Cholinergic stimulation of AP-1 and NFκB transcription factors is differentially sensitive to oxidative stress in SH-SY5Y neuroblastoma: Relationship to phosphoinositide hydrolysis

Abstract

Oxidative stress appears to contribute to neuronal dysfunction in a number of neurodegenerative conditions, notably including Alzheimer's disease, in which cholinergic receptor-linked signal transduction activity is severely impaired. To test whether oxidative stress could contribute to deficits in cholinergic signaling, responses to carbachol were measured in human neuroblastoma SH-SY5Y cells exposed to H2O2. DNA binding activities of two transcription factors that are respondent to oxidative conditions, AP- 1 and NFκB, were measured in nuclear extracts. H2O2 and carbachol individually induced dose-and time-dependent increases in AP-1 and NFκB. In contrast, when given together, H2O2 concentration dependently (30-300 μM) inhibited the increase after carbachol in AP-1. Carbachol's stimulation of NFκB was not inhibited except with a high concentration (300 μM) of H2O2, which was associated with impaired activation of protein kinase C. Lower concentrations of H2O2 (30-300 μM) inhibited carbachol-induced [3H]phosphoi nositide hydrolysis, and this inhibition correlated (r = 0.95) withthe inhibition of carbachol-induced AP-1. Activation of [3H]phosphoinositide hydrolysis by the calcium ionophore ionomycin was unaffected by H2O, indicating that phospholipase C and phosphoinositides were impervious to this treatment. In contrast, activation with NaF of G- proteins coupled to phos pholipase C was concentration dependently inhibited by H2O2, indicating impaired G-protein function. These effects of H2O2 are similar to signaling impairments reported in Alzheimer's disease brain, which involve deficits in receptor- and G-protein-stimulated phosphoinositide hydrolysis, but not phospholipase C activity. Thus, these findings indicate that oxidative stress may contribute to impaired phosphoinositide signaling in neurological disorders in which oxidative stress occurs, and that oxidative stress can differentially influence transcription factors activated by cholinergic stimulation.