Muscarinic activation of mitogen-activated protein kinase in PC12 cells

Muscarinic acetylcholine receptors (mAChRs) activate many downstream signal ing pathways, some of which can lead to mitogen-activated protein kinase (M APK) phosphorylation and activation. MAPKs play roles in regulating cell gr owth, differentiation, and synaptic plasticity. Here, the activation of MAP K was examined in PC12 cells endogenously expressing mAChRs. Western blot a nalysis using a phosphospecific MAPK antibody revealed a dose-dependent and atropine-sensitive increase in MAPK phosphorylation in cells stimulated wi th carbachol (CCh). The maximal response occurred after 5 min and was rapid ly reduced to baseline. To investigate the receptors responsible for CCh ac tivation of MAPK in PC12 cells, the mAChR subtypes present were determined using RT-PCR and immunoprecipitation. RT-PCR was used to amplify fragments of the appropriate sizes for m1, m4, and m5, and the identities of the band s were confirmed with restriction digests. Immunoprecipitation using subtyp e-specific antibodies showed that similar to 95% of the expressed receptors were m4, whereas the remaining similar to 5% were mi and m5. A highly spec ific mi toxin completely blocked MAPK phosphorylation in response to CCh st imulation. The mAChR-induced MAPK activation was abolished by protein kinas e C down-regulation and partially inhibited by pertussis toxin. Although mi represents a small proportion of the total mAChR population, pharmacologic al evidence suggests that mi is responsible for MAPK activation in PC12 cel ls.