J. Physiol. (1975), 244, pp. 1-14 With 7 text-ftgurem Printed in Great Britain
LIGHT SCATTERING ASSOCIATED WITH TENSION CHANGES IN THE SHORTRANGE ELASTIC COMPONENT OF RESTING FROG'S MUSCLE
By F. W. FLITNEY From the Department of Physiology, University of St Andrews, St Andrews
(Received 19 November 1973) SUMMARY
1. A study has been made of some optical and associated mechanical properties of resting frog's sartorius muscles in isotonic and hypertonic solutions. Tension and transparency changes accompanying small alterations of muscle length (< 1-5 %) were recorded simultaneously. 2. The form of the transparency change is complex. It has three phases, two of which occur during the length change and the third (delayed phase) after it is complete. Directional recording of the response reveals both scattering and diffraction components. 3. The change of light scattering is associated with tension changes in the short-range elastic component (SREC) of the muscle. Its magnitude is related to the stiffness of the SREC; both increase when the osmotic strength of the external solution is raised and when muscle length is increased. 4. The change of light scattering is not much affected by the velocity of the applied length change. This is true also of the mechanical stiffness of the SREC. 5. The origin of the diffraction change is not known. 6. It is concluded that the scattering effect is caused by conformational changes in the SREC. INTRODUCTION
The experiments to be described are concerned with certain of the optical and mechanical properties of resting frog's muscle. It has been known for many years (A. V. Hill, 1949) that a frog's sartorius at rest exerts a small force, even when held at lengths far below its standard length in the body. D. K. Hill (1968) has shown that part of this resting tension, the filamentary resting tension (or FRT), is due to a weak form of 1-2
F. W. FLITNEY interaction between the actin and myosin filaments; the tension is generated by a small population of activated cross-bridges which form relatively stable linkages between the filaments, and which endow the muscle with short-range elastic properties. He referred to them collectively as the short-range elastic component, or SREC. The purpose of the present study was to examine the possibility that the existence of comparatively long-lived linkages between the overlapping filaments might have some bearing on the transparency changes produced by small stretches of a resting muscle (D. K. Hill, 1949, 1953). A preliminary account of this work was presented to the Physiological Society (Flitney, 1973). 2
METHODS were made with All experiments the sartorius muscle of Rana temporaria. The muscles were kept in oxygenated Ringer solution having the following composition (mm): NaCl 96; KC1, 5; CaCl2, 4; sodium phosphates, 5, pH 6-9. The majority of experiments were with muscles bathed in solutions made hypertonic by the addition of sucrose. The osmotic strength of these solutions is expressed throughout as a multiple of the value for the above solution (e.g. Ringer solution containing 5 % (w/v) sucrose is referred to as 1-65 x NR, with 8% sucrose, 2-0 x NR, etc.). Stimulation was effected through two platinum electrodes (dimensions, 5 mmn x 20 mm) spaced 12 mm apart, using square pulses of 1 ms duration and strength 10 V/cm. A conditioning stimulus was given to the muscle 2-3 min prior to applying the length changes.
Apparatus Muscle chamber. The muscle is suspended vertically in a cylindrical glass chamber enclosed in a tightly fitting aluminium jacket (Fig. 1). Its tendon is attached to a tension recorder by a fine chain, and the pelvic bone is retained by a loop formed at one end of a platinum rod. The rod is connected at its lower end to an electromagnetic displacement transducer. The chamber is cooled by three thermoelectric modules (type 12-15G, De La Rue Frigistors Ltd) clamped to the aluminium jacket. Length changes. Changes of muscle length are produced by a moving-coil electromagnetic unit (Ling-Altec vibrator, model 201) controlled by a servo amplifier with positional and velocity feed-back. Displacement is monitored by a photo-electric device comprising a light source, a vane attached to the connecting rod and two silicon photodiodes (Ferranti, type MS 1AE). The system is driven by voltage signals of the 'ramp-and-hold' type, supplied by a Servomex LF 141 waveform generator. Tension recorder. A variable capacitance gauge similar to that described by A. F. Huxley & Simmons (1968) is used to record muscle tension. The compliance of the unloaded transducer at the point of attachment of the muscle is 0 3 ,sm mN-1. Tension is displayed on one channel of a dual beam oscilloscope.
Optical system An area of muscle (approx. 1 mm diam., and located 10 mm from the pelvic end) is illuminated with light from a tungsten-halogen lamp (12 V, 100 W) run off a heavyduty battery. The light is passed through a lens to give a nearly parallel beam (divergence, 30) and then collimated by a circular aperture. Light emerging from the muscle reaches the photodetector through a two-stage fibre-optics light guide. The first part is a rigid, coherent bundle of fibres (type IG 1;
LIGHT SCATTERING BY SREC OF FROG'S MUSCLE
3
Barr & Stroud Ltd) mounted on a micrometer-actuated sliding stage (Research Instruments Ltd). Its distal end is immersed in the fluid bathing the muscle and it can be accurately positioned to collect light from any desired direction. The spatial distribution of light entering this first stage is preserved, owing to the coherence of the glass fibres, and an image is formed at the proximal end of the bundle where it joins the second section. The latter is a flexible, non-coherent collection of fibres (type LG 5, Barr & Stroud Ltd) which leads to the photo-detector housing.
L
E
M
L
T
CB R
I
Fig. 1. Muscle chamber and optical system for recording changes of transparency and tension of frog's muscle. The glass chamber (C) is supported in an aluminium block (CB) cooled by three thermomodules (T) clamped to its surface. The muscle (M) is attached at its pelvic end to a rod (R) connected to the moving element of an electromagnetic length transducer (L) through a seal (S) in the floor of the chamber. The tibial tendon is connected to the tension recorder (P) with a length of fine chain. A rigid light guide (LG) mounted on a moving stage (ST) collects the light and transmits it along a second, flexible section (F) to the photodetector. An aperture (A) is interposed between the two sections of light guide to limit the light reaching the photomultiplier. Two platinum stimulating electrodes (E) flank the muscle.
The cone of light reaching the photomultiplier is limited by a circular aperture located between the two sections of the light guide. A small aperture, 200 jtm diameter, is employed where high angular definition is required, as for example, when locating the intensity maxima of the diffracted rays (see below), and a larger one, up to 3 mm diameter, when the light intensity is low and resolution is not the limiting factor. Intensity changes are detected with a 12 stage photomultiplier tube (9558C, EMI Electronics Ltd), operated from a Brandenburg 472R stabilized power supply, and displayed on the oscilloscope. In all records an upward deflexion of the trace indicates an increase of light intensity; the change of intensity is expressed as a percentage of the resting (transmitted) light level.
