SPECTROSCOPIC DETERMINATION OF SARCOPLASMIC-RETICULUM CA2+ UPTAKE ANDCA2+ RELEASE

In this report we describe the application of spectroscopic methods to the study of Ca2+ release by isolated native sarcoplasmic reticulum ( SR) membranes from rabbit skeletal muscle. To date, dual-wavelength sp ectroscopy of arsenazo III and antipyrylazo III difference absorbance have been the most common spectroscopic methods for the assay of SR Ca 2+ transport. The utility of these methods is the ability to manipulat e intraluminal Ca2+ loading of SR vesicles. These methods have also be en useful for studying the effect of both agonists and antagonists upo n SR Ca2+ release and Ca2+ uptake. In this study, we have developed th e application of Calcium Green-2, a long-wavelength excitable fluoresc ent indicator, for the study of SR Ca2+ uptake and release. With this method we demonstrate how ryanodine receptor Ca2+ channel opening and closing is regulated in a complex manner by the relative distribution of Ca2+ between extraluminal and intraluminal Ca2+ compartments. Intra luminal Ca2+ is shown to be a key regulator of Ca2+ channel opening. H owever, these methods also reveal that the intraluminal Ca2+ threshold for Ca2+-induced Ca2+ release varies as a function of extraluminal Ca 2+ concentration. The ability to study how the relative distribution o f a finite pool of Ca2+ across the SR membrane influences Ca2+ uptake and Ca2+ release may be useful for understanding how the ryanodine rec eptor is regulated, in vivo.