method for measuring sarcoplasmic reticulum calcium uptake in the skeletal muscle using Fura-2.

Warmington SA, et al. Cell Calcium. 1996.

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Abstract

We have presented an assay for measuring the rate of sarcoplasmic reticulum (SR) Ca2+ uptake and Ca2+ release in skeletal muscle homogenates using the fluorescent Ca2+ probe Fura-2. Using this assay, we investigated the effects of an elevated temperature (40 degrees C) and lowered pH (6.8), two factors proposed to be involved in skeletal muscle fatigue, on SR Ca2+ uptake. The EDL muscle was found to have a higher rate of Ca2+ uptake than the soleus (34%). Exposure of the muscles to a raised temperature, but not a reduced pH, resulted in a reduction in the rate of Ca2+ uptake in both the EDL and soleus homogenates. This uptake process was blocked by cyclopiazonic acid (CPA) a specific inhibitor of the major transport protein of the sarcoplasmic reticulum, the Ca(2+)-ATPase. Calcium release was induced using AgNO3 after loading of the vesicles during the uptake process. It was found that AgNO3 was only effective in producing Ca2+ release in the EDL muscles. The soleus muscles did not release Ca2+ under varying [Mg2+] or with Hg2+ substitution for Ag+, suggesting that fast- and slow-twitch muscle fibres require different conditions for maximum Ca2+ release, or that different isoforms of the Ca2+ release channels are present in the different fibres

You cannot measure intracellular calcium levels using Fura-2 and muscle cryosections. For one, sectioning disrupts the plasmalemma (and organelle membranes, e.g., sarcoplasmic reticulum) and you lose the extracellular-intracellular calcium gradient, which is several hundred fold. This definitely occurs if the section is ever exposed to an aqueous solution, e.g., as in when the Fura-2 is loaded.