4
F. W. FLITNEY
Sarcomere lengths. The A and I bands within its sarcomeres causes a muscle to act as a diffraction grating and the light beam is split up into a series of diffracted rays (Sandow, 1936). At the start of each experiment the sarcomere length is measured directly with the muscle set up in the chamber at its standard length. This is done by measuring the vertical separation of the 1st and 0 order spectra at two specified distances from the muscle, moving the tip of the light guide vertically in the plane containing the incident beam until the intensity maxima corresponding with the two rays are located. The angle of diffraction is obtained from the micrometer readings and used to calculate the sarcomere length (Blinks, 1965). Terminology. The mechanical stiffness of the short-range elastic component, denoted by E, is expressed throughout in the form of a Young's modulus (N.m-2). The standard length of the muscle in the body is denoted by 1l. RESULTS
The form of the optical response is determined by changes in the amount of light scattered by the muscle and in that diffracted by the striations. The ability to distinguish between these two components is crucial for interpreting the records and the method by which the distinction is made will be outlined before presenting the results. Reference is also made to the effects of hypertonicity on the optical and mechanical properties of a resting muscle, since the majority of the experiments were made using solutions made hypertonic by the addition of sucrose. These two points are discussed below. Directional recording of the optical change. When a frog's sartorius is illuminated with a parallel beam, both the diffracted and non-diffracted light suffers scattering, predominantly in the planes which lie at right angles to the long axis of the fibres. There is very little true absorption of light in the visible part of the spectrum, at wave-lengths greater than about 250 nm (Hill, 1959b), and absorption changes are not likely to affect the response to an appreciable extent. Hill was able to recognize scattering and diffraction components in his optical traces by recording changes of light intensity from selected regions of the diffraction pattern. His method of 'directional' recording, and the terminology he employed, is used here also. Reference should be made to his paper for a detailed explanation. Briefly, the distinction is made by comparing records taken in the following four positions. Zero order, direct. The tip of the light guide is placed directly in line with the incident beam and so receives undeviated light. In this position an increase in either the scattering or diffracting power of the muscle causes the light intensity to fall. Zero order, lateral. The light guide is moved laterally to a point along the zero order line. An increase of diffraction has the same sign as at position zero, direct, but an increase of light scattering shows reversal at some critical angle, and at this and greater lateral angles the two effects oppose one another. First order, direct. The light guide is positioned to record from the first order spectrum and is located in the vertical plane which contains the incident beam. Scattering and diffraction effects have a different sign: an increase of diffraction causes the light intensity to increase, whereas an increase of scattering causes it to fall. First order, lateral. The light guide is positioned at some lateral angle along the first order diffraction line. Scattering and diffraction changes have the same sign, and the combined effect is to give a response which is the inverse of that seen at position zero, direct.
LIGHT SCATTERING BY SREC OF FROG'S MUSCLE
5
Effect of hypertonicity on optical and mechanical properties of resting muscle. In hypertonic solution the FRT and stiffness of the SREC are both increased (Hill, 1968). The optical changes accompanying stretch or release of a muscle are also enhanced in hypertonic solution, and for this reason the majority of the experiments were made using Ringer solution containing varying amounts of sucrose. The changes in both the optical and mechanical properties of a muscle induced by hypertonic solutions are entirely reversible.
Characteristic tension and optical responses to imposed length changes Fig. 2A shows the tension and transparency changes resulting from a linear 'ramp-and-hold' stretch (not displayed) of 400 /tm. Tension record. The increase of tension is due largely to the elastic resistance of the SREC. The early part of the record is dominated by this A
10 mN[
;
200 msec
t
2%[ 400 msec 2. the a effect of stretch (400 /sm; velocity, 1600 Am.s-1) on the Fig. A, tension (upper trace) and transparency (lower trace) of a frog's sartorius. Arrows indicate onset and end of length changes. Spots on record indicate final levels for tension and light intensity. Light guide in position zero, direct, located 15 mm away from the muscle. Aperture, 3 mm. Muscle sarcomere length, 2-75 /sm. Temp. 20 C. B, transparency changes due to stretch (S) and release (R) of a muscle. Tension change not displayed. Sarcomere length, 2 5 /am. Applied length change, ± 200 jum, velocity 1000 ,um . s-1. Temp. 10 C. Osmotic strength for A and B, 1-65xNR.
6 F. W. FLITNEY component. The 'elastic limit' of the SREC, defined arbitrarily as the point at which the slope of the tension record falls to 25 % of its initial value, is reached for a displacement of 60 jpm, equivalent to a relative sliding movement of the filaments of 3 40 nm per half sarcomere. When the length change is complete tension falls, rapidly at first and then more slowly, towards a value lying between the initial resting tension and that reached at the end of the stretch.
ImN[
A
B
1,+3 mm
I,+5 mm
C
1I+6mm Fig. 3. Tension (upper trace) and transparency changes (lower trace) resulting from stretch (207 /sm, velocity, 100 /t. s-1) at three different muscle lengths. Light guide, zero, direct. Temp. 00 C. Osmotic strength, 1*65 x NR. Vertical arrows, onset and end of stretch. Records traced from original chart recordings.
Optical record. The change of transparency was recorded with the light guide in position zero order, direct. There are three phases which correspond with the three phases of the tension record. Two are seen during the stretch and the third after it is complete. First, there is a decrease of transparency which parallels closely the build-up of tension in the SREC. Secondly, at about the point where the SREC reaches its elastic limit, there is an abrupt change of direction and the light intensity begins to increase. Thirdly, when the stretch is over there is a further increase of transparency and the light intensity ultimately reaches a level which is greater than the initial base line value. Effect of muscle length on the form of the transparency change. The three phases referred to above are always seen, although the exact form of the response is variable. For a given set of experimental conditions, it depends primarily on the initial muscle length. Fig. 3 shows three records made at different muscle lengths. At the shortest length (A) the second phase shows a continuing decrease of transparency, but the slope is less than during
7 LIGHT SCATTERING BY SREC OF FROG'S MUSCLE the early part of the stretch; note that the discontinuity again coincides with the elastic limit of the SREC. At the intermediate length there is no change of transparency during the second phase (B) while at the longer length (C) the transparency increases during the latter part of the stretch (as seen also in Fig. 2). These same records show that the light intensity level ultimately reached may be greater or less than the initial level.
