Pharmacological characterization of human recombinant melatonin mt(1) and MT(2) receptors

Abstract

We have pharmacologically characterized recombinant human mt(1) and MT(2) receptors, stably expressed in Chinese hamster ovary cells (CHO-mt(1) and CHO-MT(2)), by measurement of [(3)H]-melatonin binding and forskolin-stimulated cyclic AMP (cAMP) production. [3H]-melatonin bound to mt(1) and MT(2) receptors with pK(D) values of 9.89 and 9.56 and B(max) values of 1.20 and 0.82 pmol mg(-1) protein, respectively. Whilst most melatonin receptor agonists had similar affinities for mt(1) and MT(2) receptors, a number of putative antagonists had substantially higher affinities for MT(2) receptors, including luzindole (11 fold), GR128107 (23 fold) and 4-P-PDOT (61 fold). In both CHO-mt(1) and CHO-MT(2) cells, melatonin inhibited forskolin-stimulated accumulation of cyclic AMP in a concentration-dependent manner (pIC(50) 9.53 and 9.74, respectively) causing 83 and 64% inhibition of cyclic AMP production at 100 nM, respectively. The potencies of a range of melatonin receptor agonists were determined. At MT(2) receptors, melatonin, 2-iodomelatonin and 6-chloromelatonin were essentially equipotent, whilst at the mt(1) receptor these agonists gave the rank order of potency of 2-iodomelatonin>melatonin>6-chloromelatonin. In both CHO-mt(1) and CHO-MT(2) cells, melatonin-induced inhibition of forskolin-stimulated cyclic AMP production was antagonized in a concentration-dependent manner by the melatonin receptor antagonist luzindole, with pA(2) values of 5.75 and 7.64, respectively. Melatonin-mediated responses were abolished by pre-treatment of cells with pertussis toxin, consistent with activation of G(i)/G(o) G-proteins. This is the first report of the use of [(3)H]-melatonin for the characterization of recombinant mt(1) and MT(2) receptors. Our results demonstrate that these receptor subtypes have distinct pharmacological profiles.

Figure 1

Time-courses of association (A, B) and dissociation (C, D) of [³H]-melatonin binding to the human recombinant mt₁ (A, C) and MT₂ (B, D) receptors. Data are the mean of duplicate points and representative of three similar experiments. For association experiments, upper asymptote (mean±s.e.mean)=0.37±0.05 pmol mg⁻¹ (mt₁) and 0.39±0.01 pmol mg⁻¹ (MT₂). For dissociation experiments, binding at time t₀ (mean±s.e.mean)=0.38±0.03 pmol mg⁻¹ (mt₁) and 0.40±0.03 pmol mg⁻¹ (MT₂).

Figure 2

[³H]-melatonin saturation binding to human recombinant mt₁ (A) and MT₂ (MT₂) receptors. Data are mean of duplicate points and representative of three separate experiments.

Figure 3

Competition for [³H]-melatonin binding at human recombinant mt₁ (A) and MT₂ (B) receptors. Data are mean of duplicate points and representative of at least three separate experiments. Binding in the absence of competitor (mean±s.e.mean)=0.42±0.02 pmol mg⁻¹ (mt₁) and 0.35±0.02 pmol mg⁻¹ (MT₂).

Figure 4

The effect of pertussis toxin (100 ng ml⁻¹) on melatonin-induced inhibition of forskolin-stimulated cyclic AMP production in CHO-mt₁ (A) and CHO-MT₂ (B) cells. Data are mean±s.e.mean of three separate experiments. Mean basal cyclic AMP levels were 0.30 pmol well⁻¹ (range 0.18–0.35); forskolin-stimulated cyclic AMP levels were 27 pmol well⁻¹ (range 14–40). Similar values were obtained in subsequent experiments.

Figure 5

Agonist concentration-effect curves for inhibition of forskolin-stimulated cyclic AMP in CHO-mt₁ (A) and CHO-MT₂ cells. The data are mean of 3–11 separate experiments.

Figure 6

Antagonism by luzindole of melatonin-induced inhibition of forskolin-stimulated cyclic AMP production in CHO-mt₁ (A) and CHO-MT₂ (B) cells. Data are mean±s.e.mean of 3–4 separate experiments. Inset: Schild plots depicting antagonism of melatonin responses by luzindole. The line through the data was plotted using the parameters obtained from the analysis described in the Methods.

Figure 7

The effect of melatonin, luzindole and 4-P-PDOT on forskolin-stimulated cyclic AMP production in CHO-mt₁ (A) and CHO-MT₂ (B) cells. Data are mean±s.e.mean of 3–4 separate experiments.