J. Physiol. (1975), 248, pp. 207-230 With 2 plates and 7 text-figure8 Printed in Great Britain

207

STRUCTURAL, MECHANICAL AND MYOTHERMIC PROPERTIES OF RABBIT RECTOCOCCYGEUS MUSCLE

By D. F. DAVEY,* C. L. GIBBS AND H. C. MCKIRDYt From the Department of Physiology, Monash University, Clayton, Victoria, Australia

(Received 28 October 1974) SUMMARY

1. The fine structure of rabbit rectococcygeus muscle has been studied with the electron microscope. 2. The mechanical performance and the heat production of this muscle has been investigated during tetanic contractions at 27° C. 3. In isometric contractions a force of 164 + 27 mN/mm2 (mean + S.D., = n 17) is developed and the heat production is linearly related to the force. 4. There is a relationship between the duration of stimulation (t) and the total heat production (H) of the type H = A +bt, where A and b are constants. 5. After-loaded isotonic experiments show that the relationship between force and velocity can be fitted by the 'characteristic equation' of Hill (1938). 6. The value of a/P0 (0-302 + 0'093, mean + S.D.) is slightly higher than in frog skeletal muscle but the constant b is about 50 times smaller. 7. The ratio of work/total energy production, for the stimulus conditions employed, was maximally 0x185. 8. The ratio of total enthalpy to initial enthalpy is difficult to measure accurately but is probably about 2. INTRODUCTION

Although investigations into the energetics of invertebrate smooth muscle have been carried out by several workers (Bozler, 1930, 1936; Abbot & Lowry, 1958; Baguet & Gillis, 1967, 1968) no detailed examination of mammalian smooth muscle has been reported. There are several reasons * Present address: Department of Physiology, University of Sydney, N.S.W. 2006, Australia. t Leverhulme Overseas Student. Present address: Institute of Physiology, University of Glasgow, Glasgow G12 8QQ.

208 D. F. DAVEY, C. L. GIBBS AND H. C. McKIRDY for this. The spontaneous activity of most commonly used mammalian smooth-muscle preparations makes it difficult to establish a myothermic base line. The mechanical response is often complicated by the presence of longitudinal and circular muscle layers. In addition to these problems smooth-muscle heat production is low in comparison to that measured in skeletal muscle. Smooth muscles are known to be considerably more 'economic' than skeletal muscles in maintaining tension (Bozler, 1930) yet there has been no myothermic investigation of their isotonic efficiency. Several authors (Mashima, 1969; Gordon & Siegman, 1971; Hellstrand & Johansson, 1974) report high values for the ratio, a/PO (obtained by fitting the force-velocity data with Hill's (1938) equation) and a high value, according to the results of Woledge (1968), would predict low mechanical efficiency. This apparent contradiction between high isometric economy and low predicted mechanical efficiency could be clarified by a myothermic investigation of smoothmuscle isotonic efficiency. Furthermore, data on smooth muscle energetics may aid the unravelling of the controversy which surrounds its mechanism of contraction (Lowy, Vibert, Haselgrove & Poulsen, 1973; Somlyo, Devine, Somlyo & Rice, 1973). We have therefore investigated the properties of the rabbit rectococcygeus muscle. This preparation was described by Langley & Anderson (1895, 1896) and more recently by McKirdy (1972). It has the advantage that the muscle is made up of parallel longitudinal bundles and at 270 C the preparation is normally quiescent for 2-5 hr. Moreover when stimulated tetanically it produces tension that compares favourably with that of mammalian skeletal muscle. A preliminary report of this work has already been published (Gibbs & McKirdy, 1974). METHODS Rabbits of both sexes weighing 2-3 kg were killed by exsanguination following i.v. Nembutal anaesthesia. The rectococcygeus muscle (Langley & Anderson, 1895) was ligated at the rectal origin and at a few millimetres from the coccygeal insertion using Ethicon 000 thread and was then excised. Electron microscopy Muscles destined for electron microscopic examination were mounted on Perspex rods, stretched to slightly in excess of body length, and bathed for 1 hr in a modified Krebs solution at room temperature before fixation in a manner similar to that used by Davey (1973). The muscles, still mounted on the rods, were fixed (1 hr, 40 C) by immersion in distilled acrolein (4 %, v/v) dissolved in a vehicle buffered with Hepes (N-2-hydroxyethyl-piperazine-N'-2-ethanesulphonic acid) adjusted to pH 7-4 with NaOH (Good, Winget, Winter, Connolly, Izawa & Singh, 1966) and having similar cation composition to the Krebs solution (namely 142.8 mm-NaCl, 5*93 mM-KCl, 2-54 mM-CaCl2, 1'18 mm-MgSO4, 10 mm glucose, 5 mm Hepes). They were then given

SMOOTH-MUSCLE ENERGETICS

209

three 10 min rinses in the same vehicle before post-fixation (1-5 hr, 4 C) with Os04 (1 % mass/vol.) dissolved in the same vehicle without the Hepes buffer. The muscles were then rinsed at room temperature in several changes of maleic acid buffer (0.05 M, pH adjusted to 5-2 with NaOH) over a period of 30 min. During this period small blocks suitable for embedding were cut from the muscles. These blocks were then stained (1 hr, room temperature) using uranyl nitrate (0 5 % mass/vol.) dissolved in a similar buffer adjusted to a final pH 5-2 (Karnowsky, 1967), dehydrated through an ethanol series, and embedded in Spurr's (1969) resin. Thin sections exhibiting silver interference colours were stained with Reynold's (1963) lead citrate. In some experiments the acrolein fixative was dissolved in a solution similar to that used by Birks (1971, 1974) containing a high concentration of Mg2+ ions (110 mM-MgCl2, 5-5 mm-KCl, 10 mm-Hepes, pH adjusted to 7-4 with NaOH). All subsequent steps were as outlined above, except that the blocks were stained overnight in uranyl nitrate. Morphometry was performed on cross-sections using the method of Weibel (1972). It was assumed that structures measured were isotropic in the transverse plane. Compression was disregarded.

Energetic measurements The preparations were tied with Ethicon 000 silk in situ and were then transferred to the thermopile. The thread tied to the coccygeal end of the muscle was clamped about 3 mm below the thermopile whilst the other thread was connected via a light stainless-steel tube to the length-tension transducer. The total system compliance (transducer, tube and ties) was 0-42 ,um/mN. The quick release technique (Wilkie, 1956) was used with five muscles to estimate the internal work in a tetanic response. The length-tension transducer consisted of a magnesium-alloy lever mounted to the armature shaft of a Brush, Mark 200, 80 mm penmotor. The muscle was loaded by passing constant current through the coils of the penmotor. Micrometer stops were used to limit the excursion of the lever. In quick release experiments the constant current could be switched electrically to simulate any desired load. Muscle displacement was measured by utilizing the angular position transducer which forms part of the feed-back circuitary to the Brush penmotor. Tension development was monitored using two Ether 350 il P-type strain-gauges bonded to the lever as described by Jewell, Kretzschmar & Woledge (1967). The preparations had a weight of 53-2 ± 16-0 mg (mean + S.D.). The length at which maximum tetanic tension was developed was designated lo and averaged 17-8 ± 3-4 mm (mean + S.D.) No attempt was made to locate 10 until the excised muscle had been in solution for at least 15 min under a load of 1-0 g. The muscle was then lengthened in 1-0 mm steps and tetanic tension was measured 1 min after each length change. For the majority of muscles, maximum active tension was generated at extensions 2-4 mm past their length under the 1-0 g load. All experiments commenced at 270 C, but in four cases where spontaneous activity was evident earlier than usual the temperature was lowered to 240 C in an attempt to produce inhibition (see Axelsson & BUlbring, 1961). The muscles were bathed in modified Krebs solution of the following composition: NaCl, 118-0 mM; KCl, 4-75mM; MgSO4.7H20, 1 -18mM; KH2PO4, 1 -18mM; CaCl2.2H20, 2-54mM; NaHCO3, 24-8 mm; glucose, 11-0 mm. The solution was bubbled with 95 % 02:5 % CO2 to maintain the pH at 7-4.

