Tumor necrosis factor reduces cAMP production in rat microglia.
cAMP has been reported to exert a neuroprotective role in several in vivo and in vitro models of brain pathologies, mainly by regulating microglial activation and orienting these cells toward a neuroprotective phenotype. In order to elucidate the intracellular pathways regulated by tumor necrosis factor (TNF) in glial cells, I have studied the modulation of cAMP accumulation by TNF in microglia and astrocyte cultures obtained from the neonatal rat brain. Pre-treatment of microglia with TNF reduced in a dose- and time-dependent manner cAMP accumulation induced by forskolin (FSK), in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). The TNF inhibitory action was 90% reverted by a neutralizing polyclonal anti-TNF antibody and was not prevented by a 16 h pre-treatment of microglial cultures with the Gi protein inhibitor pertussis toxin (PTx). These results suggest that TNF acts at a step of the cAMP transduction pathway other than receptors, G proteins, and phosphodiesterases. The target of TNF appeared to be adenylyl cyclase, whose ability to synthesize cAMP was markedly reduced (up to 50%) in membranes prepared from TNF-treated microglial cells, both in basal conditions and after stimulation with FSK. TNF induced a time-dependent degradation of IkappaB-alpha in microglial cells that was reverted by two inhibitors of nuclear factor kappaB activation, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-CBZ-Leu-Leu-Leu-al (MG132). The same inhibitors also markedly prevented the reduction of FSK-evoked cAMP accumulation by TNF, suggesting the involvement of NFkappaB in the regulation of adenylyl cyclase by TNF in microglia. Conversely, cAMP accumulation in astrocytes was not affected by TNF. Based on these findings, it is proposed that the ability of TNF to inhibit cAMP synthesis in microglia may exacerbate its response and contribute to cell damage in neuroinflammation and neurodegeneration, possibly through enhanced release of proinflammatory and/or cytotoxic factors.
Patrizio
Mario
M
Department of Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy. patrizio@iss.it
D000262:Adenylyl Cyclases / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D018920:Coculture Techniques; D000242:Cyclic AMP / Q000096:biosynthesis / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects* / Q000502:physiology; D004660:Encephalitis / Q000139:chemically induced / Q000378:metabolism* / Q000503:physiopathology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000187:drug effects / Q000378:metabolism; D020672:I-kappa B Proteins / Q000187:drug effects / Q000378:metabolism; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D009410:Nerve Degeneration / Q000139:chemically induced / Q000378:metabolism* / Q000503:physiopathology; D037342:Pertussis Toxin / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; D011500:Protein Synthesis Inhibitors; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000262:Adenylyl Cyclases