title: Involvement of store-operated Ca(2+) entry in activation of AMP-activated protein kinase and stimulation of glucose uptake by M3 muscarinic acetylcholine receptors in human neuroblastoma cells.
abstract: Gq/11-coupled muscarinic acetylcholine receptors (mAChRs) belonging to M1, M3 and M5 subtypes have been shown to activate the metabolic sensor AMP-activated protein kinase (AMPK) through Ca(2+)/calmodulin-dependent protein kinase kinase-β (CaMKKβ)-mediated phosphorylation at Thr172. However, the source of Ca(2+) required for this response has not been yet elucidated. Here, we investigated the involvement of store-operated Ca(2+) entry (SOCE) in AMPK activation by pharmacologically defined M3 mAChRs in human SH-SY5Y neuroblastoma cells. In Ca(2+)-free medium the cholinergic agonist carbachol (CCh) caused a transient increase of phospho-Thr172 AMPK that rapidly ceased within 2min. Conversely, in the presence of extracellular Ca(2+) CCh-induced AMPK phosphorylation lasted for at least 180min. The SOCE modulator 2-aminoethoxydiphephenyl borate (2-APB), at a concentration (50μM) that suppressed CCh-induced intracellular Ca(2+) ([Ca(2+)]i) plateau, inhibited CCh-induced AMPK phosphorylation. CCh triggered the activation of the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule (STIM) 1, as indicated by redistribution of STIM1 immunofluorescence into puncta, and promoted the association of STIM1 with the SOCE channel component Orai1. Cell depletion of STIM1 by siRNA treatment reduced both CCh-induced [Ca(2+)]i plateau and AMPK activation. M3 mAChRs increased glucose uptake and this response required extracellular Ca(2+) and was inhibited by 2-APB, STIM1 knockdown, CaMKKβ and AMPK inhibitors, and adenovirus infection with dominant negative AMPK. Thus, the study provides evidence that SOCE is required for sustained activation of AMPK and stimulation of downstream glucose uptake by M3 mAChRs and suggests that SOCE is a critical process connecting M3 mAChRs to the control of neuronal energy metabolism.
authors.affiliation: Laboratory of Cellular and Molecular Pharmacology, Section of Neurosciences, Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Olianas
authors.affiliation: Laboratory of Cellular and Molecular Pharmacology, Section of Neurosciences, Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Dedoni
authors.affiliation: Laboratory of Cellular and Molecular Pharmacology, Section of Neurosciences, Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy. Electronic address: onali@unica.it.
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Onali
keywords: AMP-activated protein kinase; Glucose uptake; Human neuroblastoma cell; M(3) muscarinic acetylcholine receptor; Store-operated Ca(2+) entry; Stromal interaction molecule 1