Characterization of adenosine A1 receptors in intact DDT1 MF-2 smooth muscle cells

P Gerwins, C Nordstedt and BB Fredholm

Department of Pharmacology, Karolinska Institutet, Stockholm, Sweden.

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/10(6) 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 greater than 8-cyclopentyl-1,3- dimethylxanthine greater than 8-(p-sulfophenyl)-theophylline greater than 3-isobutyl-1-methylxanthine greater than 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 degrees and 25 degrees. However, short term incubations (1-4 min) at 25 degrees showed biphasic binding curves in intact cells. The equilibrium KD values for intact cells were similar to the low affinity KD values in membranes (KL). The order of potency was N6-cyclopentyladenosine greater than or equal to (- )-(R)-N6-phenylisopropyladenosine[(R)-PIA] greater than or equal to N6- cyclohexyl adenosine greater than 5'-N-ethylcarboxamidoadenosine (NECA) greater than 2-chloroadenosine greater than adenosine (intact cells only) greater than 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 microM) 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 receptors, 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Volume 38, Issue 5, pp. 660-666, 11/01/1990
Copyright © 1990 by American Society for Pharmacology and Experimental Therapeutics

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