When the anterior byssal retractor muscle (ABRM) of Mytilus edulis was subjected to a sudden reduction in temperature during a contractionrelaxation cycle, a tension increment, the cold-induced contracture (CIC), was observed. The CIC could be obtained in a muscle stimulated by the application of acetylcholine (ACh), caffeine or high-K+ solutions, but could not be elicited from a muscle at rest or in ‘catch’. When the muscle was released from ‘catch’ by the application of 5-hydroxytryptamine (5-HT), a CIC could once again be produced. The CIC tension elicited after the application of ACh was shown to follow a log-dose relationship with respect to ACh concentration but no CIC was observed at an ACh concentration at which the initial tension was 10% or less of maximum. The CIC tension decreased with time after the application of ACh and appeared to be dependent on both the initial and final temperatures and on the size of the temperature drop. The significance of these results and possible explanations are considered.

The effect of subjecting muscles to rapid reductions in temperature has been extensively studied and cold-induced increments in tension have been observed in both resting vertebrate and invertebrate muscles.

Rapid cooling in physiological saline has been reported to result in the initiation of a tonic contracture in the retractor penis muscle, cat nictitating membrane, pig stomach and taenia coli (Perkins et al. 1950; Magaribuchi, Ito & Kuriyama, 1973; Ercoli & Guzzon, 1952). Resting vertebrate skeletal muscle and the anterior byssal retractor muscle (ABRM) of Mytilus edulis, however, belong to a group of muscles which must be pretreated with sub-threshold caffeine or sub-threshold high-K+ solutions in order to produce a tension increment in response to rapid cooling (Sakai, 1965; Guttman & Katz, 1953; Guttman & Gross, 1956; Sakai & Yoshioka, 1973).

The rapid cooling response appears to vary with the type of muscle and the composition of the bathing medium. When toad and frog skeletal muscles are rapidly cooled in the presence of sub-threshold caffeine, for example, they show tonic contractures which relax either spontaneously or on rewarming, depending on the caffeine concentration (Sakai, 1965; Yoshioka, 1976), while the ABRM shows a phasic response which is fully relaxed in 10 min (Guttman & Katz, 1953; Guttman & Gross, 1956; Guttman, Dowling & Ross, 1957; Guttman & Ross, 1958). Untreated frog muscles, on the other hand, have been reported to exhibit a slight relaxation in response to rapid cooling (Hill, 1970a,EXBIO_119_1_133C11b).

To date, the effects of rapid cooling have been studied in resting muscles since, in general, the time course of the contractile response of most preparations is too short to permit such studies to be made on contracting muscles. However, the ABRM can maintain tension for prolonged periods and the present study investigates the effects of rapid cooling during a contraction-relaxation cycle.

Animals

Specimens of Mytilus edulis were obtained from the Gatty Marine Laboratory, University of St Andrews. They were stored either in aerated sea water, or in a refrigerator at 4°C for up to 4 days before use.

Dissection

The ABRM was dissected as described by Twarog (1954). The muscle was stripped of all connective and nervous tissue and trimmed to obtain a bundle of fibres of approximately 1 mm in diameter. A hook was tied around its base, anterior to the foot and byssal organ.

All experiments were conducted at a standard muscle length, 80% of the in situ length (Lo).

Apparatus

The muscle was suspended vertically. A small piece of shell left attached to the muscle during dissection held one end of the muscle in a glass stirrup, and a silver chain attached the other end to the E and M Linear Core Isometric Transducer (range 0–1 kg). The transducer was mounted on an adjustable micrometer, which allowed the muscle length to be varied to within 0·01 mm. The whole unit was supported on a moveable stand, allowing it to be raised and lowered into solutions of varying temperatures within 5–7 s.

The temperature of the experimental solutions was maintained within acceptable limits by polystyrene insulation (Linehan, 1982).

Procedure

After dissection the muscle was allowed to equilibrate in a sea water solution for at least 1 h before experimentation commenced. Tension was induced by the application of 10−3moll−1 acetylcholine chloride (ACh) for 1 min. At this concentration a reproducible tension response, approximately 90–100% of maximum, was obtained, and repeated contractures could be elicited without any visible adverse effects on the muscle. Tension was abolished with 10−6 moll−1 5-hydroxytryptamine creatine sulphate complex (5-HT). These drugs were either made up directly in sea water or as concentrated stock solutions in distilled water (which were diluted as required in the experimental solutions so that the ionic strength of the sea water was not reduced by more than 1%). The concentrations of 5-HT and ACh were only varied in some control experiments.

