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

Abstract

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 lM) generated a rapid elevation in cytoplasmic Ca 2+ that was partially and additively inhibited (40%) by a7 and a3b2* nAChR subtype selective antagonists; a3b4* nAChR probably account for the remaining response (60%). A substantial blockade (80%) by CdCl 2 (100 lM) indicates that voltage-operated Ca 2+ channels (VOCC) mediate most of the nicotine-evoked response, although the a7 selective antagonist a-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 lM) and the inositoltriphosphate (IP 3 )-receptor antagonist, xestospongin-c (10 lM). 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 a7, but not a3b2* 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.

Ryanodine receptors are involved in muscarinic antinociception in mice

“…Pretreatment with calcium, BAYK8644 and thapsigargin, compounds that increase the intracellular calcium levels, potentated nicotineinduced antinociception, whereas agents that decrease intracellular calcium blocked the increase of the pain threshold induced by nicotine [6]. Furthermore, the nicotine-evoked calcium response in SH-SY5Y is blocked by ryanodine and xestospongin C (an antagonist of InsP 3 R) indicating that it involves calcium release from ryanodine and InsP 3 -dependent intracellular stores [5]. However, ryanodine, in our experimental conditions prevented not only physostigmine-induced but also oxotremorine-induced increase of pain threshold suggesting that the release of Ca 2+ from intracellular stores produced by RyR activation is necessary to induce muscarinic antinociception.…”

Nicotinic acetylcholine receptor-mediated calcium signaling in the nervous system

“…In neurons of the substantia nigra pars compacta, depletion of internal calcium stores inhibits the increase in cytoplasmic calcium levels induced by nicotine and the α7 nAChR-selective agonist choline [12] . Blockade of ryanodine receptors in neuroblastoma cells also significantly reduces the increase in cytoplasmic calcium induced by activation of β2 and α7 subunit-containing nAChRs [14] . Functional coupling between α7 nAChRs and ryanodine receptors has also been observed in cultured hippocampal astrocytes, where α7 nAChRmediated calcium signals arise primarily from CICR through ryanodine receptors [13] .…”

“…There are three types of cytoplasmic calcium signals initiated by neuronal nAChR activation; (1) direct calcium influx through the nAChR [6,11] , (2) indirect calcium influx through voltage-dependent calcium channels (VDCCs) which are activated by the nAChR-mediated depolarization [6,11] , and (3) calcium-induced calcium release (CICR) (triggered by the two sources listed above) from the endoplasmic reticulum (ER) through the ryanodine receptors [12][13][14] and inositol (1,4,5)-triphosphate receptors (IP 3 Rs) [13][14][15] .…”

“…The nAChRs that contain α3 and/or β2 subunits in brain and ganglionic neuronal preparations are associated predominantly with calcium signals that are mediated by depolarization and the activation of VDCCs [12,14,24] , as well as the α7 nAChRs [25,26] . Calcium influx through VDCCs augments the primary calcium signals generated by the direct influx through nAChRs [11,14] . These two mechanisms may be physiologically complementary; calcium entry through inwardly rectifying nAChRs will be robust under either resting or hyperpolarized potentials, whereas calcium influx through VDCCs will occur mainly at more depolarized potentials (-40 mV) [27] .…”

“…In particular, calcium release from intracellular stores (via CICR) can have a crucial role in defining calcium responses [28] . The activation of α7 nAChRs can generate calcium transients via entry through the channel pore itself (independently of VDCCs), which can then activate CICR from ryanodine-dependent stores [12][13][14] . In neurons of the substantia nigra pars compacta, depletion of internal calcium stores inhibits the increase in cytoplasmic calcium levels induced by nicotine and the α7 nAChR-selective agonist choline [12] .…”