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1The relative abilities of caffeine and transverse tubular (T‐) system depolarisation to induce Ca2+release in mammalian skeletal muscle were compared in mechanically skinned fibres of the rat, in order to determine whether normal excitation‐contraction (E‐C) coupling is achieved by up‐regulating the Ca2+‐induced Ca2+release process, as caffeine is known to do.2Caffeine triggered Ca2+release in soleus (slow‐twitch) fibres at much lower concentrations than in extensor digitorum longus (EDL) (fast‐twitch) fibres when the sarcoplasmic reticulum (SR) of each type was loaded with Ca2+at close to endogenous levels. The difference in caffeine sensitivity resulted at least in part from the SR being loaded endogenously at near maximal capacity in soleus fibres but at less than half of maximal capacity in EDL fibres. The caffeine sensitivity could be reversed by reversing the relative level of SR loading.3The ability of caffeine to induce Ca2+release was markedly reduced by lowering the level of SR loading or by raising the free [Mg2+] from 1 to 3 mm. Caffeine, even at 30 mm, triggered little or no Ca2+release in EDL fibres (a) at 1 mm(physiological) Mg2+when the SR was loaded at two‐thirds or less of the endogenous level, and (b) at 3 mmMg2+when the SR was loaded at close to the endogenous level. In contrast, depolarisation potently elicited Ca2+release under these conditions in the same fibres.4The inability of 30 mmcaffeine to induce Ca2+release under certain conditions was not attributable to desensitisation or inactivation of the release channels, because there was no response even upon initial exposure to caffeine and depolarisation always remained able to trigger Ca2+release. It instead appeared that caffeine was a relatively ineffectual stimulus in EDL fibres except under conditions where (a) the SR was heavily loaded, (b) the free [Mg2+] was low, or (c) a high [Cl−] was present.5These results show that the normal E‐C coupling mechanism in mammalian skeletal muscle does not involve just enhancing Ca2+‐induced Ca2+release, and evidently requires the removal or bypassing of the inhibitory effect of Mg2+on the Ca2+release channels.

1. Force responses from mechanically skinned fibres of rat skeletal muscles (extensor digitorum longus and soleus) were measured at different temperatures in the range 3‐35 degrees C following sudden changes in Ca2+ concentration in the preparations. 2. At all temperatures there were characteristic differences between the slow‐ and fast‐twitch muscle fibres with respect to the relative steady‐state force‐[Ca2+] relation: such as a lower [Ca2+] threshold for activation and a less steep force‐pCa curve in slow‐twitch muscle fibres. 3. At 3‐5 degrees C the force changes in both types of muscle fibres lagged considerably behind the estimated changes in [Ca2+] within the preparations and this enabled us to perform a comparative analysis of the Ca2+ kinetics in the process of force development in both muscle fibre types. This analysis suggest that two and six Ca2+ ions are involved in the regulatory unit for contraction of slow‐ and fast‐twitch muscle fibres respectively. 4. The rate of relaxation following a sudden decrease in [Ca2+] was much lower in the slow‐twitch than in the fast‐twitch muscle at 5 degrees C, suggesting that properties of the contractile apparatus could play an essential role in determining the rate of relaxation in vivo. 5. There was substantial variation in Ca2+ sensitivity between muscle fibres of the same type from different animals at each temperature. However the steepness of the force‐[Ca2+] relation was essentially the same for all fibres of the same type. 6. A change in temperature from 5 to 25 degrees C had a statistically significant effect on the sensitivity of the fast‐twitch muscle fibres, rendering them less sensitive to Ca2+ by a factor of 2. However a further increase in temperature from 25 to 35 degrees C did not have any statistically significant effect on the force‐[Ca2+] relation in fast‐twitch muscle fibres. 7. The effect of temperature on the Ca2+ sensitivity of slow‐twitch muscle fibres was not statistically significant, mainly because of the large variation in sensitivity amongst these preparations at room temperature. 8. Two types of oscillatory processes not associated with intracellular membranes were observed in the force response of all slow‐twitch muscle fibres when submaximally activated (less than 60% maximum force) at 25 and 35 degrees C, but never at 3‐5 degrees C. The frequency of oscillations increased with temperature. 9. Maximum Ca2+‐activated force in both muscle fibre types was greatly dependent upon temperature over the range 0‐25 degrees C, but increased only slightly above 25 degrees C. 10. Experiments on the rigor state suggest that the number of possible actomyosin interacting sites diminishes considerably as temperature is decreased below 25 degrees C.

