title: Nobiletin, a citrus flavonoid, activates vasodilator-stimulated phosphoprotein in human platelets through non-cyclic nucleotide-related mechanisms.
abstract: Nobiletin, a bioactive polymethoxylated flavone, has been described to possess a diversity of biological effects through its antioxidant and anti-inflammatory properties. Vasodilator-stimulated phosphoprotein (VASP) is a common substrate for cyclic AMP and cyclic GMP-regulated protein kinases [i.e., cyclic AMP-dependent protein kinase (PKA; also known as protein kinase A) and cyclic GMP-dependent protein kinase (PKG; also known as protein kinase G)] and it has been shown to be directly phosphorylated by protein kinase C (PKC). In the present study, we demonstrate that VASP is phosphorylated by nobiletin in human platelets via a non-cyclic nucleotide-related mechanism. This was confirmed by the use of inhibitors of adenylate cyclase (SQ22536) and guanylate cyclase [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)], since they prevented VASP phosphorylation induced by nobiletin. Furthormore, this event was also not affected by specific inhibitors of PKA (H-89), PKG (KT5823) and PKC (Ro318220), representing cyclic nucleotide-dependent pathways upon nobiletin-induced VASP phosphorylation. Similarly, inhibitors of p38 mitogen-activated protein kinase (MAPK; SB203580), extracellular signal-regulated kinase 2 (ERK2; PD98059), c-Jun N-terminal kinase 1 (JNK1; SP600125), Akt (LY294002) and nuclear factor-κB (NF-κB; Bay11-7082) did not affect nobiletin‑induced VASP phosphorylation. Moreover, electron spin resonance, dichlorofluorescein fluorescence and western blotting techniques revealed that nobiletin did not affect hydroxyl radicals (OH•), intracellular reactive oxygen species (ROS) and on protein carbonylation, respectively. Furthermore, the nobiletin‑induced VASP phosphorylation was surprisingly reversed by the intracellular antioxidant, N-acetylcysteine (NAC), but not by the inhibitor of NADPH oxidase, diphenyleneiodonium chloride (DPI). It was surprising to observe the differential effects of nobiletin and NAC on VASP phosphorylation in human platelets, since they both have been reported to have antioxidant properties. The likely explanation for this discrepancy is that NAC may bind to allosteric sites on the receptor different from those that nobiletin binds to in human platelets. Taken together, our findings suggest that nobiletin induces VASP phosphorylation in human platelets through non-cyclic nucleotide-related mechanisms. Nevertheless, the exact mechanisms responsible for these effects need to be further confirmed in future studies.
authors.affiliation: Department of Pharmacology, College of Medicine, Taipei Medical University, Taipei, Taiwan, R.O.C.
authors.forename: Thanasekaran
authors.initials: T
authors.lastname: Jayakumar
authors.affiliation: Department of Pharmacology, College of Medicine, Taipei Medical University, Taipei, Taiwan, R.O.C.
authors.forename: Kao-Chang
authors.initials: KC
authors.lastname: Lin
authors.affiliation: Department of Pharmacology, College of Medicine, Taipei Medical University, Taipei, Taiwan, R.O.C.
authors.forename: Wan-Jung
authors.initials: WJ
authors.lastname: Lu
authors.affiliation: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan, R.O.C.
authors.forename: Chia-Ying
authors.initials: CY
authors.lastname: Lin
authors.affiliation: Department of Animal Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India.
authors.forename: Geraldine
authors.initials: G
authors.lastname: Pitchairaj
authors.affiliation: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan, R.O.C.
authors.forename: Jiun-Yi
authors.initials: JY
authors.lastname: Li
authors.affiliation: Department of Pharmacology, College of Medicine, Taipei Medical University, Taipei, Taiwan, R.O.C.
authors.forename: Joen-Rong
authors.initials: JR
authors.lastname: Sheu
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002957:Citrus / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D047309:Flavones / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D017665:Hydroxyl Radical / Q000378:metabolism; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009712:Nucleotides, Cyclic / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D050050:Protein Carbonylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D000098662:Vasodilator-Stimulated Phosphoprotein
chemical_list: D015815:Cell Adhesion Molecules; D047309:Flavones; D005419:Flavonoids; D008840:Microfilament Proteins; D016328:NF-kappa B; D009712:Nucleotides, Cyclic; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D017665:Hydroxyl Radical; C008661:nobiletin; D019255:NADPH Oxidases; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D000111:Acetylcysteine