Activation mechanisms of platelet-activating factor in U937 cells: possible involvement of protein kinase C.
We have previously demonstrated that platelet-activating factor (PAF) binds specifically on cell membranes isolated from U937 cells. We now describe biological evidence showing that the effect of PAF on U937 cells is a receptor-mediated event. myo-[3H]Inositol-labeled U937 cells were used to investigate the possible role of phosphoinositide metabolism in these cells after binding of PAF. Formation of inositol phosphates (IP1, IP2, and IP3) in response to PAF was increased two- to threefold more than in vehicle control in U937 cells. The effect of PAF on endogenous protein phosphorylation was also studied by using 32PO4-labeled cells. PAF stimulates the phosphorylation of a 45-kDa protein in a time-dependent and dose-related fashion. Since the phospholipase C-generated diglyceride is an important activator of protein kinase C, the phosphorylated 45-kDa protein could be the substrate of protein kinase C. In this regard, we were able to demonstrate that phorbol ester enhances the phosphorylation of the same 45-kDa protein band. In addition, sphingosine, a protein kinase C inhibitor, inhibits the phosphorylation of the same 45-kDa protein band. Down-regulation of the protein kinase C also inhibits the 45-kDa protein phosphorylation. These results suggest that protein kinase C is involved in the PAF-U937 cell interaction.
Ong
R C
RC
Department of Medicine, University of Tennessee, Memphis 38104.
Yoo
T J
TJ
Chiang
T M
TM
D004075:Diglycerides / Q000494:pharmacology; D015536:Down-Regulation; D006801:Humans; D066298:In Vitro Techniques; D008264:Macrophages / Q000502:physiology*; D008970:Molecular Weight; D010703:Phorbol Esters / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins; D011493:Protein Kinase C / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled; D015398:Signal Transduction; D013110:Sphingosine / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D004075:Diglycerides; D010703:Phorbol Esters; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D011493:Protein Kinase C; D013110:Sphingosine