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Keywords:

  • cyclopiazonic acid;
  • caffeine;
  • ryanodine;
  • Na+/Ca2+ exchanger;
  • Na+ pump;
  • store-operated channels

Abstract

Signaling by two classes of endoplasmic reticulum (ER) Ca2+ stores was studied in primary cultured rat astrocytes. Cytosolic and intra-ER Ca2+ concentrations ([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+ stores (CPA inhibits ER Ca2+ pumps). Agonist-evoked release was abolished by prior treatment with CPA but was unaffected by prior depletion of caffeine/ryanodine (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 not the CAF/RY-sensitive stores, promoted Ca2+ entry through “store-operated channels.” Resting [Ca2+]ER averaged 153 μM (based on in situ calibration of Furaptra: KD = 76 μM, vs 53 μM 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 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 CPA-sensitive and CAF/RY-sensitive ER Ca2+ stores. GLIA 31:15–28, 2000. © 2000 Wiley-Liss, Inc.