α3* and α7 nAChR-mediated Ca2+ transient generation in IMR-32 neuroblastoma cells

α3-Containing (α3*) and α7 nicotinic acetylcholine receptors (nAChRs) are expressed in human IMR-32 neuroblastoma cells and implicated in Ca2+ signaling. In this study, we investigated the intracellular Ca2+ transient generation evoked by selective activation of α3* (agonist potency rank order: epibatidine > varenicline > nicotine ≈ cytisine) and α7 (rank order in the presence of α7 positive allosteric modulator or PAM: A-795723 > NS6784 ≈ PNU-282987) using, respectively, varenicline and NS6784 (+α7 PAM) by Ca2+ imaging. Effects of inhibitors of nAChRs (MLA and mecamylamine), ER Ca2+ ATPase pump (CPA and thapsigargin), Ca2+-induced Ca2+ release (ryanodine and dantrolene), Ca2+ channels (nitrendipine, diltiazem, and Cd2+), and removal of extracellular Ca2+ were examined. α7 PAMs, when tested in the presence of NS6784, were more active when added first, followed by the agonist, than in the reverse order. Removal of extracellular Ca2+ – but not CPA, thapsigargin, ryanodine, dantrolene, nitrendipine, diltiazem, or Cd2+ – diminished the α7 agonist-evoked Ca2+ transients. In contrast, only diltiazem and nitrendipine and removal of extracellular Ca2+ inhibited the α3*-mediated Ca2+ transients. The differential effect of diltiazem and nitrendipine versus Cd2+ was due to direct inhibition of α3* nAChRs as revealed by Ca2+ imaging in HEK-293 cells expressing human α3β4 nAChRs and patch clamp in IMR-32 cells. In summary, this study provides evidence that α3* and α7 nAChR agonist-evoked global Ca2+ transient generation in IMR-32 cells does not primarily involve voltage-dependent Ca2+ channels, intracellular Ca2+ stores, or Ca2+-induced Ca2+ release. These mechanisms may, however, be still involved in other forms of nAChR-mediated Ca2+ signaling.