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Background information Pancreatic stellate cells play a key role in the fibrosis that develops in diseases such as pancreatic cancer. In the growing tumour, a hypoxia condition develops under which cancer cells are able to proliferate. The growth of fibrotic tissue contributes to hypoxia. In this study, the effect of hypoxia (1% O2 ) on pancreatic stellate cells physiology was investigated. Changes in intracellular free-Ca2+ concentration, mitochondrial free-Ca2+ concentration and mitochondrial membrane potential were studied by fluorescence techniques. The status of enzymes responsible for the cellular oxidative state was analyzed by quantitative reverse transcription-polymerase chain reaction, high-performance liquid chromatography, spectrophotometric and fluorimetric methods and by Western blotting analysis. Cell viability and proliferation were studied by crystal violet test, 5-bromo-2-deoxyuridine cell proliferation test and Western blotting analysis. Finally, cell migration was studied employing the wound healing assay. Results Hypoxia induced an increase in intracellular and mitochondrial free-Ca2+ concentration, whereas mitochondrial membrane potential was decreased. An increase in mitochondrial reactive oxygen species production was observed. Additionally, an increase in the oxidation of proteins and lipids was detected. Moreover, cellular total antioxidant capacity was decreased. Increases in the expression of superoxide dismutase 1 and 2 were observed and superoxide dismutase activity was augmented. Hypoxia evoked a decrease in the oxidized/reduced glutathione ratio. An increase in the phosphorylation of nuclear factor erythroid 2-related factor and in expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 were detected. The expression of cyclin A was decreased, whereas expression of cyclin D and the content of 5-bromo-2-deoxyuridine were increased. This was accompanied by an increase in cell viability. The phosphorylation state of c-Jun NH2 -terminal kinase was increased, whereas that of p44/42 and p38 was decreased. Finally, cells subjected to hypoxia maintained migration ability. Conclusions and significance Hypoxia creates pro-oxidant conditions in pancreatic stellate cells to which cells adapt and leads to increased viability and proliferation.

TRPC6 channel is widely expressed in most human tissues and participates in a number of physiological processes. TRPC6 belongs to the DAG-activated subfamily of channels, but has also been postulated as a mediator in the store-operated calcium entry pathway. The recent characterization of TRPC6 crystal structure has granted a wonderful tool to finally dissect and understand TRPC6 physiological and biophysical properties. Growing evidences have demonstrated that the pattern of expression of TRPC6 proteins is upregulated in several pathophysiological conditions, including breast cancer. However, the real role of TRPC6 in breast cancer persists still unknown. Here we present the current state of the art concerning the function and significance of TRPC6 in this disease. Future investigations should be focus in the creation and identification of compounds that specifically target the channel to ameliorate TRPC6-related diseases.

In this work we have studied the effects of pharmacological concentrations of melatonin (1 µM–1 mM) on pancreatic stellate cells (PSC). Cell viability was analyzed by AlamarBlue test. Production of reactive oxygen species (ROS) was monitored following CM-H2DCFDA and MitoSOX Red-derived fluorescence. Total protein carbonyls and lipid peroxidation were analyzed by HPLC and spectrophotometric methods respectively. Mitochondrial membrane potential (ψm) was monitored by TMRM-derived fluorescence. Reduced (GSH) and oxidized (GSSG) levels of glutathione were determined by fluorescence techniques. Quantitative reverse transcription-polymerase chain reaction was employed to detect the expression of Nrf2-regulated antioxidant enzymes. Determination of SOD activity and total antioxidant capacity (TAC) were carried out by colorimetric methods, whereas expression of SOD was analyzed by Western blotting and RT-qPCR. The results show that melatonin decreased PSC viability in a concentration-dependent manner. Melatonin evoked a concentration-dependent increase in ROS production in the mitochondria and in the cytosol. Oxidation of proteins was detected in the presence of melatonin, whereas lipids oxidation was not observed. Depolarization of ψm was noted with 1 mM melatonin. A decrease in the GSH/GSSG ratio was observed, that depended on the concentration of melatonin used. A concentration-dependent increase in the expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 was detected in cells incubated with melatonin. Finally, decreases in the expression and in the activity of superoxide dismutase were observed. We conclude that pharmacological concentrations melatonin modify the redox state of PSC, which might decrease cellular viability.

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. However, evidence suggests that this compound could exert deleterious actions on cell physiology. In this study, we have analyzed the effect of ebselen on rat pancreatic AR42J cells. Cytosolic free-Ca2+ concentration ([Ca2+ ]c ), cellular oxidative status, setting of endoplasmic reticulum stress, and phosphorylation of major mitogen-activated protein kinases were analyzed. Our results show that ebselen evoked a concentration-dependent increase in [Ca2+ ]c . The compound induced an increase in the generation of reactive oxygen species in the mitochondria. We also observed an increase in global cysteine oxidation in the presence of ebselen. In the presence of ebselen an impairment of cholecystokinin-evoked amylase release was noted. Moreover, involvement of the unfolded protein response markers, ER chaperone and signaling regulator GRP78/BiP, eukaryotic translation initiation factor 2α and X-box binding protein 1 was detected. Finally, increases in the phosphorylation of SAPK/JNK, p38 MAPK, and p44/42 MAPK in the presence of ebselen were also observed. Our results provide evidences for an impairment of cellular oxidative state and enzyme secretion, the induction of endoplasmic reticulum stress and the activation of crucial mitogen-activated protein kinases in the presence of ebselen. As a consequence ebselen exerts a potential toxic effect on AR42J cells.

Store-operated Ca2+ entry (SOCE) is a major mechanism for the regulation of intracellular Ca2+ homeostasis and cellular function. Emerging evidence has revealed that altered expression and function of the molecular determinants of SOCE play a critical role in the development or maintenance of several cancer hallmarks, including enhanced proliferation and migration. Here we show that, in the acute myeloid leukemia cell line HL60, Orai2 is highly expressed at the transcript level, followed by the expression of Orai1. Using fluorescence Ca2+ imaging we found that Orai2 silencing significantly attenuated thapsigargin-induced SOCE, as well as knockdown of Orai1, while silencing the expression of both channels almost completely reduced SOCE, thus suggesting that SOCE in these cells is strongly dependent on Orai1 and Orai2. On the other hand, the expression of TRPC1, TRPC3 and TRPC6 is almost absent at the transcript and protein level. Bromodeoxyuridine cell proliferation assay revealed that Orai1 and Orai2 expression silencing significantly reduced HL60 cell proliferation. Furthermore, knockdown of Orai1 and Orai2 significantly attenuated the ability of HL60 to migrate in vitro as determined by transwell migration assay, probably due to the impairment of FAK tyrosine phosphorylation. These findings provide evidence for a role for Orai1 and Orai2, in SOCE and migration in the human HL60 promyeloblastic cell line. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

In this study, the effects of melatonin (1 μM–1 mM) on pancreatic stellate cells (PSC) have been examined. Cell viability and proliferation, caspase-3 activation, and the expression of cyclin A and cyclin D were analyzed. Our results show that melatonin decreased PSC viability in a time- and concentration-dependent manner. This effect was not inhibited by treatment of cells with MT1, MT2, calmodulin, or ROR-alpha inhibitors prior to melatonin addition. Activation of caspase-3 in response to melatonin was detected. The expression of cyclin A and cyclin D was decreased in cells treated with melatonin. Finally, changes in BrdU incorporation into the newly synthesized DNA of proliferating cells were also observed in the presence of melatonin. We conclude that melatonin, at pharmacological concentrations, modulates proliferation of PSC through activation of apoptosis and involving crucial regulators of the cell cycle. These actions might not require specific melatonin receptors. Our observations suggest that melatonin, at high doses, could potentially exert anti-fibrotic effects and, thus, could be taken into consideration as supportive treatment in the therapy of pancreatic diseases.

