Store-operated Calcium Entry In Cells Research Articles

Development of Chemical Tools Based on GSK-7975A to Study Store-Operated Calcium Entry in Cells

Many physiological functions, such as cell differentiation, proliferation, muscle contraction, neurotransmission and fertilisation, are regulated by changes of Ca2+ levels. The major Ca2+ store in cells is the endoplasmic reticulum (ER). Certain cellular processes induce ER store depletion, e.g. by activating IP3 receptors, that in turn induces a store refilling process known as store-operated calcium entry (SOCE). This refilling process entails protein-protein interactions between Ca2+ sensing stromal interaction molecules (STIM) in the ER membrane and Orai proteins in the plasma membrane. Fully assembled STIM/Orai complexes then form highly selective Ca2+ channels called Ca2+ release activated Ca2+ Channels (CRAC) through which Ca2+ ions flow into the cytosol and subsequently are pumped into the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Abnormal SOCE has been associated with numerous human diseases and cancers, and therefore key players STIM and Orai have attracted significant therapeutic interest. Several potent experimental and clinical candidate compounds have been developed and have helped to study SOCE in various cell types. We have synthesized multiple novel small-molecule probes based on the known SOCE inhibitor GSK-7975A. Here we present GSK-7975A derivatives, which feature photo-caging, photo-crosslinking, biotin and clickable moieties, and also contain deuterium labels. Evaluation of these GSK-7975A probes using a fluorometric imaging plate reader (FLIPR)-Tetra-based Ca2+ imaging assay showed that most synthetic modifications did not have a detrimental impact on the SOCE inhibitory activity. The photo-caged GSK-7975A was also used in patch-clamp electrophysiology experiments. In summary, we have developed a number of active, GSK-7975A-based molecular probes that have interesting properties and therefore are useful experimental tools to study SOCE in various cells and settings.

176-OR: STIM1 Deletion Leads to Loss of Beta-Cell Identity in High-Fat Diet-Fed Female Mice

STIM1 is an endoplasmic reticulum (ER) calcium (Ca2+) sensor that replenishes ER Ca2+ stores in a process known as store operated calcium entry (SOCE). Previously, we have shown that SOCE is impaired in the diabetic pancreatic β cell; however, the consequences of reduced β cell SOCE have not been well studied. To this end, mice with pancreatic β cell specific deletion of STIM1 (STIM1Δβ) were generated by crossing STIM1flox/flox with Ins-Cre mice and fed a high fat diet (HFD) beginning at 8 wk of age. Male STIM1Δβ mice did not show changes in glucose tolerance or body composition. In contrast, female HFD-fed STIM1Δβ mice were heavier (25.1 vs. 28.3 g, p<0.05), had increased fat mass (4.9 vs. 8.2g, P<0.01), and exhibited significant glucose intolerance (AUC IPGTT 22461 vs. 29245 mg/dL*min; p<0.0001) with impaired insulin secretion by in-vivo GSIS (p<0.05) without differences insulin tolerance, energy expenditure or food intake. Bulk RNA-sequencing of wild-type and STIM1Δβ female islets revealed reduced expression of MafA, UCN3, and Ins1 and Ins2 in STIM1Δβ islets, indicating potential loss of β cell identity. Pathway analysis showed modulation of pathways related to 17-beta estradiol (E2) signaling, with downregulation of a G-protein coupled estrogen receptor 1 (GPER), while differentially expressed genes were enriched for functions related to apoptosis, lipid metabolism, and epithelial cell differentiation. Consistently, STIM1Δβ female mice had lower β cell mass (2.984 mg vs. 1.349 mg, p<0.05) and higher alpha cell mass (0.1822 mg vs. 0.4463 mg, p<0.05). Proteomics analysis performed in STIM1-deficient INS-1 cells revealed significantly increased glucagon expression. Further, lower mobilization of intracellular cAMP levels was noted in response to treatment with E2 or G1, an agonist of GPER. These findings suggest a role for STIM1 in the maintenance of pancreatic β cell identity and indicate a potential interaction between SOCE and E2 signaling in female mice. Disclosure P. Sohn: None. P. Krishnan: None. C. Lee: None. T. Kono: None. C. Evans-molina: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; Dompe, Other Relationship; Self; Bristol-Myers Squibb Company, Nimbus Pharmaceuticals, Pfizer Inc. Funding National Institutes of Health (5T32DK064466-17, 5F30DK123996); U.S. Department of Veterans Affairs (2-I01BX001733); National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK093954-06A1); Indiana University School of Medicine; Gates Millennium Scholars Program

