Department of Anesthesiology and Pain Medicine, Gyeongsang National University College of Medicine, Gyeongsang National University Hospital, 15 Jinju-daero 816 beon-gil, Jinju-si, Gyeongsangnam-do 52727, Republic of Korea.
Department of Pharmacology, Hypoxia-Related Disease Research Center, Inha Research Institute for Medical Sciences, Inha University College of Medicine, Inha-ro 100, Incheon 22212, Republic of Korea.
Eur J Pharmacol. 2019 Jun 15;853:121-128. doi: 10.1016/j.ejphar.2019.03.026. Epub 2019 Mar 15.
This study examined the mechanism associated with the endothelium-dependent attenuation of vasoconstriction induced by bupivacaine (BPV), with a particular focus on the upstream cellular signaling pathway of endothelial nitric oxide synthase (eNOS) phosphorylation induced by BPV in human umbilical vein endothelial cells (HUVECs). BPV concentration-response curves were investigated in the isolated rat aorta. The effects of Nω-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), methylene blue, calmidazolium, the Src kinase inhibitor 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and the combination of L-arginine and L-NAME on BPV-induced contraction in endothelium-intact aorta preparations were examined. The effects of BPV alone and in combination with PP2 on the phosphorylation of eNOS (at Ser1177 or Thr495), caveolin-1 and Src kinase were examined in HUVECs. BPV-induced contraction was lower in endothelium-intact aortae than in endothelium-denuded aortae. L-NAME, ODQ, methylene blue and calmidazolium increased BPV-induced contraction in endothelium-intact aortae, whereas PP2 alone and combined treatment with L-arginine and L-NAME inhibited BPV-induced contraction. Low-concentration BPV (30 µM) induced both stimulatory (Ser1177) and inhibitory (Thr495) phosphorylation of eNOS in HUVECs. However, high-concentration BPV (150 µM) induced only stimulatory (Ser1177) eNOS phosphorylation. Additionally, phosphorylation of Src kinase, caveolin-1 and inhibitory eNOS (Thr495) induced by low-concentration BPV was inhibited by PP2. These results suggest that contraction induced by low-concentration BPV is attenuated by endothelial nitric oxide release, which is modulated both stimulatory (Ser1177) and inhibitory eNOS phosphorylation (Thr495). BPV-induced phosphorylation of eNOS (Thr495) is indirectly mediated by an upstream cellular signaling pathway involving Src kinase (Tyr416) and caveolin-1 (Tyr14).
本研究旨在探讨布比卡因（BPV）诱导的血管收缩减弱与内皮依赖性相关的机制，特别关注 BPV 诱导人脐静脉内皮细胞（HUVEC）内皮型一氧化氮合酶（eNOS）磷酸化的上游细胞信号通路。在分离的大鼠主动脉中研究了 BPV 浓度-反应曲线。研究了 Nω-硝基-L-精氨酸甲酯（L-NAME）、1H-[1,2,4]恶二唑[4,3-a]喹喔啉-1-酮（ODQ）、亚甲蓝、西咪替丁、Src 激酶抑制剂 4-氨基-3-(4-氯苯基)-1-(叔丁基)-1H-吡唑并[3,4-d]嘧啶、4-氨基-5-(4-氯苯基)-7-(叔丁基)吡唑并[3,4-d]嘧啶（PP2）以及 L-精氨酸和 L-NAME 联合应用对内皮完整主动脉制备中 BPV 诱导收缩的影响。研究了 BPV 单独作用以及与 PP2 联合作用对 HUVEC 中 eNOS（Ser1177 或 Thr495）、 caveolin-1 和 Src 激酶磷酸化的影响。与去内皮主动脉相比，内皮完整的主动脉中 BPV 诱导的收缩较低。L-NAME、ODQ、亚甲蓝和西咪替丁增加了内皮完整的主动脉中 BPV 诱导的收缩，而 PP2 单独和与 L-精氨酸和 L-NAME 的联合治疗抑制了 BPV 诱导的收缩。低浓度 BPV（30 μM）诱导 HUVEC 中 eNOS 的刺激（Ser1177）和抑制（Thr495）磷酸化。然而，高浓度 BPV（150 μM）仅诱导刺激（Ser1177）eNOS 磷酸化。此外，低浓度 BPV 诱导的 Src 激酶、caveolin-1 和抑制性 eNOS（Thr495）磷酸化被 PP2 抑制。这些结果表明，低浓度 BPV 诱导的收缩通过内皮一氧化氮释放来减弱，这种释放受刺激（Ser1177）和抑制性 eNOS 磷酸化（Thr495）的双重调节。BPV 诱导的 eNOS（Thr495）磷酸化是通过涉及 Src 激酶（Tyr416）和 caveolin-1（Tyr14）的上游细胞信号通路间接介导的。