Working hypothesis It is necessary to introduce a working hypothesis to serve as a basis for a quantitative study of the effect. The one offered accounts satisfactorily for the form of the records obtained with the light guide in position zero order, direct, and it is consistent with recordings taken from the other positions mentioned earlier. To facilitate description, the effect of a stretch will be considered only. Three assumptions are made: First, there is an increase in the light scattering power of the muscle which coincides with the steep rise of tension in the SREC. The increased scattering is maximal at the point where the SREC reaches its elastic limit and it is maintained at this level until the stretch ends. It then reverses, and it is assumed for simplicity that its decay follows an exponential time course. Secondly, there is an increase in the diffracting power of the striations throughout the period of stretch. This partially reverses when the length change ends, and again the decay is assumed to be exponential, but with a rate constant which is smaller than that for reversal of the scattering effect. Thirdly, there is a non-specific decrease of scattering, due to the muscle becoming thinner as it is extended. This is a function of muscle length, and is maintained without decay when the stretch ends. The recorded traces represent the nett effect of these three changes on the light intensity. The curves of Fig. 4 show how the characteristic form of the responses recorded at position zero order, direct are generated by varying the relative contribution of each component. Directional recording of the optical response. The form of the delayed transparency change. If the above assumptions are correct, then the delayed transparency change accompanying the fall of tension after the stretch is a composite one, made up of a relatively fast scattering decay superimposed upon a slower decrease of diffraction. Its form should therefore alter when one of these two components is made to reverse in sign without the other, a condition which is met by appropriately positioning the light guide (p. 2). Furthermore, the form of the- initial part of the response, seen during the stretch, should also change in a way which can be predicted from the above assumptions. Fig. 5 shows the expected form for records taken with the light guide in position first order, direct (C) and zero order, lateral (D), with the corresponding oscilloscope traces (A, B respectively). The significant points to note are: first, in both kinds of record the delayed change is biphasic; and second, the initial part of the response (seen during the length change) is inverted when recording from position zero order, lateral. Both features are consistent with the working hypothesis.
F. W. FLITNEY
8
B
A D
Si
~B C
_L/11._
S2
------
Zero order, direct
Fig. 4. Computed form of optical responses at zero, direct, for three different muscle lengths. D, diffraction change; S1, specific scattering change; S21 non-specific scattering change. The relative contribution of each component is selected arbitrarily to give curves resembling those obtained experimentally (Fig. 3), but no attempt is made to reproduce them exactly. See text for further details. A l0ImN
10 mN
400 ms
A D
S
LJ11
n_~~
_
1%
200 MS
D
Da
SI
S2 S2_
_/_0
_
_
_-
_
_
_
Fig. 5. Tension (upper trace) and transparency (lower trace) changes recorded from positionfir8t order, direct (A) and zero order, lateral (B), with computed curves showing the expected form of the records (C, D). Note (a) the biphasic, delayed change of transparency, and (b) inversion of the initial phase seen in the record made at position zero order, lateral. D, S, and S2 as for Fig. 4.
LIGHT SCATTERING BY SREC OF FROG'S MUSCLE
Quantitative analysis of the optical records The scattering change associated with the SREC is the component of the optical response which is of physiological interest. The agreement between the expected form ofthe records and those obtained experimentally supports the hypothesis and affords a basis for a quantitative study of the effect. The combined effect of the non-specific scattering change and the diffraction change (during the period of stretch) is equivalent to working on a 'sliding' base line, and the amplitude of the SREC scattering change 10 B 4
16
A
o6 E 3
4
12
~~~~~2
%_
~2
-
+
-BE i i
1,
I 1.00
+
U. +
0
+ +
.
1
2
3
Ait/~%
+
0
4
1-04
1-08 1-12 1-16 Muscle length (xI*)
1-20
Fig. 6. A, plot showing length dependence of elastic modulus, E, of SREC (crosses) and AI./It (filled circles). Osmotic strength, 1 65 x NR. Muscle length was increased in steps of 0 5 mm. Stretches were applied 5 min after a single conditioning stimulus at each length. Stretch, 207 j#m; velocity 100 #. 8-1. B, plot of E against AIJ/IL for same set of records used in A. Line fitted by regression analysis.
is measured by extrapolating the slope of the second phase to the point where the length change commences. In what follows, the change of light intensity attributable to the specific scattering effect, denoted by A8I,, is expressed as a percentage of the resting (transmitted) light intensity, It. Dependence of E and AI.5/It on initial muscle length. It is found that the amplitude of the scattering change is closely related to the stiffness of the SREC. Hill (1968) showed that there is little change in the value of E with increasing muscle length, but when E is related to the amount of overlap between the filaments it is seen to increase approximately 3 x in the range
10 F. W. FLITNEY of lengths corresponding with sarcomere spacings of 20-3-0 pam (osmotic strength, 2416 x NR). Fig. 6A shows values of E (crosses) per unit overlap of the filaments at different lengths for a muscle in solution 1-65 x NR. E rises from 2-8 to 10 8 x 105N . m-2 in the range of lengths corresponding with sarcomeres of 24-3 0 #sm. The values of AI1/It per unit overlap of the filaments at each length are also shown (filled circles), and these show a similar dependence on muscle length. Fig. 6B shows the same values of E and AI,/It plotted one against the other. The extrapolated region of the regression line passes close to the origin. 10
1-8xN, 1*65xNR *
8
~
6
-a
4
19xNR
* 1*6xNR *
414xNR 2 //
0
0
2
4 6 8 Relative E Fig. 7. Effect of hypertonicity on E and AI./It. Normalized values of both parameters increase progressively as the osmotic strength is raised. Osmotic strength of solutions marked near points. Continuous line fitted by regression analysis.
Dependence of E and AI./It on osmotic strength. It is found that both E and AIs/It increase with increasing osmotic strength. The results of Fig. 7 are from six experiments in which optical and tension records were made in solutions of differing tonicity, ranging from NR to 2-3 x NR. A period of 30-40 min was allowed for the muscle to equilibrate after a change of solution. The values of both parameters are normalized by expressing them as multiples of those obtained in NR. In this series of experiments, E increased approximately 8 x on going from NR to 2-3 x NR, and the corresponding increase of AIIIt was about lOx .
Effect of velocity of length change on E and AI'/It. Hill (1968) showed that the short-range stiffness of a resting muscle is not much affected by the
LIGHT SCATTERING BY SREC OF FROG'S MUSCLE 11 velocity of the length change, and it has been shown that AI8/It is also only slightly dependent upon the velocity of stretch. The results from two experiments serve to illustrate the point. In the first stretches were made at speeds of 6-200 tm. s-1, and Al,/It remained roughly constant. In the second experiment stretches of 75-4500 ,m .s-1 were applied and AII/It increased by about 2 5 x . E was not measured, but reference to Hill's results (his Fig. 6, p. 649; 1968) shows that it increases by a similar amount (3 x ) over the same range (750 x ) of velocities. DISCUSSION
It has been shown that a change of transparency is produced by stretching a resting muscle, and that an identifiable component of the response, an increase of light scattering, is associated with the development of tension in the SREC. The results are presented in such a way as to emphasize the close association between the amplitude of the scattering change and the mechanical stiffness of the SREC: both E and AI8/It increase when the osmotic strength of the external solution is raised, and also when sarcomere length is increased. Hill (1968) attributed the short-range stiffness of a resting muscle to the flexural rigidity of the population of cross-bridges involved in generating the FRT, and the proven association between MI./It and E under different experimental conditions suggests strongly that the scattering effect is generated by a conformational change in these cross-bridges. Is it possible to be more precise as to the origin of the scattering change? The phenomenon of light scattering is due to the re-radiation of incident light resulting from induced oscillations of charged particles within the scattering medium. The intensity of the light deviated in this way is proportional to the amplitude of the induced oscillations, and the latter is in turn related to the dipole moment of the scattering particles (Stokes, 1963). It follows that a change in the distribution of charge within the particles would be expected to alter their scattering properties. H. E. Huxley (1969) has postulated that part of the myosin molecule, the elongated S2 sub-fragment of heavy meromyosin (S2-HMM), is joined at one end through a flexible link to the light meromyosin component which forms the backbone of the thick filament, and at its distal end, through a similar link, to the S, fragment of HMM, in which the actin binding and ATP-ase activity resides. Huxley & Simmons (1971, 1972) have shown that the mechanical responses of contracting muscle to imposed length changes can be explained if it is supposed that this part of the cross-bridge is not inextensible, as was proposed originally by Huxley, but that it constitutes instead a compliant linkage, in series with an element which
F. W. FLITNEY has both viscous and elastic properties. In view of what is said above about the factors which affect the ability of particles to scatter light, an elastic connexion, such as the one they propose, is clearly a strong candidate for the component which generates the scattering change. The possibility that the filaments themselves also contribute to the effect must be considered. The elastic force generated in the linkages and shared with the filaments could conceivably produce a scattering change which would display a similar dependence on tension. It is known from X-ray diffraction studies of contracting and passively stretched muscle that the axial periodicity of the filaments remains almost constant (Huxley & Brown, 1967), and in a resting muscle where the forces involved are very small, they can be regarded as virtually inextensible. They are, however, relatively large compared to the size of the cross-bridges, and it is well known that large particles scatter more strongly than small ones: a small change in their length might contribute substantially to the overall effect,and it is difficult to see how this possibility could be tested experimentally. No explanation has been offered to account for the observation that both E and A18/It increase with decreasing filament overlap. This is an unexpected result, because the number of cross-bridges potentially capable of contributing to the effect is directly proportional to the amount of filament overlap. However, Endo (1972a, b) has reported that the isometric force produced by 'skinned' fibres in activating solutions containing low concentrations of calcium, comparable to those encountered in a resting muscle, also increase with increasing sarcomere length, and the indications are that the behaviour of the cross-bridges is different under these conditions. It is possible that the decrease in lateral separation of the filaments at longer sarcomere lengths may enhance mechanical interaction between the filaments. According to Endo (1972b), this does not seem to be the cause of the stretch-induced activation seen in his experiments, but it would seem premature to dismiss this possibility altogether. The proximity of the filaments might turn out to be an important factor in determining the degree of interaction between actin and myosin in an intact, resting muscle. The dependence of E and MI./It on the tonicity of the external solution solution is of interest. Hypertonicity is known to increase the resting metabolism of a muscle: oxygen consumption (Sekine, Iijima, Genba, Tanaka & Kanai, 1957) ,heat production (Yamada, 1970) and break-down of phosphate esters (Daemers-Lambert, Debrun, Dethier & Manil, 1966) are all enhanced. In addition, it has been shown to mobilize intracellular calcium (Isaacson, 1969; Homsher & Briggs, 1968) and the multiple effects 12
LIGHT SCATTERING BY SREC OF FROG'S MUSCLE 13 of hypertonicity can be attributed directly or indirectly to a raised level of this ion. The marked increase of E and AIJ/It in hypertonic solution is consistent with this interpretation. I thank Dr D. K. Hill for helpful discussion and Mr M. J. McDonnell for expert technical assistance. Dr W. G. S. Stephens kindly designed and constructed the servo-control system for the length transducer. This work was assisted by grants from the Medical Re3earch Council and Action Research for the Crippled Child. It was done while the author was in receipt of a Beit Memorial Fellowship. REFERENCES
BLINKS, J. R. (1965). Influence of osmotic strength on cross-section and volume of isolated single muscle fibres. J. Physiol. 177, 42-57. DAEMERS-LAMBERT, C., DEBRuN, F. M., DETHIER, G. & MANui, J. (1966). M6tabolisme des esters phosphores dans le sartorius de Rana temporaria trait par une solution de Ringer hypertonique. Arch8 int. Physiol. 74, 374-396. ENDO, M. (1972a). Stretch-induced increase in activation of skinned muscle fibres by calcium. Nature, Lond. 237, 211-213. ENDO, M. (1972b). Length dependence of activation of skinned muscle fibres by calcium. aoid Spring Harb. Symp. quant. Biol. 37, 505-510. FLITNEY, F. W. (1973). Light scattering associated with the short-range elastic component of resting frog's muscle. J. Physiol. 238, 40-41 P. HILL, A. V. (1949). Is relaxation an active process? Proc. R. Soc. B 136, 420435. HILL, D. K. (1949). Changes in transparency of muscle during a twitch. J. Physiol. 108, 292-302. HILL, D. K. (1953). The optical properties of resting striated muscle. The effect of rapid stretch on the scattering and diffraction of light. J. Physiol. 119, 489-500. HILL, D. K. (1959b). The ultra-violet dichroism of living frog's muscle. J. Physiol. 148, 379-392. HTiT, D. K. (1968). Tension due to interaction between the sliding filaments in resting striated muscle. The effect of stimulation. J. Physiol. 199, 637-684. HuLxEy, A. F. & SiMMoNs, R. (1968). A capacitance gauge tension transducer.
J. Physiol. 197, 12P. HuxLEY, A. F. & SnoNs, R. (1971). Proposed mechanism of force generation in striated muscle. Nature, Land. 233, 533-538. HuxLEY, A. F. & SnmmoNs, R. (1972). Mechanical transients and the origin of
muscular force. Cold Spring Harb. Symp. quaint. Biol. 37, 669-680. HUEY, H. E. & BROWN, W. (1967). The low angle X-ray diagram of vertebrate striated muscle and its behaviour during contraction and rigor. J. molec. Biol. 30, 383-434. HuxLEY, H. E. (1969). The mechanism of muscular contraction. Science, N.Y. 164, 1356-1366. HOMSHR, E. & BRIGGS, N. (1968). Effects of hypertonicity on calcium fluxes in frog sartorius muscle. Fedn Proc. 27, 375. IsAAcOsoN, A. (1969). Caffeine-induced contractures and related calcium movements of muscle in hypertonic media. Experientia 25, 1263-1265. SANDow, A. (1936). Diffraction patterns of the frog sartorius and sarcomere behaviour under stretch. J. cell. comp. Physiol. 9, 37-54.
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SEKrNE, T., IIGIMA, J., GENBA, T., TANAKA, K. &; KANAI, M. (1957). About the increase of respiration during the muscle activity. Conference on the Chemistry of Muscular Contraction. Tokyo: Igaku Shoin. STOKES, A. R. (1963). The Theory of the Optical Properties of Inhomogeneou Materials. London: Spon Ltd. YAMADA, K. (1970). The increase in the rate of heat production of frog's skeletal muscle caused by hypertonic solutions. J. Physiol. 208, 49-64.
