Treatment of RGCs with insulin or C2 ceramide alone increased survival rate by 30%. Adding both insulin and C2 ceramide increased survival rate by 80%. Protein phosphatase 2A (PP2A) inhibitor okadaic acid (OA) eliminated the effect of C2 ceramide, but not that of insulin. Protein kinase inhibitor K252a decreased the effect of C2 ceramide in a dose-dependent manner, but the effect of insulin was not changed. Treatment of RGCs with bFGF increased survival rate by 36%. Adding both bFGF and C2 ceramide increased survival rate by 102%. OA did not alter the effect of bFGF, whereas K252a increased survival rate in a dose-dependent manner. Inhibition of C2 ceramide by CIA suggests that PP2A activation is involved in its pathway, whereas PP2A is not involved in the insulin- and bFGF-activated pathway. Elimination of the effect of C2 ceramide by K252a suggests that sphingomyelin cycle activation is mediated by a protein kinase not important in the insulin-activated pathway. Moreover, the increased effect of bFGF and dose-dependently decreased effect of C2 ceramide by K252a suggest that different protein kinases are important in bFGF- and ceramide-mediated enhancement of RGC survival rate. (C) 2003 Elsevier Science (USA). All rights reserved.