Fig. 8.

Model for eNOS regulation of CaMKKβ/AMPK pathway via H₂O₂. This figure presents a plausible if speculative model for eNOS modulation of CaMKKβ/AMPK via H₂O₂ in endothelial cells. The present studies have shown that H₂O₂ promotes the phosphorylation of AMPK in endothelial cells (Fig. 1); the response is blocked by the CaMKKβ inhibitor STO-609 (Fig. 2) or by siRNA-mediated CaMKKβ knockdown (Fig. 3). Conversely, the NOS inhibitor L-NAME potentiates AMPK phosphorylation (Fig. 2), as does siRNA-mediated eNOS knockdown (Fig. 4); both of these effects are inhibited by CaMKKβ inhibition (Fig. 4). siRNA-mediated eNOS knockdown or enzyme inhibition with L-NAME leads to a marked increase in intracellular H₂O₂ generation (Fig. 5); the increase in intracellular H₂O₂ generation is blocked by PEG-catalase, which also suppresses the increase in AMPK phosphorylation. Since previous work have shown that eNOS modulates mitochondrial function, we propose that suppression of the eNOS-NO pathway (by siRNA or enzyme inhibition) enhances H₂O₂ production and thereby leads to CaMKKβ -dependent phosphorylation of AMPK. We propose that H₂O₂ directly or indirectly activates CaMKKβ. The phosphorylated AMPK in turn phosphorylates and activates eNOS, representing a feedback mechanism controlling this pathway.