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

Abstract

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 Mel1b 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.