DEMONSTRATION OF BOTH A1-ADENOSINE AND A2-ADENOSINE RECEPTORS IN DDT1 MF-2 SMOOTH-MUSCLE CELLS

  • 1 February 1990
    • journal article
    • research article
    • Vol. 37  (2) , 149-156
Abstract
Adenosine receptors of the A1 and A2 subtypes were characterized in membranes from DDT1 MF-2 smooth muscle cells. These cells possess a high density of A1 adenosine receptors (Bmax = 0.8-0.9 pmol/mg of protein), as measured by both agonist and antagonist radioligands. Agonists compete for [125I]N6-[2-(4-amino-3-iodophenyl)ethyl]-adenosine (A1 receptor-selective radioligand) binding with the following potency series: (R)-phenylisopropyladenosine [(R)-PIA] > 5''-N-ethylcarboxamide adenosine (NECA) > (S)-PIA, indicative of their interaction with A1 adenosine receptors. Agonist competition for [3H]8-{4-[{{[(2-aminoethyl)amino]carbonyl}methyl}oxy]phenyl}-1,3-dipropylxanthine ([3H]XAC) (an antagonist radioligand for the A1 adenosine receptor) was described by a two-state model of 1.3 nM (high affinity state, Kk) and 370 nM (low affinity state, KL), with 70% of the receptors in the high affinity state (RH). Addition of guanosine 5''-[.beta., .alpha.-imido]triphosphate (100 .mu.M) shifted the (R)-PIA competition curves to the right to lower affinities. Photoaffinity labeling with the agonist photoprobe [125I]N6-[2-(4-amino-3-iodophenyl)ethyl]adenosine indicates that the A1 adenosine receptor binding subunit is a Mr 38,000 protein. Adenosine receptor agonists [(R)-PIA, NECA, and (S)-PIA] inhibited isoproterenol-stimulated adenylate cyclase activity in DDT1 MF-2 cell membrane with IC50 values of 62, 538, and 750 nM, respectively. Inhibition of adenylate cyclase by (R)-PIA was attenuated by the A1 receptor antagonist XAC and following inactivation of Gi with pertussis toxin (100 ng/ml). Using a recently developed A2 adenosine receptor agonist radioligand 2-[4-(2-{[4-aminophenyl]methylcarbonyl}ethyl)phenyl]ethylamino-5''-N-ethylcarboxamido adenosine (125I-PAPA-APEC), we have demonstrated the presence of A2 adenosine receptors in this cell line. Saturation curves with 125I-PAPA-APEC indicated the Bmax and Kd values to be 0.21 pmol/mg of protein and 4.0 nM, respectively. In competition experiments, NECA was more potent at inhibiting 125I-PAPA-APEC binding than (R)-PIA, with their respective IC50 values being 5.6 and 351 nM. The photolabeled A2 adenosine receptor migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an Mr of 42,000. Finally, adenosine receptor agonists stimulated adenylate cyclase activity by .apprx.2-3-fold with the following potency series: PAPA-APEC .gtoreq. NECA > (R)-PIA, indicative of their interaction at A2 receptors. These data represent the first demonstration of the presence of both A1 and A2 receptors in a single cell line, DDT, MF-2 smooth muscle cells.