Abstract
The effect of Ca2+ on the time course of force generation in frog skinned muscle fibres has been investigated using laser flash photolysis of the caged-calcium, either nitr-5 or DM-Nitrophen. Gradations in the rate and extent of contraction could be achieved by changing the energy of the laser pulse, which varied the amount of caged Ca2+ photolysed and hence the amount of calcium released. The half-time for force development at 12°C was noticeably calcium-sensitive when small amounts of calcium were released (low energy pulses) but did not change appreciably for calcium releases which produced a final tension of more than 50% of the maximal tension at pCa 4.5. This result is unlikely to be due to calcium binding to the regulatory sites of troponin C when on the thin filament, as this process is considered rapid (kon 108 M-1 s-1, koff 100 s-1). Our experimental results show that force develops relatively rapidly at intermediate Ca2+ which produce only partial activation (i.e. 50% Pmax or greater). This would not be the case if the affinity of the regulatory sites changes slowly with crossbridge attachment. The kinetics of calcium exchange with the regulatory sites may be much more rapid than crossbridge cycling, so that if calcium binding to a particular functional unit induces crossbridge attachment and force production, the force producing state may be maintained long after calcium has dissociated from that particular functional unit. The relaxation of skinned muscle fibres has also been successfully studied following the rapid uptake of Ca2+ by a photolabile chelator Diazo-2, a photolabile derivative of BAPTA, which is rapidly (> 2000 s-1) converted from a chelator of low Ca2+ affinity (Kd 2.2 μM) to a high affinity chelator (Kd 0.073 μM). We have used single skinned muscle fibres from both frog (actin regulated) and scallop striated muscle (myosin regulated), to study the time course of muscle relaxation. This procedure has enabled us to examine the effects of the intracellular metabolites, ADP, Pi and H+ upon the rate of relaxation. Single skinned muscle fibres from the semitendinosis muscle of the frog Rana temporaria to relax with a mean half-time of 56.0 ± 4.1 ms (range 30–100 ms, n = 18) at 12°C, which is faster than the relaxation observed in the intact muscles (half-time 133 ms at 14°C) and similar to the rate of the fast phase of tension decay in intact single fibres (20 s-1 at 10°C). Thé presence of 6.3 mM free ADP led to an increase in the mean half time of relaxation to 123 ± 8 ms (n = 7).
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Ashley, C.C., Lea, T.J., Mulligan, I.P., Palmer, R.E., Simnett, S.J. (1993). Activation and Relaxation Mechanisms in Single Muscle Fibres. In: Sugi, H., Pollack, G.H. (eds) Mechanism of Myofilament Sliding in Muscle Contraction. Advances in Experimental Medicine and Biology, vol 332. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-2872-2_9
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