ucn-1028-c and edelfosine

The activation of phospholipases is one of the earliest key events in receptor-mediated cellular responses to a number of extracellular signaling molecules. Lipopolysaccharide (LPS) is a principle component of the outer membrane of Gram-negative bacteria and a prime target for recognition by the innate immune system. In the present study, we evaluated the role of specific phospholipase in the activation of a chicken macrophage cell line HD11 by LPS. Activation of HD11 cells by LPS results in induction of nitric oxide (NO). Using selective inhibitors, we have identified that phosphatidylinositol (PI)-phospholipase C (PI-PLC), but not phosphatidylcholine (PC)-phospholipase C (PC-PLC) nor PC-phospholipase D (PC-PLD), was required for LPS-induced NO production. Preincubation with PI-PLC selective inhibitors (U-73122 and ET-18-OCH3) abrogated LPS-induced NO production in HD11 cells, whereas PC-PLC inhibitor (D609), phosphatide phosphohydrolase inhibitor (propranolol), and PC-PLD inhibitor (n-butanol) had no inhibitory effects. We also showed that inhibition of protein kinase C (PKC) by selective inhibitors Ro 31-8220 and calphostin C and chelating intracellular Ca2+ by BAPTA-AM significantly reduced NO production in LPS-stimulated HD11 cells. Our results demonstrate that PI-PLC plays a critical role, most likely through activation of PKC pathway, in TLR4 mediated immune responses of avian macrophage cells to LPS.

Topics: Animals; Cell Line; Chickens; Estrenes; Indoles; Lipopolysaccharides; Macrophages; Naphthalenes; Nitric Oxide; Nitrites; Phosphatidylinositol Diacylglycerol-Lyase; Phosphodiesterase Inhibitors; Phosphoinositide Phospholipase C; Phospholipid Ethers; Protein Kinase C; Pyrrolidinones; Salmonella enteritidis; Toll-Like Receptor 4

Macrophages can adapt to the absence of oxygen by switching to anaerobic glycolysis. In this study, we investigated (a) the roles of fructose 2,6-bisphosphate (Fru-2,6-P2) and ribose 1,5-bisphosphate (Rib-1,5-P2), potent activators of phosphofructokinase, (b) the enzymes responsible for the synthesis of Rib-1,5-P2, and (c) the mechanisms of regulation of these enzymes in H36.12j macrophages during the initial phase of hypoxia. Within 1 min after initiating hypoxia, glycolysis was activated through activation of phosphofructokinase. Over the same period, Fru-2,6-P2 decreased 50% and recovered completely upon reoxygenation. Similar changes in cAMP levels were observed. In contrast, the Rib-1,5-P2 concentration rapidly increased to a maximum level of 8.0 +/- 0.9 nmol/g cell 30 s after hypoxia. Thus, Rib-1,5-P2 was the major factor increasing the rate of glycolysis during the initial phase of hypoxia. Moreover, we found that Rib-1,5-P2 was synthesized by two steps: the ribose-phosphate pyrophosphokinase (5-phosphoribosyl-1-pyrophosphate synthetase; PRPP synthetase) reaction (EC ) catalyzing the reaction, Rib-5-P + ATP --> PRPP + AMP and a new enzyme, "PRPP pyrophosphatase" catalyzing the reaction, PRPP --> Rib-1,5-P2 + P(i). Both PRPP synthetase and PRPP pyrophosphatase were significantly activated 30 s after hypoxia. Pretreatment with 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine and calphostin C prevented the activation of ribose PRPP synthetase and PRPP pyrophosphatase as well as increase in Rib-1,5-P2 and activation of phosphofructokinase 30 s after hypoxia. These data suggest that the activation of the above enzymes was mediated by protein kinase C acting via activation of phosphatidylinositol specific phospholipase C in the macrophages during hypoxia.

Topics: Adenosine Monophosphate; Adenosine Triphosphate; Animals; Cell Line; Cyclic AMP; Enzyme Activation; Enzyme Inhibitors; Fructosediphosphates; Hot Temperature; Hypoxia; Kinetics; Macrophages; Mice; Models, Biological; Naphthalenes; Oxygen; Pentosephosphates; Phospholipid Ethers; Protein Kinase C; Ribose-Phosphate Pyrophosphokinase; Temperature; Time Factors

Covalent modification by phosphorylation is a characteristic of the P-glycoproteins expressed in multidrug-resistant cells. This report describes analysis of P-glycoprotein phosphorylation in multidrug-resistant human KB-V1 cells and a study of the relationship of phosphorylation and drug accumulation. In isolated membranes, phosphorylation of P-glycoprotein by purified protein kinase C (PKC) was rapid, and time-dependent dephosphorylation was inhibited by okadaic acid, an inhibitor of type 1 and type 2A protein phosphatases. In 32P-labeled intact KB-V1 cells, P-glycoprotein phosphorylation was stimulated by both 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, and okadaic acid. Two-dimensional thin layer tryptic phosphopeptide maps indicated that the sites of phosphorylation were similar in control, TPA-treated, and okadaic acid-treated cells and that they corresponded to those phosphorylated by PKC in vitro. The protein kinase inhibitor staurosporine, and the PKC-selective inhibitors calphostin C and the alkyl-lysophospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, inhibited P-glycoprotein phosphorylation in vitro and in intact cells. Drug accumulation assays demonstrated that in KB-V1 cells TPA caused a decrease, whereas staurosporine and calphostin C caused an increase, in accumulation of [3H]vinblastine. These compounds did not significantly alter [3H]vinblastine levels in drug-sensitive KB-3 cells. These results suggest that PKC is chiefly responsible for P-glycoprotein phosphorylation in KB-V1 cells, that membrane-associated protein phosphatases 1 and 2A are active in dephosphorylation of P-glycoprotein, and that phosphorylation of P-glycoprotein may be an important mechanism for modulation of drug-pumping activity.

Topics: Alkaloids; ATP Binding Cassette Transporter, Subfamily B, Member 1; Drug Resistance; Ethers, Cyclic; Humans; KB Cells; Membrane Glycoproteins; Naphthalenes; Okadaic Acid; Peptide Mapping; Phenotype; Phospholipid Ethers; Phosphopeptides; Phosphoprotein Phosphatases; Phosphorylation; Polycyclic Compounds; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate; Verapamil; Vinblastine