Joad J P
Department of Pediatrics, University of California, Davis.
Am J Respir Cell Mol Biol. 1990 Feb;2(2):193-8. doi: 10.1165/ajrcmb/2.2.193.
Because adenosine narrows asthmatic airways, is released during hypoxia and by mast cells, and is antagonized by theophylline, it may play a role in asthma. I characterized the first step in pulmonary responses to adenosine: its adenosine receptor. Plasma membranes, prepared from macroscopically normal human peripheral lung, were incubated with 10 nM 5'-N-ethylcarboxamido[3H]adenosine ([3H]NECA) and various concentrations of competing ligand under experimentally determined optimal conditions: 4 degrees C, pH 7.4, 5 mM MgCl2, 1.8 mg protein/ml, 30-min incubation time. Bound and free ligand were separated by rapid vacuum filtration, and the radioactive counts were analyzed using a weighted, non-linear, least-squares curve-fitting program, LIGAND. Analyzed together, the data from the lungs of 6 patients revealed a single binding site with a dissociation constant (Kd) for NECA of 200 nM +/- 14% and a receptor concentration of 543 fmol/mg protein +/- 13%. Analyzed separately, the individual Kds ranged from 133 to 430 nM and the receptor concentrations from 338 to 811 fmol/mg protein. Adenosine receptor ligands competed with NECA in an A2 rank order of potency: NECA greater than 8-phenyltheophylline greater than 3-isobutyl-1-methylxanthine greater than theophylline greater than N6-L-phenylisopropyladenosine greater than N6-D-phenylisopropyladenosine greater than N6-cyclohexyladenosine. Theophylline bound to the receptor with an inhibition constant (Ki = 70.9 microM +/- 28%) near the therapeutic range (28 to 56 microM). Cromolyn also bound with high affinity (Ki = 5.42 microM +/- 47%). I conclude that human lung adenosine receptors: (1) are single-site receptors, probably of the A2 subtype and (2) bind to both theophylline and cromolyn.
由于腺苷会使哮喘气道变窄，在缺氧时以及由肥大细胞释放，且可被茶碱拮抗，因此它可能在哮喘中发挥作用。我对肺部对腺苷反应的第一步进行了表征：其腺苷受体。从宏观正常的人外周肺制备的质膜，在实验确定的最佳条件下（4℃、pH 7.4、5 mM氯化镁、1.8 mg蛋白质/毫升、30分钟孵育时间），与10 nM 5'-N-乙基羧酰胺基[3H]腺苷（[3H]NECA）和各种浓度的竞争性配体一起孵育。通过快速真空过滤分离结合的和游离的配体，并使用加权、非线性、最小二乘曲线拟合程序LIGAND分析放射性计数。综合分析6名患者肺部的数据显示，存在一个单一结合位点，NECA的解离常数（Kd）为200 nM±14%，受体浓度为543 fmol/毫克蛋白质±13%。单独分析时，个体Kd范围为133至430 nM，受体浓度范围为338至811 fmol/毫克蛋白质。腺苷受体配体与NECA竞争的效力顺序为A2型：NECA＞8-苯基茶碱＞3-异丁基-1-甲基黄嘌呤＞茶碱＞N6-L-苯基异丙基腺苷＞N6-D-苯基异丙基腺苷＞N6-环己基腺苷。茶碱与受体结合的抑制常数（Ki = 70.9 microM±28%）接近治疗范围（28至56 microM）。色甘酸钠也以高亲和力结合（Ki = 5.42 microM±47%）。我的结论是，人肺腺苷受体：（1）是单一位点受体，可能是A2亚型；（2）与茶碱和色甘酸钠都能结合。
