P2Y receptors activate MAPK/ERK through a pathway involving PI3K/PDK1/PKC-zeta in human vein endothelial cells.
OBJECTIVE
In this study we investigated the effects of P2 receptors in the regulation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) in human umbilical vein endothelial cells (HUVEC).


METHODS
Cytosolic Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2/AM, and MAPK/ ERK phosphorylation using Western blot analysis.


RESULTS
ATP, 2-meSATP, UTP and UDP cause a rapid and transitory increase in the phosphorylation of MAPK/ERK. In contrast, negligible response was seen for a,Beta-meATP, a general P2X receptors agonist. ATP-dependent activation of MAPK/ERK was prevented by pretreatment of HUVEC with pertussis toxin or MEK inhibitor PD98059. In addition, activation of the MAPK/ ERK cascade by ATP was blocked in cells pretreated with wortmannin and LY294002, but not by U73122, BAPTA or a Ca(2+)-free medium. Furthermore, an inhibition of ATP-dependent MAPK/ERK phosphorylation was observed in HUVEC pretreated with high doses of GF109203X or myristoylated PKC- zeta pseudosubstrate. Similar results were observed when cells were pretreated with the Src tyrosine kinase inhibitor PP2. However, ATP-stimulated MAPK/ERK activation was unaffected in cells pretreated with AG1478 or perillic acid. We also found that ATP stimulates both the phosphorylation of 3- phosphoinositide-dependent protein kinase-1 (PDK1) and its translocation to plasma membrane in a time-dependent manner.


CONCLUSIONS
These observations suggest that the effects mediated by ATP in HUVEC occur via PTX-sensitive G-protein-coupled P2Y receptors through PI3K-dependent mechanisms, in which PDK1 and PKC-zeta are two key molecules within signal cascade leading to MAPK/ERK activation.
Montiel
Mercedes
M
Department of Biochemistry and Molecular Biology, Malaga University, Malaga, Spain.
de la Blanca
Enrique Pérez
EP
Jiménez
Eugenio
E
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors; D014530:Uridine Diphosphate / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000494:pharmacology
D005419:Flavonoids; D018048:Receptors, Purinergic P2; D014530:Uridine Diphosphate; D000255:Adenosine Triphosphate; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D014544:Uridine Triphosphate