科研之友
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摘要
Bundles of intact fibres from soleus muscles of adult mice were isolated by dissection and one fibre within a bundle was micro-injected with either furaptra or mag-fluo-4, two low-affinity rapidly responding Ca(2+) indicators. Fibres were activated by action potentials to elicit changes in indicator fluorescence (Delta F), a monitor of the myoplasmic free Ca(2+) transient (Delta[Ca(2+)]), and changes in fibre tension. All injected fibres appeared to be slow-twitch (type I) fibres as inferred from the time course of their tension responses. The full-duration at half-maximum (FDHM) of Delta F was found to be essentially identical with the two indicators; the mean value was 8.4 +/- 0.3 ms (+/-SEM) at 16 degrees C and 5.1 +/- 0.3 ms at 22 degrees C. The value at 22 degrees C is about one-third that reported previously in enzyme-dissociated slow-twitch fibres that had been AM-loaded with mag-fluo-4: 12.4 +/- 0.8 ms and 17.2 +/- 1.7 ms. We attribute the larger FDHM in enzyme-dissociated fibres either to an alteration of fibre properties due to the enzyme treatment or to some error in the measurement of Delta F associated with AM loading. Delta F in intact fibres was simulated with a multi-compartment reaction-diffusion model that permitted estimation of the amount and time course of Ca(2+) release from the sarcoplasmic reticulum (SR), the binding and diffusion of Ca(2+) in the myoplasm, the re-uptake of Ca(2+) by the SR Ca(2+) pump, and Delta[Ca(2+)] itself. In response to one action potential at 16 degrees C, the following estimates were obtained: 107 mu M for the amount of Ca(2+) release; 1.7 ms for the FDHM of the release flux; 7.6 mu M and 4.9 ms for the peak and FDHM of spatially averaged Delta[Ca(2+)]. With five action potentials at 67 Hz, the estimated amount of Ca(2+) release is 186 mu M. Two important unknown model parameters are the on- and off-rate constants of the reaction between Ca(2+) and the regulatory sites on troponin; values of 0.4 x 10(8) M(-1) s(-1) and 26 s(-1), respectively, were found to be consistent with the Delta F measurements.
- 出版日期2012-2
