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Extracellular ATP Triggers Tumor Necrosis Factor‐α Release from Rat Microglia

Abstract: Brain microglia are a major source of inflammatory cytokines, such as tumor necrosis factor‐α (TNF‐α), which have been implicated in the progression of neurodegenerative diseases. Recently, microglia were revealed to be highly responsive to ATP, which is released from nerve terminals, activated immune cells, or damaged cells. It is not clear, however, whether released ATP can regulate TNF‐α secretion from microglia. Here we demonstrate that ATP potently stimulates TNF‐α release, resulting from TNF‐α mRNA expression in rat cultured brain microglia. The TNF‐α release was maximally elicited by 1 mM ATP and also induced by a P2X7 receptor‐selective agonist, 2′‐ and 3′‐O‐(4‐benzoylbenzoyl)adenosine 5′‐triphosphate, suggesting the involvement of P2X7 receptor. ATP‐induced TNF‐α release was Ca2+‐dependent, and a sustained Ca2+ influx correlated with the TNF‐α release in ATP‐stimulated microglia. ATP‐induced TNF‐α release was inhibited by PD 098059, an inhibitor of extracellular signal‐regulated protein kinase (ERK) kinase 1 (MEK1), which activates ERK, and also by SB 203580, an inhibitor of p38 mitogen‐activated protein kinase. ATP rapidly activated both ERK and p38 even in the absence of extracellular Ca2+. These results indicate that extracellular ATP triggers TNF‐α release in rat microglia via a P2 receptor, likely to be the P2X7 subtype, by a mechanism that is dependent on both the sustained Ca2+ influx and ERK/p38 cascade, regulated independently of Ca2+ influx.

Production and release of neuroprotective tumor necrosis factor by P2X7 receptor-activated microglia

After a brain insult, ATP is released from injured cells and activates microglia. The microglia that are activated in this way then release a range of bioactive substances, one of which is tumor necrosis factor (TNF). The release of TNF appears to be dependent on the P2X7 receptor. The inhibitors 1,4-diamino-2,3-dicyano-1,4-bis[2-amino-phenylthio]butadiene (U0126), anthra[1,9-cd]pyrazol-6(2H)-one (SP600125), and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)IH-imidazole (SB203580), which target MEK (mitogen-activated protein kinase kinase), JNK (c-Jun N-terminal kinase), and p38, respectively, all potently suppress the production of TNF in ATP-stimulated microglia, whereas the production of TNF mRNA is strongly inhibited by U0126 and SP600125. SB203580 did not affect the increased levels of TNF mRNA but did prevent TNF mRNA from accumulating in the cytoplasm. The ATP-provoked activation of JNK and p38 [but not extracellular signal-regulated kinase (ERK)] could be inhibited by brilliant blue G, a P2X7 receptor blocker, and by genistein and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, which are general and src-family-specific tyrosine kinase inhibitors, respectively. Most important, we found that treatment of the microglia in neuron-microglia cocultures with the P2X7 agonist 2′-3′-O-(benzoyl-benzoyl) ATP led to significant reductions in glutamate-induced neuronal cell death, and that either TNF-α converting enzyme inhibitor or anti-TNF readily suppressed the protective effect implied by this result. Together, these findings indicate that both ERK and JNK are involved in the regulation of TNF mRNA expression, that p38 is involved in the nucleocytoplasmic transport of TNF mRNA, and that a PTK (protein tyrosine kinase), possibly a member of the src family, acts downstream of the P2X7 receptor to activate JNK and p38. Finally, our data suggest that P2X7 receptor-activated microglia protect neurons against glutamate toxicity primarily because they are able to release TNF.

