Alzheimer's disease cybrid cells produced by replacing endogenous mitochondria in human neuroblastoma SH-SY5Y cells with platelet mitochondria from subjects with Alzheimer's disease have higher levels of reactive oxygen species than do cybrid cells with mitochondria from control subjects. These cells were used to test if this chronic mild increase in reactive oxygen species affects muscarinic receptor-coupled signaling activities. Basal and carbachol-stimulated phosphoinositide hydrolysis were higher, and there was less inhibition by glutathione depletion, in Alzheimer's disease than control cybrid cells. Elevated phosphoinositide hydrolysis in Alzheimer's disease cybrid cells also was evident upon direct activation of G-proteins (Gq/11) linked to phosphoinositide signaling or of phospholipase C, but immunoblot analyses revealed equivalent levels of Gq/11 and phospholipase C in both cell lines. These results indicate that there is up-regulation of phosphoinositide signaling in Alzheimer's disease cybrid cells in association with chronic mild oxidative stress, although treatment of cells with H(2)O(2) to induce greater acute oxidative stress caused decreases in carbachol-stimulated phosphoinositide hydrolysis that were similar in Alzheimer's disease and control cybrid cells. In contrast to phosphoinositide hydrolysis, carbachol-stimulated AP-1 DNA binding activity was lower in Alzheimer's disease than control cybrid cells, and this deficit was associated with deficient protein kinase C-mediated activation of AP-1. Overall, these results demonstrate that chronically elevated reactive oxygen species in Alzheimer's disease cybrid cells are associated with a more robust phosphoinositide signaling system, but lower signaling to activation of AP-1. These alterations may represent adaptations to exposure to oxidants, which precede more widespread deficits in signaling associated with more severe oxidative stress.