Olah M E, Gallo-Rodriguez C, Jacobson K A, Stiles G L
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Mol Pharmacol. 1994 May;45(5):978-82.
The rat A3 adenosine receptor (AR) is a recently characterized AR subtype cloned from testis and brain cDNA libraries. N6-2-(4-Amino-3-[125I]iodophenyl)ethyladenosine, a high affinity A1AR agonist, has served as the only radioligand available for study of the A3AR. The relatively low affinity of N6-2-(4-amino-3-[125I] iodophenyl)ethyladenosine for the A3AR and its greater A1AR selectivity necessitate the development of more appropriate radioligands for A3AR analysis. This report characterizes 125I-4-aminobenzyl-5'-N-methylcarboxamidoadenosine (125I-AB-MECA), a high affinity radioligand for the A3AR, in two cell lines that express this AR subtype. Membranes from Chinese hamster ovary (CHO) cells expressing the rat A3AR and from the rat mast cell line RBL-2H3 bound 125I-AB-MECA with Kd values of 1.48 +/- 0.33 nM and 3.61 +/- 0.30 nM, respectively. As determined by 125I-AB-MECA binding, levels of A3AR expression in the A3AR-CHO cell line and RBL-2H3 cells were 3.06 +/- 0.21 pmol/mg and 1.02 +/- 0.13 pmol/mg, respectively. Binding of 125I-AB-MECA was characterized in competition assays. In the A3AR-CHO cell line a potency order of cyclohexyl-5'-N-ethylcarboxamidoadenosine (cyclohexyl-NECA) = benzyl-NECA > (-)-N6-[(R)-phenylisopropyl]adenosine = NECA was observed, and in RBL-2H3 cells (-)-N6-[(R)-phenylisopropyl]adenosine and NECA were equipotent. Xanthine amine congener (XAC) and 8-cyclopentyl-1,3-dipropylxanthine did not significantly inhibit 125I-AB-MECA binding. The parent compound, AB-MECA, dose-dependently inhibited forskolin-stimulated adenylyl cyclase activity in A3AR-CHO cell membranes. 125I-AB-MECA bound to the rat A1AR and canine A2aAR expressed in COS-7 cells with Kd values of 3.42 +/- 0.43 nM and 25.1 +/- 12.6 nM, respectively. This binding was significantly reduced in the presence of 1 microM XAC. In RBL-2H3 cells, XAC had no effect on 125I-AB-MECA affinity and reduced the level of radioligand binding by approximately 5%.
大鼠A3腺苷受体（AR）是最近从睾丸和脑cDNA文库中克隆得到并鉴定的一种AR亚型。N6-2-（4-氨基-3-[125I]碘苯基）乙基腺苷是一种高亲和力的A1AR激动剂，是目前唯一可用于研究A3AR的放射性配体。N6-2-（4-氨基-3-[125I]碘苯基）乙基腺苷对A3AR的亲和力相对较低，且对A1AR的选择性更高，因此需要开发更合适的放射性配体用于A3AR分析。本报告在两种表达该AR亚型的细胞系中对125I-4-氨基苄基-5'-N-甲基羧酰胺腺苷（125I-AB-MECA）进行了鉴定，它是一种对A3AR具有高亲和力的放射性配体。表达大鼠A3AR的中国仓鼠卵巢（CHO）细胞和大鼠肥大细胞系RBL-2H3的细胞膜与125I-AB-MECA结合，其解离常数（Kd）值分别为1.48±0.33 nM和3.61±0.30 nM。通过125I-AB-MECA结合测定，A3AR-CHO细胞系和RBL-2H3细胞中A3AR的表达水平分别为3.06±0.21 pmol/mg和1.02±0.13 pmol/mg。在竞争结合实验中对125I-AB-MECA的结合特性进行了研究。在A3AR-CHO细胞系中观察到环己基-5'-N-乙基羧酰胺腺苷（环己基-NECA）=苄基-NECA > （-）-N6-[(R)-苯异丙基]腺苷 = NECA的效价顺序，而在RBL-2H3细胞中（-）-N6-[(R)-苯异丙基]腺苷和NECA的效力相当。黄嘌呤胺类似物（XAC）和8-环戊基-1,3-二丙基黄嘌呤对125I-AB-MECA的结合没有显著抑制作用。母体化合物AB-MECA能剂量依赖性地抑制A3AR-CHO细胞膜中福斯可林刺激的腺苷酸环化酶活性。125I-AB-MECA与COS-7细胞中表达的大鼠A1AR和犬A2aAR结合，其Kd值分别为3.42±0.43 nM和25.1±12.6 nM。在1 μM XAC存在的情况下，这种结合显著减少。在RBL-2H3细胞中，XAC对125I-AB-MECA的亲和力没有影响，但使放射性配体的结合水平降低了约5%。