To initiate phasic contractions, a high-K+ artificial sea water (ASW) solution was used, in which 320 mmol 1−1 NaCl was replaced by KC1 (Nagai & Hagiwara, 1970).

Control experiments showed that, in the majority of cases, a reproducible tension response could be obtained at temperatures up to approximately 35°C. However, at temperatures above 25°C muscles were stimulated only once since an irreversible effect on muscle proteins could not then be excluded.

Cold-induced contractures

A tension increment could be produced by removing an ABRM from a solution of ACh in which it was undergoing contraction and rapidly transferring the muscle to an ordinary sea water solution at a lower temperature. This phenomenon was investigated with respect to time after the application of ACh, ACh concentration of the initial activating solution and the size of the temperature drop applied. In experiments to test the effects of time after ACh stimulation and ACh concentration, the temperature of the initial solution (T1) was kept constant at 20°C and the temperature of the cold sea water solution (Ta) was kept constant at 2°C. The temperature drop (ΔT) was, therefore, constant at 18°C.

In experiments to test the effect of altering the size of the temperature drop, the time of application of the cold sea water was kept constant at to (time of application of ACh) plus 2min (t0+ 2 min). In the first set of these experiments (Method 1), the temperature of the ACh solution (T1) was kept constant at 20°C and the temperature of the cold sea water solution (T2) was varied between 2 and 15°C. In the second set of experiments (Method 2), T1 was varied between 5 and 45°C while T2 was kept constant at 2°C.

The size of the cold-induced contracture was found by subtracting the tension response obtained when the muscle was kept at a constant temperature (T1) from that obtained when the temperature was lowered (T1 to T2). The difference in tension was measured at 20-s intervals and plotted. This enabled the size and the time course of the CIC to be accurately determined.

Solutions

Fresh or artificial sea water was used in all experiments. No difference was detected between the two in terms of the response of the muscle. The artificial sea water used was that of Nagai & Hagiwara (1970): (in mmol 1−1) NaCl, 450; KC1, 10; CaCl2, 10; MgSO4, 51; Tris (hydroxymethyl) aminomethane, 50 (pH7·3–7·4).

Recordings

Tension responses were recorded on a Bryans (27000) chart recorder.

Analysis of tension responses

Unless otherwise stated, tension is expressed in absolute terms (kgcm−1). Muscle length is expressed as a multiple of the in situ length (Lo). The parameter dP/dT (the rate of tension development) was obtained by drawing the tangent to the tension increment and calculating the slope; dP/dT is expressed in kg cm−1 s−1. The rate of relaxation was estimated by taking the time required for the muscle tension to decrease to 50% of maximum. The relaxation T1/2 is expressed as a percentage of the maximum T1/2 obtained during a series of experiments.

Statistics

Results are expressed as mean ± 1 standard error of the mean. Statistical analysis was performed using Student’s t-test or linear regression where appropriate.

Reagents

Acetylcholine chloride and 5-hydroxytryptamine creatinine sulphate complex were obtained from the Sigma Chemical Company, St Louis, U.S.A. All other reagents used were of the analytical grade.

Time after acetylcholine stimulus

A CIC was produced by suddenly reducing the ambient temperature of an ABRM to 2°C at 2, 4 and 30 min after the initial application of an ACh solution at 20°C. The size of the tension increment decreased as this delay was increased (Figs 1,2). The CIC tension was observed to be maximal (1-36 kg cm−1) at the earliest time investigated (to + 1 min), and was observed to decrease to 0·6 kg cm−1 at to + 7 min. From to + 7 to t0 + 33·5 min, the CIC tension did not alter significantly.

Fig. 1.

Typical cold-induced contracture traces. The CIC was elicited in response to the application of sea water at 2°C (▴) for 2, 4 and 30min (traces A, B and C, respectively) after the addition of 10−3 mol 1−1 ACh at 20°C (△). Muscle length: 0·8L0.

Fig. 1.

Typical cold-induced contracture traces. The CIC was elicited in response to the application of sea water at 2°C (▴) for 2, 4 and 30min (traces A, B and C, respectively) after the addition of 10−3 mol 1−1 ACh at 20°C (△). Muscle length: 0·8L0.

Fig. 2.

