Rhynchophylline Ameliorates Endothelial Dysfunction via Src-PI3K/Akt-eNOS Cascade in the Cultured Intrarenal Arteries of Spontaneous Hypertensive Rats

Abstract

Objectives: To examine the protective effect of Rhynchophylline (Rhy) on vascular endothelial function in spontaneous hypertensive rats (SHRs) and the underlying mechanism. Methods: Intrarenal arteries of SHRs and Wistar rats were suspended in myograph for force measurement. Expression and phosphorylation of endothelial nitric oxide (NO) synthase (eNOS), Akt, and Src kinase (Src) were examined by Western blotting. NO production was assayed by ELISA. Results: Rhy time- and concentration-dependently improved endothelium-dependent relaxation in the renal arteries from SHRs, but had no effect on endothelium-independent relaxation in SHR renal arteries. Wortmannin (an inhibitor of phosphatidylinositol 3-kinase) or PP2 (an inhibitor of Src) inhibited the improvement of relaxation in response to acetylcholine by 12 h-incubation with 300 μM Rhy. Western blot analysis revealed that Rhy elevated phosphorylations of eNOS, Akt, and Src in SHR renal arteries. Moreover, wortmannin reversed the increased phosphorylations of Akt and eNOS induced by Rhy, but did not affect the phosphorylation of Src. Furthermore, the enhanced phosphorylations of eNOS, Akt, and Src were blunted by PP2. Importantly, Rhy increased NO production and this effect was blocked by inhibition of Src or PI3K/Akt. Conclusion: The present study provides evidences for the first time that Rhy ameliorates endothelial dysfunction in SHRs through the activation of Src-PI3K/Akt-eNOS signaling pathway.

Keywords: PI3K/Akt; Src kinase; endothelial function; hypertension; renal artery.

Figure 1

Endothelial function is impaired in SHR renal arteries. Endothelium-dependent relaxation (A, n = 6), eNOS phosphorylation (B, n = 4) and nitric oxide (NO) production (C, n = 4) in renal arteries from Wistar rats (WT) and SHRs. ^*p < 0.05 vs. WT.

Figure 2

Rhy improves endothelial function of SHRs in a time-dependent manner. Endothelium-dependent relaxations in response to ACh in SHR renal arteries incubated with Rhy (300 μM) for 1 h (A), 4 h (B), 8 h (C), and 12 h (D). n = 4; ^*p < 0.05 vs. vehicle.

Figure 3

Rhy restores endothelial function concentration-dependently in SHR renal arteries. (A) Endothelium-dependent relaxation in response to ACh in the arteries from SHRs incubated with Rhy (30, 100, or 300 μM) or vehicle for 12 h. (B) Endothelium-independent relaxation response to SNP in SHR renal arteries. (C) Effects of Rhy on eNOS phosphorylation in the renal arteries from SHRs. (D) Effects of Rhy on NO production in the renal arteries from SHRs. n = 6; ^*p < 0.05 vs. vehicle.

Figure 4

Rhy ameliorates endothelial dysfunction in SHR renal arteries. (A) Effects of 12 h-incubation with Rhy (300 μM) on endothelium-dependent relaxation in response to ACh in the renal arteries from Wistar and SHRs. (B) Effects of 12 h-incubation with Rhy (300 μM) on endothelium-independent relaxation in response to SNP in the renal arteries from Wistar and SHRs. (C) Effects of Rhy (12 h, 300 μM) on eNOS phosphorylation in the rat renal arteries. (D) Effects of Rhy (12 h, 300 μM) on NO production in the rat renal arteries. n = 6 (A,B), n = 4 (C,D); ^*p < 0.05 vs. vehicle in WT; #p < 0.05 vs. vehicle in SHRs.

Figure 5

Endothelial function improved by Rhy is Src- and Akt-dependent in SHR renal arteries. (A) Effects of 12 h-incubation with Rhy (30, 100, or 300 μM) or vehicle on Akt phosphorylation in the arteries from SHRs (n = 4). (B) Effects of wortmannin (a PI3K inhibitor, 10 μM) on the Rhy-improved relaxation (n = 6). (C) Effects of wortmannin (a PI3K inhibitor, 10 μM) on the Rhy-increased eNOS phosphorylation in SHR renal arteries (n = 4). (D) Effects of incubation with Rhy (30, 100, or 300 μM) or vehicle for 12 h on Src phosphorylation in the arteries from SHRs (n = 4). (E) Effects of PP2 (a Src inhibitor, 10 μM) on the Rhy-improved endothelium-dependent relaxation in SHR renal arteries (n = 6). (F) Effects of PP2 (a Src inhibitor, 10 μM) on the Rhy-increased eNOS phosphorylation in SHR renal arteries (n = 4). ^*p < 0.05 vs. vehicle; #p < 0.05 vs. Rhy.

Figure 6

Rhy improves endothelial function via Src/Akt pathway in the renal arteries from SHRs. (A) Effects of wortmannin (10 μM) and PP2 (10 μM) on the Rhy (300 μM, 12 h)-enhanced phosphorylations of Src in SHR renal arteries. (B) Effects of wortmannin (10 μM) and PP2 (10 μM) on the Rhy (300 μM, 12 h)-enhanced phosphorylations of Akt in SHR renal arteries. (C) Effects of wortmannin (10 μM) and PP2 (10 μM) on the Rhy (300 μM, 12 h)-elevated NO production in SHR renal arteries. n = 4; ^*p < 0.05 vs. vehicle; #p < 0.05 vs. Rhy.