1. Steady‐state force—pCa relations were determined for mechanically skinned fibres of fast‐ and slow‐twitch rat skeletal muscle, extensor digitorum longus (e.d.l.) and soleus respectively, at varied sarcomere lengths and temperatures (3‐35 °C).

Single mechanically skinned muscle fibres of different types (fast‐ and slow‐twitch mammalian; slow and twitch amphibian) were successively activated in solutions of various Ca2+ concentrations at different temperatures. An increase in temperature from 5 to 22 degrees C reversibly shifted the isometric steady‐state force‐pCa curves towards higher Ca2+ concentration for individual fibres of each of the muscle types. A further increase in temperature to 35 degrees C in mammalian fibres resulted in an additional decrease in Ca2+ sensitivity. The temperature dependence of Ca2+ sensitivity was greater in the 'faster' fibre types: fast‐twitch greater than slow‐twitch; twitch greater than slow. The maximum isometric force response, P0, of both rat and toad skinned fibres was found to be strongly dependent on temperature below 22 degrees C. No detectable force could be induced by Ca2+ in mammalian muscle fibres at 0‐1 degree C while in toad fibres P0 decreased by about 90% when temperature dropped from 20 to 0 degree C. Since in mechanically skinned fibres of other amphibians (Bufo bufo, Rana species) P0 is only marginally affected it is likely that the P0‐temperature relations are indicative of the range of temperature over which the muscles are normally functional. The P0‐temperature relations of skinned muscle fibres closely resembled the P0‐temperature relations of tetanically stimulated intact muscle preparations from the same species of animals suggesting that the contractile apparatus is mainly responsible for the variation of force response with temperature in intact muscle.

A study was carried out to investigate the effects of ionic strength and monovalent cations on isometric, Ca2+‐activated force and rigor responses in mechanically skinned muscle fibres. Three types of skeletal muscle fibres were used: rat fast‐ and slow‐twitch fibres and toad twitch fibres. The contractile apparatus of rat slow‐twitch fibres was affected differently from that of rat fast‐twitch and amphibian twitch fibres when changing the ionic strength (expressed either in terms of ionic equivalents as I or formally as gamma/2) and [K+]. Thus, the apparent sensitivity to Ca2+ decreased substantially more in slow‐twitch fibres (by a factor of 20) than in the other fibre types (by a factor of 12) when I and [K+] were increased from 94 to 354 mM and from 56 to 316 mM respectively. Maximum Ca2+‐activated force, however, declined only by a factor of 2.2 in slow‐twitch fibres compared with 4.2 in the other fibre types, when I was increased from 154 to 354 mM. In slow‐twitch fibres the force oscillations of myofibrillar origin were found to increase substantially in amplitude, duration and frequency at low values of I and almost disappeared at high ionic strength. At low values of I, it was also discovered that ca. 50% of the fast‐twitch fibres responded with myofibrillar force oscillations when submaximally activated. The characteristics of these oscillations were different from those of slow‐twitch fibres. Rigor force levels were found to decline markedly with increasing iota and [K+] in all fibre types. Unexpectedly, once rigor force was established in a certain ionic environment, the level of force was stable regardless of further changes in ionic strength and monovalent cation concentration. These results indicate that the rigor cross‐bridges can be formed in different stable positions and that the probability of attachment in certain positions (rather than the total number of cross‐bridges that can be formed) is influenced by the ionic conditions. Further experimental evidence provided in this study shows that the increase in [K+] is mainly responsible for the decrease of the Ca2+‐sensitivity of the contractile apparatus and that ionic strength (expressed as I rather than gamma/2) influences markedly the maximal Ca2+‐activated force, the maximum steepness of the pCa‐force relations and the oscillatory processes of myofibrillar origin.

