Imaging of changes in sarcoplasmic reticulum [Ca(2+)] using Oregon Green BAPTA 5N and confocal laser scanning...

We describe experiments in which the low affinity indicator Oregon Green BAPTA 5N was used to record the spatially resolved changes in [Ca(2+)] from intracellular stores in rat gastric myocytes. Cells were loaded with the membrane permeant form of the indicator and imaged using a confocal scanning laser microscope. In doubly stained cells the Oregon Green signal colocalized with BIODIPY 558/568 Brefeldin A, a label for the endo/sarcoplasmic reticulum (SR) and Golgi apparatus. Oregon Green BAPTA 5N was calibrated in gastric myocytes, giving an in situ K(d) of 90 microM. The resting free [Ca(2+)] within the SR averaged 65 microM. A reversible decrease in Oregon Green fluorescence was observed on bath application of Inositol triphosphate (IP(3)) (10 microM) to permeabilized cells. Similar changes were also observed when cyclopiazonic acid (5 microM) was applied to intact myocytes, again with recovery of store [Ca(2+)] following drug washout. Identical patterns of Ca(2+) depletion were seen when caffeine (1 microM) and carbachol (10 microM) were applied sequentially to the same cells, suggesting that activation of ryanodine and IP(3)-sensitive channels can result in the release of Ca(2+) from the same regions of the SR.