Intracellular Ca2+ signals evoked by stimulation of nicotinic acetylcholine receptors in SH-SY5Y cells: contribution of voltage-operated Ca2+ channels and Ca2+ stores

Neuronal nicotinic acetylcholine receptors (nAChR) can regulate several neuronal processes through Ca2+-dependent mechanisms. The versatility of nAChR-mediated responses presumably reflects the spatial and temporal characteristics of local changes in intracellular Ca2+ arising from a variety of sources. The aim of this study was to analyse the components of nicotine-evoked Ca2+, signals in SH-SY5Y cells, by monitoring fluorescence changes in cells loaded with fluo-3 AM. Nicotine (30 muM) generated a rapid elevation in cytoplasmic Ca2+ that was partially and additively inhibited (40%) by alpha7 and alpha3beta2* nAChR subtype selective antagonists; alpha3beta4* nAChR probably account for the remaining response (60%). A substantial blockade (80%) by CdCl2 (100 muM) indicates that voltage-operated Ca2+ channels (VOCC) mediate most of the nicotine-evoked response, although the alpha7 selective antagonist alpha-bungarotoxin (40 nM) further decreased the CdCl2-resistant component. The elevation of intracellular Ca2+ levels provoked by nicotine was sustained for at least 10 min and required the persistent activation of nAChR throughout the response. Intracellular Ca2+, stores were implicated in both the initial and sustained nicotine-evoked Ca2+ responses, by the blockade observed after ryanodine (30 muM) and the inositoltriphosphate (IP3)-receptor antagonist, xestospongin-c (10 muM). Thus, nAChR subtypes are differentially coupled to specific sources of Ca2+ : activation of nAChR induces a sustained elevation of intracellular Ca2+ levels which is highly dependent on the activation of VOCC, and also involves Ca2+ release from ryanodine and IP3-dependent intracellular stores, Moreover, the alpha7, but not alpha3beta2* nAChR, are responsible for a fraction of the VOCC-independent nicotine-evoked Ca2+ increase that appears to be functionally coupled to ryanodine sensitive Ca2+ stores.