Background and purpose
Neuronal nicotinic acetylcholine receptors (nAChR) can modulate cell survival and memory processing. The involvement of specific nAChR subtypes in downstream signalling events has been ill defined thus far, because of a lack of subtype-selective ligands. In this study, we investigated activation and modulation of alpha7 nAChR-mediated phosphorylation of extracellular signal-regulated kinases (ERK1/2) in PC12 cells, using selective agonists and positive allosteric modulators. Experimental approach: We used undifferentiated PC12 cells endogenously expressing alpha7 nAChR for both biochemical and functional studies. ERK phosphorylation changes were measured by using a novel In-Cell Western procedure. alpha7 nAChR-mediated Ca(2+) signalling was determined by using the fluorometric imaging plate reader assay. Key
Purpose
Neuronal nicotinic acetylcholine receptors (nAChR) can modulate cell survival and memory processing. The involvement of specific nAChR subtypes in downstream signalling events has been ill defined thus far, because of a lack of subtype-selective ligands. In this study, we investigated activation and modulation of alpha7 nAChR-mediated phosphorylation of extracellular signal-regulated kinases (ERK1/2) in PC12 cells, using selective agonists and positive allosteric modulators. Experimental approach: We used undifferentiated PC12 cells endogenously expressing alpha7 nAChR for both biochemical and functional studies. ERK phosphorylation changes were measured by using a novel In-Cell Western procedure. alpha7 nAChR-mediated Ca(2+) signalling was determined by using the fluorometric imaging plate reader assay. Key
Results
Robust induction of ERK phosphorylation followed exposure of PC12 cells to the selective agonist PNU-282987 in the presence of the alpha7 nAChR modulator PNU-120596. ERK phosphorylation was transient and was attenuated by the selective antagonist methyllycaconitine. Consistent with allosteric modulation of alpha7 nAChRs, PNU-120596 enhanced both the agonist potency and efficacy in activating ERK. Moreover, alpha7 nAChR agonists could be quantitatively differentiated based on their potency in activating ERK signalling. The rank order of potencies correlated fairly well with the corresponding binding K(i) values of these alpha7 nAChR agonists. Conclusions and implications: The present work extends previous observations demonstrating the involvement of alpha7 nAChRs in ERK1/2 phosphorylation in PC12 cells. The In-Cell Western procedure allowed a detailed investigation of alpha7 nAChR function and downstream ERK signalling in response to agonist and allosteric modulators.