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Role of calcium influx in process extension of oligodendrocytes Yoo, Andrew


The effects of phorbol ester, which activates protein kinase C (PKC), and free cytosolic Ca²+ were studied on their role in process extension in cultured murine oligodendrocytes. Previous studies have shown that PKC activation by phorbol esters greatly enhances process formation in cultured oligodendrocytes (OLG), an important step for myelination. Because of the possible involvement of PKC and Ca²+ in process extension in OLGs, we studied the effect of PKC activation on changes in intracellular free Ca²+ concentration ([Ca²+]i). We found that PKC activation by phorbol esters induced a sustained rise in [Ca²+]i of OLGs. This rise was due to transmembrane influx of Ca²+ since omission of extracellular Ca²+ failed to trigger the rise in [Ca²+]i. Changes in [Ca²+]i were also produced by modifying the extracellular [Ca²+] where increasing extracellular [Ca²+] led to a rise in [Ca²+]i. In order to establish the relationship between Ca²+ influx and OLG process formation, OLGs were incubated in media containing various [Ca²+] with or without phorbol ester treatment. After 72 hours, OLGs were immunostained using an antibody against proteolipid protein (PLP). The correlation between the process formation of OLGs and extracellular [Ca²+] was assessed by obtaining the percentage of OLGs with longer processes in the OLG population, the number of primary process per cell body and the area covered by PLP-positive OLG processes. Our results indicate that the degree of OLG process extension is related to the [Ca²+] present in the culture media. We found that increased extracellular [Ca²+] alone, without concurrent phorbol ester application, resulted in increased OLG process extension. When OLGs were treated with phorbol ester, positive correlations between increasing extracellular [Ca²+] and some aspects of OLG process extension were seen, as suggested by the results from analysis of covariance (ANCOVA). In addition, blocking the intracellular Ca²+ signalling by BAPTA as well as inhibiting PKC by RO-31 leads to retraction o f membrane sheath o f OLGs . Our results demonstrate that increasing [Ca²+]i stimulates OLG process outgrowth and suggest that intracellular Ca²+ signalling following either phorbol ester treatment or increasing extracellular [Ca²+] may be an important mediator for OLG process extension.

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