title: Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain.
abstract: O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2 ± 2 vs 7.9 ± 1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4 ± 2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3 ± 2 vs 7.5 ± 2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1 ± 2 vs 7.4 ± 2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca(2+)/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.
authors.affiliation: Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso, Barra do Garças, MT, Brasil.
authors.forename: V V
authors.initials: VV
authors.lastname: Lima
authors.affiliation: Curso de Medicina, Setor de Fisiologia Humana, Universidade Federal de Goiás, Jataí, GO, Brasil.
authors.forename: N S
authors.initials: NS
authors.lastname: Lobato
authors.affiliation: Curso de Medicina, Setor de Fisiologia Humana, Universidade Federal de Goiás, Jataí, GO, Brasil.
authors.forename: F P
authors.initials: FP
authors.lastname: Filgueira
authors.affiliation: Department of Physiology, Georgia Regents University, Augusta, GA, USA.
authors.forename: R C
authors.initials: RC
authors.lastname: Webb
authors.affiliation: Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil.
authors.forename: R C
authors.initials: RC
authors.lastname: Tostes
authors.affiliation: Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso, Barra do Garças, MT, Brasil.
authors.forename: F R
authors.initials: FR
authors.lastname: Giachini
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D000215:Acylation / Q000187:drug effects / Q000502:physiology; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001013:Aorta, Thoracic; D001381:Azepines / Q000494:pharmacology; D015153:Blotting, Western; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007004:Hypoglycemic Agents / Q000494:pharmacology; D008297:Male; D008387:Marine Toxins; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D018994:Myosin Light Chains / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D010080:Oxazoles / Q000494:pharmacology; D010091:Oximes / Q000494:pharmacology; D048448:Phenylcarbamates / Q000494:pharmacology; D010656:Phenylephrine / Q000819:agonists; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017208:Rats, Wistar; D012265:Ribonucleotides / Q000494:pharmacology; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology*; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors
chemical_list: D001381:Azepines; D004791:Enzyme Inhibitors; D007004:Hypoglycemic Agents; D008387:Marine Toxins; D018994:Myosin Light Chains; D010080:Oxazoles; D010091:Oximes; D048448:Phenylcarbamates; D012265:Ribonucleotides; D014662:Vasoconstrictor Agents; C056218:ML 9; C068836:N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime; D010656:Phenylephrine; D000620:Aminoimidazole Carboxamide; C059041:calyculin A; D009219:Myosin-Light-Chain Kinase; D043263:Myosin-Light-Chain Phosphatase; C110628:hexosaminidase C; D001619:beta-N-Acetylhexosaminidases; C031143:AICA ribonucleotide; D000117:Acetylglucosamine