Ca 21 Compartments in Saponin - skinned Cultured Vascular Smooth Muscle Cells

Amethod for saponin skinning of primary cultured rat aortic smooth muscle cells was established . The Saponin-treated cells could be stained with trypan blue and incorporated more "Ca" than the nontreated cells under the same conditions . At low free Ca" concentration, >85% of "Ca" uptake into the skinned cells was dependent on the extracellularly supplied MgATP. In the intact cells, both caffeine and norepinephrine increased "Ca" efflux . In the skinned cells, caffeine increased "'Ca" efflux, whereas norepinephrine did not. The caffeine-releasable "Ca" uptake fraction in the skinned cells appeared at 3 x 10' M Ca", increased gradually with the increase in free Ca" concentration, and reached a plateau at 1 x 10-5 M Ca". The "Ca' uptake fraction, which was significantly suppressed by sodium azide, appeared at 1 x 10-5 M Ca" and increased monotonically with increasing free Ca"concentration. The results suggest that the caffeine-sensitive Ca' store, presumably the sarcoplasmic reticulum, plays a physiological role by releasing Ca' in response to norepinephrine or caffeine and by buffering excessive Cat+. The"Ca" uptake by mitochondria appears too insensitive to be important under physiological