Nitric oxide (NO) is a ubiquitous intercellular messenger molecule synthesized from the amino acid L-arginine by NO synthases in diverse cells and tissues. NO is synthesized in vascular endothelial cells and appears to play an important role in the control of blood pressure and platelet aggregation. A detailed understanding of the regulation of NO synthesis by endothelial cells has been hampered by the lack of molecular clones for endothelial NO synthase; the isolation and characterization ofsuch clones is reported herein. The constitutive NO synthases present in endothelial cells and in brain share common biochemical and pharmacologic features. We purified NO synthase from bovine brain and determined the amino acid sequence of several tryptic peptides. The sequence ofthe bovine brain peptides is nearly identical to the deduced amino acid sequence previously determined for the rat brain NO synthase.These sequence data were utilized to design PCR-generated NO synthase cDNA probes, which were used to isolate clones encoding NO synthase from a bovine aortic endothelial cell (BAEC) cDNA library. A full-length NO synthase cDNA clone was isolated, representing a protein of 1205 amino acids with a molecular mass of 133 kDa; transfection of this clone in a heterologous expression system demonstrated the expected enzymatic activity. The deduced amino acid sequence of the BAEC NO synthase cDNA differs at numerous residues from the sequence determined for the purified bovine brain protein and shows 50-60% sequence identity with recently isolated molecular clones for murine macrophage and rat brain NO synthase isoforms. Bovine genomic Southern blots probed with bovine brain and BAEC NO synthase cDNA probes identify distinct bands, indicating that these cDNAs are the products of different genes. Prolonged treatment of BAECs with the cytokine tumor necrosis factor a, which we have previously shown to result in a marked increase in NO synthase activity, is associated with a decrease in the abundance of the 4.8-kilobase BAEC NO synthase transcript. The increase in BAEC NO synthase activity induced by tumor necrosis factor a is thus likely to involve posttranscriptional mechanisms or the induction of a distinct endothelial NO synthase isoform.The vascular endothelium forms the lining of the circulatory system in all vertebrates and comprises a dynamic monocellular interface between the surrounding vascular cells and the soluble and cellular components of the blood. In response to diverse stimuli, the vascular endothelium synthesizes and secretes vasoactive agents that control vascular tone (1, 2). In 1980, a labile endothelium-derived compound was discovered to play an important role in relaxing vascular smooth muscle and was termed endothelium-derived relaxing factor (EDRF) (3). EDRF was subsequently shown to be equivalent to nitric oxide (NO) or a NO-containing compound (for review, see ref. 4). NO synthesis is being discovered in a striking diversity of tissues and cultured cells (5).Catalysis by NO synthase, in all e...

In the vascular endothelium, diverse cell surface receptors are coupled to the Ca2+/calmodulin-dependent activation of nitric oxide (NO) synthase. We now report that, in intact cultured endothelial cells, several drugs and agonists are associated with increased serine phosphorylation of the endothelial NO synthase. We biosyntheticaily labeled bovine aortic endothelial cells with [32PJorthophosphoric acid, exposed the cells to various drugs and hormones, and then immunoprecipitated the enzyme from cell extracts using a highly specific anti-peptide antibody. The marked endothelial NO synthase phosphorylation induced by bradykinin is maximal only after 5 min of agonist exposure and is stable for at least 20 min. Basal and agonist-induced phosphorylation of the NO synthase in endothelial cells is completely inhibited by the calmodulin antagonist compound W-7. We prepared subcel- nine-labeled NO synthase is now found in the cytosolic fraction, associated with a marked increase in the level of cytosolic enzyme phosphorylation. We propose that agonist-induced phosphorylation of NO synthase is associated with translocation of the enzyme from membrane to cytosol and may thereby regulate the biological effects of endothelial NO synthesis in situ.Nitric oxide (NO) is a ubiquitous intercellular signaling molecule and is synthesized in diverse mammalian tissues by a family of related NO synthase enzymes. In all tissues thus far characterized, the NO synthase isoforms share a common overall catalytic scheme for the oxidation of L-arginine to form NO plus L-citrulline and appear to share similar cofactor requirements (reviewed in refs. 1-3). The different tissuespecific NO synthase isoforms subserve disparate biological functions in diverse mammalian cells and appear to be encoded by distinct genes (4).The endothelial NO synthase plays an important role in the control of blood pressure and platelet aggregation (5). In bovine endothelial cells, the NO synthase is transiently activated by increases in intracellular calcium induced by the activation of diverse G-protein-coupled cell surface receptors, including bradykinin. Our analysis of the primary structure of the endothelial NO synthase cDNA identified consensus sequences for posttranslational modifications of the enzyme, including N-terminal myristoylation as well as phosphorylation (4). We have recently shown that the endothelialThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.NO synthase undergoes N-terminal myristoylation, and this covalent modification is essential for the association of the enzyme with the particulate subcellular fraction (6). Some myristoylated proteins implicated in intracellular signaling pathways have been shown to regulated by reversible phosphorylation, and phosphorylation further appears to modulate their subcellular localization (reviewed in refs. 7 and 8).The deduced primary stru...

The endothelial isoform of nitric-oxide synthase (eNOS), a key determinant of vascular homeostasis, is a calcium/calmodulindependent phosphoprotein regulated by diverse cell surface receptors. Vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) stimulate eNOS activity through Akt/phosphoinositide 3-kinase and calcium-dependent pathways. AMP-activated protein kinase (AMPK) also activates eNOS in endothelial cells; however, the molecular mechanisms linking agonist-mediated AMPK regulation with eNOS activation remain incompletely understood. We studied the role of AMPK in VEGF-and S1P-mediated eNOS activation and found that both agonists led to a striking increase in AMPK phosphorylation in pathways involving the calcium/calmodulin-dependent protein kinase kinase ␤. Treatment with tyrosine kinase inhibitors or the phosphoinositide 3-kinase inhibitor wortmannin demonstrated differential effects of VEGF versus S1P. Small interfering RNA (siRNA)-mediated knockdown of AMPK␣1 or Akt1 impaired the stimulatory effects of both VEGF and S1P on eNOS activation. AMPK␣1 knockdown impaired agonist-mediated Akt phosphorylation, whereas Akt1 knockdown did not affect AMPK activation, thus suggesting that AMPK lies upstream of Akt in the pathway leading from receptor activation to eNOS stimulation. Importantly, we found that siRNA-mediated knockdown of AMPK␣1 abrogates agonist-mediated activation of the small GTPase Rac1. Conversely, siRNA-mediated knockdown of Rac1 decreased the agonist-mediated phosphorylation of AMPK substrates without affecting that of AMPK, implicating Rac1 as a molecular link between AMPK and Akt in agonist-mediated eNOS activation. Finally, siRNA-mediated knockdown of caveolin-1 significantly enhanced AMPK phosphorylation, suggesting that AMPK is negatively regulated by caveolin-1. Taken together, these results suggest that VEGF and S1P differentially regulate AMPK and establish a central role for an agonist-modulated AMPK 3 Rac1 3 Akt axis in the control of eNOS in endothelial cells.