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.
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.
Doronzo
Gabriella
G
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
Russo
Isabella
I
Del Mese
Paola
P
Viretto
Michela
M
Mattiello
Luigi
L
Trovati
Mariella
M
Anfossi
Giovanni
G
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
D016194:Receptors, N-Methyl-D-Aspartate; D006710:Homocysteine; D019869:Phosphatidylinositol 3-Kinases; D020928:Mitogen-Activated Protein Kinases