Microglial α7 nicotinic acetylcholine receptors drive a phospholipase C/IP3 pathway and modulate the cell activation toward a neuroprotective role

Microglia perform both neuroprotective and neurotoxic functions in the brain, with this depending on their state of activation and their release of mediators. Upon P2X7 receptor stimulation, for example, microglia release small amounts of TNF, which protect neurons, whereas LPS causes massive TNF release leading to neuroinflammation. Here we report that, in rat primary cultured microglia, nicotine enhances P2X7 receptor‐mediated TNF release, whilst suppressing LPS‐induced TNF release but without affecting TNF mRNA expression via activation of α7 nicotinic acetylcholine receptors (α7 nAChRs). In microglia, nicotine elicited a transient increase in intracellular Ca2+ levels, which was abolished by specific blockers of α7 nAChRs. However, this response was independent of extracellular Ca2+ and blocked by U73122, an inhibitor of phospholipase C (PLC), and xestospongin C, a blocker of the IP3 receptor. Repeated experiments showed that currents were not detected in nicotine‐stimulated microglia. Moreover, nicotine modulation of LPS‐induced TNF release was also blocked by xestospongin C. Upon LPS stimulation, inhibition of TNF release by nicotine was associated with the suppression of JNK and p38 MAP kinase activation, which regulate the post‐transcriptional steps of TNF synthesis. In contrast, nicotine did not alter any MAP kinase activation, but enhanced Ca2+ response in P2X7 receptor‐activated microglia. In conclusion, microglial α7 nAChRs might drive a signaling process involving the activation of PLC and Ca2+ release from intracellular Ca2+ stores, rather than function as conventional ion channels. This novel α7 nAChR signal may be involved in the nicotine modification of microglia activation towards a neuroprotective role by suppressing the inflammatory state and strengthening the protective function.

Eumenitin, a novel antimicrobial peptide from the venom of the solitary eumenine wasp Eumenes rubronotatus.

A novel antimicrobial peptide, eumenitin, was isolated from the venom of the solitary eumenine wasp Eumenes rubronotatus. The sequence of eumenitin, Leu-Asn-Leu-Lys-Gly-Ile-Phe-Lys-Lys-Val-Ala-Ser-Leu-Leu-Thr, was mostly analyzed by mass spectrometry together with Edman degradation, and corroborated by solid-phase synthesis. This peptide has characteristic features of cationic linear alpha-helical antimicrobial peptides, and therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the CD spectra of eumenitin in the presence of TFE or SDS showed a high content of alpha-helical conformation. Eumenitin exhibited inhibitory activity against both Gram-positive and Gram-negative bacteria, and moderately stimulated degranulation from the rat peritoneal mast cells and the RBL-2H3 cells, but showed no hemolytic activity against human erythrocytes. This antimicrobial peptide in the eumenine wasp venom may play a role in preventing potential infection by microorganisms during prey consumption by their larvae.

Hypoxic stress activates chaperone-mediated autophagy and modulates neuronal cell survival

Autophagy is a conserved mechanism responsible for the continuous clearance of unnecessary organelles or misfolded proteins in lysosomes. Three types of autophagy have been reported in the difference of substrate delivery to lysosome: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Among these types, CMA is a unique autophagy system that selectively degrades substrates detected by heat shock cognate protein 70 (HSC70). Recently, autophagic cell death has been reported to be involved in neuronal death following brain ischemia; however, the contribution of CMA to neuronal death/survival after ischemic stress has not been addressed. In the present study, we determined whether quantitative alterations in LAMP-2A, which is the key molecule in CMA, would modulate neuronal cell survival under hypoxic conditions. Incubation of Neuro2A cells in a hypoxic chamber (1% O(2), 5% CO(2)) increased the level of LAMP-2A and induced accumulation of LAMP-2A-positive lysosomes in the perinuclear area, which is a hallmark of CMA activation. The activation of CMA in response to hypoxia was also confirmed by the GAPDH-HaloTag CMA indicator system at the single cell level. Next, we asked whether CMA was involved in cell survival during hypoxia. Blocking LAMP-2A expression with siRNA increased the level of cleaved caspase-3 and the number of propidium iodide-positive cells after hypoxic stress regardless of whether macroautophagy could occur, whereas the administration of mycophenolic acid, a potent CMA activator, rescued hypoxia-mediated cell death. Finally, we asked whether CMA was activated in the neurons after middle cerebral artery occlusion in vivo. The expression of LAMP-2A was significantly increased in the ischemic hemisphere seven days after brain ischemia. These results indicate that CMA is activated during hypoxia and contributes to the survival of cells under these conditions.