This article belongs to the Special Issue Calcium Signaling Remodeling and Functional Role in Cancer Cells., [Simple Summary] Breast cancer accounts as the most extended disease among women worldwide. Store-operated calcium entry (SOCE), a major mechanism that allows calcium entry from the extracellular region through the plasma membrane, is required for several physiological processes. In recent years, it has been revealed that several breast cancer types present dysregulated calcium homeostasis, which contribute to their malignancy. Here we show the role of two important regulators of SOCE, SARAF and EFHB, which are necessary for cell viability, proliferation, and migration in breast cancer and pre-neoplastic cells, respectively, thus suggesting that these regulators play a key function in breast cancer development and progression., [Abstract] Breast cancer is among the most common malignancies in women. From the molecular point of view, breast cancer can be grouped into different categories, including the luminal (estrogen receptor positive (ER+)) and triple negative subtypes, which show distinctive features and, thus, are sensitive to different therapies. Breast cancer cells are strongly dependent on Ca2+ influx. Store-operated Ca2+ entry (SOCE) has been found to support a variety of cancer hallmarks including cell viability, proliferation, migration, and metastasis. The Ca2+ channels of the Orai family and the endoplasmic reticulum Ca2+ sensor STIM1 are the essential components of SOCE, but the extent of Ca2+ influx is fine-tuned by several regulatory proteins, such as the STIM1 modulators SARAF and EFHB. Here, we show that the expression and/or function of SARAF and EFHB is altered in breast cancer cells and both proteins are required for cell proliferation, migration, and viability. EFHB expression is upregulated in luminal and triple negative breast cancer (TNBC) cells and is essential for full SOCE in these cells. SARAF expression was found to be similar in breast cancer and pre-neoplastic breast epithelial cells, and SARAF knockdown was found to result in enhanced SOCE in pre-neoplastic and TNBC cells. Interestingly, silencing SARAF expression in ER+ MCF7 cells led to attenuation of SOCE, thus suggesting a distinctive role for SARAF in this cell type. Finally, we used a combination of approaches to show that molecular knockdown of SARAF and EFHB significantly attenuates the ability of breast cancer cells to proliferate and migrate, as well as cell viability. In aggregate, SARAF and EFHB are required for the fine modulation of SOCE in breast cancer cells and play an important role in the maintenance of proliferation, migration, and viability in these cells., This research was funded by Agencia Estatal de Investigación PID2019-104084GB-C21/AEI/10.13039/501100011033 and PID2019-104084GB-C22/AEI/10.13039/501100011033, MICINN (Grants BFU2016-74932-C2) and Junta de Extremadura-Fondo Europeo de Desarrollo Regional (FEDER; Grants IB20007 and GR18061) to J.A.R. and T.S. I.J. and J.J.L. are supported by contracts from Junta de Extremadura (TA18054 and TA18011).

Pancreatic stellate cells (PSC) play a major role in the formation of fibrotic tissue in pancreatic tumors. On its side, melatonin is a putative therapeutic agent for pancreatic cancer and inflammation. In this work, the actions of melatonin on PSC subjected to hypoxia were evaluated. Reactive oxygen species (ROS) generation reduced (GSH) and oxidized (GSSG) levels of glutathione, and protein and lipid oxidation were analyzed. The phosphorylation of nuclear factor erythroid 2-related factor (Nrf2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), and the regulatory protein nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha (IκBα) was studied. The expression of Nrf2-regulated antioxidant enzymes, superoxide dismutase (SOD) enzymes, cyclooxygenase 2 (COX-2), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were also studied. Total antioxidant capacity (TAC) was assayed. Finally, cell viability was studied. Under hypoxia and in the presence of melatonin generation of ROS was observed. No increases in the oxidation of proteins or lipids were detected. The phosphorylation of Nrf2 and the expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1, heme oxygenase-1, SOD1, and of SOD2 were augmented. The TAC was increased. Protein kinase C was involved in the effects of melatonin. Melatonin decreased the GSH/GSSG ratio at the highest concentration tested. Cell viability dropped in the presence of melatonin. Finally, melatonin diminished the phosphorylation of NF-kB and the expression of COX-2, IL-6, and TNF-α. Our results indicate that melatonin, at pharmacological concentrations, modulates the red-ox state, viability, and the expression of proinflammatory mediators in PSC subjected to hypoxia.

An endoplasmic reticulum (ER)-resident protein that regulates cytosolic and ER free-Ca2+ concentration by induction of store-operated calcium entry: that is the original definition of STIM2 and its function. While its activity strongly depends on the amount of calcium stored in the ER, its function goes further, to intracellular signalling and gene expression. Initially under-studied owing to the prominent function of STIM1, STIM2 came to be regarded as vital in mice, gradually emerging as an important player in the nervous system, and cooperating with STIM1 in the immune system. STIM2 has also been proposed as a relevant player in pathological conditions related to ageing, Alzheimer's and Huntington's diseases, autoimmune disorders and cancer. The discovery of additional functions, together with new splicing forms with opposite roles, has clarified existing controversies about STIM2 function in SOCE. With STIM2 being essential for life, but apparently not for development, newly available data demonstrate a complex and still intriguing behaviour that this review summarizes, updating current knowledge of STIM2 function.

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. Because of its properties, it may be protective against injury to the nervous tissue. However, evidence suggests that its glutathione peroxidase activity could underlie certain deleterious actions on cell physiology. In this study we have analyzed the effect of ebselen on rat hippocampal astrocytes in culture. Cellular oxidative status, cytosolic free-Ca(2+) concentration ([Ca(2+)]c), setting of endoplasmic reticulum stress and phosphorylation of glial fibrillary acidic protein and major mitogen-activated protein kinases were analyzed. Our results show that ebselen induced a concentration-dependent increase in the generation of reactive oxygen species in the mitochondria. We observed a concentration-dependent increase in global cysteine oxidation and in the level of malondialdehyde in the presence of ebselen. We also detected increases in catalase, glutathione S-transferase and glutathione reductase activity. Ebselen also evoked a concentration-dependent increase in [Ca(2+)]c. Moreover, we observed a concentration-dependent increase in the phosphorylation of the unfolded protein response markers, eukaryotic translation initiation factor 2α and X-box binding protein 1. Finally, ebselen also induced an increase in the phosphorylation of glial fibrillary acidic protein, SAPK/JNK, p38 MAPK and p44/42 MAPK. Our results provide strong evidence that implicate endoplasmic reticulum stress and activation of crucial mitogen-activated protein kinases in an oxidative damage of cells in the presence of ebselen. The compound thus might exert deleterious actions on astrocyte physiology that could compromise their function.

The store-operated Ca(2+)entry-associated regulatory factor (SARAF) has recently been identified as a STIM1 regulatory protein that facilitates slow Ca(2+)-dependent inactivation of store-operated Ca(2+)entry (SOCE). Both the store-operated channels and the store-independent arachidonate-regulated Ca(2+)(ARC) channels are regulated by STIM1. In the present study, we show that, in addition to its location in the endoplasmic reticulum, SARAF is constitutively expressed in the plasma membrane, where it can interact with plasma membrane (PM)-resident ARC forming subunits in the neuroblastoma cell line SH-SY5Y. Using siRNA-based and overexpression approaches we report that SARAF negatively regulates store-independent Ca(2+)entry via the ARC channels. Arachidonic acid (AA) increases the association of PM-resident SARAF with Orai1. Finally, our results indicate that SARAF modulates the ability of AA to promote cell survival in neuroblastoma cells. In addition to revealing new insight into the biology of ARC channels in neuroblastoma cells, these findings provide evidence for an unprecedented location of SARAF in the plasma membrane.

Arachidonic acid (AA) is a phospholipase A2 metabolite that has been reported to mediate a plethora of cellular mechanisms involved in healthy and pathological states such as platelet aggregation, lymphocyte activation, and tissue inflammation. AA has been described to activate Ca2+ entry through the arachidonate-regulated Ca2+-selective channels (ARC channels). Here, the analysis of the changes in the intracellular Ca2+ homeostasis revealed that, despite MDA-MB-231 cells expressing the ARC channel components Orai1, Orai3, and STIM1, AA does not evoke Ca2+ entry in these cells. We observed that AA evokes Ca2+ entry in MDA-MB-231 cells transiently expressing ARC channels. Nevertheless, MDA-MB-231 cell treatment with AA reduces cell proliferation and migration while inducing cell death through apoptosis. The latter mostly likely occurs via mitochondria membrane depolarization and the activation of caspases-3, -8, and -9. Altogether, our results indicate that AA exerts anti-tumoral effects on MDA-MB-231 cells, without having any effect on non-tumoral breast epithelial cells, by a mechanism that is independent on the activation of Ca2+ influx via ARC channels.

Stromal interaction molecule 1 (STIM1) is one of the key elements for the activation of store-operated Ca2+ entry (SOCE). Hence, identification of the relevant phosphorylatable STIM1 residues with a possible role in the regulation of STIM1 function and SOCE is of interest. By performing a computational analysis, we identified that the Y316 residue is susceptible to phosphorylation. Expression of the STIM1-Y316F mutant in HEK293, NG115-401L and MEG-01 cells resulted in a reduction in STIM1 tyrosine phosphorylation, SOCE and the Ca2+ release-activated Ca2+ current (ICRAC). STIM1-Orai1 colocalization was reduced in HEK293 cells transfected with YFP-STIM1-Y316F compared to in cells with wild-type (WT) YFP-tagged STIM1. Additionally, the Y316F mutation altered the pattern of interaction between STIM1 and SARAF under resting conditions and upon Ca2+ store depletion. Expression of the STIM1 Y316F mutant enhanced slow Ca2+-dependent inactivation (SCDI) as compared to STIM1 WT, an effect that was abolished by SARAF knockdown. Finally, in NG115-401L cells transfected with shRNA targeting SARAF, expression of STIM1 Y316F induced greater SOCE than STIM1 WT. Taken together, our results provide evidence supporting the idea that phosphorylation of STIM1 at Y316 plays a relevant functional role in the activation and modulation of SOCE.

We studied the effects of the tartrazine-metabolite sulfanilic acid on the physiology of pancreatic AR42J cells. Sulfanilic acid (1 μM-1 mM) induced a slow and progressive increase in intracellular free-calcium concentration that reached a plateau. The effect of sulfanilic acid was not concentration-dependent. Stimulation of cells with thapsigargin (1 μM) after treatment with sulfanilic acid (1 mM) induced a smaller Ca2+ response compared with that obtained with thapsigargin alone. Sulfanilic acid induced a concentration-dependent production of reactive oxygen species; however, this effect was not Ca2+-dependent. Depolarization of mitochondrial membrane potential was observed at the concentration of 1 mM sulfanilic acid. In the presence of the compound a decrease in the GSH/GSSG ratio was observed. A decrease in the expression of superoxide dismutase 2 was noted. Finally, stimulation of cells with CCK-8 led to a concentration-dependent increase of trypsin secretion that was impaired by pretreatment of cells with sulfanilic acid. Preincubation of cells with the antioxidant melatonin (100 μM) reduced the effect of sulfanilic acid on trypsin secretion. We conclude that sulfanilic acid might induce oxidative stress, which could alter Ca2+ signaling and enzyme secretion in pancreatic AR42J cells. This creates a situation potentially leading to damage of the exocrine pancreas.

Use of cooled and frozen semen is becoming increasingly prevalent in the equine industry. However, these procedures cause harmful effects in the sperm cell resulting in reduced cell lifespan and fertility rates. Apoptosis and necrosis-related events are increased during semen cryopreservation. However, a third type of cell death, named autophagy, has not been studied during equine semen storage. Light chain (LC)3 protein is a key component of the autophagy pathway. Under autophagy activation, LC3-I is lipidated and converted to LC3-II. The ratio of LC3-II/LC3-I is widely used as a marker of autophagy activation. The main objective of this study was to investigate whether LC3 is processed during cooling, freezing and the stressful conditions associated with these technologies. A secondary objective was to determine if LC3 processing can be modulated and if that may improve the quality of cryopreserved semen. LC3 processing was studied by Western blot with a specific antibody that recognized both LC3-I and LC3-II. Viability was assessed by flow cytometry. Modulation of LC3-I to LC3-II was studied with known autophagy activators (STF-62247 and rapamycin) or inhibitors (chloroquine and 3-MA) used in somatic cells. The results showed that conversion of LC3-I to LC3-II increased significantly during cooling at 4°C, freezing/thawing and each of the stressful conditions tested (UV radiation, oxidative stress, osmotic stress and changes in temperature). STF-62247 and rapamycin increased the LC3-II/LC3-I ratio and decreased the viability of equine sperm, whereas chloroquine and 3-MA inhibited LC3 processing and maintained the percentage of viable cells after 2 h of incubation at 37°C. Finally, refrigeration at 4°C for 96 h and freezing at -196°C in the presence of chloroquine and 3-MA resulted in higher percentages of viable cells. In conclusion, results showed that an 'autophagy-like' mechanism may be involved in the regulation of sperm viability during equine semen cryopreservation. Modulation of autophagy during these reproductive technologies may result in an improvement of semen quality and therefore in higher fertility rates.

Non-capacitative calcium entry (NCCE) contributes to cell activation in response to the occupation of G protein-coupled membrane receptors. Thrombin administration to platelets evokes the synthesis of diacylglycerol downstream of PAR receptor activation. Diacylglycerol evokes NCCE through activating TRPC3 and TRPC6 in human platelets. Although it is known that immunophilins interact with TRPCs, the role of immunophilins in the regulation of NCCE remains unknown. Platelet incubation with FK506, an immunophilin antagonist, reduced OAG-evoked NCCE in a concentration-dependent manner, an effect that was independent on the inactivation of calcineurin (CaN). FK506 was unable to reduce NCCE evoked by OAG in platelets from TRPC6−/− mice. In HEK-293 cells overexpressing TRPC6, currents through TRPC6 were altered in the presence of FK506. We have found interaction between FKBP38 and other FKBPs, like FKBP25, FKBP12, and FKBP52 that were not affected by FK506, as well as with calmodulin (CaM). FK506 modified the pattern of association between FKBP25 and TRPCs as well as impaired OAG-evoked TRPC3 and TRPC6 coupling in both human and mouse platelets. By performing biotinylation experiments we have elucidated that FKBP25 and FKBP38 might be found at different cellular location, the plasma membrane and the already described intracellular locations. Finally, FKBP25 and FKBP38 silencing significantly inhibits OAG-evoked NCCE in MEG-01 and HEK293 cells, while overexpression of FKBP38 does not modify NCCE in HEK293 cells. All together, these findings provide strong evidence for a role of immunophilins, including FKBP25 and FKBP38, in NCCE mediated by TRPC6.

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 Ca(2+) 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 Ca(2+) 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.

SummaryThe function of the mammalian target of rapamycin (mTOR) is upregulated in response to cell stimulation with growing and differentiating factors. Active mTOR controls cell proliferation, differentiation and death. Since mTOR associates with different proteins to form two functional macromolecular complexes, we aimed to investigate the role of the mTORI and mTOR2 complexes in MEG-01 cell physiology in response to thrombopoietin (TPO). By using mTOR antagonists and overexpressing FKBP38, we have explored the role of both mTOR complexes in proliferation, apoptosis, maturation-like mechanisms, endoplasmic reticulum-stress and the intracellular location of both active mTOR complexes during MEG-01 cell stimulation with TPO. The results demonstrate that mTOR1 and mTOR2 complexes play different roles in the physiology of MEG-01 cells and in the maturation-like mechanisms; hence, these findings might help to understand the mechanism underlying generation of platelets.

In this study we studied Ca2+ responses to different stimuli in pancreatic acinar cells subjected to culture conditions. Cells were isolated from adult mice pancreas and were subjected to culture conditions along one week. Changes in intracellular free Ca2+ concentration were monitored by single cell fluorescence analysis of fura-2-loaded cells. Mitochondrial distribution was analyzed by confocal microscopy study of MitoTracker Green FM-loaded cells. Expression of amylase-containing cytoplasmic vesicles was analyzed by confocal microscopy study of cells transfected with a plasmid encoding amylase linked to a green fluorescent protein. Cell viability was analyzed employing the AlamarBlue test. Our results show that pancreatic cells in culture retain a mitochondrial network and amylase-positive vesicles. However, cells dropped their ability to mobilize Ca2+ in response to activation of cell membrane receptors. Ca2+ mobilization in response to the sarcoendoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin was not altered. Cell viability was not affected by treatment with cholecystokinin, but was reduced in the presence of thapsigargin or hydrogen peroxide. We conclude that primary culture of pancreatic cells may be a suitable model to be used in studies where the involvement of mechanisms linked to the activation of specific cell membrane receptors is not required.

Cytosolic-free Ca(2+) plays a crucial role in blood platelet function and is essential for thrombosis and hemostasis. Therefore, cytosolic-free Ca(2+) concentration is tightly regulated in this cell. TRPC6 is expressed in platelets, and an important role for this Ca(2+) channel in Ca(2+) homeostasis has been reported in other cell types. The aim of this work is to study the function of TRPC6 in platelet Ca(2+) homeostasis. The absence of TRPC6 resulted in an 18.73% decreased basal [Ca(2+)]c in resting platelets as compared to control cells. Further analysis confirmed a similar Ca(2+) accumulation in wild-type and TRPC6-deficient mice; however, passive Ca(2+) leak rates from agonist-sensitive intracellular stores were significantly decreased in TRPC6-deficient platelets. Biotinylation studies indicated the presence of an intracellular TRPC6 population, and subcellular fractionation indicated their presence on endoplasmic reticulum membranes. Moreover, the presence of intracellular calcium release in platelets stimulated with 1-oleoyl-2-acetyl-sn-glycerol further suggested a functional TRPC6 population located on the intracellular membranes surrounding calcium stores. However, coimmunoprecipitation assay confirmed the absence of STIM1-TRPC6 interactions in resting conditions. This findings together with the absence of extracellular Mn(2+) entry in resting wild-type platelets indicate that the plasma membrane TRPC6 fraction does not play a significant role in the maintenance of basal [Ca(2+)]c in mouse platelets. Our results suggest an active participation of the intracellular TRPC6 fraction as a regulator of basal [Ca(2+)]c, controlling the passive Ca(2+) leak rate from agonist-sensitive intracellular Ca(2+) stores in resting platelets.

