Mechanism of Purinergic Activation of Endothelial Nitric Oxide Synthase in Endothelial Cells

Overview

Journal Circulation

Specialty Cardiology & Vascular Diseases

Date 2009 Feb 4

PMID 19188511

Citations 47

Authors

Cleide Goncalves da Silva

Anke Specht

Barbara Wegiel

Christiane Ferran

Elzbieta Kaczmarek

Affiliations

Soon will be listed here.

Abstract

Background: Decreased endothelial nitric oxide (NO) synthase (eNOS) activity and NO production are critical contributors to the endothelial dysfunction and vascular complications observed in many diseases, including diabetes mellitus. Extracellular nucleotides activate eNOS and increase NO generation; however, the mechanism of this observation is not fully clarified.

Methods And Results: To elucidate the signaling pathway(s) leading to nucleotide-mediated eNOS phosphorylation at Ser-1177, human umbilical vein endothelial cells were treated with several nucleotides, including ATP, UTP, and ADP, in the presence or absence of selective inhibitors. These experiments identified P2Y1, P2Y2, and possibly P2Y4 as the purinergic receptors involved in eNOS phosphorylation and demonstrated that this process was adenosine independent. Nucleotide-induced eNOS phosphorylation and activity were inhibited by BAPTA-AM (an intracellular free calcium chelator), rottlerin (a protein kinase Cdelta inhibitor), and protein kinase Cdelta siRNA. In contrast, blockade of AMP-activated protein kinase, calcium/calmodulin-dependent kinase II, calcium/calmodulin-dependent kinase kinase, serine/threonine protein kinase B, protein kinase A, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase did not affect nucleotide-mediated eNOS phosphorylation.

Conclusions: The present study indicates that extracellular nucleotide-mediated eNOS phosphorylation is calcium and protein kinase Cdelta dependent. This newly identified signaling pathway opens new therapeutic avenues for the treatment of endothelial dysfunction.

Citing Articles

Impact of Estrogen on Purinergic Signaling in Microvascular Disease.

Cassavaugh J, Longhi M, Robson S Int J Mol Sci. 2025; 26(5).

PMID: 40076726 PMC: 11900469. DOI: 10.3390/ijms26052105.

Extracellular purines in lung endothelial permeability and pulmonary diseases.

Gerasimovskaya E, Patil R, Davies A, Maloney M, Simon L, Mohamed B Front Physiol. 2024; 15():1450673.

PMID: 39234309 PMC: 11372795. DOI: 10.3389/fphys.2024.1450673.

Total Arterial Revascularization in Diabetic Patients Undergoing Coronary Artery Bypass Graft Surgery: A Systematic Review and Meta-Analysis.

Liao G, Liu T, Li Y, Bai L, Ye Y, Chen X Rev Cardiovasc Med. 2024; 24(6):183.

PMID: 39077537 PMC: 11264116. DOI: 10.31083/j.rcm2406183.

Indole-3-Propionic Acid, a Gut Microbiota-Derived Tryptophan Metabolite, Promotes Endothelial Dysfunction Impairing Purinergic-Induced Nitric Oxide Release in Endothelial Cells.

Geddo F, Antoniotti S, Gallo M, Querio G Int J Mol Sci. 2024; 25(6).

PMID: 38542360 PMC: 10970397. DOI: 10.3390/ijms25063389.

Red Blood Cell Adenylate Energetics Is Related to Endothelial and Microvascular Function in Long COVID.

Romanowska-Kocejko M, Jedrzejewska A, Braczko A, Stawarska K, Krol O, Franczak M Biomedicines. 2024; 12(3).

PMID: 38540167 PMC: 10968064. DOI: 10.3390/biomedicines12030554.

References

Michel J, Feron O, Sacks D, Michel T . Reciprocal regulation of endothelial nitric-oxide synthase by Ca2+-calmodulin and caveolin. J Biol Chem. 1997; 272(25):15583-6. DOI: 10.1074/jbc.272.25.15583. View

Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher A . Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999; 399(6736):601-5. DOI: 10.1038/21224. View

Matsubara M, Hayashi N, Jing T, Titani K . Regulation of endothelial nitric oxide synthase by protein kinase C. J Biochem. 2003; 133(6):773-81. DOI: 10.1093/jb/mvg099. View