LIGHT SCATTERING ASSOCIATED WITH TENSION CHANGES IN THE SHORTRANGE ELASTIC COMPONENT OF RESTING FROG'S MUSCLE
By F. W. FLITNEY From the Department of Physiology, University of St Andrews, St Andrews
(Received 19 November 1973) SUMMARY
1. A study has been made of some optical and associated mechanical properties of resting frog's sartorius muscles in isotonic and hypertonic solutions. Tension and transparency changes accompanying small alterations of muscle length (< 1-5 %) were recorded simultaneously. 2. The form of the transparency change is complex. It has three phases, two of which occur during the length change and the third (delayed phase) after it is complete. Directional recording of the response reveals both scattering and diffraction components. 3. The change of light scattering is associated with tension changes in the short-range elastic component (SREC) of the muscle. Its magnitude is related to the stiffness of the SREC; both increase when the osmotic strength of the external solution is raised and when muscle length is increased. 4. The change of light scattering is not much affected by the velocity of the applied length change. This is true also of the mechanical stiffness of the SREC. 5. The origin of the diffraction change is not known. 6. It is concluded that the scattering effect is caused by conformational changes in the SREC. INTRODUCTION
The experiments to be described are concerned with certain of the optical and mechanical properties of resting frog's muscle. It has been known for many years (A. V. Hill, 1949) that a frog's sartorius at rest exerts a small force, even when held at lengths far below its standard length in the body. D. K. Hill (1968) has shown that part of this resting tension, the filamentary resting tension (or FRT), is due to a weak form of 1-2
F. W. FLITNEY interaction between the actin and myosin filaments; the tension is generated by a small population of activated cross-bridges which form relatively stable linkages between the filaments, and which endow the muscle with short-range elastic properties. He referred to them collectively as the short-range elastic component, or SREC. The purpose of the present study was to examine the possibility that the existence of comparatively long-lived linkages between the overlapping filaments might have some bearing on the transparency changes produced by small stretches of a resting muscle (D. K. Hill, 1949, 1953). A preliminary account of this work was presented to the Physiological Society (Flitney, 1973). 2
METHODS were made with All experiments the sartorius muscle of Rana temporaria. The muscles were kept in oxygenated Ringer solution having the following composition (mm): NaCl 96; KC1, 5; CaCl2, 4; sodium phosphates, 5, pH 6-9. The majority of experiments were with muscles bathed in solutions made hypertonic by the addition of sucrose. The osmotic strength of these solutions is expressed throughout as a multiple of the value for the above solution (e.g. Ringer solution containing 5 % (w/v) sucrose is referred to as 1-65 x NR, with 8% sucrose, 2-0 x NR, etc.). Stimulation was effected through two platinum electrodes (dimensions, 5 mmn x 20 mm) spaced 12 mm apart, using square pulses of 1 ms duration and strength 10 V/cm. A conditioning stimulus was given to the muscle 2-3 min prior to applying the length changes.
Apparatus Muscle chamber. The muscle is suspended vertically in a cylindrical glass chamber enclosed in a tightly fitting aluminium jacket (Fig. 1). Its tendon is attached to a tension recorder by a fine chain, and the pelvic bone is retained by a loop formed at one end of a platinum rod. The rod is connected at its lower end to an electromagnetic displacement transducer. The chamber is cooled by three thermoelectric modules (type 12-15G, De La Rue Frigistors Ltd) clamped to the aluminium jacket. Length changes. Changes of muscle length are produced by a moving-coil electromagnetic unit (Ling-Altec vibrator, model 201) controlled by a servo amplifier with positional and velocity feed-back. Displacement is monitored by a photo-electric device comprising a light source, a vane attached to the connecting rod and two silicon photodiodes (Ferranti, type MS 1AE). The system is driven by voltage signals of the 'ramp-and-hold' type, supplied by a Servomex LF 141 waveform generator. Tension recorder. A variable capacitance gauge similar to that described by A. F. Huxley & Simmons (1968) is used to record muscle tension. The compliance of the unloaded transducer at the point of attachment of the muscle is 0 3 ,sm mN-1. Tension is displayed on one channel of a dual beam oscilloscope.
Optical system An area of muscle (approx. 1 mm diam., and located 10 mm from the pelvic end) is illuminated with light from a tungsten-halogen lamp (12 V, 100 W) run off a heavyduty battery. The light is passed through a lens to give a nearly parallel beam (divergence, 30) and then collimated by a circular aperture. Light emerging from the muscle reaches the photodetector through a two-stage fibre-optics light guide. The first part is a rigid, coherent bundle of fibres (type IG 1;
LIGHT SCATTERING BY SREC OF FROG'S MUSCLE
3
Barr & Stroud Ltd) mounted on a micrometer-actuated sliding stage (Research Instruments Ltd). Its distal end is immersed in the fluid bathing the muscle and it can be accurately positioned to collect light from any desired direction. The spatial distribution of light entering this first stage is preserved, owing to the coherence of the glass fibres, and an image is formed at the proximal end of the bundle where it joins the second section. The latter is a flexible, non-coherent collection of fibres (type LG 5, Barr & Stroud Ltd) which leads to the photo-detector housing.
L
E
M
L
T
CB R
I
Fig. 1. Muscle chamber and optical system for recording changes of transparency and tension of frog's muscle. The glass chamber (C) is supported in an aluminium block (CB) cooled by three thermomodules (T) clamped to its surface. The muscle (M) is attached at its pelvic end to a rod (R) connected to the moving element of an electromagnetic length transducer (L) through a seal (S) in the floor of the chamber. The tibial tendon is connected to the tension recorder (P) with a length of fine chain. A rigid light guide (LG) mounted on a moving stage (ST) collects the light and transmits it along a second, flexible section (F) to the photodetector. An aperture (A) is interposed between the two sections of light guide to limit the light reaching the photomultiplier. Two platinum stimulating electrodes (E) flank the muscle.
The cone of light reaching the photomultiplier is limited by a circular aperture located between the two sections of the light guide. A small aperture, 200 jtm diameter, is employed where high angular definition is required, as for example, when locating the intensity maxima of the diffracted rays (see below), and a larger one, up to 3 mm diameter, when the light intensity is low and resolution is not the limiting factor. Intensity changes are detected with a 12 stage photomultiplier tube (9558C, EMI Electronics Ltd), operated from a Brandenburg 472R stabilized power supply, and displayed on the oscilloscope. In all records an upward deflexion of the trace indicates an increase of light intensity; the change of intensity is expressed as a percentage of the resting (transmitted) light level.
