Melatonin reduces proliferation in many different cancer cell lines. Thus, melatonin is considered a promising antitumor agent, promoting apoptosis in tumor cells while preserving viability of normal cells. Herein, we examined the effects of melatonin on the pancreatic AR42J tumor cell line. We have analyzed cytosolic-free Ca(2+) concentration ([Ca(2+) ](c) ), mitochondrial-free Ca(2+) concentration ([Ca(2+) ](m) ), mitochondrial membrane potential (Ψm), mitochondrial flavin adenine dinucleotide (FAD) oxidative state, cellular viability and caspase-3 activity. Our results show that melatonin induced transient changes in [Ca(2+) ](c) and [Ca(2+) ](m) . Melatonin also induced depolarization of Ψm and led to a reduction in the level of oxidized FAD. In addition, melatonin reduced AR42J cell viability. Finally, we found a Ca(2+) -dependent caspase-3 activation in response to melatonin. Collectively, these data support the likelihood that melatonin reduces viability of tumor AR42J cells via its action on mitochondrial activity and caspase-3 activation.

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 μm and 1 mm) 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 μm 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.

Background: A significant percentage of patients with pancreatitis often presents a history of excessive alcohol consumption. Nevertheless, the patho-physiological effect of ethanol on pancreatitis remains poorly understood. In the present study, we have investigated the early effects of acute ethanol exposure on CCK-8-evoked Ca 2+ signals in mouse pancreatic acinar cells. Changes in [Ca 2+ ] i and ROS production were analyzed employing fluorescence techniques after loading cells with fura-2 or CM-H 2 DCFDA, respectively.

The effects of alcohol on Ca(2+) signalling remains poorly understood. Here we have investigated the effects of acute ethanol exposure on Ca(2+) influx in mouse pancreatic acinar cells. Cells were loaded with fura-2 and the changes in fluorescence were monitored by spectrofluorimetry and imaging analysis. Stimulation of cells with 20 pM cholecystokinin evoked an oscillatory pattern in [Ca(2+)](c), both in the presence and in the absence of extracellular Ca(2+). Stimulation of cells with cholecystokinin in the presence of 50 mM ethanol led to a transformation of physiological oscillations into a single transient increase in [Ca(2+)](c). This effect was observed when Ca(2+) was present in the extracellular medium, and did not appear in its absence. Addition of 1 mM CaCl(2) to the extracellular medium, following release of Ca(2+) from intracellular stores by stimulation of cells with 1 nM cholecystokinin or 1 microM thapsigargin in the absence of extracellular Ca(2+), was followed by an increase in [Ca(2+)](c). Ca(2+) influx was increased in the presence of 50 mM ethanol. The anti-oxidant cinnamtannin B-1 (10 microM) or inhibition of alcohol dehydrogenase by 4-MP (1 mM), significantly reduced Ca(2+) influx evoked by cholecystokinin in the presence of ethanol. In summary, intoxicating concentrations of ethanol may lead to over stimulation of pancreatic acinar cells by cholecystokinin. This might be partially explained by the generation of reactive oxygen species and an increased Ca(2+) entry in the presence of ethanol. Potentially ethanol might lead to Ca(2+) overload, which is a common pathological precursor that is implicated in pancreatitis.

SUMMARY1. Cinnamtannin B-1 is a naturally occurring A-type proanthocyanidin that belongs to a class of polyphenols widely distributed throughout the plant kingdom and exhibiting anti-oxidant properties.2. In the present study, we examined the effects of cinnamtannin B-1 on cholecystokinin octapeptide (CCK-8)-evoked Ca Cholecystokinin-8 had a biphasic effect on amylase secretion, producing maximum at a concentration of 0.1 nmol ⁄ L and reducing secretion at higher concentrations. Pre-incubation of cells with 10 lmol ⁄ L cinnamtannin B-1 significantly attenuated the inhibition of enzyme secretion in response to high concentrations of )10 mol ⁄ L). Finally, the anti-oxidant protected acinar cells against CCK-8-induced cell death. 4. The beneficial effects of cinnamtannin B-1 appear to be mediated by a reduction in intracellular Ca 2+ overload, ROS production and intracellular accumulation of digestive enzymes, which is a common pathological precursor that mediates pancreatitis.