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Melatonin induces reactive oxygen species generation and changes in glutathione levels and reduces viability in human pancreatic stellate cells

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Abstract

In this study, the effects of pharmacological concentrations of melatonin (1 μM–1 mM) on human pancreatic stellate cells (HPSCs) have been examined. Cell type–specific markers and expression of melatonin receptors were analyzed by western blot analysis. Changes in intracellular free Ca2+ concentration were followed by fluorimetric analysis of fura-2–loaded cells. Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were determined by fluorescence techniques. Production of reactive oxygen species (ROS) was monitored following 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester and MitoSOX™ Red–derived fluorescence. Cell viability was studied using the AlamarBlue® test. Cultured cells expressed markers typical of stellate cells. However, cell membrane receptors for melatonin could not be detected. Thapsigargin, bradykinin, or melatonin induced changes in intracellular free Ca2+ concentration. In the presence of the indole, a decrease in the GSH/GSSG ratio was observed that depended on the concentration of melatonin used. Furthermore, the indole evoked a concentration-dependent increase in ROS production in the mitochondria and in the cytosol. Finally, melatonin decreased HPSC viability in a time and concentration-dependent manner. We conclude that melatonin, at pharmacological concentrations, induces changes in the oxidative state of HPSC. This might regulate cellular viability and could not involve specific plasma membrane receptors.

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Abbreviations

[Ca2+]c :

Intracellular free Ca2+ concentration

CM-H2DCFDA:

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

ER:

Endoplasmic reticulum

Fura-2-AM:

Fura-2-acetoxymethyl ester

GSH:

Reduced glutathione

GSSG:

Oxidized glutathione

HBSS:

Hank’s balanced salts

HPSCs:

Human pancreatic stellate cells

H2O2 :

Hydrogen peroxide

ROS:

Reactive oxygen species

RPSCs:

Rat pancreatic stellate cells

SERCA:

Sarcoendoplasmic reticulum Ca2+-ATPase

Tps:

Thapsigargin

α-sma:

Alpha-smooth muscle actin

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Funding

This study was partly funded by the Ministerio de Economía y Competitividad (BFU2016-79259-R; UNEX13-1E-1608) and Junta de Extremadura-FEDER (IB16006). The funding sources had no role in the study design; in the collection, analysis, and interpretation of the data; in the writing of the report; and in the decision to submit the paper for publication.

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Authors and Affiliations

  1. Department of Physiology, Institute of Molecular Pathology Biomarkers, University of Extremadura, Avenida Universidad s/n, 10003, Cáceres, Spain

    Matias Estaras, Noelia Moreno, Gines M. Salido & Antonio Gonzalez

  2. Centre de Recherche en Cancérologie de Marseille, INSERM U1068, CNRS UMR 7258, Aix-Marseille Université and Institut Paoli-Calmettes, Parc Scientifique et Technologique de Luminy, Marseille, France

    Patricia Santofimia-Castaño & Juan L. Iovanna

  3. Unit of Toxicology, Veterinary Faculty, University of Extremadura, Cáceres, Spain

    Salome Martinez-Morcillo

  4. Unit of Histology and Pathological Anatomy, Veterinary Faculty, University of Extremadura, Cáceres, Spain

    Vicente Roncero

  5. Hepatobiliary-Pancreatic Surgery and Liver Transplant Unit, Infanta Cristina Hospital, Badajoz, Spain

    Gerardo Blanco & Diego Lopez

  6. Department of Gastroenterology, San Pedro de Alcantara Hospital, Cáceres, Spain

    Miguel Fernandez-Bermejo & Jose M. Mateos

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  1. Matias Estaras
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Correspondence to Antonio Gonzalez.

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Estaras, M., Moreno, N., Santofimia-Castaño, P. et al. Melatonin induces reactive oxygen species generation and changes in glutathione levels and reduces viability in human pancreatic stellate cells. J Physiol Biochem 75, 185–197 (2019). https://doi.org/10.1007/s13105-019-00671-x

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