title: Role of NMDA receptor in homocysteine-induced activation of mitogen-activated protein kinase and phosphatidyl inositol 3-kinase pathways in cultured human vascular smooth muscle cells.
abstract: BACKGROUND
  Exposure of vascular smooth muscle cells (VSMC) to homocysteine, at concentrations associated with an increased risk of cardiovascular events, enhances synthesis and secretion of Matrix Metalloproteinase-2 (MMP-2), which is involved in atherosclerotic plaque instabilization. This effect was prevented by inhibitors of Mitogen Activated Protein Kinase (MAPK) and Phosphatidylinositol 3-Kinase (PI3-K) pathways, allowing to hypothesize that homocysteine activates both these pathways, likely via a receptor-mediated mechanism. One possible receptor is N-methyl-D-aspartate receptor (NMDAr), which is expressed in VSMC and is involved in homocysteine effects in other cell types.
  
  
  METHODS
  VSMC exposed to DL-homocysteine or NMDA (100 micromol/L for both; 5 min-8 hours), were investigated by measuring: i) phosphorylation of ERK1/2, p38MAPK (signaling molecules of MAPK pathway) and Akt and p70S6K (signaling molecules of PI3-K pathway) by western blot; ii) synthesis and secretion of MMP-2 (western blot); iii) activation of MMP-2 (gelatin zimography). To evaluate NMDAr involvement in the homocysteine effects, the experiments were repeated in the presence of a non-competitive NMDAr-antagonist MK-801 (50 micromol/L) or L-glycine (10 micromol/L), which inhibits NMDAr function by promoting its internalization.
  
  
  RESULTS
  DL-homocysteine and NMDA time-dependently increased: i) the phosphorylation of ERK1/2, p38 MAPK, Akt and p70S6K (ANOVA, p<0.0001); ii) the synthesis, secretion and activation of MMP-2. DL-homocysteine and NMDA effects were prevented by VSMC pre-incubation with MK-801 or high L-glycine concentrations.
  
  
  CONCLUSIONS
  In human VSMC homocysteine-at concentrations associated with increased cardiovascular risk- activates MAPK and PI3-K pathways and MMP-2 synthesis and secretion through NMDA receptor, a potential mechanism involved in intracellular signaling in response to homocysteine in VSMC.
authors.affiliation: Metabolic Disease and Diabetes Unit, Department of Clinical and Biological Sciences of the University of Turin, San Luigi Gonzaga Hospital, Orbassano (Turin), Italy. gabriella.doronzo@unito.it
authors.forename: Gabriella
authors.initials: G
authors.lastname: Doronzo
authors.forename: Isabella
authors.initials: I
authors.lastname: Russo
authors.forename: Paola
authors.initials: P
authors.lastname: Del Mese
authors.forename: Michela
authors.initials: M
authors.lastname: Viretto
authors.forename: Luigi
authors.initials: L
authors.lastname: Mattiello
authors.forename: Mariella
authors.initials: M
authors.lastname: Trovati
authors.forename: Giovanni
authors.initials: G
authors.lastname: Anfossi
mesh_terms: D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D006710:Homocysteine / Q000494:pharmacology*; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D013997:Time Factors
chemical_list: D016194:Receptors, N-Methyl-D-Aspartate; D006710:Homocysteine; D019869:Phosphatidylinositol 3-Kinases; D020928:Mitogen-Activated Protein Kinases