Molecular characterization of the lipopolysaccharide/platelet activating factor- and zymosan-induced pathways leading to prostaglandin production in P388D1 macrophages

Membrane Prostaglandin E Synthase-1: A Novel Therapeutic Target

“…Dexamethasone completely suppressed the effect of both cytokines . Numerous studies have since confirmed these findings (e.g., Han et al, 2002;Schaloske et al, 2005) and p38 and extracellular signalregulated kinase mitogen-activated kinases were shown to be important for induction (Han et al, 2002). TNF-␣ up-regulation of COX-2 and mPGES-1 was attenuated and augmented by interferon-␥, respectively (Wright et al, 2004).…”

TLR-4 and Sustained Calcium Agonists Synergistically Produce Eicosanoids Independent of Protein Synthesis in RAW264.7 Cells

“…Nonreceptor-mediated ionophores such as ionomycin, and purinergic receptor activation by high levels of adenosine triphosphate (ATP), can produce sustained changes in intracellular Ca 2ϩ levels that induce eicosanoid production (21,23,24). Furthermore, the response to Ca 2ϩ agonists has been shown to be synergistically increased by priming cells with a 60-min dose of a TLR activator (21,(25)(26)(27)(28)(29), producing more eicosanoid release than the additive amount of TLR and Ca 2ϩ stimulation alone. Here we employed LC/MS/MS methodology to specifically investigate these mechanisms of eicosanoid generation, identifying two distinct eicosanoid profiles.…”

PGE2 release is independent of upregulation of Group V phospholipase A2 during long-term stimulation of P388D1 cells with LPS

“…To determine which mRNAs are expressed in P388D 1 cells, we performed PCR using cDNA from either nontreated cells or cells treated with LPS for 18 h. The reactions contained 100 ng of cDNA, 1 M of gene-specific primers (described in Ref. 21) and the SYBR Green PCR Master Mix in a total volume of 25 l. PCR was performed on a BioRad iCycler Thermal Cycler. PCR cycles were identical to those used for real-time Q-PCR as described below.…”

“…The real-time Q-PCR consisted of an initial hold at 95ЊC for 10 min, and then 40 cycles of 95ЊC for 15 s and 61ЊC for 1 min. The amount of template cDNA and the primer concentration that was used are described in reference (21). Gene expression was normalized to the house-keeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).…”

“…All the primers used are listed in reference (21) and were selected using the Primer 3 software (www-genome.wi.mit.edu/cgibin/primer/primer3_www.cgi). They encompassed at least one intron of the gene.…”