Cell Biol Toxicol (2015) 31:95–110 DOI 10.1007/s10565-015-9297-6
ORIGINAL RESEARCH
Melatonin induces calcium mobilization and influences cell proliferation independently of MT1/MT2 receptor activation in rat pancreatic stellate cells Patricia Santofimia-Castaño & Lourdes Garcia-Sanchez & Deborah Clea Ruy & Beatriz Sanchez-Correa & Miguel Fernandez-Bermejo & Raquel Tarazona & Gines M. Salido & Antonio Gonzalez
Received: 14 July 2014 / Accepted: 26 February 2015 / Published online: 13 March 2015 # Springer Science+Business Media Dordrecht 2015
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 exam ined. 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-H2DCFDAderived fluorescence. Determination of the activation of p44/42 mitogen-activated protein kinase (MAPK), SAPK/JNK and p38 was measured by Western blot P. Santofimia-Castaño : L. Garcia-Sanchez : G. M. Salido : A. Gonzalez (*) Cell Physiology Research Group, Department of Physiology, University of Extremadura, Avenida Universidad s/n, E-10003, Caceres, Spain e-mail: [email protected] D. C. Ruy Facultade de Agronomia & Medicina Veterinaria, Universidade de Brasilia, 70900-100 Brasilia, DF, Brasil B. Sanchez-Correa : R. Tarazona Immunology Unit, Department of Physiology, University of Extremadura, Caceres, Spain M. Fernandez-Bermejo Department of Gastroenterology, San Pedro de Alcantara Hospital, 10003 Caceres, Spain
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. Keywords Melatonin . Calcium . Red-ox state . Proliferation . Pancreatic stellate cells Abbreviations CCK-8 Cholecystokinin octapeptide [Ca2+]c Intracellular free Ca2+ concentration CM5-(and-6)-Chloromethyl-2′,7′H2DCFDA 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
96
Tps
Cell Biol Toxicol (2015) 31:95–110
Sarcoendoplasmic reticulum Ca2+ATPase Thapsigargin
Introduction It is well known that most cellular activity of the exocrine pancreas is initiated by the activation of specific receptors located at the plasma membrane of the cells. In this tissue, changes in intracellular free Ca2+ concentration ([Ca2+]c) represent a crucial signaling mechanism, and the major physiological processes occur downstream to Ca2+ mobilization (Petersen 2004). Additionally, the transmission of extracellular signals into their intracellular targets is mediated by a network of interacting proteins, which regulate a large number of cellular processes (Dabrowski 2003). The interest in the role of melatonin in cellular physiology is increasing. Membrane-bound melatonin receptors include types 1 (MT1) and 2 (MT2) receptors (Slominski et al. 2012). Melatonin receptors are widely distributed throughout the body, and their expression differs among various organs. The presence of melatonin MT1- and MT2-type receptors in the pancreas has been reported (González et al. 2011; Mühlbauer et al. 2009). Binding of melatonin to a cytosolic receptor and to nuclear orphan receptors from the RORα/RZR family has also been proposed (Chen et al. 2011; Slominski et al. 2012). The existing evidences indicate that melatonin plays a major role in the function of the exocrine pancreas. In general, it has been suggested that the indole exerts a regulatory action on enzyme secretion (Jaworek et al. 2004; Santofimia-Castaño et al. 2013) and on bicarbonate secretion (Aust et al. 2006). In addition, melatonin possesses antioxidant (Jaworek et al. 2003; Muñoz-Casares et al. 2006) and antiinflammatory properties in the exocrine pancreas (Gülben et al. 2010; Jaworek et al. 2003, 2010; Qi et al. 1999). Moreover, melatonin induces antitumor effects in the gland (Bazwinsky-Wutschke et al. 2012; González et al. 2011; Leja-Szpak et al. 2010). Its actions may be direct (Aust et al. 2006; Santofimia-Castaño et al. 2013) or indirect, via stimulation of vagal nerves (Nawrot-Porąbka et al. 2013) or through the release of gut hormones that reach the pancreas via blood supply (Jaworek et al. 2004).
