Fig. 3. Effect of shear stress on the association of eNOS with PECAM-1 and the phosphorylation of eNOS, Akt and AMPK and the generation of cyclic GMP. (A) Human endothelial cells were exposed to fluid shear stress (12 dynes cm^–2) for the times indicated. Thereafter, PECAM-1 was immunoprecipitated and the co-precipitated eNOS was detected by a specific antibody. The bar graph summarizes data obtained in three independent experiments. (B) Human endothelial cells were exposed to fluid shear stress (12 dynes cm^–2, 30 minutes) in the absence (CTL) and presence of PP1 (30 µmol l^–1) or its inactive analogue PP3 (30 µmol l^–1). Thereafter, the Triton X-100-soluble cell fraction was subjected to SDS-PAGE and the phosphorylation of eNOS (Ser¹¹⁷⁷), Akt (Ser⁴⁷³) and AMPK (Thr¹⁷²) was determined using phospho-specific antibodies. The blots shown are representative of data obtained in three additional experiments. (C) Cells were incubated with solvent (CTL), wortmannin (40 nmol l^–1) or PP1 (30 µmol l^–1) in the presence of IBMX (50 µmol l^–1) and either maintained under static conditions or exposed to shear stress (12 dynes cm^–2, 30 minutes). Intracellular cyclic GMP levels were determined by radioimmunoassay and the bar graph summarizes data obtained in four independent experiments (each performed in duplicate); ^*P<0.05, ^**P<0.01 versus static conditions.