Figure 1

The influence of cholinergic activation on cytosolic calcium levels in CHO-M3 cells. CHO cells stably transfected with the gene for human M3 muscarinic acetylcholine receptor (CHO-M3) were loaded with fura-2 AM dye for 60 min at room temperature. The loading dye was removed and replaced with basal salt solution (BSS) and the cells were incubated for an additional 30 min. The BSS solution was replaced by fresh BSS or BSS without calcium immediately before measuring intracellular calcium levels. a Control responses measured in the presence of 2 mM extracellular calcium. Carbamylcholine (10 μM) was added at the time indicated by the first dashed vertical line. EDTA (5 mM) was added at the time indicated by the second dashed vertical line. b Control responses measured in the absence of extracellular calcium. Calcium was introduced into the extracellular medium (final concentration, 2 mM) at the point indicated by the second dashed vertical line. The average and standard deviation of the calcium concentrations from 22 (Figure 1a) or 20 (Figure 1b) cells from a single experiment are shown. These results are representative of responses from more than 20 independent experiments. In all of the experiments related in this report, the resting calcium level was 27 ± 8 nM (N = 19, including measurements from >350 cells). Two components of the calcium response to extracellular calcium are evident: a rapid initial response reflecting release from the endoplasmic reticulum and a plateau response dependent on the influx of extracellular calcium.