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Molecular and Cellular Biochemistry

, Volume 397, Issue 1–2, pp 75–86 | Cite as

Pharmacological dose of melatonin reduces cytosolic calcium load in response to cholecystokinin in mouse pancreatic acinar cells

  • Patricia Santofimia-Castaño
  • Deborah Clea Ruy
  • Miguel Fernandez-Bermejo
  • Gines M. Salido
  • Antonio GonzalezEmail author
Article

Abstract

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.

Keywords

Melatonin Calcium Cholecystokinin Ion transport Pancreas 

Abbreviations

CCK-8

Cholecystokinin octapeptide

[Ca2+]c

Intracellular free Ca2+ concentration

EGTA

Ethylene glycol-bis(2-aminoethylether)-N,N,NN′-tetraacetic acid

ER

Endoplasmic reticulum

FCCP

Carbonyl cyanide p-trifluoromethoxy-phenylhydrazone

Fura-2/AM

Fura-2 acetoxymethyl ester

LaCl3

Lanthanum chloride

PMCA

Plasma membrane calcium ATPase

SERCA

Sarcoendoplasmic reticulum Ca2+-ATPase

Tps

Thapsigargin

Notes

Acknowledgements

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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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Patricia Santofimia-Castaño
    • 1
  • Deborah Clea Ruy
    • 2
  • Miguel Fernandez-Bermejo
    • 3
  • Gines M. Salido
    • 1
  • Antonio Gonzalez
    • 1
    Email author
  1. 1.Department of Physiology (Cell Physiology Research Group)University of ExtremaduraCaceresSpain
  2. 2.Facultade de Agronomia & Medicina VeterinariaUniversidade de BrasiliaBrasíliaBrazil
  3. 3.Department of GastroenterologySan Pedro de Alcantara HospitalCaceresSpain

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