However, extracellular ATP is rapidly hydrolyzed by a cascade of ectonucleotidases resulting in an enhanced level of adenosine. Cor respondingly, excitotoxic conditions LDK378 such as ischemia, hypoxia, seizure and head injury are known to induce a rapid increase in extracellular adenosine concentrations, up to 100 times that of the Inhibitors,Modulators,Libraries resting concentration. There is abundant evidence for immune regulation by adenosine including expression and release of growth factors and cytokines such as nerve growth factor, S100beta, IL 6 and CCL2 in glial cells. However, it is not known whether adenosine can induce LIF expression in astrocytes. In the present study, we investigated the potential in fluence of adenosine receptor activity on LIF release from cultured astrocytes.
Methods Chemicals and reagents Neurobasal media, Hanks balanced salt solution, phosphate buffered saline, Inhibitors,Modulators,Libraries sodium pyruvate, L glutamine, penicillin Inhibitors,Modulators,Libraries streptomycin, hydroxyethyl pipera zineethanesulfonic acid, glutaMAX 1 and B27 supplement were obtained from Gibco. Dulbeccos modified Eagles medium and fetal calf serum were obtained from PAA Laboratories. Trypsin was ob tained from Life Technologies. L leucine methyl ester and the remaining cell medium components were purchased from Sigma Aldrich. Recombinant mouse LIF was obtained from Milli pore. Brefeldin A, caffeine, L glutamate, adenosine A2B receptor antagonist, protein kinase A inhibitor, protein kinase C inhibitor, p38 mitogen activated protein kinases inhibitor, and adenosine analog were obtained from Sigma Aldrich.
Non hydrolysable ATP, adenosine A2A receptor antagonist, adenosine A2A receptor agonist and MEK1 2 inhibitor were obtained from Tocris Bioscience. NF kB inhibitor and c Jun N terminal kinase inhibitor were obtained from Calbiochem. Reagents used in immunoblotting experiments were purchased from Bio Rad Laboratories with the Inhibitors,Modulators,Libraries exception of the polyvinylidene fluoride membranes that were obtained from Millipore. Animals Wild type C57BL 6 J mice were obtained from Central Laboratory Animal Facility. Adenosine A2B receptor knock out mice with the same genetic background were kindly pro vided by Professor Marco Idzko. Wild type C57BL 6 J mice were obtained from Harlan. All procedures were in accordance with the regulation of the Ethical Committee for the use of experimental animals of the University of Groningen, The Netherlands.
Animals were housed in standard MakrolonTM cages and maintained on a 12 hour light dark cycle. They received food and water ad libitum. Primary neuronal culture Primary culture of cortical neurons from mouse embryo was established as described Inhibitors,Modulators,Libraries previously. Briefly, cortices from embryonic brains were dissected in ice cold HBSS supplemented with 30% selleck glucose. Menin ges were removed, and the tissues were treated with trypsin before they were gently dissociated by trituration in neuronal culture media. The cell suspension was filtered using cell strainer before centrifuga tion.