Isolated pancreatic cells are a useful model to investigate the direct effects of certain drugs. Whereas in vivo, there are numerous indirect mechanisms involved in the regulation of pancreatic function, in vitro, the contribution of factors derived from external sources (other cells/tissues), which might influence pancreatic cell physiology, will be avoided. Furthermore, in order to study proliferation and the pathways involved, cell culture techniques have been developed. Currently, there is a lot of interest in the physiology of pancreatic stellate cells (PSCs). This is a cell type that is responsible for the progressive fibrosis and for the accumulation of extracellular matrix that occurs in chronic pancreatitis and in pancreatic cancer. The involvement of PSCs in pancreatic tumor progression has been documented (Hwang et al. 2008), playing stroma an important role in tumor growth and angiogenesis (Zha et al. 2014; Zhu et al. 2014). Following activation of PSCs, tumor progression and chemoresistance is enhanced, and a major contributing factor is the characteristic extensive stromal or fibrotic reaction (McCarroll et al. 2014). The results obtained employing cultures of PSCs will be useful to analyze the effects of different drugs acting directly on the cells. Special attention can be paid to study the processes responsible for the outcome of pancreatic fibrosis, including cell proliferation, because any drug with anti-inflammatory, antifibrotic, and antitumor properties could be applied in therapy. Bearing in mind that most observations suggest that melatonin exerts protective effects in the healthy exocrine pancreas, whereas it induces death of abnormal or transformed (cancer) cells, we sought to investigate whether melatonin could exert any effect on PSCs physiology. The interest was based on the assumption that PSCs have been pointed out as the cell type that mainly produces extracellular matrix in inflammation and cancer. Our objective was to shed more light on the mechanisms involved in the actions of melatonin on the exocrine pancreatic function and its putative role in the prevention of disease.
Materials and methods Animals and chemicals Newborn Wistar rats (7–9 days) were used in the present study. Animals were obtained from the animal house of
Cell Biol Toxicol (2015) 31:95–110
97
the University of Extremadura (Caceres, Spain). Animals were humanely handled and sacrificed in accordance to the institutional Bioethical Committee. Collagenase CLSPA was obtained from Worthington Biochemical (Lakewood, NJ, USA). Cell lysis reagent for cell lysis and protein solubilization, ethylene glycolbis(2-aminoethylether)-N,N,N′N′-tetraacetic acid (EGTA), melatonin, Tween®-20, and thapsigargin were obtained from Sigma Chemicals (Madrid, Spain). AlamarBlue® was purchased from AbD serotec (bioNova Científica, Madrid, Spain). 5-(and-6)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA), fura-2-AM, hydrogen peroxide (H2O2), medium 199, and horse serum were obtained from Invitrogen (Barcelona, Spain). Fetal bovine serum (FBS) was purchased from HyClone (Thermo Scientific, Erembodegen, Belgium). Penicillin/ streptomycin was obtained from BioWhittaker (Lonza, Basel, Switzerland). LY294002 was purchased from Alomone Labs. (Jerusalem, Israel). Bradford reagent, Tris/glycine/sodium dodecyl sulfate (SDS) buffer (10×) and Tris/glycine buffer (10×) were from BioRad (Madrid, Spain). Enhanced chemiluminescence detection reagents were obtained from Pierce (Fisher Scientific, Madrid, Spain). Antibodies against MEL-1A-R, MEL-1B-R, and αtubulin were purchased from Santa Cruz Biotechnology (Quimigranel, Madrid, Spain). Antibodies against phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), and phospho-SAPK/JNK (Thr183/Tyr185) were purchased from Cell Signaling (IZASA Biolabs, Barcelona, Spain). Antibodies against vimentin and cytokeratin 7, secondary antibodies rabbit anti-goat immunoglobulin G (IgG)-horse radish peroxidase (HRP) and goat anti-rabbit IgG-HRP, and goat anti-rabbit IgG fluorescein isothiocyanate (FITC)-conjugate secondary antibody were purchased from Pierce (Fisher Scientific). GFAP rabbit polyclonal IgG antibody and secondary antibody goat anti-mouse IgGHRP were obtained from Santa Cruz Biotechnology (Quimigranel). All other analytical grade chemicals used were obtained from Sigma Chemicals.
