Pharmacological dose of melatonin reduces cytosolic calcium load in response to cholecystokinin in mouse pancreatic acinar cells
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Intracellular Ca2+ overload has been considered a common pathological precursor of pancreatic injury. In this study, the effects of melatonin on Ca2+ mobilization induced by cholecystokinin octapeptide (CCK-8) in freshly isolated mouse pancreatic acinar cells have been examined. Changes in intracellular free Ca2+ concentration were followed by single cell fluorimetry. For this purpose, cells were loaded with the Ca2+-sensitive fluorescent dye fura-2-acetoxymethyl ester. In order to evaluate the contribution of Ca2+ transport at the plasma membrane, at the endoplasmic reticulum (ER) or at the mitochondria, cells were incubated with CCK-8 alone or in combination with LaCl3, thapsigargin (Tps), or FCCP to, respectively, uncouple Ca2+ transport at these localizations. The experiments were performed in the absence or in the presence of melatonin in combination with the stimuli mentioned. Our results show that the total Ca2+ mobilization evoked by CCK-8 was attenuated by a 30 % in the presence of 100 µM melatonin compared with the responses induced by CCK-8 alone. Upon inhibition of Ca2+ transport into the ER by Tps, Ca2+ mobilization was also reduced in the presence of melatonin. In the presence of LaCl3 plus melatonin, the total Ca2+ mobilization induced by CCK-8 was significantly decreased, compared with the response obtained without melatonin but in the presence of LaCl3. No major differences were found when the cells were incubated with CCK-8 or Tps alone or in combination with LaCl3 plus melatonin and FCCP, compared with the responses obtained in the absence of FCCP. The initial Ca2+ release from intracellular stores evoked by CCK-8 or Tps was not significantly reduced in the presence of melatonin. The effect of melatonin could be explained on the basis of a stimulated Ca2+ transport out of the cell through the plasma membrane and by a stimulation of Ca2+ reuptake into the ER. Accumulation of Ca2+ into mitochondria might not be a major mechanism stimulated by melatonin. We conclude that melatonin alleviates intracellular Ca2+ accumulation, a situation potentially leading to cell damage in the exocrine pancreas.
KeywordsMelatonin Calcium Cholecystokinin Ion transport Pancreas
Intracellular free Ca2+ concentration
Ethylene glycol-bis(2-aminoethylether)-N,N,N′N′-tetraacetic acid
Carbonyl cyanide p-trifluoromethoxy-phenylhydrazone
Fura-2 acetoxymethyl ester
Plasma membrane calcium ATPase
Sarcoendoplasmic reticulum Ca2+-ATPase
The authors declare that there is no conflict of interest. This work was supported by Junta de Extremadura-FEDER (GR10010). Patricia Santofimia-Castaño was granted a fellowship from Junta de Extremadura (Consejería de Economía, Comercio e Innovación) and European Social Fund. The authors would like to thank Mrs. Mercedes Gomez Blázquez for her excellent technical support.
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