The endothelial nitric oxide synthase (eNOS), the expression of which is regulated by a range of transcriptional and posttranscriptional mechanisms, generates nitric oxide (NO) in response to a number of stimuli. The physiologically most important determinants for the continuous generation of NO and thus the regulation of local blood flow are fluid shear stress and pulsatile stretch. Although eNOS activity is coupled to changes in endothelial cell Ca²⁺levels, an increase in Ca²⁺alone is not sufficient to affect enzyme activity because the binding of calmodulin (CaM) and the flow of electrons from the reductase to the oxygenase domain of the enzyme is dependent on protein phosphorylation and dephosphorylation. Two amino acids seem to be particularly important in regulating eNOS activity and these are a serine residue in the reductase domain (Ser¹¹⁷⁷) and a threonine residue (Thr⁴⁹⁵) located within the CaM-binding domain. Simultaneous alterations in the phosphorylation of Ser¹¹⁷⁷and Thr⁴⁹⁵in response to a variety of stimuli are regulated by a number of kinases and phosphatases that continuously associate with and dissociate from the eNOS signaling complex. eNOS associated proteins, such as caveolin, heat shock protein 90, eNOS interacting protein, and possibly also motor proteins provide the scaffold for the formation of the protein complex as well as its intracellular localization.

—The activity of the endothelial nitric oxide synthase (eNOS) can be regulated independently of an increase in Ca ²⁺ by the phosphorylation of Ser ¹¹⁷⁷ but results only in a low nitric oxide (NO) output. In the present study, we assessed whether the agonist-induced (Ca ²⁺ -dependent, high-output) activation of eNOS is associated with changes in the phosphorylation of Thr ⁴⁹⁵ in the calmodulin (CaM)-binding domain. eNOS Thr ⁴⁹⁵ was constitutively phosphorylated in porcine aortic endothelial cells and was rapidly dephosphorylated after bradykinin stimulation. In the same cells, bradykinin enhanced the phosphorylation of Ser ¹¹⁷⁷ , which was maximal after 5 minutes, and abolished by the CaM-dependent kinase II (CaMKII) inhibitor KN-93. Bradykinin also enhanced the association of CaMKII with eNOS. Phosphorylation of Thr ⁴⁹⁵ was attenuated by the protein kinase C (PKC) inhibitor Ro 31-8220 and after PKC downregulation using phorbol 12-myristate 13-acetate. The agonist-induced dephosphorylation of Thr ⁴⁹⁵ was completely Ca ²⁺ -dependent and inhibited by the PP1 inhibitor calyculin A. Little CaM was bound to eNOS immunoprecipitated from unstimulated cells, but the agonist-induced dephosphorylation of Thr ⁴⁹⁵ enhanced the association of CaM. Mutation of Thr ⁴⁹⁵ to alanine increased CaM binding to eNOS in the absence of cell stimulation, whereas the corresponding Asp ⁴⁹⁵ mutant bound almost no CaM. Accordingly, NO production by the Ala ⁴⁹⁵ mutant was more sensitive to Ca ²⁺ /CaM than the aspartate mutant. These results suggest that the dual phosphorylation of Ser ¹¹⁷⁷ and Thr ⁴⁹⁵ determines the activity of eNOS in agonist-stimulated endothelial cells. Moreover, the dephosphorylation of Thr ⁴⁹⁵ by PP1 precedes the phosphorylation of Ser ¹¹⁷⁷ by CaMKII. The full text of this article is available at http://www.circresaha.org.

Aging is considered to be the major risk factor for the development of atherosclerosis and, therefore, for coronary artery disease. Apart from age-associated remodeling of the vascular wall, which includes luminal enlargement, intimal and medial thickening, and increased vascular stiffness, endothelial function declines with age. This is most obvious from the attenuation of endothelium-dependent dilator responses, which is a consequence of the alteration in the expression and/or activity of the endothelial NO synthase, upregulation of the inducible NO synthase, and increased formation of reactive oxygen species. Aging is also associated with a reduction in the regenerative capacity of the endothelium and endothelial senescence, which is characterized by an increased rate of endothelial cell apoptosis.