Feoktistov I, Biaggioni I
Department of Medicine, Vanderbilt University, Nashville, TN 37232-2195, USA.
Biochem Pharmacol. 1998 Mar 1;55(5):627-33. doi: 10.1016/s0006-2952(97)00512-1.
Characterization of A2B receptors is hampered by the lack of selective pharmacological probes and often relies on their relative affinity to agonists that are selective at other receptor types. This approach is limited because the affinity of A2B receptors for putative A3 agonists has not been determined. Using the human erythroleukemia cell line HEL as a cellular model for A2B-mediated adenylate cyclase activation, we found the following potencies (pD2) for the non-selective agonist 5'-N-ethylcarboxamidoadenosine (NECA) (5.65 +/- 0.04), the putative A3 agonists N6-benzyl-NECA (4.17 +/- 0.06) and N6-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (IB-MECA) (3.7 +/- 0.02), and the A2A agonist 4-[(N-ethyl-5'-carbamoyladenos-2-yl)-aminoethyl]-phenylpropionic acid (CGS21680) (2.8 +/- 0.1). Because of the lack of a selective agonist, characterization of A2B receptor function is difficult in cells co-expressing A2A receptors. In the human mast cell line HMC-1, NECA induced cAMP accumulation with a concentration-response relationship best fitted to a two-sited model (pD2 7.69 +/- 0.42 and 5.92 +/- 0.21 for high- and low-affinity sites), suggesting the presence of both A2A and A2B receptors in these cells. We demonstrated that A2B receptors can be selectively activated with NECA in the presence of the selective A2A antagonist 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c ]pyrimidine (SCH 58261). Under these conditions, the concentration-response relationship of NECA for cyclic AMP accumulation was now best fitted to a one-site model (pD2 5.68 +/- 0.03, Hill slope 0.93 +/- 0.06, 95% confidence intervals 0.8 to 1.06) corresponding to selective activation of A2B receptors. Using the approaches developed in this study, we determined that A2B, and not A2A or A3, receptors account for all the calcium mobilization induced by NECA in HMC-1 cells.
缺乏选择性药理学探针阻碍了对A2B受体的特性描述，其特性描述通常依赖于它们对在其他受体类型上具有选择性的激动剂的相对亲和力。这种方法存在局限性，因为尚未确定A2B受体对假定的A3激动剂的亲和力。我们使用人红白血病细胞系HEL作为A2B介导的腺苷酸环化酶激活的细胞模型，发现非选择性激动剂5'-N-乙基甲酰胺基腺苷（NECA）（5.65±0.04）、假定的A3激动剂N6-苄基-NECA（4.17±0.06）和N6-（3-碘苄基）-N-甲基-5'-氨基甲酰腺苷（IB-MECA）（3.7±0.02）以及A2A激动剂4-[(N-乙基-5'-氨基甲酰腺苷-2-基)-氨基乙基]-苯丙酸（CGS21680）（2.8±0.1）具有以下效价（pD2）。由于缺乏选择性激动剂，在共表达A2A受体的细胞中很难对A2B受体功能进行特性描述。在人肥大细胞系HMC-1中，NECA诱导cAMP积累，其浓度-反应关系最符合双位点模型（高亲和力位点和低亲和力位点的pD2分别为7.69±0.42和5.92±0.21），这表明这些细胞中同时存在A2A和A2B受体。我们证明，在选择性A2A拮抗剂5-氨基-7-（苯乙基）-2-（2-呋喃基）-吡唑并[4,3-e]-1,2,4-三唑并[1,5-c]嘧啶（SCH 58261）存在的情况下，A2B受体可以被NECA选择性激活。在这些条件下，NECA诱导环化AMP积累的浓度-反应关系现在最符合一位点模型（pD2 5.68±0.03，希尔斜率0.93±0.06，95%置信区间0.8至1.06），这对应于A2B受体的选择性激活。使用本研究中开发的方法，我们确定在HMC-1细胞中，是A2B受体而非A2A或A3受体介导了NECA诱导的所有钙动员。
