DIRECT MEASUREMENT OF CA2-RETICULUM OF SAPONIN PERMEABILIZED ISOLATEDSMOOTH-MUSCLE CELLS( UPTAKE AND RELEASE BY THE SARCOPLASMIC)

TO make direct measurements of Ca2+ uptake and release by the sarcopla smic reticulum (SR) of isolated smooth muscle cells, a fluorometric me thod for monitoring Ca2+ uptake by striated muscle SR vesicles (Kargac in, M. E., C. R. Scheid, and T. W. Honeyman. 1988. American Journal of Physiology. 245:C694-C698) was modified. With the method, it was poss ible to make continuous measurements of SR function in saponin-skinned smooth muscle cells in suspension. Calcium uptake by the SR was inhib ited by thapsigargin and sequestered Ca2+ could be released by Br-A231 87 and thapsigargin. From the rate of Ca2+ uptake by the skinned cells and the density of cells in suspension, it was possible to calculate the Ca2+ uptake rate for the SR of a single cell. Our results indicate that the SR Ca2+ pump in smooth muscle cells can remove Ca2+ at a rat e that is 45-75% of the rate at which Ca2+ is removed from the cytopla sm of intact cells during transient Ca2+ signals. From estimates of SR volume reported by others and our measurements of the amount of Ca2taken up by the skinned cells, we conclude that the SR of a single cel l can store greater than 10 times the amount of Ca2+ needed to elicit a single transient contractile response.