ucn-1028-c and Gram-Negative-Bacterial-Infections

Protein kinase C (PKC) has been implicated in lipopolysaccharide (LPS)-induced endothelial cell (EC) monolayer permeability. Myristoylated alanine-rich C kinase substrate (MARCKS), as a specific PKC substrate, appears to mediate PKC signaling by PKC-dependent phosphorylation of MARCKS and subsequent modification of the association of MARCKS with filamentous actin and calmodulin (CaM). Therefore, in the present study, we investigated LPS-induced MARCKS phosphorylation in bovine pulmonary artery EC (BPAEC). LPS potentiated MARCKS phosphorylation in BPAEC in a time- and dose-dependent manner. The PKC inhibitor, calphostin C, significantly decreased LPS-induced phosphorylation of MARCKS. In addition, downregulation of PKC with phorbol 12-myristate 13-acetate (PMA) did not affect the LPS-induced MARCKS phosphorylation, suggesting that LPS and PMA activate different isoforms of PKC. Pretreatment with SB203580, a specific inhibitor of p38 MAP kinase, or genistein, a tyrosine kinase inhibitor, prevented LPS-induced MARCKS phosphorylation. Phosphorylation at appropriate sites will induce translocation of MARCKS from the cell membrane to the cytosol. However, LPS, in contrast to PMA, did not generate MARCKS translocation in BPAEC, suggesting that MARCKS translocation may not play a role in LPS-induced actin rearrangement and EC permeability. LPS also enhanced both thrombin- and PMA-induced phosphorylation of MARCKS, suggesting that LPS was able to prime these signaling pathways in BPAEC. Because the CaM-dependent phosphorylation of myosin light chains (MLC) results in EC contraction, we studied the effect of LPS on MLC phosphorylation in BPAEC. LPS induced diphosphorylation of MLC in a time-dependent manner, which occurred at lower doses of LPS, than those required to induce MARCKS phosphorylation. In addition, there was no synergism between LPS and thrombin in the induction of MLC phosphorylation. These data indicate that MLC phosphorylation is independent of MARCKS phosphorylation. In conclusion, LPS stimulated MARCKS phosphorylation in BPAEC. This phosphorylation appears to involve activation of PKC, p38 MAP kinase, and tyrosine kinases. Further studies are needed to explore the role of MARCKS phosphorylation in LPS-induced actin rearrangement and EC permeability.

Topics: Animals; Cattle; Cell Membrane; Cells, Cultured; Cytosol; Endothelium, Vascular; Enzyme Inhibitors; Genistein; Gram-Negative Bacterial Infections; Imidazoles; Intracellular Signaling Peptides and Proteins; Lipopolysaccharides; MAP Kinase Signaling System; Membrane Proteins; Mitogen-Activated Protein Kinases; Myosin Light Chains; Myristoylated Alanine-Rich C Kinase Substrate; Naphthalenes; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Protein Transport; Protein-Tyrosine Kinases; Proteins; Pulmonary Artery; Pyridines; Respiratory Distress Syndrome; Tetradecanoylphorbol Acetate; Thrombin