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

✍ Scribed by Vera A. Golovina; Mordecai P. Blaustein

• Publisher: John Wiley and Sons
• Year: 2000
• Tongue: English
• Weight: 452 KB
• Volume: 31
• Category: Article
• ISSN: 0894-1491
• DOI: 10.1002/(sici)1098-1136(200007)31:1<15::aid-glia20>3.0.co;2-h

✦ Synopsis

Signaling by two classes of endoplasmic reticulum (ER) Ca 2ϩ stores was studied in primary cultured rat astrocytes. Cytosolic and intra-ER Ca 2ϩ concentrations ([Ca 2ϩ ] CYT and [Ca 2ϩ ] ER ) were measured with, respectively, Fura-2 and Furaptra, in separate experiments. The agonists, glutamate and ATP, released Ca 2ϩ primarily from cyclopiazonic acid (CPA)-sensitive ER Ca 2ϩ stores (CPA inhibits ER Ca 2ϩ pumps). Agonist-evoked release was abolished by prior treatment with CPA but was unaffected by prior depletion of caffeine/ryanodine (CAF/RY)-sensitive ER Ca 2ϩ stores. Conversely, prior depletion of the CPA-sensitive stores did not interfere with Ca 2ϩ release or reuptake in the CAF/RY-sensitive stores. Unloading of the CPA-sensitive stores, but not the CAF/RY-sensitive stores, promoted Ca 2ϩ entry through "store-operated channels." Resting [Ca 2ϩ ] ER averaged 153 M (based on in situ calibration of Furaptra: K D ϭ 76 M, vs 53 M in solution). The releasable Ca 2ϩ in both types of ER Ca 2ϩ stores was increased by Na ϩ pump inhibition with 1 mM ouabain or K ϩ -free medium. Using high spatial resolution imaging and image subtraction methods, we observed that some regions of the ER (45-58% of 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 removed. The overlap between these two classes of ER was only 10 -18%. These data indicate that there are two structurally separate, independent components of the ER and that they are responsible for the functional independence of the CPAsensitive and CAF/RY-sensitive ER Ca 2ϩ stores.