Inducible nitric oxide synthase-dependent stimulation of PKGI and phosphorylation of VASP in human embryonic kidney cells.
Inducible nitric oxide synthase (iNOS) production of nitric oxide (NO) has been mostly associated with so-called nitrosative stress or interaction with superoxide anion. However, recent investigations have indicated that, as for the other isoenzymes producing NO, guanylyl cyclase (GC) is a very sensitive target of iNOS activity. To further investigate this less explored signaling, the NO-cyclic guanosine 3'-5'-monophosphate (NO-cGMP)-induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation on serine 239 was investigated in human embryonic kidney 293 cells (HEK cells). First, the expression and activity of alpha2 and beta1 NO-sensitive GC subunits was determined by Western blot analysis, reverse transcription-polymerase chain reaction and NO donors administration. Then, the expression of a functional cGMP-dependent protein kinase I (PKGI) was verified by addition of 8-Br-cGMP followed by determination of phosphorylation of VASP on serine 239. Finally, iNOS activation of this signaling pathway was characterized after transfection of HEK cells with human iNOS cDNA. Altogether our data show that iNOS-derived NO activates endogenous NO-sensitive GC and leads to VASP phosphorylation in HEK cells.
André
Muriel
M
Pharmacology and Toxicology Department, Rue du Bugnon 27, 1005 Lausanne, Switzerland.
Latado
Hélia
H
Felley-Bosco
Emanuela
E
D000595:Amino Acid Sequence; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D006801:Humans; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000096:biosynthesis; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000502:physiology*; D052247:Nitric Oxide Synthase Type II; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D015398:Signal Transduction; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; D020030:Nitric Oxide Donors; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP