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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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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DOI: https://doi.org/10.1007/s13105-019-00671-x