Unloading and refilling of two classes of spatially resolved endoplasmic reticulum Ca(2+) stores in astrocytes.
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 microM (based on in situ calibration of Furaptra: K(D) = 76 microM, vs 53 microM 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.
Golovina
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VA
Department of Physiology, University of Maryland School of Medicine, Baltimore 21201, USA. vgolovin@umaryland.edu
Blaustein
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MP
D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000819:agonists / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D007211:Indoles / Q000494:pharmacology; D051381:Rats; D012433:Ryanodine / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors
D015220:Calcium Channels; D005456:Fluorescent Dyes; D007211:Indoles; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D012433:Ryanodine; D002110:Caffeine; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium; D016257:Fura-2; C000543:cyclopiazonic acid