DIFFERENTIAL CALCIUM SIGNALING BY M2 AND M3 MUSCARINIC ACETYLCHOLINE-RECEPTORS IN A SINGLE-CELL TYPE

We have compared muscarinic acetylcholine receptor (mAChR) coupling to phospholipase C (PLC) and increases in cytoplasmic Ca2+ concentration [Ca2+](i), in human embryonic kidney (HEK) cells, stably expressing e ither the human m3 or m2 receptor subtype. In m3 mAChR-expressing cell s, carbachol stimulated inositol phosphate (InsP) formation and increa sed [Ca2+](i), with EC(50) values of about 2 mu M and 30 nM, respectiv ely. Maximal inositol 1,4,5-trisphosphate (InsP(3)) production (about fourfold) was rapid (15 s) and stable for 2 min. Maximal increases in [Ca2+](i) were 300-350 nM and mainly, almost 90%, due to influx of ext racellular Ca2+. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was not significantly different from that of carbachol. All m3 mAChR-mediated responses were pertussis toxin (PTX)-insensitiv e. In m2 mAChR-expressing cells, carbachol stimulated InsP formation a nd increased [Ca2+](i) with EC(50) values of about 20 mu M and 7 mu M, respectively. Maximal InsP formation was only 10-15% of that observed in m3 mAChR-expressing cells, whereas maximal elevations of [Ca2+](i) were similar in both cell types. Formation of InsP(3) was rapid (15 s to 2 min) and about twofold above basal. In contrast to m3 mAChR acti vation, [Ca2+](i) increases induced by m2 mAChR activation were exclus ively due to Ca2+ mobilization from intracellular stores. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was 50% and 20 % of the efficacy of carbachol, respectively. PTX treatment did not af fect m2 mAChR-induced PLC stimulation, but reduced the m2 mAChR-mediat ed increases in [Ca2+](i) to 50%. In conelusion, m3 and m2 mAChRs stab ly expressed in HEK cells can induce similar cellular responses; howev er, they do so by activating apparently distinct signalling pathways. While coupling of m2 mAChR to PLC occurs in a PTX-insensitive manner, coupling to mobilization of Ca2+ from intracellular stores is partly P TX-sensitive and this may occur at least partly independent of PLC act ivation.