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Activation of G protein-coupled receptors is known to stimulate cell migration, but receptor-mediated signals inhibiting cell migration have not been identified. We investigated the ability of transfected human M3 muscarinic acetylcholine receptors (mAChR) to regulate the migration of Chinese hamster ovary (CHO) cells. Single cells migrated on colloidal gold applied to fibronectin-coated plates, and videomicroscopy was used to measure cell spreading and migration. Activation of M3 mAChR with the agonist carbachol was found to inhibit cell migration, whereas direct activation of protein kinase C (PKC) with PMA was found to stimulate migration. The amount of cell adhesion and spreading was found to be equivalent for carbachol- and PMA-treated cells. Selective inactivation of conventional PKC isoforms with Go6976 (C24H18N4O) abolished the PMA-mediated increase in cell migration. In contrast, the mAChR-mediated decrease in migration was not altered by Go6976, but was abolished when both novel and conventional PKC isoforms were inactivated by calphostin C or chelerythrine. These findings suggest involvement of conventional PKC isoforms in the stimulation of migration and of novel PKC isoforms in the inhibition of migration. Carbachol- but not PMA-treated cells exhibited an elongated morphology reminiscent of migrating cells that cannot detach their trailing edges from the substratum. Similarly, carbachol-treated cells detached less readily from fibronectin than control or PMA-treated cells when integrin activity was diminished by the chelation of Ca2+ and Mg2+. Finally, the carbachol-induced diminution of cell detachment was preserved after inhibition of the conventional PKC isoforms with Go6976, but was abrogated by treatment with either calphostin C or chelerythrine. These findings suggest that mAChR activation diminishes the ability of cells to detach from the substratum, resulting in diminished migration. This is in contrast to the direct activation of PKC with PMA, which stimulates migration. [Copyright &y& Elsevier]