Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Jan 15;361(Pt 2):193–201. doi: 10.1042/0264-6021:3610193

Endothelial nitric oxide synthase activity is linked to its presence at cell-cell contacts.

Roland Govers 1, Lonneke Bevers 1, Petra de Bree 1, Ton J Rabelink 1
PMCID: PMC1222299  PMID: 11772391

Abstract

The enzyme endothelial nitric oxide synthase (eNOS) is essential for vascular integrity. Many studies have demonstrated a link between the localization and activity of eNOS. Here, we studied the influence of cell-cell contact on this link in the microvascular endothelial bEnd.3 cell line. By immunofluorescence microscopy, eNOS localization at the plasma membrane was found to be dependent on cell-cell contact. In particular, eNOS was highly enriched at the intercellular contact sites. Further analysis showed that the pattern of eNOS localization at the plasma membrane resembled that of PECAM-1 (platelet endothelial cell adhesion molecule 1), but not that of the adherens junction proteins VE (vascular endothelial)-cadherin and plakoglobin. eNOS that was localized at the contact sites was, in part, Triton X-100-insoluble, in contrast with eNOS at the Golgi complex, which may indicate an association of eNOS with the actin cytoskeleton. Interestingly, eNOS activity was up-regulated in confluent monolayers compared with subconfluent cells, while there was no difference in eNOS expression. This correlation between cell confluence and eNOS activity was also found when primary bovine aortic endothelial cells were studied. These data imply that cell-cell contact induces the localization of eNOS at intercellular junctions, which is required for agonist-induced eNOS activation.

Full Text

The Full Text of this article is available as a PDF (455.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alexander J. S., Alexander B. C., Eppihimer L. A., Goodyear N., Haque R., Davis C. P., Kalogeris T. J., Carden D. L., Zhu Y. N., Kevil C. G. Inflammatory mediators induce sequestration of VE-cadherin in cultured human endothelial cells. Inflammation. 2000 Apr;24(2):99–113. doi: 10.1023/a:1007025325451. [DOI] [PubMed] [Google Scholar]
  2. Andries L. J., Brutsaert D. L., Sys S. U. Nonuniformity of endothelial constitutive nitric oxide synthase distribution in cardiac endothelium. Circ Res. 1998 Feb 9;82(2):195–203. doi: 10.1161/01.res.82.2.195. [DOI] [PubMed] [Google Scholar]
  3. Arnal J. F., Yamin J., Dockery S., Harrison D. G. Regulation of endothelial nitric oxide synthase mRNA, protein, and activity during cell growth. Am J Physiol. 1994 Nov;267(5 Pt 1):C1381–C1388. doi: 10.1152/ajpcell.1994.267.5.C1381. [DOI] [PubMed] [Google Scholar]
  4. Ayalon O., Sabanai H., Lampugnani M. G., Dejana E., Geiger B. Spatial and temporal relationships between cadherins and PECAM-1 in cell-cell junctions of human endothelial cells. J Cell Biol. 1994 Jul;126(1):247–258. doi: 10.1083/jcb.126.1.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baldwin A. L., Thurston G., al Naemi H. Inhibition of nitric oxide synthesis increases venular permeability and alters endothelial actin cytoskeleton. Am J Physiol. 1998 May;274(5 Pt 2):H1776–H1784. doi: 10.1152/ajpheart.1998.274.5.H1776. [DOI] [PubMed] [Google Scholar]
  6. Bobryshev Y. V., Cherian S. M., Inder S. J., Lord R. S. Neovascular expression of VE-cadherin in human atherosclerotic arteries and its relation to intimal inflammation. Cardiovasc Res. 1999 Sep;43(4):1003–1017. doi: 10.1016/s0008-6363(99)00125-x. [DOI] [PubMed] [Google Scholar]
  7. Carmeliet P., Lampugnani M. G., Moons L., Breviario F., Compernolle V., Bono F., Balconi G., Spagnuolo R., Oosthuyse B., Dewerchin M. Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis. Cell. 1999 Jul 23;98(2):147–157. doi: 10.1016/s0092-8674(00)81010-7. [DOI] [PubMed] [Google Scholar]