MgCl2, 1.2 mM KH2PO4, 10 mM glucose, 10 mM HEPES, 0.01 % trypsin inhibitor (soybean), and 0.2 % bovine serum albumin (pH 7.4 adjusted with NaOH). This buffer was supplemented with 30 units⁄ml collagenase CLSPA from Worthington. The tissue was incubated for 50 min at 37 °C under constant oxygenation. The enzymatic digestion of the tissue was followed by gently pipetting the cell suspension through tips of decreasing diameter, for mechanical dissociation of the cells. After centrifugation at 30×g for 5 min at 4 °C, cells were resuspended in culture medium. The culture medium employed was a Medium 199 supplemented with 4 % horse serum, 10 % FBS, antibiotics (0.1 mg/ml streptomycin, 100 IU penicillin), and 1 mM NaHCO3. This medium was prepared under sterile conditions. Finally, cells were seeded (≈30,000 cells) on glass coverslips placed in independent dishes (35-mm diameter) or were seeded in multiwell cell culture polystyrene plates (12 wells; PAA Laboratories, Pasching, Austria). The cells were allowed to grow in culture medium at 37 °C and 5 % CO2, employing a humidified incubator. After 5 days of culture, 90–95 % confluence was reached. Experiments were performed at room temperature (23–25 °C), and different batches of cells, obtained from different preparations, were used for the studies.
Preparation of pancreatic stellate cells (PSCs) cultures
This procedure was employed to determine phosphorylated MAPKs localization and glial fibrillary acidic protein (GFAP) expression. Briefly, the cells, attached to the cover slip, were fixed with 4 % paraformaldehyde and permeabilized with 0.1 % Triton×100. Cells were
Following sacrifice of the animals, pancreata were placed in a physiological Na-HEPES solution containing: 130 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl2, 1 mM
Measurement of total amylase content Cellular amylase content was measured employing the Phadebas blue starch method, as described previously (Jensen et al. 1982). Cells were isolated, seeded in 12well cell culture plates and cultured during 1 week. Data show the mean (±S.E.M.) total amylase content expressed as percentage of the total content of amylase of cells at day 1 (n), where n is the number of independent experiments. Data were previously normalized with respect to total protein content (determined employing Bradford’s method). Immunocytochemistry
98
then incubated with the specific primary antibody and the corresponding FITC-labeled secondary antibody. Monitorization of fluorescence signals was performed employing an epifluorescence microscope (Nikon Eclipse TE300 microscope; Nikon Instruments). Cells were observed with a 60× objective under green illumination (excitation at 450–490 nm, emission at 520 nm). The absence of nonspecific staining was assessed by processing the samples without primary antibody.
Cell Biol Toxicol (2015) 31:95–110
and sonicated in lysis buffer. Protein lysates (30 μg/ well) were fractionated by SDS-PAGE using 10 % polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were incubated with the specific primary and the corresponding IgG-HRP conjugated secondary antibodies. The intensity and molecular weight of the bands which appear were quantified using the software Image J (http://imagej.nih.gov/ij/). The experiments were carried out employing different batches of cells, harvested on different days.