Relationship between CIC tension and time between initial addition of 10−3 mol 1−1 ACh and application of cold shock solution. The CIC tension (P) is expressed in terms of the corresponding ACh-induced tension (P0) and decreases with increasing time between the addition of 10−3moll ACh (20°C) and application of cold shock (2°C). In absolute terms, 0·2P/Po = 0·9kgcm−2 approximately. The filled circle represents the time at which cold shock was routinely applied in other experiments. Each point represents the mean of six observations and the vertical bars are the standard error of the mean. Muscle length: 0·8L0.

Fig. 2.

Relationship between CIC tension and time between initial addition of 10−3 mol 1−1 ACh and application of cold shock solution. The CIC tension (P) is expressed in terms of the corresponding ACh-induced tension (P0) and decreases with increasing time between the addition of 10−3moll ACh (20°C) and application of cold shock (2°C). In absolute terms, 0·2P/Po = 0·9kgcm−2 approximately. The filled circle represents the time at which cold shock was routinely applied in other experiments. Each point represents the mean of six observations and the vertical bars are the standard error of the mean. Muscle length: 0·8L0.

The rate of tension development (Fig. 3) and relaxation rate (Fig. 4) were also shown to decrease in a similar manner with respect to time after stimulus.

Fig. 3.

Relationship between the rate of CIC tension development and the time between the addition of initial 10−3moll−3 ACh (20°C) and the application of cold shock solution (2°C). As the time interval increased from 60 s to 420 s, the rate of tension development decreased markedly; thereafter, the reduction in the rate of tension development was less marked. Each point is the mean of five observations and the vertical bars represent the standard error of the mean. Muscle length: 0·8L0.

Fig. 3.

Relationship between the rate of CIC tension development and the time between the addition of initial 10−3moll−3 ACh (20°C) and the application of cold shock solution (2°C). As the time interval increased from 60 s to 420 s, the rate of tension development decreased markedly; thereafter, the reduction in the rate of tension development was less marked. Each point is the mean of five observations and the vertical bars represent the standard error of the mean. Muscle length: 0·8L0.

Fig. 4.

Relationship between CIC relaxation half-time and the time between the addition of 10−3 mol 1−1 ACh (20°C) and the application of cold shock solution (2°C). Relaxation half-time increased four-fold as the time interval was increased from 60 to 1800 s. Each point represents the mean of five observations and the vertical bars are the standard error of the mean. The curve was fitted by eye. Muscle length: L0.

Fig. 4.

Relationship between CIC relaxation half-time and the time between the addition of 10−3 mol 1−1 ACh (20°C) and the application of cold shock solution (2°C). Relaxation half-time increased four-fold as the time interval was increased from 60 to 1800 s. Each point represents the mean of five observations and the vertical bars are the standard error of the mean. The curve was fitted by eye. Muscle length: L0.

At t0+ 2 min, the muscle was observed to have achieved maximum tension in response to ACh and the CIC was large enough to be measured accurately. For these reasons, the time of application of cold sea water was kept constant at to + 2 min in all experiments reported below.

Size of the temperature drop

Method 1: constant starting temperature (T1)

In these experiments, T1 was constant at 20°C and T2 was varied between 15 and 2°C. The CIC tension was observed to increase exponentially (log-linear slope = 0·069 ± 0·01) as the temperature step was increased (Fig. 5).

Fig. 5.

Relationship between CIC tension and (ΔT). Method 1: constant T1 (initial temperature = 20°C) variable T2 (temperature of cold shock solution = 15-2°C). The CIC tension increased from 0·2 ± –0 0·5 kg cm−2 at a ΔT of 5°C to 0·825 ± 0·2 kg cm−2 at a AT of 18°C. Each point represents the mean of five observations and the vertical bars are the standard error of the mean. The curve was fitted by eye. Muscle length: 0·8L0.

Fig. 5.

Relationship between CIC tension and (ΔT). Method 1: constant T1 (initial temperature = 20°C) variable T2 (temperature of cold shock solution = 15-2°C). The CIC tension increased from 0·2 ± –0 0·5 kg cm−2 at a ΔT of 5°C to 0·825 ± 0·2 kg cm−2 at a AT of 18°C. Each point represents the mean of five observations and the vertical bars are the standard error of the mean. The curve was fitted by eye. Muscle length: 0·8L0.

The rate of tension development (Fig. 6) was observed to increase and the relaxation rate (expressed as a percentage of the maximum T1/2 ) was seen to decrease as ΔT was increased and T2 was decreased (Fig. 7). It is important to note that the rate of relaxation decreases as the relaxation half-time increases.

Fig. 6.

