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Unloading and refilling of two classes of spatially resolved endoplasmic reticulum Ca 2+ stores in astrocytes
Author(s) -
Golovina Vera A.,
Blaustein Mordecai P.
Publication year - 2000
Publication title -
glia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.954
H-Index - 164
eISSN - 1098-1136
pISSN - 0894-1491
DOI - 10.1002/(sici)1098-1136(200007)31:1<15::aid-glia20>3.0.co;2-h
Subject(s) - cyclopiazonic acid , endoplasmic reticulum , ryanodine receptor , biology , biophysics , fura 2 , cytosol , calcium , medicine , microbiology and biotechnology , biochemistry , enzyme
Signaling by two classes of endoplasmic reticulum (ER) Ca 2+ stores was studied in primary cultured rat astrocytes. Cytosolic and intra‐ER Ca 2+ concentrations ([Ca 2+ ] CYT and [Ca 2+ ] ER ) were measured with, respectively, Fura‐2 and Furaptra, in separate experiments. The agonists, glutamate and ATP, released Ca 2+ primarily from cyclopiazonic acid (CPA)‐sensitive ER Ca 2+ stores (CPA inhibits ER Ca 2+ 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 2+ stores. Conversely, prior depletion of the CPA‐sensitive stores did not interfere with Ca 2+ release or reuptake in the CAF/RY‐sensitive stores. Unloading of the CPA‐sensitive stores, but not the CAF/RY‐sensitive stores, promoted Ca 2+ entry through “store‐operated channels.” Resting [Ca 2+ ] ER averaged 153 μM (based on in situ calibration of Furaptra: K D = 76 μM, vs 53 μM in solution). The releasable Ca 2+ in both types of ER Ca 2+ 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 Ca 2+ stores. GLIA 31:15–28, 2000. © 2000 Wiley‐Liss, Inc.