1248-P: Histone Deacetylase Inhibition Improves Store-Operated Calcium Entry and Glucose-Stimulated Insulin Secretion in Cytokine-Stressed Pancreatic ß-Cells

Store-operated calcium entry (SOCE) functions to refill endoplasmic reticulum (ER) Ca2+ stores in response to ER Ca2+ depletion through gating of plasmalemmal Orai channels by the ER Ca2+ sensor STIM1. Dysfunctional β cell SOCE has been observed in response to cytokine-mediated stress, whereas select histone deacetylase (HDAC) inhibitors are able to protect against cytokine-mediated β cell death and have been shown to improve glucose homeostasis in diabetic mice. Here, we tested the relationship between HDAC inhibition and SOCE in the pancreatic β cell. To this end, we investigated the effects of the HDAC inhibitor sodium butyrate (NaB) on Ca2+ signaling, insulin secretion, and gene expression in cytokine-treated mouse islets, human islets, and STIM1 knockout (KO) 832/13 INS-1 β cells. In mouse islets treated with IL-1β, IFN-γ and TNF-⍺, NaB increased glucose-induced phase Ca2+ responses and restored Ca2+ oscillations. In IL-1β-treated INS-1 β cells, NaB rescued SOCE and ER Ca2+ store depletion, and increased glucose-stimulated insulin secretion (GSIS), while additionally improving cell viability and reducing apoptosis. Chronic (24hrs) but not acute (8hrs) NaB treatment prevented IL-1β-mediated reduction in the SOCE response, suggesting changes in gene expression may underlie these effects. To test this, RT-qPCR for SOCE molecular components was performed and revealed increased STIM1 expression in cytokine-treated mouse islets and INS-1 cells, while no change was observed in Orai1/2 levels. Consistent with these results, NaB was unable to restore SOCE in IL-1β-treated STIM1 KO INS-1 cells. Lastly, NaB treatment in human islets from donors with type 2 diabetes significantly increased levels of GSIS. Taken together, these data suggest that HDAC inhibition restores insulin secretion under pro-inflammatory conditions through improved SOCE and STIM1 upregulation. Disclosure C. Lee: None. T. Kono: None. S. A. Weaver: None. P. Sohn: None. C. Evans-molina: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; Dompe, Other Relationship; Self; Bristol-Myers Squibb Company, Nimbus Pharmaceuticals, Pfizer Inc.

2074-P: Histone Deacetylase Inhibitors Improve Store-Operated Calcium Entry and Glucose-Stimulated Insulin Secretion in Cytokine-Stressed Pancreatic ß-Cells

