Measurement of Sarcoplasmic Reticulum Function in Mammalian Skeletal Muscle: Technical Aspects

Abstract

Two recently developed methods for measuring the maximal rate of Ca2+ uptake and Ca2+ ATPase activity (EC 3.1.6.38) in vitro use muscle homogenate rather than isolated sarcoplasmic reticulum (SR). In this study we investigated technical aspects of these assays, including specificity and variability of the assays, the effect of different freezing treatments on maximal Ca2+ uptake and Ca2+ ATPase activity of human and rat muscle homogenate, stability of the homogenate, and the modification of the Ca2+ uptake assay to measure Ag+-induced Ca2+ release. Addition of cyclopiazonic acid (20 μm) blocked Ca2+ uptake, demonstrating specificity of the assay. Using frozen muscle homogenate, the inter- and intraassay variation for both assays was less than 9%. Whereas homogenates were stable to freezing for both Ca2+ uptake and Ca2+ ATPase activity, there was a significant (P < 0.05) decrease in activity when muscle was freeze-dried or quickly frozen in small pieces. After 1 h on ice, rat muscle homogenate Ca2+ uptake and Ca2+ ATPase activity had decreased by 6.0% (ns) and 3.6% (P < 0.05), respectively; after 3 h, activity had decreased by 15.3 and 14.7%, respectively (P < 0.01). The Ca2+ uptake assay was modified to allow measurement of Ag+-induced Ca2+ release. Following homogenate addition, after the SR vesicles were loaded with Ca2+ and [Ca2+] had declined to a plateau, AgNO3 (141 μm) was added, initiating release of Ca2+ into the assay solution. Addition of dithiothreitol (4 mM) blocked the Ag+-induced Ca2+ release, demonstrating specificity of the assay.