ABSTRACT
Free fatty acid releases are triggered by PLA2 activation and are substrates for many enzymes such as cyclooxygenases. These reactions are responsible for the production of many prostaglandins implicated in the inflammation yet many purinergic receptors have been implicated in diseases characterised by chronic inflammation. The role of P2X receptors was evaluated in LPS-primed murine peritoneal macrophages which were labelled with either [(3)H]-oleic acid or [(3)H]-arachidonic acid. Ten µmolar thapsigargin and 1mM ATP stimulated the release of both unsaturated acids. ATP had no effect at 10 µM and ivermectin had no effect on the response to ATP. The response to ATP was inhibited by magnesium and was not observed with cells from P2X(7)(-/-) mice. The response to ATP was not affected by the removal of extracellular calcium and was inhibited by arachidonyltrifluoromethyl ketone and bromoenol lactone but not by pyrrophenone. The release of the [(3)H]-fatty acids by ATP and thapsigargin was diminished by PD-98058, an inhibitor of MEK-1. It was concluded that in LPS-primed macrophages, P2X(7) receptors, not P2X(4) receptors, activated an iPLA(2) and promoted the release of unsaturated fatty acids secondary to the activation of a kinase. This response might contribute to the inflammation provoked by extracellular ATP.
Subject(s)
Macrophages, Peritoneal/drug effects , Phospholipases A2, Calcium-Independent/metabolism , Purinergic P2 Receptor Agonists/pharmacology , Receptors, Purinergic P2X7/metabolism , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Animals , Arachidonic Acid/biosynthesis , Arachidonic Acid/metabolism , Calcium/metabolism , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Gene Deletion , Ivermectin/pharmacology , Lipopolysaccharides/pharmacology , MAP Kinase Kinase 1/antagonists & inhibitors , MAP Kinase Kinase 1/metabolism , Macrophages, Peritoneal/cytology , Macrophages, Peritoneal/enzymology , Magnesium/metabolism , Magnesium/pharmacology , Mice , Mice, Knockout , Oleic Acid/biosynthesis , Oleic Acid/metabolism , Organic Chemicals/pharmacology , Receptors, Purinergic P2X7/genetics , Thapsigargin/pharmacology , TritiumABSTRACT
The regulation of interleukin (IL)-1 expression and secretion by salivary glands and macrophages in response to lipopolysaccharides (LPS) was compared. In wild-type mice, injection of LPS significantly decreased the volume of saliva stimulated by pilocarpine and increased its protein and amylase concentration. It did not modify the salivary concentration of IL-1ß. The cytokine was expressed by submandibular acini and ducts. Macrophages also expressed IL-1ß but at lower concentration than salivary glands. The pre-incubation of macrophages with LPS increased the phosphorylation of IκB and the expression of IL-1ß. Adenosine triphosphate also promoted the secretion of the cytokine by these cells. These responses were absent in submandibular gland cells. These glands expressed CD14, TLR4 and MyD88. P2X(7)-KO mice secreted a lower volume of saliva which contained less proteins and amylase. In conclusion, IL-1ß is constitutively expressed by submandibular glands and its secretion is not regulated by a P2X(7) agonist. In these cells, LPS do not activate the nuclear factor-κB-pro-IL-1ß axis in spite of the expression of the proteins involved in their recognition.
Subject(s)
Interleukin-1beta/immunology , Lipopolysaccharides/immunology , Macrophages/metabolism , Saliva/metabolism , Salivary Glands/immunology , Adenosine Triphosphate/pharmacology , Animals , Cells, Cultured , Gene Expression Regulation/drug effects , I-kappa B Kinase/metabolism , Interleukin-1beta/genetics , Macrophages/drug effects , Macrophages/immunology , Macrophages/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation/drug effects , Pilocarpine/administration & dosage , Receptors, Purinergic P2X7/genetics , Salivary Glands/drug effects , Salivary Glands/pathology , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/immunologyABSTRACT
BACKGROUND: Agonists of P2X7 receptors increase the production of reactive oxygen species (ROS) in immunocytes. In this work we tested this response and its effect on mitochondrial inner membrane potential (Deltapsi(m)) in exocrine glands. METHODS: The production of ROS by rat submandibular glands was investigated by measuring the oxidation of dichlorodihydrofluorescein (DCFH), a fluorescent probe. The Deltapsi(m) was estimated with tetramethylrhodamine. RESULTS: Activation of P2X7 receptors by ATP or Bz-ATP increased the production of ROS. This response was not modified by inhibitors of phospholipase A2 or of various kinases. The effect of ATP was calcium-dependent and was blocked by diphenyliodonium, an inhibitor of flavoproteins. It was not affected by rotenone, an inhibitor of the complex I of the mitochondrial electron transfer chain. Scavengers of ROS had no effect on the dissipation of Deltaψ(m) by ATP. CONCLUSIONS: We conclude that, in rat submandibular glands, P2X7 receptors stimulate in a calcium-dependent manner an oxidase generating ROS, suggesting the involvement of the dual oxidase Duox2. The production of ROS does not contribute to the depolarization of mitochondria by purinergic agonists. GENERAL SIGNIFICANCE: Purinergic receptors could be regulators of the bactericidal properties of saliva by promoting both the secretion of peroxidase from acinar cells and by activating Duox2.