This study was performed to determine whether Viso (the maximum speed of shortening extrapolated from force‐velocity curves) equalled Vu (the unloaded speed of shortening determined by the slack test) in both living fibres from Rana temporaria and mechanically skinned fibres from Rana pipiens. In living fibres (R. temporaria) we obtained improved estimates of Viso by performing force clamps (isotonic) and length ramps (isovelocity) down to very low loads (0.005 isometric tension, P0). Force‐velocity characteristics determined by force clamps and length ramps were the same. The hyperbolic Hill curves deviated from the force‐velocity data at both high and low loads and underestimated Viso by varying degrees. A better estimate of Viso was obtained by linear extrapolation of data at loads from 0.005‐0.02 P0 and the mean Viso at 7.5 degrees C was 4.08 muscle lengths/s +/‐ 0.11 (mean +/‐ S.E., n = 14). Improved estimates of Vu in living fibres were obtained by photographically calibrating the slack test. The mean Vu was 4.05 muscle lengths/s +/‐ 0.13 (mean +/‐ S.E., n = 14) and the intercept was 0.0156 fibre lengths (L0) +/‐ 0.0013 (mean +/‐ S.E., n = 14). The step‐ramp photographic method, in which the motor speed is matched to Vu, was developed as an independent way to measure Vu in living fibres. Vu measured in this way agreed well with Vu measured by the slack test. In all living fibres, the improved estimates of Vu agreed well with the improved estimates of Viso. Vu/Viso = 0.99 +/‐ 0.01 (mean +/‐ S.E., n = 14). In mechanically skinned R. pipiens fibres, force clamps were performed down to loads of 0.01 mN. The force‐velocity curve of the skinned fibres differed in shape from that of the living fibres. Although there was significant deviation from the Hill equation at low loads, the data at high loads were well fitted by the Hill curve. Viso determined by extrapolating the Hill equation to zero load was 5.87 muscle lengths/s +/‐ 0.38 (mean +/‐ S.E., n = 9) at 7.5 degrees C. In five fibres, the linear extrapolation of low loads (0.01‐0.05 P0) showed that the Hill equation underestimated the true Viso by 6%. The slack test with mechanically skinned fibres was calibrated by taking a series of photographic exposures of the fibre at various times following each length step. Vu = 6.12 muscle lengths/s +/‐ 0.44 (mean +/‐ S.E., n = 10) and the intercept was 0.0585 L0 +/‐ 0.0069 (mean +/‐ S.E., n = 10).(ABSTRACT TRUNCATED AT 400 WORDS)

The ability of the sarcoplasmic reticulum of skinned cardiac muscle of the rat to accumulate and release Ca2+ was studied in the presence and absence of procaine. Ca2+ accumulation was estimated from the magnitude of the caffeine‐ (30 mM) induced force transient in a weakly Ca2+ buffered solution. The relative area under the caffeine‐induced force transient was up to 4‐fold greater when 5 mM‐procaine had been present during the preceding period of Ca2+ loading, than that after an equivalent period of Ca2+ loading in the absence of procaine. Procaine antagonized the caffeine‐induced release of Ca2+ when present in the Ca2+ releasing solution, however, the ability of procaine to attenuate the caffeine‐induced Ca2+ release diminished as the extent to which the sarcoplasmic reticulum was loaded with Ca2+ increased. In the presence of 1 mM‐Mg2+ procaine also markedly attenuated the small spontaneous force oscillations (5‐10% P0) associated with the cyclic release and reuptake of Ca2+ by the sarcoplasmic reticulum. When the Mg2+ concentration was reduced to 0.1 mM, procaine initially suppressed the small spontaneous oscillations in force, however, large force oscillations (40‐80% P0) of lower frequency were invariably initiated after 20‐60 s exposure to 5 mM‐procaine. Procaine (5 mM) produced a slight shift (approximately 0.04 pCa unit) of the force‐pCa relation toward lower Ca2+ concentrations. This effect is too small to influence in any substantial way the apparent effects of procaine on the sarcoplasmic reticulum. The results indicate that whilst procaine is indeed able to suppress Ca2+ release under certain circumstances, in its presence the net accumulation of Ca2+ by the sarcoplasmic reticulum can be markedly enhanced.

Differences in contractile activation by Ca2+ and Sr2+ between various types of normal and dystrophic murine muscle fibres were investigated using mechanically skinned fibres derived from soleus and extensor digitorum longus (e.d.l.) muscles of normal and dystrophic mice of strain 129ReJ. In terms of contractile activation, the normal e.d.l. muscle was found to consist of one relatively homogeneous population of muscle fibres characterized by steep force‐pCa and force‐pSr curves, low sensitivity to Ca2+ and very low sensitivity to Sr2+. Normal soleus muscles contained two fibre populations of similar size which could be distinguished on the basis of their contractile activation properties. The first fibre population was characterized mainly by its shallow force‐pCa and force‐pSr curves, high Ca2+ sensitivity, high Sr2+ sensitivity and the occurrence of large, slow force oscillations of myofibrillar origin. The second fibre population was characterized by force‐pCa and force‐pSr curves of steepness intermediate between those of normal e.d.l. and those of the first fibre population of normal soleus, by faster myofibrillar force oscillations and by low sensitivity to Ca2+ and Sr2+. The dystrophic e.d.l. fibre population had contractile characteristics which were distinct from those of the three types of normal fibre populations. However, some characteristics of the dystrophic e.d.l. fibres were very similar to those of the normal e.d.l. fibre population. Of all the fibre types investigated, dystrophic e.d.l. fibres were the least sensitive to Ca2+. Dystrophic soleus muscle contained a single homogeneous population of fibres which shared some common contractile activation characteristics with both of the fibre populations present in normal soleus muscle. However, of all fibre types investigated, the dystrophic soleus fibres were the most sensitive to Ca2+. Because of this characteristic, these fibres formed a distinct population. The maximum tensions induced by Ca2+ and Sr2+ were usually smaller in dystrophic fibres than in normal fibres obtained equivalent muscles. In conclusion, various normal murine muscle fibre types can be identified on the basis of differences in the mechanism of force activation by Ca2+ and Sr2+. Furthermore, it is possible to detect significant physiological differences in the mechanism of force activation brought about by murine muscular dystrophy.

The contractile apparatus of mechanically skinned muscle fibres of frog can be reversibly activated by Ba2+ and Cd2+. The maximum force induced by both Ba2+ and Cd2+ is the same as that induced by Ca2+ and Sr2+. The ionic concentrations of the divalent cations required to induce 50% of the maximum activated force at 1 mM‐Mg2+, pH 7.10, 22 degrees C and 250 mM ionic strength are about 8 X 10(‐7) M for Ca2+, 5 X 10(‐6) M for Cd2+, 2.6 X 10(‐5) M for Sr2+ and 7 X 10(‐4) M for Ba2+. Exposure of the skinned fibre to relatively low Ni2+ concentrations (between 10(‐6) and 10(‐5) M) resulted in a transient force response accompanied by an irreversible change in the ability of the preparation to develop force. The Ba2+‐ and Cd2+‐activation curves are considerably flatter than the corresponding curves for Ca2+ and Sr2+. An increase in Mg2+ concentration from 1 to 3 mM decreased the sensitivity of the contractile apparatus to Ba2+ by a factor of about 1.5 without affecting the maximum force response. The Ca2+‐activation curve was modified in the presence of subthreshold concentrations of Ba2+ and the results indicate that Ba2+ could have both an activating and an inhibitory action on the Ca2+‐activated force. A kinetic model which can quantitatively explain the results for activation of contraction by Ba2+ and Ca2+, is described.