MEASUREMENT OF SR FREE CA2-MUSCLE CELLS WITH USE OF FURAPTRA( AND MG2+ IN PERMEABILIZED SMOOTH)

The concentrations of intrasarcoplasmic reticulum (SR) free Ca2+ ([Ca2 +](SR)) and Mg2+ ([Mg2+](SR)) were measured in furaptra-loaded saponin -permeabilized cultured aortic smooth muscle (A7r5) cells. Ca2+-indepe ndent fluorescence emitted by furaptra trapped within organelles, exci ted at 346 nm (isosbestic point), decreased with a half time of 30 min . All Ca2+ measurements appeared to be from SR, because the apparent C a2+ distribution within permeabilized cells was uniform and therefore inconsistent with furaptra loading into mitochondria. Moreover, thapsi gargin-induced SR Ca2+-adenosinetriphosphatase inhibition caused near- total depletion of Ca2+, and the metabolic poisons oligomycin and rote none had no effect. Calibration curves relating 370 nm-to-346 nm ratio s to [Ca2+] and to [Mg2+] were calculated in situ; dissociation consta nts for Ca2+ and Mg2+ binding were 49 mu M and 6.8 mM, respectively. R esting [Ca2+](SR) was 75-130 mu M, with a mean of 97.2 +/- 2.2 mu M (n = 376), whereas [Mg2+](SR), estimated in the absence of Ca2+, was 1.0 mM. Stimulation with inositol 1,4,5-trisphosphate resulted in time-de pendent declines in [Ca2+](SR), and pretreatment with guanosine 5'-tri phosphate caused a large increase in the rate of inositol 1,4,5-trisph osphate-evoked SR Ca2+ release, although guanosine 5'-triphosphate had no effect by itself. These observations indicate that furaptra will b e a valuable tool with which to directly study [Ca2+](SR) and SR funct ion.