Vanadate Is a Potent Activator of Endothelial Nitric-Oxide Synthase: Evidence for the Role of the Serine/Threonine Kinase Akt and the 90-kDa Heat Shock Protein
- Andreas Papapetropoulos,
- David Fulton1,
- Michelle I. Lin,
- Jason Fontana,
- Timothy J. McCabe2,
- Stefan Zoellner,
- Guillermo García-Cardeña3,
- Zongmin Zhou,
- Jean-Phillipe Gratton4 and
- William C. Sessa
- Department of Pharmacology and Vascular Cell Signaling and Therapeutics Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut (A.P., D.F., M.I.L., J.F., S.F., G.G.-C., J.-P.G., W.C.S.) and “George P. Livanos” Laboratory, Evangelismos Hospital, Critical Care Department and Pulmonary Services, University of Athens, Athens, Greece (A.P., Z.Z.)
- Address correspondence to:
Dr. Andreas Papapetropoulos, “George P. Livanos” Laboratory, Department of Critical Care and Pulmonary Services, University of Athens, Ploutarchou 3, Athens, Greece 10675. E-mail: apapapet{at}upatras.gr
Abstract
We investigated the molecular mechanisms of sodium vanadate (vanadate)-induced nitric oxide (NO) production. Exposure of bovine lung microvascular cells (BLMVEC) to vanadate increased the release of biologically active NO in endothelium/smooth muscle cocultures, as measured by the accumulation of its surrogate marker, cGMP. This release was sensitive to NO synthase (NOS) inhibition and was greater than that observed with ionomycin. Although calcium chelators (BAPTA, EGTA) inhibited basal and ionomycin-induced NO production, they failed to inhibit vanadate-induced NO release. Moreover, in the absence of calcium/calmodulin, cell lysates from vanadate-treated cells exhibited greater NOS activity compared with control cells. Vanadate activates the phosphoinositide3-kinase (PI3-K)/Akt pathway, which is known to increase endothelial NOS (eNOS) activity by direct phosphorylation of Ser-1179. Treatment of BLMVEC with vanadate resulted in phosphorylation of both Akt and endothelial NOS. In addition, wortmannin, a PI3-K inhibitor, blocked both the vanadate-induced phosphorylation of eNOS and the increase in cGMP accumulation. Similarly, adenovirus-mediated gene transfer of an activation deficient form of Akt (AA-Akt) blocked the release of NO brought about by vanadate. To further investigate the mechanism of action of vanadate, eNOS was immunoprecipitated and its association with proteins that alter eNOS activity was tested. Immunoblots demonstrated that the eNOS-caveolin interaction remained unaffected by vanadate, whereas vanadate promoted recruitment of the 90-kDa heat shock protein (hsp90) to eNOS. We conclude that vanadate causes NO release via a mechanism that involves Akt-induced eNOS phosphorylation and increased binding of the activator protein hsp90 to eNOS.
Footnotes
-
ABBREVIATIONS: NO, nitric oxide; eNOS, endothelial nitric-oxide synthase; NOS, nitric-oxide synthase; hsp90, 90-kDa heat shock protein; vanadate, sodium vanadate; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; AM, acetoxymethyl ester; Ab, antibody; IBMX, 3-isobutyl-1-methylxanthine; l-NAME, Nω-nitro-l-arginine methyl ester; BLMVEC, bovine lung microvascular endothelial cells; HBSS, Hanks' balanced salt solution; PAGE, polyacrylamide gel electrophoresis; RASM, rat aortic smooth muscle cells; β-gal, β-galactosidase; PI3-K, phosphatidylinositol-3-kinase.
-
This work was supported by National Institutes of Health grants R01-HL57665, HL61371, and HL64793 (to W.C.S.), T32-HL10183 (to D.F.) and grants from the Greek Secretariat of Research and Technology (PENED 2001/01EΔ67), the University of Athens (70/4/5918 to A.P.), and by the Thorax Foundation. J.P.G. is the recipient of a fellowship from the Canadian Institutes of Health Research.
-
A.P. and D.F. contributed equally to this study.
-
↵1 Present address: Vascular Biology Center, Medical College of Georgia, Augusta, Georgia.
-
↵2 Present address: Department of Research Technologies, Pharmaceutical Division, Bayer Corporation. West Haven, Connecticut.
-
↵3 Present address: Center for Excellence in Vascular Biology, Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts.
-
↵4 Present address: Laboratory of Endothelial Cell Biology, Institut de Recherches Cliniques de Montreal (IRCM), Montreal, Quebec, Canada.
-
- Received June 13, 2003.
- Accepted September 29, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
