Glutathione depletion exacerbates impairment by oxidative stress of phosphoinositide hydrolysis, AP-1, and NF-κB activation by cholinergic stimulation

Abstract

Oxidative stress appears to contribute to neuronal dysfunction associated with Alzheimer's disease and other CNS neurodegenerative disorders. This investigation examined if oxidative stress might contribute to impairments in cholinergic receptor-linked signaling systems and if intracellular glutathione levels modulated responses to oxidative stress. To do this the activation of the AP-1 and NF-κB transcription factors and of the phosphoinositide second-messenger system was measured in human neuroblastoma SH-SY5Y cells after exposure to the oxidants H 2O 2 or diamide, with or without prior depletion of cellular glutathione. H 2O 2 concentration-dependently inhibited carbachol-stimulated AP-1 activation and this inhibition was potentiated in glutathione-depleted cells. Carbachol-stimulated NF-κB activation was unaffected by H 2O 2 unless glutathione was depleted, in which case there was a H 2O 2 concentration-dependent inhibition. Glutathione depletion also potentiated the inhibition by H 2O 2 of carbachol- or G-protein (NaF)-stimulated phosphoinositide hydrolysis, whereas phospholipase C activated by the calcium ionophore ionomycin was not inhibited. The thiol-oxidizing agent diamide also inhibited phosphoinositide hydrolysis stimulated by carbachol or NaF, and glutathione depletion potentiated the diamide concentration-dependent inhibition. Unlike H 2O 2, diamide also inhibited ionomycin-stimulated phosphoinositide hydrolysis. Activation of both AP-1 and NF-κB stimulated by carbachol was inhibited by diamide, and glutathione depletion potentiated the inhibitory effects of diamide. Thus, diamide inhibited a wider range of signaling processes than did H 2O 2, but glutathione depletion increased the susceptibility of phosphoinositide hydrolysis and of transcription factor activation to inhibition by both H 2O 2 and diamide. These results demonstrate that the vulnerability of signaling systems to oxidative stress is influenced by intracellular glutathione levels, indicating that cell-selective susceptibility to inhibition of signal transduction systems by oxidative stress can arise from cellular variations in antioxidant capacity.