Mechanism of thrombin mediated eNOS phosphorylation in endothelial cells is dependent on ATP levels after stimulation

Abstract

Conflicting results have been reported concerning the role of AMP-activated protein kinase (AMPK) in mediating thrombin stimulation of endothelial NO-synthase (eNOS). We examined the involvement of two upstream kinases in AMPK activation in cultured human umbilical endothelial cells, LKB1 stimulated by a rise in intracellular AMP/ATP ratio, and Ca(+2)/CaM kinase kinase (CaMKK) responding to elevation of intracellular Ca(+2). We also studied the effects of AMPK activation on the downstream target eNOS. In culture medium 1640 the level of intracellular ATP was unchanged after thrombin stimulation and the CaMKK inhibitor STO-609 totally inhibited phosphorylation of AMPK and acetyl coenzyme A carboxylase (ACC) but not eNOS. In Morgan's medium 199 thrombin caused a significant lowering of intracellular ATP and STO-609 only partially inhibited the phosphorylation of AMPK, ACC and eNOS. Inhibition of AMPK by Compound C or AMPK downregulation using siRNA partially inhibited the phosphorylation of eNOS in medium 199 but not in 1640, underscoring a clear difference in the pathways mediating thrombin-stimulated eNOS phosphorylation in different culture media. Thus, conditions subjecting endothelial cells to a fall in ATP after thrombin stimulation facilitate activation of pathways partly dependent on AMPK causing downstream phosphorylation of eNOS. In contrast, under culture conditions that do not facilitate a fall in ATP after stimulation, AMPK activation is exclusively mediated by CaMKK and does not contribute to the phosphorylation of eNOS.

Salidroside improves endothelial function and alleviates atherosclerosis by activating a mitochondria-related AMPK/PI3K/Akt/eNOS pathway

“…Mitochondrial membrane depolarization is expected to lower cellular ATP levels and thereby increase the AMP: ATP ratio [38,52]. The mechanism proposed in this study is similar to the mechanism of histamine and thrombin-induced eNOS activation [13,14]. Meanwhile, this acute elevation of AMP: ATP ratio recovered within 24 h which was also in accordance with the aforementioned transient activation of AMPK in in-vitro conditions.…”

“…In response to several stimuli, including metformin [11], adiponectin [12], thrombin and histamine [13,14], and vascular endothelial growth factor [15], AMPK is activated and in turn acts as a direct activator of eNOS [16] by phosphorylating its serine 1177 site. There are also evidences that AMPK lies upstream of the PI3K/Akt pathway, which also leads to eNOS activation and NO production [15,17,18].…”

Activation of AMP-activated Protein Kinase by Vascular Endothelial Growth Factor Mediates Endothelial Angiogenesis Independently of Nitric-oxide Synthase

“…These data indicate that AMPK activation and eNOS phosphorylation may be dissociated when AMPK is activated by metabolic stress. Moreover, eNOS did not become an AMPK substrate when cells were stimulated with VEGF in the presence of 2-deoxyglucose as suggested previously (65). The combined addition of LKB1-siRNA and STO-609 completely prevented the phosphorylation of AMPK induced by VEGF plus 2-deoxyglucose but had FIGURE 3.…”

“…The situation may differ depending on the cell type and the specific circumstances of stimulation. A recent study, for example, suggests that eNOS may become AMPKresponsive under conditions of ATP depletion (65); these authors show that thrombin phosphorylates eNOS via the CaMKK␤/AMPK pathway only when cellular ATP is lowered, for instance when cells are preincubated with 2-deoxyglucose. Another study reports that serine 1177 phosphorylation of eNOS by AMPK occurs only in hypoxic but not in normoxic conditions (10).…”

Dilation of Porcine Retinal Arterioles to Cilostazol: Roles of eNOS Phosphorylation via cAMP/Protein Kinase A and AMP-Activated Protein Kinase and Potassium Channels

“…9,10 In the second series of studies, to examine the involvement of protein kinase A (PKA) and AMP-activated protein kinase (AMPK), we studied the cilostazolinduced response after incubation with the PKA inhibitor, Rp-8-BrcAMPS (100 lM), 19 and the AMPK inhibitor, compound C (10 lM). 20 …”