Ginés Salido | Universidad de Extremadura (original) (raw)
Papers by Ginés Salido
Molecular and Cellular Biochemistry, 2008
This study investigated the effects of extracellular Mg2+ ([Mg2+]o) on basal and acetylcholine (A... more This study investigated the effects of extracellular Mg2+ ([Mg2+]o) on basal and acetylcholine (ACh)-evoked amylase secretion and intracellular free Ca2+ ([Ca2+]i) in rat parotid acinar cells. In a medium containing 1.1 mM [Mg2+]o, ACh evoked significant increases in amylase secretion and [Ca2+]i. Either low (0 mM) or elevated (5 and 10 mM) [Mg2+]o attenuated ACh-evoked responses. In a nominally Ca2+ free medium, elevated [Mg2+]o attenuated basal and ACh-evoked amylase secretion and [Ca2+]i. In parotid acinar cells incubated with either 0, 1.1, 5 or 10 mM [Mg2+]o, ACh evoked a gradual decrease in [Mg2+]i. These results indicate that the ACh-evoked Mg2+ efflux is an active process since Mg2+ has to move against its gradient. Either lidocaine, amiloride, N-methyl-d-glucamine, quinidine, dinitrophenol or bumetanide can elevate [Mg2+]i above basal level. In the presence of these membrane transport inhibitors, ACh still evoked a decrease in [Mg2+]i but the response was less pronounced with either [Na+]o removal or in the presence of either amiloride or quinidine. These results indicate marked interactions between Ca2+ and Mg2+ signalling in parotid acinar cells and that ACh-evoked Mg2+ transport was not dependent upon [Na+]o.
Biochemical Journal, 2012
Homer is a family of cytoplasmic adaptor proteins that play different roles in cell function, inc... more 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.
Thrombosis and Haemostasis, 2015
The function of the mammalian target of rapamycin (mTOR) is upregulated in response to cell stimu... more The 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 mTOR1 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.
Thrombosis Research, 2014
ABSTRACT Changes in cytosolic calcium concentration ([Ca2+]c) regulate phosphorylation of importa... more ABSTRACT Changes in cytosolic calcium concentration ([Ca2+]c) regulate phosphorylation of importante intracellular signaling players, cytoskeleton reorganization, granule secretion and shape change in platelets among others, being all of them important mechanisms that are required for platelet activation and aggregation. Thrombin (Thr) activates protease activated receptors (PAR1 and PAR4) and promotes platelet stimulation, resulting in the differential release of calcium (Ca2+) located into different intracellular stores, which in turn activate a mechanism of Ca2+ entry from the extracellular medium into the cell, referred as store-operated Ca2+ entry (SOCE). SOCE is considered the main mechanism of Ca2+ entry into platelets and is essential from platelet function. A crucial step during platelet function is the release of physiologic agonists stored inside granules located in the cytosol to the extracellular compartment during activation. Aim. Study the role of Ca2+ mobilization from different intracellular stores or entry from extracellular medium in platelet granule secretion.
Journal of Cellular and Molecular Medicine, 2013
The use of the mammal target of rapamycin (mTOR) inhibitors has been consolidated as the therapy ... more 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.
