Capacitative Ca²⁺ entry is involved in regulating soluble amyloid precursor protein (sAPPα) release mediated by muscarinic acetylcholine receptor activation in neuroblastoma SH-SY5Y cells

Previous studies have demonstrated that stimulation of phospholipase C-linked G-protein-coupled receptors, including muscarinic M₁ and M₃ receptors, increases the release of the soluble form of amyloid precursor protein (sAPPα) by α-secretase cleavage. In this study, we examined the involvement of capacitative Ca²⁺ entry (CCE) in the regulation of muscarinic acetylcholine receptor (mAChR)-dependent sAPPα release in neuroblastoma SH-SY5Y cells expressing abundant M3 mAChRs. The sAPPα release stimulated by mAChR activation was abolished by EGTA, an extracellular Ca²⁺ chelator, which abolished mAChR-mediated Ca ²⁺ influx without affecting Ca²⁺ mobilization from intracellular stores. However, mAChR-mediated sAPPα release was not inhibited by thapsigargin, which increases basal [Ca²⁺]i by depletion of Ca²⁺ from intracellular stores. While these results indicate that the mAChR-mediated increase in sAPPα release is regulated largely by Ca²⁺ influx rather than by Ca²⁺ mobilization from intracellular stores, we further investigated the Ca²⁺ entry mechanisms regulating this phenomenon. CCE inhibitors such as Gd³⁺, SKF96365, and 2-aminoethoxydiphenyl borane (2-APB), dose dependently reduced both Ca²⁺ influx and sAPPα release stimulated by mAChR activation, whereas inhibition of voltage-dependent Ca²⁺ channels, Na⁺/Ca²⁺ exchangers, or Na⁺-pumps was without effect. These results indicate that CCE plays an important role in the mAChR-mediated release of sAPPα.