Prostaglandins and nitric oxide (NO) are among the numerous substances released by activated microglial cells, the brain resident macrophages, and they mediate several important microglial functions. We have previously shown that cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS), the two key enzymes in prostaglandin and NO synthesis, respectively, are rapidly co-induced in rat neonatal microglial cultures activated by bacterial endotoxin (lipopolysaccharide [LPS]) and that COX-2 expression appears to be under the negative control of endogenous as well as exogenous NO. In this study we show that exogenous prostaglandin E2 (PGE2), which is known to increase cyclic adenosine monophosphate (cAMP) levels in microglial cells, downregulates LPS-induced iNOS expression in a dose-dependent manner. The involvement of cAMP in the PGE2-dependent inhibition of iNOS is supported by several pieces of evidence. First, iNOS expression was also inhibited by agents such as isoproterenol and forskolin, which cause an elevation of cAMP levels, and by dibutyryl cAMP (dbcAMP), a cAMP stable analogue. Second, the inhibitory effect of PGE2 was mimicked by 11-deoxy-16,16-dm PGE2, a selective agonist at the PGE2 receptor subtype EP2, coupled to the activation of adenylyl cyclase, but not by sulprostone, a potent agonist at receptor subtypes EP3 and EP1, associated with an inhibition of adenylyl cyclase activity and intracellular Ca2+ elevation, respectively. Third, the inhibitory effect of PGE2 on NO synthesis was blocked by SQ 22,536, a specific inhibitor of adenylyl cyclase. Interestingly, the abrogation of endogenous prostanoid production by several COX inhibitors caused a reduction of iNOS expression, suggesting a positive modulatory effect of endogenous prostanoids of iNOS expression, as opposed to the inhibitory effect of exogenous PGE2.
1 Activated microglial cells are believed to play an active role in most brain pathologies, during which they can contribute to host defence and repair but also to the establishment of tissue damage. These actions are largely mediated by microglial secretory products, among which are prostaglandins (PGs) and nitric oxide (NO). 2 The anti-in¯ammatory protein, lipocortin 1 (LC1) was reported to have neuroprotective action and to be induced by glucocorticoids in several brain structures, with a preferential expression in microglia. In this paper we tested whether the neuroprotective eect of LC1 could be explained by an inhibitory eect on microglial activation. 3 We have previously shown that bacterial endotoxin (LPS) strongly stimulates PGE 2 and NO production in rat primary microglial cultures, by inducing the expression of the key enzymes cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), respectively. 4 Dexamethasone (DEX, 1 ± 100 nM) and LC1-derived N-terminus peptide (peptide Ac2-26, 1 ± 100 mg ml 71) dose-dependently inhibited the production of both PGE 2 and NO from LPS-stimulated microglia. The inhibitory eects of DEX on NO and of the peptide on NO and PGE 2 synthesis were partially abrogated by a speci®c antiserum, raised against the N-terminus of human LC1. The peptide Ac2-26 did not aect arachidonic acid release from control and LPS-stimulated microglial cultures. 5 Western blot experiments showed that the LPS-induced expression of COX-2 and iNOS was eectively down-regulated by DEX (100 nM) and peptide Ac2-26 (100 mg ml 71 ). 6 In conclusion, our ®ndings support the hypothesis that LC1 may foster neuroprotection by limiting microglial activation, through autocrine and paracrine mechanisms.
Cyclooxygenase-2, the inducible isoform of cyclooxygenase, is highly expressed in microglial cells activated by bacterial lipopolysaccharide and is a major regulatory factor in the synthesis of prostanoids, such as prostaglandins, prostacyclin and thromboxanes. Since prostanoids are potent modulators of inflammation, immune responses and neurotoxicity, the regulation of their synthesis may be crucial for balancing microglial neuroprotective and neurotoxic activities. The present study shows that expression of cyclooxygenase-2 and prostanoid production in cultured rat microglia activated by lipopolysaccharide is up-regulated by cyclic AMP (cAMP), as indicated by experiments performed in the presence of adenylyl cyclase activators, cAMP analogues and protein kinase A-specific inhibitors. Exogenous prostaglandin E2 (PGE2), which elevates the cAMP level in microglial cells, also increased the lipopolysaccharide-induced expression of cyclooxygenase-2 and production of thromboxane in a dose- and time-dependent manner. The observations that the lipopolysaccharide-induced prostanoid production was specifically increased by 11-deoxy-16,16-dm PGE2, a selective agonist at the PGE2 receptor EP2 coupled to the activation of adenylyl cyclase, and that the enhancing effect of PGE2 was partially prevented by specific inhibitors of adenylyl cyclase and protein kinase A, suggest that the up-regulation of cyclooxygenase-2 expression by PGE2 is mediated by cAMP, through a putative microglial EP2 receptor. Unexpectedly, non-steroidal anti-inflammatory drugs such as indomethacin and 6-methoxy naphthalene acetic acidic, which inhibit cyclooxygenase enzymatic activity and abrogate prostanoid synthesis, caused a moderate but consistent up-regulation of cyclooxygenase-2 expression. In conclusion, while the strong up-regulation of cyclooxygenase-2 expression by exogenous PGE2 appears to be mediated by EP2 receptors and cAMP, the limited down-regulation caused by anti-inflammatory drug treatments may be either due to arachidonic acid metabolites other than PGE2, or to PGE2 itself, acting through a distinct cAMP-independent signalling pathway.
Epidemiological studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) are neuroprotective, although the mechanisms underlying their beneficial effect remain largely unknown. Given their well-known adverse effects, which of the NSAIDs is the best for neurodegenerative disease management remains a matter of debate. Paracetamol is a widely used analgesic/antipyretic drug with low peripheral adverse effects, possibly related to its weak activity as inhibitor of peripheral cyclooxygenase (COX), the main target of NSAIDs. As microglia play an important role in CNS inflammation and pathogenesis of neurodegenerative diseases, we investigate the effect of paracetamol on rat microglial cultures. Although less potent than other NSAIDs, (indomethacin approximately NS-398 > flurbiprofen approximately piroxicam > paracetamol approximately acetylsalicylic acid), paracetamol completely inhibited the synthesis of prostaglandin E(2) (PGE(2)) in lipopolysaccharide-stimulated microglia, when used at concentrations comparable to therapeutic doses. The drug did not affect the expression of the enzymes involved in PGE(2) synthesis, i.e., COX-1, COX-2, and microsomal PGE synthase, or the release of the precursor arachidonic acid (AA). Paracetamol inhibited the conversion of exogenous AA, but not PGH(2), into PGE(2) indicating that the target of the drug is COX activity. Consistently, paracetamol inhibited with similar IC(50) the synthesis of PGF(2alpha) and thromboxane B(2), two other COX metabolites. Finally, none of the NSAIDs affected the productions of nitric oxide and tumor necrosis factor(alpha), two inflammatory mediators released by activated microglia. As paracetamol was reported to inhibit PG synthesis in peripheral macrophages with an IC(50) at least three orders of magnitude higher than in microglia, we suggest that this drug represents a good tool for treating brain inflammation without compromising peripheral PG synthesis.
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