2-[125I]Iodomelatonin binding sites in the quail heart: Characteristics, distribution and modulation by guanine nucleotides and cations

Abstract To investigate whether melatonin has a direct action on the cardiovascular system, putative melatonin receptors were studied in quail heart membrane preparations using the specific melatonin agonist 2-[ 125 I]iodomelatonin ([ 125 I]Mel) as the radioligand. The [ 125 I]Mel binding demonstrated in the mature quail heart was saturable, highly specific and reversible, of picomolar affinity and femtomolar density (Kd = 35.2 ±5.2 pM; Bmax = 1.32 ± 0.25 fmol/mg protein; n = 8). The linear Scatchard plots and the close to unity Hill coefficient indicated a single class of binding sites. The pharmacological profile was in the affinity order of 2-iodomelatonin = 2-phenylmelatonin > melatonin > 6-chloromelatonin ⪢ 6-hydroxymelatonin > 6-sulphatoxymelatonin ⪢ N-acetylserotonin ⋙ 5-hydroxytryptamine. Guanosine 5′- riphosphate and guanosine 5'-O-(3-thiotriphosphate) (GTPγS) dose dependently inhibited the binding. Ten μM GTPγS lowered the binding affinity by 50% in saturation studies. The order of potency of inhibition by cations was: Ca 2+ > Mg 2+ > Li + > Na + > K + > choline chloride . Contrary to most other melatonin binding sites, millimolar concentrations of Ca 2+ and Mg 2+ did not promote binding in the quail heart membranes. In vitro autoradiography indicated homogenous labeling in the heart. Our results demonstrated [ 125 I]Mel binding sites in the quail heart. That guanine nucleotides and Na + inhibited the binding indicated that these putative melatonin receptors are coupled to guanine nucleotide-binding proteins (G-proteins).