Characterization of the human peripheral lung adenosine receptor.
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.
Joad
J P
JP
Department of Pediatrics, University of California, Davis.
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism; D019830:Adenosine-5'-(N-ethylcarboxamide); D000293:Adolescent; D000368:Aged; D001667:Binding, Competitive; D004205:Cromolyn Sodium / Q000378:metabolism*; D005260:Female; D006801:Humans; D008168:Lung / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D011983:Receptors, Purinergic / Q000378:metabolism*; D013806:Theophylline / Q000378:metabolism*
D011983:Receptors, Purinergic; D019830:Adenosine-5'-(N-ethylcarboxamide); D013806:Theophylline; D000241:Adenosine; D004205:Cromolyn Sodium