Intracellular Ca(2+) signals evoked by stimulation of nicotinic acetylcholine receptors in SH-SY5Y cells: contribution of voltage-operated Ca(2+) channels and Ca(2+) stores.

摘要

Neuronal nicotinic acetylcholine receptors (nAChR) can regulate several neuronal processes through Ca(2+)-dependent mechanisms. The versatility of nAChR-mediated responses presumably reflects the spatial and temporal characteristics of local changes in intracellular Ca(2+) arising from a variety of sources. The aim of this study was to analyse the components of nicotine-evoked Ca(2+) signals in SH-SY5Y cells, by monitoring fluorescence changes in cells loaded with fluo-3 AM. Nicotine (30 microm) generated a rapid elevation in cytoplasmic Ca(2+) 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 CdCl(2) (100 microm) indicates that voltage-operated Ca(2+) channels (VOCC) mediate most of the nicotine-evoked response, although the alpha7 selective antagonist alpha-bungarotoxin (40 nm) further decreased the CdCl(2)- resistant component. The elevation of intracellular Ca(2+) levels provoked by nicotine was sustained for at least 10 min and required the persistent activation of nAChR throughout the response. Intracellular Ca(2+) stores were implicated in both the initial and sustained nicotine-evoked Ca(2+) responses, by the blockade observed after ryanodine (30 microm) and the inositoltriphosphate (IP(3))-receptor antagonist, xestospongin-c (10 microm). Thus, nAChR subtypes are differentially coupled to specific sources of Ca(2+): activation of nAChR induces a sustained elevation of intracellular Ca(2+) levels which is highly dependent on the activation of VOCC, and also involves Ca(2+) release from ryanodine and IP(3)-dependent intracellular stores. Moreover, the alpha7, but not alpha3beta2* nAChR, are responsible for a fraction of the VOCC-independent nicotine-evoked Ca(2+) increase that appears to be functionally coupled to ryanodine sensitive Ca(2+) stores.