Identification of Nitric Oxide as an Endogenous Activator of the AMP-activated Protein Kinase in Vascular Endothelial Cells

Abstract

2؉ chelator, indicating that NO-induced AMPK activation is guanylyl cyclase-mediated and calcium-dependent. Exposure of HUVECs or isolated mice aortas to either calcium ionophore A23187 or bradykinin significantly increased AMPK Thr-172 phosphorylation, which was abolished by N-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase. Finally, A23187-or bradykinin-enhanced AMPK activation was significantly greater in aortas from wild type mice than those in the aortas of endothelial nitric oxide synthase knock-out mice. Taken together, we conclude that NO might act as an endogenous AMPK activator.The AMP-activated protein kinase (AMPK) 2 is a key enzyme in an important energy-sensing/signaling system by which cells sense and decode changes in energy status. The overall effect of AMPK activation is to switch off ATP-consuming pathways such as lipogenesis or gluconeogenesis, whereas switching on ATP-producing pathways such as fatty acid and glucose oxidation. AMPK is a protein consisting of three subunits designated ␣, ␤, and ␥. The ␣ subunit is the catalytic subunit containing the kinase domain, which transfers a phosphate from ATP to the target protein. The ␤ and ␥ subunits are considered regulatory components (1-3). All three subunits are required for expression of full activity (4).Phosphorylation of Thr-172 on the ␣ subunit by upstream kinases, AMPK kinases, results in AMPK activation. Several upstream kinases have been identified, including a complex of the tumor suppressor protein LKB1 and two accessory subunits, termed STRAD and MO25 (5, 6), and Ca 2ϩ /calmodulindependent protein kinase kinase (CaMKK), especially the CaMKK ␤ isoform (7-9). AMP also allosterically promotes phosphorylation at Thr-172 by LKB1 (10, 11). Any situation that causes an increase in cytoplasmic Ca 2ϩ will create a subsequent demand for ATP because Ca 2ϩ is immediately pumped out of the cytoplasm using ATP-driven pumps in the plasma membrane and endoplasmic reticulum. Activation of AMPK under these circumstances may represent a mechanism to anticipate the demand for ATP created by Ca 2ϩ entry. Nitric oxide (NO) is a small, highly reactive, diffusible free radical that has been implicated in many physiological and pathophysiological processes. NO exerts its effects through many ways including activation of the cGMP/protein kinase G pathway and through S-nitrosylation of proteins (12). There is an emerging body of evidence indicating a relationship between NO and AMPK expression/activity in cells (13). Such a relationship cooperatively promotes glucose and fatty acid oxidation. There is ample evidence showing that AMPK regulates NO production in cells. For example, endothelial nitric oxide synthase (eNOS) is phosphorylated by AMPK at position Ser-*

Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells

“…These findings contrast with the conclusions of a recent publication, which reported that nitric oxide donors promote an increase in AMPK phosphorylation (38). The conclusions of this previous paper were largely based on experiments in which treatment of cultured cells with pharmacological NO donors, such as SNP, increased AMPK phosphorylation in HeLa cells and in human umbilical vein endothelial cells.…”

IRE1-Dependent Activation of AMPK in Response to Nitric Oxide

“…These findings, which suggest that the regulation of Akt phosphorylation in response to nitric oxide is independent of mTOR, are consistent with previous reports showing that Akt is a direct target for inhibitory S-nitrosation by nitric oxide (58). Overall, these findings confirm that nitric oxide activates AMPK (2,34,61) and that AMPK activation correlates with Raptor phosphorylation and the inhibition of mTORC1 signaling (13). These studies also suggest that nitric oxide may alter mTOR signaling in part through AMPK activation.…”

“…While nitric oxide stimulates cellular damage, it also activates a number of signaling pathways that limit additional cellular damage and repair existing damage. In pancreatic ␤-cells, the protective responses activated by nitric oxide include (i) JNK-dependent induction of GADD45␣ (growth arrest and DNA damage-inducible protein 45␣) and DNA repair, (ii) activation of AMP-activated protein kinase (AMPK), resulting in enhanced metabolic recovery, and (iii) activation of the unfolded-protein response (UPR) (25,34,38,54,57,61).…”

“…While nitric oxide stimulates cellular damage, it also activates a number of signaling pathways that limit additional cellular damage and repair existing damage. In pancreatic ␤-cells, the protective responses activated by nitric oxide include (i) JNK-dependent induction of GADD45␣ (growth arrest and DNA damage-inducible protein 45␣) and DNA repair, (ii) activation of AMP-activated protein kinase (AMPK), resulting in enhanced metabolic recovery, and (iii) activation of the unfolded-protein response (UPR) (25,34,38,54,57,61).AMPK is a conserved heterotrimeric (␣, ␤, and ␥ subunits) serine/threonine kinase involved in sensing and responding to the energetic demand within eukaryotic cells (15). AMPK is activated by phosphorylation at threonine 172 in the catalytic ␣ subunit (19) in a constitutive fashion by the upstream kinase LKB1; however, this phosphorylation is rapidly removed by a phosphatase to maintain low basal activity (18,43).…”

Urocortin 2 autocrine/paracrine and pharmacologic effects to activate AMP-activated protein kinase in the heart

“…Interestingly, AMPK is also activated during ischemic preconditioning (40,41), the physiologic phenomenon by which preceding short durations of ischemia decrease the susceptibility to necrosis during more prolonged and potentially injurious ischemia. AMPK is also activated by other treatment regimens that prevent ischemic injury, such as heat shock (10), adrenergic stimulation (42), and nitric oxide (43). AMPK activation is sufficient to precondition the heart, based the results of recent pharmacologic studies with a direct AMPK activator (41).…”