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Melatonin plays a pleiotropic role in the immune system of mammals and birds. Endogenous and exogenous melatonin modulates lymphocyte proliferation via specific MT1, MT2 and Mel1c membrane receptors, although the mechanisms behind this process are poorly understood. The diurnal changes in the expression and function of melatonin membrane receptors within the immune system have so far received little attention. We investigated the day/night differences in melatonin membrane receptor mRNA expression in chicken lymphoid organs and cultured splenocytes and examined the in vitro effect of melatonin and 2-iodomelatonin on the intracellular Ca2+ concentration ([Ca2+]i) in chicken splenocytes. In whole organs, expression of all subtypes of Mel membrane receptors was observed, and the level did not change significantly with the time of day. Interestingly, we observed a significant increase in the expression of the transcripts of all receptor subtypes in cultured splenocytes isolated at night compared with cells obtained during the day. In chicken spleen leukocytes isolated during the day, melatonin and 2-iodomelatonin increased [Ca2+]i, with only 2-iodomelatonin being effective in the ‘night’ cells. Luzindole modulated the [Ca2+]i increase caused by melatonin receptor agonists: it potentiated the stimulatory effect of melatonin during the day, but counteracted that evoked by 2-iodomelatonin at night. The results of this study demonstrate that melatonin can induce changes in [Ca2+]i in chicken spleen leukocytes that should modulate proliferation. The effect of melatonin on [Ca2+]i is less pronounced at night, possibly caused by receptor desensitization.