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.
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 nitric-oxide synthase (type I) (nNOS). The peptide inhibited formation of L-citrulline by eNOS and nNOS with IC(50) values of 10.6 +/- 0.4 microM and 7.1 +/- 0.6 microM, 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 IC(50) of 3.7 +/- 0.6 microM, but the cytochrome c reductase activity of the enzyme was much less susceptible to inhibition (IC(50) >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 heme 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.
Golser
R
R
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria.
Gorren
A C
AC
Leber
A
A
Andrew
P
P
Habisch
H J
HJ
Werner
E R
ER
Schmidt
K
K
Venema
R C
RC
Mayer
B
B
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002956:Citrulline / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D051381:Rats; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors
D010455:Peptides; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D011994:Recombinant Proteins; D002956:Citrulline; D019001:Nitric Oxide Synthase