MODULATION OF 2 FUNCTIONALLY DISTINCT CA2- ROLE OF THE PLASMALEMMAL NA( STORES IN ASTROCYTES )CA EXCHANGER/

Mechanisms that regulate the amount of releasable Ca2+ in intracellula r stores of cultured mouse astrocytes were investigated using digital imaging of fura-2 loaded cells. At rest, the cytoplasmic Ca2+ concentr ation, [Ca2+](cyt), was about 110 nM. In the absence of extracellular Ca2+, cyclopiazonic acid (CPA), an inhibitor of the endoplasmic reticu lum (ER) Ca2+-ATPase, induced a transient, four-fold increase in [Ca2](cyt) due to the release of Ca2+ from inositol triphosphate (IP3) sen sitive stores. Caffeine (CAF), which releases Ca2+ from Ca2+-sensitive stores, induced a two-fold increase in [Ca2+](cyt). The CPA- and CAF- sensitive stores could be released independently. Changes in the ampli tudes of the Ca2+ transients were taken as a measure of changes in sto re content. Removal of extracellular Na+ or addition of ouabain, which inhibit Ca2+ extrusion and promote Ca2+ entry across the plasmalemma via the Na/Ca exchanger, caused minimal increases in resting [Ca2+](cy t) but greatly potentiated both CPA- and CAF-induced Ca2+ transients. The amount of Ca2+ releasable from the IP3 (CPA) sensitive store was d irectly proportional to cytosolic Na+ concentration (i.e., inversely p roportional to the transmembrane Na+ electrochemical gradient). Under these reduced Na+ gradient conditions, little, if any, Ca2+ destined f or the ER stores enters the cells through voltage-dependent Ca2+ chann els. These results demonstrate that mouse astrocytes contain two disti nct ER Ca2+ stores, the larger, IP3- (CPA-) sensitive, and the smaller , Ca2+- (CAF-) sensitive. The Ca2+ content of both ER stores can be re gulated by the Na/Ca exchanger. Thus, the magnitude of cellular respon ses to signals that are mediated by Ca2+ release induced by the two se cond messengers, IP3 and Ca2+, can be modulated by factors that affect the net transport of Ca2+ across the plasmalemma. (C) 1996 Wiley-Lies , Inc.