CHARACTERIZATION OF ADENOSINE-A1-RECEPTORS IN INTACT DDT1 MF-2 SMOOTH-MUSCLE CELLS

  • 1 November 1990
    • journal article
    • research article
    • Vol. 38  (5) , 660-666
Abstract
Adenosine receptors in the smooth muscle cell line DDT1 MF-2 were studied by radioligand binding using the A1 receptor-selective antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H] DPCPX) as the ligand. Binding characteristics were similar in intact cells and in membranes (KD value of approximately 1 nM). The maximum binding amounted to 183 fmol/106 intact cells or 344 fmol/mg of membranes. To characterize the receptor, competition experiments were performed by inhibiting [3H]DPCPX binding with several adenosine agonists and antagonists. Adenosine receptor antagonists appeared to bind to a single class of binding site, both in membranes and intact cells. The order of potency was DPCPX = CGS 15943A > 8-cyclopentyl-1,3-dimethylxanthine > 8-(p-sulfophenyl)-theophylline > 3-isobutyl-1-methylxanthine > theophylline. Competition curves with adenosine agonists in membranes were best described by a two-site rather than a one-site model. At equilibrium in intact cells, only a single site was detected at both 4.degree. and 25.degree.. However, short term incubations (1-4 min) at 25.degree. showed biphasic binding curves in intact cells. The equilibrium KD values for intact cells were simlar to the low affinity KD values in membranes (KL). The order of potency was N6-cyclopentyladenosine .gtoreq. (-)-(R)-N6-phenylisopropyladenosine[(R)-PIA] .gtoreq. N6-cyclohexyl adenosine > 5''-N-ethylcarboxamidoadenosine (NECA) > 2-chloroadenosine > adenosine (intact cells only) > 2-phenylaminoadenosine (CV 1808). Treatment of cells with pertussis toxin ADP-ribosylated GTP-binding proteins and eliminated the high affinity agonist binding in membranes but did not affect binding to intact cells. The addition of GTP (100 .mu.M) also shifted the competition curves from bi- to monophasic curves in membranes. Adenosine receptor agonists inhibited the formation of cAMP induced by isoprenaline (IC50 for (R)-PIA, 0.4 nM). This inhibition could be prevented with adenosine receptor antagonists. Pretreatment with pertussis toxin also reversed these effects and actually revealed functional A2 recetpors, as shown by the formation of cAMP induced by NECA. In conclusion, the equilibrium binding of A1 receptor agonists to intact smooth muscle cells is similar to the low affinity binding observed in membranes. In addition, it is suggested that agonists may transiently convert the A1 receptor from a "resting" low affinity state to a high affinity state coupled to a GTP-binding protein. DDT1 MF-2 cells should prove useful for studying regulation of A1 receptor signaling in intact cells.

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