The endoplasmic reticulum as one continuous Ca2+ pool: visualization of rapid Ca2+ movements and equilibration

We investigated whether the endoplasmic reticulum (ER) is a functionally co nnected Ca2+ store or is composed of separate subunits by monitoring moveme nts of Ca2+ and small fluorescent probes in the ER lumen of pancreatic acin ar cells, using confocal microscopy, local bleaching and uncaging, We obser ved rapid movements and equilibration of Ca2+ and the probes. The bulk of t he ER at the base was not connected to the granules in the apical part, but diffusion into small apical ER extensions occurred, The connectivity of th e ER Ca2+ store was robust, since even supramaximal acetylcholine (ACh) sti mulation for 30 min did not result in functional fragmentation. ACh could e licit a uniform decrease in the ER Ca2+ concentration throughout the cell, but repetitive cytosolic Ca2+ spikes, induced by a low ACh concentration, h ardly reduced the ER Ca2+ level, We conclude that the ER is a functionally continuous unit, which enables efficient Ca2+ liberation, Ca2+ released fro m the apical ER terminals is quickly replenished from the bulk of the rough ER at the base.