目的:研究橙皮素诱导的人脐静脉去极化收缩后血管舒张功能,阐明L型Ca2+通道(LTCC)及其相关信号通路的作用。
方法:在用K预收缩的HUV环中进行等距张力记录。使用LTCC开放剂(BayK8644)和环核苷酸和磷酸二酯酶(PDE)的阻断剂研究了橙皮素的松弛机制。A7r5细胞的全细胞膜片钳,大鼠血管平滑肌细胞系,进行研究橙皮素对LTCC电流的影响。
结果:去极化前收缩后,橙皮素诱导HUV舒张浓度依赖性和内皮依赖性;与匹配载体相比,1mmol/L橙皮素可将裸露的HUV环张力降低68.7%±4.3%,渗透压,和时间控制(P<0.0001)。重要的是,橙皮素竞争性抑制BayK8644诱导的收缩,将BayK8644反应的半最大有效浓度从载体对照的1.08nmol/L[95%置信区间(CI)0.49-2.40]转变为橙皮素的11.30nmol/L(95%CI5.45-23.41)(P=0.0001)。此外,橙皮素在用可溶性鸟苷酸环化酶抑制剂预处理的裸露HUV环中引起进一步的血管舒张,腺苷酸环化酶,PDE3、PDE4和PDE5(P<0.01),而用PDE1抑制剂预处理的环不能被橙皮素放松(P>0.05)。然而,同时应用可溶性鸟苷酸环化酶和腺苷酸环化酶抑制剂均不能抑制橙皮素的作用(P>0.05)。在全细胞膜片钳中,橙皮素使A7r5细胞中的LTCC电流迅速降低至66.7%±5.8%(P=0.0104)。
结论:橙皮素通过抑制LTCC减少人血管平滑肌的去极化收缩,而不是环核苷酸或PDE。我们的证据支持直接的LTCC相互作用,并为橙皮素及其前体橙皮苷作为血管扩张剂的使用提供了额外的基础,并可能导致未来的血管扩张剂药物开发作为心血管疾病的替代治疗方法。
OBJECTIVE: To study hesperetin-induced vasorelaxation after depolarizing contraction in human umbilical veins (HUVs) to elucidate the role of L-type Ca2+ channel (LTCC) and related signaling pathway.
METHODS: Isometric tension recording was performed in HUV rings pre-contracted with K+. Hesperetin relaxing mechanism was investigated using a LTCC opener (BayK8644) and blockers of cyclic nucleotides and phosphodiesterases (PDEs). Whole-cell patch-clamping in A7r5 cells, a rat vascular smooth muscle cell line, was performed to study the effect of hesperetin on LTCC current.
RESULTS: After depolarizing precontraction, hesperetin induced HUV relaxation concentration-dependently and endothelium-independently; 1 mmol/L hesperetin reduced denuded HUV ring tension by 68.7% ± 4.3% compared to matching vehicle, osmolality, and time controls (P<0.0001). Importantly, hesperetin competitively inhibited BayK8644-induced contraction, shifting the half maximal effective concentration of BayK8644 response from 1.08 nmol/L [95% confidence interval (CI) 0.49-2.40] in vehicle control to 11.30 nmol/L (95% CI 5.45-23.41) in hesperetin (P=0.0001). Moreover, hesperetin elicited further vasorelaxation in denuded HUV rings pretreated with inhibitors of soluble guanylyl cyclase, adenylyl cyclase, PDE3, PDE4, and PDE5 (P<0.01), while rings pretreated with PDE1 inhibitors could not be relaxed by hesperetin (P>0.05). However, simultaneously applying inhibitors of soluble guanylyl cyclase and adenylyl cyclase could not inhibit hesperetin\'s effect (P>0.05). In whole-cell patch-clamping, hesperetin rapidly decreased LTCC current in A7r5 cells to 66.7% ± 5.8% (P=0.0104).
CONCLUSIONS: Hesperetin diminishes depolarizing contraction of human vascular smooth muscle through inhibition of LTCC, and not cyclic nucleotides nor PDEs. Our evidence supports direct LTCC interaction and provides additional basis for the use of hesperetin and its precursor hesperidin as vasodilators and may lead to future vasodilator drug development as a treatment alternative for cardiovascular diseases.