Interleukin‐1β‐induced substance P release from rat cultured primary afferent neurons driven by two phospholipase A2 enzymes: secretory type IIA and cytosolic type IV

We previously described that recombinant interleukin‐1β (IL‐1β) induced the significant release of substance P (SP) via a cyclooxygenase (COX) pathway in primary cultured rat dorsal root ganglion (DRG) cells. In the present study, we examined the involvement of two types of phospholipase A2 (PLA2) enzymes, which lie upstream of COX in the prostanoid‐generating pathway, in the IL‐1β‐induced release of SP from DRG cells. The expression of type ΙΙΑ secretory PLA2 (sPLA2‐IIA) mRNA was undetectable by ribonuclease protection assay in non‐treated DRG cells, while in DRG cells incubated with 1 ng/mL of IL‐1β, the expression was induced in a time‐dependent manner. On the other hand, type IV cytosolic PLA2 (cPLA2) mRNA was constitutively expressed in the non‐treated DRG cells, and treatment with 1 ng/mL of IL‐1β for 3 h significantly increased the levels of cPLA2 mRNA. The IL‐1β‐induced SP release was significantly inhibited by the sPLA2 inhibitor, thioetheramide phosphorylcholine (TEA‐PC), and the cPLA2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3). Furthermore AACOCF3 suppressed the induction of sPLA2‐IIA mRNA expression induced by IL‐1β. These observations suggested that two types of PLA2, sPLA2‐IIA and cPLA2, were involved in the IL‐1β‐induced release of SP from DRG cells, and that the functional cross‐talk between the two enzymes might help to control their activity in the prostanoid‐generating system in DRG cells. These events might be key steps in the inflammation‐induced hyperactivity in primary afferent neurons of spinal cord.

Nitric oxide synergistically potentiates interleukin-1β-induced increase of cyclooxygenase-2 mRNA levels, resulting in the facilitation of substance P release from primary afferent neurons: involvement of cGMP-independent mechanisms

We previously demonstrated that cultured rat dorsal root ganglion (DRG) cells respond to stimulation with interleukin-1 beta (IL-1 beta) by releasing substance P (SP), and this response is regulated via the cyclooxygenase (COX)-2 pathway. In this study, to ascertain the interaction between nitric oxide (NO) and prostaglandins in primary afferent neurons, we investigated the effect of NO on the IL-1 beta-induced release of SP in cultured DRG cells. An NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), did not in itself evoke SP release. However, it potentiated the IL-1 beta-induced release of SP. Similarly, while SNAP did not elicit the expression of COX-2 mRNA, it potentiated the expression induced by IL-1 beta in cultured DRG cells, and this potentiation was significantly suppressed by the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO). Moreover, SNAP also potentiated the expression of COX-2 protein induced by IL-1 beta in cultured DRG cells. The stimulatory effect of SNAP on the IL-1 beta-induced release of SP was completely inhibited on co-incubation with a selective COX-2 inhibitor, NS-398. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a potent inhibitor of soluble guanylate cyclase, did not suppress, and a membrane-permeable cGMP analogue, 8-Br-cGMP, did not mimic the stimulatory effects of SNAP in DRG cells. These results suggest that in cultured DRG cells, NO potentiates the IL-1 beta-induced increase in COX-2 expression via a soluble guanylate cyclase-cGMP-independent pathway, resulting in facilitation of SP release. The interaction between NO and COX in primary afferent neurons might contribute to the change in nociceptive perception in inflammatory hyperalgesia.