Determination of intracellular free Ca2+ concentration ([Ca2+]c) Cell viability assay Cells, growing on coverslips, were incubated in the presence of fura-2/AM (4 μM) at room temperature (23–25 °C) for 40 min. Monitoring of [Ca2+]c was performed employing an image acquisition and analysis system for video microscopy as previously described (Del Castillo-Vaquero et al. 2010). Results are expressed as the ratio of fluorescence emitted by fura-2 (previously normalized to the resting fluorescence). All fluorescence measurements were made from areas considered individual cells. Stimuli were dissolved in the extracellular Na-HEPES buffer and applied directly to the cells in the perfusion chamber. In the experiments where Ca2+-free medium was employed, Ca2+ was omitted from the extracellular solution, and 250 μM EGTA was added to the medium.
Analysis of cell viability under the different treatments applied was performed using AlamarBlue® test, following previously described methods (Santofimia-Castaño et al. 2013). Data show the mean reduction of
Determination of oxidation For the determination of the red-ox state, cells were incubated in the presence of CM-H2DCFDA (10 μM) for 40 min at room temperature (23–25 °C). Red-ox state of cells was determined by measuring cellular fluorescence at 530 nm/590 nm (exc, Citation/ emission) following previously described methods (González et al. 2007). Data show the mean increase of fluorescence expressed in percentage±S.E.M. (n) with respect to control (nonstimulated) cells, where n is the number of independent experiments. Western blot analysis Activation of crucial components in MAPKs pathway was detected by Western blot analysis as previously described (Garcia-Sanchez et al. 2012). Briefly, after the corresponding treatments, cells were detached, centrifuged washed with phosphate-buffered saline (PBS),
Fig. 1 Pancreatic cells growing in culture. a Picture taken 96 h after seeding of cells. b Growing cell observed at higher magnification. Viability of cells was not decreased under culture conditions and in the absence of stimulus. Images are representative of four to six different preparations
Cell Biol Toxicol (2015) 31:95–110
AlamarBlue® expressed in percentage±S.E.M. (n) with respect to control (nontreated) cells, where n is the number of independent experiments.
Statistical analysis Statistical analysis of data was performed by one-way analysis of variance (ANOVA) followed by Tukey post hoc test, and only P values
ORIGINAL RESEARCH
Melatonin induces calcium mobilization and influences cell proliferation independently of MT1/MT2 receptor activation in rat pancreatic stellate cells Patricia Santofimia-Castaño & Lourdes Garcia-Sanchez & Deborah Clea Ruy & Beatriz Sanchez-Correa & Miguel Fernandez-Bermejo & Raquel Tarazona & Gines M. Salido & Antonio Gonzalez
Received: 14 July 2014 / Accepted: 26 February 2015 / Published online: 13 March 2015 # Springer Science+Business Media Dordrecht 2015
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 exam ined. 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-H2DCFDAderived fluorescence. Determination of the activation of p44/42 mitogen-activated protein kinase (MAPK), SAPK/JNK and p38 was measured by Western blot P. Santofimia-Castaño : L. Garcia-Sanchez : G. M. Salido : A. Gonzalez (*) Cell Physiology Research Group, Department of Physiology, University of Extremadura, Avenida Universidad s/n, E-10003, Caceres, Spain e-mail: [email protected] D. C. Ruy Facultade de Agronomia & Medicina Veterinaria, Universidade de Brasilia, 70900-100 Brasilia, DF, Brasil B. Sanchez-Correa : R. Tarazona Immunology Unit, Department of Physiology, University of Extremadura, Caceres, Spain M. Fernandez-Bermejo Department of Gastroenterology, San Pedro de Alcantara Hospital, 10003 Caceres, Spain
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. Keywords Melatonin . Calcium . Red-ox state . Proliferation . Pancreatic stellate cells Abbreviations CCK-8 Cholecystokinin octapeptide [Ca2+]c Intracellular free Ca2+ concentration CM5-(and-6)-Chloromethyl-2′,7′H2DCFDA 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
96
Tps
Cell Biol Toxicol (2015) 31:95–110
Sarcoendoplasmic reticulum Ca2+ATPase Thapsigargin