D. F. DAVEY, C. L. GIBBS AND H. C. McKIRDY

210

Stimulation The muscles were always stimulated transversely as described by Gibbs & Gibson (1972) and as shown in Text-fig. 1. The stimulus duration was kept at 2 msec and the voltage was just supramaximal. It appears that the stimulus predominantly excited nerve endings, since atropine (1 0 jug/ml.) greatly reduced the response. The stimulus frequency was in the range 5-5-7-5 Hz and the usual period of stimulation was close to 4 sec. A correction was required for the stimulus heat as this averaged 20-5 % of the total heat in a tetanus. The magnitude of the correction was determined by reducing the stimulus duration to a subthreshold value, 0-02 msec. The preparation was then stimulated for a longer period of time at a higher than usual frequency and the resulting stimulus heat was measured. In two preparations, atropine (1 -0 jggml.) administered at the end ofthe experimental protocol completely abolished mechanical activity and allowed the stimulus heat to be measured without change of the stimulus parameters. Integrating thermopile

B Top view t To transducers

A Cross-section Silver

Thermopile

Reference junctions

Muscle Active junctions Metal-filled epoxy

Calibration

Silver

wire in groove

Top electrode

Stainless-steel

frame

Muscle

Bottom electrode (insulated from ---~silver) E~J

Clamp

Text-fig. 1. Integrating thermopile (A). Cross-section showing two conventional thermopiles, bent as described in text, so that their active junctions make contact with the strip of silver on which the muscle lies. (B) Top view showing the muscle on the dished silver heat sink. The transverse stimulating electrodes are shown together with the calibration wire used for heating the muscle thermopile system. Force-velocity curves The velocity of shortening was always measured in after-loaded contractions and in three preparations the quick-release method used by Jewell & Wilkie (1960) was employed. The release took place during the plateau period of a 6 sec tetanus and the experimental data were fitted by the method of least squares to the straight line rearrangement of the Hill equation in which (Po - P)fv is plotted against P (where P is the load, V is the velocity of shortening and P0 is the isometric tetanic tension).

SMOOTH-MUSCLE ENERGETICS

211

Heat measurements Two thermopiles were used in the present experiments. One has already been described in detail (Gibbs & Gibson, 1972); the other, shown in Text-fig. 1, is a modification of the integrating thermopile described by Wilkie (1968). Two electroplated thermopiles (Ricchiuti & Mommaerts, 1965) were bent at an angle of about 30° along the line of their active junctions. A strip of silver was bonded to the active junctions using metal-filled epoxy. A fine constantan wire was located in a groove cut into the silver and for calibration purposes current could be passed through it. The thermopile possessed the following characteristics: output of 11-36 mV/0C; heat loss coefficient 40 9 + 2-2 mW/0C (mean + S.D.); thermal capacity equivalent to 72-9 + 15-7 mg muscle (mean+ S.D., n = 14). In all but four of the seventeen experiments the integrating thermopile was used, since its calibration is independent of the thermal capacity of the muscle, thermopile and any adhering solution. The four experiments with the conventional thermopile were carried out to check that the integrating thermopile was not seriously distorting the time course of heat liberation. With either thermopile, the heat loss was exponential and was corrected for electrically. Resting heat production was measured as described by Hill (1965) and Yamada (1970). Recovery heat was routinely measured taking the precaution necessary for accurate recording as reported previously (Gibbs & Gibson, 1972). Two methods were used in an attempt to assess initial and recovery energies in isometric tetani. In the first method the heat up to the end of mechanical relaxation defined arbitrarily as the point when tension had declined to 10 % of its P0 value was assumed to be the initial heat plus stimulus heat. The balance of heat production was assumed to be recovery heat. In the second method, total heat production was measured in the usual way then the muscle was bathed in solution containing 1 mmKCN and bubbled with 95 % N2: 5 % CO2 for 20 min. The heat production in a tetanus was then measured in an atmosphere of 95 % N2:5 % C02. This heat was assumed to be free of oxidative recovery heat.

Experimental procedure Because the rectococcygeus muscles eventually became spontaneously active we decided to complete certain basic experiments and to add to these if the preparation stayed quiescent or if the spontaneous mechanical response was infrequent and of low magnitude. Initially five after-loaded isotonic contractions and one isometric contraction were studied. The stimulus duration was the same in each case (about 4 sec); the loads were selected to cover the range 0-05-0-8 P0. After a 20 min rest period the same sequence of experiments was repeated but in the reverse order. The results were averaged and the external work and enthalpy versus load curves were constructed. If a preparation was still quiescent, either a heat vs. tension or a heat vs. stimulus duration experiment was carried out. The former relation was obtained by gradually shortening the muscle; the stimulation period remained at 4 sec. The heat-duration relationship was obtained by varying the stimulus duration between 0 3 and 8-0 see; the muscle length remained constant. The experiments in nitrogen were always the last ones attempted. In some preparations where occasional spontaneous contractions occurred early in the experimental sequence, the isometric experiments were not attempted and the recoverylinitial heat measurements commenced immediately after the isotonic series. At the conclusion of the above experiments each preparation was exposed to atropine and the stimulus heat was measured.

212

D. F. DAVEY, C. L. GIBBS AND H. C. McKIRDY RESULTS

Ultrastructure Electron microscopic examination of thin sections covering the whole cross-section of one rectococcygeus muscle showed that it contained exclusively smooth-muscle fasciculi; no skeletal muscle fibres were present. The irregularly shaped bundles varied in size to the extent that transverse sections of whole bundles, such as shown in PI. 1, fig. 1, might include anywhere between 75 and 300 fibres. The muscle fibres themselves (see P1. 1, figs. 3, 4) varied substantially in cross-sectional area, even within one bundle, as is evident in P1. 1, fig. 1. Some of the apparent variation arises because of the lack of longitudinal register amongst all the fibres; some cells are sectioned near their tapering ends and display smaller cross-sections. Longitudinal sections of the muscle cells showed that nuclei were never located near the ends of the fibres, hence the presence of either a nucleus or a central accumulation of mitochondria and endoplasmic reticulum (see PI. 1, fig. 4), which extends for a short distance longitudinally from each nucleus, can be used to select a population of cell cross-sections which excludes those through tapering fibre ends. The mean cross-sectional area of such a population was found to be 14-6 + 4-5 ,/m2 (mean + S.D., n = 46). It is evident from the magnitude of the standard deviation of this population, that even in their central regions, the fibres vary substantially in cross-sectional area. The fibres, like the bundles, tend to be irregularly shaped, and the membranes appear wrinkled, particularly in cross-sections. The surface to volume ratio was found to be 1.57 'um2/#lm3 (see also Table 3). This indicates that there is sufficient membrane to cover right circular cylinders of 2 5 ,tm diameter. This is further evidence of wrinkling, because such cylinders have a cross-sectional area of only 5.1 ,tm2, whereas the fibre cross-sectional area was determined to be about 14 um2 as stated above (note that the surface density of cylinders decreases as their diameter increases). An investigation of the type required to accurately determine fibre lengths (such as carried out by Merrillees, 1968) was not undertaken. However, in longitudinal sections 1 jtm thick examined in the light microscope, many fibres were observed to remain in the plane of section for more than 300 ,tm, so the mean fibre length is probably in excess of this value. Small axon bundles were observed to run longitudinally along many fasciculi. These bundles were common in sections from blocks which had been cut from regions of the whole muscles (see Methods) to which small nerve branches had been traced using a dissecting stereomicroscope. In

