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-[125I]iodomelatonin ([125I]Mel) as the radioligand. The [125I]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: Ca2+ > Mg2+ > Li+ > Na+ > K+ > cholinechloride. Contrary to most other melatonin binding sites, millimolar concentrations of Ca2+ and Mg2+ did not promote binding in the quail heart membranes. In vitro autoradiography indicated homogenous labeling in the heart. Our results demonstrated [125I]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).