Melatonin receptor-mediated stimulation of phosphoinositide breakdown in chick brain slices

The direct effect of melatonin and related agonists on Li⁺-amplified phosphoinositide breakdown was studied in chick brain slices prelabeled with myo-[2-³H]inositol. The melatonin receptor agonist 6-chloromelatonin (10-100 μM) increased, in a concentration-dependent manner, the accumulation of inositol phosphates (IP) in chick brain slices. This effect of 6-chloromelatonin (10 μM) was rapid as transient increases in IP₃/IP₄ (maximal increase, 29% at 20 s) and IP₂ levels (maximal in- crease, 36% at 1 min) were observed, followed by a slower but sustained increase in IP₁ level (30% at 5 min), when the amount of IP₃/IP₄ and IP₂ had already been decreased to the control level. The phosphoinositide response elicited by 6-chloromelatonin (10 μM) was dependent on the presence of extracellular calcium. Direct stimulation of membrane phospholipase C by 6-chloromelatonin (10 μM) in isolated myo-[2- ³H]inositol-prelabeled optic tectum membranes was dependent on the presence of guanosine-5'-O-(3-thio)triphosphate (1 μM), thus suggesting that G protein(s) link melatonin receptor activation to phospholipase C stimulation. The competitive melatonin receptor antagonist luzindole (10- 100 μM) inhibited in a concentration-dependent manner the IP₁ accumulation stimulated by 6-chloromelatonin (10-100 μM); however, it did not affect the accumulation stimulated by 5-hydroxytryptamine (10 μM). By contrast, methysergide (10 μM) completely inhibited 5-hydroxytryptamine (10 μM)-, but not 6-chloromelatonin (10 μM)-, induced IP₁ accumulation. Melatonin receptor agonists increased IP₁ accumulation in a concentration-dependent manner reaching different maximal responses. N- Acetyl-5-hydroxytryptamine was more potent than melatonin in increasing IP₁ accumulation, suggesting activation of a melatonin receptor site other than the ML-1 melatonin receptor (i.e., N-acetyl-5- hydroxytryptamine ≥ melatonin). In conclusion, these results demonstrate that activation of a melatonin receptor with pharmacological characteristics different from those of the ML-1 subtype leads to activation of the phospholipase C-mediated signal transduction pathway.