Carbachol-induced secretion from rat basophilic leukemia (RBL-2H3) cells transfected with human m3 muscarinic acetylcholine receptors

• Published in: Japanese Journal of Pharmacology 1994, Vol.64 (suppl.2), p.304-304
• Main Authors: Rika Inoue, Akihiro Sakurai, Hirofumi Tsuga, Kazuhiko Oishi, Masaatsu K. Uchida
• Format: Article
• Language: Japanese
• ISSN: 0021-5198

Muscarmnic acetylcholine receptors (mAChRs) are members of a superfamily of receptors that couple heterotrimeric GTP-binding proteins. Activation of m3 subtype results in the hydrolysis of inositol phospholipids by phospholipase C. In order to investigate the mechanisms underlying the exocytotic response mediated through m3 subtype mAChRs/GTP-binding protein pathway, RBL-2H3 cells, which secrete inflammatory mediators by aggregation of the high affinity IgE receptor but not by carbachol, were stably transfected with cDNA encoding the human m3 mAChRs. The transfected cells used here expressed mAChRs levels of Bmax. 5.9 pmol/mg of membrane protein and Kd 0.083 nM, as assessed in saturation binding analysis using 「^^3 H」QNB. Like antigen, carbachol produced secretion from the transfected cells in a dose-dependent manner; 100 μM carbachol gave maximal secretion from spontaneous levels to 74%. Atropine (10 nM) completely inhibited carbachol-induced secretion, indicating that the secretion was m3 mAChRs-specific. mAChRs as well as other GTP-binding protein-linked receptors are known to be desensitized following exposure to agonists. Exposure of the cells to 100 μM carbachol for 30 min in Ca^2+ -free medium inhibited the secretion induced by the addition of 10 μM carbachol plus Ca^2+ , indicating that m3 mAChRs were desensitized. These results suggest that m3 mAChRs mediate secretion in RBL-2H3 cells transfected with m3 cDNA.