High Density Distribution of Endoplasmic Reticulum Proteins and Mitochondria at Specialized Ca2+ Release Sites in Oligodendrocyte Processes

Abstract

In oligodendrocyte processes, methacholine-evoked Ca2+ waves propagate via regions of specialized Ca2+ release kinetics (wave amplification sites) at which the amplitude and rate of rise of local Ca2+ signals are markedly higher than in surrounding areas (Simpson, P. B., and Russell, J. T. (1996) J. Biol. Chem. 271, 33493-33501). In the present study we have examined the effects of other phosphoinositide-coupled agonists on Ca2+ in these cells, and the structural specializations underlying regenerative wave amplification sites. Both bradykinin and norepinephrine evoke Ca2+ 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 (InsP3R2) and calreticulin identify expression of these proteins in oligodendrocyte membranes in Western blots. Immunocytochemistry followed by high resolution fluorescence microscopy revealed that both InsP3R2 and calreticulin are expressed in high intensity patches along processes. Cross-correlation analysis of the profiles of local Ca2+ release kinetics during a Ca2+ 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(R), 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 Ca2+ release in oligodendrocyte processes, including high levels of calreticulin, InsP3R2 Ca2+ release channels, and mitochondria.