Contribution of intracellular stores to the increase in cytoplasmic calcium after nAChR activation in SH-SY5Y cells

The capacity of nicotinic acetylcholine receptors (nAChR) to mediate changes in cytoplasmic calcium concentrations (cyt[Ca²⁺]) is well established. However, the diverse routes through which calcium ions gain access to the cytoplasm after nAChR activation are debated. Calcium release from intracellular stores is emerging as a key aspect of the nicotinic modulation of cyt[Ca²⁺] ^[1].
We have examined cyt[Ca²⁺] in SH-SY5Y cells, loaded with the calcium sensitive dye Fluo-3. Nicotine (30 μM) produced a significant and long term (20 min) increase in fluorescence, that was almost completely prevented by mecamylamine (10 μM), and partially blocked by α-Bgt (40 nM) or α-CtxMII (200 nM). Cadmium (100 μM), cyclopiazonic acid (20 μM) and ryanodine (30 μM) significantly reduced the nicotine evoked increase in fluorescence, implicating VOCCs, calcium stores and ryanodine receptors respectively. In addition, a minor component of nicotine's increase in cyt[Ca²⁺] was independent of VOCC activation, but instead relied on the activation of α7 nAChR and ryanodine receptors. Xestospongin-c (10 μM), an inhibitor of IP-3 receptors, reduced nicotine's short term effect by 60 % and almost completely prevented the long term increase in cyt[Ca²⁺] evoked by nicotine.
Thus, the application of a variety of pharmacological tools to dissect sources of cyt[Ca²⁺] increase after nicotine, suggests that nAChR activation leads to calcium entry through nAChR and VOCCs, with additional calcium release from intracellular calcium stores via ryanodine and IP-3 receptors.