Endothelial Nitric-oxide Synthase (Type III) Is Activated and Becomes Calcium Independent upon Phosphorylation by Cyclic Nucleotide-dependent Protein Kinases

Butt, Elke; Bernhardt, Manfred; Smolenski, Albert; Kotsonis, Peter; Fröhlich, L.; Sickmann, Albert; Meyer, Helmut E.; Lohmann, Suzanne M.; Schmidt, Harald

doi:10.1074/jbc.275.7.5179

Cited by 271 publications

(209 citation statements)

References 54 publications

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“…Of the known phosphorylation sites on eNOS responsible for its activation, serine 1179 has been characterized most extensively (Chen et al, 1999;Fulton et al, 2002). Serine 1179 phosphorylation by PKA leads to activation of eNOS (Butt et al, 2000). We hypothesize that the mechanisms by which NO-cGMP-PKA pathway in the endothelium mediates vascular smooth muscle relaxation involve NO production through eNOS phosphorylation by PKA.…”

Section: Discussionmentioning

confidence: 95%

Dynamic Association of Nitric Oxide Downstream Signaling Molecules with Endothelial Caveolin-1 in Rat Aorta

Linder¹,

McCluskey²,

Cole³

et al. 2005

J Pharmacol Exp Ther

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Classically, nitric oxide (NO) formed by endothelial NO synthase (eNOS) freely diffuses from its generation site to smooth muscle cells where it activates soluble guanylyl cyclase (sGC), producing cGMP. Subsequently, cGMP activates both cGMPand cAMP-dependent protein kinases [cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA), respectively], leading to smooth muscle relaxation. In endothelial cells, eNOS has been localized to caveolae, small invaginations of the plasma membrane rich in cholesterol. Membrane cholesterol depletion impairs acetylcholine (ACh)-induced relaxation due to alteration in caveolar structure. Given the nature of NO to be more soluble in a hydrophobic environment than in water, and assuming that colocalization of components in a signal transduction cascade seems to be a critical determinant of signaling efficiency by eNOS activation, we hypothesize that sGC, PKA, and PKG activation may occur at the plasma membrane caveolae. In endothelium-intact rat aortic rings, the relaxation induced by ACh, by the sGC activator 3-(5Ј-hydroxymethyl-2Јfuryl)-1-benzyl indazole (YC-1), and by 8-bromocGMP was impaired in the presence of methyl-␤-cyclodextrin, a drug that disassembles caveolae by sequestering cholesterol from the membrane. sGC, PKG, and PKA were colocalized with caveolin-1 in aortic endothelium, and this colocalization was abolished by methyl-␤-cyclodextrin. Methyl-␤-cyclodextrin efficiently disassembled caveolae in endothelium. In summary, our results provide evidence of compartmentalization of sGC, PKG, and PKA in endothelial caveolae contributing to NO signaling cascade, giving new insights by which the endothelium mediates vascular smooth muscle relaxation.

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Section: Discussionmentioning

confidence: 95%

Dynamic Association of Nitric Oxide Downstream Signaling Molecules with Endothelial Caveolin-1 in Rat Aorta

Linder¹,

McCluskey²,

Cole³

et al. 2005

J Pharmacol Exp Ther

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“…Pretreatment of B-TECs with the membrane-permeable PKA inhibitory peptide myristoylated PKI [14][15][16][17][18][19][20][21][22] (10 minutes, 20 μmol/L) completely abolished AA-induced calcium entry in 99% of the cells tested (n = 84; Fig. 1; Table 1), suggesting a critical role of endogenous PKA in the activation of calcium signals by AA.…”

Section: Pka Is Required For Aa-induced Calcium Entry In B-tecsmentioning

confidence: 99%

“…PKA regulates eNOS activity through serine phosphorylation, and it has been recently reported that forskolin, a widely used adenylyl cyclase activator, is able to trigger proangiogenic effects through the PI3K, Akt, and eNOS pathways in human umbilical vascular ECs (21,22). In addition, it is worth noting that a number of different endothelial calcium-permeable channels are substrates for PKA phosphorylation, including some members of the transient receptor potential (TRP) superfamily of proteins.…”

