Melatonin regulates splenocytes proliferation via IP3-dependent intracellular Ca2+ release in seasonally breeding bird, Perdicula asiatica

Melatonin plays an important role in the immune regulation of birds. Both endogenous and exogenous melatonin modulates lymphocyte proliferation via its specific membrane receptors, Mel(1a), Mel(1b) and Mel(1c), though the mechanisms behind this process are poorly understood. We investigated the differences in melatonin membrane receptor Mel(1a), Mel(1b) and Mel(1c) expression by western blot and reverse transcription reaction and the in vitro effect of melatonin on the intracellular Ca2+ concentration ([Ca2+]i) in splenocytes of the Indian Jungle Bush Quail, Perdicula asiatica. We used a non-selective melatonin receptor antagonist for Mel(1a) and Mel(1b), luzindole, and the selective Mel(1b) blocker, 4P-PDOT to check the specific role of melatonin receptor on ([Ca2+]i). The expression of Mel(1a), Mel(1b) and Mel(1c) receptors mRNA and protein was upregulated by melatonin (10(-7) M) with a significant high rise in ([Ca2+]i), which was differentially blocked by supplementation of antagonist, luzindole (10(-7) M) and 4P-PDOT (10(-7) M). Furthermore, we noted in vitro effect of melatonin and 2-aminoethoxydiphenyl borate (2-APB), a cell-permeable antagonist of inositol 1, 4, 5-trisphosphate (IP3) receptor to check the rise in ([Ca2+]i) through the IP3 pathway. Significantly low ([Ca2+]i) was noted in melatonin and 2-APB pretreated splenocytes when compared with splenocytes where 2-APB was absent. Thus, our data suggest that melatonin through its membrane receptor induced the elevation of ([Ca2+]i) via IP3-dependent pathway for splenocyte proliferation in P. asiatica.