Figure 6. EP4 increases eNOS phosphorylation at Ser1177 via the AMPK pathway.

(A and B) The AMPK inhibitor AraA reduced the phosphorylation of eNOS at Ser1177 in HUVECs treated by PGE₁-OH (100 nM) (A). The ratio of p-eNOS to total eNOS and p-AMPK to total AMPK was quantified (B). **P < 0.01 vs. control; ^#P < 0.05, and ^##P < 0.01 vs. PGE₁-OH, n = 3. (C and D) AraA decreased eNOS phosphorylation at Ser1177 in HUVECs treated with CAY10580 (1 μM) (C). Quantification of the ratio of p-eNOS to total eNOS and p-AMPK to total AMPK was performed (D). **P < 0.01 vs. control; ^#P < 0.05 vs. CAY10580, n = 4–5. (E and F) AMPK inhibition via the infection with an adenovirus containing an AMPK dominant-negative construct (Ad-AMPK-DN) diminished PGE₁-OH–induced eNOS phosphorylation at Ser1177 in HUVECs (E). Quantification of the ratio of p-eNOS to total eNOS is also shown (F). ***P < 0.001 vs. GFP; ^##P < 0.01 vs. GFP + PGE₁-OH, n = 3–4. Data are represented as mean ± SEM; 1-way ANOVA followed by Tukey’s multiple comparisons tests for B and D. Two-way ANOVA followed by Tukey’s multiple comparisons test for F.