Role of heat shock protein 90 in bradykinin-stimulated endothelial nitric oxide release

M. Brennan Harris, Hong Ju, Virginia J. Venema, Michele Blackstone, Richard C. Venema

Research output: Contribution to journalArticle

55 Citations (Scopus)

Abstract

Previously we described ENAP-1, a 90-kDa protein that is tyrosine-phosphorylated in endothelial cells in response to bradykinin (BK) stimulation and is associated with endothelial nitric oxide synthase (eNOS). Subsequently, other investigators demonstrated that eNOS interacts with heat shock protein 90 (Hsp90) following stimulation of endothelial cells with vascular endothelial growth factor (VEGF), histamine, or fluid shear stress. Therefore, we tested the hypotheses that ENAP-1 and Hsp90 are the same protein and that BK activation of eNOS is dependent on Hsp90. Immunoblotting of immunoprecipitated Hsp90 with anti-phosphotyrosine antibody shows that Hsp90 is tyrosine-phosphorylated in response to BK stimulation of bovine aortic endothelial cells (BAECs). Coimmunoprecipitation of Hsp90 with anti-eNOS antibody reveals a Hsp90-eNOS complex in endothelial cells under basal conditions that is increased following BK stimulation. Taken together with the tyrosine phosphorylation data, these data suggest that ENAP-1 is Hsp90. BK-stimulated nitric oxide (NO) release is completely blocked by pretreatment with geldanamycin, a specific inhibitor of Hsp90, illustrating the importance of the Hsp90-eNOS interaction. In vitro binding assays with Hsp90-glutathione-S-transferase fusion proteins show direct binding of eNOS with the middle domain (residues 259-615) of Hsp90.

Original languageEnglish (US)
Pages (from-to)165-170
Number of pages6
JournalGeneral Pharmacology: The Vascular System
Volume35
Issue number3
DOIs
StatePublished - Dec 1 2000

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HSP90 Heat-Shock Proteins
Bradykinin
Nitric Oxide
Nitric Oxide Synthase Type III
Endothelial Cells
Tyrosine
Phosphotyrosine
Proteins
Glutathione Transferase
Immunoblotting
Histamine
Vascular Endothelial Growth Factor A
Anti-Idiotypic Antibodies

Keywords

  • Bradykinin
  • Geldanamycin
  • Hsp90
  • Nitric oxide
  • Tyrosine phosphorylation
  • eNOS

ASJC Scopus subject areas

  • Pharmacology

Cite this

Role of heat shock protein 90 in bradykinin-stimulated endothelial nitric oxide release. / Harris, M. Brennan; Ju, Hong; Venema, Virginia J.; Blackstone, Michele; Venema, Richard C.

In: General Pharmacology: The Vascular System, Vol. 35, No. 3, 01.12.2000, p. 165-170.

Research output: Contribution to journalArticle

Harris, M. Brennan ; Ju, Hong ; Venema, Virginia J. ; Blackstone, Michele ; Venema, Richard C. / Role of heat shock protein 90 in bradykinin-stimulated endothelial nitric oxide release. In: General Pharmacology: The Vascular System. 2000 ; Vol. 35, No. 3. pp. 165-170.
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