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

Motley E, Eguchi K, Patterson M, Palmer P, Suzuki H, Eguchi S . Mechanism of endothelial nitric oxide synthase phosphorylation and activation by thrombin. Hypertension. 2007; 49(3):577-83. DOI: 10.1161/01.HYP.0000255954.80025.34. View

Stahmann N, Woods A, Carling D, Heller R . Thrombin activates AMP-activated protein kinase in endothelial cells via a pathway involving Ca2+/calmodulin-dependent protein kinase kinase beta. Mol Cell Biol. 2006; 26(16):5933-45. PMC: 1592798. DOI: 10.1128/MCB.00383-06. View

Erb L, Liao Z, Seye C, Weisman G . P2 receptors: intracellular signaling. Pflugers Arch. 2006; 452(5):552-62. DOI: 10.1007/s00424-006-0069-2. View

Fulton D, Gratton J, Sessa W . Post-translational control of endothelial nitric oxide synthase: why isn't calcium/calmodulin enough?. J Pharmacol Exp Ther. 2001; 299(3):818-24. View

Sessa W . Regulation of endothelial derived nitric oxide in health and disease. Mem Inst Oswaldo Cruz. 2005; 100 Suppl 1:15-8. DOI: 10.1590/s0074-02762005000900004. View

10.

Wang L, Karlsson L, Moses S, Hultgardh-Nilsson A, Andersson M, Borna C . P2 receptor expression profiles in human vascular smooth muscle and endothelial cells. J Cardiovasc Pharmacol. 2002; 40(6):841-53. DOI: 10.1097/00005344-200212000-00005. View

11.

Thors B, Halldorsson H, Thorgeirsson G . Thrombin and histamine stimulate endothelial nitric-oxide synthase phosphorylation at Ser1177 via an AMPK mediated pathway independent of PI3K-Akt. FEBS Lett. 2004; 573(1-3):175-80. DOI: 10.1016/j.febslet.2004.07.078. View

12.

Naruse K, Rask-Madsen C, Takahara N, Ha S, Suzuma K, Way K . Activation of vascular protein kinase C-beta inhibits Akt-dependent endothelial nitric oxide synthase function in obesity-associated insulin resistance. Diabetes. 2006; 55(3):691-8. DOI: 10.2337/diabetes.55.03.06.db05-0771. View

13.

Kaczmarek E, Erb L, Koziak K, Jarzyna R, Wink M, Guckelberger O . Modulation of endothelial cell migration by extracellular nucleotides: involvement of focal adhesion kinase and phosphatidylinositol 3-kinase-mediated pathways. Thromb Haemost. 2005; 93(4):735-42. PMC: 2830093. DOI: 10.1160/TH04-09-0576. View

14.

Lin M, Fulton D, Babbitt R, Fleming I, Busse R, Pritchard Jr K . Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production. J Biol Chem. 2003; 278(45):44719-26. DOI: 10.1074/jbc.M302836200. View

15.

Sessa W . eNOS at a glance. J Cell Sci. 2004; 117(Pt 12):2427-9. DOI: 10.1242/jcs.01165. View

16.

Parker P, Murray-Rust J . PKC at a glance. J Cell Sci. 2003; 117(Pt 2):131-2. DOI: 10.1242/jcs.00982. View

17.

Fleming I, Busse R . Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol. 2002; 284(1):R1-12. DOI: 10.1152/ajpregu.00323.2002. View

18.

Schneider J, El Kebir D, Chereau C, Lanone S, Huang X, de Buys Roessingh A . Involvement of Ca2+/calmodulin-dependent protein kinase II in endothelial NO production and endothelium-dependent relaxation. Am J Physiol Heart Circ Physiol. 2003; 284(6):H2311-9. DOI: 10.1152/ajpheart.00932.2001. View

19.

Mathie R, Ralevic V, Alexander B, Burnstock G . Nitric oxide is the mediator of ATP-induced dilatation of the rabbit hepatic arterial vascular bed. Br J Pharmacol. 1991; 103(2):1602-6. PMC: 1908371. DOI: 10.1111/j.1476-5381.1991.tb09834.x. View

20.

Anter E, Thomas S, Schulz E, Shapira O, Vita J, Keaney Jr J . Activation of endothelial nitric-oxide synthase by the p38 MAPK in response to black tea polyphenols. J Biol Chem. 2004; 279(45):46637-43. DOI: 10.1074/jbc.M405547200. View