SMOOTH-MUSCLE ENERGETICS 213 sections from blocks cut distant from such nerve branches, axon bundles were less commonly observed and some fairly large fasciculi contained no signs of a nerve supply. Even near the nerve branches, most fasciculi contained only one axon bundle. The small axon bundles were enveloped by a single Schwann cell, and usually contained 3-10 axons (see P1. 1, figs. 1, 2). Axon varicosities, containing synaptic vesicles, were usually naked around approximately one quarter of their perimeter as viewed in cross-section (see P1. 1, fig. 2) and a distance of about 150 nm separated them from the nearest muscle fibres. The axon bundles generally ran along the surface of the fasciculi, although some bundles were observed one or two muscle fibres deep, usually at the bottom of a cleft in the fascicular surface; bundles were never observed deep in the fasciculi. Only one single axon was observed in this investigation and like the axon bundles, it was partly surrounded by a Schwann cell, and separated from the nearest adjacent muscle fibre by a gap of 150 nm. Hence in the terminology adopted by Bennett (1972) the nerve supply was generally in the form of 'innervation by small axon bundles'; 'close contact varicosities' were rarely observed. An example of the latter is shown in P1. 2. Thus the pattern of innervation appears fairly typical of gastrointestinal smooth muscle, although the nerve supply did appear to be somewhat sparse, and many cells are probably not close enough to axon varicosities to be stimulated directly. Intercellular gap junctions such as the one shown in P1. 1, fig. 3 were frequently observed between adjacent cells and probably represent sites of electrical coupling which result in a whole bundle being the excited unit. The intracellular features of the muscle fibres are not significantly different to those of other gastrointestinal smooth muscles (see, for example, Lane, 1965). There is a limited amount of intracellular membrane which may be analogous to skeletal muscle sarcoplasmic reticulum, whereas the mitochondrial content, found to be 3-9 % of fibre volume, lies between the values for fast- and slow-twitch mammalian skeletal muscles. These differences are quantitated in Table 3, which will be considered in more detail in the discussion. The distribution of thick, intermediate and thin filaments (see PI. 2) is also typical of gastrointestinal muscle. As noted in the methods, two slightly different fixation methods were used in this study: the primary aldehyde fixative was dissolved either in a modified Krebs solution or in a solution with an unphysiologically high concentration of Mg2+ ions. The latter method resulted in a slightly different over-all appearance of the tissue, which was regarded subjectively as indicative of improved fixation. As previously reported by Birks (1971, 1974) for the cat superior cervical ganglion, there was an increased number of synaptic vesicles in the axon varicosities. The subplasmalemmal spaces

214 D. F. DAVEY, C. L. GIBBS AND H. C. McKIRDY (arrows P1. 1, fig. 3) evident in most cells fixed using the Krebs vehicle, were absent in cells fixed in the presence of a high Mg2+ concentration, and the over-all filament distribution appeared more uniform. It is noteworthy that a few fibres having a similar appearance were observed in bundles fixed using the modified Krebs vehicle, but because of their rarity they tended to be regarded as atypical. Resting heat production The resting heat was measured at the start of the myothermic experiments with the muscles at their lo length. In eleven preparations it had a value of 0604 + 0-218 mW/g muscle (mean + S.D.). No attempt was made to determine the effects of muscle length on the resting heat. 160

E

80

::;

E

I 0

E 240

Zs E C

I

201

C

1!0 Text-fig. 2. Tension development (bottom trace) and heat production (top trace) in a tetanic contraction. The time course of the stimulus heat, which is included in the heat record, is shown superimposed on the heat record. The small blips on the rising phase of the heat record are stimulus artifacts. The middle trace provides the time calibration; defiexions are 1 sec apart. There is a change in time base during the measurement of recovery heat.

Isometric responses

During the equilibration period the mechanical response to a single supramaximal stimulus was recorded; the time to contraction peak averaged 2-7 sec and the contraction duration averaged 9-4 sec. The maximal rate of tetanic tension development was achieved with stimulus rates between 5-5 and 7*5 Hz and the average developed tetanic tension was 164 + 27 mN/mm2 (force-cross-sectional area, mean + SD_, n = 17). The twitch: tetanic tension ratio averaged 0-25. Five preparations whose mean

SMOOTH-MUSCLE ENERGETICS 215 tetanic tension was 187 mN/mm2 had their internal work measured by the quick release technique (Wilkie, 1956); internal work averaged 4*47 + 1.05 mJ/g muscle (mean + S.D.). An experimental trace showing heat production and tension development in an isometric tetanus is shown in Text-fig. 2. After correction for the stimulus heat, the heat production up to the contraction peak is approximately equal to the heat recorded during relaxation. Indeed in all the muscles examined the heat measured at the contraction peak averaged only 45 % of the heat measured up to the end of relaxation (defined as the 08 07

-

06

x A

AA

05

I0AI

x

A

0.1 -~~~~1

0.2

0o 01

0

1

2

3 5 4 Duration of stimulus (sec)

6

7

8

Text-fig. 3. The relationship between heat-tension ratio (H/P0 lo) and stimulus duration in four muscles identified by the symbols (*, x, 1, A). Individual regression lines were fitted to the data on the right of the vertical dashed line.

point where tension had declined to 10 % P0). This value has not been underestimated through the use of the integrating thermopile, for comparable results were obtained when using the more rapidly responding conventional thermopile. Measurements were made of the rate of heat production during tension

216 D. F. DAVEY, C. L. GIBBS AND H. C. McKIRDY development. In four experiments with the conventional thermopile the rate was 6-40 + 0 90 mW/g muscle and in thirteen experiments with the integrating thermopiles it was 4-98 + 1-46 mW/g muscle (mean + S.D.). In all experiments one measurement of the heat production rate was made during a well-defined tension plateau. In some muscles this necessitated the use of a 6 see stimulus period. This measurement allowed calculation of the heat-rate: tension ratio, (dH/dt)/PO lo, which is a measure of the 'economy' with which muscles can maintain tension (Feng, 1931; Hill & Woledge, 1962). The mean heat-rate: tension ratio was 0-032 + 0.011 sec-i (mean+S.D., n =17). 80

X

H=22-7+0-286P

X

(r=0 88)

E

I.E

40 _S

10 I

0

0'

0

'

40

'

'

80 120 Tension (mN/mm2)

160

I

200

Text-fig. 4. Relationship between total heat production and isometric tension development in four tetani at different muscle lengths. Data from four muscles ([O. A, *, x ) are shown.

In four preparations H, the total heat (initial and recovery), was determined as a function of the stimulus duration and H/P014 (the heat/ tension ratio) was calculated. The results are shown in Text-fig. 3 and can be compared with data from frog muscle (cf. Fig. 3, Hill & Woledge, 1962). For stimulus durations of 2 see and above, the data for each muscle is adequately fitted by a straight line (r > 0.98). The data from the four muscles show that HIPo1o = 0 304 + 0 070 (S.D.) +0-046 + 0*011 (s.D.).t, where t is the stimulus time in seconds. The first term on the right is a measure of the energy cost of tension development and relaxation and the second term is a measure of the cost of tension maintenance (Hartree & Hill, 1921).

217 SMOOTH-MUSCLE ENERGETICS The relationship between total heat production (mJ/g) and tension development (mN/mm2) was obtained by shortening muscles to different lengths below lo. In order to completely prevent tension development muscles had to be shortened to about 20 % 4o. Results from four muscles are shown in Text-fig. 3. Regression analysis shows that the data can be fitted by a straight line (r = 0.88) such that H = 22*7 + 0*286P. Data from some of the muscles appear to show a curvilinear relationship; similar results have been reported when skeletal muscle (Hill, 1925; Hill, 1965; Chapman & Gibbs, 1972) and cardiac muscle (Gibbs, Mommaerts & Ricchiuti, 1967) is shortened. Unfortunately the rapid onset of spontaneous activity at high resting tensions prevented us from examining the heat: tension relationship in stretched muscles. It seems clear from Text-fig. 4 that the isometric heat production can be divided into a tensionindependent and a tension-dependent component.

Isotonic responses There were two aims in the isotonic experiments: to see whether the force-velocity data could be fitted by the Hill (1938) equation; and to measure both the work and total energy production in order to calculate the maximum mechanical efficiency. In Text-fig. 5 the force-velocity data from one muscle is shown; the straight-line fit of the data with the Hill equation is shown below, Textfig. 5B. The data from most muscles fitted the Hill equation well, judging by eye, but there was a tendency in about half of the muscles for the fit to be less satisfactory with heavy loads. A similar result has been reported by McIntyre (1965) for the guinea-pig ductus arteriosus. Any such discrepancy will tend to raise the value of a/PO and increase b in a leastsquares analysis. In Table 1 the computer-fitted results are presented since they are less subject to experimental bias; however, it may be worth while to remember that the results will have been influenced by any deviations from linearity at high loads. It is obvious, however, that the rectococcygeus muscle has, compared to skeletal muscle, a very low maximum shortening velocity. This means that the constant b of the Hill equation has a very low value. On the other hand it seems probable that the ratio, a/P0, is higher than in skeletal muscle even allowing for the problem mentioned above. In three preparations, force-velocity data obtained from after-load experiments were compared with quick release data. As can be seen from Table 1, the agreement is satisfactory. The apparently increased mechanical performance in the release experiments is primarily caused by one of the quick-release experiments having been

D. F. DAVEY, C. L. GIBBS AND H. C. McKIRDY

218

5 * A 4

-

3

F

0) -

E

0

._

0

2 0 0

1

S 0

0 600

I

I

I

al

_

l

B

500 EU LI

0

400

0

z E

300

I.-,

of

200 100

0L 0

100

200

300 400 500 Force (mN)

600

700

Text-fig. 5. Relationship between velocity of shortening and load. Data from a typical experiment (A) and the straight line fit of the data with the Hill equation (B). The muscle weighed 49-9 mg and its tetanie tension was 666 mN. The constants A and b had values of 209 mN and 1-51 mm/sec respectively.

carried out after an equilibration period in nitrogen. A period of anoxia always increased mechanical performance (see below). The results of the myothermic experiments to determine mechanical efficiency (external work/total enthalpy) are presented in Text-fig. 6. Data from seventeen muscles are shown and it should be pointed out that a stimulus duration of 4-0 see was chosen so that the mechanical record only just reached a plateau when medium-to-heavy loads were lifted. The

SMOOTH-MUSCLE ENERGETICS

219

TABLE 1. Summary (mean + S.D.) of force-velocity parameters obtained by fitting afterloaded (A) and quick-release (R) data with the Hill equation

Polo/M*

lo

A At Rt

VMax/10

b/i0

(mN/mm2) (sec-1) 17-8+3-4 164+27 0-072 + 0-031 16-8 + 2-4 173 + 11 0-062 + 0-017 16-8 ± 2-4 185 + 17 0*089 + 0-034 (mm)

a/1PO

(sec-,)

n

0-302 ± 0-093 0-224 + 0-018 0-273 + 0-032

0*247 + 0-079 0-277 + 0-080 0-318 + 0-091

17 3

3

Tension/cross-sectional area calculated assuming area = Mfplo, where M is the mass (g) and p is the density (g/ml.), which was assumed to be unity. t Force velocity data obtained from the same three muscles using the afterloaded (A) and the quick-release (R) method. *

100 _

75

-

0 E

00

50 F

I-

Ln LU

25

0

,

0

I

I

0-2

0-4

I

0-6

0-8

1-0

P/Po Text-fig. 6. The relationship between total energy output, external work and load, where loads are expressed as fractions of peak tetanic tension (PO). The data represent the mean + s.E. for seventeen muscles. The top continuous line gives the energy production and the bottom continuous line represents the external work. The top dashed line is obtained by subtracting twice the external work from the total energy curve. The bottom dashed line represents the expected tension dependent heat obtained by using data from Text-fig. 4. The shaded area represents heat production that could be associated with muscle shortening.

220 D. F. DAVEY, (. L. GIBBS AND H. C. McKIRDY mechanical efficiency so determined should be close to the maximum value attainable. If a longer stimulus duration is employed the external work does not increase but heat production continues and hence lowers the calculated mechanical efficiency. Using the data in Text-fig. 6, a maximum efficiency value of 18-5 % is reached at a load near 0 4 PO. Abbott & Lowy (1958) reported a shortening heat component in molluscan smooth muscle. In the present experiments we have not directly attempted to determine whether or not a shortening heat component contributes to isotonic heat production. However, upon analysis of the data in Text-fig. 6 it is evident that in an isotonic contraction more energy is liberated than would be expected in an isometric contraction where the muscles developed a force equal to the isotonic load. The excess energy is present both as external work and as additional heat, and if we employ the working definition of shortening heat proposed by Gibbs et at. (1967) and Homsher, Mommaerts & Ricchiuti (1974) then the excess heat can be interpreted as being shortening related heat. Thus if the heat-versustension relationship of Text-fig. 4 is superimposed upon Text-fig. 6 and twice the external work is subtracted from the total enthalpy curve there is excess heat, shown as a shaded area in Text-fig. 6. Twice the external work is subtracted because there is presumably recovery metabolism equivalent to this component (Hill, 1939), but if such an assumption was incorrect it would only increase the shaded area and hence increase the excess heat. TABLE 2. Comparison of the total heat production in a 4 sec tetanus with the initial heat. The initial heat was estimated either by taking the heat up to the end of the mechanical response in oxygen (see Methods) or as the heat remaining in an anaerobic environment and in the presence of KCN (designated N2 + KCN). In the latter case heat tended to fall with repeated periodic trials, despite the fact that the mechanical response was maintained Total