Store-operated calcium entry (SOCE) is a mechanism that functions to refill endoplasmic reticulum (ER) Ca2+ stores in response to ER Ca2+ depletion, through gating of plasmallemal Ora channels by the ER Ca2+ sensor STIM1. Dysfunctional β cell SOCE has been observed in response to cytokine-mediated stress and in models of diabetes. To identify compounds capable of modulating SOCE, a screening was performed in INS-1 cells treated with interleukin-1β (IL-1β). Two HDAC inhibitors (HDACi), sodium butyrate (NaB) and MS275, rescued SOCE in a dose-dependent manner in IL-1β-treated INS-1 cells. While dysregulation of HDACs has been linked with metabolic disease, the relationship between HDACs and SOCE has not been tested in the pancreatic β cell. To this end, we explored the effect of HDACi on Ca2+ signaling, insulin secretion, and gene expression in cytokine-treated islets and β cells. In mouse islets treated with IL-1 β and interferon-𝛾;, NaB and MS275 increased first phase Ca2+ responses in response to glucose, while NaB restored glucose-induced Ca2+ oscillations. Consistent with this, NaB, but not MS275, restored GSIS in IL-1β-treated INS-1 cells and in human islets from donors with type 2 diabetes. Chronic (24 hrs) but not acute (8 hrs) HDACi treatment restored cytokine-mediated reductions in SOCE, raising the possibility that changes in gene expression may underlie these effects. To test this, qRT-PCR for SOCE molecular components was performed. Both NaB and MS275 restored STIM1 expression but had no effect on Orai1, Orai2 and Orai3 levels. Consistent with this, HDACi were unable to restore SOCE in IL-1β-treated STIM1 knockout INS-1 cells. Taken together, these data suggest that HDAC inhibition restores insulin secretion under pro-inflammatory conditions through improved SOCE and upregulated STIM1 expression. Thus, our findings identify a novel pathway through which HDAC inhibition exerts protective effects in the diabetic β cell. Disclosure C. Lee: None. T. Kono: None. S.A. Weaver: None. P. Sohn: None. C. Evans-Molina: Consultant; Self; Bristol-Myers Squibb. Funding National Institute of Diabetes and Digestive and Kidney Diseases (2R01DK093954)

Interplay between ER Ca2+ Binding Proteins, STIM1 and STIM2, Is Required for Store-Operated Ca2+ Entry.

Store-operated calcium entry (SOCE), a fundamentally important homeostatic and Ca2+ signaling pathway in many types of cells, is activated by the direct interaction of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum (ER) Ca2+-binding protein, with Ca2+-selective Orai1 channels localized in the plasma membrane. While much is known about the regulation of SOCE by STIM1, the role of stromal interaction molecule 2 (STIM2) in SOCE remains incompletely understood. Here, using clustered regularly interspaced short palindromic repeats -CRISPR associated protein 9 (CRISPR-Cas9) genomic editing and molecular imaging, we investigated the function of STIM2 in NIH 3T3 fibroblast and αT3 cell SOCE. We found that deletion of Stim2 expression reduced SOCE by more than 90% in NIH 3T3 cells. STIM1 expression levels were unaffected in the Stim2 null cells. However, quantitative confocal fluorescence imaging demonstrated that in the absence of Stim2 expression, STIM1 did not translocate or form punctae in plasma membrane-associated ER membrane (PAM) junctions following ER Ca2+ store depletion. Fluorescence resonance energy transfer (FRET) imaging of intact, living cells revealed that the formation of STIM1 and Orai1 complexes in PAM nanodomains was significantly reduced in the Stim2 knockout cells. Our findings indicate that STIM2 plays an essential role in regulating SOCE in NIH 3T3 and αT3 cells and suggests that dynamic interplay between STIM1 and STIM2 induced by ER Ca2+ store discharge is necessary for STIM1 translocation, its interaction with Orai1, and activation of SOCE.

Reduced Membrane Cholesterol Following Chronic Hypoxia Limits Orai1 ‐Mediated Pulmonary Endothelial Ca2+ Entry