TRPA1 is a non-selective Ca(2+) permeable channel located in the plasma membrane that functions as a cellular sensor detecting mechanical, chemical and thermal stimuli, being a component of neuronal, epithelial, blood and smooth muscle tissues. TRPA1 has been shown to influence a broad range of physiological processes that involve Ca(2+)-dependent signaling pathways. Here we report that TRPA1 is expressed in MEG01 but not in platelets at the protein level. MEG01 cells maturation induced by PMA results in attenuation of TRPA1 protein expression and enhances thapsigargin-evoked Ca(2+) entry without altering the release of Ca(2+) from intracellular stores. Inhibition of TRPA1 by HC-030031 results in enhancement of both thrombin- and thapsigargin-stimulated Ca(2+) entry. Co-immunoprecipitation experiments revealed that TRPA1 associates with STIM1, as well as Orai1, TRPC1 and TRPC6. Downregulation of TRPA1 expression by MEG01 maturation, as well as pharmacological inhibition of TRPA1 by HC-030031, results in enhancement of the association between STIM1 and Orai1. Altogether, these findings provide evidence for a new and interesting function of TRPA1 in cellular function associated to the regulation of agonist-induced Ca(2+) entry by the modulation of STIM1/Orai1 interaction.

G protein-coupled receptors can be directly modulated by changes in transmembrane voltage in a variety of cell types. Here we show that, while changes in the membrane voltage itself do not induce detectable modifications in the cytosolic Ca 2+ concentration, platelet stimulation with thrombin or the PAR-1 and PAR-4 agonist peptides SFLLRN and AYPGKF, respectively, results in Ca 2+ release from intracellular stores that is sensitive to the membrane depolarisation. Direct activation of G proteins or phospholipase C by AlF 4 − and m -3M3FBS, respectively, leads to Ca 2+ release that is insensitive to changes in the membrane potential. Thapsigargin-, as well as OAG-induced Ca 2+ entry are affected by the membrane voltage, probably as a result of the modification in the driving force for Ca 2+ influx; however, hyperpolarisation does not enhance thrombin- or OAG-evoked Ca 2+ entry probably revealing the presence of a voltage-sensitive regulatory mechanism. Transmembrane voltage also modulates the activity of the plasma membrane Ca 2+ -ATPase (PMCA) most likely due to a decrease in the phosphotyrosine content of the pump. Thrombin-stimulated platelet aggregation is modulated by membrane depolarisation by a mechanism that is, at least partially, independent of Ca 2+ . These observations indicate that PAR-1 and PAR-4 receptors are modulated by the membrane voltage in human platelets.

The use of the mammal target of rapamycin (mTOR) inhibitors has been consolidated as the therapy of election for preventing graft rejection in kidney transplant patients, despite their immunosuppressive activity is less strong than anti-calcineurin agents like tacrolimus and cyclosporine A. Furthermore, as mTOR is widely expressed, rapamycin (a macrolide antibiotic produced by Streptomyces hygroscopicus) is recommended in patients presenting neoplasia due to its antiproliferative actions. Hence, we have investigated whether rapamycin presents side effects in the physiology of other cell types different from leucocytes, such as platelets. Blood samples were drawn from healthy volunteers and kidney transplant patients long-term medicated with rapamycin: sirolimus and everolimus. Platelets were either loaded with fura-2 or directly stimulated, and immunoassayed or fixed with Laemmli's buffer to perform the subsequent analysis of platelet physiology. Our results indicate that rapamycin evokes a biphasic time-dependent alteration in calcium homeostasis and function in platelets from kidney transplant patients under rapamycin regime, as demonstrated by the reduction in granule secretion observed and subsequent impairment of platelet aggregation in these patients compared with healthy volunteers. Platelet count was also reduced in these patients, thus 41% of patients presented thrombocytopenia. All together our results show that long-term administration of rapamycin to kidney transplant patients evokes alteration in platelet function.

Macroautophagy (hereafter autophagy) is an evolutionarily highly conserved cellular process that participates in the maintenance of intracellular homeostasis through the degradation of most long-lived proteins and entire organelles. Autophagy participates in some reproductive events; however, there are not reports regarding the role of autophagy in the regulation of sperm physiology. Hence, the aim of this study was to investigate whether autophagy-related proteins are present and functionally active in human spermatozoa. Proteins related to autophagy/mitophagy process (LC3, Atg5, Atg16, Beclin 1, p62, m-TOR, AMPKα 1/2 and PINK1) were present in human spermatozoa. LC3 colocalized with p62 in the middle piece of the spermatozoa. Autophagy activation induced a significant increase in motility and a decrease in PINK1, TOM20 expression and caspase 3/7 activation. In contrast, autophagy inhibition resulted in decreased motility, viability, ATP and intracellular calcium concentration whereas PINK1, TOM20 expression, AMPK phosphorylation and caspase 3/7 activation were significantly increased. In conclusion our results show that autophagy related proteins and upstream regulators are present and functional in human spermatozoa. Modification of mitochondrial proteins expression after autophagy activation/inhibition may be indicating that a specialized form of autophagy named mitophagy may be regulating sperm function such as motility and viability and may be cooperating with apoptosis.

Canonical transient receptor potential-1 (TRPC1) is an almost ubiquitously expressed channel that plays a relevant role in cell function. As other TRPC members, TRPC1 forms receptor-operated cation channels that exhibit both STIM1-dependent and store-independent behaviour. The STIM1 inhibitor SARAF (for store-operated Ca2+ entry (SOCE)-associated regulatory factor) modulates SOCE by interaction with the STIM1 region responsible for Orai1 activation (SOAR). Furthermore, SARAF modulates Ca2+ entry through the arachidonate-regulated Ca2+ (ARC) channels, consisting of Orai1 and Orai3 heteropentamers and plasma membrane-resident STIM1. While a role for STIM1–Orai1-mediated signals has been demonstrated, the possible role of SARAF in TRPC1 function remains unknown. Here, we provide evidence for the interaction of SARAF with TRPC1, independently of STIM1 both in STIM1-deficient NG115-401L cells and SH-SY5Y cells endogenously expressing STIM1. Silencing of SARAF expression in STIM1-deficient cells demonstrated that SARAF plays a negative regulatory role in TRPC1-mediated Ca2+ entry. The interaction of SARAF with TRPC1 in STIM1-deficient cells, as well as with the TRPC1 pool not associated with STIM1 in STIM1-expressing cells was enhanced by stimulation with the physiological agonist ATP. In contrast with TRPC1, we found that the interaction between SARAF and TRPC6 was constitutive rather than inducible by agonist stimulation. Furthermore, we found that SARAF expression silencing was without effect on Ca2+ entry evoked by agonists in TRPC6 overexpressing cells, as well as in Ca2+ influx evoked by the TRPC6 activator Hyp9. These findings provide evidence for a new regulator of TRPC1 channel function and highlight the relevance of SARAF in intracellular Ca2+ homeostasis.

Ca2+ influx by store-operated Ca2+ channels is a major mechanism for intracellular Ca2+ homeostasis and cellular function. Here we present evidence for the dynamic interaction between the SOCE-associated regulatory factor (SARAF), STIM1 and Orai1. SARAF overexpression attenuated SOCE and the STIM1-Orai1 interaction in cells endogenously expressing STIM1 and Orai1 while RNAi-mediated SARAF silencing induced opposite effects. SARAF impaired the association between Orai1 and the Orai1-activating small fragment of STIM1 co-expressed in the STIM1-deficient NG115-401L cells. Cell treatment with thapsigargin or physiological agonists results in direct association of SARAF with Orai1. STIM1-independent interaction of SARAF with Orai1 leads to activation of this channel. In cells endogenously expressing STIM1 and Orai1, Ca2+ store depletion leads to dissociation of SARAF with STIM1 approximately 30s after treatment with thapsigargin, which paralleled the increase in SARAF-Orai1 interaction, followed by reinteraction with STIM1 and dissociation from Orai1. Co-expression of SARAF and either Orai1 or various N-terminal deletion Orai1 mutants did not alter SARAF-Orai1 interaction; however, expression of C-terminal deletion Orai1 mutants or blockade of the C-terminus of Orai1 impair the interaction with SARAF. These observations suggest that SARAF exerts an initial positive role in the activation of SOCE followed by the facilitation of SCDI of Orai1., Supported by MINECO (Grants BFU2013-45564-C2-1-P, BFU2013-45564-C2-2-P, BFU2011-24365 and RD12-0043-0016 (RETICEF)) and Junta de Extremadura-FEDER. JJL and LA are supported by Juan de la Cierva Program and MINECO fellowship BES-2011-043356, respectively.

Two-pore channels (TPCs) are NAADP-sensitive receptor channels that conduct Ca 2 + efflux from the intracellular stores. Discharge of the internal Ca 2 + pools results in the activation of store-operated Ca 2 + entry (SOCE); however, the role of TPCs in the modulation of SOCE remains unexplored. Mammalian cells express three TPCs: TPC1, TPC2 and TPC3, a pseudogene in humans. Here we report that MEG01 and HEK293 cells endogenously express TPC1 and TPC2. Silencing TPC2 expression results in attenuation of the rate and extent of thapsigargin (TG)-evoked SOCE both in MEG01 and HEK293 cells, without having any effect on the ability of cells to accumulate Ca 2 + into the TG-sensitive stores. Similarly, silencing of native TPC2 expression reduced thrombin-induced Ca 2 + entry in MEG01 cells. In contrast, silencing of TPC1 expression was without effect either on TG or thrombin-stimulated Ca 2 + entry both in MEG01 and HEK293 cells. Biotinylation analysis revealed that TPC1 and TPC2 are expressed in internal membranes. Finally, co-immunoprecipitation experiments indicated that endogenously expressed TPC2, but not TPC1, associates with STIM1 and Orai1, but not with TRPC1, in MEG01 cells with depleted intracellular Ca 2 + stores, but not in resting cells. These results provide strong evidence for the modulation of SOCE by TPC2 involving de novo association between TPC2 and STIM1, as well as Orai1, in human cells.

Discharge of the intracellular Ca(2+) stores activates Ca(2+) entry through store-operated channels (SOCs). Since the recent identification of STIM1 and STIM2, as well as the Orai1 homologs, Orai2 and Orai3, the protein complexes involved in Ca(2+) signaling needs re-evaluation in native cells. Using real time PCR combined with Western blotting we have found the expression of the three Orai isoforms, STIM1, STIM2 and different TRPCs in human platelets. Depletion of the intracellular Ca(2+) stores with thapsigargin, independently of changes in cytosolic Ca(2+) concentration, enhanced the formation of a signaling complex involving STIM1, STIM2, Orai1, Orai2 and TRPC1. Furthermore, platelet treatment with the dyacylglicerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) resulted in specific association of Orai3 with TRPC3. Treatment of platelets with arachidonic acid enhanced the association between Orai1 and Orai3 in human platelets and overexpression of Orai1 and Orai3 in HEK293 cells increased arachidonic acid-induced Ca(2+) entry. These results indicate that Ca(2+) store depletion results in the formation of exclusive signaling complexes involving STIM proteins, as well as Orai1, Orai2 and TRPC1, but not Orai3, which seems to be involved in non-capacitative Ca(2+) influx in human platelets.

Homer is a family of cytoplasmic adaptor proteins that play different roles in cell function, including the regulation of G-protein-coupled receptors. These proteins contain an Ena (Enabled)/VASP (vasodilator-stimulated phosphoprotein) homology 1 domain that binds to the PPXXF sequence motif, which is present in different Ca 2+ -handling proteins such as IP 3 (inositol 1,4,5-trisphosphate) receptors and TRPC (transient receptor potential canonical) channels. In the present study we show evidence for a role of Homer proteins in the STIM1 (stromal interaction molecule 1)–Orai1 association, as well as in the TRPC1–IP 3 RII (type II IP 3 receptor) interaction, which might be of relevance in platelet function. Treatment of human platelets with thapsigargin or thrombin results in a Ca 2+ -independent association of Homer1 with TRPC1 and IP 3 RII. In addition, thapsigargin and thrombin enhanced the association of Homer1 with STIM1 and Orai1 in a Ca 2+ -dependent manner. Interference with Homer function by introduction of the synthetic PPKKFR peptide into cells, which emulates the proline-rich sequences of the PPXXF motif, reduced STIM1–Orai1 and TRPC1– IP 3 RII associations, as compared with the introduction of the inactive PPKKRR peptide. The PPKKFR peptide attenuates thrombin-evoked Ca 2+ entry and the maintenance of thapsigargin-induced store-operated Ca 2+ entry. Finally, the PPKKFR peptide attenuated thrombin-induced platelet aggregation. The findings of the present study support an important role for Homer proteins in thrombin-stimulated platelet function, which is likely to be mediated by the support of agonist-induced Ca 2+ entry.

Sperm plasma membrane is a very important structure that functions to protect sperm against extracellular injuries and to respond to physiological challenges. It plays a crucial role during sperm capacitation, in sperm-egg interaction and, finally, in fertilization. Concerning sperm technology, possibly the most important factors causing damage in mammalian spermatozoa membranes are initiated by the osmotic stress generated by dehydration of the cells during freezing and thawing. These changes are rapidly derived to the plasma and organelle membranes that gradually experiment loss of membrane architecture, causing unbalanced production of reactive oxygen species and increased lipid peroxidation. Other procedures such as sperm sorting or liquid storage of sperm also induce harmful changes in the integrity of the membrane. The specific composition of lipids of the sperm membranes may provide clues for understanding the mechanisms behind the differences found in the response to stress in different species. In the present review, we deal with the composition, architecture and organization of the sperm plasma membrane, emphasizing the factors that can affect membrane integrity. The intracellular signalling pathways related with membrane reorganization during capacitation and acrosome reaction are also reviewed.

Stromal interaction molecule 1 (STIM1) is a key element of the store-operated Ca 2+ entry mechanism (SOCE). Recently, regulation of STIM1 by glycosylation and phosphorylation on serine/threonine or proline residues has been described; however other modes of phosphorylation that are important for activating SOCE in platelets, such as tyrosine phosphorylation, have been poorly investigated. Here we investigate the latency of STIM1 phosphorylation on tyrosine residues during the first steps of SOCE activation. Human platelets were stimulated and fixed at desired times using rapid kinetic assays instruments, and immunoprecipitation and western blotting techniques were then used to investigate the pattern of STIM1 tyrosine phosphorylation during the first steps of SOCE activation. We have found that maximal STIM1 tyrosine phosphorylation occurred 2.5 s after stimulation of human platelets with thapsigargin (Tg). STIM1 localized in the plasma membrane were also phosphorylated in platelets stimulated with Tg. By using chemical inhibitors that target different members of the Src family of tyrosine kinases (SKFs), two independent signaling pathways involved in STIM1 tyrosine phosphorylation during the first steps of SOCE activation were identified. We finally conclude that STIM1 tyrosine phosphorylation is a key event for the association of STIM1 with plasma membrane Ca 2+ channels such as Orai1, hence it is required for conducting SOCE activation.

This work was designed in order to gain an insight on the mechanisms by which antioxidants prevent pancreatic disorders. We have examined the properties of cinnamtannin B-1, which belongs to the class of polyphenols, against the effect of hydrogen peroxide (H(2)O(2)) in mouse pancreatic acinar cells. We have studied Ca(2+) mobilization, oxidative state, amylase secretion, and cell viability of cells treated with cinnamtannin B-1 in the presence of various concentrations of H(2)O(2). We found that H(2)O(2) (0.1-100 μM) increased CM-H(2)DCFDA-derived fluorescence, reflecting an increase in oxidation. Cinnamtannin B-1 (10 μM) reduced H(2)O(2)-induced oxidation of CM-H(2)DCFDA. CCK-8 induced oxidation of CM-H(2)DCFDA in a similar way to low micromolar concentrations of H(2)O(2), and cinnamtannin B-1 reduced the oxidant effect of CCK-8. In addition, H(2)O(2) induced a slow and progressive increase in intracellular free Ca(2+) concentration ([Ca(2+)](c)). Cinnamtannin B-1 reduced the effect of H(2)O(2) on [Ca(2+)](c), but only at the lower concentrations of the oxidant. H(2)O(2) inhibited amylase secretion in response to cholecystokinin, and cinnamtannin B-1 reduced the inhibitory action of H(2)O(2) on enzyme secretion. Finally, H(2)O(2) reduced cell viability, and the antioxidant protected acinar cells against H(2)O(2). In conclusion, the beneficial effects of cinnamtannin B-1 appear to be mediated by reducing the intracellular Ca(2+) overload and intracellular accumulation of digestive enzymes evoked by ROS, which is a common pathological precursor that mediates pancreatitis. Our results support the beneficial effect of natural antioxidants in the therapy against oxidative stress-derived deleterious effects on cellular physiology.

Background A novel family of intracellular Ca 2+ -release channels termed two-pore channels (TPCs) has been presented as the receptors of NAADP (nicotinic acid adenine dinucleotide phosphate), the most potent Ca 2+ mobilizing intracellular messenger. TPCs have been shown to be exclusively localized to the endolysosomal system mediating NAADP-evoked Ca 2+ release from the acidic compartments. Objectives The present study is aimed to investigate NAADP-mediated Ca 2+ release from intracellular stores in the megakaryoblastic cell line MEG01. Methods Changes in cytosolic and intraluminal free Ca 2+ concentrations were registered by fluorimetry using fura-2 and fura-ff, respectively; TPC expression was detected by PCR. Results Treatment of MEG01 cells with the H + /K + ionophore nigericin or the V-type H + -ATPase selective inhibitor bafilomycin A1 revealed the presence of acidic Ca 2+ stores in these cells, sensitive to the SERCA inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). NAADP releases Ca 2+ from acidic lysosomal-like Ca 2+ stores in MEG01 cells probably mediated by the activation of TPC1 and TPC2 as demonstrated by TPC1 and TPC2 expression silencing and overexpression. Ca 2+ efflux from the acidic lysosomal-like Ca 2+ stores or the endoplasmic reticulum (ER) results in ryanodine-sensitive activation of Ca 2+ -induced Ca 2+ release (CICR) from the complementary Ca 2+ compartment. Conclusion Our results show for the first time NAADP-evoked Ca 2+ release from acidic compartments through the activation of TPC1 and TPC2, and CICR, in a megakaryoblastic cell line.

