Intracellular ascorbate tightens the endothelial permeability barrier through Epac1 and the tubulin cytoskeleton

Am J Physiol Cell Physiol. 2016 Oct 1;311(4):C652-C662. doi: 10.1152/ajpcell.00076.2016. Epub 2016 Sep 7.


Vitamin C, or ascorbic acid, both tightens the endothelial permeability barrier in basal cells and also prevents barrier leak induced by inflammatory agents. Barrier tightening by ascorbate in basal endothelial cells requires nitric oxide derived from activation of nitric oxide synthase. Although ascorbate did not affect cyclic AMP levels in our previous study, there remains a question of whether it might activate downstream cyclic AMP-dependent pathways. In this work, we found in both primary and immortalized cultured endothelial cells that ascorbate tightened the endothelial permeability barrier by ∼30%. In human umbilical vein endothelial cells, this occurred at what are likely physiologic intracellular ascorbate concentrations. In so doing, ascorbate decreased measures of oxidative stress and also flattened the cells to increase cell-to-cell contact. Inhibition of downstream cyclic AMP-dependent proteins via protein kinase A did not prevent ascorbate from tightening the endothelial permeability barrier, whereas inhibition of Epac1 did block the ascorbate effect. Although Epac1 was required, its mediator Rap1 was not activated. Furthermore, ascorbate acutely stabilized microtubules during depolymerization induced by colchicine and nocodazole. Over several days in culture, ascorbate also increased the amount of stable acetylated α-tubulin. Microtubule stabilization was further suggested by the finding that ascorbate increased the amount of Epac1 bound to α-tubulin. These results suggest that physiologic ascorbate concentrations tighten the endothelial permeability barrier in unstimulated cells by stabilizing microtubules in a manner downstream of cyclic AMP that might be due both to increasing nitric oxide availability and to scavenging of reactive oxygen or nitrogen species.

Keywords: Epac1; ascorbate; endothelial permeability; microtubules; paracellular transport.

MeSH terms

  • Ascorbic Acid / metabolism*
  • Cell Line
  • Cyclic AMP / metabolism
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Cytoskeleton / metabolism*
  • Cytoskeleton / physiology
  • Endothelium / metabolism*
  • Endothelium / physiology*
  • Guanine Nucleotide Exchange Factors / metabolism*
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Microtubules / metabolism
  • Nitric Oxide / metabolism
  • Oxidative Stress / physiology
  • Permeability
  • Tubulin / metabolism*


  • Guanine Nucleotide Exchange Factors
  • RAPGEF3 protein, human
  • Tubulin
  • Nitric Oxide
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases
  • Ascorbic Acid