Diminished Ca2+‐dependent production of endothelium‐derived vasodilators like nitric oxide is observed in chronic hypoxia (CH)‐induced pulmonary hypertension. Our previous studies show that endothelial store‐operated calcium entry (SOCE) as well as membrane cholesterol (Chol) are reduced in pulmonary arteries from rats exposed to CH (4 wk, 380 Torr) compared to their normoxic controls. Interestingly, Chol supplementation restores both membrane Chol and endothelial SOCE in cells from CH rats. Substitution of membrane Chol with epicholesterol (EpiChol, epimeric form of Chol) in freshly isolated pulmonary artery endothelial cell (PAEC) sheets mimics the effect of CH to blunt endothelial SOCE. These data suggest that reduced membrane Chol following CH limits the activity of ion channels required for endothelial SOCE. However, the ion channels involved in Chol‐sensitive SOCE have not been identified. We hypothesized that membrane Chol facilitates Orai1‐mediated endothelial Ca2+ entry, and that reduced Ca2+ entry following CH is due to loss of membrane Chol. To test this hypothesis, we pretreated PAEC sheets isolated from control and CH rats with Chol (2 mM) or EpiChol (2 mM), and administered the Orai1 inhibitor AnCoA4 (20 μM), or vehicle. Ca2+ influx in response store depletion with cyclopiazonic acid (CPA, 10 μM) was assessed by Mn2+‐quenching of fura‐2 fluorescence. Whereas Chol supplementation restored diminished SOCE in PAEC sheets from CH rats, both EpiChol and AnCoA4 significantly reduced endothelial SOCE in normoxic controls, but not in cells from CH rats. However, the Orai1 inhibitor had no further effect in cells pretreated with EpiChol. Additional experiments examined SOCE following Orai1 knockdown using siRNA in cultured rat pulmonary microvascular endothelial cells (PMVEC). We found that both EpiChol and Orai1 siRNA significantly inhibited SOCE compared to vehicle or non‐targeting siRNA, respectively. EpiChol treatment had no additional effect in cells following knockdown of Orai1. Furthermore, using a proximity ligation assay, we observed that EpiChol treatment significantly reduced the STIM1‐Orai1 interaction required for SOCE when compared to either vehicle or Chol treated groups. We conclude that membrane Chol is required for STIM1‐Orai1 interaction necessary to elicit endothelial SOCE. Furthermore, reduced Ca2+ entry after CH is due to loss of membrane Chol thereby limiting Orai1 activity.Support or Funding InformationNIH R01 HL132883, NIH R01 HL111084, AHA 16GRNT27700010, AHA 15GRNT21080001,

CMT-linked loss-of-function mutations in GDAP1 impair store-operated Ca2+ entry-stimulated respiration

GDAP1 is an outer mitochondrial membrane protein involved in Charcot-Marie-Tooth (CMT) disease. Lack of GDAP1 gives rise to altered mitochondrial networks and endoplasmic reticulum (ER)-mitochondrial interactions resulting in a decreased ER-Ca2+ levels along with a defect on store-operated calcium entry (SOCE) related to a misallocation of mitochondria to subplasmalemmal sites. The defect on SOCE is mimicked by MCU silencing or mitochondrial depolarization, which prevent mitochondrial calcium uptake. Ca2+ release from de ER and Ca2+ inflow through SOCE in neuroblastoma cells result in a Ca2+-dependent upregulation of respiration which is blunted in GDAP1 silenced cells. Reduced SOCE in cells with CMT recessive missense mutations in the α-loop of GDAP1, but not dominant mutations, was associated with smaller SOCE-stimulated respiration. These cases of GDAP1 deficiency also resulted in a decreased ER-Ca2+ levels which may have pathological implications. The results suggest that CMT neurons may be under energetic constraints upon stimulation by Ca2+ mobilization agonists and point to a potential role of perturbed mitochondria-ER interaction related to energy metabolism in forms of CMT caused by some of the recessive or null mutations of GDAP1.

Microscopic analysis of Orai-mediated store-operated calcium entry in cells with experimentally altered levels of amyloid precursor protein

Familial Alzheimer's disease (FAD)-causing mutations in presenilins were shown to alter intracellular calcium dynamics, including store-operated calcium entry (SOCE). However, the involvement of FAD-linked amyloid precursor protein (APP) in SOCE remains controversial. Here, we used gain-of-function and loss-of-function approaches to shed light on this issue. We found that Jurkat cells, which exhibit prominent SOCE mediated by Orai channels, maintain low APP levels. The ectopic expression of APP, either with wildtype sequence or FAD-causing Swedish mutation, had no effect on SOCE induced by calcium store depletion with cyclopiazonic acid (CPA). The overproduction of C99 fragments, mimicking amyloidogenic processing of APP, also had no effect. Moreover, there was no alteration in the CPA-evoked SOCE upon APP knockdown in HeLa cells, which natively express 100-fold more APP than Jurkat cells. Consistently, we found no evidence for APP-dependent changes in the mRNA or protein levels of main SOCE components. Altogether, these results suggest that APP does not modulate Orai-dependent SOCE following quantitative calcium store depletion.

