Effects of sphingosine on phorbol ester-mediated changes in astrocyte morphology and protein phosphorylation

Previous studies indicate that phorbol myristate acetate (PMA) can induce morphological changes in astrocytes cultured from the rat neocortex. PMA also increased 32P incorporation into several proteins, including glial fibrillary acidic protein (GFAP), vimentin, and proteins with molecular weights of 80,000 (pI 4.5), 50,000 (pI 4.9), and 30,000 (pI 5.5). The present studies were conducted to determine if the morphological effect and the phosphorylation effect of PMA could be blocked by treatment with sphingosine, a protein kinase C inhibitor. Treatment with 15 microM sphingosine inhibited the effect of PMA on astrocyte morphology. This agent also inhibited the increase in phosphorylation mediated by PMA. The percent inhibition ranged from approximately 20% for the 30,000-Mr protein to 70% for GFAP. Analysis of phosphorylation sites on GFAP and vimentin using two-dimensional tryptic mapping techniques indicate that the partial inhibition of phosphorylation is likely the consequence of partial inhibition of protein kinase C rather than a selective inhibition at some phosphorylation sites and not others. In addition to increasing 32P incorporation into various proteins, PMA also decreased 32P incorporation in several 20,000-Mr proteins (pI values of 6.7, 6.4, 6.2, 4.9). However, this effect was not blocked by treatment with sphingosine. This suggests that the actions of PMA to increase and decrease 32P incorporation are mediated by different mechanisms.