Relationship between CIC tension development and ΔT. Method 1: T1 constant at 20°C and T2 varied between 15 and 2°C. The rate of tension development increased from 0·012 ± 0·0017 kg cm−2 s−1 at a ΔT of 5°C to 0·0177 ± 0·0015 kg cm−2 s−1 at a ΔT of 18°C. Each point represents the mean of six observations and the vertical bars are the standard error of the mean. The curve was fitted by eye. Muscle length: 0·8L0.

Fig. 6.

Relationship between CIC tension development and ΔT. Method 1: T1 constant at 20°C and T2 varied between 15 and 2°C. The rate of tension development increased from 0·012 ± 0·0017 kg cm−2 s−1 at a ΔT of 5°C to 0·0177 ± 0·0015 kg cm−2 s−1 at a ΔT of 18°C. Each point represents the mean of six observations and the vertical bars are the standard error of the mean. The curve was fitted by eye. Muscle length: 0·8L0.

Fig. 7.

The effect of ΔT on the CIC relaxation half-time. Method 1: T, constant at 20°C and T1 varied between 2 and 15°C. The ordinate shows the relaxation half-time expressed as a percentage of the maximum value (obtained at ΔT = 18°C). The time to 50% CIC relaxation decreases in absolute terms from 760 ± 70 s at a ΔT of 18°C to 438 ± 54 s at a ΔT of 5°C. Each point represents the mean of five observations and the vertical bars are the standard error of the mean. The line was fitted by eye. Muscle length: 0·8L0.

Fig. 7.

The effect of ΔT on the CIC relaxation half-time. Method 1: T, constant at 20°C and T1 varied between 2 and 15°C. The ordinate shows the relaxation half-time expressed as a percentage of the maximum value (obtained at ΔT = 18°C). The time to 50% CIC relaxation decreases in absolute terms from 760 ± 70 s at a ΔT of 18°C to 438 ± 54 s at a ΔT of 5°C. Each point represents the mean of five observations and the vertical bars are the standard error of the mean. The line was fitted by eye. Muscle length: 0·8L0.

Method 2: constant final temperature (T2)

In this set of experiments, T1 was varied between 5 and 45°C and T2 was kept constant at 2°C. Under these conditions, the CIC tension was observed to increase exponentially over the range T1 = 5·35°C (Fig. 8). The rate of tension development also increased over this range (Fig. 9). However, when T1was greater than 25°C, the rate of tension development increased sharply until above 30°C, when no further increase was observed.

Fig. 8.

Relationship between ΔT and CIC tension. Method 2: T1, was varied between 5 and 45°C and T2 remained constant at 2°C. The size of the CIC increases from 0·2 ± 0·6 kg cm−2 at a AT of 3°C to 3·0 ± 0·25 kg cm−2 at a AT of 32°C. As T1 increased above 35°C, the response declined. Each point represents the mean of five observations and the vertical bars are the standard error of the mean. Muscle length: 0·8L0.

Fig. 8.

Relationship between ΔT and CIC tension. Method 2: T1, was varied between 5 and 45°C and T2 remained constant at 2°C. The size of the CIC increases from 0·2 ± 0·6 kg cm−2 at a AT of 3°C to 3·0 ± 0·25 kg cm−2 at a AT of 32°C. As T1 increased above 35°C, the response declined. Each point represents the mean of five observations and the vertical bars are the standard error of the mean. Muscle length: 0·8L0.

Fig. 9.

Relationship between ΔT and the rate of CIC tension development. Method 2: T1 was varied between 5 and 40°C and T2 was kept constant at 2°C. The rate of tension development increased from a value of 0·0077 ± 0·0006 kg cm−2 s−1 at a ΔT of 3°C to 0·034 ± 0·004 kg cm−2 s−1 at a AT of 28°C. Each point represents the mean of five observations and the vertical bars are the standard error of the mean. Muscle length: 0·8L0.

Fig. 9.

Relationship between ΔT and the rate of CIC tension development. Method 2: T1 was varied between 5 and 40°C and T2 was kept constant at 2°C. The rate of tension development increased from a value of 0·0077 ± 0·0006 kg cm−2 s−1 at a ΔT of 3°C to 0·034 ± 0·004 kg cm−2 s−1 at a AT of 28°C. Each point represents the mean of five observations and the vertical bars are the standard error of the mean. Muscle length: 0·8L0.

The rate of relaxation expressed as a percentage of the T1/2 was also observed to increase as the temperature difference increased (Fig. 10).

