SPECIALIZED DISTRIBUTIONS OF MITOCHONDRIA AND ENDOPLASMIC-RETICULUM PROTEINS DEFINE CA2+ WAVE AMPLIFICATION SITES IN CULTURED ASTROCYTES

This study was undertaken to examine the expression and role of the en doplasmic reticulum (ER) proteins calreticulin and ryanodine receptors , and mitochondria, in cultured astrocytes. Using several lines of inv estigation, we have identified a key role for mitochondria in astrocyt e Ca2+ signalling: (1) a significant correlation was found between sit es of regenerative Ca2+ wave amplification (possessing high amplitude ER Ca2+ release) and the location of mitochondria in the cell; (2) nor epinephrine (2 mu M) caused a rapid-onset increase in rhod 2 fluoresce nce in 34% of astrocyte mitochondria, indicating that cytosolic Ca2+ r esponses result in mitochondrial Ca2+ elevation; and (3) pretreatment with the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydra zone to inhibit mitochondrial activity markedly reduced the amplitude of subsequent norepinephrine-evoked cytosolic Ca2+ responses, We then investigated the roles of several ER proteins in Ca2+ signalling by im munocytochemistry, Ryanodine receptors and calreticulin were found to be expressed in heterogeneous patterns in astrocytes. The expression p attern of calreticulin corresponded closely,vith the distribution of m itochondria, whereas the expression of ryanodine receptors was not sim ilar to that of either of these cellular factors. We measured Ca2+ wav e kinetics in a single astrocyte, then assessed protein distribution b y immunocytochemistry in the same cell. Cross-correlation between nore pinephrine-evoked Ca2+ wave amplitude and calreticulin distribution in dicated a close spatial relationship between this Ca2+-binding protein and sites of regenerative wave amplification. These results demonstra te that amplification sites for Ca2+ waves in astrocytes are identifia ble by accumulations of calreticulin (and type 2 InsP(3)Rs), and by th e presence of mitochondria, which may regulate the ER Ca2+ release pro cess, (C) 1998 Wiley-Liss, Inc.