Mechanism of vascular smooth muscle cells activation by hydrogen peroxide: role of phospholipase C gamma.
BACKGROUND
Hydrogen peroxide (H2O2) formation is a critical factor in processes involving ischaemia/ reperfusion. However, the precise mechanism by which reactive oxygen species (ROS) induce vascular damage are insufficiently known. Specifically, activation of phospholipase C gamma (PLCgamma) is a probable candidate pathway involved in vascular smooth muscle cells (VSMC) activation by H2O2.


METHODS
The activation of human venous VSMC was measured as cytosolic free calcium mobilization, shape change and protein phosphorylation, focusing on the role of tyrosine phosphorylation-activated PLCgamma.


RESULTS
The exposure of VSMC to exogenous H2O2 caused a rapid increase in cytosolic free calcium concentration ([Ca2+]i), and induced a significant VSMC shape change. Both effects were dependent on a tyrosine kinase-mediated mechanism, as determined by the blockade of short-term treatment of VSMC with the protein tyrosine kinase inhibitor, genistein. Giving further support to the putative role of phospholipase C (PLC)-dependent pathways, the [Ca2+]i and VSMC shape change response were equally inhibited by the specific PLC blocker, 1-(6-((17-beta-methoxyestra-1,3,5(10)trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). In addition, U73122 had a protective effect against the deleterious action (24 h) of H2O2 on non-confluent VSMC. As a further clarification of the specific pathway involved, the exposure to H2O2 significantly stimulated the tyrosine phosphorylation of PLCgamma with a concentration- and time-profile similar to that of [Ca(2+)](i) mobilization.


CONCLUSIONS
The present study reveals that H(2)O(2) activates PLCgamma on VSMC through tyrosine phosphorylation and that this activation has a major role in rapid [Ca(2+)](i) mobilization, shape-changing actions and damage by H(2)O(2) in this type of cells. These findings have direct implications for understanding the mechanisms of the vascular actions of H(2)O(2) and may help to design pharmacologically protective strategies.
González-Pacheco
Francisco R
FR
Servicio de Nefrología and Servicio de Inmunología, Fundación Jiménez Díaz, Universidad Autónoma, Madrid, Spain.
Caramelo
Carlos
C
Castilla
Maria Angeles
MA
Deudero
Juan J P
JJ
Arias
Javier
J
Yagüe
Susana
S
Jiménez
Sonsoles
S
Bragado
Rafael
R
Alvarez-Arroyo
Maria Victoria
MV
D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000633:toxicity*; D007527:Isoenzymes / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D051966:Phospholipase C gamma; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D007527:Isoenzymes; D017382:Reactive Oxygen Species; D014443:Tyrosine; D008345:Manganese; D006861:Hydrogen Peroxide; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium