FIGURE 6.

NO-dependent AMPK activation is guanylyl cyclase and calcium-dependent. A and B, HeLa S3 cells were pretreated with ODQ (20 μm, 30 min) (A) or BAPTA-AM (20 μm, 30 min) (B) prior to stimulation with SNP (50 μm, 1 h). C, HUVECs were stimulated with AICAR (2 mm, 2 h) with or without pretreatment with BAPTA (20 μm, 30 min). Cell lysates were subjected to Western blot analysis using antibodies against phospho-AMPK (Thr-172), AMPK-α, or β-actin. Representative blots and densitometric analysis are shown (mean ± S.E., n >= 3). D, SNP (50 μm) was added to HUVECs, and intracellular Ca²⁺ was measured as described under “Experimental Procedures.” E, HUVECs were stimulated with 8-Br-cGMP (1 mm) for 1 h, and cell lysates were subjected to Western blot analysis using antibodies against phospho-AMPK (Thr-172), AMPK-α, or β-actin. Representative blots are shown, n >= 3. *, p < 0.05 versus vehicle. F, effects of 8-bromo-GMP on intracellular Ca²⁺ in HUVEC. HUVECs were stimulated with SNP (50 μm) or 8-Br-cGMP (100 μm) for 10 min. n >3, *, p < 0.05 versus vehicle.