Trans-forming endothelial nitric oxide synthase in hypertension: more than meets the eye.

Endothelium exerts diverse effects within the vessel wall and on nearby target cells.1 Through these effects, endothelial cells inhibit vascular tone and vascular growth and protect against thrombosis and progression of atherosclerosis, as well as increases in blood pressure.1,2 Much of the influence of endothelium on other cells is mediated by intercellular signals carried by diffusible factors. Of these endothelium-derived signals, NO produced by the endothelial form of NO synthase (eNOS) plays a particularly prominent role in both large and small blood vessels in diverse experimental models and normal humans.1,3–5 Impairment of eNOSdriven signaling underlies vascular dysfunction in a broad spectrum of diseases and is a risk factor for cardiovascular events and stroke.1,2 The function of eNOS is regulated at many levels, including expression of mRNA and protein, posttranslational modifications, subcellular trafficking, substrate and cofactor availability, and associations with other protein regulators, as well as interactions of NO with superoxide.1,6 A key set of mechanisms that regulate activity of eNOS involves reversible phosphorylation of specific amino acid residues (mainly serine, threonine, and tyrosine residues), resulting in increases or decreases in eNOS activity. Kinase-dependent phosphorylation and phosphatase-mediated dephosphorylation of these residues are common elements of cell signaling in general. Within the eNOS molecule, there are multiple phosphorylation sites, including Ser 116 (Ser 114 and 116 in human and bovine eNOS, respectively) in the oxygenase domain of the enzyme. Phosphorylation of Ser 116 inhibits activity of eNOS, whereas dephosphorylation of the same residue increases activity (Figure).7 A major posttranslational modification of eNOS involves proline-directed protein kinases that recognize and phosphorylate a Ser/Thr-Pro motif (Figure).8 Proline has unique stereochemistry where it can exist in cis or trans conformations. The conversion between these 2 conformations is catalyzed by peptidyl-prolyl cis/trans isomerases, which may act as a “molecular switch,” where enzymatic activity is conformation specific (ie, trans on, cis off). While in the “on” state, local structural changes may occur and allow other posttranslational modifications to further affect enzyme activity. Pin1 is a prolyl-peptidyl cis/trans isomerase that regulates the dephosphorylation of proteins through isomerization of peptide bonds that link phosphoserine or phosphothreonine to proline.8 The results of this interaction are phosphorylation-dependent conformation changes. In relation to eNOS specifically, a very recent study provided evidence that Pin1 binds with and regulates activity of eNOS through site-specific phosphorylation of Ser 116,7 resulting in changes to both basal and agonist-induced NO production (Figure). These conclusions were based on pharmacological and molecular approaches using cultured endothelium and isolated aorta. In this issue of Hypertension, Chiasson et al9 interrogate this concept further, while adding new approaches. Using a series of complementary methods, they studied endothelium and aorta in culture and performed experiments designed to evaluate the impact of Pin1 in vivo. There were several major findings. First, they confirmed the interaction between Pin1 and eNOS and then found that knockdown (using RNA interference) or pharmacological inhibition (using juglone [5-hydroxyl-1,4-napthoquinone]) of Pin1 increased eNOS Ser 116 phosphorylation, reduced NO production, and decreased endothelium-dependent relaxation to acetylcholine. These findings are consistent with those of Ruan et al7 that highlight the complexity of eNOS regulation and support the concept that Pin1 affects basal and agonist-induced eNOS activity (Figure). Second, Chiasson et al9 found that systemic treatment of normal mice with juglone for 2 weeks had effects on eNOS activity in aorta that were similar to those seen in analogous culture experiments. Importantly, juglone treatment also caused a progressively developing hypertension. To examine this concept further, blood vessels from mice with genetic deletion of Pin1 were studied. Aorta from Pin1 knockout mice (homozygous knockouts) had increased Ser 116 phosphorylation, reduced NO production, and endothelial dysfunction. Pin1-deficient mice were also hypertensive, exhibiting increases in arterial pressure that were similar in magnitude to those seen after systemic treatment with juglone. Because juglone may exert effects unrelated to Pin1, the observations that genetic knockout of Pin1 and systemic treatment with juglone produced similar effects on aortic The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Departments of Pharmacology and Internal Medicine, Division of Cardiovascular Diseases, Cardiovascular Center, University of Iowa Carver College of Medicine, Iowa City, IA. Correspondence to Frank M. Faraci, Department of Internal Medicine, Division of Cardiovascular Diseases, E315-GH, University of Iowa, Carver College of Medicine, Iowa City, IA 52242-1081. E-mail [email protected] (Hypertension. 2011;58:359-360.) © 2011 American Heart Association, Inc.