Thrombin Activates AMP-Activated Protein Kinase in Endothelial Cells via a Pathway Involving Ca 2 /Calmodulin-Dependent

identified as LKB1 or Ca 2 /calmodulin-dependent protein kinase kinase (CaMKK). Our study of human endothelial cells shows that AMPK is activated by thrombin through a Ca 2 -dependent mechanism involving the thrombin receptor protease-activated receptor 1 and Gq-protein-mediated phospholipase C activation. Inhibition of CaMKK with STO-609 or downregulation of CaMKK using RNA interference decreased thrombin-induced AMPK activation significantly, indicating that CaMKK was the responsible AMPK kinase. In contrast, downregulation of LKB1 did not affect thrombin-induced AMPK activation but abolished phosphorylation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside. Thrombin stimulation led to phosphorylation of acetyl coenzyme A carboxylase (ACC) and endothelial nitric oxide synthase (eNOS), two downstream targets of AMPK. Inhibition or downregulation of CaMKK or AMPK abolished phosphorylation of ACC in response to thrombin but had no effect on eNOS phosphorylation, indicating that thrombin-stimulated phosphorylation of eNOS is not mediated by AMPK. Our results underline the role of Ca 2 as a regulator of AMPK activation in response to a physiologic stimulation. We also demonstrate that endothelial cells possess two pathways to activate AMPK, one Ca 2 /CaMKK dependent and one AMP/LKB1 dependent.