Ca2+ compartments in saponin-skinned cultured vascular smooth muscle cells

Abstract

A method 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 45Ca2+ than the nontreated cells under the same conditions. At low free Ca2+ concentration, >85% of 45Ca2+ uptake into the skinned cells was dependent on the extracellularly supplied MgATP. In the intact cells, both caffeine and norepinephrine increased 45Ca2+ efflux. In the skinned cells, caffeine increased 45Ca2+ efflux, whereas norepinephrine did not. The caffeine-releasable 45Ca2+ uptake fraction in the skinned cells appeared at 3 × 10-7 M Ca2+, increased gradually with the increase in free Ca2+ concentration, and reached a plateau at 1 × 10-5 MCa2+. The 45Ca2+ uptake fraction, which was significantly suppressed by sodium azide, appeared at 1 × 10-5 M Ca2+ and increased monotonically with increasing free Ca2+ concentration. The results suggest that the caffeine-sensitive Ca2+ store, presumably the sarcoplasmic reticulum, plays a physiological role by releasing Ca2+ in response to norepinephrine or caffeine and by buffering excessive Ca2+. The45Ca2+ uptake by mitochondria appears too insensitive to be important under physiological conditions. © 1986, Rockefeller University Press., All rights reserved.