7 beta-hydroxysterol is cytotoxic to neonatal rat astrocytes in primary culture when cAMP levels are increased

We have shown previously that 7 beta-hydroxycholesterol (7 beta OHCH) and 7 beta-hydroxycholesteryl-3-oleate (7 beta OHCH-3-OL) are potent inhibitors of lesion-induced astrogliosis in the rat cortex or spinal cord; these subs tances reduce reactive astrocyte proliferation and hypertrophy. In this stu dy, we employed cultured newborn rat astrocytes with increased cAMP levels as an in vitro model of reactive astrocytes, Treatment with either dibutyry l-cAMP (dbcAMP) or isoproterenol resulted in morphologic differentiation of astrocytes which became fibrous. Concomitant incubation with 30 mu M 7 bet a OHCH and dbcAMP (or isoproterenol) provoked the cells to retract and was cytotoxic. When the beta-adrenergic receptor-mediated cAMP increase was abo lished by propranolol, the 7 beta OHCH cytotoxicity was inhibited. Immunocy tochemical labelling for glial fibrillary acidic protein (GFAP) and beta-tu bulin and electron microscopy suggested that intermediate filament and micr otubular organizations were modified by 7 beta OHCH, Analysis of the activi ty of cAMP-dependent protein kinase (PKA) in astrocytes treated with dbcAMP and 7 beta OHCH showed a rapid and marked inhibition of the phosphotransfe rase activity which lasted for 24 hr, We suggest that this culture system p rovides an experimental system to study the molecular mechanisms involved i n the effect of oxysterols on astrocytic hypertrophy, The cytotoxicity of 7 beta OHCH seems to be mediated by inhibition of PKA, which phosphorylates intermediate filaments and the transcription factor cyclic AMP responsive e lement binding. (C) 2000 Wiley-Liss, Inc.