Characterization of membrane melatonin receptor in mouse peritoneal macrophages: inhibition of adenylyl cyclase by a pertussis toxin-sensitive G protein.

Melatonin binding sites were characterized in mouse peritoneal macrophages. Binding of 2-[125I]melatonin by macrophages fulfills all criteria for binding to a receptor site. Thus, binding was dependent on time, temperature and cell concentration, stable, reversible, saturable and specific. Stoichiometric studies showed a high-affinity binding site with a Kd of 0.58-0.71 nM. These data are in close agreement with data obtained from kinetic studies (Kd = 0.29 nM). The affinity of these binding sites suggests that they may recognize the physiological concentrations of melatonin in serum. Moreover, binding experiments using macrophage crude membranes showed that melatonin bound specifically to the membranes. Additionally, in competition studies we observed a low-affinity binding site (Kd = 2.02 microM). Melatonin inhibited significantly forskolin-stimulated cyclic AMP accumulation in a dose-dependent manner. This effect was blocked by luzindole, an antagonist of the melatonin membrane receptor. Pretreatment of macrophages with pertussis toxin blocked the inhibitory effect of melatonin. Pertussis toxin ADP-rybosilation and Western blot experiments demonstrated both alpha(i1/2) and alpha(i3/o) G protein subunits expression in mouse peritoneal macrophages membranes. Our results demonstrate the existence of melatonin receptors in mouse peritoneal macrophages, and a pertussis toxin-sensitive melatonin signal transduction pathway that involves the inhibition of adenylyl cyclase.