A homozygous STIM1 mutation impairs store-operated calcium entry and natural killer cell effector function without clinical immunodeficiency

A homozygous STIM1 mutation impairs store-operated calcium entry and natural killer cell effector function without clinical immunodeficiency

The Sigma1 Receptor Competes with STIM1 to Bind Orai1 to Regulate Store Operated Calcium Entry (SOCE)

The Sigma1 receptor is an ER chaperone protein targeted to mitochondrial associated ER membrane regions (MAMs). An increase in its expression has been observed in a variety of cancer cells and it exerts an anti-apoptotic action, promoting cell proliferation and inhibiting the apoptotic response to tumor suppressor genes such as p53 and Bax. Although its mechanism of action remains unclear, there is evidence to suggest that Sigma1R regulates inter-organelle Ca2+ signaling thereby controlling cellular bioenergetics, and is also a regulator of plasma membrane ion channels. We have investigated its role in the regulation of store operated Ca2+ entry (SOCE) triggered by ER Ca2+ store depletion. Increased expression of Sigma1R in HEK293 cells profoundly inhibited SOCE, whereas knockdown of endogenous Sigma1R in CHO cells increased SOCE. An agonist at Sigma1R, SKF10047, further inhibited SOCE whereas the antagonists, BD1047, relieved this inhibition.Co-immunoprecipitation experiments showed a novel interaction between Sigma1R and STIM1 which is ligand dependent, SKF10047 promoted the interaction and BD1047 reduced it. Total internal reflection fluorescence (TIRF) microscopy showed the recruitment of Sigma1R to ER-plasma membrane junctions was STIM1 dependent, whereas elevated Sigma1R expression slowed STIM1 recruitment. Plasma membrane biotinylation experiments also showed that STIM1 promoted Sigma1R recruitment to a surface complex while Sigma1R reduced the levels of STIM1 in the complex. Increased expression of STIM1 but not Orai1 partially rescued the inhibition of SOCE in cells over-expressing Sigma1R. Imaging of purified plasma membrane Orai1 channels complexes by atomic force microscopy (AFM) showed hexameric Orai1 decorated by both Sigma1R and STIM1. Overall our results suggest that Sigma1R inhibits SOCE in two ways; first, by binding STIM1 and reducing its recruitment to surface Orai1 channels and second, by directly modulating the STIM1-Orai1 complex.

The boron-oxygen core of borinate esters is responsible for the store-operated calcium entry potentiation ability

BackgroundStore-Operated Calcium Entry (SOCE) is the major Ca2+ ion entry pathway in lymphocytes and is responsible of a severe combined immunodeficiency (SCID) when deficient. It has recently been observed or highlighted in other cell types such as myoblasts and neurons, suggesting a wider physiological role of this pathway. Whereas Orai1 protein is considered to be the channel allowing the SOCE in T cells, it is hypothesized that other proteins like TRPC could associate with Orai1 to form SOCE with different pharmacology and kinetics in other cell types. Unraveling SOCE cell functions requires specific effectors to be identified, just as dihydropyridines were crucial for the study of Ca2+ voltage-gated channels, or spider/snake toxins for other ion channel classes. To identify novel SOCE effectors, we analyzed the effects of 2-aminoethyl diphenylborinate (2-APB) and its analogues. 2-APB is a molecule known to both potentiate and inhibit T cell SOCE, but it is also an effector of TRP channels and endoplasmic reticulum Ca2+-ATPase.ResultsA structure-function analysis allowed to discover that the boron-oxygen core present in 2-APB and in the borinate ester analogues is absolutely required for the dual effects on SOCE. Indeed, a 2-APB analogue where the boron-oxygen core is replaced by a carbon-phosphorus core is devoid of potentiating capacity (while retaining inhibition capacity), highlighting the key role of the boron-oxygen core present in borinate esters for the potentiation function. However, dimesityl borinate ester, a 2-APB analogue with a terminal B-OH group showed an efficient inhibitory ability, without any potentiating capacity. The removal or addition of phenyl groups respectively decrease or increase the efficiency of the borinate esters to potentiate and inhibit the SOCE. mRNA expression revealed that Jurkat T cells mainly expressed Orai1, and were the more sensitive to 2-APB modulation of SOCE.ConclusionsThis study allows the discovery of new boron-oxygen core containing compounds with the same ability as 2-APB to both potentiate and inhibit the SOCE of different leukocyte cell lines. These compounds could represent new tools to characterize the different types of SOCE and the first step in the development of new immunomodulators.

