Rutaecarpine Increases Nitric Oxide Synthesis via eNOS Phosphorylation by TRPV1-Dependent CaMKII and CaMKKβ/AMPK Signaling Pathway in Human Endothelial Cells

Abstract

Rutaecarpine (RUT) is a bioactive alkaloid isolated from the fruit of Evodia rutaecarpa that exerts a cellular protective effect. However, its protective effects on endothelial cells and its mechanism of action are still unclear. In this study, we demonstrated the effects of RUT on nitric oxide (NO) synthesis via endothelial nitric oxide synthase (eNOS) phosphorylation in endothelial cells and the underlying molecular mechanisms. RUT treatment promoted NO generation by increasing eNOS phosphorylation. Additionally, RUT induced an increase in intracellular Ca²⁺ concentration and phosphorylation of Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ), AMP-activated protein kinase (AMPK), and Ca²⁺/calmodulin-dependent kinase II (CaMKII). Inhibition of transient receptor potential vanilloid type 1 (TRPV1) attenuated RUT-induced intracellular Ca²⁺ concentration and phosphorylation of CaMKII, CaMKKβ, AMPK, and eNOS. Treatment with KN-62 (a CaMKII inhibitor), Compound C (an AMPK inhibitor), and STO-609 (a CaMKKβ inhibitor) suppressed RUT-induced eNOS phosphorylation and NO generation. Interestingly, RUT attenuated the expression of ICAM-1 and VCAM-1 induced by TNF-α and inhibited the inflammation-related NF-κB signaling pathway. Taken together, these results suggest that RUT promotes NO synthesis and eNOS phosphorylation via the Ca²⁺/CaMKII and CaM/CaMKKβ/AMPK signaling pathways through TRPV1. These findings provide evidence that RUT prevents endothelial dysfunction and benefit cardiovascular health.

Keywords: AMPK; Ca2+; CaMKII; TRPV1; eNOS; rutaecarpine.

Figure 1

Effects of RUT on viability of, and cytotoxicity in, endothelial cells. (A) Chemical structure of RUT. (B) Cell viability was assessed by MTT assay. (C) Cytotoxicity was assessed by LDH assay. * Significantly different from control at p < 0.01.

Figure 2

Effects of RUT on eNOS phosphorylation and NO generation. Cells were treated with (A) 10 µM RUT for 10–60 min or (B) 1–10 µM RUT for 30 min and assessed by Western blotting. (C) Cells were pretreated with 100 µM L-NAME for 1 h and then 10 µM RUT for 90 min. NO generation was detected using DAF-2DA. * Significantly different from control at p < 0.01. ^# Significantly different from RUT-treated cells at p < 0.01.

Figure 3

Effects of RUT on levels of intracellular calcium and phosphorylation of CaMKII, CaMKKβ, and AMPK. (A) Intracellular calcium was detected using the fluorescent calcium indicator, Fluo-4-AM. Fluo-4-AM-treated cells were stimulated with 1–10 µM RUT for 5 min. * Significantly different from control at p < 0.01. Cells were treated with (B) 10 µM RUT for 5–60 min or (C) 1–10 µM RUT for 60 min and assessed by Western blotting.

Figure 4

Effects of TRPV1 blockade on levels of intracellular calcium and phosphorylation of CaMKKβ, CaMKII, AMPK, and eNOS in response to RUT. (A) The effects of RUT on intracellular calcium influx in endothelial cells are mediated via TRPV1 channels. Cells were pretreated with 10 μM SB366791 (SB), 10 μM BAPTA-AM (BAPTA), or 10 μM EDTA for 30 min before Fluo-4-AM treatment. Then, the Fluo-4-AM-treated cells were stimulated with 10 µM RUT for 5 min. * Significantly different from control at p < 0.01. ^# Significantly different from RUT-treated cells at p < 0.01. (B) Effect of TRPV1 and calmodulin inhibition on eNOS phosphorylation by RUT. Cells were treated with 10 μM SB and 10 μM W7 for 30 min and then stimulated with 10 μM RUT for 30 min. (C) Cells were treated with 10 μM SB and 10 μM W7 for 30 min and then stimulated with 10 μM RUT for 15 min. Western blotting was performed to assess phosphorylation of CaMKKβ, CaMKII, AMPK, and eNOS.

Figure 5

Roles of CaMKII activation and the CaMKKβ/AMPK signaling pathway in RUT-induced eNOS activation and NO generation. (A) Effects of CaMKII inhibition on eNOS phosphorylation induced by RUT. Cells were pretreated with 10 μM KN-62 (KN) for 30 min and then incubated with 10 μM RUT for a further 30 min. (B) Effects of AMPK inhibition on eNOS phosphorylation induced by RUT. Cells were pretreated with 10 μM Compound C (CC) for 30 min and then incubated with 10 μM RUT for a further 30 min. (C) Effects of CaMKKβ inhibition on eNOS phosphorylation induced by RUT. Cells were pretreated with 10 μM STP-609 (STO) for 30 min and then incubated with 10 μM RUT for a further 30 min. Western blotting was performed to assess phosphorylation of CaMKKβ, CaMKII, AMPK, and eNOS. (D) Effects of CaMKII, AMPK, and CaMKKβ inhibition on increased NO generation by RUT. Cells were pretreated with 10 μM STO, CC, or KN for 30 min prior to incubation with DAF-2DA for 30 min and treatment with 10 μM RUT for a further 90 min. * Significantly different from control at p < 0.01. ^# Significantly different from RUT-treated cells at p < 0.01.

Figure 6

Inhibitory effect of RUT on the expression of adhesion molecules and adhesion of monocytes to endothelial cells. (A) Endothelial cells were treated with 1–10 μM RUT for 3 h, and TNF-α was added at 10 ng/mL for an additional 12 h. Endothelial cells were co-cultured with THP-1 monocytes for 1 h, and the adherence of endothelial cells to monocytes was assessed by fluorescence microscopy. * Significantly different from control at p < 0.01. ^# Significantly different from RUT-treated cells at p < 0.01. (B) Cells were treated with various concentrations (1–10 μM) of RUT for 3 h, followed by incubation with 10 ng/mL TNF-α for 12 h. (C) Cells were pretreated with 100 μM L-NAME or 10 μM SB366791 (SB) for 1 h, and then treated with 10 μM RUT for an additional 3 h, followed by incubation with 10 ng/mL TNF-α for 12 h. Western blotting was performed to assess expression of ICAM-1 and VCAM-1.

Figure 7

Inhibitory effect of RUT on the TNF-α-induced NF-κB signaling pathway in human endothelial cells. (A) Cells were treated with various concentrations (1–10 μM) of RUT for 1 h, followed by incubation with 10 ng/mL TNF-α for 30 min. (B) Cells were pretreated with 100 μM L-NAME or 10 μM SB366791 (SB) for 1 h, and then treated with 10 μM RUT for an additional 1 h, followed by incubation with 10 ng/mL TNF-α for 30 min. (C) Cells were pretreated with 10 μM RUT for 1 h, followed by incubation with 10 ng/mL TNF-α for 3 h. Cell lysates were analyzed by Western blotting using specific antibodies.