Method 1 2 2

02

N2+KCN (1st trial) N2+KCN (3rd trial)

heat/initial heat ratio 1X94 + 0.25

1.69+±019 2.13+±029

n 17 9 6

Recovery heat As outlined in the Methods, recovery heat production was estimated in two ways and the results are shown in Table 2. In the simplest case the heat production up to the end of relaxation minus the stimulus heat was taken to be the initial heat. The balance of the total heat production was assumed to be the recovery heat. This method gives a total enthalpy/initial enthalpy ratio of 1-94. In the second method an attempt was made to

SMOOTH-MUSCLE ENERGETICS 221 duplicate the procedure of Hill (1928) and to record the heat production in the presence of oxygen and then in the presence of nitrogen. Even after long periods in nitrogen-equilibrated physiological solution the initial heat production remained unexpectedly high, and in order to rule out the possibility of some oxidative metabolism contributing to the measured heat, 1.0 mM-KCN was routinely added to the nitrogen-equilibrated Krebs solutions. We are indebted to Dr J. B. Chapman for confirming fluorometrically that the KCN, even in oxygenated solution, rapidly inhibited mitochondrial metabolism. In spite of these procedures the initial enthalpy remained higher than expected. It should be pointed out that the nitrogencyanide treatment did not adversely effect the mechanical response. 120

60

_ -

I

200 E E

E 1000 0 Z~~~~~~~~~~~~~ C

0L I min Text-fig. 7. Tension development and heat production in the presence of oxygen (1st record) and after 20 min in nitrogen-bubbled Krebs solution containing 1 mM-KCN (second record). For comparative purposes the nitrogen results have been superimposed upon the oxygen ones. The heat records contain stimulus heat and the size of this artifact is indicated by the height of the horizontal bar. Notice the effect of N2 + KCN on maximum tension and contraction duration. The time calibration marks are 1-0 sec apart.

Indeed the reverse was true: in not one preparation did tension development fall. Most preparations showed about 10 % increment in tetanic tension and an increase in the contractile duration, i.e. relaxation was slower than usual. Thus in part some of the heat recorded under anaerobic conditions might be associated with the increased mechanical performance. One other effect was discovered: when the anaerobic preparations were subject to repeated tetanization at about 4 min intervals their mechanical performance was maintained but their heat production tended to fall. An

222 D. F. DAVEY, C. L. GIBBS AND H. C. McKIRDY additional two to five tetani were therefore recorded under anaerobic conditions and in Table 2 data from the first tetanus (1st trial) and from the third tetanus (3rd trial) are given. Repeated tetanization increased the total enthalpy/initial enthalpy ratio from 1 69 to 2 13. In Text-fig. 7 typical mechanical and myothermic responses are shown recorded in oxygen and then in nitrogen. DISCUSSION

Energetics The resting heat production of quiescent rectococcygeus muscle was 0-60 mW/g muscle at 270 C. In oxygen consumption studies on guinea-pig taenia coli at 350 C the resting level seems to be about 5 4d. 02/g per minute, which is equivalent to a resting heat production of 1-58 mW/g muscle (Biilbring & Golenhofen, 1967; Casteels, Raeymaekers & Wuytack, 1973). Using the same preparation but working at 230 C Mulvany & Woledge (1972) report a resting heat production of 0 3 mW/g muscle in calcium free solution. Interestingly, invertebrate smooth muscle such as the anterior byssus retractor muscle of Mytilu8 edulis (ABRM), which can maintain tension at a much lower energy cost than the rectococcygeus muscle, still has a similar resting heat (Baguet & Gillis, 1967). These resting heat values are all high in comparison to amphibian skeletal muscle. If resting heat is in part related to resting Ca2+ influx (the resulting active Ca2+ extrusion and the possibility of some activation of the contractile proteins causing subthreshold cross-bridge cycling), the comparatively high resting heat rate could be due to the large surface to volume ratio of smooth muscle cells (see Table 3). However, this explanation cannot be accepted without some reservation; mammalian skeletal muscle fibres have similar resting heat (Wendt & Gibbs, 1974) even though their surfaceto-volume ratio is low (Eisenberg, Kuda & Peter, 1974; see Table 3). However, considered from the point of view of total membrane potentially involved in Ca2+ turnover, the sarcolemma and internal membrane systems, the rectococcygeus is not dissimilar to skeletal muscles (see Table 3). Isometric results In 1921, Hartree & Hill showed that the heat production (H) of frog sartorius muscle can be fitted by an equation of the type H = A+ bt, where A and b are constants and t is the stimulus duration. Bozler (1930), using the retractor pharynx muscle of the snail Helix pomatia, plotted the heat-tension ratio (HI/P01) against (t) and reported a similar result. His values for A and b were 0-14 and 0-15 respectively and can be compared with values of 0 304 and 0046 reported in this study. Note, however, that

SMOOTH-MUSCLE ENERGETICS

223

TABLE 3. Morphometric composition of rectococcygeus and mammalian fast- and slow-twitch skeletal muscle

Surface density Sarcoplasmic reticulum density Transverse tubular system density Total potential activatingmembrane densityll Mitochondrial volume *

Units flm2/100 /tm3

/Zm2/100 /Zm3

Rabbit rectococcygeus 157 38

/Um2/100 /M3

tm2/I100 /sms um3/100 fum3

195

3-9

Guineapig soleus* 12§ 97

Guinea-pig white vastust 8§ 131

6

15

115

154

4-9

2-1

Eisenberg, Kuda & Peter (1974).

t Eisenberg (1973). t These estimates exclude sub-sarcolemmal vesicles. For the rectococcygeus, inclusion of the vesicles would increase the surface density of 245,um2/100 /Lm3. (Distinction of the vesicles from reticulum was somewhat arbitrary. See P1. 1, fig. 4). § Light microscopic observations (Eisenberg & Kuda, 1975) suggest these figures underestimate the surface area by about 20%. 1I The sum of surface, reticular and tubular densities. TABLE 4. Comparison of heat tension ratio (HfPO O) as a function of stimulus duration (t) in several species TemperaReference Relation Preparation ture (0C) Bozler (1930) Snail pharynx retractor 15 H/POl0 = 0-14+0-15.t Hill & Woledge Frog sartorius 17 H1P010 = 0-16+0-79.t (1962) = H/POlO 0-20+0-003.t Baguet & Gillis 20 Mussell ABRM (1967) 27 Rabbit rectococcygeus H/POIO* = 0-15+0-023.t *

Assuming initial enthalpy = recovery heat.

