1. The Ca2+ content of single mammalian skeletal muscle fibres was determined using a novel technique. Mechanically skinned fibres were equilibrated with varying amounts of the Ca2+ buffer BAPTA and were then lysed in a detergent-paraffin oil emulsion. The subsequent myofilament force response was used to estimate the additional amount of Ca2+ bound to BAPTA following lysis of intracellular membranes. 2. The total endogenous Ca2± content (corrected for endogenous Ca2+ buffering) of fast-twitch (FT) and slow-twitch (ST) fibres at a myoplasmic pCa (-log [Ca2+]) of 7-15 was 1 32 + 0-02 and 1P35 + 0-08 mm per fibre volume, respectively. The sarcoplasmic reticulum (SR) component of these estimates was calculated as 1P01 and 1P14 mm, respectively, which normalized to SR volume corresponds to resting SR Ca2P contents of 11 and 21 mm, respectively.3. Equilibration of 'resting' fibres with low myoplasmic [Ca2+] (pCa 7 67-9 00) elicited a timedependent decrease in Ca2P content in both fibre types. Equilibration of resting fibres with higher myoplasmic [Ca2+] (pCa 5 96-6 32) had no effect on the Ca2P content of ST fibres but increased the Ca2+ content of FT fibres. The maximum steady-state total Ca2P content (3-85 mM) was achieved in FT fibres after 3 min equilibration at pCa 5-96. Equilibration at higher myoplasmic [Ca2+] was less effective, probably due to Ca2+-induced Ca2+ release from the SR. 4. Exposure of fibres to either caffeine (30 mm, pCa -8, 2 min) or low myoplasmic [Mg2+] (0 05 mm, pCa -9, 1 min) released approximately 85 % of the resting SR Ca2P content. The ability of caffeine to release SR Ca2+ was dependent on the myoplasmic Ca2P buffering conditions.5. The results demonstrate that the SR of ST fibres is saturated with Ca2P at resting myoplasmic [Ca2+] while the SR of FT fibres is only about one-third saturated with Ca2P under equivalent conditions. These differences suggest that the rate of SR Ca2P uptake in FT fibres is predominantly controlled by myoplasmic [Ca2+] while that of ST fibres is more likely to be limited by the [Ca2+] within the SR lumen.The time course of the contraction-relaxation cycle of skeletal muscle is greatly influenced by the function of the sarcoplasmic reticulum (SR), which acts to both release and to re-sequester intracellular calcium ions. Some aspects of SR function in mammalian skeletal muscle have been ascertained from the simultaneous measurement of the myoplasmic free calcium ion concentration ([Ca2+]i) and force output of intact fibres (Fryer & Neering, 1986;Westerblad & Allen, 1991 ([Ca2K]SR), which is a key determinant of: (i) the rate of Ca2+ loss through SR Ca2+ release channels (Feher & Briggs, 1982;Sitsapesan & Williams, 1995), and (ii) the activity of the SR Ca2+ pump (Inesi & De Meis, 1989 (Fryer & Stephenson, 1993a,b;. METHODSSkinned muscle fibre preparation Skinned fibres were prepared using methods previously decribed in detail (Fink, Stephenson & Williams, 1986). Male Long-Evans hooded rats (Rattus norvegicus; 5-12 months old) were killed by dieth...

