Inhibition of Akt phosphorylation by thrombin, histamine and lysophosphatidylcholine in endothelial cells

Differential role of protein kinase C
  • Brynhildur Thors
    Institute of Pharmacy, Pharmacology and Toxicology, University of Iceland, P.O. Box 8216, 128 Reykjavik, Iceland
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  • Haraldur Halldórsson
    Institute of Pharmacy, Pharmacology and Toxicology, University of Iceland, P.O. Box 8216, 128 Reykjavik, Iceland

    Department of Medicine, Landspitalinn-University Hospital, 101 Reykjavik, Iceland
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  • Gudrun D. Clarke
    Institute of Pharmacy, Pharmacology and Toxicology, University of Iceland, P.O. Box 8216, 128 Reykjavik, Iceland
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  • Gudmundur Thorgeirsson
    Corresponding author. Present address: Landspitalinn-University Hospital, 101 Reykjavik, Iceland. Tel.: +354-568-0866; fax: +354-543-6467
    Institute of Pharmacy, Pharmacology and Toxicology, University of Iceland, P.O. Box 8216, 128 Reykjavik, Iceland

    Department of Medicine, Landspitalinn-University Hospital, 101 Reykjavik, Iceland
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      The protein kinase Akt is involved in embryonic vascular development and neoangiogenesis as well as in several endothelial cell functions, including activation of endothelial NO-synthase (eNOS) and promotion of endothelial cell survival. We have examined the effects of G-protein activators thrombin and histamine as well as lysophosphatidylcholine (LPC) on Akt phosphorylation in cultured human umbilical vein endothelial cells (HUVEC). Akt phosphorylation was analyzed with the phosphospecific Akt (Ser473) antibody by Western blotting. While epidermal growth factor (EGF) was a potent stimulator of Akt phosphorylation histamine, thrombin and LPC blocked its activation when used in cotreatment with EGF. Following inhibition or downregulation of protein kinase C (PKC), the inhibitory effect of both histamine and thrombin on the endothelial response to EGF was prevented. Furthermore, stimulation of PKC, using short-term 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, markedly inhibited the stimulatory effects of EGF on Akt phosphorylation. Rottlerin, an inhibitor of the PKCδ, but not Gö6976, which is an inhibitor of α, β, γ and isoforms, reversed the inhibitory effects of histamine. Conversely, inhibition or downregulation of PKC did not prevent the inhibitory effect of LPC. Akt phosphorylation was also increased by sphingosine 1-phosphate (S1P) treatment and this activity was influenced by the various cotreatments in the same way as the activation by EGF. Overall, this study demonstrated that the G-protein activators thrombin and histamine inhibited both EGF- and S1P-mediated Akt phosphorylation in HUVEC by activation of PKCδ, while the inhibitory effects of LPC were independent of PKCδ.


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