Interaction of endothelial and neuronal nitric-oxide synthases with the bradykinin B2 receptor. Binding of an inhibitory peptide to the oxygenase domain blocks uncoupled NADPH oxidation

Regina Golser, Antonius C.F. Gorren, Andrea Leber, Penelope Andrew, Hans Jörg Habisch, Ernst R. Werner, Kurt Schmidt, Richard C Venema, Bernd Mayer

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Abstract

Endothelial nitric-oxide synthase (type III) (eNOS) was reported to form an inhibitory complex with the bradykinin receptor B2 (B2R) from which the enzyme is released in an active form upon receptor activation (Ju, H., Venema, V. J., Marrero, M. B., and Venema, R. C. (1998) J. Biol. Chem. 273, 24025-24029). Using a synthetic peptide derived from the known inhibitory sequence of the B2R (residues 310-329) we studied the interaction of the receptor with purified eNOS and neuronal nitricoxide synthase (type I) (nNOS). The peptide inhibited formation of L-citrulline by eNOS and nNOS with IC50 values of 10.6 ± 0.4 μM and 7.1 ± 0.6 μM, respectively. Inhibition was not due to an interference of the peptide with L-arginine or tetrahydrobiopterin binding. The NADPH oxidase activity of nNOS measured in the absence of L-arginine was inhibited by the peptide with an IC50 of 3.7 ± 0.6 μM, but the cytochrome c reductase activity of the enzyme was much less susceptible to inhibition (IC50 >0.1 mM). Steady-state absorbance spectra of nNOS recorded during uncoupled NADPH oxidation showed that the heme remained oxidized in the presence of the synthetic peptide consisting of amino acids 310-329 of the B2R, whereas the reduced oxyferrous heme complex was accumulated in its absence. These data suggest that binding of the B2R 310-329 peptide blocks flavin to heine electron transfer. Co- immunoprecipitation of B2R and nNOS from human embryonic kidney cells stably transfected with human nNOS suggests that the B2R may functionally interact with nNOS in vivo. This interaction of nNOS with the B2R may recruit the enzyme to allow for the effective coupling of bradykinin signaling to the nitric oxide pathway.

Original languageEnglish (US)
Pages (from-to)5291-5296
Number of pages6
JournalJournal of Biological Chemistry
Volume275
Issue number8
DOIs
StatePublished - Feb 25 2000

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Bradykinin B2 Receptors
Oxygenases
Nitric Oxide Synthase Type I
Nitric Oxide Synthase Type III
NADP
Oxidation
Peptides
Inhibitory Concentration 50
Heme
Arginine
Enzymes
Cytochrome Reductases
Glycogen Synthase
Citrulline
NADPH Oxidase
Bradykinin
Cytochromes c
Immunoprecipitation
Nitric Oxide
Electrons

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Interaction of endothelial and neuronal nitric-oxide synthases with the bradykinin B2 receptor. Binding of an inhibitory peptide to the oxygenase domain blocks uncoupled NADPH oxidation. / Golser, Regina; Gorren, Antonius C.F.; Leber, Andrea; Andrew, Penelope; Habisch, Hans Jörg; Werner, Ernst R.; Schmidt, Kurt; Venema, Richard C; Mayer, Bernd.

In: Journal of Biological Chemistry, Vol. 275, No. 8, 25.02.2000, p. 5291-5296.

Research output: Contribution to journalArticle

Golser, Regina ; Gorren, Antonius C.F. ; Leber, Andrea ; Andrew, Penelope ; Habisch, Hans Jörg ; Werner, Ernst R. ; Schmidt, Kurt ; Venema, Richard C ; Mayer, Bernd. / Interaction of endothelial and neuronal nitric-oxide synthases with the bradykinin B2 receptor. Binding of an inhibitory peptide to the oxygenase domain blocks uncoupled NADPH oxidation. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 8. pp. 5291-5296.
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AU - Golser, Regina

AU - Gorren, Antonius C.F.

AU - Leber, Andrea

AU - Andrew, Penelope

AU - Habisch, Hans Jörg

AU - Werner, Ernst R.

AU - Schmidt, Kurt

AU - Venema, Richard C

AU - Mayer, Bernd

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