Reduced basal and lipopolysaccharide-stimulated adenosine A1 receptor expression in the brain of nuclear factor-kappaB p50-/- mice.
Adenosine promotes cytoprotection under conditions of infection, ischemic preconditioning and oxidative stress. Previous studies from our laboratory indicate that the expression of the adenosine A1 receptor (A1AR) is induced by oxidative stress via activation of nuclear factor (NF)-kappaB. The prototypic transcription factor is composed of homo- or heterodimers of p50 and p65 subunits. To determine the role of NF-kappaB in the regulation of the A1AR in vivo, we compared the A1AR RNA and protein levels in the brains of mice lacking the p50 subunit of NF-kappaB (p50-/- mice) and age-matched B6129PF2/J (F2) controls. Radioligand binding assays in the cortex revealed a significantly lower number of A(1)AR (maximal binding capacity, Bmax) in the cortex of p50-/- mice (151+/-62 fmol/mg protein) versus 479+/-181 fmol/mg protein in the F2 (N=5 per strain, P<0.05), but no change in the equilibrium dissociation constant. Similar reductions in A1AR were measured in the hippocampus, brain stem and hypothalamus and in peripheral tissues, such as the adrenal gland, kidney and spleen. Estimation of the A1AR following purification by antibody affinity columns also indicated reduced A1AR in the p50-/- mice cortex, as compared with the F2 mice. A1AR immunocytochemistry indicates distinct neuronal labeling in the F2 cortex, which was substantially reduced in similar sections obtained from p50-/- mice. The p50-/- mice expressed lower levels of A1AR mRNA than F2 mice, as determined by real time PCR. Quantitation of the A1AR transducing G proteins by Western blotting show significantly less Galphai3, no change in Galphai1, but higher levels of Galphao and Gbeta in the cortices of p50-/-, as compared with F2 mice. Administration of bacterial lipopolysaccharide (LPS), an activator of NF-kappaB, increased A1AR expression in the cortices of F2 mice but not p50-/- mice. Cortical neurons cultures prepared from p50-/- mice showed a greater degree of apoptosis, compared with neurons from F2 mice. Activation of the A1AR reduced apoptosis with greater efficacy in cultures from F2 than p50-/- mice. Taken together, these data support a role for NF-kappaB in determining both the basal and LPS-stimulated A1AR expression in vivo which could contribute to neuronal survival.
Jhaveri
K A
KA
Department of Pharmacology, Southern Illinois University School of Medicine, PO Box 19629, Springfield, IL 62794-9629, USA.
Reichensperger
J
J
Toth
L A
LA
Sekino
Y
Y
Ramkumar
V
V
D000704:Analysis of Variance; D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D047468:Immunoprecipitation / Q000379:methods; D020287:In Situ Nick-End Labeling / Q000379:methods; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D052002:NF-kappa B p50 Subunit / Q000172:deficiency*; D011485:Protein Binding / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D011869:Radioligand Assay / Q000379:methods; D043682:Receptor, Adenosine A1 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014970:Xanthines / Q000493:pharmacokinetics
D008070:Lipopolysaccharides; D052002:NF-kappa B p50 Subunit; D012333:RNA, Messenger; D043682:Receptor, Adenosine A1; D014970:Xanthines; C051360:1,3-dipropyl-8-cyclopentylxanthine; D019204:GTP-Binding Proteins