期刊
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
卷 311, 期 4, 页码 C652-C662出版社
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpcell.00076.2016
关键词
ascorbate; endothelial permeability; Epac1; paracellular transport; microtubules
资金
- National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [DK-050435, T32-DK-007061]
- Cell Culture Core of the Vanderbilt Diabetes Research and Training Center [DK-020593]
- National Institutes of Health [CA-68485, DK-20593, DK-58404, DK-59637, EY-08126]
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 similar to 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 alpha-tubulin. Microtubule stabilization was further suggested by the finding that ascorbate increased the amount of Epac1 bound to alpha-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.
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