Melatonin receptors are present in adult rat Leydig cells and are coupled through a pertussis toxin-sensitive G-protein

Previous studies have suggested that melatonin (MLT) acts directly on rat Leydig cells by modulating androgen production. In the present study, the site of action of MLT was investigated. The binding of 2-[I-125]iodomelatonin (I-125-MLT; 7-240 pmol/l) to Leydig cell membrane fragments was tested in the presence or absence of guanosine 5'-0-(3-thiotriphosphate) (GTP-gamma-S; 50 mu mol/l). Saturation studies and Scatchard analysis revealed the existence of a high-affinity binding site with a B-max of 46.70 +/- 3.50 fmol/mg protein and a K-d of 88.70+/-6.20 pmol/l; treatment with GTP-gamma-S reduced the concentration of I-125-MLT binding sites (B-max 34.03+/-4.50), while increasing the K-d to 106.5 +/- 2.61 pmol/l. Pretreatment of the cells with pertussis toxin (PTX; 10 ng/ml for 16 h) resulted in a decreased binding of I-125-MLT and a lack of effect of GTP-gamma-S. Moreover, the effect of MLT on testosterone secretion induced by LH (30 mIU/ml), forskolin (1 mu mol/l) and LHRH (100 nmol/l) was studied after 3-h incubation of cells which had been precultured with or without PTX. The inhibition of testosterone secretion due to MLT administration was eliminated by PTX pretreatment during forskolin and LH, but not during LHRH administration. However, 17-hydroxyprogesterone levels were higher in all groups incubated in the presence of MLT, irrespective of PTX pretreatment. Our data suggest that: (a) MLT receptors are present on the membranes of adult rat Leydig cells; (b) they couple through PTX-sensitive G-protein-coupled binding sites; (c) the mechanism by which MLT blocks 17-20 desmolase enzymatic activity (thus leading to increased 17-hydroxyprogesterone levels), and testosterone secretion during LHRK stimulation is likely to depend on one or more different mechanism(s) of action.