Melatonin induces calcium release from CCK-8- and thapsigargin- sensitive cytosolic stores in pancreatic AR42J cells Introduction In pancreatic acinar cells, activation of phospholipase C (PLC)-linked receptors by agonists, such as acetylcholine or cholecystokinin (CCK), produces a cytosolic increase in the concentration of inositol 1,4,5-trisphosphate (IP 3 ). IP 3 in turn releases calcium (Ca 2+ ) from cytoplasmic stores leading to an increase in cytosolic free calcium concentra- tion ([Ca 2+ ] c ) [1]. Ca 2+ signals are not only a result of its release from intracellular stores but also a co-ordinate influx from the extracellular space [2], Ca 2+ extrusion across the plasma membrane [3] as well as Ca 2+ uptake into intracellular organelles [4]. A rise in [Ca 2+ ] c is an important early signal by which physiological secretagogues elicit the release of digestive enzymes from pancreatic acinar cells. The exocrine pancreas is highly specialized for the synthesis, storage and exocrine secretion of digestive enzymes [5], and gastrointestinal hormones such as CCK, gastrin, neurotensin and bombesin/gastrin-releasing pep- tide are potent regulatory peptides that modulate pancre- atic function. Its function is thus subject to regulation and to interdigestive ÔrestingÕ periods [6–8]. In mammals, melatonin has been implicated in the regulation of many physiological processes. The hormone is produced in the mammalian pineal gland and retina following a circadian rhythm with high levels being released from the pineal in the blood at night. The circadian secretion of melatonin is controlled by the suprachiasmatic nucleus of the hypothalamus and synchronized to a 24 hr cycle by the daily photoperiod [9, 10]. Melatonin, in turn, acts via G-protein-coupled receptors to synchronize these clock-generated circadian rhythms [11]. In addition to the pineal gland, melatonin is also released from the gastroin- testinal tract, where the amount of this indole is greater than the content of melatonin in the central nervous system [12]. Moreover, the indole is present in plant foods [13]. Melatonin MT1 and MT2 receptors are G-protein- coupled receptors, which are expressed in various parts of the CNS (suprachiasmatic nuclei, hippocampus, cerebellar cortex, prefrontal cortex, basal ganglia, substantia nigra, ventral tegmental area, nucleus accumbens and retinal horizontal, amacrine and ganglion cells) and in peripheral organs (blood vessels, mammary gland, gastrointestinal tract, liver, kidney and bladder, ovary, testis, prostate, skin and the immune system) [14]. The existing data support that the actions of the indoleamine are related to its effects on intracellular cAMP [15], reactive oxygen species (ROS) production [16] or via the cGMP signalling pathway [17]. Recently, it has been shown that exogenous melatonin has an effect on [Ca 2+ ] c in pancreatic islets [18]. Formerly, Bach et al. [19] showed the involvement of the second messengers IP 3 and [Ca 2+ ] c in Abstract: Melatonin is produced following circadian rhythm with high levels being released at night and has been implicated in the regulation of physiological processes in major tissues, including the pancreas. The aim of our study was to examine the effects of melatonin on intracellular free Ca 2+ concentration ([Ca 2+ ] c ) in AR42J pancreatic cells. Our results show that stimulation of cells with 1 nm cholecystokinin (CCK)-8 led to a transient increase in [Ca 2+ ] c followed by a decrease towards a value close to the prestimulation level. Melatonin (at the concentrations 1, 10, 100 lm and 1mm) induced changes in [Ca 2+ ] c that consisted of single or short lasting spikes in the form of oscillations or slow transient increases followed by a slow reduction towards a value close to the resting level. Depletion of intracellular Ca 2+ stores by stimulation of cells with 1 nm CCK-8 or 1 lm thapsigargin (Tps) blocked Ca 2+ responses evoked by melatonin in the majority of cells. Conversely, prior stimulation of cells with 1 mm melatonin in the absence of extracellular Ca 2+ inhibited Ca 2+ mobilization in response to a secondary application of CCK-8 or Tps. In summary, our results show that melatonin releases Ca 2+ from intracellular stores and can therefore modulate the responses of the pancreas to CCK-8. The source for Ca 2+ mobilization most probably is the endoplasmic reticulum. These data raise the possibility that melatonin also involves Ca 2+ signalling, in addition to other intracellular messengers, to modulate cellular function. Angel del Castillo-Vaquero, Gines M. Salido and Antonio Gonzalez Department of Physiology (Cell Physiology Research Group), University of Extremadura, Caceres, Spain Key words: AR42J, calcium, cholecystokinin, fluorescence, melatonin, pancreas Address reprint requests to Antonio Gonzalez, Department of Physiology, Faculty of Veteri- nary Sciences, University of Extremadura, Avenida Universidad s/n, E-10071 Caceres, Spain. E-mail: agmateos@unex.es Received March 31, 2010; accepted May 25, 2010. J. Pineal Res. 2010; 49:256–263 Doi:10.1111/j.1600-079X.2010.00790.x Ó 2010 The Authors Journal of Pineal Research Ó 2010 John Wiley & Sons A/S Journal of Pineal Research 256 Molecular, Biological, Physiological and Clinical Aspects of Melatonin