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Evidence for cross‐talk between M 2 and M 3 muscarinic acetylcholine receptors in the regulation of second messenger and extracellular signal‐regulated kinase signalling pathways in Chinese hamster ovary cells
Author(s) -
Hornigold David C,
Mistry Rajendra,
Raymond Pamela D,
Blank Jonathan L,
John Challiss R A
Publication year - 2003
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0705178
Subject(s) - muscarinic acetylcholine receptor , chinese hamster ovary cell , receptor , mapk/erk pathway , endocrinology , muscarinic acetylcholine receptor m2 , second messenger system , medicine , biology , forskolin , pertussis toxin , signal transduction , microbiology and biotechnology , chemistry , g protein , stimulation
We have examined possible mechanisms of cross‐talk between the G q/11 ‐linked M 3 muscarinic acetylcholine (mACh) receptor and the G i/o ‐linked M 2 mACh receptor by stable receptor coexpression in Chinese hamster ovary (CHO) cells. A number of second messenger (cyclic AMP, Ins(1,4,5)P 3 ) and mitogen‐activated protein kinase (ERK and JNK) responses stimulated by the mACh receptor agonist methacholine were examined in CHO‐m2m3 cells and compared to those stimulated in CHO‐m2 and CHO‐m3 cell‐lines, expressing comparable levels of M 2 or M 3 mACh receptors. Based on comparisons between cell‐lines and pertussis toxin (PTx) pretreatment to eliminate receptor‐G i/o coupling, evidence was obtained for (i) an M 2 mACh receptor‐mediated contribution to the predominantly M 3 mACh receptor‐mediated Ins(1,4,5)P 3 response and (ii) a facilitation of the inhibitory effect of M 2 mACh receptor on forskolin‐stimulated cyclic AMP accumulation by M 3 mACh receptor coactivation at low agonist concentrations (MCh 10 −9 –10 −6 M ). The most profound cross‐talk effects were observed with respect to ERK activation. Thus, while MCh stimulated ERK activation in both CHO‐m2 and CHO‐m3 cells (pEC 50 values: 5.64±0.09 and 5.57±0.16, respectively), the concentration–effect relation was approx 50‐fold left‐shifted in CHO‐m2m3 cells (pEC 50 : 7.17±0.07). In addition, the ERK response was greater and more sustained in CHO‐m2m3 cells. In contrast, only minor differences were seen in the time‐courses and concentration‐dependencies of JNK activation in CHO‐m3 and CHO‐m2m3 cells. Costimulation of endogenous P2Y 2 purinoceptors also caused an approx 10‐fold left‐shift in the MCh‐stimulated ERK response in CHO‐m2 cells, suggesting that the G q/11 /G i/o interaction to affect ERK activation is not specific to muscarinic receptors. PTx pretreatment of cells had unexpected effects on ERK activation by MCh in both CHO‐m2m3 and CHO‐m3 cells. Thus, in CHO‐m3 cells PTx pretreatment caused a marked left‐shift in the MCh concentration–effect curve, while in PTx‐treated CHO‐m2m3 cells the maximal responsiveness was decreased, but the potency of MCh was only slightly affected. The data presented here strongly suggest that cross‐talk between M 2 and M 3 mACh receptors occurs at the level of both second messenger and ERK regulation. Further, these data provide novel insights into the involvement of G i/o proteins in both positive and negative modulation of ERK responses evoked by G protein‐coupled receptors.British Journal of Pharmacology (2003) 138 , 1340–1350. doi: 10.1038/sj.bjp.0705178