Department of Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A & M University, College Station, Texas;
Department of Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A & M University, College Station, Texas; College of Life Science, Henan Normal University, Xinxiang, Henan Province, China; and.
Am J Physiol Endocrinol Metab. 2014 Aug 15;307(4):E398-407. doi: 10.1152/ajpendo.00534.2013. Epub 2014 Jul 8.
Activation of GPER exerts a protective effect in hypertension and ischemia-reperfusion models and relaxes arteries in vitro. However, our understanding of the mechanisms of GPER-mediated vascular regulation is far from complete. In the current study, we tested the hypothesis that GPER-induced relaxation of porcine coronary arteries is mediated via cAMP/PKA signaling. Our findings revealed that vascular relaxation to the selective GPER agonist G-1 (0.3-3 μM) was associated with increased cAMP production in a concentration-dependent manner. Furthermore, inhibition of adenylyl cyclase (AC) with SQ-22536 (100 μM) or of PKA activity with either Rp-8-CPT-cAMPS (5 μM) or PKI (5 μM) attenuated G-1-induced relaxation of coronary arteries preconstricted with PGF2α (1 μM). G-1 also increased PKA activity in cultured coronary artery smooth muscle cells (SMCs). To determine downstream signals of the cAMP/PKA cascade, we measured RhoA activity in cultured human and porcine coronary SMCs and myosin-light chain phosphatase (MLCP) activity in these artery rings by immunoblot analysis of phosphorylation of myosin-targeting subunit protein-1 (p-MYPT-1; the MLCP regulatory subunit). G-1 decreased PGF2α-induced p-MYPT-1, whereas Rp-8-CPT-cAMPS prevented this inhibitory effect of G-1. Similarly, G-1 inhibited PGF2α-induced phosphorylation of MLC in coronary SMCs, and this inhibitory effect was also reversed by Rp-8-CPT-cAMPS. RhoA activity was downregulated by G-1, whereas G36 (GPER antagonist) restored RhoA activity. Finally, FMP-API-1 (100 μM), an inhibitor of the interaction between PKA and A-kinase anchoring proteins (AKAPs), attenuated the effect of G-1 on coronary artery relaxation and p-MYPT-1. These findings demonstrate that localized cAMP/PKA signaling is involved in GPER-mediated coronary vasodilation by activating MLCP via inhibition of RhoA pathway.
GPER 的激活在高血压和缺血再灌注模型中发挥保护作用，并在体外使动脉松弛。然而，我们对 GPER 介导的血管调节机制的理解还远远不够。在本研究中，我们检验了以下假设：GPER 诱导的猪冠状动脉松弛是通过 cAMP/PKA 信号转导介导的。我们的发现表明，选择性 GPER 激动剂 G-1（0.3-3 μM）引起的血管松弛与 cAMP 产生的浓度依赖性增加有关。此外，用 SQ-22536（100 μM）抑制腺苷酸环化酶（AC）或用 Rp-8-CPT-cAMPS（5 μM）或 PKI（5 μM）抑制 PKA 活性，均可减弱预先用 PGF2α（1 μM）收缩的冠状动脉对 G-1 的松弛反应。G-1 还增加了培养的冠状动脉平滑肌细胞（SMCs）中的 PKA 活性。为了确定 cAMP/PKA 级联反应的下游信号，我们通过免疫印迹分析磷酸化肌球蛋白靶向亚基蛋白-1（p-MYPT-1；MLCP 调节亚基），测量了培养的人源和猪源冠状动脉 SMC 中的 RhoA 活性和这些动脉环中的肌球蛋白轻链磷酸酶（MLCP）活性。G-1 降低了 PGF2α 诱导的 p-MYPT-1，而 Rp-8-CPT-cAMPS 阻止了 G-1 的这种抑制作用。同样，G-1 抑制了冠状动脉 SMC 中 PGF2α 诱导的 MLC 磷酸化，而 Rp-8-CPT-cAMPS 也逆转了这种抑制作用。RhoA 活性被 G-1 下调，而 G36（GPER 拮抗剂）恢复了 RhoA 活性。最后，PKA 和锚定蛋白激酶 anchoring proteins（AKAPs）之间相互作用的抑制剂 FMP-API-1（100 μM）减弱了 G-1 对冠状动脉松弛和 p-MYPT-1 的作用。这些发现表明，局部 cAMP/PKA 信号转导通过抑制 RhoA 途径激活 MLCP，参与了 GPER 介导的冠状动脉舒张。
