title: Activation of the adenosine A3 receptor in RAW 264.7 cells inhibits lipopolysaccharide-stimulated tumor necrosis factor-alpha release by reducing calcium-dependent activation of nuclear factor-kappaB and extracellular signal-regulated kinase 1/2.
abstract: Bacterial lipopolysaccharide (LPS) activates the immune system and promotes inflammation via Toll-like receptor (TLR) 4, which regulates the synthesis and release of tumor necrosis factor (TNF)-alpha and other inflammatory cytokines. Previous studies have shown that the nucleoside adenosine suppresses LPS-stimulated TNF-alpha release in human UB939 macrophages by activating an adenosine A(3) receptor (A(3)AR) subtype on these cells. In this study, we examined the mechanism(s) underlying A(3)AR-dependent inhibition of TNF-alpha release in a mouse (RAW 264.7) cell line. Treatment of RAW 264.7 cells with LPS (3 mug/ml) increased TNF-alpha release, which was reduced in a dose-dependent manner by adenosine analogs N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) and R-phenylisopropyladenosine and reversed by selective A(3)AR blockade. The increase in TNF-alpha release was preceded by an increase in intracellular Ca(2+) levels. Inhibition of intracellular Ca(2+) release by IB-MECA, a selective agonist of the A(3)AR, or with BAPTA-AM, an intracellular Ca(2+) chelator, reduced LPS-stimulated TNF-alpha release. Activation of the A(3)AR or inhibition of intracellular Ca(2+) release also reduced LPS-stimulated nuclear factor-kappaB (NF-kappaB) activation and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Similar inhibition by A(3)AR was observed for LPS-stimulated inducible nitric-oxide synthase. These data support the contention that inhibition of LPS-stimulated release of inflammatory molecules, such as TNF-alpha and NO via the A(3)AR, involves suppression of intracellular Ca(2+)signaling, leading to suppression of NF-kappaB and ERK1/2 pathways.
authors.affiliation: Dept. of Pharmacology, Southern Illinois University School of Medicine, P.O. Box 19230, Springfield, IL 62794-1222, USA.
authors.forename: Lynn
authors.initials: L
authors.lastname: Martin
authors.forename: Sandeep C
authors.initials: SC
authors.lastname: Pingle
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Hallam
authors.forename: Leonard P
authors.initials: LP
authors.lastname: Rybak
authors.forename: Vickram
authors.initials: V
authors.lastname: Ramkumar
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D001711:Biotransformation / Q000187:drug effects; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis; D010766:Phosphorylation; D011869:Radioligand Assay; D043684:Receptor, Adenosine A3 / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000378:metabolism*
chemical_list: D008070:Lipopolysaccharides; D016328:NF-kappa B; D043684:Receptor, Adenosine A3; D014409:Tumor Necrosis Factor-alpha; D052247:Nitric Oxide Synthase Type II; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium