Aim: 

In the present study we have evaluated the effect of melatonin on caspase activity evoked by increases in cytosolic free calcium concentration ([Ca²⁺]_c) in human neutrophils.

Methods: 

Caspase-3 and -9 activities were determined from the cleavage of the respective specific fluorogenic substrates and measured with a fluorescence spectrophotometer. For [Ca²⁺]_c determination, the cells, once isolated, were loaded with the fluorescent ratiometric calcium indicator Fura-2 AM (2 mM, 30 min, room temperature).

Results: 

Our results show that treatment of neutrophils with the physiological agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP) (10 nM) or the specific inhibitor of calcium reuptake thapsigargin (TG) (1 mM) induced a transient increase in [Ca²⁺]_c due to calcium release from internal stores. Similar results were obtained when the neutrophils were treated with H₂O₂ (100 mM-1 mM). Our results also show that fMLP and TG were able to increase the caspase-3 and -9 activities. The effect of fMLP and TG on caspase activation was time dependent, reaching a maximal caspase activity after 120 min of stimulation. However, when neutrophils were pretreated with 1 mM melatonin for 60 min, caspase activity induced by fMLP and TG was significantly reduced. Melatonin alone had no effect on caspase activation. We have obtained similar results in human lymphocytes.

Conclusion: 

Our results suggest that melatonin is able to reduce caspase-3 and -9 activities induced by increases in cytosolic free calcium concentration ([Ca²⁺]_c) in human neutrophils and lymphocytes.

Supported by MEC-DGI and Junta de Extremadura grants BFU2007-60091 and PRI07-A024, respectively. I. Bejarano was beneficiary of grant by Junta de Extremadura PRE06070.