Melatonin binding sites are functionally coupled to phosphoinositide hydrolysis in Syrian hamster RPMI 1846 melanoma cells

Recent pharmacological studies demonstrate that Syrian hamster melanoma (RPMI 1846) cells possess a nanomolar-affinity binding site for melatonin. Inhibition of 2-[125I]-iodomelatonin binding to RPMI membranes by melatonergic ligands exhibit a rank order relationship similar to that observed in hamster hypothalamus. Biochemical studies indicate that the melatonin binding site in RPMI 1846 cells is not coupled in either a stimulatory or inhibitory fashion to adenylate cyclase as a second messenger. We now report that stimulation of RPMI 1846 melanoma cells with melatonergic agonists induces phosphoinositide hydrolysis in a concentration-dependent manner (EC50: N-acetylserotonin = 0.29 microM; 2-I-Melatonin = 0.39 microM; 6-Cl-Melatonin = 0.38 microM; Melatonin = 0.45 microM). Further, phosphoinositide hydrolysis induced by 2-I-melatonin and N-acetylserotonin was blocked by pre-incubation with the melatonin antagonist N-acetyltryptamine and prazosin, an antagonist which exhibits potency at the nanomolar affinity melatonin binding site. 2-I-melatonin and N-acetylserotonin-induced phosphoinositide hydrolysis were not blocked by the serotonin type 2 antagonist ketanserin or alpha-adrenergic antagonist, phentolamine. These data suggest that melatonin binding sites on RPMI 1846 cells are linked to phosphoinositide hydrolysis as a second messenger.