Bozler was recording initial heat only and our results should therefore be divided by 2 to make them comparable. It is obvious that values for A are similar and values for b are markedly different. Several authors have pointed out that the A term which related to the energy cost of tension development and relaxation is similar for all muscles but that the constant b which relates to the energy cost of tension maintenance varies widely in different preparations. We summarize a few results in Table 4 to illustrate this point. Note that the rectococcygeus muscle expends about 30 times less energy than a frog sartorius muscle in maintaining tetanic tension. 8

PHY 248

224 D. F. DAVEY, C. L. GIBBS AND H. C. McKIRDY (In fact this is an underestimate as the sartorius data were obtained at 170 C.) In confirmation of this result the mean heat-rate: tension ratio at 270 C was 0-032 sec-', which is widely different from the value of 0-561 for frog sartorius muscle at 170 C (Hill & Woledge, 1962). In mammalian skeletal muscles at 270 C values of 041 and 0-6 have been obtained for rat soleus and rat extensor digitorum longus respectively (C. L. Gibbs, unpublished results). The heat rate measured during tension development was not much higher than that measured during maintained tension. This result is similar to that seen in mammalian skeletal muscle (Gibbs & Gibson, 1972; Wendt & Gibbs, 1973) and in chicken latissimus dorsi (Canfield, 1971) and differs substantially from results obtained in amphibian skeletal muscle (Hill, 1938; Aubert, 1956). The relationship between heat production and tension development is similar to that reported in amphibian skeletal muscle (Hill, 1925; Aubert, 1956; Sandberg & Carlson, 1966). In view of the results of Smith (1972) and of Homsher, Mommaerts, Ricchiuti & Wallner (1972) it would be desirable to examine this relationship in stretched preparations. This may be possible if lower temperatures are employed to inhibit the spontaneous activity that normally accompanies stretching.

Isotonic results The inverse relationship between load and shortening velocity could be adequately described by the Hill equation although in about half the muscles examined the fit was not perfectly satisfactory with loads greater than 0-6Po. This phenomenon may reflect the fact that it is difficult to measure the shortening velocity accurately when heavy loads are being lifted but the measured velocity consistently seemed to be too high (see also McIntyre, 1965). Alternatively we may have been underestimating P0 under aerobic conditions (see recovery heat results), or difficulties of the type that exist in cardiac muscle (Brady, 1968) may influence the fit. Gordon & Siegman (1971) have shown that the dynamic constants from the Hill equation are independent of initial muscle length in rabbit taenia coli at 220 C, Their a/PO value of 0-331 is not far different from 0-302 value found for the rectococcygeus but their value for b/Il is considerably smaller: 0.010 compared to 0-072 sec-l. Even so the rectococcygeus muscle has a b/IO value which would be at least 50 times smaller than that expected in frog sartorius muscle at an equivalent temperature. As might be expected the maximal shortening velocity of these two muscles when normalized for length, i.e. Vmax/4o, shows a similar forty- to fifty-fold difference. Strictly speaking, the term efficiency should be restricted to the ratio of work done to free energy liberated (Wilkie, 1960), but we have used the

SMOOThI-MUSCLE ENERGETICS

225 phrase 'mechanical efficiency' in the present paper to denote external work/total enthalpy. In a complete cycle of contraction and recovery where glycogen is the oxidized substrate, enthalpy liberation is about equal to free energy liberation (Burk, 1929) and it should be valid to compare the mechanical efficiencies of different muscles. The maximum mechanical efficiency estimated from Text-fig. 7 is about 18-5%. This value is quite comparable with values obtained in mammalian skeletal muscle (Wendt & Gibbs, 1974) and with reported values for amphibian skeletal muscle (Hill, 1939) if allowance is made for the magnitude of recovery heat production. In a comparative biochemical study of muscle efficiency, guinea-pig taenia coli was found to have an efficiency similar to that found in mammalian slow-twitch muscles (Awan, Frearson, Goldspink & Waterson, 1972). In 1968 Woledge suggested that differences in the curvature of the forcevelocity relationship might be expected to alter the ratio of work to the enthalpy liberation. He found that tortoise skeletal muscle had a low a/P ratio (0.072) and a high mechanical efficiency (35 %). Additional evidence in support of his suggestion has recently been found in mammalian skeletal muscle where fast-twitch muscles, which have higher a/P0 ratios than slow-twitch muscles, have a lower mechanical efficiency. It is perhaps surprising therefore that the rectococcygeus muscle, which has a high a/PO ratio, has a mechanical efficiency that is -comparable with frog sartorius. It may be argued that the difference in a/P0 ratios is relatively small, 0-25 in frog and 0 30 in rectococcygeus muscle, so that only small changes in mechanical efficiency would be expected. Alternatively the lack of mechanical constraint, imposed by sarcomere Z-lines and NI-lines in skeletal muscle, may allow smooth muscle to maintain a particular force level over a greater range of muscle lengths. Thus the rectococcygeus muscle can shorten to values about 20 % 10 whereas amphibian muscle can only reach about 60 % 10. Gibbs & Chapman (1974) have shown that the mechanical efficiency of frog and toad sartorii is dependent upon the muscle length from which shortening takes place. When the possible shortening distance is increased, by operating from lengths greater than 10, the mechanical efficiency rises.

Recovery heat On balance it appears as if the recovery heat production is approximately equal to the initial heat, as in frog sartorius muscle (Hill, 1939). In invertebrate smooth muscle Bozler (1930) came to the same conclusion. With the direct method the total enthalpy/initial enthalpy ratio of 1 91 is somewhat less than the 2 1 ratio reported by Hill, but this discrepancy is probably caused by an arbitrary decision as to where to read off the initial

D. F. DAVEY, a. L. GIBBS AND H. C. McKIRDY 226 heat (see Methods). It is quite likely that when recorded the measured heat includes some recovery heat as well as the initial heat. It is less easy to account for the effects of anoxia and KCN. The low ratio, particularly evident in the first tetanus under anaerobic conditions, may reflect a large anaerobic delayed heat component (Hartree & Hill, 1928). If so it is difficult to understand why repeated activity would subsequently lower the contribution of such a component. Alternatively a low ratio might result from a vapour pressure effect of the type reported by Hill & Kupalov (1930). When a muscle is stimulated in the absence of oxygen, metabolic end products accumulate and raise the osmotic pressure of the muscle. This in turn lowers its vapour pressure and allows water vapour present in the thermopile chamber to condense upon the muscle with the attendant production of heat. This vapour-pressure effect is generally easy to detect as the new heat base line is maintained at an elevated level (Gibbs, 1969) whereas in the present experiments the heat production returned to the reference level. As a check we carried out similar tetanic experiments with frog sartorius muscle at 200 C and rat soleus at 270 C and never obtained any evidence of a base-line shift. The fact that the initial enthalpy falls with repeated tetanization also argues against an osmotic pressure artifact. It is interesting that Bozler (1930) reported a somewhat similar phenomenon in invertebrate smooth muscle. With repetitive activity, in the presence of oxygen, Bozler found a progressive increase in the 'economy' with which muscles could maintain tension. Over a period of minutes he reported increases of six to ten times in the absence of any signs of muscle deterioration except for prolongation of relaxation. These observations suggest that metabolism associated with Ca2+ turnover may significantly contribute to the recovery heat. If intracellular Ca2+ concentration is normally substantially lower than the mechanical threshold, a rise in resting level following repeated activity might be mechanically undetectable. Reduced Ca2+ influx would then still bring about full activation; resulting reduced Ca2+ extrusion would utilize less energy, and could explain the prolonged activation. A similar mechanism may explain the reduced Ca2+ transient measured with aequorin (Taylor & Rudel, 1973) during a post-tetanic potentiated twitch in frog skeletal muscle, particularly if the aequorin technique does not detect subthreshold shifts in Ca2+ concentration. The improved peak mechanical response and the slower relaxation produced by anoxia may indicate that mitochondria have an important role in regulating calcium levels in smooth muscle (Goodford & Wolowyk, 1972; Devine, Somlyo & Solyo, 1973).

