No abstract

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...

Intracellular acidification of skeletal muscles is commonly thought to contribute to muscle fatigue. However, intracellular acidosis also acts to preserve muscle excitability when muscles become depolarized, which occurs with working muscles. Here, we show that this process may be mediated by decreased chloride permeability, which enables action potentials to still be propagated along the internal network of tubules in a muscle fiber (the T system) despite muscle depolarization. These results implicate chloride ion channels in muscle function and emphasize that intracellular acidosis of muscle has protective effects during muscle fatigue.

1. The effects on normal excitation‐contraction (E‐C) coupling of two important intracellular ions, H+ and Mg2+, were examined in skinned fibres from the extensor digitorum longus muscle of rat. 2. A single depolarization (2‐3 s duration) in the presence of 1 mM Mg2+ (pH 7.1, 23 degrees C) released most of the available Ca2+ in the sarcoplasmic reticulum (SR), but a similar depolarization in the presence of 10 mM Mg2+ was unable to release almost any Ca2+. Thus, raised [Mg2+] potently inhibits depolarization‐induced Ca2+ release in mammalian muscle. 3. Depolarization at pH 6.2 (1 mM Mg2+, 23 degrees C) induced a large force response, which was on average 78 +/‐ 2%, n = 6, of the depolarization‐induced response at pH 7.1; this reduction resulted from a corresponding reduction in maximum Ca(2+)‐activated force at pH 6.2. Similar results were obtained at 37 degrees C. Also, a single depolarization at pH 6.2 caused almost complete depletion of the releasable Ca2+ in the SR. Thus, low pH does not prevent depolarization‐induced Ca2+ release in mammalian muscle. 4. Lowering the free [Mg2+] from 1 mM to 15 microM caused massive release of Ca2+, and depletion of the SR, at both pH 7.1 and 6.2, indicating that over this pH range, H+ did not readily substitute for Mg2+ at its inhibitory site on the Ca2+ release channel.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Raising the intracellular [Ca2+] for 10 s at 23 degrees C abolished depolarization‐induced force responses in mechanically skinned muscle fibres of toad and rat (half‐maximal effect at 10 and 23 microM, respectively), without affecting the ability of caffeine or low [Mg2+] to open the ryanodine receptor (RyR)/Ca2+ release channels. Thus, excitation‐contraction coupling was lost, even though the Ca2+ release channels were still functional. Coupling could not be restored in the duration of an experiment (up to 1 h). 2. The Ca(2+)‐dependent uncoupling had a Q10 > 3.5, and was three times slower at pH 5.8 than at pH 7.1. Sr2+ caused similar uncoupling at twenty times higher concentration, but Mg2+, even at 10 mM, was ineffective. Uncoupling was not noticeably affected by removal of ATP or application of protein kinase or phosphatase inhibitors. 3. Confocal laser scanning microscopy showed that the transverse tubular system was sealed in its entirety in mechanically skinned fibres and that its integrity was maintained in uncoupled fibres. Electron microscopy revealed distorted or severed triad junctions and Z‐line aberrations in uncoupled fibres. 4. Only when uncoupling was induced at a relatively slow rate (e.g. over 60 s with 2.5 microM Ca2+) could it be prevented by the protease inhibitor leupeptin (1 mM). Immunostaining of Western blots showed no evidence of proteolysis of the RyR, the alpha 1‐subunit of dihydropyridine receptor (DHPR) or triadin in uncoupled fibres. 5. Fibres which, whilst intact, were stimulated repeatedly by potassium depolarization with simultaneous application of 30 mM caffeine showed reduced responsiveness after skinning to depolarization but not to caffeine. Rapid release of endogenous Ca2+, or raised [Ca2+] under conditions which minimized the loss of endogenous diffusible myoplasmic molecules from the skinned fibre, caused complete uncoupling. Taken together, these results suggest that Ca(2+)‐dependent uncoupling can also occur in intact fibres. 6. This Ca(2+)‐dependent loss of depolarization‐induced Ca2+ release may play an important feedback role in muscle by stopping Ca2+ release in localized areas where it is excessive and may be responsible for long‐lasting muscle fatigue after severe exercise, as well as contributing to muscle weakness in various dystrophies.