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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:1571.)
© 2001 American Heart Association, Inc.

Vascular Biology

Induction of Endothelial NO Synthase by Hydrogen Peroxide via a Ca²⁺/Calmodulin-Dependent Protein Kinase II/Janus Kinase 2–Dependent Pathway

Hua Cai; Michael E. Davis; Grant R. Drummond; David G. Harrison

From the Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Ga.

Correspondence to David G. Harrison, MD, Division of Cardiology, Emory University School of Medicine, Suite 319, Woodruff Memorial Research Building, 1639 Pierce Dr, Atlanta, GA 30322. E-mail dharr02{at}emory.edu

Abstract—— We have recently demonstrated that hydrogen peroxide (H₂O₂) is an extremely potent stimulus of endothelial NO synthase (eNOS) gene expression. The present study was designed to identify the signaling mechanisms mediating this response. Induction of eNOS expression by H₂O₂ was found to be Ca²⁺ dependent, inasmuch as it was blocked by BAPTA-AM. Further studies have indicated that Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) plays a critical role in mediating this response. Immunocytochemical staining with an anti-CaM kinase II antibody confirmed the expression of CaM kinase II in cultured bovine aortic endothelial cells. H₂O₂ induced autophosphorylation of CaM kinase II and increased the activity of the enzyme, as assessed by an in-gel kinase assay. A specific inhibitor for CaM kinase II, KN93, and a calmodulin antagonist, W-7, attenuated eNOS induction by H₂O₂. Further studies have indicated that janus kinase 2 is important in mediating increased eNOS expression in response to H₂O₂ and likely is downstream from CaM kinase II. In conclusion, these data provide the first evidence that CaM kinase II plays a critical role in endothelial redox signaling. Regulation of eNOS via this pathway may represent an important vascular adaptation to oxidant stress.

Key Words: Ca²⁺/calmodulin-dependent protein kinase II • janus kinase 2 • endothelial NO synthase • hydrogen peroxide • gene regulation

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