Negative regulation of melatonin secretion by melatonin receptors in ovine pinealocytes

Abstract

Melatonin (MLT) is a biological modulator of circadian and seasonal rhythms and reproduction. The photoperiodic information is detected by retinal photoreceptors and transmitted through nerve transmissions to the pineal gland, where MLT is synthesized and secreted at night into the blood. MLT interacts with two G protein-coupled receptors, MT1 and MT2. The aim of our work was to provide evidence for the presence of MLT receptors in the ovine pineal gland and define their involvement on melatonin secretion. For the first time, we identified the expression of MLT receptors with the specific 2-[125I]-MLT agonistic radioligand in ovin pinealocytes. The values of Kd and Bmax are 2.24 ± 1.1 nM and 20 ± 6.8 fmol/mg. MLT receptors are functional and inhibit cAMP production and activate ERK1/2 through pertussis toxin-sensitive Gi/o proteins. The MLT receptor antagonist/ inverse agonist luzindole increased cAMP production (189 ± 30%) and MLT secretion (866 ± 13%). The effect of luzindole on MLT secretion was additive with the effect of well-described activators of this pathway such as the β-adrenergic agonist isoproterenol and the α-adrenergic agonist phenylephrine. Co-incubation of all three compounds increased MLT secretion by 1236 ± 199%. These results suggest that MLT receptors are involved in the negative regulation of the synthesis of its own ligand in pinealocytes. While adrenergic receptors promote MLT secretion, MLT receptors mitigate this effect to limit the quantity of MLT secreted by the pineal gland.

Fig 1. Cytological characterization of primary culture of ovine pineal gland cells.

Five (A) and eight days (B) primary culture of ovine pineal glands were observed by optical microscopy. Cells from ovine pineal glands were immunolabelled with anti-tryptophan hydroxylase (TPH, C) or anti- glial fibrillary acidic protein (GFAP, D) antibodies and detected by a peroxydase-coupled secondary antibody. Non immun serum (NIS) was used as a negative control (Fig 1E). Magnification was x400 in A and C, x200 in B and x100 in D. Pinealocyte were labelled with an anti-TPH antibody (G) detected by a cyanin-coupled secondary antibody, while astrocytes were labelled with an anti-GFAP antibody (H) detected by fluorescein-coupled secondary antibody. Nuclei were stained with DAPI (F). The merge of the three labelings is shown in I. Maginifcation is x10. Scale bar 50 μm. Astrocytes (arrow).

Fig 2. Binding properties of MT₁ and MT₂ in ovine pinealocytes.

Saturation curves performed with 2-[^125I]-MLT (A) and [^125I]-S70254 (C) on primary culture of ovine pineal gland cells at 37°C for 1 h. Specific binding is represented. Scatchard plot is associated with the saturation curves (B and D). The experiments are performed in duplicates and are repeated five times.

Fig 3. Coupling of MT receptor to Gi proteins in pinealocytes.

(A) Effect of 10⁻⁷ M MLT on cAMP production induced by 1 μM of Forskolin (Fsk). Effect of pre-treatment with Pertussis toxin (PTX; 5 ng/ml; 3 h at 37°C) on cAMP accumulation following stimulation with 10⁻⁷ M MLT and 1 μM of Fsk (1 h at 37°C). Each histogram is presented as a percentage of the maximal response to 1 μM of forskolin (100%). B) Effect of 10⁻⁷ M MLT or of a pre-treatment with Pertussis toxin (PTX; 5 ng/ml; 3 h at 37°C) and then 10⁻⁷ M MLT (5 min) on the level of phosphorylated ErK 1/2. (C) The Western blot is a representative experiment. Columns and bars represent mean ± s.e.m. values of data obtained from duplicate determinations of cAMP in 5 different experiments (A) and 3 assays for the phospho-Erk1/2 level (B and C). *P<0.05 in Kruskal-Wallis tests followed by the Dunn’s multiple comparison tests.

Fig 4. Inhibition of cAMP production mainly mediated by MT receptors.

Effect of 1 μM Forskolin or 1 μM luzindole on basal cAMP production. Each histogram is presented as a percentage of the basal cAMP accumulation (control; 100%). Columns and bars represent mean ± s.e.m. values of data obtained from duplicate determinations of cAMP in 5 different experiments. *P<0.05 in Kruskal-Wallis tests followed by the Dunn’s multiple comparison tests.

Fig 5. Regulation of MLT synthesis.

Effects of adrenergic and/or MT receptors on MLT synthesis. Receptor activity was stimulated with isoproterenol for β- adrenergic receptors (A), phenylephrine for α1-adrenergic receptors (B), both isoproterenol and phenylephrine (C), luzindole for MT_1/2, both luzindol and isoproterenol for MT and β- adrenergic receptors (E), both luzindol and phenylephrine for MT and α1-adrenergic receptors (F), and both luzindol, isoproterenol and phenylephrine for MT and β- and α1-adrenergic receptors (G). Each histogram is presented as a percentage of the control MLT secretion (Control, 100%). Columns and bars represent mean ± s.e.m. values of data obtained from duplicate determinations of cAMP in 5 different experiments. *P<0.05 in Kruskal-Wallis tests followed by the Dunn’s multiple comparison tests.