4
F. W. FLITNEY
Sarcomere lengths. The A and I bands within its sarcomeres causes a muscle to act as a diffraction grating and the light beam is split up into a series of diffracted rays (Sandow, 1936). At the start of each experiment the sarcomere length is measured directly with the muscle set up in the chamber at its standard length. This is done by measuring the vertical separation of the 1st and 0 order spectra at two specified distances from the muscle, moving the tip of the light guide vertically in the plane containing the incident beam until the intensity maxima corresponding with the two rays are located. The angle of diffraction is obtained from the micrometer readings and used to calculate the sarcomere length (Blinks, 1965). Terminology. The mechanical stiffness of the short-range elastic component, denoted by E, is expressed throughout in the form of a Young's modulus (N.m-2). The standard length of the muscle in the body is denoted by 1l. RESULTS
The form of the optical response is determined by changes in the amount of light scattered by the muscle and in that diffracted by the striations. The ability to distinguish between these two components is crucial for interpreting the records and the method by which the distinction is made will be outlined before presenting the results. Reference is also made to the effects of hypertonicity on the optical and mechanical properties of a resting muscle, since the majority of the experiments were made using solutions made hypertonic by the addition of sucrose. These two points are discussed below. Directional recording of the optical change. When a frog's sartorius is illuminated with a parallel beam, both the diffracted and non-diffracted light suffers scattering, predominantly in the planes which lie at right angles to the long axis of the fibres. There is very little true absorption of light in the visible part of the spectrum, at wave-lengths greater than about 250 nm (Hill, 1959b), and absorption changes are not likely to affect the response to an appreciable extent. Hill was able to recognize scattering and diffraction components in his optical traces by recording changes of light intensity from selected regions of the diffraction pattern. His method of 'directional' recording, and the terminology he employed, is used here also. Reference should be made to his paper for a detailed explanation. Briefly, the distinction is made by comparing records taken in the following four positions. Zero order, direct. The tip of the light guide is placed directly in line with the incident beam and so receives undeviated light. In this position an increase in either the scattering or diffracting power of the muscle causes the light intensity to fall. Zero order, lateral. The light guide is moved laterally to a point along the zero order line. An increase of diffraction has the same sign as at position zero, direct, but an increase of light scattering shows reversal at some critical angle, and at this and greater lateral angles the two effects oppose one another. First order, direct. The light guide is positioned to record from the first order spectrum and is located in the vertical plane which contains the incident beam. Scattering and diffraction effects have a different sign: an increase of diffraction causes the light intensity to increase, whereas an increase of scattering causes it to fall. First order, lateral. The light guide is positioned at some lateral angle along the first order diffraction line. Scattering and diffraction changes have the same sign, and the combined effect is to give a response which is the inverse of that seen at position zero, direct.
LIGHT SCATTERING BY SREC OF FROG'S MUSCLE
5
Effect of hypertonicity on optical and mechanical properties of resting muscle. In hypertonic solution the FRT and stiffness of the SREC are both increased (Hill, 1968). The optical changes accompanying stretch or release of a muscle are also enhanced in hypertonic solution, and for this reason the majority of the experiments were made using Ringer solution containing varying amounts of sucrose. The changes in both the optical and mechanical properties of a muscle induced by hypertonic solutions are entirely reversible.
Characteristic tension and optical responses to imposed length changes Fig. 2A shows the tension and transparency changes resulting from a linear 'ramp-and-hold' stretch (not displayed) of 400 /tm. Tension record. The increase of tension is due largely to the elastic resistance of the SREC. The early part of the record is dominated by this A
10 mN[
;
200 msec
t
2%[ 400 msec 2. the a effect of stretch (400 /sm; velocity, 1600 Am.s-1) on the Fig. A, tension (upper trace) and transparency (lower trace) of a frog's sartorius. Arrows indicate onset and end of length changes. Spots on record indicate final levels for tension and light intensity. Light guide in position zero, direct, located 15 mm away from the muscle. Aperture, 3 mm. Muscle sarcomere length, 2-75 /sm. Temp. 20 C. B, transparency changes due to stretch (S) and release (R) of a muscle. Tension change not displayed. Sarcomere length, 2 5 /am. Applied length change, ± 200 jum, velocity 1000 ,um . s-1. Temp. 10 C. Osmotic strength for A and B, 1-65xNR.
6 F. W. FLITNEY component. The 'elastic limit' of the SREC, defined arbitrarily as the point at which the slope of the tension record falls to 25 % of its initial value, is reached for a displacement of 60 jpm, equivalent to a relative sliding movement of the filaments of 3 40 nm per half sarcomere. When the length change is complete tension falls, rapidly at first and then more slowly, towards a value lying between the initial resting tension and that reached at the end of the stretch.
ImN[
A
B
1,+3 mm
I,+5 mm
C
1I+6mm Fig. 3. Tension (upper trace) and transparency changes (lower trace) resulting from stretch (207 /sm, velocity, 100 /t. s-1) at three different muscle lengths. Light guide, zero, direct. Temp. 00 C. Osmotic strength, 1*65 x NR. Vertical arrows, onset and end of stretch. Records traced from original chart recordings.
Optical record. The change of transparency was recorded with the light guide in position zero order, direct. There are three phases which correspond with the three phases of the tension record. Two are seen during the stretch and the third after it is complete. First, there is a decrease of transparency which parallels closely the build-up of tension in the SREC. Secondly, at about the point where the SREC reaches its elastic limit, there is an abrupt change of direction and the light intensity begins to increase. Thirdly, when the stretch is over there is a further increase of transparency and the light intensity ultimately reaches a level which is greater than the initial base line value. Effect of muscle length on the form of the transparency change. The three phases referred to above are always seen, although the exact form of the response is variable. For a given set of experimental conditions, it depends primarily on the initial muscle length. Fig. 3 shows three records made at different muscle lengths. At the shortest length (A) the second phase shows a continuing decrease of transparency, but the slope is less than during
7 LIGHT SCATTERING BY SREC OF FROG'S MUSCLE the early part of the stretch; note that the discontinuity again coincides with the elastic limit of the SREC. At the intermediate length there is no change of transparency during the second phase (B) while at the longer length (C) the transparency increases during the latter part of the stretch (as seen also in Fig. 2). These same records show that the light intensity level ultimately reached may be greater or less than the initial level.