  8. Cho M. M., Ziats N. P., Pal D., Utian W. H., Gorodeski G. I. Estrogen modulates paracellular permeability of human endothelial cells by eNOS- and iNOS-related mechanisms. Am J Physiol. 1999 Feb;276(2 Pt 1):C337–C349. doi: 10.1152/ajpcell.1999.276.2.C337. [DOI] [PubMed] [Google Scholar]
  9. Cines D. B., Pollak E. S., Buck C. A., Loscalzo J., Zimmerman G. A., McEver R. P., Pober J. S., Wick T. M., Konkle B. A., Schwartz B. S. Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood. 1998 May 15;91(10):3527–3561. [PubMed] [Google Scholar]
  10. Corson M. A., James N. L., Latta S. E., Nerem R. M., Berk B. C., Harrison D. G. Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress. Circ Res. 1996 Nov;79(5):984–991. doi: 10.1161/01.res.79.5.984. [DOI] [PubMed] [Google Scholar]
  11. Dejana E., Corada M., Lampugnani M. G. Endothelial cell-to-cell junctions. FASEB J. 1995 Jul;9(10):910–918. [PubMed] [Google Scholar]
  12. Del Maschio A., Zanetti A., Corada M., Rival Y., Ruco L., Lampugnani M. G., Dejana E. Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions. J Cell Biol. 1996 Oct;135(2):497–510. doi: 10.1083/jcb.135.2.497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dimmeler S., Fleming I., Fisslthaler B., Hermann C., Busse R., Zeiher A. M. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999 Jun 10;399(6736):601–605. doi: 10.1038/21224. [DOI] [PubMed] [Google Scholar]
  14. Dorudi S., Hart I. R. Mechanisms underlying invasion and metastasis. Curr Opin Oncol. 1993 Jan;5(1):130–135. [PubMed] [Google Scholar]
  15. Ehringer W. D., Edwards M. J., Miller F. N. Mechanisms of alpha-thrombin, histamine, and bradykinin induced endothelial permeability. J Cell Physiol. 1996 Jun;167(3):562–569. doi: 10.1002/(SICI)1097-4652(199606)167:3<562::AID-JCP20>3.0.CO;2-4. [DOI] [PubMed] [Google Scholar]
  16. Esser S., Lampugnani M. G., Corada M., Dejana E., Risau W. Vascular endothelial growth factor induces VE-cadherin tyrosine phosphorylation in endothelial cells. J Cell Sci. 1998 Jul;111(Pt 13):1853–1865. doi: 10.1242/jcs.111.13.1853. [DOI] [PubMed] [Google Scholar]
  17. Fischer S., Clauss M., Wiesnet M., Renz D., Schaper W., Karliczek G. F. Hypoxia induces permeability in brain microvessel endothelial cells via VEGF and NO. Am J Physiol. 1999 Apr;276(4 Pt 1):C812–C820. doi: 10.1152/ajpcell.1999.276.4.C812. [DOI] [PubMed] [Google Scholar]
  18. Fulton D., Gratton J. P., McCabe T. J., Fontana J., Fujio Y., Walsh K., Franke T. F., Papapetropoulos A., Sessa W. C. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature. 1999 Jun 10;399(6736):597–601. doi: 10.1038/21218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. García-Cardeña G., Oh P., Liu J., Schnitzer J. E., Sessa W. C. Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: implications for nitric oxide signaling. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6448–6453. doi: 10.1073/pnas.93.13.6448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gerez L., Mohrmann K., van Raak M., Jongeneelen M., Zhou X. Z., Lu K. P., van Der Sluijs P. Accumulation of rab4GTP in the cytoplasm and association with the peptidyl-prolyl isomerase pin1 during mitosis. Mol Biol Cell. 2000 Jul;11(7):2201–2211. doi: 10.1091/mbc.11.7.2201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ghigo D., Aldieri E., Todde R., Costamagna C., Garbarino G., Pescarmona G., Bosia A. Chloroquine stimulates nitric oxide synthesis in murine, porcine, and human endothelial cells. J Clin Invest. 1998 Aug 1;102(3):595–605. doi: 10.1172/JCI1052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ghigo D., Arese M., Todde R., Vecchi A., Silvagno F., Costamagna C., Dong Q. G., Alessio M., Heller R., Soldi R. Middle T antigen-transformed endothelial cells exhibit an increased activity of nitric oxide synthase. J Exp Med. 1995 Jan 1;181(1):9–19. doi: 10.1084/jem.181.1.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gronowski A. M., Bertics P. J. Modulation of epidermal growth factor receptor interaction with the detergent-insoluble cytoskeleton and its effects on receptor tyrosine kinase activity. Endocrinology. 1995 May;136(5):2198–2205. doi: 10.1210/endo.136.5.7720669. [DOI] [PubMed] [Google Scholar]
