SARAF overexpression impairs thrombin-induced Ca2+ homeostasis in neonatal platelets.

Neonatal platelets present a reduced response to the platelet agonist, thrombin (Thr), thus resulting in a deficient Thr-induced aggregation. These alterations are more pronounced in premature newborns. Here, our aim was to uncover the causes underneath the impaired Ca2+ homeostasis described in neonatal platelets. Both Ca2+ mobilization and Ca2+ influx in response to Thr are decreased in neonatal platelets compared to maternal and control woman platelets. In neonatal platelets, we observed impaired Ca2+ mobilization in response to the PAR-1 agonist (SFLLRN) or by blocking SERCA3 function with tert-butylhydroquinone. Regarding SOCE, the STIM1 regulatory protein, SARAF, was found overexpressed in neonatal platelets, promoting an increase in STIM1/SARAF interaction even under resting conditions. Additionally, higher interaction between SARAF and PDCD61/ALG2 was also observed, reducing SARAF ubiquitination and prolonging its half-life. These results were reproduced by overexpressing SARAF in MEG01 and DAMI cells. Finally, we also observed that pannexin 1 permeability is enhanced in response to Thr in control woman and maternal platelets, but not in neonatal platelets, hence, leading to the deregulation of the Ca2+ entry found in neonatal platelets. Summarizing, we show that in neonatal platelets both Ca2+ accumulation in the intracellular stores and Thr-evoked Ca2+ entry through either capacitative channels or non-selective channels are altered in neonatal platelets, contributing to deregulated Ca2+ homeostasis in neonatal platelets and leading to the altered aggregation observed in these subjects.

CAPN1 (Calpain1)-Dependent Cleavage of STIM1 (Stromal Interaction Molecule 1) Results in an Enhanced SOCE (Store-Operated Calcium Entry) in Human Neonatal Platelets.

BACKGROUND: Altered intracellular Ca2+ homeostasis in neonatal platelets has been previously reported. This study aims to examine the changes in the Ca2+ entry through the store-operated calcium entry (SOCE) mechanism in neonatal platelets. METHODS: Human platelets from either control women, mothers, and neonates were isolated and, following, were fixed after being treated as required. Platelet samples were analyzed by Western blotting, qRT-PCR, and MALDITOF/TOF. Ca2+ homeostasis was also determined. Culture cells were used as surrogated of platelets to overexpress the proteins of interest to reproduce the alterations observed in platelets. RESULTS: Altered TG (thapsigargin)-evoked SOCE, alternative molecular weight form of STIM1 (stromal interaction molecule 1; s-STIM1 [short STIM1 isoform (478 aa)], around 60 kDa) and overexpression of SARAF (SOCE-associated regulatory factor) were found in neonatal platelets as compared to maternal and control women platelets. s-STIM1 may result due to CAPN1 (calpain1)-dependent processing, as confirmed in platelets and MEG01 cells by using calpeptin and overexpressing CAPN1, respectively. In HEK293 (STIM1 and STIM2 [stromal interaction molecule 2] double knockout) cells transfected either with c-STIM1 (canonical STIM1 [685 aa]), s-STIM1 (478), STIM1B (540), and CAPN1 overexpression plasmids, we found s-STIM1 and c-STIM1, except in cells overexpressing s-STIM1 (478) that lacked CAPN1 target residues. These results and the in silico analysis, lead us to conclude that STIM1 is cleaved at Q496 by CAPN1. Ca2+ imaging analysis and coimmunoprecipitation assay using MEG01 and HEK293 cells overexpressing SARAF together with s-STIM1 (478) reported a reduced slow Ca2+-dependent inactivation, so reproducing the Ca2+-homeostasis pattern observed in neonatal platelets. CONCLUSIONS: CAPN1 may cleave STIM1 in neonatal platelets, hence, impairing SARAF coupling after SOCE activation. s-STIM1 may avoid slow Ca2+-dependent inactivation and, subsequently, results in an enhanced TG-evoked SOCE as observed in neonatal platelets.

New Insights into Adipokines as Potential Biomarkers for Type-2 Diabetes Mellitus.

A large number of studies have been focused on investigating serum biomarkers associated with risk or diagnosis of type-2 diabetes mellitus. In the last decade, promising studies have shown that circulating levels of adipokines could be used as a relevant biomarker for diabetes mellitus progression as well as therapeutic future targets. Here, we discuss the possible use of recently described adipokines, including apelin, omentin-1, resistin, FGF-21, neuregulin-4 and visfatin, as early biomarkers for diabetes. In addition, we also include recent findings of other well known adipokines such as leptin and adiponectin. In conclusion, further studies are needed to clarify the pathophysiological significance and clinical value of these biological factors as potential biomarkers in type-2 diabetes and related dysfunctions.

Stanniocalcin 2 Regulates Non-capacitative Ca2+ Entry and Aggregation in Mouse Platelets.

Stanniocalcin 2 (STC2) is a fish protein that controls body Ca2+ and phosphate metabolism. STC2 has also been described in mammals, and as platelet function highly depends on both extracellular and intracellular Ca2+, we have explored its expression and function in these cells. STC2-/- mice exhibit shorter tail bleeding time than WT mice. Platelets from STC2-deficient mice showed enhanced aggregation, as well as enhanced Ca2+ mobilization in response to the physiological agonist thrombin (Thr) and the diacylglycerol analog, OAG, a selective activator of the non-capacitative Ca2+ entry channels. Interestingly, platelets from STC2-/- mice exhibit attenuated interaction between STIM1 and Orai1 in response to Thr, thus suggesting that STC2 is required for Thr-evoked STIM1-Orai1 interaction and the subsequent store-operated Ca2+ entry (SOCE). We have further assessed possible changes in the expression of the most relevant channels involved in non-capacitative Ca2+ entry in platelets. Then, protein expression of Orai3, TRPC3 and TRPC6 were evaluated by Western blotting, and the results revealed that while the expression of Orai3 was enhanced in the STC2-deficient mice, others like TRPC3 and TRPC6 remains almost unaltered. Summarizing, our results provide for the first time evidence for a role of STC2 in platelet physiology through the regulation of agonist-induced Ca2+ entry, which might be mediated by the regulation of Orai3 channel expression.

Ethanol impairs CCK-8-evoked amylase secretion through Ca2+-mediated ROS generation in mouse pancreatic acinar cells.

In the present study, we have investigated the effect of ethanol on amylase release in response to cholecystokinin octapeptide (CCK-8). We have also studied the effect of ethanol on cytosolic free Ca(2+) concentration ([Ca(2+)](c)) and reactive oxygen species (ROS) production by loading of cells with fura-2 and 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H(2)DCFDA), respectively. Our results show that stimulation of pancreatic acinar cells with CCK-8 induced a dose-dependent amylase secretion, resulting in a maximum at 0.3nM of 19.39+/-2.73% of the total content of amylase. Treatment of pancreatic acini with ethanol did not induce any significant effect on amylase release at a wide range of concentrations (1-50mM). In contrast, incubation of cells with 50mM ethanol clearly reduced amylase release stimulated by CCK-8. The inhibitory effect of ethanol on CCK-8-induced amylase secretion was abolished by dithiothreitol, a sulfhydryl reducing agent. Ethanol induced an increase in [Ca(2+)](c) resulting in a level higher than the prestimulation level both in the presence and in the absence of extracellular Ca(2+). Additionally, ethanol led to an increase in fluorescence of CM-H(2)DCFDA, reflecting an increase in oxidation. A decrease in oxidation was observed in the absence of extracellular Ca(2+) and in the presence of ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid. Similarly, when the cells were challenged in the presence of the intracellular Ca(2+) chelator 1,2-Bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and in the absence of extracellular Ca(2+), the responses to ethanol were reduced, although not completely inhibited. Taken together, our results suggest that ethanol induces generation of ROS by a Ca(2+)-dependent mechanism and reduces CCK-8-evoked amylase secretion in exocrine pancreatic cells.

Changes in mitochondrial activity evoked by cholecystokinin in isolated mouse pancreatic acinar cells.

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 show that stimulation of cells with cholecystokinin led to release of Ca2+ from intracellular stores that then accumulated into mitochondria. In the presence of the hormone a depolarization of mitochondrial membrane potential was observed, which partially recovered; in addition a transient increase in FAD autofluorescence could be observed. Similarly, treatment of cells with thapsigargin induced increases in mitochondrial Ca2+ and FAD autofluorescence, and depolarized mitochondria. Pretreament of cells with thapsigargin blocked cholecystokinin-evoked changes. Similar results were obtained when the cells were incubated in the presence of rotenone, which blocks the mitochondrial electron transport chain. Our findings are consistent with changes in mitochondrial activity in response to stimulation of pancreatic acinar cells with cholecystokinin. Following stimulation, mitochondria take up Ca2+ that could in turn activate the mitochondrial machinery that may match the energy supply necessary for the cell function during secretion, suggesting that Ca2+ can act as a regulator of mitochondrial activity.

Role of mTOR1 and mTOR2 complexes in MEG-01 cell physiology.

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.

Generation of ROS in response to CCK-8 stimulation in mouse pancreatic acinar cells.

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 by following changes in fluorescence in the cuvette of a spectrofluorimeter. The results show that stimulation of cells with CCK-8 and/or the sarco-endoplasmic reticulum Ca2+ pump inhibitor, thapsigargin (Tps), both induced changes in cytosolic free Ca2+ concentration and led to an increase in fluorescence of CM-H2DCF-DA, reflecting an increase in oxidation. In the presence of Tps, addition of CCK-8 did not significantly increase fluorescence compared to that evoked by the SERCA inhibitor. Similar results were obtained in the absence of extracellular Ca2+ and in the presence of EGTA. When the cells were challenged in the presence of the intracellular Ca2+ chelator BAPTA and in the absence of extracellular Ca2+ the responses to both CCK-8 and Tps were reduced although not completely inhibited. The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and the inhibitor of the electron transport chain, antimycin, evoked a marked increase in CM-H2DCF-DA fluorescence and completely inhibited CCK-8 and Tps-evoked responses, indicating that ROS are generated in the mitochondria. In summary, stimulation of mouse pancreatic acinar cells with CCK-8 leads to generation of ROS, and this effect may be derived from Ca2+ mobilization from intracellular stores and involves mitochondrial metabolism.

Dietary virgin olive oil enhances secretagogue-evoked calcium signaling in rat pancreatic acinar cells.

OBJECTIVE: We evaluated the long-term effects of a fat-enriched diet (virgin olive oil) on calcium mobilization and amylase secretion induced by cholecystokinin-octapeptide (CCK-8) in rat pancreatic acinar cells. Olive oil is a major component of the Mediterranean diet, and its role in human health is actively being debated. METHODS: Weaning male Wistar rats (21 d old) were assigned to one of two experimental groups and fed for 8 wk with a commercial chow (control group) or an experimental diet (olive group) containing 100 g/kg of virgin olive oil as dietary fat. Intracellular free calcium [Ca(2+)](i) levels were determined by loading the pancreatic cells with the fluorescent ratio-metric calcium indicator Fura-2 on an inverted fluorescent microscope. For measurement of amylase secretion, cells were incubated with the appropriate secretagogue for 30 min, and amylase activities in the supernatant were determined by the Phadebas blue starch method. Analysis of variance was used to test differences between groups. RESULTS: Compared with the control group, the CCK-8-induced increase in [Ca(2+)](i) occurred in cells from rats in the olive group (P < 0.05). This stimulatory effect of dietary virgin olive oil was observed in calcium oscillations and large [Ca(2+)](i) transients induced by low (20 pM/L) and high (10 nM/L) concentrations of CCK-8, respectively. In addition to the effects of dietary virgin olive oil on calcium mobilization, it increased (P < 0.05) amylase secretion in response to CCK-8. Olive oil treatment did not significantly alter resting [Ca(2+)](i) or amylase release values compared with the control group. Similar results were obtained when pancreatic acinar cells were stimulated with a high concentration of acetylcholine (10 microM/L). CONCLUSION: The present results demonstrate that a diet supplemented with virgin olive oil can modify pancreatic cell function as assessed by [Ca(2+)](i) mobilization and amylase release evoked by secretagogues in rat pancreatic acinar cells. A role for fatty acids in calcium signaling is suggested.

H2O2 mobilizes Ca2+ from agonist- and thapsigargin-sensitive and insensitive intracellular stores and stimulates glutamate secretion in rat hippocampal astrocytes.

The effect of hydrogen peroxide (H2O2) on cytosolic free calcium concentration ([Ca2+]c) as well as its effect on glutamate secretion in rat hippocampal astrocytes have been the aim of the present research. Our results show that 100 microM H2O2 induces an increase in [Ca2+]c, that remains at an elevated level while the oxidant is present in the perfusion medium, due to its release from intracellular stores as it was observed in the absence of extracellular Ca2+, followed by a significant increase in glutamate secretion. Ca2+-mobilization in response to the oxidant could only be reduced by thapsigargin plus FCCP, indicating that the Ca2+-mobilizable pool by H2O2 includes both endoplasmic reticulum and mitochondria. We conclude that ROS in hippocampal astrocytes might contribute to an elevation of resting [Ca2+]c which, in turn, could lead to a maintained secretion of the excitatory neurotransmitter glutamate, which has been considered a situation potentially leading to neurotoxicity in the hippocampus.

Dose-dependent effect of hydrogen peroxide on calcium mobilization in mouse pancreatic acinar cells.

We have employed confocal laser scanning microscopy to investigate how intracellular free calcium concentration ([Ca2+]i) is influenced by hydrogen peroxide (H2O2) in collagenase-dispersed mouse pancreatic acinar cells. In the absence of extracellular calcium, treatment of cells with increasing concentrations of H2O2 resulted in an increase in [Ca2+]i, indicating the release of calcium from intracellular stores. Micromolar concentrations of H2O2 induced an oscillatory pattern, whereas 1 mmol H2O2/L caused a slow and sustained increase in [Ca2+]i. H2O2 abolished the typical calcium release stimulated by thapsigargin or by the physiological agonist cholecystokinin octapeptide (CCK-8). Depletion of either agonist-sensitive or mitochondrial calcium pools was unable to prevent calcium release induced by 1 mmol H2O2/L, but depletion of both stores abolished it. Additionally, lower H2O2 concentrations were able to release calcium only after depletion of mitochondrial calcium stores. Treatment with either the phospholipase C inhibitor U-73122 or the inhibitor of the inositol 1,4,5-trisphosphate (IP3) receptor xestospongin C did not modify calcium release from the agonist-sensitive pool induced by 100 micromol H2O2/L, suggesting the involvement of a mechanism independent of IP3 generation. In addition, H2O2 reduced amylase release stimulated by CCK-8. Finally, either the H2O2-induced calcium mobilization or the inhibitory effect of H2O2 on CCK-8-induced amylase secretion was abolished by dithiothreitol, a sulphydryl reducing agent. We conclude that H2O2 at micromolar concentrations induces calcium release from agonist-sensitive stores, and at millimolar concentrations H2O2 can also evoke calcium release from the mitochondria. The action of H2O2 is mediated by oxidation of sulphydryl groups of calcium ATPases independently of IP3 generation.

Involvement of ryanodine-operated channels in tert-butylhydroperoxide-evoked Ca2+ mobilisation in pancreatic acinar cells.

Reactive oxygen species and related oxidative damage have been implicated in the initiation of acute pancreatitis, a disease characterised in its earliest stages by disruption of intracellular Ca2+ homeostasis. The present study was carried out in order to establish the effect of the organic pro-oxidant, tert-butylhydroperoxide (tBHP), on the mobilisation of intracellular Ca2+ stores in isolated rat pancreatic acinar cells and the mechanisms underlying this effect. Cytosolic free Ca2+ concentrations ([Ca2+]c) were monitored using a digital microspectrofluorimetric system in fura-2 loaded cells. In the presence of normal extracellular Ca2+ concentrations ([Ca2+]o), perfusion of pancreatic acinar cells with 1 mmol l-1 tBHP caused a slow sustained increase in [Ca2+]c. This increase was also observed in a nominally Ca2+-free medium, indicating a release of Ca2+ from intracellular stores. Pretreatment of cells with tBHP abolished the typical Ca2+ response of both the physiological agonist CCK-8 (1 nmol l-1) and thapsigargin (TPS, 1 micromol l-1), an inhibitor of the SERCA pump, in the absence of extracellular Ca2+. Similar results were observed with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 0.5 micromol l-1), a mitochondrial uncoupler. In addition, depletion of either agonist-sensitive Ca2+ pools by CCK-8 or TPS or mitochondrial Ca2+ pools by FCCP were unable to prevent the tBHP-induced Ca2+ release. By contrast, simultaneous administration of TPS and FCCP clearly abolished the tBHP-induced Ca2+ release. These results show that tBHP releases Ca2+ from agonist-sensitive intracellular stores and from mitochondria. On the other hand, simultaneous application of FCCP and of 2-aminoethoxydiphenylborane (2-APB), a blocker of IP3-mediated Ca2+ release, was unable to suppress the increase in [Ca2+]c induced by tBHP, while the application of 50 micromol l-1 of ryanodine (which is able to block the ryanodine channels) inhibits tBHP-evoked Ca2+ mobilisation. These findings indicate that tBHP releases Ca2+ from non-mitochondrial Ca2+ pools through ryanodine channels.

H2O2-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells.

This study employed confocal laser scanning microscopy to monitor the effect of H2O2 on cytosolic as well as mitochondrial calcium (Ca2+) concentrations, mitochondrial inner membrane potential (psi m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H2O2, in the absence of extracellular Ca2+ ([Ca2+],) led to an increase either in cytosolic and in mitochondrial Ca2+ concentration. Additionally, H2O2 induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H2O2 on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H2O2. However, the H2O2-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca2+ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H2O2-evoked changes in mitochondrial Ca2+ concentration but not those in membrane potential and FAD state. The present results have indicated that H2O2 can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H2O2. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas.

Effect of H2O2 on CCK-8-evoked changes in mitochondrial activity in isolated mouse pancreatic acinar cells.

BACKGROUND INFORMATION: This paper studies the effect of H(2)O(2) on mitochondrial responses evoked by CCK-8 (cholecystokinin 8) in mouse pancreatic acinar cells. Cytosolic ([Ca(2+)](c)) and mitochondrial ([Ca(2+)](m)) free-calcium concentrations, mitochondrial inner membrane potential (psi(m)) and FAD autofluorescence were monitored using confocal laser scanning microscopy. RESULTS: CCK-8 induced an increase in [Ca(2+)](m) that slowly declined towards the prestimulation level. Depolarization of psi(m) that partially recovered, as well as increases in FAD autofluorescence, could also be observed in response to the hormone. Pretreatment of cells with 1 mM H(2)O(2) alone resulted in marked changes in mitochondrial parameters and, moreover, H(2)O(2) inhibited the CCK-8-evoked changes in [Ca(2+)](m), psi(m) and FAD autofluorescence. The results of the present study have demonstrated that CCK-8 can evoke marked changes in pancreatic acinar cell mitochondrial activity and that CCK-8-evoked responses are blocked by H(2)O(2). Additionally, H(2)O(2) releases Ca(2+) from intracellular stores and inhibits pancreatic acinar cell responses to CCK-8. CONCLUSION: The effects observed reflect an impairment of mitochondrial activity in the presence of H(2)O(2) that could represent some of its mechanisms of action to induce cellular damage leading to cell dysfunction and generation of pathologies.