Endothelial nitric oxide synthase interactions with G-protein-coupled receptors

The endothelial nitric oxide synthase (eNOS) is activated in response to st imulation of endothelial cells by a number of vasoactive substances includi ng, bradykinin (BK), angiotensin II (Ang II), endothelin-1 (ET-1) and ATP. In the present study we have used in vitro activity assays of purified eNOS and in vitro binding assays with glutathione S-transferase fusion proteins to show that the capacity to bind and inhibit eNOS is a common feature of membrane-proximal regions of intracellular domain 4 of the BK B2, the Ang I I AT1 and the ET-1 ETB receptors, but not of the ATP P2Y2 receptor. Phospho rylation of serine or tyrosine residues in the eNOS-interacting region of t he B2 receptor results in a loss of eNOS inhibition due to a decrease in th e binding affinity of the receptor domain for the eNOS enzyme. Furthermore, the B2 receptor is transiently phosphorylated on tyrosine residues in cult ured endothelial cells in response to BK stimulation. Phosphorylation occur s during the time in which eNOS transiently dissociates from the receptor a ccompanied by a transient increase in nitric oxide production. Taken togeth er, these data support the hypotheses that eNOS is regulated in endothelial cells by reversible and inhibitory interactions with G-protein-coupled rec eptors and that these interactions can be modulated by receptor phosphoryla tion.