title: Colocalization of eNOS and the catalytic subunit of PKA in endothelial cell junctions: a clue for regulated NO production.
abstract: Localization and coordinate phosphorylation/dephosphorylation of endothelial nitric oxide synthase (eNOS) are critical determinants for the basal and stimulated production of nitric oxide. Several phosphorylation sites in eNOS have been identified as targets of the cAMP-dependent protein kinase A (PKA). Basal eNOS activity is also regulated by interaction with caveolin-1, the major coat protein of caveolae. In the present study we have examined in rat aorta endothelium the subcellular steady-state distribution of eNOS, the catalytic subunit of PKA (PKA-c), and caveolin-1. Basal eNOS expression was found in two distinct locations, the endothelial cell surface and the Golgi complex. Cell surface eNOS was equally distributed over caveolar and non-caveolar membranes but was 2.5-fold enriched on luminal lamellipodia located at endothelial cell contacts. PKA-c colocalized with eNOS in the lamellipodia, whereas caveolin-1 was absent from these membrane domains. PKA-c was also found associated with cell surface caveolae and with tubulovesicular membranes of Golgi complex and endosomes. The topological proximity of eNOS with the catalytic subunit of PKA in restricted intracellular locations may provide mechanisms for differential PKA-mediated eNOS regulation.
authors.affiliation: Thrombosis and Haemostasis Laboratory, Department of Biology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. h.f.g.heijnen@azu.nl
authors.forename: Harry F G
authors.initials: HF
authors.lastname: Heijnen
authors.forename: Sandra
authors.initials: S
authors.lastname: Waaijenborg
authors.forename: James D
authors.initials: JD
authors.lastname: Crapo
authors.forename: Russell P
authors.initials: RP
authors.lastname: Bowler
authors.forename: Jan-Willem N
authors.initials: JW
authors.lastname: Akkerman
authors.forename: Jan W
authors.initials: JW
authors.lastname: Slot
mesh_terms: D000818:Animals; D001011:Aorta / Q000201:enzymology; D020134:Catalytic Domain; D021941:Caveolae / Q000201:enzymology; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000201:enzymology* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000201:enzymology; D007365:Intercellular Junctions / Q000201:enzymology*; D016253:Microscopy, Immunoelectron; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D051381:Rats
chemical_list: C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D017868:Cyclic AMP-Dependent Protein Kinases