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Noradrenaline and adrenaline are high affinity agonists at dopamine D4 receptors

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Abstract

The activity of monoamine neurotransmitters was examined at dopamine D4 receptors. In competition binding with [3H]spiperone, noradrenaline and adrenaline exhibited a high affinity binding component (KH=12.1 nM and 5.0 nM ,respectively), similar to that of dopamine (KH=2.6 nM), whereas serotonin (5-hydroxytryptamine, 5-HT) and histamine had low affinity (Ki>1000 nM). Noradrenaline and adrenaline acted as agonists at dopamine D4 receptors, stimulating receptor-mediated [35S]guanylyl-γ-thiotriphosphate ([35S]GTPγS) binding (EC50=7.8 and 5.8 μM, respectively, versus 0.1 μM for dopamine). The dopamine D4 receptor-selective ligand, 3-(4-[4-chlorophenyl]piperazin-1-yl)methyl-1H-pyrrolo[2,3b]-pyridine (L 745,870) and the dopaminergic antagonists, spiperone, haloperidol and clozapine, inhibited noradrenaline-stimulated [35S]GTPγS binding whereas α1-, α2- and β-adrenoceptor antagonists did not. These results indicate that dopamine D4 receptors are activated by noradrenaline and adrenaline, although at 50–100-fold higher concentrations than dopamine.

Introduction

The dopamine D4 receptor is of interest for several reasons. First, it is localised in limbic structures associated with regulation of mood and cognition, such as cerebral cortex and hippocampus (Lahti et al., 1996; Mrzljak et al., 1996). Second, the atypical antipsychotic, clozapine, has significant affinity at the dopamine D4 receptor (Van Tol et al., 1991) and, third, dopamine D4-like receptor upregulation in postmortem schizophrenic brain has been observed by some researchers (Seeman et al., 1995; Murray et al., 1995) – although not by others (Reynolds, 1996). The dopamine D4 receptor may therefore represent a novel target for the understanding of the mechanisms underlying psychosis. However, its precise physiological significance is yet to be clarified. Indeed, whilst dopamine D4 receptors expressed in COS 7 (African green monkey kidney) cells were found to display high affinity for dopamine (Van Tol et al., 1991) some preliminary evidence suggests that dopaminergic receptors may also exhibit high affinity for other neurotransmitters (Odagaki et al., 1995; Van der Graaf et al., 1995; Lanau et al., 1995). The present study therefore investigated the relative affinities of monoamine neurotransmitters at recombinant human dopamine D4 receptors. Two receptor isoforms (D4.4 and D4.2) and two heterologous expression systems (Chinese hamster ovary and Sf9 cells) were used. Further, we investigated the agonist activity of monoamines by their ability to induce dopamine D4 receptor-mediated stimulation of [35S]guanylyl-γ-thiotriphosphate ([35S]GTPγS) binding. Finally, a range of antagonists, including the novel, selective, dopamine D4 receptor antagonist, 3-(4-[4-chlorophenyl]piperazin-1-yl)methyl-1H-pyrrolo[2,3b]-pyridine (L 745,870; Kulagowski et al., 1996), was used to demonstrate the involvement of dopamine D4 receptors versus other dopaminergic or adrenergic receptors. The results present evidence of a potent interaction of both noradrenaline and adrenaline, in addition to dopamine, at dopamine D4 receptors.

Section snippets

Competition binding at dopamine D4 receptors

CHO-D4.4, CHO-D4.2 (Receptor Biology, Baltimore, MD, USA) or Sf9-D4.2 (BioSignal, Montreal, Canada) cell membranes were incubated with [3H]spiperone (0.5 nM) and competing ligands in buffer A (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2 and 0.1% (w/v) ascorbic acid) for 1 h at 22°C. Non-specific binding was defined using haloperidol (10 μM). Membranes of mouse A9L-D2 cells (Receptor Biology) were incubated with [125I]iodosulpride (0.1 nM) and competing ligands in buffer A supplemented with bovine serum

Competition binding at dopamine D4 receptors

The dopamine D4 receptor antagonist, L 745,870, and the antagonists, spiperone, haloperidol and clozapine, inhibited [3H]spiperone binding to dopamine D4.4 receptors monophasically (pseudo-Hill coefficients, nH, close to unity) with Ki values of 1.58±0.64, 0.22±0.02, 1.79±0.67 and 62.8±11.8 nM respectively. In contrast, noradrenaline, adrenaline and dopamine inhibited [3H]spiperone binding to CHO-D4.4 CHO-D4.2 and Sf9-D4.2 receptors biphasically (Fig. 1A) yielding KH values for the high

Discussion

The present study shows that both noradrenaline and adrenaline, as well as dopamine, inhibited [3H]spiperone binding to human recombinant CHO-D4.4 receptors with high affinity (KH=12.1, 5.0 and 2.6 nM, respectively). These KH values are similar to the affinitites of noradrenaline and adrenaline at α1-, α2- and β-adrenoceptors (Hieble et al., 1995). Nanomolar KH values were also observed for a further dopamine D4 receptor isoform (D4.2) and in a different expression system (baculovirus-infected

Acknowledgements

We thank C. Chaput, L. Verrièle, V. Pasteau and S. Aubry for excellent technical assistance.

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