Fig. 10.

Relationship between ΔT and the CIC relaxation half-time. Method 2: T1 was varied from 5 to 40°C and T2 was kept constant at 2°C. The ordinate shows the relaxation half-time expressed as a percentage of the initial value obtained at a ΔT of 3°C. Relaxation half-time decreased, in absolute terms, from 900 ± 63 s at a ΔT of 3°C to 180 ± 60s at a ΔT of 38°C. Each point represents the mean of five observations and the vertical bars are the standard error of the mean. The curve was fitted by eye. Muscle length: 0·8L0.

Fig. 10.

Relationship between ΔT and the CIC relaxation half-time. Method 2: T1 was varied from 5 to 40°C and T2 was kept constant at 2°C. The ordinate shows the relaxation half-time expressed as a percentage of the initial value obtained at a ΔT of 3°C. Relaxation half-time decreased, in absolute terms, from 900 ± 63 s at a ΔT of 3°C to 180 ± 60s at a ΔT of 38°C. Each point represents the mean of five observations and the vertical bars are the standard error of the mean. The curve was fitted by eye. Muscle length: 0·8L0.

Requirement for activation

The dose-response curve of the CIC tension to ACh concentration in the initial activating solution was investigated using a T1 of 20°C and a T2 of 2°C (ΔT = 18°C) at to + 2 min. The CIC showed a typical dose response curve which was very similar to that shown by the initial ACh tension (Fig. 11). The threshold concentration for a CIC was, however, greater than that for the initial ACh tension, since at ACh concentrations which produced approximately 10% of maximum tension no CIC was observed.

Fig. 11.

Acetylcholine dose-response curve for active tension (●) and CIC tension (◯). When ACh tension had fallen to approximately 10% of its maximum, the CIC tension was no longer measurable. Each point represents the mean of five observations and the vertical bars are the standard error of the mean. The curves were fitted by eye. Muscle length: 0·8L0.

Fig. 11.

Acetylcholine dose-response curve for active tension (●) and CIC tension (◯). When ACh tension had fallen to approximately 10% of its maximum, the CIC tension was no longer measurable. Each point represents the mean of five observations and the vertical bars are the standard error of the mean. The curves were fitted by eye. Muscle length: 0·8L0.

These two observations suggested that the CIC was in some way related to the level of activity of the muscle rather than to an alteration in the sensitivity of the muscle to ACh which might result from a sudden drop in temperature. This hypothesis was partially confirmed by the finding that the CIC could not be produced in a muscle which had been put into ‘catch’ by repeated exposure to ACh at 10-min intervals (Twarog, 1967a). Furthermore, when a muscle was brought out of this ‘catch’ state by the application of 5-HT, a CIC could once again be produced (Fig. 12). A CIC-type response was also observed in muscles undergoing contraction in response to high-K+ solutions or caffeine (unpublished observations).

Fig. 12.

The effect of cold shock on a muscle undergoing a 5-hydroxytryptamine (5-HT) induced relaxation. A solution of 10−3 moll−1 ACh at 20°C was added (A) followed by 10−4 moll−1 5-HT at 20°C (▴) and a cold shock solution at 2°C (▾). The shaded area indicates the tension increment in response to cold shock. Muscle length: 0·8L0.

Fig. 12.

The effect of cold shock on a muscle undergoing a 5-hydroxytryptamine (5-HT) induced relaxation. A solution of 10−3 moll−1 ACh at 20°C was added (A) followed by 10−4 moll−1 5-HT at 20°C (▴) and a cold shock solution at 2°C (▾). The shaded area indicates the tension increment in response to cold shock. Muscle length: 0·8L0.

The observed relationship between parameters of the CIC and the size of the temperature drop (ΔT) showed a distinct break at 25°C. In addition, the relationship between CIC tension and ΔT showed a further break at approximately 32°C. This may be related to the effect of temperature on the various components of the ACh tension response. The rate of tension development in response to ACh has been demonstrated to show three linear phases, from 2–25, 25–35 and 35–40°C, while the reduction in peak tension with temperature also exhibits three distinct phases from 2–25, 25–40 and above 40°C (Linehan, 1982). The significance of these observations is not clear, although alterations in the contractile response of the ABRM above 25°C have previously been explained in terms of a paramyosin phase change (Johnson, 1966), an alteration in the kinetics of an activator or the release of 5-HT from nerve terminals within the muscle (Twarog, 1967a).

