Pharmacology & Toxicology Poster Session
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Introduction
Melatonin (MEL) has been described to be a potent calmodulin antagonist in several non lymphoid cell lines (1,2), and has been suggested to modulate multiple cellular functions via this mechanism of action (3,4). Calmodulin, a ubiquitous transducer protein of the intracellular calcium signal, plays a key role in activation processes of lymphocytes, such as the production of IL-2 (5). We therefore studied the influence of MEL in comparison to the well established calmodulin antagonists trifluoperazine and W7 on the production of IL-2 in the lymphoblastoid Jurkat cell line.
Materials and Methods
The human T-cell line Jurkat E6.1 (obtained from ECACC) was cultured in RPMI1640/10% FCS at a concentration of 5 x 10^5 cells/ml. Different concentrations of MEL (100 mM stock solution in ethanol), trifluoperazine or W7 were added together with the stimulatory agents ionomycin + PMA or Con A + PMA. Culture supernatants were taken after 24 hours or at different time points to examine the time profile of IL-2 production. Cell viability was determined by trypan blue dye exclusion. IL-2 was measured by an ELISA with primary and secondary monoclonal antibodies (Genzyme) and IL-2 standard (Boehringer Mannheim).
Results
While in non-toxic dosages TFP and W7 inhibited IL-2 production down to 42% and 44%, respectively, MEL did not affect IL-2 production of stimulated Jurkat cells. TFP significantly decreased IL-2 production of activated Jurkat cells already after 4 hours, but MEL did not affect it at any time point of incubation. Two-way ANOVA revealed a significant (p<0,001) dose-dependent decreasing effect of TFP on IL-2 production in activated Jurkat cells, whereas MEL pretreatment had no effect. In isolated human lymphocytes TFP significantly decreased proliferation, whereas MEL did not affect PBML mitogenesis.
Discussion and Conclusion
Taken together, our data clearly show that IL-2 production after T-cell receptor dependent or pharmacologic activation of Jurkat T-cells by Con A + PMA or ionomycin + PMA, respectively, is markedly inhibited by calmodulin antagonists. In contrast to that MEL did not affect IL-2 production at any time point neither in physiological nor in pharmacological dosages proving that MEL is not acting as a calmodulin antagonist in this model. Therefore, the concept of MEL being a general antagonist to calmodulin has to be questioned, and the mechanisms of MEL affecting calmodulin-dependent cellular processes need further, more differentiated investigation.
References
1) Benitez-King G. Huerto-Delgadillo L, Anton-Tay F. BRAIN RES 557 (1991): 289-292
2) Benitez-King G, Huerto-Delgadillo L. Anton-Tay F. LIFE SCI 53 (1993): 201-207
3) Pozo D, Reiter RJ, Calvo JR, Guerrero JM. LIFE SCI 55 (1994): PL 455-460
4) Anton-Tay F, Huerto-Delgadillo L, Ortega-Corona B, Benitez-King G. in: Melatonin and the pineal gland- from basic science to clinical application. Excerpta medica (1993)
5) Cheung RK, Grinstein S, Gelfand EW. J. IMMUNOL. 131 (1983): 2291-2295
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