1. Mechanically skinned fast-twitch (FT) and slow-twitch (ST) muscle fibres of the rat were used to investigate the effects of fatigue-like changes in creatine phosphate (CP) and inorganic phosphate (Pi) concentration on Ca2+-activation properties of the myofilaments as well as Ca2+ movements into and out of the sarcoplasmic reticulum (SR). 2. Decreasing CP from 50 mm to zero in FT fibres increased maximum Ca2+-activated tension (Tmax) by 16 + 2 % and shifted the mid-point of the tension-pCa relation (pCa50) to the left by 0'28 + 0 03 pCa units. In ST fibres, a decrease of CP from 25 mm to zero increased Tmax by 9 + 1 % and increased the pCa50 by 0 16 + 0 01 pCa units. The effect of CP on Tmax was suppressed in both fibre types by prior treatment with 0 3 mm FDNB (1-fluoro-2,4-dinitrobenzene), suggesting that these effects may occur via changes in creatine kinase activity. 3. Increases of Pi in the range 0-50 mm reduced the pCa50 and Tmax in both fibre types.These effects were more pronounced in ST fibres than in FT fibres in absolute terms. However, normalization of the results to resting Pi levels appropriate to both fibre types (1 mm for FT and 5 mm for ST fibres) revealed similar decreases in Tmax (% 39% at 25 mM Pi and -48% at 50 mm Pi) and pCa50 (0 25

SUMMARY1. The effects of caffeine (0-2-20 mmol l-1) have been examined on calcium transients (measured with aequorin) and isometric force in intact bundles of fibres from soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles of the rat.2. At 25°C, threshold caffeine concentration for an observable increase in resting[Ca2+]i was 0-2 and 1-0 mmol 1-1 for soleus and EDL muscles respectively. Increases in resting force were first detectable at about 0 5 mmol 1-1 caffeine for soleus muscles and 5 0 mmol 1-1 caffeine for EDL muscles and occurred in the range 02-0-4 ,amol 1-l [Ca2+], for soleus and 0-7-09 jumol 1-1 for EDL.3. Caffeine potentiated the twitch responses of soleus and EDL in a dose-related manner. The soleus was more sensitive in this respect, with 50 % potentiation occurring at 1 mmol 1-1 caffeine compared with 3-5 mmol 1-1 for the EDL. Concentrations of caffeine below 2 mmol 1-1 potentiated Ca2+ transients associated with twitches in both soleus and EDL muscles with no apparent change in the decay rate constant.4. High concentrations of caffeine (> 2 mmol 1-1) further potentiated peak Ca2+ in the EDL but depressed it in the soleus. The rate of decay of the Ca2+ transient in high caffeine was significantly prolonged in the soleus but remained unaffected in the EDL.

1. The relationship between the total Ca2' content of a muscle fibre and the magnitude of the force response to depolarization was examined in mechanically skinned fibres from the iliofibularis muscle of the toad and the extensor digitorum longus muscle of the rat. The response to depolarization in each skinned fibre was assessed either at the endogenous level of Ca2' content or after depleting the fibre of Ca2! to some degree. Ca2W content was determined by a fibre lysing technique. 2. In both muscle types, the total Ca2+ content could be reduced from the endogenous level of -1 3 mmol F' (expressed relative to intact fibre volume) to -0 25 mmol by either depolarization or caffeine application in the presence of Ca2P chelators, showing that the great majority of the Ca2+ was stored in the sarcoplasmic reticulum (SR). Chelation of Ca2+ in the transverse tubular (T-) system, either by exposure of fibres to EGTA before skinning or by permeabilizing the T-system with saponin after skinning, reduced the lower limit of Ca2+ content to 6 0-12 mmol F7', indicating that 10-20% of the total fibre Ca2+ resided in the T-system. 3. In toad fibres, both the peak and the area (i.e. time integral) of the force response to depolarization were reduced by any reduction in SR Ca2+ content, with both decreasing to zero in an approximately linear manner as the SR Ca2+ content was reduced to < 15 % of the endogenous level. In rat fibres, the peak size of the force response was less affected by small decreases in SR content, but both the peak and area of the response decreased to zero with greater depletion. In partially depleted toad fibres, inhibition of SR Ca2P uptake potentiated the force response to depolarization almost 2-fold. 4. The results show that in this skinned fibre preparation: (a) T-system depolarization and caffeine application can each virtually fully deplete the SR of Ca!+ irrespective of any putative inhibitory effect of SR depletion on channel activation; (b) all of the endogenous level of SR Ca2+ must be released in order to produce a maximal response to depolarization; and (c) a substantial part (-40 %) of the Ca2P released by a depolarization is normally taken back into the SR before it can contribute to force production.It is well known that the release of Ca2+ from the sarcoplasmic reticulum (SR) of a vertebrate skeletal muscle fibre is required for the activation of the contractile apparatus (Ashley, Mulligan & Lea, 1991; Melzer, Herrmann-Frank & Liittgau, 1995). Electron probe analysis has shown that there is -I5 mmol (1 fibre water)`o f Ca2P (i.e. 1 -2 mmol F-', when expressed relative to fibre volume) in the SR terminal cisternae in frog fibres at rest and that -40 % of this is present at the end of a 1-2 s tetanus (Somlyo, GonzalezSerratos, Shuman, McClellan & Somlyo, 1981). However, it is not clear whether the Ca2P still present in the terminal cisternae could be released by depolarization or indeed whether some of it had been already released and re-sequestered by that time. Other studies have used Ca...

