Melatonin Reduces Apoptosis Induced by Calcium Signaling
in Human Leukocytes: Evidence for the Involvement
of Mitochondria and Bax Activation
Pedro C. Redondo
Juan A. Rosado
Russel J. Reiter
Ana B. Rodrı
Received: 29 October 2009 / Accepted: 11 January 2010 / Published online: 4 February 2010
Ó Springer Science+Business Media, LLC 2010
Abstract We have evaluated the effect of melatonin on
apoptosis evoked by increases in [Ca
in human leu-
kocytes. Our results show that treatment of neutrophils
with the calcium mobilizing agonist FMLP or the speciﬁc
inhibitor of calcium reuptake thapsigargin induced a tran-
sient increase in [Ca
. Our results also show that FMLP
and thapsigargin increased caspase-9 and -3 activities and
the active forms of both caspases. The effect of FMLP and
thapsigargin on caspase activation was time-dependent.
Similar results were obtained when lymphocytes were
stimulated with thapsigargin. This stimulatory effect was
accompanied by induction of mPTP, activation of the
proapoptotic protein Bax and release of cytochrome c.
However, when leukocytes were pretreated with melatonin,
all of the apoptotic features indicated above were signiﬁ-
cantly reversed. Our results suggest that melatonin reduces
caspase-9 and -3 activities induced by increases in [Ca
in human leukocytes, which are produced through the
inhibition of both mPTP and Bax activation.
Keywords Melatonin Á Caspases Á Cytochrome c Á
Mitochondria Á Apoptosis Á Leukocytes
Apoptosis is a gene-regulated form of cell death that is
critical for normal development and tissue homeostasis. A
major component of the apoptotic machinery involves a
family of aspartic acid-directed cysteine proteases, called
‘‘caspases’’ (cysteinyl aspartate-speciﬁc proteinases),
which cleave multiple protein substrates en masse, leading
to the loss of cellular structure and function and ultimately
resulting in cell death (Stennicke and Salvesen 1997).
From a functional point of view, caspases involved in
apoptosis act either as initiators (caspases 8, 9 and 10) or as
effectors (caspases 3, 6 and 7) (Earnshaw et al. 1999).
Caspase-8 was identiﬁed as the most important initiator
enzyme of the Fas/CD95 pathway (Kischkel et al. 1995).
Caspase-9 interacts with many other regulators and trans-
ducers, such as cytochrome c, in intrinsic pathways (Shi
2002). Both initiator caspases are activators of downstream
caspases. Caspase-3, the most important among them,
executes the ﬁnal disassembly of the cell by cleaving a
variety of cell structure proteins and generating DNA
strand breaks (Enari et al. 1998).
Traditionally, two general apoptotic pathways have been
described. The ﬁrst is the extrinsic pathway, triggered by the
binding of an extracellular death ligand, such as factor
activating ExoS ligand (FasL), to its cell-surface death
receptor, such as Fas (Ashkenazi and Dixit 1998). The sec-
ond is the intrinsic pathway, which is mediated by mito-
chondrial alterations. In response to apoptotic stimuli,
several proteins are released from the mitochondrial inter-
membrane space into the cytoplasm (Green and Reed 1998).
Some of the well-characterized proteins include cytochrome
c, which mediates the activation of caspase-9 (Li et al.
1997), triggering a cascade of caspase activation, including
caspase-3, which promotes cellular self-destruction.
J. Espino Á I. Bejarano Á P. C. Redondo Á
J. A. Rosado Á C. Barriga Á J. A. Pariente (&) Á A. B. Rodrı
Department of Physiology, University of Extremadura,
06071 Badajoz, Spain
R. J. Reiter
Department of Cellular and Structural Biology,
The University of Texas Health Sciences Center,
7703 Floyd Curl Drive, San Antonio, TX 78229, USA
J Membrane Biol (2010) 233:105–118