Melatonin induces reactive oxygen species generation and changes in glutathione levels and reduces viability in human pa
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ORIGINAL ARTICLE
Melatonin induces reactive oxygen species generation and changes in glutathione levels and reduces viability in human pancreatic stellate cells Matias Estaras 1 & Noelia Moreno 1 & Patricia Santofimia-Castaño 2 & Salome Martinez-Morcillo 3 & Vicente Roncero 4 & Gerardo Blanco 5 & Diego Lopez 5 & Miguel Fernandez-Bermejo 6 & Jose M. Mateos 6 & Juan L. Iovanna 2 & Gines M. Salido 1 & Antonio Gonzalez 1 Received: 26 October 2018 / Accepted: 26 February 2019 # University of Navarra 2019
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 concentrationdependent 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. Keywords Melatonin . Calcium . Glutathione . Cell viability . Human pancreatic stellate cells
Abbreviations * Antonio Gonzalez [email protected] 1
Department of Physiology, Institute of Molecular Pathology Biomarkers, University of Extremadura, Avenida Universidad s/n, 10003 Cáceres, Spain
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
3
Unit of Toxicology, Veterinary Faculty, University of Extremadura, Cáceres, Spain
4
Unit of Histology and Pathological Anatomy, Veterinary Faculty, University of Extremadura, Cáceres, Spain
5
Hepatobiliary-Pancreatic Surgery and Liver Transplant Unit, Infanta Cristina Hospital, Badajoz, Spain
6
Department of Gastroenterology, San Pedro de Alcantara Hospital, Cáceres, Spain
[Ca2+]c C M H2DCFDA ER Fura-2-AM GSH GSSG HBSS HPSCs H2O2 ROS RPSCs SERCA Tps α-sma
Intracellular free Ca2+ concentration 5-(and-6)-Chloromethyl-2′,7′dichlorodihydrofluorescein
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