AIMS: 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. CONCLUSION: 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.
AIMS: 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. CONCLUSION: 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.
Authors: B K Manne; P Münzer; R Badolia; B Walker-Allgaier; R A Campbell; E Middleton; A S Weyrich; S P Kunapuli; O Borst; M T Rondina Journal: J Thromb Haemost Date: 2018-05-08 Impact factor: 5.824
Authors: Taras Lyubchenko; Heather Woodward; Kristopher D Veo; Nana Burns; Hala Nijmeh; Ganna A Liubchenko; Kurt R Stenmark; Evgenia V Gerasimovskaya Journal: Am J Physiol Cell Physiol Date: 2010-10-20 Impact factor: 4.249