Elsevier

Biochemical Pharmacology

Volume 58, Issue 6, 15 September 1999, Pages 1035-1045
Biochemical Pharmacology

Molecular and Cellular Pharmacology
Reciprocal interactions between adenosine A2A and dopamine D2 receptors in Chinese hamster ovary cells co-transfected with the two receptors

https://doi.org/10.1016/S0006-2952(99)00184-7Get rights and content

Abstract

Human adenosine A2A and rat dopamine D2 receptors (A2A and D2 receptors) were co-transfected in Chinese hamster ovary (CHO) cells to study the interactions between two receptors that are co-localized in striatopallidal γ-aminobutyric acid-(GABA)ergic neurons. Membranes from transfected cells showed a high density of D2 (3.6 pmol per mg protein) and A2A receptors (0.56 pmol per mg protein). The D2 receptors were functional: an agonist, quinpirole, could stimulate GTPγS binding and reduce stimulated adenylyl cyclase activity. The A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS 21680) decreased high-affinity binding of the agonist dopamine at D2 receptors. Activation of adenosine A2A receptors shifted the dose–response curve for quinpirole on adenosine 3′,5′-cyclic monophosphate (cAMP) to the right. However, CGS 21680 did not affect dopamine D2 receptor-induced GTPγS binding, but did cause a concentration-dependent increase in cAMP accumulation. The maximal cAMP response was decreased by the D2 agonist quinpirole in a concentration-dependent manner, but there was no change in ec50 and no effect in cells transfected only with adenosine A2A receptors. A2A receptor activation also increased phosphorylation of cAMP response element-binding protein and expression of c-fos mRNA. These effects were also strongly counteracted by quinpirole. These results show that the antagonistic actions between adenosine A2A and dopamine D2 receptors noted previously in vivo can also be observed in CHO cells where the two receptors are co-transfected. Thus, no brain cell-specific factors are required for such interactions. Furthermore, the interaction at the second messenger level and beyond may be quantitatively more important than A2A receptor-mediated inhibition of high affinity D2 agonist binding to the receptor.

Section snippets

Chemicals

Adenosine deaminase was obtained from Boehringer Mannheim. Cyclic AMP, bacitracin, BSA, 1-10 phenanthroline, GDP, Gpp(NH)p, dithiothreitol, 2-chloroadenosine, NECA, and forskolin were purchased from Sigma. Dopamine, quinpirole, and CGS 21680 were obtained from Research Biochemicals International. SCH 58261 was a gift from Dr. E. Ongini, Schering-Plough Research Institute, Milan, Italy. [3H]CGS 21680, [3H]raclopride, and [3H]cAMP were obtained from Du Pont NEN. [35S]GTPγS was from New England

Saturation binding experiments

The expression of dopamine D2 and adenosine A2A receptors was determined using radioligand-binding experiments (Fig. 1). The cells showed high specific binding of the dopamine D2 receptor antagonist radioligand [3H]raclopride (Bmax 3600 ± 140 fmol mg−1 protein; Kd 7.4 ± 0.6 nM; mean ± SEM, N = 3) (Fig. 1) with a better fit for one binding site than for two sites (data not shown). Non-specific binding was less than 10% at concentrations of [3H]raclopride close to the Kd value. The adenosine A2A

Discussion

The present results show reciprocal interactions between adenosine A2A and dopamine D2 receptors co-transfected in CHO cells. We have confirmed that stimulation of adenosine A2A receptors can decrease agonist affinity at dopamine D2 receptors and demonstrate that this is related to a small change in the potency of a dopamine D2 receptor agonist to induce some functional responses. Conversely, we show that activation of dopamine D2 receptors effectively reduces signaling induced by adenosine A2A

Acknowledgements

These studies were supported by the Swedish Medical Research Council (project nr 2553, 715, 12707), Knut och Alice Wallenbergs stiftelse, and by the European Commission (Biomed II: adenosine-dopamine interactions).

References (43)

  • P. Svenningsson et al.

    Regional differences in the ability of caffeine to affect haloperidol-induced striatal c-fos mRNA expression in the rat

    Neuropharmacology

    (1998)
  • B. Kull et al.

    Differences in the order of potency for agonists, but not antagonists, at human and rat adenosine A2A receptors

    Biochem Pharmacol

    (1999)
  • C. Nordstedt et al.

    A modification of a protein-binding method for rapid quantification of cAMP in cell-culture supernatants and body fluid

    Anal Biochem

    (1990)
  • B. Johansson et al.

    Differences in the regional and cellular localization of c-fos mRNA induced by amphetamine, cocaine and caffeine in the rat

    Neuroscience

    (1994)
  • T. Kenakin

    Differences between natural and recombinant G protein-coupled receptor systems with varying receptor/G protein stoichiometry

    Trends Pharmacol Sci

    (1997)
  • P.J. Gebicke-Haerter et al.

    Both adenosine A1- and A2-receptors are required to stimulate microglial proliferation

    Neurochem Int

    (1996)
  • L.M. Robertson et al.

    Regulation of c-fos expression in transgenic mice requires multiple interdependent transcription control elements

    Neuron

    (1995)
  • P. Sun et al.

    An inactivating point mutation demonstrates that interaction of cAMP response element binding protein (CREB) with the CREB binding protein is not sufficient for transcriptional activation

    J Biol Chem

    (1995)
  • M. Morelli et al.

    Adenosine A2 receptors interact negatively with dopamine D1 and D2 receptors in unilaterally 6-hydroxydopamine-lesioned rats

    Eur J Pharmacol

    (1994)
  • P. Svenningsson et al.

    Caffeine mimics the effect of a dopamine D2/3 receptor agonist on the expression of immediate early genes in globus pallidus

    Neuropharmacology

    (1997)
  • M.E. Olah et al.

    Adenosine receptor subtypesCharacterization and therapeutic regulation

    Annu Rev Pharmacol Toxicol

    (1995)
  • Cited by (0)

    View full text