SMOOTH-MUSCLE ENERGETICS

227

REFERENCES

ABBOTT, B. C. & LoWRY, J. (1958). Contraction in molluscan smooth muscle. J. Physiol. 141, 385-397. AUBERT, X. (1956). Le Couplage energitique de la contraction mwsculaire, pp. 95, 99, 130, 162. Bruxelles: Editions Arscia. AwAN, M. Z., FREARSON, N., GOLDSPINK, G. & WATERSON, S. E. (1972). Biochemical efficiency of smooth muscle and different types of striated muscle. J. Mechanochem. & cell motility 1, 225-232. AxELSSON, J. & BULBRING, E. (1961). Metabolic factors affecting the electrical activity of intestinal smooth muscle. J. Physiol. 156, 344-356. BAGUET, F. & GILLIS, J. M. (1967). The respiration of the anterior byssus retractor muscle of Mytilus edulis (ABRM) after a phasic contraction. J. Physiol. 188, 67-82. BAGUET, F. & GILLIs, J. M. (1968). Energy cost of tonic contraction in a lamellibranch catch muscle. J. Physiol. 198, 127-143. BENNETT, M. R. (1 972). Autonomic Neuromuscular Transmission. London: Cambridge University Press. BIRKs, R. I. (1971). Effects of stimulation on synaptic vesicles in sympathetic ganglia, as shown by fixation in the presence of Mg2+. J. Physiol. 216, 26-28P. BIRKS, R. I. (1974). The relationship of transmitter release and storage to fine structure in a sympathetic ganglion. J. Neurocytol. 3, 133-160. BOZLER, E. (1930). The heat production of smooth muscle. J. Physiol. 69, 442-462. BOZLER, E. (1936). The energy changes of smooth muscle during relaxation. J. cell. comp. Physiol. 8, 419-438. BRADY, A. J. (1968). Active state in cardiac muscle. Physiol. Rev. 48, 570-600. BULBRING, E. & GOLENHOFEN, K. (1967). Oxygen consumption ofthe isolated smooth muscle of guinea-pig taenia coli. J. Physiol. 193, 213-224. BURK, D. (1929). The free energy of glycogen-lactic acid breakdown in muscle. Proc. R. Soc. B 104, 153-170. CANFIELD, S. P. (1971). The mechanical properties and heat production of chicken latissimus dorsi muscles during tetanic contractions. J. Physiol. 219, 281-302. CASTEELS, R., RAEYMAEKERS, L. & WUYTACK, F. (1973). Metabolism of smooth muscle cells under aerobic and anaerobic conditions. J. Physiol. 232, 27-29P. CHAPMAN, J. B. & GIBBS, C. L. (1972). Energetics of isometric and isotonic twitches in toad sartorius. Biophys. J. 12, 215-226. DAVEY, D. F. (1973). The effect of fixative tonicity on the myosin filament lattice volume of frog muscle fixed following exposure to normal or hypertonic Ringer. Histochem. J. 5, 87-104. DEVINE, C. E., SomLyo, A. V. & SomLyo, A. P. (1973). Sarcoplasmic reticulum and mitochondria as cation accumulation sites in smooth muscle. Phil. Trans. R. Soc. B 265, 17-23. EISENBERG, B. R. (1973). Stereological analysis of the fast-glycolytic fibers of skeletal muscle. J. cell Biol. 59 (2, pt. 2) 89a. EISENBERG, B. R., KUDA, A. M. & PETER, J. B. (1974). Stereological analysis of mammalian skeletal muscle. I. Soleus muscle of the adult guinea-pig. J. cell Biol. 60, 732-754. EISENBERG, B. R. & KUDA, A. M. (1975). Stereological analysis of mammalian skeletal muscle. II. White vastus muscle of the adult guinea-pig. J. Ultrastruct. Res. (In the Press.) FENG, T. P. (1931). The heat-tension ratio in prolonged tetanic contractions. Proc. R. Soc. B 108, 522-537.

228

D. F. DAVEY, C. L. GIBBS AND H. C. McKIRDY

GIBBS, C. L. (1969). The energy output of normal and anoxic cardiac muscle. In Comparative Physiology of the Heart: Current Trends, ed. MCCANN, F. V. Basel: Birkhauser Verlag. GIBBS, C. L. & CHAPMAN, J. B. (1974). The effect of stimulus conditions, temperature and length upon the energy output of frog and toad sartorii. Am. J. Physiol. 227, 964-971. GIBBS, C. L. & GIBSON, W. R. (1972). Energy production of rat soleus muscle. Am.

J. Physiol. 223, 864-871. GIBBS, C. L. & McKIRDY, H. (1974). Energetics of rabbit rectococcygeus muscle. Proc. Aust. Physiol. Pharmac. Soc. 5, 72. GIBBS, C. L., MOMMAERTS, W. F. H. M. & RIcCHIUTI, N. V. (1967). Energetics of cardiac contractions. J. Physiol. 191, 25-46. GOOD, N. E., WINGET, G. D., WINTER, W., CONNOLLY, T. N., IZAWA, W. & SINGH, R. M. M. (1966). Hydrogen ion buffers for biological research. Biochemistry, N.Y. 5, 467-477. GOODFORD, P. J. & WALOWYK, M. W. (1972). Localization of cation interactions in the smooth muscle of the guinea-pig taenia coli. J. Physiol. 224, 521-535. GORDON, A. R. & SIEGMAN, M. J. (1971). Mechanical properties of smooth muscle. I. Length-tension and force-velocity relations. Am. J. Physiol. 221, 1243-1249. HARTREE, W. & HILL, A. V. (1921). The regulation of the supply of energy in

muscular contraction. J. Physiol. 55, 133-158. HELLSTRAND, P. & JOHANSSON, B. (1974). The force-velocity relation in venous smooth muscle. Acta physiol. scand. 91, 45A. HILL, A. V. (1925). Length of muscle, and the heat and tension developed in an isometric contraction. J. Physiol. 60, 237-263. HILL, A. V. (1928). The anaerobic delayed heat after a tetanus. Proc. R. Soc. B 103, 207-217. HILL, A. V. (1928). The recovery heat production in oxygen after a series of muscle twitches. Proc. R. Soc. B 103, 183-191. HILL, A. V. (1938). The heat of shortening and the dynamic constants of muscle. Proc. R. Soc. B 126, 136-195. HILL, A. V. (1939). Recovery heat in muscle. Proc. R. Soc. B 127, 297-307. HILL, A. V. (1965). Trails and Trials in Physiology, pp. 143, 259. London: Arnold. HILL, A. V. & KUPALOV, P. S. (1930). The vapour pressure of muscle. Proc. R. Soc. B 67, 152-165. HILL, A. V. & WOLEDGE, R. C. (1962). An examination of absolute values in myo. thermic measurements. J. Physiol. 162, 311-333. HOMSHER, E., MOMMAERTS, W. F. H. M. & RIcCHIUTI, N. V. (1974). Energetics of shortening muscles in twitches and tetanic contractions. J. gen. Physiol. 62. 667-692. HOMSHER, E., MOMMAERTS, W. F. H. M., RICCHIUTI, N. V. & WALLNER, A. (1972). Activation heat, activation metabolism and tension-related heat in frog semitendinosus muscles. J. Physiol. 220, 601-625. JEWELL, B. R., KRETZSCHMAR, M. & WOLEDGE, R. C. (1967). Length and tension transducers. J. Physiol. 191, lOP. JEWELL, B. R. & WILKIE, D. R. (1 960). The mechanical properties of relaxing muscle. J. Physiol. 152, 30-47. KARNOVSKY, M. J. (1967). The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J. cell Biol. 35, 213-236. LANE, B. P. (1965). Alterations in the cytological detail of intestinal smooth muscle cells in various stages of contraction. J. cell Biol. 27, 199-213.