Working hypothesis It is necessary to introduce a working hypothesis to serve as a basis for a quantitative study of the effect. The one offered accounts satisfactorily for the form of the records obtained with the light guide in position zero order, direct, and it is consistent with recordings taken from the other positions mentioned earlier. To facilitate description, the effect of a stretch will be considered only. Three assumptions are made: First, there is an increase in the light scattering power of the muscle which coincides with the steep rise of tension in the SREC. The increased scattering is maximal at the point where the SREC reaches its elastic limit and it is maintained at this level until the stretch ends. It then reverses, and it is assumed for simplicity that its decay follows an exponential time course. Secondly, there is an increase in the diffracting power of the striations throughout the period of stretch. This partially reverses when the length change ends, and again the decay is assumed to be exponential, but with a rate constant which is smaller than that for reversal of the scattering effect. Thirdly, there is a non-specific decrease of scattering, due to the muscle becoming thinner as it is extended. This is a function of muscle length, and is maintained without decay when the stretch ends. The recorded traces represent the nett effect of these three changes on the light intensity. The curves of Fig. 4 show how the characteristic form of the responses recorded at position zero order, direct are generated by varying the relative contribution of each component. Directional recording of the optical response. The form of the delayed transparency change. If the above assumptions are correct, then the delayed transparency change accompanying the fall of tension after the stretch is a composite one, made up of a relatively fast scattering decay superimposed upon a slower decrease of diffraction. Its form should therefore alter when one of these two components is made to reverse in sign without the other, a condition which is met by appropriately positioning the light guide (p. 2). Furthermore, the form of the- initial part of the response, seen during the stretch, should also change in a way which can be predicted from the above assumptions. Fig. 5 shows the expected form for records taken with the light guide in position first order, direct (C) and zero order, lateral (D), with the corresponding oscilloscope traces (A, B respectively). The significant points to note are: first, in both kinds of record the delayed change is biphasic; and second, the initial part of the response (seen during the length change) is inverted when recording from position zero order, lateral. Both features are consistent with the working hypothesis.
F. W. FLITNEY
8
B
A D
Si
~B C
_L/11._
S2
------
Zero order, direct
Fig. 4. Computed form of optical responses at zero, direct, for three different muscle lengths. D, diffraction change; S1, specific scattering change; S21 non-specific scattering change. The relative contribution of each component is selected arbitrarily to give curves resembling those obtained experimentally (Fig. 3), but no attempt is made to reproduce them exactly. See text for further details. A l0ImN
10 mN
400 ms
A D
S
LJ11
n_~~
_
1%
200 MS
D
Da
SI
S2 S2_
_/_0
_
_
_-
_
_
_
Fig. 5. Tension (upper trace) and transparency (lower trace) changes recorded from positionfir8t order, direct (A) and zero order, lateral (B), with computed curves showing the expected form of the records (C, D). Note (a) the biphasic, delayed change of transparency, and (b) inversion of the initial phase seen in the record made at position zero order, lateral. D, S, and S2 as for Fig. 4.
LIGHT SCATTERING BY SREC OF FROG'S MUSCLE
Quantitative analysis of the optical records The scattering change associated with the SREC is the component of the optical response which is of physiological interest. The agreement between the expected form ofthe records and those obtained experimentally supports the hypothesis and affords a basis for a quantitative study of the effect. The combined effect of the non-specific scattering change and the diffraction change (during the period of stretch) is equivalent to working on a 'sliding' base line, and the amplitude of the SREC scattering change 10 B 4
16
A
o6 E 3
4
12
~~~~~2
%_
~2
-
+
-BE i i
1,
I 1.00
+
U. +
0
+ +
.
1
2
3
Ait/~%
+
0
4
1-04
1-08 1-12 1-16 Muscle length (xI*)
1-20
Fig. 6. A, plot showing length dependence of elastic modulus, E, of SREC (crosses) and AI./It (filled circles). Osmotic strength, 1 65 x NR. Muscle length was increased in steps of 0 5 mm. Stretches were applied 5 min after a single conditioning stimulus at each length. Stretch, 207 j#m; velocity 100 #. 8-1. B, plot of E against AIJ/IL for same set of records used in A. Line fitted by regression analysis.
is measured by extrapolating the slope of the second phase to the point where the length change commences. In what follows, the change of light intensity attributable to the specific scattering effect, denoted by A8I,, is expressed as a percentage of the resting (transmitted) light intensity, It. Dependence of E and AI.5/It on initial muscle length. It is found that the amplitude of the scattering change is closely related to the stiffness of the SREC. Hill (1968) showed that there is little change in the value of E with increasing muscle length, but when E is related to the amount of overlap between the filaments it is seen to increase approximately 3 x in the range
10 F. W. FLITNEY of lengths corresponding with sarcomere spacings of 20-3-0 pam (osmotic strength, 2416 x NR). Fig. 6A shows values of E (crosses) per unit overlap of the filaments at different lengths for a muscle in solution 1-65 x NR. E rises from 2-8 to 10 8 x 105N . m-2 in the range of lengths corresponding with sarcomeres of 24-3 0 #sm. The values of AI1/It per unit overlap of the filaments at each length are also shown (filled circles), and these show a similar dependence on muscle length. Fig. 6B shows the same values of E and AI,/It plotted one against the other. The extrapolated region of the regression line passes close to the origin. 10
1-8xN, 1*65xNR *
8
~
6
-a
4
19xNR
* 1*6xNR *
414xNR 2 //
0
0
2
4 6 8 Relative E Fig. 7. Effect of hypertonicity on E and AI./It. Normalized values of both parameters increase progressively as the osmotic strength is raised. Osmotic strength of solutions marked near points. Continuous line fitted by regression analysis.
Dependence of E and AI./It on osmotic strength. It is found that both E and AIs/It increase with increasing osmotic strength. The results of Fig. 7 are from six experiments in which optical and tension records were made in solutions of differing tonicity, ranging from NR to 2-3 x NR. A period of 30-40 min was allowed for the muscle to equilibrate after a change of solution. The values of both parameters are normalized by expressing them as multiples of those obtained in NR. In this series of experiments, E increased approximately 8 x on going from NR to 2-3 x NR, and the corresponding increase of AIIIt was about lOx .
