FIGURE 7.

Differential effects on ADP-mediated responses of siRNA-mediated knockdown of AMPK or CaMKKβ versus inhibition of kinase activity. A, BAEC were treated for 30 min with the AMPK inhibitor compound C (20 μm), and eNOS enzyme activity (left panel) was assayed using the l-[³H]arginine-[³H]citrulline assay following the addition of ADP (50 μm) for 20 min. Cell extracts were processed to quantitate formation of l-[³H]citrulline, as described under “Experimental Procedures.” Shown here are data from three separate experiments that yielded similar results. The right panel shows data from cells treated with compound C (20 μm, 30 min) prior to ADP stimulation (50 μm, 5 min). This experiment was performed at the same time as the eNOS activity assay was being determined in cells from the same passage. Cell lysates were resolved by SDS-PAGE, and the immunoblot was probed with antibodies against phospho-AMPK and phospho-ACC. Equal loading was determined in immunoblots probed with antibodies against AMPK, ACC, and eNOS, as shown. B, BAEC were transfected with control or AMPK siRNA and treated with 50 μm ADP for 20 min. The left panel shows quantitation of eNOS activity data pooled from three different experiments that yielded equivalent results. siRNA-mediated knockdown of AMPK was determined by immunoblot analysis (right panel) of cells harvested at the time of the eNOS activity assay. C, BAEC were preincubated with the CaMKKβ inhibitor STO-609 (10 μm) for 30 min and then treated with ADP for 20 min. eNOS enzyme activity is shown on the left with each bar representing the mean value ± S.E. of five independent experiments with similar results. Similar cells were treated with STO-609 (10 μm, 30 min) and then stimulated with ADP (50 μm, 5 min) at the time of eNOS activity measurement. A representative immunoblot probed for phospho-AMPK, AMPK, phospho-ACC, ACC, and eNOS is shown on the right. D, cells were transfected with control or CaMKKβ siRNA, and eNOS activity was measured by quantitating the formation of [³H]citrulline from l-[³H]arginine after adding ADP (50 μm) for 20 min. Shown on the left are the pooled results analyzing data from three independent experiments. Specific CaMKKβ knockdown was confirmed with an immunoblot probed for CaMKKβ and eNOS (right panel). In all graphs, * indicates p < 0.05, ** indicates p < 0.01, and *** represents p < 0.001. p, phospho; NS, not significant.