title: Peptide-stimulation enhances compartmentalization and the catalytic activity of lung endothelial NOS.
abstract: We reported that an 11 amino acid synthetic peptide (P1) activates lung endothelial cell nitric oxide synthase (eNOS) independent of its change in expression and/or phosphorylation. Since caveolae/eNOS dissociation is known to enhance the catalytic activity of eNOS, we examined whether P1-mediated increase of eNOS activity is associated with caveolae/cholesterol modulation, increased caveolin-1 phosphorylation, and intracellular compartmentalization of eNOS in pulmonary artery endothelial cells (PAEC). PAEC were incubated with or without (control) P1 or cholesterol modulators/caveolae disruptors, cholesterol oxidase (CHOX) and methyl-beta-cyclodextrin (CD), for 1 h at 37 degrees C. After incubation cells were used for: i) immunoprecipitation, ii) isolation of plasma membrane (PM)-, Golgi complex (GC)-, and non-Golgi complex (NGC)-enriched fractions, iii) immunofluorescence confocal imaging, and iv) electron microscopy for localization and/or eNOS activity. P1, CHOX, and CD-stimulation caused dissociation of eNOS from PM with increased localization to GC and/or NGC. P1 and CHOX significantly increased eNOS activity in PM and GC and CD-stimulation increased eNOS activity localized only in GC. P1 increased phosphorylation of caveolin-1 in intact cells and GC fraction. Immunofluorescence and/or immunogold labeled imaging/electron microscopy analysis of P1-, CHOX-, and CD-stimulated intact cells confirmed eNOS/caveolae dissociation and translocation of eNOS to GC. These results suggest that: i) P1-stimulation translocates eNOS to GC and enhances the catalytic activity of eNOS in both the PM and GC fractions of PAEC, ii) CHOX- but not CD-mediated caveolae and/or cholesterol modulation mimics the effect of P1-stimulated compartmentalization and activation of eNOS in PAEC, and iii) P1-stimulated caveolae/cholesterol modulation, phosphorylation of caveolin-1, and activation of eNOS is physiologically relevant since P1 is known to enhance NO/cGMP-dependent vasorelaxation in the pulmonary circulation.
authors.affiliation: Malcom Randall Department of Veterans Affairs Medical Center, Gainesville, FL 32608-1197, USA.
authors.forename: Tarun E
authors.initials: TE
authors.lastname: Hutchinson
authors.forename: Sudeep
authors.initials: S
authors.lastname: Kuchibhotla
authors.forename: Edward R
authors.initials: ER
authors.lastname: Block
authors.forename: Jawaharlal M
authors.initials: JM
authors.lastname: Patel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1 / Q000378:metabolism; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002789:Cholesterol Oxidase / Q000494:pharmacology; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D008168:Lung / Q000201:enzymology*; D052250:Nitric Oxide Synthase Type III / Q000032:analysis / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology*; D010766:Phosphorylation; D013552:Swine; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D051242:Caveolin 1; D010455:Peptides; D047392:beta-Cyclodextrins; D002789:Cholesterol Oxidase; D052250:Nitric Oxide Synthase Type III; C031215:betadex