Mammalian cells accumulate Ca2+ into agonist-sensitive acidic organelles, vesicles that possess a vacuolar proton- ATPase. Acidic Ca2+ stores include secretory granules and lysosome-related organelles. Current evidence clearly indicates that acidic Ca2+ stores participate in cell signaling and function, including the activation of store-operated Ca2+ entry in human platelets upon depletion of the acidic stores, although the mechanism underlying the activation of store-operated Ca2+ entry controlled by the acidic stores remains unclear. STIM1 has been presented as the endoplasmic reticulum Ca2+ sensor, but its role sensing intraluminal Ca2+ concentration in the acidic stores has not been investigated. Here we report that STIM1 and STIM2 are expressed in the lysosome-related organelles and dense granules in human platelets isolated by immunomagnetic sorting. Depletion of the acidic Ca2+ stores using the specific vacuolar proton-ATPase inhibitor, bafilomycin A1, enhanced the association between STIM1 and STIM2 as well as between these proteins and the plasma membrane channel Orai1. Depletion of the acidic Ca2+ stores also induces time-dependent co-immunoprecipitation of STIM1 with the TRPC proteins hTRPC1 and hTRPC6, as well as between Orai1 and both TRPC proteins. In addition, bafilomycin A1 enhanced the association between STIM2 and SERCA3. These findings demonstrate the location of STIM1 and STIM2 in the acidic Ca2+ stores and their association with Ca2+ channels and ATPases upon acidic stores discharge.

Melatonin reduces proliferation in many different cancer cell lines. Thus, melatonin is considered a promising antitumor agent, promoting apoptosis in tumor cells while preserving viability of normal cells. Herein, we examined the effects of melatonin on the pancreatic AR42J tumor cell line. We have analyzed cytosolic-free Ca(2+) concentration ([Ca(2+) ](c) ), mitochondrial-free Ca(2+) concentration ([Ca(2+) ](m) ), mitochondrial membrane potential (Ψm), mitochondrial flavin adenine dinucleotide (FAD) oxidative state, cellular viability and caspase-3 activity. Our results show that melatonin induced transient changes in [Ca(2+) ](c) and [Ca(2+) ](m) . Melatonin also induced depolarization of Ψm and led to a reduction in the level of oxidized FAD. In addition, melatonin reduced AR42J cell viability. Finally, we found a Ca(2+) -dependent caspase-3 activation in response to melatonin. Collectively, these data support the likelihood that melatonin reduces viability of tumor AR42J cells via its action on mitochondrial activity and caspase-3 activation.

A number of disorders, such as Alzheimer disease and diabetes mellitus, have in common the alteration of the redox balance, resulting in an increase in reactive oxygen species (ROS) generation that might lead to the development of apoptosis and cell death. It has long been known that ROS can significantly alter Ca 2+ mobilization, an intracellular signal that is involved in the regulation of a wide variety of cellular functions. Cells have a limited capability to counteract the effects of oxidative stress, but evidence has been provided supporting the beneficial effects of exogenous ROS scavengers. Here, we review the effects of oxidative stress on intracellular Ca 2+ homeostasis and the role of antioxidants in the prevention and treatment of disorders associated to abnormal Ca 2+ mobilization induced by ROS. Keywords: reactive oxygen species; scavengers; antioxidants; intracellular Ca 2+ homeostasis 1. Calcium Homeostasis In eukaryotic cells, Ca 2+ is the most versatile signal involved in the control of cellular processes and functions [1-3]. This versatility derives from the fact that Ca

1. Cinnamtannin B-1 is a naturally occurring A-type proanthocyanidin that belongs to a class of polyphenols widely distributed throughout the plant kingdom and exhibiting anti-oxidant properties. 2. In the present study, we examined the effects of cinnamtannin B-1 on cholecystokinin octapeptide (CCK-8)-evoked Ca(2+) mobilization, reactive oxygen species (ROS) production and amylase secretion in the exocrine pancreas. 3. Stimulation of cells with 1 nmol/L CCK-8 led to a transient increase in the cytosolic free calcium concentration ([Ca(2+) ](c) ), followed by a decrease towards a value close to the prestimulation level. In the presence of 10 μmol/L cinnamtannin B-1, stimulation of cells with CCK-8 resulted in a smaller [Ca(2+) ](c) peak response, a faster rate of decay of [Ca(2+) ](c) and lower values for the steady state of [Ca(2+) ](c) , compared with the effect of CCK-8 alone. Cinnamtannin B-1 decreased Ca(2+) influx after depletion of intracellular stores by either CCK-8 or thapsigargin (1 μmol/L). Conversely, CCK-8 increased the fluorescence of 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H(2) DCFDA), reflecting an increase in oxidation. Cinnamtannin B-1 reduced CCK-8-induced oxidation of CM-H(2) DCFDA. Cholecystokinin-8 had a biphasic effect on amylase secretion, producing maximum at a concentration of 0.1 nmol/L and reducing secretion at higher concentrations. Pre-incubation of cells with 10 μmol/L cinnamtannin B-1 significantly attenuated the inhibition of enzyme secretion in response to high concentrations of CCK-8 (i.e. >10(-10) mol/L). Finally, the anti-oxidant protected acinar cells against CCK-8-induced cell death. 4. The beneficial effects of cinnamtannin B-1 appear to be mediated by a reduction in intracellular Ca(2+) overload, ROS production and intracellular accumulation of digestive enzymes, which is a common pathological precursor that mediates pancreatitis.

Melatonin is produced following circadian rhythm with high levels being released at night and has been implicated in the regulation of physiological processes in major tissues, including the pancreas. The aim of our study was to examine the effects of melatonin on intracellular free Ca(2+) concentration ([Ca(2+) ](c)) in AR42J pancreatic cells. Our results show that stimulation of cells with 1 nm cholecystokinin (CCK)-8 led to a transient increase in [Ca(2+) ](c) followed by a decrease towards a value close to the prestimulation level. Melatonin (at the concentrations 1, 10, 100 μm and 1 mm) induced changes in [Ca(2+) ](c) that consisted of single or short lasting spikes in the form of oscillations or slow transient increases followed by a slow reduction towards a value close to the resting level. Depletion of intracellular Ca(2+) stores by stimulation of cells with 1 nm CCK-8 or 1 μm thapsigargin (Tps) blocked Ca(2+) responses evoked by melatonin in the majority of cells. Conversely, prior stimulation of cells with 1 mm melatonin in the absence of extracellular Ca(2+) inhibited Ca(2+) mobilization in response to a secondary application of CCK-8 or Tps. In summary, our results show that melatonin releases Ca(2+) from intracellular stores and can therefore modulate the responses of the pancreas to CCK-8. The source for Ca(2+) mobilization most probably is the endoplasmic reticulum. These data raise the possibility that melatonin also involves Ca(2+) signalling, in addition to other intracellular messengers, to modulate cellular function.

