Cysteinyl‐leukotrienes are released from astrocytes and increase astrocyte proliferation and glial fibrillary acidic protein via cys‐LT₁ receptors and mitogen‐activated protein kinase pathway

Abstract

Cysteinyl-leukotrienes (cys-LTs), potent mediators in inflammatory diseases, are produced by nervous tissue, but their cellular source and role in the brain are not very well known. In this report we have demonstrated that rat cultured astrocytes express the enzymes (5'-lipoxygenase and LTC(4) synthase) required for cys-LT production, and release cys-LTs in resting condition and, to a greater extent, in response to calcium ionophore A23187, 1 h combined oxygen-glucose deprivation or 2-methyl-thioATP, a selective P2Y(1)/ATP receptor agonist. MK-886, a LT synthesis inhibitor, prevented basal and evoked cys-LT release. In addition, 2-methyl-thioATP-induced cys-LT release was abolished by suramin, a P2 receptor antagonist, or by inhibitors of ATP binding cassette proteins involved in cys-LT release. We also showed that astrocytes express cys-LT(1) and not cys-LT(2) receptors. The stimulation of these receptors by LTD(4) activated the mitogen-activated protein kinase (MAPK) pathway. This effect was: (i) insensitive to inhibitors of receptor-coupled Gi protein (pertussis toxin) or tyrosine kinase receptors (genistein); (ii) abolished by MK-571, a cys-LT(1) selective receptor antagonist, or PD98059, a MAPK inhibitor; (iii) reduced by inhibitors of calcium/calmodulin-dependent kinase II (KN-93), Ca(2+)-dependent and -independent (GF102903X) or Ca(2+)-dependent (Gö6976) protein kinase C isoforms. LTD(4) also increased astrocyte proliferation and glial fibrillary acidic protein content, which are considered hallmarks of reactive astrogliosis. Both effects were counteracted by cell pretreatment with MK-571 or PD98059. Thus, cys-LTs released from astrocytes might play an autocrine role in the induction of reactive astrogliosis that, in brain injuries, contributes to the formation of a reparative glial scar.

Activation of Microglia by Amyloid β Requires P2X7 Receptor Expression

“…Release of ATP was fully abrogated by the P2X blocker oATP. Maximal ATP release occurred at 40 M A␤ [25][26][27][28][29][30][31][32][33][34][35] , but even an A␤ [25][26][27][28][29][30][31][32][33][34][35] concentration of 20 M triggered near maximal ATP secretion. In both wtN13 and R-N13, there was a basal ATP release in the absence of A␤ [25][26][27][28][29][30][31][32][33][34][35] stimulation, slightly higher in R-N13 than in wtN13.…”

“…When indicated, CaCl 2 was omitted and 500 M EGTA added. Primary mouse microglial cells were isolated from 2-to 4-day-old postnatal mice as described by Ciccarelli et al (32). More than 98% of the cultured cells were identified as microglia using a macrophage cell-specific F4/80 biotinylated mAb (Serotec) followed by staining with Oregon Green 488 goat anti-rat IgG (Molecular Probes) (33).…”

“…Large P2X 7 pore formation was assessed by means of the standard YO-PRO uptake measurement. An incubation time of 6 h in the presence of A␤ [25][26][27][28][29][30][31][32][33][34][35] was chosen because at this time point A␤ 25-35 -stimulated ATP release was near maximal. Incubation in the presence of A␤ [25][26][27][28][29][30][31][32][33][34][35] made wtN13 cells permeable to YO-PRO (Fig.…”

“…Increase in extracellular LDH was negligible at all time points (not shown). The A␤ [25][26][27][28][29][30][31][32][33][34][35] peptide is the shortest fragment that retains the toxicity of full length A␤. Although the biological effects of the short and full length A␤ peptide overlap, we also investigated the activity of A␤ .…”

“…Cultures were used for experiments 24 h after plating. A␤ [25][26][27][28][29][30][31][32][33][34][35] , A␤ (iA␤), and oxidized ATP (oATP) were purchased from Sigma-Aldrich; A␤ and A␤ 42-1 (iA␤) were purchased from Bachem. All experiments were performed at 37°C.…”