Jhaveri K A, Reichensperger J, Toth L A, Sekino Y, Ramkumar V
Department of Pharmacology, Southern Illinois University School of Medicine, PO Box 19629, Springfield, IL 62794-9629, USA.
Neuroscience. 2007 Apr 25;146(1):415-26. doi: 10.1016/j.neuroscience.2006.12.035. Epub 2007 Mar 9.
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.
在感染、缺血预处理和氧化应激条件下，腺苷可促进细胞保护作用。我们实验室之前的研究表明，氧化应激通过激活核因子（NF）-κB诱导腺苷A1受体（A1AR）的表达。典型的转录因子由p50和p65亚基的同二聚体或异二聚体组成。为了确定NF-κB在体内对A1AR调节中的作用，我们比较了缺乏NF-κB p50亚基的小鼠（p50-/-小鼠）和年龄匹配的B6129PF2/J（F2）对照小鼠大脑中的A1AR RNA和蛋白质水平。皮质中的放射性配体结合试验显示，p50-/-小鼠皮质中的A(1)AR数量（最大结合容量，Bmax）显著低于F2小鼠（分别为151±62 fmol/mg蛋白质和479±181 fmol/mg蛋白质，每个品系N = 5，P<0.05），但平衡解离常数没有变化。在海马体、脑干、下丘脑以及肾上腺、肾脏和脾脏等外周组织中也测量到了类似的A1AR减少情况。通过抗体亲和柱纯化后对A1AR进行评估，结果也表明与F2小鼠相比，p50-/-小鼠皮质中的A1AR减少。A1AR免疫细胞化学显示F2皮质中有明显的神经元标记，而从p50-/-小鼠获得的类似切片中这种标记明显减少。通过实时PCR测定，p50-/-小鼠表达的A1AR mRNA水平低于F2小鼠。通过蛋白质印迹法对转导G蛋白的A1AR进行定量分析显示，与F2小鼠相比，p50-/-小鼠皮质中的Gαi3明显减少，Gαi1没有变化，但Gαo和Gβ水平较高。给予细菌脂多糖（LPS）（一种NF-κB激活剂）可增加F2小鼠皮质中的A1AR表达，但对p50-/-小鼠无效。与F2小鼠的神经元相比，从p50-/-小鼠制备的皮质神经元培养物显示出更高程度的细胞凋亡。在F2小鼠的培养物中，A1AR激活对细胞凋亡的减少作用比p50-/-小鼠更有效。综上所述，这些数据支持NF-κB在决定体内基础和LPS刺激的A1AR表达中起作用，这可能有助于神经元存活。