Cholecystokinin acts in a concentration-dependent fashion on its binding sites in pancreatic acinar cells, and results in the generation of different second messengers in the signal cascades that lead to enzymatic secretion (Gonzalez et al. 1999). The activation of phospholipase C-linked receptors by cholecystokinin produces an increase in the concentration of inositol 1,4,5-trisphosphate (IP3) in the cytosol. IP3 in turn releases calcium (Ca2+) from cytoplasmic stores leading to an increase in cytosolic free calcium concentration ([Ca2+]c) (Streb et al. 1983). Ca2+ signals are not only a result of release from intracellular stores but also a coordinate influx from the extracellular space (Putney 1988), Ca2+ extrusion across the plasma membrane (Carafoli 1991) as well as Ca2+ uptake into intracellular organelles (Gonzalez et al. 1997a). A rise in [Ca2+]c is an important early signal by which physiological secretagogues elicit the release of digestive enzymes from pancreatic acinar cells. In pancreatic acinar cells, cholecystokinin regulates secretion via generation of repetitive local cytosolic Ca2+ signals in the apical pole. In addition, the spatiotemporal pattern of agonist-induced Ca2+ signals is of critical importance for exocytosis of enzymes (Habara & Kanno 1994). Cytosolic Ca2+ overload may result from excessive loss of Ca2+ from the intracellular stores by its release through specific channels and by the inhibition of Ca2+ pumps, and/or followed by entry of extracellular Ca2+ (Petersen & Sutton 2006; Gonzalez et al. 2008). Therefore, it is important to know how Ca2+ homeostasis is regulated because, despite being one of the initial steps involved in cellular function, abnormally elevated [Ca2+]c is a shared phenomenon that could induce trypsin premature activation (Ding et al. 2006). This is a previous step that can trigger acute pancreatitis and necrosis (Ward et al. 1995; Petersen & Sutton 2006). Although cholecystokinin is a major physiological regulator of secretion by the exocrine pancreas, an over stimulation can cause injury to the pancreas which may lead to dysfunction of the gland and even to the activation of death signalling pathways involving caspases (Saluja et al. 1999; Gukovskaya et al. 2002). It has been long recognized that a significant percentage of patients with pancreatitis often presents a history of excessive alcohol consumption. Alcohol is involved in approximately 40% of cases of acute pancreatitis (Kristiansen et al. 2008; Ellis et al. 2009). Nevertheless, the mechanisms underlying alcohol-derived deleterious effects are not completely understood. The exocrine pancreas can metabolize ethanol mainly via an oxidative pathway involving the enzymes alcohol dehydrogenase (ADH) and cytochrome P4502E1, although a non-oxidative pathway involving fatty acid ethyl ester synthases has also been proposed (Criddle et al. 2004). Metabolism of ethanol by pancreatic acinar cells and the consequent generation of toxic metabolites are postulated to play an important role in the development of alcohol-related pancreatic injury (Petersen & Sutton 2006; Lawrencia et al. 2009). In these terms, ethanol may induce its deleterious effects in pancreatic acinar cells through the generation of oxidative and non-oxidative metabolites (Criddle et al. 2006; Gonzalez et al. 2006; Palmieri et al. 2007). One of the early events leading to alcoholic pancreatitis seems to be the effect of ethanol on stimulus-secretion coupling mechanisms. It has been suggested that ethanol acts to sensitize the pancreas to the deleterious effects of other stimuli such as the physiological agonist cholecystokinin octapeptide (CCK-8), which leads to an inflammatory response and pancreatitis. This effect is in part mediated by augmenting activation of proinflamatory factors (Pandol et al. 2003), although a decrease in the levels of prostaglandin E2 by ethanol could be involved in alcohol-induced injury in the pancreas too (Siegmund et al. 2003). Furthermore, it has been proposed that ethanol induces generation of oxygen radicals in pancreatic acinar cells (Wittel et al. 2003; Gonzalez et al. 2006; Lawrencia et al. 2009), impairs CCK-8-evoked secretion of digestive enzymes (Gonzalez et al. 2006) and leads to a delayed or reduced Ca2+ extrusion from the cytosol towards the extracellular space or into the cytosolic stores (Gonzalez et al. 2008). Despite the great amount of research carried out to understand the action of ethanol on cell physiology, the involvement of Ca2+ metabolism in ethanol-evoked effects needs to be further studied, because most cellular activity is initiated by changes in [Ca2+]c. In this study, we have investigated the early effects of acute ethanol exposure on CCK-8-evoked Ca2+ influx in mouse pancreatic acinar cells. Our objective is to shed more light on the effects of ethanol on CCK-8 physiological actions. The findings will contribute to a better understanding of the mechanisms by which ethanol causes pancreatic disorders.

Objective— The role of cyclophilins (chaperones that are widely expressed in different cell types, including human platelets) was explored in sarcoendoplasmic calcium (Ca 2+ ) adenosine triphosphatase (SERCA) activity. Methods and Results— Cyclophilin inhibition by cyclosporin A (CsA) evoked a time- and concentration-dependent reduction of Ca 2+ uptake by SERCA2b. However, other Ca 2+ -adenosine triphosphatases expressed in platelets, such as SERCA3 and plasma membrane Ca 2+ adenosine triphophatase, remained unaltered after CsA treatment. Cypermethrin, a non–CsA-related calcineurin inhibitor, did not alter SERCA2b activity. Furthermore, SERCA2b was affected by other CsA analogues, which do not interfere with calcineurin, such as PKF-211-811-NX5 (NIM811) and sanglifehrin A. Inhibition of the immunophilin family members using FK506 (tacrolimus) did not alter SERCA2b ability to sequester Ca 2+ into the dense tubular system. Coimmunoprecipitation experiments confirmed that cyclophilin A associates with SERCA2b and stromal interaction molecule-1 in resting platelets. This interaction is attenuated by the physiological agonist thrombin but enhanced by treatment with CsA or sanglifehrin A. Conclusion— Cyclophilin A is a regulator of SERCA2b in human platelets.

SummaryDiabetes mellitus is a disease characterised by hyperglycaemia and associated with several cardiovascular disorders, including angiopathy and platelet hyperactivity, which are major causes of morbidity and mortality in type 2 diabetes mellitus. In type 2 diabetic patients, homo-cysteine levels are significantly increased compared with healthy subjects. Hyperhomocysteinaemia is an independent risk factor for macro-and microangiopathy and mortality. The present study is aimed to investigate the effect of homocysteine on platelet apoptosis. Changes in cytosolic or intraluminal free Ca2+ concentration were determined by fluorimetry. Caspase activity and phosphorylation of the eukaryotic initiation factor 2α (eIF2α) were explored by Western blot. Our results indicate that homocysteine releases Ca2+ from agonist sensitive stores, enhances eIF2α phosphorylation at Ser51 and activates caspase-3 and -9 independently of extracellular Ca2+. Homocysteine induced activation of caspase-3 and -9 was abolished by salubrinal, an agent that prevents endoplasmic reticulum (ER) stress-induced apoptosis. Homo-cysteine-induced platelet effects were significantly greater in type 2 diabetics than in healthy subjects. These findings demonstrate that homocysteine induces ER stress-mediated apoptosis in human platelets, an event that is enhanced in type 2 diabetic patients, which might be involved in the pathogenesis of cardiovascular complications associated with type 2 diabetes mellitus.

A phenylpropanoid 1, a slovenolide 2, and two germacranes bearing a methylthiopropenoate moiety, 3 and 4, along with twenty known metabolites have been isolated from the roots of THAPSIA VILLOSA var. VILLOSA L. The structures of two known phenylpropanoids 5 and 6 have been corrected. Compounds 7 and 8 showed activity as potential inhibitors of the sarco- and endoplasmic Ca 2+ -dependent ATPases (SERCA) pump. Compounds 9, 10 and 11 increased significantly the cytoplasmic free calcium concentration ([Ca 2+ ] c ) in human platelets in a concentration-dependent manner.

Protein tyrosine kinases have important roles in spermatozoa; however, little is known about the presence and regulation in these cells of their counterparts in signaling, namely, protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs). The objectives of the present study were to identify PTPs and DSPs in boar, stallion, and dog spermatozoa; to characterize their subcellular distribution; and to investigate the roles of tyrosine phosphatases in maintenance of protein tyrosine phosphorylation level and in sperm motility. Using Western blotting with specific antibodies in boar and stallion sperm lysates, we unequivocally identified two PTPs (PTPRB and PTPN11) and two DSPs (DUSP3 and DUSP4). In dog sperm lysates, only PTPN11, DUSP3, and DUSP4 were detected. In all these species, we did not detect the specific signal with anti-PTPRC (CD45), CDKN3, DUSP1, DUSP2, DUSP6, DUSP9, PTPN1, PTPN3, PTPN6, PTPN7, PTPN13, PTPRA, PTPRG, PTPRJ, PTPRK, or PTPRZ antibodies. Positive matches were further investigated by indirect immunofluorescence and confocal microscopy. Results showed that PTPRB was associated with the plasma membrane in the head and tail of boar and stallion spermatozoa. In agreement with Western blotting results, PTPRB antibodies did not show immunoreactivity in dog sperm analyzed by immunofluorescence. In the three species, DUSP4 was mainly found in the tail of spermatozoa, with little or no immunoreactivity in the head. PTPN11 was mainly located in the postacrosomal region in the head, whereas DUSP3 immunoreactivity was extended within the acrosome. PTPN11 and DUSP3 showed immunoreactivity in the tail that was restricted to the midpiece. Finally, we incubated boar, stallion, and dog spermatozoa with pervanadate and sodium orthovanadate, two PTP inhibitors, and analyzed overall protein tyrosine phosphorylation and assessed sperm motility. Sodium orthovanadate and pervanadate showed concentration-dependent inhibition of sperm motility that was rapid and reversible. Pervanadate also increased tyrosine phosphorylation of different proteins in capacitated and noncapacitated spermatozoa. Results showed that the phosphatases PTPN11, DUSP4, and DUSP3 are present in boar, stallion, and dog spermatozoa. PTPRB is also present in boar and stallion spermatozoa but was not detected in dog. The subcellular distribution of the identified phosphatases is diverse, suggesting that they likely have specific roles in sperm. Finally, PTP activity has a positive role in the regulation of motility and is involved in protein tyrosine phosphorylation in mammalian sperm.

