Abstract 364 Poster Session II, Sunday, 5/2 (poster 22)

In utero exposure to cocaine may result in the altered neuronal development. Our previous studies indicate cocaine inhibits neurite outgrowth in NGF-induced PC12 cells. These effects were dose and time-dependent and did not affect cell viability. More recently we documented that cocaine mediated its inhibitory effects through dopamine. NGF exerts its biological effects through interaction with the receptor tyrosine kinase which activates a cascade of intracellular signaling proteins such as ras, a GTP-binding protein, raf, and MAP kinase pathways. These early signaling events lead to transcriptional activation of immediate early genes and to induction of the late genes, such that their protein products are responsible for the neuronal phenotype. We showed cocaine alters IEG expression which affects neuronal differentiation. To characterize further the stage in NGF signaling where cocaine interferes, we used PC12-derived transfected cells, GSras1, which inducibly express activated forms of Ras. The GSras1 cell line was transfected by molecular-biology techniques with a plasmid that encodes the oncogenic variant of Ras under the control of a dexamethasone-inducible promoter. Morphological differentiation was quantified by counting cells bearing neurites greater than one cell diameter after 72 hr exposure to: dexamethasone 0.5µM, dexamethasone +cocaine or dopamine (10 µg/ml). NGF (20 ng/ml), NGF +cocaine or dopamine (10-20 µg/ml), NGF +dexamethasone and NGF+ dexamethasone+cocaine (10-20 µg/ml). Cocaine and dopamine inhibited neurite outgrowth significantly in cells that encode for the dominant form of Ras, cells that were treated with NGF alone, and in cells that were treated with NGF and dexamethasone, similar to the effects of these agents on NGF-induced wild type PC12 cells. Preliminary results showed that biochemical differentiation as measured by GAP-43 expression, using western blot analysis, is also affected by cocaine. The results suggest that cocaine and dopamine affect the NGF signaling downstream or independent of Ras. Cocaine is not affecting the early events of the NGF signaling, but rather is involved in late occurring events in this pathway as suggested by our previous studies, leading to altered morphological and biochemical differentiation.