Decoralin, a novel linear cationic α-helical peptide from the venom of the solitary eumenine wasp Oreumenes decoratus

A novel peptide, decoralin, was isolated from the venom of the solitary eumenine wasp Oreumenes decoratus. Its sequence, Ser-Leu-Leu-Ser-Leu-Ile-Arg-Lys-Leu-Ile-Thr, was determined by Edman degradation and corroborated by solid-phase synthesis. This sequence has the characteristic features of linear cationic alpha-helical peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, it can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the CD spectra of decoralin in the presence of TFE or SDS showed a high alpha-helical conformation content. In a biological evaluation, decoralin exhibited a significant broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. A synthetic analog with C-terminal amidation showed a much more potent activity in all the biological assays.

Substance P release evoked by capsaicin or potassium from rat cultured dorsal root ganglion neurons is conversely modulated with bradykinin

To clarify the molecular mechanism of substance P (SP) release from dorsal root ganglion (DRG) neurons, we investigated the involvement of several intracellular effectors in the regulation of SP release evoked by capsaicin, potassium or/and bradykinin. Bradykinin‐evoked SP release from cultured adult rat DRG neurons was attenuated by either the mitogen‐activated protein kinase kinase (MEK) inhibitor (U0126) or cycloheximide. As the long‐term exposure of DRG neurons to bradykinin (3 h) resulted in extracellular signal‐regulated kinase (ERK) phosphorylation at an early stage and thereafter induced cyclooxygenase‐2 (COX‐2) protein expression, which both contribute to the SP release triggered by bradykinin B2 receptor. The long‐term exposure of DRG neurons to bradykinin enhanced the SP release by capsaicin, but attenuated that by potassium. Interestingly, the inositol 1,4,5‐triphosphate (IP3)‐induced calcium release blocker [2‐aminoethyl diphenylborinate (2‐APB)] not only inhibited the potassium‐evoked SP release, but also completely abolished the enhancement of capsaicin‐induced SP release by bradykinin from cultured DRG neurons. Together, these findings suggest that the molecular mechanisms of SP release by bradykinin involve the activation of MEK, and also require the de novo protein synthesis of COX‐2 in DRG neurons. The IP3‐dependent calcium release could be involved in the processes of the regulation by bradykinin of capsaicin‐triggered SP release.

Protein kinase C‐α mediates TNF release process in RBL‐2H3 mast cells

1 To clarify the mechanism of mast cell TNF secretion, especially its release process after being produced, we utilized an antiallergic drug, azelastine (4‐(p‐chlorobenzyl)‐2‐(hexahydro‐1‐methyl‐1H‐azepin‐4‐yl)‐1‐(2H)‐ phthalazinone), which has been reported to inhibit TNF release without affecting its production in ionomycin‐stimulated RBL‐2H3 cells. 2 Such inhibition was associated with the suppression of an ionomycin‐induced increase in membrane‐associated PKC activity rather than the suppression of Ca2+ influx, suggesting that PKC might be involved in TNF release process. 3 To see whether conventional PKC family (cPKCs) are involved, we investigated the effects of a selective cPKC inhibitor (Gö6976) and an activator (thymeleatoxin) on TNF release by adding them 1 h after cell stimulation. By this time, TNF mRNA expression had reached its maximum. Gö6976 markedly inhibited TNF release, whereas thymeleatoxin enhanced it, showing a key role of cPKC in TNF post‐transcriptional process, possibly its releasing step. 4 To determine which subtype of cPKCs could be affected by azelastine, Western blotting and live imaging by confocal microscopy were conducted to detect the translocation of endogenous cPKC (α, βI and βII) and transfected GFP‐tagged cPKC, respectively. Both methods clearly demonstrated that 1 μM azelastine selectively inhibits ionomycin‐triggered translocation of αPKC without acting on βI or βIIPKC. 5 In antigen‐stimulated cells, such a low concentration of azelastine did not affect either αPKC translocation or TNF release, suggesting a functional link between αPKC and the TNF‐releasing step. 6 These results suggest that αPKC mediates the TNF release process and azelastine inhibits TNF release by selectively interfering with the recruitment of αPKC in the pathway activated by ionomycin in RBL‐2H3 cells.