  24. Guillot P. V., Guan J., Liu L., Kuivenhoven J. A., Rosenberg R. D., Sessa W. C., Aird W. C. A vascular bed-specific pathway. J Clin Invest. 1999 Mar;103(6):799–805. doi: 10.1172/JCI6017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. He P., Liu B., Curry F. E. Effect of nitric oxide synthase inhibitors on endothelial [Ca2+]i and microvessel permeability. Am J Physiol. 1997 Jan;272(1 Pt 2):H176–H185. doi: 10.1152/ajpheart.1997.272.1.H176. [DOI] [PubMed] [Google Scholar]
  26. Kojima H., Nakatsubo N., Kikuchi K., Kawahara S., Kirino Y., Nagoshi H., Hirata Y., Nagano T. Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal Chem. 1998 Jul 1;70(13):2446–2453. doi: 10.1021/ac9801723. [DOI] [PubMed] [Google Scholar]
  27. Kurose I., Kubes P., Wolf R., Anderson D. C., Paulson J., Miyasaka M., Granger D. N. Inhibition of nitric oxide production. Mechanisms of vascular albumin leakage. Circ Res. 1993 Jul;73(1):164–171. doi: 10.1161/01.res.73.1.164. [DOI] [PubMed] [Google Scholar]
  28. Lampugnani M. G., Corada M., Caveda L., Breviario F., Ayalon O., Geiger B., Dejana E. The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, beta-catenin, and alpha-catenin with vascular endothelial cadherin (VE-cadherin). J Cell Biol. 1995 Apr;129(1):203–217. doi: 10.1083/jcb.129.1.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lampugnani M. G., Resnati M., Raiteri M., Pigott R., Pisacane A., Houen G., Ruco L. P., Dejana E. A novel endothelial-specific membrane protein is a marker of cell-cell contacts. J Cell Biol. 1992 Sep;118(6):1511–1522. doi: 10.1083/jcb.118.6.1511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lewalle J. M., Bajou K., Desreux J., Mareel M., Dejana E., Noël A., Foidart J. M. Alteration of interendothelial adherens junctions following tumor cell-endothelial cell interaction in vitro. Exp Cell Res. 1997 Dec 15;237(2):347–356. doi: 10.1006/excr.1997.3799. [DOI] [PubMed] [Google Scholar]
  31. Matsumura T., Wolff K., Petzelbauer P. Endothelial cell tube formation depends on cadherin 5 and CD31 interactions with filamentous actin. J Immunol. 1997 Apr 1;158(7):3408–3416. [PubMed] [Google Scholar]
  32. McQuaid K. E., Keenan A. K. Endothelial barrier dysfunction and oxidative stress: roles for nitric oxide? Exp Physiol. 1997 Mar;82(2):369–376. doi: 10.1113/expphysiol.1997.sp004032. [DOI] [PubMed] [Google Scholar]
  33. Montesano R., Pepper M. S., Möhle-Steinlein U., Risau W., Wagner E. F., Orci L. Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene. Cell. 1990 Aug 10;62(3):435–445. doi: 10.1016/0092-8674(90)90009-4. [DOI] [PubMed] [Google Scholar]
  34. Murohara T., Horowitz J. R., Silver M., Tsurumi Y., Chen D., Sullivan A., Isner J. M. Vascular endothelial growth factor/vascular permeability factor enhances vascular permeability via nitric oxide and prostacyclin. Circulation. 1998 Jan 6;97(1):99–107. doi: 10.1161/01.cir.97.1.99. [DOI] [PubMed] [Google Scholar]
  35. Nakatsubo N., Kojima H., Kikuchi K., Nagoshi H., Hirata Y., Maeda D., Imai Y., Irimura T., Nagano T. Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: diaminofluoresceins. FEBS Lett. 1998 May 8;427(2):263–266. doi: 10.1016/s0014-5793(98)00440-2. [DOI] [PubMed] [Google Scholar]
  36. Newman P. J. The biology of PECAM-1. J Clin Invest. 1997 Dec 1;100(11 Suppl):S25–S29. [PubMed] [Google Scholar]
  37. Parton R. G., Simons K. Digging into caveolae. Science. 1995 Sep 8;269(5229):1398–1399. doi: 10.1126/science.7660120. [DOI] [PubMed] [Google Scholar]
  38. Rabiet M. J., Plantier J. L., Rival Y., Genoux Y., Lampugnani M. G., Dejana E. Thrombin-induced increase in endothelial permeability is associated with changes in cell-to-cell junction organization. Arterioscler Thromb Vasc Biol. 1996 Mar;16(3):488–496. doi: 10.1161/01.atv.16.3.488. [DOI] [PubMed] [Google Scholar]