Introduction It is well known that most cellular activity of the exocrine pancreas is initiated by the activation of specific receptors located at the plasma membrane of the cells. In this tissue, changes in intracellular free Ca2+ concentration ([Ca2+]c) represent a crucial signaling mechanism, and the major physiological processes occur downstream to Ca2+ mobilization (Petersen 2004). Additionally, the transmission of extracellular signals into their intracellular targets is mediated by a network of interacting proteins, which regulate a large number of cellular processes (Dabrowski 2003). The interest in the role of melatonin in cellular physiology is increasing. Membrane-bound melatonin receptors include types 1 (MT1) and 2 (MT2) receptors (Slominski et al. 2012). Melatonin receptors are widely distributed throughout the body, and their expression differs among various organs. The presence of melatonin MT1- and MT2-type receptors in the pancreas has been reported (González et al. 2011; Mühlbauer et al. 2009). Binding of melatonin to a cytosolic receptor and to nuclear orphan receptors from the RORα/RZR family has also been proposed (Chen et al. 2011; Slominski et al. 2012). The existing evidences indicate that melatonin plays a major role in the function of the exocrine pancreas. In general, it has been suggested that the indole exerts a regulatory action on enzyme secretion (Jaworek et al. 2004; Santofimia-Castaño et al. 2013) and on bicarbonate secretion (Aust et al. 2006). In addition, melatonin possesses antioxidant (Jaworek et al. 2003; Muñoz-Casares et al. 2006) and antiinflammatory properties in the exocrine pancreas (Gülben et al. 2010; Jaworek et al. 2003, 2010; Qi et al. 1999). Moreover, melatonin induces antitumor effects in the gland (Bazwinsky-Wutschke et al. 2012; González et al. 2011; Leja-Szpak et al. 2010). Its actions may be direct (Aust et al. 2006; Santofimia-Castaño et al. 2013) or indirect, via stimulation of vagal nerves (Nawrot-Porąbka et al. 2013) or through the release of gut hormones that reach the pancreas via blood supply (Jaworek et al. 2004).
Isolated pancreatic cells are a useful model to investigate the direct effects of certain drugs. Whereas in vivo, there are numerous indirect mechanisms involved in the regulation of pancreatic function, in vitro, the contribution of factors derived from external sources (other cells/tissues), which might influence pancreatic cell physiology, will be avoided. Furthermore, in order to study proliferation and the pathways involved, cell culture techniques have been developed. Currently, there is a lot of interest in the physiology of pancreatic stellate cells (PSCs). This is a cell type that is responsible for the progressive fibrosis and for the accumulation of extracellular matrix that occurs in chronic pancreatitis and in pancreatic cancer. The involvement of PSCs in pancreatic tumor progression has been documented (Hwang et al. 2008), playing stroma an important role in tumor growth and angiogenesis (Zha et al. 2014; Zhu et al. 2014). Following activation of PSCs, tumor progression and chemoresistance is enhanced, and a major contributing factor is the characteristic extensive stromal or fibrotic reaction (McCarroll et al. 2014). The results obtained employing cultures of PSCs will be useful to analyze the effects of different drugs acting directly on the cells. Special attention can be paid to study the processes responsible for the outcome of pancreatic fibrosis, including cell proliferation, because any drug with anti-inflammatory, antifibrotic, and antitumor properties could be applied in therapy. Bearing in mind that most observations suggest that melatonin exerts protective effects in the healthy exocrine pancreas, whereas it induces death of abnormal or transformed (cancer) cells, we sought to investigate whether melatonin could exert any effect on PSCs physiology. The interest was based on the assumption that PSCs have been pointed out as the cell type that mainly produces extracellular matrix in inflammation and cancer. Our objective was to shed more light on the mechanisms involved in the actions of melatonin on the exocrine pancreatic function and its putative role in the prevention of disease.
