Unloading and refilling of two classes of spatially resolved endoplasmic reticulum Ca2+ stores in astrocytes

Signaling by two classes of endoplasmic reticulum (ER) Ca2+ stores was stud ied in primary cultured rat astrocytes. Cytosolic and intra-ER Ca2+ concent rations ([Ca2+](CYT) and [Ca2+](ER)) were measured with, respectively, Fura -2 and Furaptra, in separate experiments. The agonists, glutamate and ATP, released Ca2+ primarily from cyclopiazonic acid (CPA)-sensitive ER Ca2+ sto res (CPA inhibits ER Ca2+ pumps). Agonist-evoked release was abolished by p rior treatment with CPA but was unaffected by prior depletion of caffeine/r yanodine (CAF/RY)-sensitive ER Ca2+ stores. Conversely, prior depletion of the CPA-sensitive stores did not interfere with Ca2+ release or reuptake in the CAF/RY-sensitive stores. Unloading of the CPA-sensitive stores, but no t the CAF/RY-sensitive stores, promoted Ca2+ entry through "store-operated channels". Resting [Ca2+](ER) averaged 153 mu M (based on in situ calibrati on of Furaptra: K-D = 76 mu M, vs 53 mu M in solution). The releasable Ca2 in both types of ER Ca2+ stores was increased by Na+ pump inhibition with 1 mM ouabain or K+-free medium. Using high spatial resolution imaging and i mage subtraction methods, we observed that some regions of the ER (45-58% o f the total ER) unloaded and refilled when CPA was added and removed. Other regions of the ER (24-38%) unloaded and refilled when CAF was added and re moved. The overlap between these two classes of ER was only 10-18%. These d ata indicate that there are two structurally separate, independent componen ts of the ER and that they are responsible for the functional independence of the CPA-sensitive and CAF/RY-sensitive ER Ca2+ stores. (C) 2000 Wiley-Li ss, Inc.