title: Inhibition of MEK/ERK1/2 signalling alters endothelial nitric oxide synthase activity in an agonist-dependent manner.
abstract: eNOS (endothelial nitric oxide synthase) activity is post-translationally regulated in a complex fashion by acylation, protein-protein interactions, intracellular trafficking and phosphorylation, among others. Signalling pathways that regulate eNOS activity include phosphoinositide 3-kinase/Akt, cyclic nucleotide-dependent kinases [PKA (protein kinase A) and PKG], PKC, as well as ERKs (extracellular-signal-regulated kinases). The role of ERKs in eNOS activation remains controversial. In the present study, we have examined the role of ERK1/2 in eNOS activation in HUVEC-CS [transformed HUVEC (human umbilical-vein endothelial cells)] as well as a widely used model for eNOS study, transiently transfected COS-7 cells. U0126 pretreatment of HUVEC-CS potentiated ATP-stimulated eNOS activity, independent of changes in intracellular Ca2+ concentration ([Ca2+]i). In COS-7 cells transiently expressing ovine eNOS, U0126 potentiated A23187-stimulated eNOS activity, but inhibited ATP-stimulated activity. Compensatory changes in phosphorylation of five key eNOS residues did not account for changes in A23187-stimulated activity. However, in the case of ATP, altered phosphorylation and changes in [Ca2+]i may partially contribute to U0126 inhibition of activity. Finally, seven eNOS alanine mutants of putative ERK1/2 targets were generated and the effects of U0126 pretreatment on eNOS activity were gauged with A23187 and ATP treatment. T97A-eNOS was the only construct significantly different from wild-type after U0126 pretreatment and ATP stimulation of eNOS activation. In the present study, eNOS activity was either potentiated or inhibited in COS-7 cells, suggesting agonist dependence for MEK/ERK1/2 signalling [where MEK is MAPK (mitogen-activated protein kinase)/ERK kinase] to eNOS and a complex mechanism including [Ca2+]i, phosphorylation and, possibly, intracellular trafficking.
authors.affiliation: Department of Obstetrics and Gynecology, The University of Wisconsin-Madison, Madison, WI 53715, USA.
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Cale
authors.forename: Ian M
authors.initials: IM
authors.lastname: Bird
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002070:Butadienes / Q000494:pharmacology*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D012756:Sheep
chemical_list: D002070:Butadienes; D009570:Nitriles; C113580:U 0126; D000001:Calcimycin; D000255:Adenosine Triphosphate; D052250:Nitric Oxide Synthase Type III; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D002118:Calcium