There are several ways in which a tension increment might be induced by the application of a sudden temperature reduction. It could be the result of a passive phenomenon such as a passive volume change. This was, however, considered unlikely since the application of cold shock to muscles in ‘catch’ was without effect, and the CIC was accompanied by an enhancement of active state (unpublished observations).

The CIC was also not a wash-out contracture as described by Ohlin & Str ömblad (1963), since the transfer of an ABRM, undergoing a contraction, from an ACh solution at 20°C to another ACh solution at 20°C produced no increase in tension.

Since a passive origin of the CIC was largely discounted, it was considered possible that any of the following steps in a typical ACh-induced contracture might be affected by the application of cold shock: (1) the rate of tension development with respect to the rate of decay, (2) ACh-receptor interaction, (3) the contractile protein interaction, (4) membrane depolarization or (5) intracellular Ca2+ levels.

  1. From the results of Method 2 the CIC does not appear to result from a differential temperature effect on the rate of tension production and decay as has been suggested for the temperature effects in frog muscle (Hill, 1970a), since, in these experiments, the rate of tension development and tension decay during a CIC both increase as the temperature drop is increased and the activation energies of the two processes are similar (49·8 kJ mol−1 and 53·3 kJ mol−1 respectively). In Method 1, it would appear that the rate of relaxation decreases as ΔT increases whilst the rate of tension development increases. The measurement of relaxation T1/2 is, however, only an indirect measure of the actual relaxation rate since it will be dependent on the size of the tension response and is a composite of the activation and relaxation processes. The initial rate of tension development is, on the other hand, thought to reflect the activation processes within the muscle. These experiments cannot, therefore, confirm or refute the suggestion that the CIC results from a differential temperature effect on the rate of tension production and decay.

  2. It appears unlikely that cold shock increases the ACh sensitivity of the muscle, since abrupt cooling in a sub-threshold ACh concentration does not result in a tension increment, and furthermore, no CIC response was observed when the ACh response was reduced to 10% of maximum (Fig. 11). Another piece of evidence which supports this view is that a CIC can also be produced during a K+ contracture or a caffeine contraction (unpublished observations).

  3. Although a reduced affinity of the regulatory light chain for Ca2+ could result in a CIC, this possibility cannot be fully considered since the temperature effects on myosin regulation have not been widely investigated. Experimental evidence, however, suggests that the CIC does not arise in this way. No CIC is observed in a muscle at rest when a decrease in the Ca-requirement of the regulatory proteins or a reduction in the Ca-sensitivity of the contractile proteins might be expected to produce a tension increase. Additionally, there is no evidence to suggest that a cold shock will decrease the Ca-requirement for actomyosin interaction and published reports suggest that a decrease in temperature actually increases this requirement (Hartshorne, Barns, Parker & Fuchs, 1972; Taniguchi & Nagai, 1970).

  4. The depolarization of the ABRM membrane observed on sudden cooling was of the order of 8–12mV (preliminary, unpublished observations), which by itself is too small to reach the threshold of 35–40 mV necessary for contraction (Twarog, 1967b). In addition, Guttman & Katz (1953); Guttman & Gross (1956); Guttman et al. (1957) and Guttman & Ross (1958) have shown that the rapid or gradual cooling of an ABRM in solutions of sub-threshold K+ produce the same reduction in membrane potential but only the rapidly cooled muscle shows a tension response.

  5. The most likely possibility still to be considered involves an increase in intracellular Ca2+ levels. It is possible that cold shock increases or initiates an influx of extracellular Ca2+ or that it releases Ca2+ from intracellular sites. Either or both of these processes might result in an increase in the myoplasmic Ca2+ levels; a situation which is thought to mediate the cold-induced effects observed in smooth, cardiac and skeletal muscle (Magaribuchi et al. 1973; Kurihara, Kuriyama & Magaribuchi, 1974) and which is suggested to be the basis of the sub-threshold K+ contractions observed in the ABRM (Guttman & Ross, 1958).

