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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 [3H]-oleic acid or [3H]-arachidonic acid. Ten μmolar thapsigargin and 1mM ATP stimulated the release of both unsaturated acids. ATP had no effect at 10μM 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 P2X7 −/− 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 [3H]-fatty acids by ATP and thapsigargin was diminished by PD-98058, an inhibitor of MEK-1. It was concluded that in LPS-primed macrophages, P2X7 receptors, not P2X4 receptors, activated an iPLA2 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. [Copyright &y& Elsevier]

Abstract: 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 (Δψm) 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 Δψm 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 Δψm 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. [ABSTRACT FROM AUTHOR]

P2X purinergic receptors are receptors which, after ATP binding, form a channel permeant to monovalent and divalent cations. Acinar and ductal cells from salivary glands express P2X4 and P2X7 receptors. The P2X4 receptor has a high affinity for ATP, rapidly desensitizes and is mostly located on the basal membrane of acinar cells. The P2X7 receptor has a very low affinity for ATP. After a sustained activation, the permeability of the channel formed by this receptor increases eventually leading to the death of the cell. This receptor is located mostly on the apical membrane of acinar and ductal cells. It is suggested that the sequential activation of the two receptors contributes to the secretory response to ATP. A low concentration of ATP released by nerve endings transiently activates the P2X4 receptors and promotes the release of secretory granules containing ATP. The local increase of the concentration of the nucleotide at the vicinity of P2X7 receptors accounts for their activation. This further increases the exocytosis., (© 2013 médecine/sciences – Inserm.)

The interaction of lipopolysaccharide-primed murine peritoneal macrophages with ivermectin, an antiparasite drug which potentiates P2X(4) receptors and dynasore which inhibits the GTPase activity of dynamin, a protein contributing to the internalization of plasma membrane proteins, was tested. Murine peritoneal macrophages express P2X(4) receptors which are mostly intracellular. In cells from P2X(7)-knockout mice (KO mice), 10 µm adenosine triphosphate (ATP) provoked a transient increase of the intracellular concentration of calcium. Ivermectin had no effect by itself but potentiated the increase of the intracellular concentration of calcium by ATP. The combination of ATP plus ivermectin also decreased the intracellular concentration of potassium and promoted the secretion of IL-1β. Concentrations of dynasore above 50 µm affected the integrity of mitochondria (MTT test) and of the plasma membrane (release of lactate dehydrogenase, LDH). At a 10 µm concentration, dynasore had no effect on the responses to ATP and on the internalization of P2X(4) receptors. By itself dynasore promoted the release of potassium and the secretion of IL-1β after activation of caspase-1. In conclusion, our results confirm that ivermectin potentiates the responses coupled to P2X(4) receptors probably by interaction with an allosteric site. We also show that this potentiation triggers the release of IL-1β by macrophages. As opposed to ivermectin, dynasore has no effect on P2X(4) receptors. This drug triggers a potassium efflux via a mechanism which does not involve purinergic receptors and generates, in consequence, the activation of caspase-1 and the secretion of IL-1β.

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-1β. The cytokine was expressed by submandibular acini and ducts. Macrophages also expressed IL-1β but at lower concentration than salivary glands. The pre-incubation of macrophages with LPS increased the phosphorylation of IκB and the expression of IL-1β. 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-1β 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-1β axis in spite of the expression of the proteins involved in their recognition.

The response to ATP of peritoneal macrophages from wild-type (WT) and P2X(7)-invalidated (KO) mice was tested. Low concentrations (1-100 μM) of ATP transiently increased the intracellular concentration of calcium ([Ca(2+)](i)) in cells from both mice. The inhibition of the polyphosphoinositide-specific phospholipase C with U73122 inhibited this response especially in WT mice suggesting that the responses coupled to P2Y receptors were potentiated by the expression of P2X(7) receptors. One millimolar ATP provoked a sustained increase in the [Ca(2+)](i) only in WT mice. The response to 10 μM ATP was potentiated and prolonged by ivermectin in both mice. One millimolar ATP increased the influx of extracellular calcium, decreased the intracellular concentration of potassium ([K(+)](i)) and stimulated the secretion of interleukin-1β (IL-1β) only in cells from WT mice. Ten micromolar ATP in combination with 3 μM ivermectin reproduced these responses both in WT and KO mice. The secretion of IL-1β was also increased by nigericin in WT mice and the secretory effect of a combination of ivermectin with ATP in KO mice was suppressed in a medium containing a high concentration of potassium. In WT mice, 150 μM BzATP stimulated the uptake of YOPRO-1. Incubation of macrophages from WT and KO mice with 10 μM ATP resulted in a small increase of YOPRO-1 uptake, which was potentiated by addition of 3 μM ivermectin. The uptake of this dye was unaffected by pannexin-1 blockers. In conclusion, prolonged stimulation of P2X(4) receptors by a combination of low concentrations of ATP plus ivermectin produced a sustained activation of the non-selective cation channel coupled to this receptor. The ensuing variations of the [K(+)](i) triggered the secretion of IL-1β. Pore formation was also triggered by activation of P2X(4) receptors. Higher concentrations of ATP elicited similar responses after binding to P2X(7) receptors. The expression of the P2X(7) receptors was also coupled to a better response to P2Y receptors.

The bactericidal activity of a cholic acid antimicrobial derivative, CSA-13, was tested against eight strains of Pseudomonas aeruginosa (both reference and clinical strains) and compared with the response to tobramycin. In planktonic cultures, the minimal inhibitory and minimal bactericidal concentrations of CSA-13 and tobramycin were in the 1-25 mg/L range except for one mucoid clinical strain which was much less sensitive to tobramycin (minimal bactericidal concentration, 65-125 mg/L). In young (24 h) biofilms, the sensitivity to CSA-13 was reduced (half-maximal concentration CSA-13 averaged 88 mg/L) and varied among the eight strains. The sensitivity to tobramycin was also very variable among the strains and some were fully resistant to the aminoglycoside. The combination of tobramycin with CSA-13 was synergistic in five strains. Only one strain showed antagonism between the two drugs at low concentrations of CSA-13. One reference and five clinical strains were tested in mature (12 days) biofilms. The effect of CSA-13 was delayed, some strains requiring 9 days exposure to the drug to observe a bactericidal effect. All the strains were tolerant to tobramycin but the addition of CSA-13 with tobramycin was synergistic in three strains. CSA-13 permeabilized the outer membrane of the bacteria (half-maximal concentration, 4.4 mg/L). At concentrations higher than 20 mg/L, it also permeabilized the plasma membrane of human umbilical vein endothelial cells. In conclusion, CSA-13 has bactericidal activity against P. aeruginosa even in mature biofilms and cationic steroid antibiotics can thus be considered as potential candidates for the treatment of chronic pulmonary infections of patients with cystic fibrosis. Considering its interaction with the plasma membrane of eukaryotic cells, less toxic derivatives of CSA-13 should be developed.