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Platelet activating factor (PAF) induces s‐nitrosylation of signalling proteins in an endothelial cell line
Author(s) -
González Francisco G,
Sánchez Fabiola A,
Velarde Victoria,
Boric Mauricio P,
Durán Walter N
Publication year - 2010
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.24.1_supplement.975.16
Subject(s) - rap1 , microbiology and biotechnology , rac1 , guanine nucleotide exchange factor , s nitrosylation , gtpase , chemistry , enos , nitric oxide , gtpase activating protein , nitrosylation , signal transduction , biology , g protein , biochemistry , nitric oxide synthase , organic chemistry , cysteine , enzyme
In endothelial cells PAF increases microvascular permeability to macromolecules through activation of endothelial nitric oxide synthase (eNOS). In these cells NO signalling determines reorganization of intercellular contacts and the consequent loss of endothelial barrier stability, whereas the action of small GTPases keeps this barrier stable. It is not known whether PAF increases the permeability by NO‐mediated protein S‐nitrosylation and/or increased guanylate cyclase (sGC) activity and whether this inflammatory ligand modifies the activity of small GTPases that could be regulated by guanine exchange protein directly activated by cAMP (Epac). We studied the effect of PAF: 1) on s‐nitrosylation of signalling proteins using the biotin switch and 2) on the activity of small GTPases Rac1 and Rap1, using fusion proteins and pull down techniques, in the cellular line ECV stably transfected with eNOS‐GFP cDNA (ECV‐eNOS‐GFP). PAF stimulation increased s‐nitrosylation of eNOS, Epac, sGC, Rap1 and Rac1, in a cyclic way, with maximum increases at 30 s and 15 min post stimulus. Rap1 and Rac1 activity did not change with PAF stimulation. These results suggest that PAF increases permeability to macromolecules by s‐nitrosylation of signalling proteins, which could represent a change in their activity, however, not involving small GTPases Rap1 and Rac1. Supported by Grants NIH‐5RO1‐HL070634 and FONDECYT 1090757.