Molecular and Cellular Biochemistry, 2008
The parotid glands are highly active secretory systems subjected to continuous stress, which in t... more The parotid glands are highly active secretory systems subjected to continuous stress, which in turn, can lead to several pathophysiological conditions. Damage of the parotid glands are caused by radical oxygen species (ROS) as by-products of oxygen metabolism. This study investigated the effect of hydrogen peroxide (H(2)O(2)) on Carbachol (CCh)-evoked secretory responses and caspase-3 activity in the isolated rat parotid gland to understand the role of oxidative stress on the function of the gland. Amylase secretion, cytosolic calcium concentration ([Ca(2+)](i)) and caspase-3 activity in parotid gland tissue were measured using fluorimetric methods. H(2)O(2) had little or no effect on amylase secretion compared to basal level. Combining H(2)O(2) with CCh resulted in an attenuation of the CCh-evoked amylase secretion compared to the effect of CCh alone. CCh can evoke a large increase in [Ca(2+)](i) comprising an initial peak followed by a plateau. In a Ca(2+)-free medium containing 1 mM EGTA, CCh evoked only the initial peak of [Ca(2+)](i). H(2)O(2) alone evoked a gradual and dose-dependent increase in [Ca(2+)](i). Combining H(2)O(2) with CCh resulted in a decrease in [Ca(2+)](i) compared to the effect of CCh alone. In a Ca(2+)-free medium, H(2)O(2) still evoked a small increase in [Ca(2+)](i), but this response was less compared to the results obtained with H(2)O(2) in normal [Ca(2+)](0). Combining H(2)O(2) with CCh resulted in only a small transient increase in [Ca(2+)](i). Following CCh stimulation, H(2)O(2) application resulted in a large increase in [Ca(2+)](i) in normal [Ca(2+)](0). This effect of H(2)O(2) was partially abolished in a nominally free Calcium medium containing EGTA. H(2)O(2) can stimulate caspase-3 activity in parotid gland tissue. Similar response was obtained with betulinic acid and thapsigargin (TPS) on caspase-3 activity compared to basal. The results have demonstrated that like CCh, H(2)O(2) can also mobilise Ca(2+) from intracellular stores and facilitate its influx into the cell from extracellular medium. This effect of H(2)O(2) may be due to its activity to induce apoptosis in the parotid gland, since H(2)O(2) can stimulate the activity of caspase-3, a marker of cellular apoptosis.
Cellular Signalling, 2012
Stromal interaction molecule 1 (STIM1) is a key element of the store-operated Ca 2+ entry mechani... more 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.
![Research paper thumbnail of Colecystokinin octapeptide CCK-8 and carbachol reduce [32P]orthophosphate labeling of phosphatidylcholine without modifying phospholipase D activity in rat pancreatic acini](https://attachments.academia-assets.com/45862474/thumbnails/1.jpg)
](We have studied phospholipase D activation in [ 32 P]orthophosphoric acid-prelabeled rat pancreat... more We have studied phospholipase D activation in [ 32 P]orthophosphoric acid-prelabeled rat pancreatic acini by measuring the formation of 32 P-phosphatidylalcohols as stimulated in the presence of ethanol or butanol. A small but significant and time-dependent basal accumulation of [ 32 P]phosphatidylethanol and [ 32 P]phosphatidylbutanol was detected, which was further stimulated by phorbol myristate acetate, orthovanadate and pervanadate. However, the secretagogues cholecystokinin octapeptide and carbachol did not enhance basal accumulation of 32 P-phosphatidylalcohol, yet they decreased [ 32 P]phosphatidylcholine content and stimulated the generation of [ 32 P]phosphatidic acid. Our results stress the need to examine the transphosphatidylation reaction as well as agonist effects on the synthesis of phosphatidylcholine in order to assess unambiguously phospholipase D activity. ß
International Review of Cell and Molecular Biology, 2015
Regulators of G-protein-signaling (RGS) proteins are a category of intracellular proteins that ha... more Regulators of G-protein-signaling (RGS) proteins are a category of intracellular proteins that have an inhibitory effect on the intracellular signaling produced by G-protein-coupled receptors (GPCRs). RGS along with RGS-like proteins switch on through direct contact G-alpha subunits providing a variety of intracellular functions through intracellular signaling. RGS proteins have a common RGS domain that binds to G alpha. RGS proteins accelerate GTPase and thus enhance guanosine triphosphate hydrolysis through the alpha subunit of heterotrimeric G proteins. As a result, they inactivate the G protein and quickly turn off GPCR signaling thus terminating the resulting downstream signals. Activity and subcellular localization of RGS proteins can be changed through covalent molecular changes to the enzyme, differential gene splicing, and processing of the protein. Other roles of RGS proteins have shown them to not be solely committed to being inhibitors but behave more as modulators and integrators of signaling. RGS proteins modulate the duration and kinetics of slow calcium oscillations and rapid phototransduction and ion signaling events. In other cases, RGS proteins integrate G proteins with signaling pathways linked to such diverse cellular responses as cell growth and differentiation, cell motility, and intracellular trafficking. Human and animal studies have revealed that RGS proteins play a vital role in physiology and can be ideal targets for diseases such as those related to addiction where receptor signaling seems continuously switched on.
Biochimica et biophysica acta, Jan 21, 2015
STIM1 is a ubiquitous Ca(2+) sensor of the intracellular, agonist-sensitive, Ca(2+) stores that c... more STIM1 is a ubiquitous Ca(2+) sensor of the intracellular, agonist-sensitive, Ca(2+) stores that communicates the filling state of the Ca(2+) compartments to plasma membrane store-operated Ca(2+) (SOC) channels. STIM1 has been presented as a point of convergence between store-operated and voltage-operated Ca(2+) influx, both inducing activation of SOC channels while suppressing Cav1.2 channels. Here we report that Homer proteins play a relevant role in the communication between STIM1 and Cav1.2 channels. HEK-293 cells transiently expressing Cav1.2 channel subunits α1, β2 and α2δ-1 exhibited a significant Ca(2+) entry upon treatment with a high concentration of KCl. In Cav1.2-expressing cells, treatment with thapsigargin (TG), to induce passive discharge of the intracellular Ca(2+) stores, resulted in Ca(2+) influx that was significantly greater than in cells not expressing Cav1.2 channels, a difference that was abolished by nifedipine and diltiazem. Treatment with TG induces co-immun...
Biochemical and Biophysical Research Communications, 1997
We have used BCECF- or Fura-2-loaded rat pancreatic acinar cells to investigate the relationship ... more We have used BCECF- or Fura-2-loaded rat pancreatic acinar cells to investigate the relationship between Ca2+mobilization and intracellular pH (pHi). Ca2+-mobilizing agonists CCK-8 and ACh induced a transient acidification totally dependent on release of Ca2+from internal stores. Employment of different physiological tools including ionomycin and thapsigargin to increase the cytosolic Ca2+concentration and capacitative calcium influx also induced cellular acidification. Application
Cellular Signalling, 2002
In fura-2 loaded isolated mouse pancreatic acinar cells, xanthine oxidase (XOD)-catalyzed reactiv... more In fura-2 loaded isolated mouse pancreatic acinar cells, xanthine oxidase (XOD)-catalyzed reactive oxygen species (ROS) generation caused an increase in the cytosolic Ca2+ concentration ([Ca2+]i) by release of Ca2+ from intracellular stores. The ROS-induced Ca2+ signals showed large variability in shape and time-course and resembled in part Ca2+ signals in response to physiological secretagogues. ROS-induced Ca2+ mobilization started at the
Transient Receptor Potential Channels, 2011
Transient receptor potential (TRP) proteins are involved in a large number of non-selective catio... more Transient receptor potential (TRP) proteins are involved in a large number of non-selective cation channels that are permeable to both monovalent and divalent cations. Two general classes of receptor-mediated Ca(2+) entry has been proposed: one of then is conduced by receptor-operated Ca(2+) channels (ROC), the second is mediated by channels activated by the emptying of intracellular Ca(2+) stores (store-operated channels or SOC). TRP channels have been presented as subunits of both ROC and SOC, although the precise mechanism that regulates the participation of TRP proteins in these Ca(2+) entry mechanisms remains unclear. Recently, TRPC proteins have been shown to associate with Orai1 and STIM1 in a dynamic ternary complex regulated by the occupation of membrane receptors in several cell models, which might play an important role in the function of TRPC proteins. The present review summarizes the current knowledge concerning the association of TRP proteins with Orai and STIM proteins and how this affects the participation of TRP proteins in store-operated or receptor-operated Ca(2+) entry.
Molecules (Basel, Switzerland), 2010
A number of disorders, such as Alzheimer disease and diabetes mellitus, have in common the altera... more 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 Ca²+ 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 Ca²+ homeostasis and the role of antioxidants in the prevention and treatment of disorders associated to abnormal Ca²+ mobilization induced by ROS.
Toxicology letters, Jan 17, 2014
Ebselen is a seleno-organic compound that causes cell death in several cancer cell types. The mec... more Ebselen is a seleno-organic compound that causes cell death in several cancer cell types. The mechanisms underlying its deleterious effects have not been fully elucidated. In this study, the effects of ebselen (1 μM-40 μM) on AR42J tumor cells have been examined. Cell viability was studied using AlamarBlue(®) test. Cell cycle phase determination was carried out by flow cytometry. Changes in intracellular free Ca(2+) concentration were followed by fluorimetry analysis of fura-2-loaded cells. Distribution of mitochondria, mitochondrial Ca(2+) concentration and mitochondrial membrane potential were monitored by confocal microscopy of cells loaded with Mitotracker Green™ FM, rhod-2 or TMRM respectively. Caspase-3 activity was calculated following the luorogenic substrate ACDEVD-AMC signal with a spectrofluorimeter. Results show that cell viability decreased in the presence of ebselen. An increase in the number of cells in the S-phase of the cell cycle was observed. Ebselen induced a con...
Chronobiology International, 1989
The effects of three anti-ulcers drugs on the temporal distribution of food intake and of the two... more The effects of three anti-ulcers drugs on the temporal distribution of food intake and of the two parameters, meal size and meal frequency, were studied in ulcerated and non-ulcerated rats exposed to light-dark (LD 12:12) cycles. Experimental ulceration with indomethacin reduces the amplitude of meal frequency and brings the acrophase forward, compared with non-ulcerated animals. These effects were reversed by the oral administration of either ranitidine, sucralfate or pirenzepine along with the food. However, the administration of either pirenzepine or sucralfate alone to non-ulcerated rats is accompanied by significant (P less than 0.05) changes in the circadian patterns of meal size and meal frequency without the total daily food intake being affected in any way (pirenzepine treatment caused large intake of food during the light period while sucralfate treatment resulted in marked food intake during the dark period). The results indicate that circadian modification of meal patterns in the ulcerated rats are attributable to indomethacin-induced gastrointestinal mucosal injury and anti-ulcer medications.
Cellular Signalling, 2003
In the present study, we have employed confocal laser scanning microscopy to investigate the effe... more In the present study, we have employed confocal laser scanning microscopy to investigate the effect that stimulation of mouse pancreatic acinar cells with the secretagogue cholecystokinin (CCK) has on mitochondrial activity. We have monitored changes in cytosolic as well as mitochondrial Ca2+ concentrations, mitochondrial membrane potential and FAD autofluorescence by loading the cells with fluo-3, rhod-2 or JC-1, respectively.Our results
Trends in cardiovascular medicine, 2008
Two separate Ca2+ stores have been reported in human platelets: the dense tubular system (DTS) an... more Two separate Ca2+ stores have been reported in human platelets: the dense tubular system (DTS) and lysosome-like acidic organelles. Recent work has reported that Ca2+ release from the DTS is mediated by the generation of inositol 1,4,5-trisphosphate, whereas Ca2+ efflux from the acidic stores is mostly linked to nicotinic acid adenine dinucleotide phosphate. Platelet agonists release Ca2+ selectively from one or both stores, which provides additional insight into the complexity of Ca2+ signaling and the cellular functions activated. Here, we review the role of multiple Ca2+ mobilizing messengers and Ca2+ stores in the activation of specific functions in platelets in response to different physiologic agonists.
Mitochondrion, 2004
In the present study we have studied the changes in the intracellular reduction–oxidation state i... more In the present study we have studied the changes in the intracellular reduction–oxidation state in mouse pancreatic acinar cells following stimulation with cholecystokinin octapeptide (CCK-8) and its dependence on Ca2+ mobilization. In our investigations cytosolic Ca2+ concentration and reactive oxygen species (ROS) production were determined by loading of cells with fura-2 and CM-H2DCF-DA, respectively. Changes in these parameters were determined
Laboratory Animals, 1983
Summary A new biliary bidirectional cannula is described which allows the study of biliary secret... more Summary A new biliary bidirectional cannula is described which allows the study of biliary secretion in conscious dogs under conditions which approach physiological normality. Materials and methods Experiments were carried out on 9 adult dogs (body weight 20-25 kg). The animals were fasted for 48 h prior to surgery. Drinking water was given ad libitum. Since the first report of
Molecular and Cellular Biochemistry, 2008
This study investigated the effects of extracellular Mg2+ ([Mg2+]o) on basal and acetylcholine (A... more This study investigated the effects of extracellular Mg2+ ([Mg2+]o) on basal and acetylcholine (ACh)-evoked amylase secretion and intracellular free Ca2+ ([Ca2+]i) in rat parotid acinar cells. In a medium containing 1.1 mM [Mg2+]o, ACh evoked significant increases in amylase secretion and [Ca2+]i. Either low (0 mM) or elevated (5 and 10 mM) [Mg2+]o attenuated ACh-evoked responses. In a nominally Ca2+ free medium, elevated [Mg2+]o attenuated basal and ACh-evoked amylase secretion and [Ca2+]i. In parotid acinar cells incubated with either 0, 1.1, 5 or 10 mM [Mg2+]o, ACh evoked a gradual decrease in [Mg2+]i. These results indicate that the ACh-evoked Mg2+ efflux is an active process since Mg2+ has to move against its gradient. Either lidocaine, amiloride, N-methyl-d-glucamine, quinidine, dinitrophenol or bumetanide can elevate [Mg2+]i above basal level. In the presence of these membrane transport inhibitors, ACh still evoked a decrease in [Mg2+]i but the response was less pronounced with either [Na+]o removal or in the presence of either amiloride or quinidine. These results indicate marked interactions between Ca2+ and Mg2+ signalling in parotid acinar cells and that ACh-evoked Mg2+ transport was not dependent upon [Na+]o.
Biochemical Journal, 2012
Homer is a family of cytoplasmic adaptor proteins that play different roles in cell function, inc... more 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.
Thrombosis and Haemostasis, 2015
The function of the mammalian target of rapamycin (mTOR) is upregulated in response to cell stimu... more The 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 mTOR1 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.
Thrombosis Research, 2014
ABSTRACT Changes in cytosolic calcium concentration ([Ca2+]c) regulate phosphorylation of importa... more ABSTRACT Changes in cytosolic calcium concentration ([Ca2+]c) regulate phosphorylation of importante intracellular signaling players, cytoskeleton reorganization, granule secretion and shape change in platelets among others, being all of them important mechanisms that are required for platelet activation and aggregation. Thrombin (Thr) activates protease activated receptors (PAR1 and PAR4) and promotes platelet stimulation, resulting in the differential release of calcium (Ca2+) located into different intracellular stores, which in turn activate a mechanism of Ca2+ entry from the extracellular medium into the cell, referred as store-operated Ca2+ entry (SOCE). SOCE is considered the main mechanism of Ca2+ entry into platelets and is essential from platelet function. A crucial step during platelet function is the release of physiologic agonists stored inside granules located in the cytosol to the extracellular compartment during activation. Aim. Study the role of Ca2+ mobilization from different intracellular stores or entry from extracellular medium in platelet granule secretion.
Journal of Cellular and Molecular Medicine, 2013
The use of the mammal target of rapamycin (mTOR) inhibitors has been consolidated as the therapy ... more 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.
Molecular and Cellular Biochemistry, 2008
The parotid glands are highly active secretory systems subjected to continuous stress, which in t... more The parotid glands are highly active secretory systems subjected to continuous stress, which in turn, can lead to several pathophysiological conditions. Damage of the parotid glands are caused by radical oxygen species (ROS) as by-products of oxygen metabolism. This study investigated the effect of hydrogen peroxide (H(2)O(2)) on Carbachol (CCh)-evoked secretory responses and caspase-3 activity in the isolated rat parotid gland to understand the role of oxidative stress on the function of the gland. Amylase secretion, cytosolic calcium concentration ([Ca(2+)](i)) and caspase-3 activity in parotid gland tissue were measured using fluorimetric methods. H(2)O(2) had little or no effect on amylase secretion compared to basal level. Combining H(2)O(2) with CCh resulted in an attenuation of the CCh-evoked amylase secretion compared to the effect of CCh alone. CCh can evoke a large increase in [Ca(2+)](i) comprising an initial peak followed by a plateau. In a Ca(2+)-free medium containing 1 mM EGTA, CCh evoked only the initial peak of [Ca(2+)](i). H(2)O(2) alone evoked a gradual and dose-dependent increase in [Ca(2+)](i). Combining H(2)O(2) with CCh resulted in a decrease in [Ca(2+)](i) compared to the effect of CCh alone. In a Ca(2+)-free medium, H(2)O(2) still evoked a small increase in [Ca(2+)](i), but this response was less compared to the results obtained with H(2)O(2) in normal [Ca(2+)](0). Combining H(2)O(2) with CCh resulted in only a small transient increase in [Ca(2+)](i). Following CCh stimulation, H(2)O(2) application resulted in a large increase in [Ca(2+)](i) in normal [Ca(2+)](0). This effect of H(2)O(2) was partially abolished in a nominally free Calcium medium containing EGTA. H(2)O(2) can stimulate caspase-3 activity in parotid gland tissue. Similar response was obtained with betulinic acid and thapsigargin (TPS) on caspase-3 activity compared to basal. The results have demonstrated that like CCh, H(2)O(2) can also mobilise Ca(2+) from intracellular stores and facilitate its influx into the cell from extracellular medium. This effect of H(2)O(2) may be due to its activity to induce apoptosis in the parotid gland, since H(2)O(2) can stimulate the activity of caspase-3, a marker of cellular apoptosis.
Cellular Signalling, 2012
Stromal interaction molecule 1 (STIM1) is a key element of the store-operated Ca 2+ entry mechani... more 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.
We have studied phospholipase D activation in [ 32 P]orthophosphoric acid-prelabeled rat pancreat... more We have studied phospholipase D activation in [ 32 P]orthophosphoric acid-prelabeled rat pancreatic acini by measuring the formation of 32 P-phosphatidylalcohols as stimulated in the presence of ethanol or butanol. A small but significant and time-dependent basal accumulation of [ 32 P]phosphatidylethanol and [ 32 P]phosphatidylbutanol was detected, which was further stimulated by phorbol myristate acetate, orthovanadate and pervanadate. However, the secretagogues cholecystokinin octapeptide and carbachol did not enhance basal accumulation of 32 P-phosphatidylalcohol, yet they decreased [ 32 P]phosphatidylcholine content and stimulated the generation of [ 32 P]phosphatidic acid. Our results stress the need to examine the transphosphatidylation reaction as well as agonist effects on the synthesis of phosphatidylcholine in order to assess unambiguously phospholipase D activity. ß
International Review of Cell and Molecular Biology, 2015
Regulators of G-protein-signaling (RGS) proteins are a category of intracellular proteins that ha... more Regulators of G-protein-signaling (RGS) proteins are a category of intracellular proteins that have an inhibitory effect on the intracellular signaling produced by G-protein-coupled receptors (GPCRs). RGS along with RGS-like proteins switch on through direct contact G-alpha subunits providing a variety of intracellular functions through intracellular signaling. RGS proteins have a common RGS domain that binds to G alpha. RGS proteins accelerate GTPase and thus enhance guanosine triphosphate hydrolysis through the alpha subunit of heterotrimeric G proteins. As a result, they inactivate the G protein and quickly turn off GPCR signaling thus terminating the resulting downstream signals. Activity and subcellular localization of RGS proteins can be changed through covalent molecular changes to the enzyme, differential gene splicing, and processing of the protein. Other roles of RGS proteins have shown them to not be solely committed to being inhibitors but behave more as modulators and integrators of signaling. RGS proteins modulate the duration and kinetics of slow calcium oscillations and rapid phototransduction and ion signaling events. In other cases, RGS proteins integrate G proteins with signaling pathways linked to such diverse cellular responses as cell growth and differentiation, cell motility, and intracellular trafficking. Human and animal studies have revealed that RGS proteins play a vital role in physiology and can be ideal targets for diseases such as those related to addiction where receptor signaling seems continuously switched on.
Biochimica et biophysica acta, Jan 21, 2015
STIM1 is a ubiquitous Ca(2+) sensor of the intracellular, agonist-sensitive, Ca(2+) stores that c... more STIM1 is a ubiquitous Ca(2+) sensor of the intracellular, agonist-sensitive, Ca(2+) stores that communicates the filling state of the Ca(2+) compartments to plasma membrane store-operated Ca(2+) (SOC) channels. STIM1 has been presented as a point of convergence between store-operated and voltage-operated Ca(2+) influx, both inducing activation of SOC channels while suppressing Cav1.2 channels. Here we report that Homer proteins play a relevant role in the communication between STIM1 and Cav1.2 channels. HEK-293 cells transiently expressing Cav1.2 channel subunits α1, β2 and α2δ-1 exhibited a significant Ca(2+) entry upon treatment with a high concentration of KCl. In Cav1.2-expressing cells, treatment with thapsigargin (TG), to induce passive discharge of the intracellular Ca(2+) stores, resulted in Ca(2+) influx that was significantly greater than in cells not expressing Cav1.2 channels, a difference that was abolished by nifedipine and diltiazem. Treatment with TG induces co-immun...
Biochemical and Biophysical Research Communications, 1997
We have used BCECF- or Fura-2-loaded rat pancreatic acinar cells to investigate the relationship ... more We have used BCECF- or Fura-2-loaded rat pancreatic acinar cells to investigate the relationship between Ca2+mobilization and intracellular pH (pHi). Ca2+-mobilizing agonists CCK-8 and ACh induced a transient acidification totally dependent on release of Ca2+from internal stores. Employment of different physiological tools including ionomycin and thapsigargin to increase the cytosolic Ca2+concentration and capacitative calcium influx also induced cellular acidification. Application
Cellular Signalling, 2002
In fura-2 loaded isolated mouse pancreatic acinar cells, xanthine oxidase (XOD)-catalyzed reactiv... more In fura-2 loaded isolated mouse pancreatic acinar cells, xanthine oxidase (XOD)-catalyzed reactive oxygen species (ROS) generation caused an increase in the cytosolic Ca2+ concentration ([Ca2+]i) by release of Ca2+ from intracellular stores. The ROS-induced Ca2+ signals showed large variability in shape and time-course and resembled in part Ca2+ signals in response to physiological secretagogues. ROS-induced Ca2+ mobilization started at the
Transient Receptor Potential Channels, 2011
Transient receptor potential (TRP) proteins are involved in a large number of non-selective catio... more Transient receptor potential (TRP) proteins are involved in a large number of non-selective cation channels that are permeable to both monovalent and divalent cations. Two general classes of receptor-mediated Ca(2+) entry has been proposed: one of then is conduced by receptor-operated Ca(2+) channels (ROC), the second is mediated by channels activated by the emptying of intracellular Ca(2+) stores (store-operated channels or SOC). TRP channels have been presented as subunits of both ROC and SOC, although the precise mechanism that regulates the participation of TRP proteins in these Ca(2+) entry mechanisms remains unclear. Recently, TRPC proteins have been shown to associate with Orai1 and STIM1 in a dynamic ternary complex regulated by the occupation of membrane receptors in several cell models, which might play an important role in the function of TRPC proteins. The present review summarizes the current knowledge concerning the association of TRP proteins with Orai and STIM proteins and how this affects the participation of TRP proteins in store-operated or receptor-operated Ca(2+) entry.
Molecules (Basel, Switzerland), 2010
A number of disorders, such as Alzheimer disease and diabetes mellitus, have in common the altera... more 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 Ca²+ 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 Ca²+ homeostasis and the role of antioxidants in the prevention and treatment of disorders associated to abnormal Ca²+ mobilization induced by ROS.
Toxicology letters, Jan 17, 2014
Ebselen is a seleno-organic compound that causes cell death in several cancer cell types. The mec... more Ebselen is a seleno-organic compound that causes cell death in several cancer cell types. The mechanisms underlying its deleterious effects have not been fully elucidated. In this study, the effects of ebselen (1 μM-40 μM) on AR42J tumor cells have been examined. Cell viability was studied using AlamarBlue(®) test. Cell cycle phase determination was carried out by flow cytometry. Changes in intracellular free Ca(2+) concentration were followed by fluorimetry analysis of fura-2-loaded cells. Distribution of mitochondria, mitochondrial Ca(2+) concentration and mitochondrial membrane potential were monitored by confocal microscopy of cells loaded with Mitotracker Green™ FM, rhod-2 or TMRM respectively. Caspase-3 activity was calculated following the luorogenic substrate ACDEVD-AMC signal with a spectrofluorimeter. Results show that cell viability decreased in the presence of ebselen. An increase in the number of cells in the S-phase of the cell cycle was observed. Ebselen induced a con...
Chronobiology International, 1989
The effects of three anti-ulcers drugs on the temporal distribution of food intake and of the two... more The effects of three anti-ulcers drugs on the temporal distribution of food intake and of the two parameters, meal size and meal frequency, were studied in ulcerated and non-ulcerated rats exposed to light-dark (LD 12:12) cycles. Experimental ulceration with indomethacin reduces the amplitude of meal frequency and brings the acrophase forward, compared with non-ulcerated animals. These effects were reversed by the oral administration of either ranitidine, sucralfate or pirenzepine along with the food. However, the administration of either pirenzepine or sucralfate alone to non-ulcerated rats is accompanied by significant (P less than 0.05) changes in the circadian patterns of meal size and meal frequency without the total daily food intake being affected in any way (pirenzepine treatment caused large intake of food during the light period while sucralfate treatment resulted in marked food intake during the dark period). The results indicate that circadian modification of meal patterns in the ulcerated rats are attributable to indomethacin-induced gastrointestinal mucosal injury and anti-ulcer medications.
Cellular Signalling, 2003
In the present study, we have employed confocal laser scanning microscopy to investigate the effe... more In the present study, we have employed confocal laser scanning microscopy to investigate the effect that stimulation of mouse pancreatic acinar cells with the secretagogue cholecystokinin (CCK) has on mitochondrial activity. We have monitored changes in cytosolic as well as mitochondrial Ca2+ concentrations, mitochondrial membrane potential and FAD autofluorescence by loading the cells with fluo-3, rhod-2 or JC-1, respectively.Our results
Trends in cardiovascular medicine, 2008
Two separate Ca2+ stores have been reported in human platelets: the dense tubular system (DTS) an... more Two separate Ca2+ stores have been reported in human platelets: the dense tubular system (DTS) and lysosome-like acidic organelles. Recent work has reported that Ca2+ release from the DTS is mediated by the generation of inositol 1,4,5-trisphosphate, whereas Ca2+ efflux from the acidic stores is mostly linked to nicotinic acid adenine dinucleotide phosphate. Platelet agonists release Ca2+ selectively from one or both stores, which provides additional insight into the complexity of Ca2+ signaling and the cellular functions activated. Here, we review the role of multiple Ca2+ mobilizing messengers and Ca2+ stores in the activation of specific functions in platelets in response to different physiologic agonists.
Mitochondrion, 2004
In the present study we have studied the changes in the intracellular reduction–oxidation state i... more In the present study we have studied the changes in the intracellular reduction–oxidation state in mouse pancreatic acinar cells following stimulation with cholecystokinin octapeptide (CCK-8) and its dependence on Ca2+ mobilization. In our investigations cytosolic Ca2+ concentration and reactive oxygen species (ROS) production were determined by loading of cells with fura-2 and CM-H2DCF-DA, respectively. Changes in these parameters were determined
Laboratory Animals, 1983
Summary A new biliary bidirectional cannula is described which allows the study of biliary secret... more Summary A new biliary bidirectional cannula is described which allows the study of biliary secretion in conscious dogs under conditions which approach physiological normality. Materials and methods Experiments were carried out on 9 adult dogs (body weight 20-25 kg). The animals were fasted for 48 h prior to surgery. Drinking water was given ad libitum. Since the first report of