Involvement of store-operated Ca2+ entry in AMP-activated protein kinase stimulation by Gq/11-coupled receptors

AMP-activated protein kinase (AMPK) regulates cell energy balance under conditions of metabolic stress and may play a role in neuroprotection. Gq/11-coupled receptors have been reported to activate AMPK through a signaling pathway involving stimulation of phospholipase C, enhancement of intracellular Ca2+ and activation of Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ), which phosphorylates AMPK at Thr172. The enhancement of cytosolic Ca2+ is due to both Ca2+ release from intracellular stores and Ca2+ entry through plasma membrane channels, but the relative contribution of these two Ca2+ pathways to AMPK stimulation by Gq/11-coupled receptors has not been previously investigated. In the present study we report that in human neuroblastoma SH-SY5Y cells endogenously expressing M3 muscarinic receptors and CHO cells transfected with the human M1 muscarinic receptor carbachol (CCh) stimulation of AMPK phosphorylation was completely dependent on the presence of extracellular Ca2+. In CHO/K1 cells, the stimulation of AMPK phosphorylation by endogenous protease-activated receptor 1 also required the presence of extracellular Ca2+. In addition, both thapsigargin (Tg) and ionomycin, which mobilize Ca2+ from intracellular stores, failed to elicit AMPK phosphorylation when Ca2+ was omitted from the extracellular medium. Assay of cytosolic free Ca2+ with Fura-2 fluorescence indicated that either activation of Gq/11-coupled receptors or treatment with Ca2+ mobilizing agents was effective in increasing Ca2+ levels in the absence of extracellular Ca2+. To investigate the involvement of store-operated Ca2+ entry in AMPK activation, cells were pretreated with Tg plus CCh in the absence of extracellular Ca2+ to deplete intracellular stores and then exposed to Ca2+ in the presence of atropine. The addition of Ca2+ increased AMPK phosphorylation in cells pretreated with Tg and CCh, but not in cells pretreated with vehicle. These data indicate that Ca2+ release from intracellular stores elicited by either Gq/11-coupled receptors or other Ca2+ mobilizing agents is insufficient to elicit AMPK phosphorylation. Conversely, store-operated Ca2+ entry seems to be the major pathway triggering AMPK activation via CaMKK