Phosphorylation of STIM1 at ERK1/2 target sites modulates store-operated calcium entry

Store-operated calcium entry (SOCE) is an important Ca2+ entry pathway that regulates many cell functions. Upon store depletion, STIM1, a transmembrane protein located in the endoplasmic reticulum (ER), aggregates and relocates close to the plasma membrane (PM) where it activates store-operated calcium channels (SOCs). Although STIM1 was early defined as a phosphoprotein, the contribution of the phosphorylation has been elusive. In the present work, STIM1 was found to be a target of extracellular-signal-regulated kinases 1 and 2 (ERK1/2) in vitro, and we have defined the ERK1/2-phosphorylated sites on the STIM1 sequence. Using HEK293 cells stably transfected for the expression of tagged STIM1, we found that alanine substitution mutants of ERK1/2 target sites reduced SOCE significantly, suggesting that phosphorylation of these residues are required to fully accomplish SOCE. Indeed, the ERK1/2 inhibitors PD184352 and PD0325901 decreased SOCE in transfected cells. Conversely, 12-O-tetradecanoylphorbol-13-acetate, which activates ERK1/2, enhanced SOCE in cells expressing wild-type tagged STIM1, but did not potentiate Ca2+ influx in cells expressing serine to alanine mutations in ERK1/2 target sites of STIM1. Alanine substitution mutations decreased Ca2+ influx without disturbing the aggregation of STIM1 upon store depletion and without affecting the relocalization in ER-PM punctae. However, our results suggest that STIM1 phosphorylation at ERK1/2 target sites can modulate SOCE by altering STIM1 binding to SOCs, because a significant decrease in FRET efficiency was observed between alanine substitution mutants of STIM1-GFP and ORAI1-CFP.

Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation

Store-operated calcium entry (SOCE) is the main Ca(2+) influx pathway involved in controlling proliferation of the human hepatoma cell lines Huh-7 and HepG2. However, the molecular nature of the calcium channels involved in this process remains unknown. Huh-7 and HepG2 cells express transient receptor potential canonical 1 (TRPC1) and TRPC6, as well as STIM1 and Orai1, and these 4 channels are the most likely candidates to account for the SOCE in these cells. We generated stable TRPC6-overexpressing or TRPC6-knockdown Huh-7 clones, in which we investigated correlations between the presence of the protein, the rate of cell proliferation, and SOCE amplitude. TRPC6-overexpressing Huh-7 cells proliferated 80% faster than did untransfected cells and their SOCE amplitude was 160% higher. By contrast, proliferation rate was 50% lower and SOCE amplitude 85% lower in TRPC6-knockdown clones than in untransfected cells. OAG (olyl acetyl glycerol)-induced calcium entry was similar in all cells, and small interfering RNA (siRNA) against TRPC1 had no effect on SOCE amplitude, highlighting the relationship among SOCE, TRPC6 and cell proliferation in Huh-7 cells. SOCE amplitude was reduced by STIM1 and Orai1 knockdowns, suggesting possible cooperation between these proteins and TRPC6 in these cells. Endothelial growth factor and hepatocyte growth factor increased TRPC6 expression and SOCE amplitude in Huh-7 cells, and cyclin D1 expression was decreased by STIM1, Orai1, and TRPC6 knockdowns. TRPC6 was very weakly expressed in isolated hepatocytes from healthy patients and expressed more strongly in tumoral samples from the liver of a cancer patient, strongly supporting a role for these calcium channels in liver oncogenesis.