Effect of velocity of length change on E and AI'/It. Hill (1968) showed that the short-range stiffness of a resting muscle is not much affected by the
LIGHT SCATTERING BY SREC OF FROG'S MUSCLE 11 velocity of the length change, and it has been shown that AI8/It is also only slightly dependent upon the velocity of stretch. The results from two experiments serve to illustrate the point. In the first stretches were made at speeds of 6-200 tm. s-1, and Al,/It remained roughly constant. In the second experiment stretches of 75-4500 ,m .s-1 were applied and AII/It increased by about 2 5 x . E was not measured, but reference to Hill's results (his Fig. 6, p. 649; 1968) shows that it increases by a similar amount (3 x ) over the same range (750 x ) of velocities. DISCUSSION
It has been shown that a change of transparency is produced by stretching a resting muscle, and that an identifiable component of the response, an increase of light scattering, is associated with the development of tension in the SREC. The results are presented in such a way as to emphasize the close association between the amplitude of the scattering change and the mechanical stiffness of the SREC: both E and AI8/It increase when the osmotic strength of the external solution is raised, and also when sarcomere length is increased. Hill (1968) attributed the short-range stiffness of a resting muscle to the flexural rigidity of the population of cross-bridges involved in generating the FRT, and the proven association between MI./It and E under different experimental conditions suggests strongly that the scattering effect is generated by a conformational change in these cross-bridges. Is it possible to be more precise as to the origin of the scattering change? The phenomenon of light scattering is due to the re-radiation of incident light resulting from induced oscillations of charged particles within the scattering medium. The intensity of the light deviated in this way is proportional to the amplitude of the induced oscillations, and the latter is in turn related to the dipole moment of the scattering particles (Stokes, 1963). It follows that a change in the distribution of charge within the particles would be expected to alter their scattering properties. H. E. Huxley (1969) has postulated that part of the myosin molecule, the elongated S2 sub-fragment of heavy meromyosin (S2-HMM), is joined at one end through a flexible link to the light meromyosin component which forms the backbone of the thick filament, and at its distal end, through a similar link, to the S, fragment of HMM, in which the actin binding and ATP-ase activity resides. Huxley & Simmons (1971, 1972) have shown that the mechanical responses of contracting muscle to imposed length changes can be explained if it is supposed that this part of the cross-bridge is not inextensible, as was proposed originally by Huxley, but that it constitutes instead a compliant linkage, in series with an element which
F. W. FLITNEY has both viscous and elastic properties. In view of what is said above about the factors which affect the ability of particles to scatter light, an elastic connexion, such as the one they propose, is clearly a strong candidate for the component which generates the scattering change. The possibility that the filaments themselves also contribute to the effect must be considered. The elastic force generated in the linkages and shared with the filaments could conceivably produce a scattering change which would display a similar dependence on tension. It is known from X-ray diffraction studies of contracting and passively stretched muscle that the axial periodicity of the filaments remains almost constant (Huxley & Brown, 1967), and in a resting muscle where the forces involved are very small, they can be regarded as virtually inextensible. They are, however, relatively large compared to the size of the cross-bridges, and it is well known that large particles scatter more strongly than small ones: a small change in their length might contribute substantially to the overall effect,and it is difficult to see how this possibility could be tested experimentally. No explanation has been offered to account for the observation that both E and A18/It increase with decreasing filament overlap. This is an unexpected result, because the number of cross-bridges potentially capable of contributing to the effect is directly proportional to the amount of filament overlap. However, Endo (1972a, b) has reported that the isometric force produced by 'skinned' fibres in activating solutions containing low concentrations of calcium, comparable to those encountered in a resting muscle, also increase with increasing sarcomere length, and the indications are that the behaviour of the cross-bridges is different under these conditions. It is possible that the decrease in lateral separation of the filaments at longer sarcomere lengths may enhance mechanical interaction between the filaments. According to Endo (1972b), this does not seem to be the cause of the stretch-induced activation seen in his experiments, but it would seem premature to dismiss this possibility altogether. The proximity of the filaments might turn out to be an important factor in determining the degree of interaction between actin and myosin in an intact, resting muscle. The dependence of E and MI./It on the tonicity of the external solution solution is of interest. Hypertonicity is known to increase the resting metabolism of a muscle: oxygen consumption (Sekine, Iijima, Genba, Tanaka & Kanai, 1957) ,heat production (Yamada, 1970) and break-down of phosphate esters (Daemers-Lambert, Debrun, Dethier & Manil, 1966) are all enhanced. In addition, it has been shown to mobilize intracellular calcium (Isaacson, 1969; Homsher & Briggs, 1968) and the multiple effects 12
LIGHT SCATTERING BY SREC OF FROG'S MUSCLE 13 of hypertonicity can be attributed directly or indirectly to a raised level of this ion. The marked increase of E and AIJ/It in hypertonic solution is consistent with this interpretation. I thank Dr D. K. Hill for helpful discussion and Mr M. J. McDonnell for expert technical assistance. Dr W. G. S. Stephens kindly designed and constructed the servo-control system for the length transducer. This work was assisted by grants from the Medical Re3earch Council and Action Research for the Crippled Child. It was done while the author was in receipt of a Beit Memorial Fellowship. REFERENCES
BLINKS, J. R. (1965). Influence of osmotic strength on cross-section and volume of isolated single muscle fibres. J. Physiol. 177, 42-57. DAEMERS-LAMBERT, C., DEBRuN, F. M., DETHIER, G. & MANui, J. (1966). M6tabolisme des esters phosphores dans le sartorius de Rana temporaria trait par une solution de Ringer hypertonique. Arch8 int. Physiol. 74, 374-396. ENDO, M. (1972a). Stretch-induced increase in activation of skinned muscle fibres by calcium. Nature, Lond. 237, 211-213. ENDO, M. (1972b). Length dependence of activation of skinned muscle fibres by calcium. aoid Spring Harb. Symp. quant. Biol. 37, 505-510. FLITNEY, F. W. (1973). Light scattering associated with the short-range elastic component of resting frog's muscle. J. Physiol. 238, 40-41 P. HILL, A. V. (1949). Is relaxation an active process? Proc. R. Soc. B 136, 420435. HILL, D. K. (1949). Changes in transparency of muscle during a twitch. J. Physiol. 108, 292-302. HILL, D. K. (1953). The optical properties of resting striated muscle. The effect of rapid stretch on the scattering and diffraction of light. J. Physiol. 119, 489-500. HILL, D. K. (1959b). The ultra-violet dichroism of living frog's muscle. J. Physiol. 148, 379-392. HTiT, D. K. (1968). Tension due to interaction between the sliding filaments in resting striated muscle. The effect of stimulation. J. Physiol. 199, 637-684. HuLxEy, A. F. & SiMMoNs, R. (1968). A capacitance gauge tension transducer.
J. Physiol. 197, 12P. HuxLEY, A. F. & SnoNs, R. (1971). Proposed mechanism of force generation in striated muscle. Nature, Land. 233, 533-538. HuxLEY, A. F. & SnmmoNs, R. (1972). Mechanical transients and the origin of
muscular force. Cold Spring Harb. Symp. quaint. Biol. 37, 669-680. HUEY, H. E. & BROWN, W. (1967). The low angle X-ray diagram of vertebrate striated muscle and its behaviour during contraction and rigor. J. molec. Biol. 30, 383-434. HuxLEY, H. E. (1969). The mechanism of muscular contraction. Science, N.Y. 164, 1356-1366. HOMSHR, E. & BRIGGS, N. (1968). Effects of hypertonicity on calcium fluxes in frog sartorius muscle. Fedn Proc. 27, 375. IsAAcOsoN, A. (1969). Caffeine-induced contractures and related calcium movements of muscle in hypertonic media. Experientia 25, 1263-1265. SANDow, A. (1936). Diffraction patterns of the frog sartorius and sarcomere behaviour under stretch. J. cell. comp. Physiol. 9, 37-54.
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F. W. FLITNEY
SEKrNE, T., IIGIMA, J., GENBA, T., TANAKA, K. &; KANAI, M. (1957). About the increase of respiration during the muscle activity. Conference on the Chemistry of Muscular Contraction. Tokyo: Igaku Shoin. STOKES, A. R. (1963). The Theory of the Optical Properties of Inhomogeneou Materials. London: Spon Ltd. YAMADA, K. (1970). The increase in the rate of heat production of frog's skeletal muscle caused by hypertonic solutions. J. Physiol. 208, 49-64.