Similar hypotheses have been put forward for the effect of rapid cooling on striated muscle, and work by Tanaguchi & Nagai (1970) and Newbold & Tume (1977) has demonstrated that the release of Ca2+ from sarcoplasmic reticulum (SR) vesicles isolated from rabbit striated muscle can be induced by a temperature drop. Newbold & Tume found that the vesicles release 45% of their stored Ca2+ in response to a drop in temperature and that this was increased when the vesicles were chemically treated or destabilized by storage. Additionally, the quantity of Ca2+ released increased as the temperature drop was increased. If, as might be expected, isolation decreased the stability of the vesicles, then Ca2+ released from stable vesicles in vivo might be too small to cause a contracture. This is supported by the findings of Tanaguchi & Nagai (1970), who calculated that the Ca2+ released from isolated SR in response to a temperature drop of 20°C would be sufficient to fully activate the muscle, yet Sakai (1965) found that rapidly cooling a resting muscle was without effect. Cooling in the presence of sub-threshold caffeine or K+, however, resulted in activation, suggesting that this treatment rendered the muscle more susceptible to cold shock in the same way as storage and chemical destabilization. Sakai & Kurihara (1974) suggest that caffeine facilitates the effect of cooling by increasing the rate and amount of Ca2+ release from the SR, while Caputo (1972) proposed that sub-threshold K+ releases small amounts of Ca2+ which, at room temperature are rapidly taken up by the SR, but at lower temperatures the uptake is inhibited resulting in contracture. Newbold & Tume (1977) suggested that sudden cooling of striated muscle would result in a release of Ca2+, since the passive outward diffusion of Ca2+ from the SR would be less temperature sensitive than the enzyme-mediated inward transport, resulting in a net Ca2+ efflux from the SR. This would seem to be confirmed to a certain extent by the observations of Gogjian & Bloomquist (1977), who showed that Ca2+ re-uptake into isolated ABRM vesicles was faster at higher temperatures.

The results of Gogjian & Bloomquist (1977) might explain why the relaxation rate of the ABRM undergoing an ACh contraction increases as the ambient temperature increases (Linehan, 1982). However, if these observations are applied to the CIC, one would expect the rate of Ca2+ uptake and hence relaxation to be related to T2. In Method 1, where T1 was kept constant and T2 was altered, the rate of relaxation was found to be directly related to T2. In Method 2, where T2 was constant at 2°C and T1 was increased, the rate of relaxation increased as T1 increased. The vesicles isolated by Gogjian & Bloomquist are therefore unlikely to be responsible for the CIC.

In addition to the vesicular Ca2+-accumulating elements, there is evidence to suggest that, in the ABRM, the mitochondria and the fibre membrane may act as intracellular Ca2+ stores (Atsumi & Sugi, 1976) and either or both of these sites may release Ca2+ in response to temperature shock; an explanation which has been put forward to account for the appearance of rapid cooling contractures (RCC) in smooth muscle preparations which also have extensive membrane-bound Ca2+ stores (Magaribuchi et al. 1973; Kurihara et al. 1974). The extracellular bound Ca2+ is a further possible source of Ca2+ ions.

Experiments are at present being undertaken in an attempt to elucidate the process whereby a sudden temperature reduction gives rise to a tension increment. It is hoped that the elucidation of this mechanism might further our understanding of catch muscle function.

I acknowledge financial support from Action Research for the Crippled Child and extend my thanks to the following: Drs Brian McCaldin, Robert Pitman and J. McCallum; Professor J. F. Lamb, Mr Ian Grieve and Mr Bob Adam.