Background/Aims The hepatitis C virus (HCV) structural core and non-structural NS5A proteins induce in liver cells a series of intracellular events, including elevation of reactive oxygen and nitrogen species (ROS/RNS). Since oxidative stress is associated to altered intracellular Ca 2+ homeostasis, we aimed to investigate the effect of these proteins on Ca 2+ mobilization in human hepatocyte-derived transfected cells, and the protective effect of quercetin treatment. Methods Ca 2+ mobilization and actin reorganization were determined by spectrofluorimetry. Production of ROS/RNS was determined by flow cytometry. Results Cells transfected with NS5A and core proteins showed enhanced ROS/RNS production and resting cytosolic Ca 2+ concentration, and reduced Ca 2+ concentration into the stores. Phenylephrine-evoked Ca 2+ release, Ca 2+ entry and extrusion by the plasma membrane Ca 2+ -ATPase were significantly reduced in transfected cells. Similar effects were observed in cytokine-activated cells. Phenylephrine-evoked actin reorganization was reduced in the presence of core and NS5A proteins. These effects were significantly prevented by quercetin. Altered Ca 2+ mobilization and increased calpain activation were observed in replicon-containing cells. Conclusions NS5A and core proteins induce oxidative stress-mediated Ca 2+ homeostasis alterations in human hepatocyte-derived cells, which might underlie the effects of both proteins in the pathogenesis of liver disorders associated to HCV infection.

Summary. Background: Thrombin is a physiological platelet agonist that activates apoptotic events, including cytochrome c release and phosphatidylserine exposure; however, the mechanisms underlying these events remain unclear. Objectives: The present study is aimed to investigate whether thrombin induces activation and mitochondrial translocation of Bid, Bax and Bak. Methods: Changes in the mitochondrial membrane potential were registered using the dye JC-1; Bid, Bax and Bak translocation to the mitochondria was detected by immunoprecipitation and Western blotting in samples from mitochondrial and cytosolic fractions. Results: Treatment of platelets with thrombin or ADP induces activation and mitochondrial association of active Bid, Bax and Bak. Translocation of Bid and Bax to the mitochondria was reduced by cytochalasin D, latrunculin A or jasplakinolide. Platelet exposure to exogenous H2O2 (10 μm) results in activation of Bid and Bax, which was found to be similar to the effect of thrombin. Thrombin evokes mitochondrial membrane depolarization, which is attenuated by catalase. Conclusion: Our results indicate that thrombin induces activation and mitochondrial translocation of Bid, Bax and Bak, which is likely to be one of the apoptotic events in human platelets.

Orai1 and hTRPC1 have been presented as essential components of store-operated channels mediating highly Ca(2+) selective I(CRAC) and relatively Ca(2+) selective I(SOC), respectively. STIM1 has been proposed to communicate the Ca(2+) content of the intracellular Ca(2+) stores to the plasma membrane store-operated Ca(2+) channels. Here we present evidence for the dynamic interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 regulated by depletion of the intracellular Ca(2+) stores, using the pharmacological tools thapsigargin plus ionomycin, or by the physiological agonist thrombin, independently of extracellular Ca(2+). In addition we report that Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the mode of activation of hTRPC1-forming Ca(2+) permeable channels. Electrotransjection of cells with anti-Orai1 antibody, directed toward the C-terminal region that mediates the interaction with STIM1, and stabilization of an actin cortical barrier with jasplakinolide prevented the interaction between STIM1 and hTRPC1. Under these conditions hTRPC1 was no longer involved in store-operated calcium entry but in diacylglycerol-activated non-capacitative Ca(2+) entry. These findings support the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of store-operated Ca(2+) entry.

Analysis of the posttranslational modification of proteins, such as phosphorylation, might yield misleading results due to the presence of other proteins with similar electrophoretic properties that coimmunoprecipitate with the target protein. The aim of the present work was to develop a reliable, easy and economical technique to completely isolate a protein from its complex. Here we present a new assay developed to fully isolate proteins from macromolecular complexes that consists of an initial SDS/PAGE (under reducing conditions), which isolates the target protein, followed by transfer of the proteins to a buffer, from which the target protein is recaptured by conventional immunoprecipitation. This technique, that we have termed "Protein Complex Immunological Separation Assay" (ProCISA), successfully separated proteins of different sizes, such as pp60Src and the IP3 receptor (IP3R), from their complexes. We show that ProCISA allows the investigation of the tyrosine phosphorylation state of isolated proteins. This technique could also be used to study other posttranslational modifications without risk of misleading results resulting from contamination with other proteins of similar electrophoretic mobility which complex with the protein of interest.

The authors investigated whether cytosolic free calcium concentration ([Ca2+]c) plays a role in hydrogen peroxide-induced pancreatic acinar AR42J cells apoptosis. We analysed mitochondrial depolarization, [Ca2+]c determination and caspase-3 activity by fluorimetric methods, and cytochrome c release by subcellular fractionation and western blotting. The data shown that hydrogen peroxide, which causes a sustained [Ca2+]c increase, induces mitochondrial depolarization and cytochrome c release, and activation of caspase-3. Dimethyl-BAPTA loading did not affect hydrogen peroxide-evoked mitochondrial apoptosis, suggesting that these responses are independent of increases in [Ca2+]c. Treatment with thapsigargin, to induce extensive calcium store depletion and subsequent increases in [Ca2+]c, also stimulates mitochondrial depolarization cytochrome c release, and caspase-3 activation. Similar results were observed in AR42J cells loaded with dimethyl-BAPTA, suggesting that activation of apoptosis by thapsigargin does not require rises in [Ca2+]c. However, the blockade of mitochondrial calcium entry by pretreating with Ru360 showed protection against hydrogen peroxide- and thapsigargin-induced mitochondrial apoptosis. These results indicate that the apoptosis evoked by hydrogen peroxide and thapsigargin is mediated by mitochondrial calcium uptake.

In this study we have investigated the effect of ethanol on [Ca2+]c by microfluorimetry and glutamate secretion using an enzyme-linked system, in rat hippocampal astrocytes in culture. Our results show that ethanol (1–200 mM) evoked a dose-dependent increase in glutamate secretion. 50 mM ethanol, a concentration within the range of blood alcohol levels in intoxicated humans, induced a release of Ca2+ from intracellular stores in the form of oscillations. Ca2+-mobilizing effect of ethanol was not prevented by preincubation of cells in the presence of 2 mM of the antioxidant dithiothreitol. Ethanol-evoked glutamate secretion was reduced when extracellular Ca2+ was omitted (medium containing 0.5 mM EGTA) and following preincubation of astrocytes in the presence of the intracellular Ca2+ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetraacetoxy-methyl ester (10 μM). Preincubation of astrocytes in the presence of 2 mM of the antioxidant dithiothreitol significantly reduced ethanol-evoked glutamate secretion. Finally, preincubation of astrocytes in the presence of bafilomycin (50 nM) significantly reduced ethanol-induced neurotransmitter release, indicating that exocytosis is involved in glutamate secretion. In conclusion, our results suggest that ethanol mobilizes Ca2+ from intracellular stores, and stimulates a Ca2+-dependent glutamate secretion, probably involving reactive oxygen species production, and therefore creating a situation potentially leading to neurotoxicity in the hippocampus.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a versatile regulator of TRP channels. We report that inclusion of a PIP2 analogue, PIP2 1,2-dioctanoyl, does not induce non-capacitative Ca2+ entry per se but enhanced Ca2+ entry stimulated either by thrombin or by selective depletion of the Ca2+ stores in platelets, the dense tubular system, using 10 nM TG, and the acidic stores, using 20 μM 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Reduction of PIP2 levels by blocking PIP2 resynthesis with Li+ or introducing a monoclonal anti-PIP2 antibody, or sequestering PIP2 using poly-lysine, attenuated Ca2+ entry induced by thrombin, TG and TBHQ, and reduced thrombin-evoked, but not TG- or TBHQ-induced, Ca2+ release from the stores. Incubation with the anti-hTRPC1 antibody did not alter the stimulation of Ca2+ entry by PIP2, whilst introduction of anti-hTRPC6 antibody directed towards the C-terminus of hTRPC6 reduced Ca2+ and Mn2+ entry induced by thrombin, TG or TBHQ, and abolished the stimulation of Ca2+ entry by PIP2. The anti-hTRPC6 antibody, but not the anti-hTRPC1 antibody or PIP2, reduced non-capacitative Ca2+ entry by the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol. In summary, hTRPC6 plays a role both in store-operated and in non-capacitative Ca2+ entry. PIP2 enhances store-operated Ca2+ entry in human platelets, most probably by stimulation of hTRPC6 channels.