Fig 1.
Epac contributes to G-1-induced relaxation of coronary arteries.
Concentration-response relationship for G-1- or 8-CPT-2Me-cAMP -induced relaxation in endothelium denuded, PGF2α (1 μM) precontracted coronary artery were observed in the presence or absence of Epac inhibitors. A & B: in the presence or absence of Epac inhibitor, BFA (A); in the presence or absence of both Epac and PKA inhibitors, BFA and PKI (B). C&D: Concentration-response relationship for Epac agonist 8-CPT-2Me-cAMP (007)-induced porcine coronary artery relaxation in the absence (C) or presence (D) of BFA pretreatment. E & F: Concentration-response relationship for G-1-induced relaxation in the presence or absence of the Epac inhibitors, ESI-09 (E) or CE3F4 (F). Each point represents the mean percent relaxation effect ± SEM., *** p<0.001, compared to G-1 group or solvent control. G. A representative Western blot of Epac1 and Epac2 expression. Well1 is the loading of porcine coronary artery tissue lysates. Well2 is the loading of rat brain tissue lysates, serving as a positive control. Beta-actin was used as a protein loading control. H. A summary bar graph of densitometry analysis of Epac1 and Epac2 Western blot bands.
Table 1.
Effects of compounds on porcine coronary artery relaxation response to G-1.
Fig 2.
G-1 stimulates Rap1 activities in porcine coronary artery.
Rap1 activity measured with a Rap1 activation kit. Porcine coronary artery SMCs (passage 4 and 5) were serum deprived for 24 hours, then treated with: BFA (50 μM) + G-1 (1 μM); G-1 (1 μM) for 0, 2.5, 5, and 15 min; 007 (100 μM) (A); PGF2α (1 μM) for 10 min, PGF2α (1 μM) for 10 min + G-1(1 μM) for 5 min (B); 007 (50 μM) for 5 min, BFA (50 μM) for 10 min + 007 (50 μM) for 5min (C); DMSO (0.1%) was used as solvent control. SMC lysate in Rap1 activation lysis buffer, upper band: cell lysate was pre-incubated with GTPγS prior to precipitation with Ral GDS-RBD; Lower band: cell lysate was pre-incubated with GDP prior to precipitation with Ral GDS-RBD. Left panel in 2A and upper panels in 2B-2C are a representative Western blot bands of Rap1-GTP and total Rap1 detected by using anti-Rap1 antibody from 3 individual assays. Rap1-GTP is the active form of Rap1. The lower panel is a summary bar graph of the Rap1 Western blot bands analyzed by densitometry. Rap1-GTP/total Rap1 ratio was normalized to control % (0 min of G-1 treatment) according to the manufacture instruction. * p<0.05, compared to control.
Fig 3.
G-1 increases phosphorylation of VASP.
Porcine coronary artery SMCs (passage 4 and 5) were serum deprived for 24 hours and then treated with: 0.01% DMSO as solvent control; 007 (50 μM); G-1 (1 μM); G-1(1 μM) + ESI-09 (10 μM) and PKI (5 μM). Left panel is a representative of Western blot for p-VASP and β–actin of three individual experiments. Right panel is a summary bar graph of the Western blot band densitometry analysis. Results are expressed as mean ± SE, * p<0.05, compared to the solvent control.
Fig 4.
Epac and Rap1 are involved in the G-1-induced phosphorylation of RhoA and the inhibition of RhoA activity in porcine CASMCs.
