Estrogens can either relax or contract arteries via rapid, nongenomic mechanisms involving classic estrogen receptors (ER). In addition to ERα and ERβ, estrogen may also stimulate G protein-coupled estrogen receptor 1 (GPER) in nonvascular tissue; however, a potential role for GPER in coronary arteries is unclear. The purpose of this study was to determine how GPER activity influenced coronary artery reactivity. In vitro isometric force recordings were performed on endothelium-denuded porcine arteries. These studies were augmented by RT-PCR and single-cell patch-clamp experiments. RT-PCR and immunoblot studies confirmed expression of GPER mRNA and protein, respectively, in smooth muscle from either porcine or human coronary arteries. G-1, a selective GPER agonist, produced a concentration-dependent relaxation of endothelium-denuded porcine coronary arteries in vitro. This response was attenuated by G15, a GPER-selective antagonist, or by inhibiting large-conductance calcium-activated potassium (BK(Ca)) channels with iberiotoxin, but not by inhibiting NO signaling. Last, single-channel patch-clamp studies demonstrated that G-1 stimulates BK(Ca) channel activity in intact smooth muscle cells from either porcine or human coronary arteries but had no effect on channels isolated in excised membrane patches. In summary, GPER activation relaxes coronary artery smooth muscle by increasing potassium efflux via BK(Ca) channels and requires an intact cellular signaling mechanism. This novel action of estrogen-like compounds may help clarify some of the controversy surrounding the vascular effects of estrogens.

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Yu X, Li F, Klussmann E, Stallone JN, Han G. G proteincoupled estrogen receptor 1 mediates relaxation of coronary arteries via cAMP/PKA-dependent activation of MLCP. Am J Physiol Endocrinol Metab 307: E398 -E407, 2014. First published July 8, 2014 doi:10.1152/ajpendo.00534.2013.-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 M) was associated with increased cAMP production in a concentration-dependent manner. Furthermore, inhibition of adenylyl cyclase (AC) with SQ-22536 (100 M) or of PKA activity with either Rp-8-CPT-cAMPS (5 M) or PKI (5 M) attenuated G-1-induced relaxation of coronary arteries preconstricted with PGF2␣ (1 M). 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 PGF2␣-induced p-MYPT-1, whereas Rp-8-CPT-cAMPS prevented this inhibitory effect of G-1. Similarly, G-1 inhibited PGF2␣-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 M), 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.

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BackgroundVascular pathology and dysfunction are direct life-threatening outcomes resulting from atherosclerosis or vascular injury, which are primarily attributed to contractile smooth muscle cells (SMCs) dedifferentiation and proliferation by re-entering cell cycle. Increasing evidence suggests potent protective effects of G-protein coupled estrogen receptor 1 (GPER) activation against cardiovascular diseases. However, the mechanism underlying GPER function remains poorly understood, especially if it plays a potential role in modulating coronary artery smooth muscle cells (CASMCs).Methodology/Principal FindingsThe objective of our study was to understand the functional role of GPER in CASMC proliferation and differentiation in coronary arteries using from humans and swine models. We found that the GPER agonist, G-1, inhibited both human and porcine CASMC proliferation in a concentration- (10−8 to 10−5 M) and time-dependent manner. Flow cytometry revealed that treatment with G-1 significantly decreased the proportion of S-phase and G2/M cells in the growing cell population, suggesting that G-1 inhibits cell proliferation by slowing progression of the cell cycle. Further, G-1-induced cell cycle retardation was associated with decreased expression of cyclin B, up-regulation of cyclin D1, and concomitant induction of p21, and partially mediated by suppressed ERK1/2 and Akt pathways. In addition, G-1 induces SMC differentiation evidenced by increased α-smooth muscle actin (α-actin) and smooth muscle protein 22α (SM22α) protein expressions and inhibits CASMC migration induced by growth medium.ConclusionGPER activation inhibits CASMC proliferation by suppressing cell cycle progression via inhibition of ERK1/2 and Akt phosphorylation. GPER may constitute a novel mechanism to suppress intimal migration and/or synthetic phenotype of VSMC.