Varani Katia, Gessi Stefania, Merighi Stefania, Iannotta Valeria, Cattabriga Elena, Pancaldi Cecilia, Cadossi Ruggero, Borea Pier Andrea
Department of Clinical and Experimental Medicine, Pharmacology Unit, University of Ferrara, via Fossato di Mortara 17-19, 44100 Ferrara, Italy.
Biochem Pharmacol. 2003 Nov 15;66(10):1897-906. doi: 10.1016/s0006-2952(03)00454-4.
The present study was designed to evaluate the binding and functional characterization of A(3) adenosine receptors in human neutrophils exposed to low frequency, low energy, pulsing electromagnetic fields (PEMFs). Great interest has grown concerning the use of PEMF in the clinical practice for therapeutic purposes strictly correlated with inflammatory conditions. Saturation experiments performed using the high affinity and selective A(3) adenosine antagonist 5N-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([3H]-MRE 3008F20) revealed a single class of binding sites with similar affinity in control and in PEMF treated human neutrophils (K(D)=2.36+/-0.16 and 2.45+/-0.15 nM, respectively). PEMFs treatment revealed that the receptor density was statistically increased (P<0.01) (B(max)=451+/-18 and 736+/-25fmolmg(-1) protein, respectively). Thermodynamic data indicated that [3H]-MRE 3008F20 binding in control and in PEMF-treated human neutrophils was entropy and enthalpy driven. Competition of radioligand binding by the high affinity A(3) receptor agonists, N(6)-(3-iodo-benzyl)-2-chloro-adenosine-5'-N-methyluronamide (Cl-IB-MECA) and N(6)-(3-iodo-benzyl)adenosine-5'-N-methyl-uronamide (IB-MECA), in the absence of PEMFs revealed high and low affinity values similar to those found in the presence of PEMFs. In both experimental conditions, the addition of GTP 100 microM shifted the competition binding curves of the agonists from a biphasic to a monophasic shape. In functional assays Cl-IB-MECA and IB-MECA were able to inhibit cyclic AMP accumulation and their potencies were statistically increased after exposure to PEMFs. These results indicate in human neutrophils treated with PEMFs the presence of significant alterations in the A(3) adenosine receptor density and functionality.
本研究旨在评估暴露于低频、低能量脉冲电磁场（PEMF）的人中性粒细胞中A(3)腺苷受体的结合及功能特性。PEMF在临床实践中用于与炎症状态密切相关的治疗目的，这已引发了极大的关注。使用高亲和力和选择性A(3)腺苷拮抗剂5N-(4-甲氧基苯基-氨基甲酰基)氨基-8-丙基-2-(2-呋喃基)吡唑并-[4,3-e]-1,2,4-三唑并[1,5-c]嘧啶（[3H]-MRE 3008F20）进行的饱和实验显示，在对照和PEMF处理的人中性粒细胞中存在一类亲和力相似的结合位点（K(D)分别为2.36±0.16和2.45±0.15 nM）。PEMF处理显示受体密度有统计学意义的增加（P<0.01）（B(max)分别为451±18和736±25 fmolmg(-1)蛋白）。热力学数据表明，对照和PEMF处理的人中性粒细胞中[3H]-MRE 3008F20的结合受熵和焓驱动。在不存在PEMF的情况下，高亲和力A(3)受体激动剂N(6)-(3-碘苄基)-2-氯腺苷-5'-N-甲基脲酰胺（Cl-IB-MECA）和N(6)-(3-碘苄基)腺苷-5'-N-甲基脲酰胺（IB-MECA）对放射性配体结合的竞争显示出与存在PEMF时相似的高亲和力和低亲和力值。在两种实验条件下，添加100 microM GTP都会使激动剂的竞争结合曲线从双相变为单相。在功能测定中，Cl-IB-MECA和IB-MECA能够抑制环磷酸腺苷（cAMP）的积累，并且在暴露于PEMF后它们的效力有统计学意义的增加。这些结果表明，在经PEMF处理的人中性粒细胞中，A(3)腺苷受体密度和功能存在显著改变。
