Drug-induced Ca2+ release from isolated sarcoplasmic reticulum. I. Use of pyrophosphate to study caffeine-induced Ca2+ release

A demonstration is made of pyrophosphate's use as a precipitating anion in studies of Ca2+ release from isolated sarcoplasmic reticulum (SR). Not only does pyrophosphate speed up the rate at which Ca2+ can be preloaded into SR, but it also allows the accumulated Ca2+ to be released in response to agents such as caffeine. Because so much Ca2+ can be preloaded into SR with pyrophosphate present, more experiments can be performed with a given amount of SR material, and even rapid Ca2+ release rates (greater than 1 mumol/mg X min) are maintained for many seconds. These rates can easily be quantified using conventional spectrophotometric and isotopic methods, without the need for expensive rapid mixing equipment. Caffeine-induced Ca2+ release is exhibited by triadic and terminal cisterna SR subfractions but not by light SR. Caffeine specifically increases the rate of unidirectional 45Ca2+ efflux. This increased efflux is blocked by ruthenium red at submicromolar concentrations and by tetracaine, 9-aminoacridine, or Ba2+ at submillimolar concentrations.