The molecular events which occur when an axolemma-enriched membrane fraction or a myelin membrane fraction stimulate a mitotically quiescent population of Schwann cells (SC) to divide will be investigated. In particular, the following metabolic events in cultured SC will be evaluated in the presence and absence of membrane mitogens: increased turnover of phosphoinositides (as revealed by radioactivity labeled lipid turnover studies), increased calcium flux and/or intracellular mobilization of calcium as revealed by radioactive calcium flux measurements and Quin-2 measurements of intracellular calcium, increased sodium influx and hydrogen ion efflux as revealed by radioactive sodium flux experiments and fluorescent dye measurements of intracelular pH; increased phosphorylation of proteins as revealed by protein labeling via P32-phosphorus. The molecules required for specific interaction of each membrane fraction with SC will be investigated by evaluating how these membrane stimulated metabolic-sequalae may be compromised as a consequence of specific perturbation of the stimulation membrane. Phorbol esters and calcium ionophores will be used to stimulate both """"""""branches"""""""" of the phosphoinositide effect to bring about mitosis. The relative efficiency and timing for the stimulation of each branch reactive to that of the membrane mitogens will be investigated as well as the nature of the proteins phosphorylation via each pathway. Finally, myelin which has been radioactively prelabeled with lipid precursors will be given to SC and the nature and timing of the lysosomal breakdown of that myelin will be evaluated relative to the onset of mitosis in order to identify potential mitogens which may be released from the myelin membrane. It is anticipated that these studies will given new information concerning the molecular events which must occur both for normal myelination to occur and for remyelination after Wallerian degeneration.
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