SMOOTH-MUSCLE

ENERGETICS

229

LANGLEY, J. N. & ANDERSON, H. K. (1895). On the innervation of the pelvic and adjoining vescera. I. The lower portion of the intestine. J. Physiol. 18, 67-105. LANGLEY, J. N. & ANDERSON, H. K. (1896). The innervation of the pelvic and adjoining viscera. J. Physiol. 20, 372-406. Lowy, J., VIBERT, P. J., HASELGROVE, J. C. & POULSEN, F. R. (1973). The structure of the myosin elements in vertebrate smooth muscles. Phil. Trans. R. Soc. B 265, 191-196. MCINTYRE, T. W. (1965). Active and mechanical properties of vascular smooth muscle. Ph.D. Dissertation, University of California, Los Angeles. McKRDY, H. C. (1972). Functional relationship of longitudinal and circular layers of the muscularis externa of the rabbit large intestine. J. Physiol. 227, 839-853. MASHIMA, H. (1969). The force-velocity and the dynamic constants of the guinea-pig taenia coli. J. physiol. Soc. Japan 31, 565-566. MERRILLEES, N. C. R. (1968). The nervous environment of individual smooth muscle cells of the guinea pig vas deferens. J. cell Biol. 37, 794-817. MULVANY, M. J. & WOLEDGE, R. C. (1972). Heat production in guinea-pig taenia coli muscles. J. Physiol. 229, 20P. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. cell Biol. 17, 208-212. RICcHIUTI, N. V. & MOMMAERTS, W. F. H. M. (1965). Technique for myothermic measurements. Physiologist, Wash. 8, 259. SANDBERG, J. A. & CARLSON, F. D. (1966). The length dependence of phosphorylcreatine hydrolysis during an isometric tetanus. Biochem. Z. 345, 212-231. SMITH, I. C. H. (1972). Energetics of activation in frog and toad muscle. J. Physiol. 220, 583-599. SOMLYO, A. P., DEVINE, C. E., SOMLYO, A. V. & RIcE, R. V. (1973). Filament organization in vertebrate smooth muscle. Phil. Trans. R. Soc. B 265, 223-230. SPuRR, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43. TAYLOR, S. R. & RUDEL, R. (1973). Aequorin luminescence during contraction of amphibian skeletal muscle. J. Physiol. 233, 5P. WENDT, I. R. & GIBBS, C. L. (1973). Energy production of rat extensor digitorum longus muscle. Am. J. Physiol. 224, 1081-1086. WENDT, I. R. & GIBBS, C. L. (1974). Energy production of mammalian fast- and slow-twitch muscles during development. Am. J. Physiol. 226, 642-647. WIEBEL, E. R. (1972). A stereological method for estimating volume and surface of sarcoplasmic reticulum. J. Microscopy 95, 229-242. WILKIE, D. R. (1956). Measurement of the series elastic component at various times during a single muscle twitch. J. Physiol. 134, 527-530. WILKIE, D. R. (1960). Thermodynamics and the interpretation of biological heat measurements. Proc. Biophys. biophys. Chem. 10, 260-298. WILKIE, D. R. (1968). Heat work and phosphorylcreatine break-down in muscle. J. Physiol. 195, 157-183. WOLEDGE, R. C. (1968). The energetics of tortoise muscle. J. Physiol. 197, 685-707. YAMADA, K. (1970). The increase in the rat of heat production of frog's skeletal muscle caused by hypertonic solutions. J. Physiol. 208, 49-64.

230

D. F. DAVEY, C. L. GIBBS AND H. C. McKIRDY EXPLANATION OF PLATES

PLATE 1

Fig. 1. Transverse section passing through an entire fasciculus of a rabbit rectococcygeus muscle. Both bundles and cells tend to be irregularly shaped. Innervation is in the form of small axon bundles and is somewhat sparse; most fasciculi have only one axon bundle, some have none. The axon bundles of this fasciculus lies within the small rectangle, and is shown at higher magnification in fig. 2. Scale bar represents 10 ,um. Fig. 2. Transverse section through a small axon bundle from the fasciculus shown in fig. 1. Note that all the axons are enclosed within a single Schwann cell. The axon on the right has been sectioned through a varicosity and synaptic vesicles can be seen. Scale bar represents 0 B5,m. Fig. 3. Transverse section through a single muscle fibre fixed using a modified Krebs solution to dissolve the aldehyde fixative (see Methods). Note the presence of empty spaces around the cell perimeter (arrows) and compare to fig. 4. An intercellular gap junction is visible (bottom centre) between this cell and its neighbour to the left. Scale bar represents 0 5 ,um. Fig. 4. Similar view to fig. 3, but from a muscle fixed in the presence of a high concentration of Mg2+ ions (see Methods). Note the lack of subplasmalemmal spaces. The accumulation of mitochondria and rough-surfaced endoplasmic reticulum near the centre of this cell indicates that it has been sectioned close to its nucleus. Smoothsurfaced endoplasmic reticulum (arrows) was distinguished from subsarcolemmal vesicles for morphometric purposed, simply by virtue of the elongated nature of the profiles (see Table 3). Scale bar represents 0 5 ,um. PLATE 2 Transverse section through part of a fasciculus showing a rare close contact junction. As in most gastrointestinal smooth muscle, three filament types can be observed: thin filaments (upper arrows), intermediate filaments (middle arrows) and thick filaments (lower arrows). Scale bar represents 0-2 jam.

Plate 1

The Journal of Physiologyq Vol. 248, No. 1 ap./m'

_

.:

__:

sA d. f l..

..

.

_.

e7

.-

14

'

A F*"0"' D. F. DAVEY, C. L. GIBBS AND H. C. McKIRDY

(Facing p. 230)

2 X'lSs-

'-.'*w|svX,o.4WX!t*w Plate 2

The Journal of Physiology, Vol. 248, No. 1

.

_F ^';:

['1

WF

.r

f_

.e

...

i:Xr.

t.

#,4


'Z'5';_' L

*

-i +

;.._''S iL

a.

f >^ Z

Fs

wS

ii > *,

w

tw ^^fi6t

AX'-8..ss '

iia

si 7

leC

IIeLtst*s EibEi s

-

D. F. DAVEY, C. L. GIBBS AND H. C. McKIRDY

.6

_.-

I

-W