Materials and methods Animals and chemicals Newborn Wistar rats (7–9 days) were used in the present study. Animals were obtained from the animal house of
Cell Biol Toxicol (2015) 31:95–110
97
the University of Extremadura (Caceres, Spain). Animals were humanely handled and sacrificed in accordance to the institutional Bioethical Committee. Collagenase CLSPA was obtained from Worthington Biochemical (Lakewood, NJ, USA). Cell lysis reagent for cell lysis and protein solubilization, ethylene glycolbis(2-aminoethylether)-N,N,N′N′-tetraacetic acid (EGTA), melatonin, Tween®-20, and thapsigargin were obtained from Sigma Chemicals (Madrid, Spain). AlamarBlue® was purchased from AbD serotec (bioNova Científica, Madrid, Spain). 5-(and-6)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA), fura-2-AM, hydrogen peroxide (H2O2), medium 199, and horse serum were obtained from Invitrogen (Barcelona, Spain). Fetal bovine serum (FBS) was purchased from HyClone (Thermo Scientific, Erembodegen, Belgium). Penicillin/ streptomycin was obtained from BioWhittaker (Lonza, Basel, Switzerland). LY294002 was purchased from Alomone Labs. (Jerusalem, Israel). Bradford reagent, Tris/glycine/sodium dodecyl sulfate (SDS) buffer (10×) and Tris/glycine buffer (10×) were from BioRad (Madrid, Spain). Enhanced chemiluminescence detection reagents were obtained from Pierce (Fisher Scientific, Madrid, Spain). Antibodies against MEL-1A-R, MEL-1B-R, and αtubulin were purchased from Santa Cruz Biotechnology (Quimigranel, Madrid, Spain). Antibodies against phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), and phospho-SAPK/JNK (Thr183/Tyr185) were purchased from Cell Signaling (IZASA Biolabs, Barcelona, Spain). Antibodies against vimentin and cytokeratin 7, secondary antibodies rabbit anti-goat immunoglobulin G (IgG)-horse radish peroxidase (HRP) and goat anti-rabbit IgG-HRP, and goat anti-rabbit IgG fluorescein isothiocyanate (FITC)-conjugate secondary antibody were purchased from Pierce (Fisher Scientific). GFAP rabbit polyclonal IgG antibody and secondary antibody goat anti-mouse IgGHRP were obtained from Santa Cruz Biotechnology (Quimigranel). All other analytical grade chemicals used were obtained from Sigma Chemicals.
MgCl2, 1.2 mM KH2PO4, 10 mM glucose, 10 mM HEPES, 0.01 % trypsin inhibitor (soybean), and 0.2 % bovine serum albumin (pH 7.4 adjusted with NaOH). This buffer was supplemented with 30 units⁄ml collagenase CLSPA from Worthington. The tissue was incubated for 50 min at 37 °C under constant oxygenation. The enzymatic digestion of the tissue was followed by gently pipetting the cell suspension through tips of decreasing diameter, for mechanical dissociation of the cells. After centrifugation at 30×g for 5 min at 4 °C, cells were resuspended in culture medium. The culture medium employed was a Medium 199 supplemented with 4 % horse serum, 10 % FBS, antibiotics (0.1 mg/ml streptomycin, 100 IU penicillin), and 1 mM NaHCO3. This medium was prepared under sterile conditions. Finally, cells were seeded (≈30,000 cells) on glass coverslips placed in independent dishes (35-mm diameter) or were seeded in multiwell cell culture polystyrene plates (12 wells; PAA Laboratories, Pasching, Austria). The cells were allowed to grow in culture medium at 37 °C and 5 % CO2, employing a humidified incubator. After 5 days of culture, 90–95 % confluence was reached. Experiments were performed at room temperature (23–25 °C), and different batches of cells, obtained from different preparations, were used for the studies.