Section: Introductionmentioning

confidence: 99%

Multiple Roles of Protein Kinase A in Arachidonic Acid–Mediated Ca2+ Entry and Tumor-Derived Human Endothelial Cell Migration

Fiorio

Genova

Pupo

et al. 2010

Molecular Cancer Research

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We recently showed that arachidonic acid (AA) triggers calcium signals in endothelial cells derived from human breast carcinoma (B-TEC). In particular, AA-dependent Ca 2+ entry is involved in the early steps of tumor angiogenesis in vitro. Here, we investigated the multiple roles of the nitric oxide (NO) and cyclic AMP/protein kinase A (PKA) pathways in AA-mediated Ca 2+ signaling in the same cells. B-TEC stimulation with 5 μmol/L AA resulted in endothelial NO synthase (NOS) phosphorylation at Ser 1177 , and NO release was measured with the fluorescent NO-sensitive probe DAR4M-AM. PKA inhibition by the use of the membrane-permeable PKA inhibitory peptide myristoylated PKI 14-22 completely prevented both AA-and NOinduced calcium entry and abolished B-TEC migration promoted by AA. AA-dependent calcium entry and cell migration were significantly affected by both the NOS inhibitor N G -nitro-L-arginine methyl ester and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, suggesting that NO release is functionally involved in the signaling dependent on AA. Moreover, pretreatment with carboxyamidotriazole, an antiangiogenic compound that interferes with agonist-activated calcium entry, prevented AA-dependent B-TEC motility. Interestingly, even in the absence of AA, enhancement of the cyclic AMP/PKA pathway with the adenylyl cyclase activator forskolin evoked a calcium entry dependent on NOS recruitment and NO release. The functional relevance of AA-induced calcium entry could be restricted to tumor-derived endothelial cells (EC) because AA evoked a smaller calcium entry in normal human microvascular ECs compared with B-TECs, and even more importantly, it was unable to promote cell motility in wound healing assay. This evidence opens an intriguing opportunity for differential pharmacologic treatment between normal and tumor-derived human ECs.

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“…The molecular mechanisms are mainly calcium-independent [20] and involve posttranslational modification by phosphorylation of eNOS at regulatory sites [21], messenger RNA (mRNA) stabilization [22], e.g., through heat shock protein 90 (hsp-90), and translocation [23]. NO produced in response to shear stress triggers vascular smooth muscle relaxation, inhibition of apoptosis [24], and inhibition of thrombocyte or monocyte adhesion [25].…”

Section: Introductionmentioning

confidence: 99%

Endothelial dysfunction in cardiovascular disease and Flammer syndrome—similarities and differences

et al. 2017

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The endothelium has increasingly been recognized as a smart barrier and a key regulator of blood flow in microand macrovascular beds. Endothelial dysfunction marks a stage of atherosclerosis and is an important prognostic marker for cardiovascular disease. Yet, some people who tend to be slim and physically active and with rather low blood pressure show a propensity to respond to certain stimuli such as emotional stress with endothelial-mediated vascular dysregulation (Flammer syndrome). This leads to characteristic vascular symptoms such as cold hands but also a risk for vascularmediated diseases such as normal-tension glaucoma. It is the aim of this review to delineate the differences between Flammer syndrome and its Bcounterpart^endothelial dysfunction in the context of cardiovascular diseases.

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Endothelial Nitric-oxide Synthase (Type III) Is Activated and Becomes Calcium Independent upon Phosphorylation by Cyclic Nucleotide-dependent Protein Kinases

Abstract: Endothelial nitric-oxide synthase (NOS-III

Cited by 271 publications

References 54 publications

Dynamic Association of Nitric Oxide Downstream Signaling Molecules with Endothelial Caveolin-1 in Rat Aorta

Dynamic Association of Nitric Oxide Downstream Signaling Molecules with Endothelial Caveolin-1 in Rat Aorta

Multiple Roles of Protein Kinase A in Arachidonic Acid–Mediated Ca2+ Entry and Tumor-Derived Human Endothelial Cell Migration

Endothelial dysfunction in cardiovascular disease and Flammer syndrome—similarities and differences

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