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-[I-125]melatonin by macrophages fulfils all criteria for bind ing to a receptor site. Thus, binding was dependent on time, temperature an d cell concentration, stable, reversible, saturable and specific. Stoichiom etric studies showed a high-affinity binding site with a K-d of 0.58-0.71 n M. These data are in close agreement with data obtained from kinetic studie s (K-d = 0.29 nM). The affinity of these binding sites suggests that they m ay recognize the physiological concentrations of melatonin in serum. Moreov er, binding experiments using macrophage crude membranes showed that melato nin bound specifically to the membranes. Additionally, in competition studi es we observed a low-affinity binding site (K-d = 2.02 mu M). Melatonin inh ibited significantly forskolin-stimulated cyclic AMP accumulation in a dose -dependent manner. This effect was blocked by luzindole, an antagonist of t he melatonin membrane receptor. Pretreatment of macrophages with pertussis toxin blocked the inhibitory effect of melatonin. Pertussis toxin ADP-rybos ilation and Western blot experiments demonstrated both alpha(i1/2) and alph a(i3/o) G protein subunits expression in mouse peritoneal macrophages membr anes. Our results demonstrate the existence of melatonin receptors in mouse peritoneal macrophages, and a pertussis toxin-sensitive melatonin signal t ransduction pathway that involves the inhibition of adenylyl cyclase. (C) 1 999 Elsevier Science B.V. All rights reserved.