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

• Published in: Journal of agricultural and food chemistry 2016-02, Vol.64 (4), p.785-791
• Main Authors: Jin, Sun Woo, Choi, Chul Yung, Hwang, Yong Pil, Kim, Hyung Gyun, Kim, Se Jong, Chung, Young Chul, Lee, Kyung Jin, Jeong, Tae Cheon, Jeong, Hye Gwang
• Format: Article
• Language: English
• Subjects: AMP-Activated Protein Kinases - genetics; AMP-Activated Protein Kinases - metabolism; Calcium - metabolism; Cell Line; Endothelial Cells - drug effects; Endothelial Cells - enzymology; Endothelial Cells - metabolism; Humans; Nitric Oxide - biosynthesis; Nitric Oxide Synthase Type III - genetics; Nitric Oxide Synthase Type III - metabolism; Phosphorylation - drug effects; Signal Transduction - drug effects; Triterpenes - pharmacology
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/acs.jafc.5b05416

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 Ca2+ and phosphorylation of Ca2+/calmodulin-dependent kinase IIα (CaMKIIα) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). Inhibition of the L-type Ca2+ channel (LTCC) and the ryanodine receptor (RyR) abolished BA-induced intracellular levels of Ca2+ 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 Ca2+/CaMKII and Ca2+/CaMKK/AMPK pathways. These findings provide further insight into the eNOS signaling pathways involved in the antiatherosclerosis effects of BA.