ACTIVATION OF NICOTINIC ACETYLCHOLINE-RECEPTORS EXPRESSED IN QUAIL FIBROBLASTS - EFFECTS ON INTRACELLULAR CALCIUM

1 The aim of these experiments was to determine the ability of the mus cle-type nicotinic acetylcholine receptor (AChR) stably expressed in q uail fibroblasts (QF18 cells) to elevate intracellular calcium ([Ca2+] (i)) upon activation. Ratiometric confocal microscopy, with the calciu m-sensitive fluorescent dye Indo-1 was used. 2 Application of the nico tinic agonist, suberyldicholine (SDC), to the transfected QF18 cells c aused an increase in [Ca2+](i). Control [Ca2+](i) levels in QF18 cells were found to be 164 +/- 22 nM (mean +/- s.e. mean; n=40 cells) risin g to 600 +/- 81 nM on addition of SDC (10 mu M; n=15 cells), whereas n o increase in [Ca2+](i) was seen in non-transfected control QT6 fibrob lasts (before: 128 +/- 9nM, n=40; after: 113 +/- 13 nM, n=15). 3 The i ncrease in [Ca2+](i) caused by application of SDC was dose-dependent, with an EC(50) value of 12.7 +/- 5.9 mu M (n=14). 4 The responses to S DC in QF18 cells were blocked by prior application of alpha-bungarotox in (200 nM), by the addition of Ca2+ (100 mu M), by removal of Na+ ion s from the extracellular solution, or by the voltage-sensitive calcium channel blockers nifedipine and omega-conotoxin, which act with IC50 values of 100 nM and 100 pM respectively. 5 We conclude that activatio n of the nicotinic AChRs leads to a Na+-dependent depolarization and h ence activation of endogenous voltage-sensitive Ca2+ channels in the p lasma membrane and an increase in [Ca2+](i). There is no significant e ntry of Ca2+ through the nicotinic receptor itself.