Preparation of pancreatic stellate cells (PSCs) cultures
This procedure was employed to determine phosphorylated MAPKs localization and glial fibrillary acidic protein (GFAP) expression. Briefly, the cells, attached to the cover slip, were fixed with 4 % paraformaldehyde and permeabilized with 0.1 % Triton×100. Cells were
Following sacrifice of the animals, pancreata were placed in a physiological Na-HEPES solution containing: 130 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl2, 1 mM
Measurement of total amylase content Cellular amylase content was measured employing the Phadebas blue starch method, as described previously (Jensen et al. 1982). Cells were isolated, seeded in 12well cell culture plates and cultured during 1 week. Data show the mean (±S.E.M.) total amylase content expressed as percentage of the total content of amylase of cells at day 1 (n), where n is the number of independent experiments. Data were previously normalized with respect to total protein content (determined employing Bradford’s method). Immunocytochemistry
98
then incubated with the specific primary antibody and the corresponding FITC-labeled secondary antibody. Monitorization of fluorescence signals was performed employing an epifluorescence microscope (Nikon Eclipse TE300 microscope; Nikon Instruments). Cells were observed with a 60× objective under green illumination (excitation at 450–490 nm, emission at 520 nm). The absence of nonspecific staining was assessed by processing the samples without primary antibody.
Cell Biol Toxicol (2015) 31:95–110
and sonicated in lysis buffer. Protein lysates (30 μg/ well) were fractionated by SDS-PAGE using 10 % polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were incubated with the specific primary and the corresponding IgG-HRP conjugated secondary antibodies. The intensity and molecular weight of the bands which appear were quantified using the software Image J (http://imagej.nih.gov/ij/). The experiments were carried out employing different batches of cells, harvested on different days.
Determination of intracellular free Ca2+ concentration ([Ca2+]c) Cell viability assay Cells, growing on coverslips, were incubated in the presence of fura-2/AM (4 μM) at room temperature (23–25 °C) for 40 min. Monitoring of [Ca2+]c was performed employing an image acquisition and analysis system for video microscopy as previously described (Del Castillo-Vaquero et al. 2010). Results are expressed as the ratio of fluorescence emitted by fura-2 (previously normalized to the resting fluorescence). All fluorescence measurements were made from areas considered individual cells. Stimuli were dissolved in the extracellular Na-HEPES buffer and applied directly to the cells in the perfusion chamber. In the experiments where Ca2+-free medium was employed, Ca2+ was omitted from the extracellular solution, and 250 μM EGTA was added to the medium.
Analysis of cell viability under the different treatments applied was performed using AlamarBlue® test, following previously described methods (Santofimia-Castaño et al. 2013). Data show the mean reduction of
Determination of oxidation For the determination of the red-ox state, cells were incubated in the presence of CM-H2DCFDA (10 μM) for 40 min at room temperature (23–25 °C). Red-ox state of cells was determined by measuring cellular fluorescence at 530 nm/590 nm (exc, Citation/ emission) following previously described methods (González et al. 2007). Data show the mean increase of fluorescence expressed in percentage±S.E.M. (n) with respect to control (nonstimulated) cells, where n is the number of independent experiments. Western blot analysis Activation of crucial components in MAPKs pathway was detected by Western blot analysis as previously described (Garcia-Sanchez et al. 2012). Briefly, after the corresponding treatments, cells were detached, centrifuged washed with phosphate-buffered saline (PBS),
Fig. 1 Pancreatic cells growing in culture. a Picture taken 96 h after seeding of cells. b Growing cell observed at higher magnification. Viability of cells was not decreased under culture conditions and in the absence of stimulus. Images are representative of four to six different preparations
Cell Biol Toxicol (2015) 31:95–110
AlamarBlue® expressed in percentage±S.E.M. (n) with respect to control (nontreated) cells, where n is the number of independent experiments.
Statistical analysis Statistical analysis of data was performed by one-way analysis of variance (ANOVA) followed by Tukey post hoc test, and only P values