Atsumi
,
S.
&
Sugi
,
H.
(
1976
).
Localization of Ca-accumulating structures in the ABRM of Mytilus edulis and their role in the regulation of active and catch contractures
.
J. Physiol., Lond
.
257
,
549
560
.
Caputo
,
C.
(
1972
).
Effect of low temperature on the excitation contracture coupling phenomena of frog single muscle fibres
.
J. Physiol., Lond
.
223
,
461
482
.
Ercoli
,
N.
&
Guzzon
,
V.
(
1952
).
Thermal stimulation of isolated organs and its inhibition by pharmacological agents
.
Science, N. Y
.
115
,
672
674
.
Gogjian
,
M.
&
Bloomquist
,
E.
(
1977
).
Ca2+ uptake by a sub-cellular membrane fraction of anterior byssus retractor muscle
.
Comp. Biochem. Physiol
.
58C
,
97
102
.
Guttman
,
M.
&
Gross
,
M. M.
(
1956
).
Relationship between electrical and mechanical changes in muscle caused by cooling
.
J. cell. comp. Physiol
.
48
,
421
433
.
Guttman
,
M.
&
Katz
,
A.
(
1953
).
Potential and mechanical changes in muscle on rapid cooling
.
XIXInternat. Physiol. Congress (Montreal)
, pp.
424
.
Guttman
,
M.
&
Ross
,
S.
(
1958
).
The effect of ions upon the response of smooth muscle to cooling
.
J. gen. Physiol
.
42
,
1
8
.
Guttman
,
R.
,
Dowung
,
J.
&
Ross
,
S.
(
1957
).
Resting potential and contractile system changes in muscle stimulated by cold and potassium
.
J. cell. camp. Physiol
.
50
,
265
276
.
Hartshorne
,
D.
,
Barns
,
E.
,
Parker
,
L.
&
Fuchs
,
F.
(
1972
).
The effect of temperature on actomyosin
.
Biochem. biophys. Acta
.
267
,
190
202
.
Hill
,
D. K.
(
1970a
).
The effect of temperature on the resting tension of frogs muscle in hypertonic solutions
.
J. Physiol., Lond
.
208
,
741
756
.
Hill
,
D. K.
(
1970b
).
The effect of temperature in the range 0–35°C on the resting tension of frogs muscle
.
J. Physiol., Lond
.
208
,
725
739
.
Johnson
,
W. H.
(
1966
).
Phase transitions in fibrous proteins as a mechanism for tonus
.
In Muscle
, (ed. edW. Paul).
Oxford
:
Pergamon Press
.
Kurihara
,
S.
,
Kuriyama
,
H.
&
Magaribuchi
,
T.
(
1974
).
Effects of rapid cooling on the electrical properties of the smooth muscle of the guinea-pig urinary bladder
.
J. Physiol., Lond
.
238
,
413
426
.
Linehan
,
C. M.
(
1982
).
The effect of temperature of the tension responses of the anterior byssal retractor muscle (ABRM) of Mytilus edulis
.
J. exp. Biol
.
97
,
375
384
.
Magaribuchi
,
T.
,
Ito
,
Y.
&
Kuriyama
,
H.
(
1973
).
Effects of rapid cooling on the mechanical and electrical activities of smooth muscles of guinea-pig stomach and taenia coli
.
J. gen. Physiol
.
61
,
323
341
.
Nagai
,
T.
&
Hagiwara
,
E.
(
1970
).
45Ca movements at rest and during potassium contracture in Mytilus ABRM. Jap
.
J. Physiol
.
20
,
72
83
.
Newbold
,
R.
&
Tume
,
R.
(
1977
).
Effect of orthophosphate and oxalate on the cold induced-release of Ca2+ from sarcoplasmic reticulum preparations from rabbit skeletal muscle
.
Aust. J. biol. Sri
.
30
,
519
526
.
Ohun
,
P.
&
Stromblad
,
R.
(
1963
).
Observations on the isolated vas deferens
.
Br.J. Pharmac
.
20
,
299
306
.
Perkins
,
J.
,
Li
,
M.
,
Nicholas
,
C. H.
,
Lassen
,
W. H.
&
Gertler
,
P.
(
1950
).
Cooling as a stimulus to smooth muscles
.
Am. J. Physiol
.
163
,
14
26
.
Sakai
,
T.
(
1965
).
The effects of temperature and caffeine on activation of the contractile mechanism in striated muscle fibres
.
Jikeikai med. J
.
12
,
88
102
.
Sakai
,
T.
&
Kurihara
,
S.
(
1974
).
The rapid cooling contracture of toad cardiac muscles
.
Jap. J. Physiol
.
24
,
649
666
.
Sakai
,
T.
&
Yoshioka
,
T.
(
1973
).
Studies on rapid cooling contractures of frog toe muscle immersed in hypertonic and hypotonic solutions
.
Jap. J. Physiol
.
23
,
135
147
.
Tanaguchi
,
M.
&
Nagai
,
T.
(
1970
).
Effect of temperature on caffeine-induced calcium release from isolated reticulum in frog skeletal muscle
.
Jap. J. Physiol
.
20
,
61
71
.
Twarog
,
B. M.
(
1954
).
Responses of a molluscan smooth muscle to ACh and 5HT
.
J. cell. comp. Physiol
.
44
,
141
164
.
Twarog
,
B. M.
(
1967a
).
Factors influencing contraction and catch inMytilus smooth muscle
.
J. Physiol., Lond
.
192
,
847
856
.
Twarog
,
B. M.
(
1967b
).
Excitation of Mytilus smooth muscle
.
J. Physiol., Lond
.
192
,
857
868
.
Yoshioka
,
T.
(
1976
).
On rapid cooling contractures of fast and slow muscle fibres
.
Jikeikai med. J
.
23
,
101
116
.