Influence of melatonin on chicken lymphocytes in vitro: involvement of membrane receptors.
OBJECTIVES: Time-dependent melatonin effects on chicken lymphocyte proliferation in vitro and the involvement of cAMP in melatonin signal transduction were examined.
MATERIALS AND METHODS: Splenocytes and peripheral blood mononuclear cells (PBMC) were cultured in vitro in the presence of melatonin, phytohemagglutinin, luzindole, dibutyrylcAMP (dbcAMP), forskolin and vasoactive intestine peptide (VIP). Proliferation was measured by [(3)H]-thymidine incorporation in cultures carried out for 24, 36, 48 and 72 h. Cyclic AMP formation was assessed by radioimmunoassay in cells incubated for 30 min. or 24 h.
RESULTS: Melatonin stimulated the spontaneous proliferation in short-term (36 and 48 h) splenocyte cultures and had no effect in 72 h cultures. It inhibited mitogen-stimulated proliferation already in 24 h cultures and this effect was observed regardless of the time of the culture. Both melatonin effects were antagonized by luzindole - membrane-bound melatonin receptor antagonist. Forskolin and dbcAMP caused a significant inhibition of proliferation of splenocytes and PBMC cultured for 24 or 72 h, respectively. Melatonin inhibited the cAMP formation (30 min. of incubation) stimulated by adenosine cyclase activators - forskolin and VIP, but added alone failed to affect the cAMP concentration. In mitogen-stimulated splenocytes cultured for 24 h Mel caused an increase in cAMP correlated with the inhibition of cell proliferation.
CONCLUSIONS: Melatonin effects on chicken splenocytes appears time- and activation-dependent: in short-term cultures it stimulates spontaneous and inhibits mitogen-activated proliferation, probably via membrane-bound, luzindole-sensitive melatonin receptors. Incubation with melatonin for 30 min. inhibits cAMP formation, but in 24 h cultures it increases cAMP concentration leading to inhibition of proliferation....