Differential calcium signalling 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 either the human m3 or m2 receptor subtype. In m3 mAChR-expressing cells, carbachol stimulated inositol phosphate (InsP) formation and increased [Ca2+]i with EC50 values of about 2 microM and 30 nM, respectively. Maximal inositol 1,4,5-trisphosphate (InsP3) 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 extracellular 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)-insensitive. In m2 mAChR-expressing cells, carbachol stimulated InsP formation and increased [Ca2+]i with EC50 values of about 20 microM and 7 microM, 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 InsP3 was rapid (15 s to 2 min) and about twofold above basal. In contrast to m3 mAChR activation, [Ca2+]i increases induced by m2 mAChR activation were exclusively 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 affect m2 mAChR-induced PLC stimulation, but reduced the m2 mAChR-mediated increases in [Ca2+]i to 50%. In conclusion, m3 and m2 mAChRs stably expressed in HEK cells can induce similar cellular responses; however, 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 PTX-sensitive and this may occur at least partly independent of PLC activation.
Schmidt
M
M
Institut für Pharmakologie, Universitätsklinikum Essen, Germany.
Bienek
C
C
van Koppen
C J
CJ
Michel
M C
MC
Jakobs
K H
KH
D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D018721:Muscarinic Agonists / Q000494:pharmacology; D037342:Pertussis Toxin; D010862:Pilocarpine / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000378:metabolism*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D018721:Muscarinic Agonists; D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D010862:Pilocarpine; D002217:Carbachol; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D002118:Calcium