CASMCs were serum deprived for 18 hours before being treated with different drugs. A: RhoA activity in CASMCs was evaluated in cells treated for 2.5 min with 0.1% DMSO, serum, serum + 007 (100 μM), serum + G-1 (100 nM), 10% serum + ESI-09 (10 μM) + G-1 (100 nM) (n = 3), * p<0.05, compared to groups as indicated. B: Western blot detection of phosphorylation of RhoA at Ser188 in CASMCs. Cells were treated for 10 minutes with: 0.1% DMSO as solvent control; 007 (100 μM); 6-Bnz-cAMP (10 μM); G-1 (1 μM); ESI-09 (10 μM) + G-1 (1 μM) and PKI (5 μM) + G-1 (1 μM) (n = 3). Upper panel: a representative Western blot phosphor-Ser188 RhoA and β-actin of three experiments. Lower panel: Bar graph of the quantitative data of the p-RhoA bands evaluated by densitometry. Sample protein amounts were normalized to β-actin which was employed as a control for protein loading, * p<0.05, compared to the groups as indicated. C: A representative phosphorylation of RhoA and siRNA knock-down of Rap1 were detected by Western blots of three independent experiments. After transfected with either scramble siRNA or Rap1 siRNA for 48 hours, CASMCs were serum deprived for 24 hours and then treated in the presence or absence of G-1 (1 μM). β-actin was served as loading control. D&E: Bar graphs showing the summary data of p-RhoA and total Rap1 knock-down normalized to total β-actin respectively, the bands were evaluated by densitometry, * p<0.05, compared to the group as indicated in the graphs.
Fig 5.
Epac is involved in the G-1 activation of MLCP in porcine coronary arteries.
A: Western blot detection of phosphorylation of MLCP at the regulatory subunit, myosin-targeting subunit protein-1 (pMYPT1 at Thr853) in porcine coronary artery rings. Rings in isometric tension studies were incubated with DMSO (solvent control, 0.1%); PGF2α (1 μM); PGF2α (1 μM) + G-1 (1 μM); PGF2α (1 μM) + ESI-09 (10 μM) +G-1 (1 μM); and PGF2α (1 μM) + 007 (100 μM). Upper panel: A representative Western blot for p-MYPT1 from three individual experiments. Lower panel: Bar graph of the quantitative data of the Western blot bands evaluated by densitometry. Tissue sample protein amounts were normalized to β-actin which was employed as a control for protein loading, * p<0.05, compared between groups as indicated. B: Upper panel: the representative p-MLC detection by Western blot from three independent experiments. Porcine CASMCs were serum deprived for 24 hours, and then treated with drugs as in porcine coronaries, except that the concentration of ESI-09 was 10 μM. Lower panel: Bar graph showing the summary data of p-MLC which was normalized to total β-actin, the bands were evaluated by densitometry, * p<0.05, compared to the group as indicated.
Fig 6.
Epac and PKA exert additive effect on G-1-induced activation of MLCP in porcine coronary arteries.
A: Western blot detection of pMYPT1 at Thr853 in artery rings. Artery rings were treated with: DMSO (solvent control, 0.1%); PGF2α (1 μM); PGF2α (1 μM) + G-1 (1 μM); PGF2α (1 μM) + ESI-09 (10 μM) +G-1 (1 μM); PGF2α (1 μM) + PKI (5 μM) + G-1 (1 μM); and PGF2α (1 μM) + ESI-09 (10 μM) + PKI (5 μM) + G-1 (1 μM). Upper panel: A representative Western blot for p-MYPT1 from three individual experiments. Lower panel: Bar graph summary of the quantitative data of the Western blot p-MYPT1 bands evaluated by densitometry. Tissue sample protein amounts were normalized to β-actin. B: Upper panel: A representative p-MLC detection by Western blots of three independent experiments. CASMCs were serum deprived for 24 hours, and then incubated with the drugs as used in arterial rings, except that 10 μM of ESI-09 was used rather than 25 μM. Lower panel: Bar graph showing the summary data of p-MLC normalized to total β-actin, the bands were evaluated by densitometry, * p<0.05, compared to the group as indicated in the graph.
Fig 7.
Proposed mechanism of Epac and PKA pathways in GPER-mediated porcine coronary artery relaxation signaling.
When GPER is activated by G-1, it activates Gs protein. Gs stimulates adenlylyl cyclase (AC) and increases cAMP generation. Cyclic AMP activates both Epac and PKA, the downstream targets. Epac activates Rap1, then RhoA is phosphorylated at serine 188 by both Rap1 signaling and PKA, thereby the activity RhoA and its effector Rho Kinase (ROCK) is inhibited, Reduced Rho kinase activity removes its inhibitory effects on MLCP by decreasing the phosphorylation of MYPT1 (pThr853), the myosin phosphatase target subunit 1 of MLCP, leading to decreased phosphorylation of MLC20 and coronary artery relaxation.