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Melatonin induces calcium mobilization and influences cell proliferation independently of MT1/MT2 receptor activation in rat pancreatic stellate cells

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Abstract

Melatonin, the product of the pineal gland, possesses antioxidant, anti-inflammatory, and antitumor properties in different tissues, in addition to its role as regulator of biological rhythms. In this study, the effects of pharmacological concentrations of melatonin (1 μM–1 mM) on pancreatic stellate cells (PSCs) have been examined. Cell viability was studied using AlamarBlue® test. Cell-type specific markers and total amylase content were analyzed by immunocytochemistry and colorimetric methods, respectively. Changes in intracellular free Ca2+ concentration were followed by fluorimetric analysis of fura-2-loaded cells. The cellular red-ox state was monitored following CM-H2DCFDA-derived fluorescence. Determination of the activation of p44/42 mitogen-activated protein kinase (MAPK), SAPK/JNK and p38 was measured by Western blot analysis. Our results show that PSCs viability decreased in the presence of 100 μM or 1 mM melatonin. However, in the presence of 1 or 10 μM melatonin, no changes in cell viability were observed. Melatonin MT1 and MT2 receptors could not be detected. Melatonin induced Ca2+ mobilization from intracellular pools. In the presence of melatonin, activation of crucial components of MAPKs pathway was noticed. Finally, the indole did not change the oxidative state of PSCs, but exerted a protective effect against H2O2-induced oxidation. We conclude that melatonin, at pharmacological concentrations, might regulate cellular proliferation of PSCs independently of specific plasma membrane receptors.

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Abbreviations

CCK-8:

Cholecystokinin octapeptide

[Ca2+]c :

Intracellular free Ca2+ concentration

CM-H2DCFDA:

5-(and-6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester

EGTA:

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

ER:

endoplasmic reticulum

Fura-2/AM:

Fura-2 acetoxymethyl ester

H2O2 :

Hydrogen peroxide

PSCs:

Pancreatic stellate cells

SERCA:

Sarcoendoplasmic reticulum Ca2+-ATPase

Tps:

Thapsigargin

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Acknowledgments

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 Gobierno de Extremadura (PD10058; Consejeria de Empleo, Empresa e Innovacion) and European Social Fund. The authors would like to thank Mrs. Mercedes Gomez Blazquez and Mr. J. J. Gomez for their excellent technical support.

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Authors and Affiliations

  1. Cell Physiology Research Group, Department of Physiology, University of Extremadura, Avenida Universidad s/n, E-10003, Caceres, Spain

    Patricia Santofimia-Castaño, Lourdes Garcia-Sanchez, Gines M. Salido & Antonio Gonzalez

  2. Facultade de Agronomia & Medicina Veterinaria, Universidade de Brasilia, 70900-100, Brasilia, DF, Brasil

    Deborah Clea Ruy

  3. Immunology Unit, Department of Physiology, University of Extremadura, Caceres, Spain

    Beatriz Sanchez-Correa & Raquel Tarazona

  4. Department of Gastroenterology, San Pedro de Alcantara Hospital, 10003, Caceres, Spain

    Miguel Fernandez-Bermejo

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  1. Patricia Santofimia-Castaño
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  2. Lourdes Garcia-Sanchez
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  3. Deborah Clea Ruy
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  7. Gines M. Salido
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  8. Antonio Gonzalez
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Correspondence to Antonio Gonzalez.

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Santofimia-Castaño, P., Garcia-Sanchez, L., Ruy, D.C. et al. Melatonin induces calcium mobilization and influences cell proliferation independently of MT1/MT2 receptor activation in rat pancreatic stellate cells. Cell Biol Toxicol 31, 95–110 (2015). https://doi.org/10.1007/s10565-015-9297-6

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