  39. Searles C. D., Miwa Y., Harrison D. G., Ramasamy S. Posttranscriptional regulation of endothelial nitric oxide synthase during cell growth. Circ Res. 1999 Oct 1;85(7):588–595. doi: 10.1161/01.res.85.7.588. [DOI] [PubMed] [Google Scholar]
  40. Sessa W. C., García-Cardeña G., Liu J., Keh A., Pollock J. S., Bradley J., Thiru S., Braverman I. M., Desai K. M. The Golgi association of endothelial nitric oxide synthase is necessary for the efficient synthesis of nitric oxide. J Biol Chem. 1995 Jul 28;270(30):17641–17644. doi: 10.1074/jbc.270.30.17641. [DOI] [PubMed] [Google Scholar]
  41. Shaul P. W., Smart E. J., Robinson L. J., German Z., Yuhanna I. S., Ying Y., Anderson R. G., Michel T. Acylation targets emdothelial nitric-oxide synthase to plasmalemmal caveolae. J Biol Chem. 1996 Mar 15;271(11):6518–6522. doi: 10.1074/jbc.271.11.6518. [DOI] [PubMed] [Google Scholar]
  42. Sheibani N., Newman P. J., Frazier W. A. Thrombospondin-1, a natural inhibitor of angiogenesis, regulates platelet-endothelial cell adhesion molecule-1 expression and endothelial cell morphogenesis. Mol Biol Cell. 1997 Jul;8(7):1329–1341. doi: 10.1091/mbc.8.7.1329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sowa G., Liu J., Papapetropoulos A., Rex-Haffner M., Hughes T. E., Sessa W. C. Trafficking of endothelial nitric-oxide synthase in living cells. Quantitative evidence supporting the role of palmitoylation as a kinetic trapping mechanism limiting membrane diffusion. J Biol Chem. 1999 Aug 6;274(32):22524–22531. doi: 10.1074/jbc.274.32.22524. [DOI] [PubMed] [Google Scholar]
  44. Sporbert A., Mertsch K., Smolenski A., Haseloff R. F., Schönfelder G., Paul M., Ruth P., Walter U., Blasig I. E. Phosphorylation of vasodilator-stimulated phosphoprotein: a consequence of nitric oxide- and cGMP-mediated signal transduction in brain capillary endothelial cells and astrocytes. Brain Res Mol Brain Res. 1999 Apr 20;67(2):258–266. doi: 10.1016/s0169-328x(99)00067-4. [DOI] [PubMed] [Google Scholar]
  45. Venema V. J., Marrero M. B., Venema R. C. Bradykinin-stimulated protein tyrosine phosphorylation promotes endothelial nitric oxide synthase translocation to the cytoskeleton. Biochem Biophys Res Commun. 1996 Sep 24;226(3):703–710. doi: 10.1006/bbrc.1996.1417. [DOI] [PubMed] [Google Scholar]
  46. Volonte D., Galbiati F., Lisanti M. P. Visualization of caveolin-1, a caveolar marker protein, in living cells using green fluorescent protein (GFP) chimeras. The subcellular distribution of caveolin-1 is modulated by cell-cell contact. FEBS Lett. 1999 Feb 26;445(2-3):431–439. doi: 10.1016/s0014-5793(99)00164-7. [DOI] [PubMed] [Google Scholar]
  47. Yan S. R., Fumagalli L., Berton G. Activation of SRC family kinases in human neutrophils. Evidence that p58C-FGR and p53/56LYN redistributed to a Triton X-100-insoluble cytoskeletal fraction, also enriched in the caveolar protein caveolin, display an enhanced kinase activity. FEBS Lett. 1996 Feb 12;380(1-2):198–203. doi: 10.1016/0014-5793(96)00029-4. [DOI] [PubMed] [Google Scholar]
  48. Yuan Y., Granger H. J., Zawieja D. C., DeFily D. V., Chilian W. M. Histamine increases venular permeability via a phospholipase C-NO synthase-guanylate cyclase cascade. Am J Physiol. 1993 May;264(5 Pt 2):H1734–H1739. doi: 10.1152/ajpheart.1993.264.5.H1734. [DOI] [PubMed] [Google Scholar]
  49. Yuan Y., Meng F. Y., Huang Q., Hawker J., Wu H. M. Tyrosine phosphorylation of paxillin/pp125FAK and microvascular endothelial barrier function. Am J Physiol. 1998 Jul;275(1 Pt 2):H84–H93. doi: 10.1152/ajpheart.1998.275.1.H84. [DOI] [PubMed] [Google Scholar]
  50. Zöllner S., Aberle S., Harvey S. E., Polokoff M. A., Rubanyi G. M. Changes of endothelial nitric oxide synthase level and activity during endothelial cell proliferation. Endothelium. 2000;7(3):169–184. doi: 10.3109/10623320009165315. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES