplasmic reticulum (SR), and is then resequestered by an active transport process located within the SR membranes. In some invertebrate skeletal muscle cells, an influx of extracellular calcium during membrane depolarization may also play a role in contractile activation. The process of contractile activation is initiated by surface membrane depolarization, and the spread of this surface depolarization to involve the specialized junctions between the SR and surface-connected membrane elements appears to be a necessary step in SR calcium release. The major unresolved problem in this activation sequence is the mechanism by which depolarization of the surface-connected junctional membranes leads to an increase in SR calcium permeability. At present, the most likely possibility is that a voltage-dependent displacement of a charged molecule within the surface-connected element of the junction controls the calcium permeability in the adjacent SR membrane. REFERENCES ADRIAN,R. H. (1964) The rubidium and potassium permeability of frog muscle membrane. J. Physiol. 175, 134159. ADRIAN,R. H. (1969) Rectification in muscle membrane. Pro#. Biophys. Molec. BioL 19, 341-369. ADRIAN, R. H. and ALMERS,W. (1974) Membrane capacity measurements on frog skeletal muscle in media of low ion content. J. Physiol. 237, 573-605. ADRIAN,R. H. and FREYGANG,W. H. (1962) The potassium and chloride conductance of frog muscle membrane. J. Physiol. 163, 61-103. ADRIAN,R. H. and PEACHEY,L~ D. 0973) Reconstruction of the action potential of frog sartorius muscle. J. Physiol. 235, 103-131. ADRIAN,R. H., CHANDLER,W. K. and HODGKIN,A. L. (1969a) The kinetics of mechanical activation in frog muscle J. Physiol. 204, 207-230. ADRIAN,R. H., COSTANTIN,L. L. and PEACHEY,L. D. (1969b) Radial spread of contraction in frog muscle fibres. J. Physiol. 204, 231-257. ADRIAN, R. H., CHANDLER,W. K. and HODGKIN, A. L. (1970a) Voltage clamp experiments in striated muscle fibres. J. Physiol. 208, 607-644. ADRIAN,R. H., CHANDLER,W. K. and HODGKIN,A. L. (1970b) Slow changes in potassium permeability in skeletal muscle. J. Physiol. 208, 645-668. ALtCmRS,W. (1972a) Potassium conductance changes in skeletal muscle and the potassium concentration in the transverse tubules. J. Physiol. 225, 33-56. ALgiERS, W. (1972b) The decline of potassium permeability during extreme hyperpolarization in frog skeletal muscle. J. Physiol. 225, 57-83. ARGIBAV,J. A. and HUTXER,O. F. (1973) Voltage-clamp experiments on the inactivation of the delayed potassium current in skeletal muscle fibres. J. Physiol. 232, 41-43P. ARMSTRONG,C. M. and BEZANILLA,F. (1973) Currents related to movement of the gating particles of the sodium channels. Nature 242, 459-461. ARMSTRONG,C. M., BEZANILLA,F. M. and HOROWICZ,P. (1972) Twitches in the presence of ethylene glycol bis (fl-aminoethyl ether)-N,N'-tetraacetic acid. Biochim. biophys. Acta 267, 605-608. ASHLEY,C. C. and RIDGWAY,E. G. (1970) On the relationships between membrane potential, calcium transient and tension in single barnacle muscle fibres. J. Physiol. 21}9, 105-130. AXELSSON,J. and THESLEFE,S. (1968) Activation of the contractile mechanism in striated muscle. Acta physiol. scand. 44, 55-66. BARRY,P. H. and ADRIAN,R. H. (1973) Slow conductance changes due to potassium depletion in the transverse tubules of frog muscle fibres during hyperpolarizing pulses. J. Memb. Biol. 14, 243-292. BASTIAN,J. and NAKAJIMA,S. (1974) Action potential in the transverse tubules and its role in the activation of skeletal muscle. J. Gen. Physiol. 63, 257-278. BEZANILLA,F., CAPUTO, C. and HOROWICZ,P. (1971) Voltage clamp activation of contraction in short striated muscle fibres of the frog. Acta Cient. Venez. 22, 72-74.

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L. and TAYLOR,S. R. (1973) Graded activation in frog muscle fibers. 2. Gen. Physiol. 61, 424-443. DULHUIqTY,A. F. and FRANZINI-ARMSTRONG,C. (1974) Caveolae as specialized structural components of the surface membrane of skeletal muscle. Fedn Proc. 33, 401. DULHUNTY,A. F. and GAGE, P. W. (1973) Differential effects of glycerol treatment on membrane capacity and excitation-contraction coupling in toad sartorius fibres. J. Physiol. 234, 373--408. DYDYSSr,A, M. and WILKIE,D. R. (1963) The osmotic properties of striated muscle fibres in hypertonic solutions. J. Physiol. 169, 312-329. EBASm, S. and EsDO, M. (1968) Calcium ion and muscle contraction. Proo. Biophys. Molec. Biol. 18, 123-183. EDWARDS,C., CHICHIBU,S. and HAGIWARA,S. (1964) Relation between membrane potential changes and tension in barnacle muscle fibers. J. Gen. Physiol. 48, 225-234. EISENBERG,B. R. (1972) Three dimensional branching of the T-system in frog sartorius muscle. J. Cell Biol. 68a. EISENBERG,B. and EISENBERG,R. S. 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(1973) Length dependence of activation of skinned muscle fibres by calcium. In The Mechanism of Muscle Contraction. Cold Spring Harb. Syrup. Quant. Biol. 37, 505-510. ESDO, M., TANAgA,M. and OGAWA,Y. (1970) Calcium induced release of calcium from the sarcoplasmic reticulure of skinned skeletal muscle fibres. Nature, Land. 228, 34-36. FAHRENBACH,W. H. (1965) Sarcoplasmic reticulum: ultrastructure of the triadic junction. Science 147, 1308-1310. FALK,G. and FATr, P. (1964) Linear electrical properties of striated muscle fibres observed with intracellular electrodes. Proc. R. Soc. B 160, 69-123. FAr'r, P. and GlsSm3RO, B. L. (1958) The ionic requirements for the production of action potentials in crustacean muscle fibres. J. Physiol. 142, 516-543. FAT'r, P. and KArz, B. (1951) An analysis of the end-plate potential recorded with an intracellular electrode. J. Physiol. 115, 320-370. FAT'r, P. and KArZ, B. (1953) The electrical properties of crustacean muscle fibres. J. Physiol. 120, 171-204. FAWCE'Ur,D. W. and McNuTr, N. S. (1969) The ultrastructure of the cat myocardium~I. Ventricular papillary muscle. J. Cell. Biol. 42, 1-45.

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