Abstract

Nefiracetam (DM-9384) is a new pyrrolidone nootropic drug being developed for the treatment of Alzheimer's type and poststroke vascular-type dementia. Because the cholinergic system plays an important role in cognitive functions and Alzheimer's disease dementia, the present study was conducted to elucidate the mechanism of action of nefiracetam and aniracetam on neuronal nicotinic acetylcholine receptors (nnAChRs). Currents were recorded from rat cortical neurons in long-term primary culture using the whole-cell, patch-clamp technique. Two types of currents were evoked by acetylcholine (ACh): α-bungarotoxin-sensitive, α7-type currents and α-bungarotoxin-insensitive, α4β2-type currents. Although nefiracetam and aniracetam inhibited α7-type currents only weakly, these nootropic agents potentiated α4β2-type currents in a very potent and efficacious manner. Nefiracetam at 1 nM and aniracetam at 0.1 nM reversibly potentiated α4β2-type currents to 200 to 300% of control. Nefiracetam at very high concentrations (∼10 μM) also potentiated α4β2-type currents but to a lesser extent, indicative of a bell-shaped dose-response relationship. Nefiracetam markedly increased the saturating responses induced by high concentrations of ACh. However, human α4β2 subunits expressed in human embryonic kidney cells were inhibited rather than potentiated by nefiracetam. The specific protein kinase A inhibitors (H-89, KT5720, and peptide 5-24) and protein kinase C inhibitors (chelerythrine, calphostin C, and peptide 19–63) did not prevent nefiracetam from potentiating α4β2-type currents, indicating that these protein kinases are not involved in nefiracetam action. The nefiracetam potentiating action was not affected by 24-h pretreatment of neurons with pertussis toxin, but was abolished by cholera toxin. Therefore, G_s proteins, but not G_i/G_o proteins, are involved in nefiracetam potentiation. These results indicate that nnAChRs are an important site of action of nefiracetam and G_s proteins may be its crucial target.