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Inhibition of eNOS internalization dissociates PAF‐induced eNOS phosphorylation at Ser1177 from NO production
Author(s) -
Sánchez Fabiola Alejandra,
Rana Roshniben,
Kim David D,
Iwahashi Toru,
Durán Walter N
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.767.5
Subject(s) - enos , caveolae , dynamin , endocytosis , phosphorylation , microbiology and biotechnology , internalization , chemistry , biology , biochemistry , signal transduction , cell , nitric oxide synthase , enzyme
We demonstrated earlier that caveolar endocytosis of eNOS regulates PAF‐induced endothelial permeability (AJP Heart 295: H1642 ‐ H1648 2008). We tested further the hypotheses that caveolar traffic is a requirement for the development of a robust hyperpermeability response and that eNOS‐derived NO production is associated to caveolar translocation. To this end, we used ECVCD8eNOS‐GFP and CVEC (bovine coronary postcapillary venular endothelial cells), as a model and PAF as the agonist. We inhibited caveolar traffic using cyclodextrin, dynamin 2K44A (negative mutant of dynamin) and cav1‐Y14F (negative mutant of caveolin1 that prevents caveolin‐1 phosphorylation at Y14). Cyclodextrin and the dominant negative mutants inhibited eNOS translocation via caveolae. eNOS phosphorylation at Ser1177 was enhanced in CD8eNOS‐GFP and CVEC when cyclodextrin or dynamin 2K44A were applied, and the response was cell line dependent when cav1‐Y14F was applied , i.e., CD8eNOS‐GFP demonstrated increased phosphorylation at Ser1177, but CVEC did not. Interestingly, NO production was partially inhibited by cav1‐Y14F and completely inhibited by dynamin 2K44A and cyclodextrin. We obtained a direct correlation between PAF‐induced eNOS traffic via caveolae and hyperpermeability. Our results demonstrate for first time an uncoupling between eNOS phosphorylation and NO production. Indeed, our results suggest a mechanism where eNOS is activated by phosphorylation at the plasma membrane and endocytosis is required to cause hyperpermeability. (Supported by 5R01 HL070634 and 1RO1 HL088479).