1 The eect of the b 2 -adrenoceptor agonist, terbutaline, was investigated on simultaneously measured force and intracellular free calcium ([Ca 2+ ] i ) in intact rat soleus muscle ®bres, and on contractile protein function and Ca 2+ content of the sarcoplasmic reticulum (SR) in skinned ®bres. 2 Terbutaline (10 mM) had no signi®cant eect on either resting force or [Ca 2+ ] i . Exposure to terbutaline increased both the integral of the indo-1 ratio transient and peak twitch force by 37%. 3 At sub-maximal (10 Hz) stimulation frequencies, terbutaline accelerated force relaxation but had highly variable eects on tetanic force amplitude. The corresponding indo-1 ratio transients were signi®cantly larger, and faster to decay than the controls. 4 Terbutaline increased tetanic force at near maximal stimulation frequencies (50 Hz) by increasing tetanic [Ca 2+ ] i . Force relaxation was accelerated at this frequency with no signi®cant change in the indo-1 ratio transient decay rate. 5 All of terbutaline's eects on force and indo-1 ratio transients in intact ®bres were completely blocked and reversed by ICI 118551 (1 mM). IntroductionA long-standing question in b-adrenoceptor pharmacology has been the mechanism of action by which sympathomimetic amines exert distinct eects on the contractility of dierent skeletal muscle types (Bowman, 1980). Twitches and unfused tetanic contractions are potentiated and slowed by b 2 -agonists in fast-twitch muscles (Bowman & Zaimis, 1958;Bowman et al., 1962;Tashiro, 1973;Holmberg & Waldeck, 1980;Cairns & Dulhunty, 1993a), whereas slow-twitch muscles show decreases (Bowman & Zaimis, 1958;Bowman et al., 1962;Holmberg & Waldeck, 1979;Tashiro, 1973), increases (Cairns & Dulhunty, 1993a) or no change (Slack et al., 1997) of contraction amplitude in the presence of a consistently accelerated relaxation.In order to determine the mechanisms underlying such dierences it is necessary to consider the factors in¯uencing the amplitude and time course of skeletal muscle contraction as well as the signal transduction pathway of b 2 -adrenoceptors. Given that SR Ca 2+ handling is a key determinant of mammalian skeletal muscle contraction, and that activation of b 2 -adrenoceptors leads to activation of the cyclic AMP/ cyclic AMP-dependent protein kinase (PKA) pathway, then it is likely that the dierences and variability in response to b 2 -agonists are related to dierences in the phosphorylation of key proteins involved in SR Ca 2+ release and uptake. Two such proteins are the SR Ca 2+ release channel/ryanodine receptor (RR), which is found in all skeletal muscles, and phospholamban (PLB), an inhibitory protein associated with the SR Ca 2+ uptake pump in slow-twitch and cardiac muscles (Kirchberger & Tada, 1976). Phosphorylation of the RR leads to an increase in its open probability (Suko et al., 1993;Hain et al., 1994), whilst phosphorylation of PLB releases its basal inhibitory eect on the SR Ca 2+ uptake pump (Kirchberger & Tada, 1976).Simultaneous measurement of force and the intracell...