Betulinic Acid Increases eNOS Phosphorylation and NO Synthesis via the Calcium-Signaling Pathway

Abstract

Betulinic acid (BA) is a naturally occurring pentacyclic triterpene that attenuates vascular diseases and atherosclerosis, but the mechanism by which it stimulates endothelial nitric oxide synthase (eNOS) is unclear. eNOS is the key regulatory enzyme in the vascular endothelium. This study examined the intracellular pathways underlying the effects of BA on eNOS activity and endothelial nitric oxide (NO) production in endothelial cells. BA treatment induced both eNOS phosphorylation at Ser1177 and NO production. It also increased the level of intracellular Ca(2+) and phosphorylation of Ca(2+)/calmodulin-dependent kinase IIα (CaMKIIα) and Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ). Inhibition of the L-type Ca(2+) channel (LTCC) and the ryanodine receptor (RyR) abolished BA-induced intracellular levels of Ca(2+) and eNOS phosphorylation. Treatment with W7 (a CaM antagonist), KN-93 (a selective inhibitor of CaMKII), and STO 609 (a selective inhibitor of CaMKK) suppressed eNOS phosphorylation and NO production. Moreover, AMP-activated protein kinase (AMPK) was induced by BA, and BA-induced eNOS phosphorylation was inhibited by compound C, an AMPK inhibitor. Taken together, these results indicate that BA activates eNOS phosphorylation and NO synthesis via the Ca(2+)/CaMKII and Ca(2+)/CaMKK/AMPK pathways. These findings provide further insight into the eNOS signaling pathways involved in the antiatherosclerosis effects of BA.

Inhibitory Effects of Betulinic Acid on LPS-Induced Neuroinflammation Involve M2 Microglial Polarization via CaMKKβ-Dependent AMPK Activation

“…However, BA not only attenuated LPS-induced AMPK activation reduction but also raised AMPK phosphorylation up to the level several-folds that of the baseline. These current data are consistent with previous reports showing that BA-induced AMPK activation conferred protection in endothelial cells and mouse models ( Lingaraju et al, 2015 ; Jin et al, 2016 ). Next, we confirmed that BA’s effects on inhibiting M1 polarization and promoting M2 polarization were dependent on AMPK activation using the AMPK inhibitor, compound C, and AMPKα siRNA to block AMPK.…”

“…Therefore, AMPK has been considered as an attractive target for the treatment of inflammatory diseases ( Amato and Man, 2011 ; O’Neill and Hardie, 2013 ). Recently, several reports also demonstrated that BA could activate AMPK pathways in various in vitro and in vivo models ( Jin et al, 2016 ; Silva et al, 2016 ; Xie et al, 2017 ). On the other hand, accumulating data indicated that the anti-inflammatory properties of some widely known anti-inflammatory agents, like salicylate, are also strongly associated with the activation of the AMPK signaling pathway ( Hawley et al, 2012 ).…”

“…In addition, BA was reported to decrease LPS-induced matrix metalloproteinase-9 expression via blocking the nuclear translocation of nuclear factor-κB in microglial cells ( Lee et al, 2011 ; Wan et al, 2013 ). Recently, several reports also demonstrated that BA could activate AMPK pathways in vitro and in vivo ( Jin et al, 2016 ; Silva et al, 2016 ; Xie et al, 2017 ). It is worth noting that BA-induced CaMKKβ-dependent AMPK activation was reported to confer protective actions in endothelial cells and in a non-alcoholic fatty liver disease mouse model ( Jin et al, 2016 ).…”

“…Recently, several reports also demonstrated that BA could activate AMPK pathways in vitro and in vivo ( Jin et al, 2016 ; Silva et al, 2016 ; Xie et al, 2017 ). It is worth noting that BA-induced CaMKKβ-dependent AMPK activation was reported to confer protective actions in endothelial cells and in a non-alcoholic fatty liver disease mouse model ( Jin et al, 2016 ). However, whether BA could promote microglial polarization to M2 phenotype and whether AMPK activation is involved in the anti-neuroinflammatory effects of BA have not been investigated.…”

Betulinic acid‑induced expression of nicotinamide adenine dinucleotide phosphate‑diaphorase in the immune organs of mice: A possible role of nitric oxide in immunomodulation

“…In addition, NO is recognized to act as a vasodilator following its release from endothelial cells ( 56 – 58 ), including BetA-treated endothelial cells ( 59 – 62 ). This function is mediated by NO inhibitory action on vascular smooth muscles.…”