In oligodendrocyte processes, methacholine-evokedIn the present study we have examined the effects of other phosphoinositide-coupled agonists on Ca 2؉ in these cells, and the structural specializations underlying regenerative wave amplification sites. Both bradykinin and norepinephrine evoke Ca 2؉ waves, which initiate at the same loci and propagate through the cell body and multiple processes via identical wave amplification sites. Antibodies against type 2 inositol 1,4,5-trisphosphate receptors (InsP 3 R2) and calreticulin identify expression of these proteins in oligodendrocyte membranes in Western blots. Immunocytochemistry followed by high resolution fluorescence microscopy revealed that both InsP 3 R2 and calreticulin are expressed in high intensity patches along processes. Cross-correlation analysis of the profiles of local Ca 2؉ release kinetics during a Ca 2؉ wave and immunofluorescence for these proteins along cellular processes showed that the domains of high endoplasmic reticulum protein expression correspond closely to wave amplification sites. Staining cells with the mitochondrial dye, MitoTracker ® , showed that mitochondria are only found in intimate association with these sites possessing high density endoplasmic reticulum proteins, and they remain in the same locations over relatively long periods of time. It appears, therefore, that multiple specializations are found at domains of elevated Ca 2؉ release in oligodendrocyte processes, including high levels of calreticulin, InsP 3 R2 Ca 2؉ release channels, and mitochondria.

This study was undertaken to examine the expression and role of the endoplasmic reticulum (ER) proteins calreticulin and ryanodine receptors, and mitochondria, in cultured astrocytes. Using several lines of investigation, we have identified a key role for mitochondria in astrocyte Ca2+ signalling: (1) a significant correlation was found between sites of regenerative Ca2+ wave amplification (possessing high amplitude ER Ca2+ release) and the location of mitochondria in the cell; (2) norepinephrine (2 microM) caused a rapid-onset increase in rhod 2 fluorescence in 34% of astrocyte mitochondria, indicating that cytosolic Ca2+ responses result in mitochondrial Ca2+ elevation; and (3) pretreatment with the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone 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 immunocytochemistry. Ryanodine receptors and calreticulin were found to be expressed in heterogeneous patterns in astrocytes. The expression pattern of calreticulin corresponded closely with the distribution of mitochondria, whereas the expression of ryanodine receptors was not similar to that of either of these cellular factors. We measured Ca2+ wave kinetics in a single astrocyte, then assessed protein distribution by immunocytochemistry in the same cell. Cross-correlation between norepinephrine-evoked Ca2+ wave amplitude and calreticulin distribution indicated a close spatial relationship between this Ca2+-binding protein and sites of regenerative wave amplification. These results demonstrate that amplification sites for Ca2+ waves in astrocytes are identifiable by accumulations of calreticulin (and type 2 InsP3Rs), and by the presence of mitochondria, which may regulate the ER Ca2+ release process.

This study was undertaken to examine the expression and role of the endoplasmic reticulum (ER) proteins calreticulin and ryanodine receptors, and mitochondria, in cultured astrocytes. Using several lines of investigation, we have identified a key role for mitochondria in astrocyte Ca2+ signalling: (1) a significant correlation was found between sites of regenerative Ca2+ wave amplification (possessing high amplitude ER Ca2+ release) and the location of mitochondria in the cell; (2) norepinephrine (2 microM) caused a rapid-onset increase in rhod 2 fluorescence in 34% of astrocyte mitochondria, indicating that cytosolic Ca2+ responses result in mitochondrial Ca2+ elevation; and (3) pretreatment with the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone 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 immunocytochemistry. Ryanodine receptors and calreticulin were found to be expressed in heterogeneous patterns in astrocytes. The expression pattern of calreticulin corresponded closely with the distribution of mitochondria, whereas the expression of ryanodine receptors was not similar to that of either of these cellular factors. We measured Ca2+ wave kinetics in a single astrocyte, then assessed protein distribution by immunocytochemistry in the same cell. Cross-correlation between norepinephrine-evoked Ca2+ wave amplitude and calreticulin distribution indicated a close spatial relationship between this Ca2+-binding protein and sites of regenerative wave amplification. These results demonstrate that amplification sites for Ca2+ waves in astrocytes are identifiable by accumulations of calreticulin (and type 2 InsP3Rs), and by the presence of mitochondria, which may regulate the ER Ca2+ release process.