Hydrogen peroxide attenuates store-operated calcium entry and enhances calcium extrusion in thyroid FRTL-5 cells

Redox modulation participates in the regulation of intracellular free calcium concentration ([Ca2+]i) in several cell types. In thyroid cells, including FRTL-5 cells, changes in [Ca2+]i regulate several important functions, including the production of H2O2 (hydrogen peroxide). As H2O2 is of crucial importance for the production of thyroid hormones, we investigated the effects of H2O2 on [Ca2+]i in thyroid FRTL-5 cells. H2O2 itself did not modulate basal [Ca2+]i. However, H2O2 attenuated store-operated calcium entry evoked by thapsigargin, both in a sodium-containing buffer and in a sodium-free buffer. The effect of H2O2 was abrogated by the reducing agent β-mercaptoethanol. H2O2 also attenuated the thapsigargin-evoked entry of barium and manganese. The effect of H2O2 was, at least in part, mediated by activation of protein kinase C (PKC), as H2O2 enhanced the binding of [3H]phorbol 12,13-dibutyrate. H2O2 also stimulated the translocation of the isoenzyme PKCε from the cytosolic fraction to the particulate fraction. Furthermore, H2O2 did not attenuate store-operated calcium entry in cells treated with staurosporine or calphostin C, or in cells with down-regulated PKC. H2O2 depolarized the membrane potential in bisoxonol-loaded cells and when patch-clamp in the whole-cell mode was used. The depolarization was attenuated in cells with down-regulated PKC. This depolarization, at least in part, explained the H2O2-evoked inhibition of calcium entry. In addition, H2O2 enhanced the extrusion of calcium from cells stimulated with thapsigargin and this effect was abolished in cells with down-regulated PKC and after treatment of the cells with the reducing agent β-mercaptoethanol. In conclusion H2O2 attenuates an increase in [Ca2+]i. As H2O2 is produced in thyroid cells in a calcium-dependent manner, our results suggest that H2O2 may participate in the regulation of [Ca2+]i in these cells via a negative-feedback mechanism involving activation of PKC.

Hydrogen peroxide attenuates store-operated calcium entry and enhances calcium extrusion in thyroid FRTL-5 cells.

Redox modulation participates in the regulation of intracellular free calcium concentration ([Ca(2+)](i)) in several cell types. In thyroid cells, including FRTL-5 cells, changes in [Ca(2+)](i) regulate several important functions, including the production of H(2)O(2) (hydrogen peroxide). As H(2)O(2) is of crucial importance for the production of thyroid hormones, we investigated the effects of H(2)O(2) on [Ca(2+)](i) in thyroid FRTL-5 cells. H(2)O(2) itself did not modulate basal [Ca(2+)](i). However, H(2)O(2) attenuated store-operated calcium entry evoked by thapsigargin, both in a sodium-containing buffer and in a sodium-free buffer. The effect of H(2)O(2) was abrogated by the reducing agent beta-mercaptoethanol. H(2)O(2) also attenuated the thapsigargin-evoked entry of barium and manganese. The effect of H(2)O(2) was, at least in part, mediated by activation of protein kinase C (PKC), as H(2)O(2) enhanced the binding of [(3)H]phorbol 12,13-dibutyrate. H(2)O(2) also stimulated the translocation of the isoenzyme PKCepsilon from the cytosolic fraction to the particulate fraction. Furthermore, H(2)O(2) did not attenuate store-operated calcium entry in cells treated with staurosporine or calphostin C, or in cells with down-regulated PKC. H(2)O(2) depolarized the membrane potential in bisoxonol-loaded cells and when patch-clamp in the whole-cell mode was used. The depolarization was attenuated in cells with down-regulated PKC. This depolarization, at least in part, explained the H(2)O(2)-evoked inhibition of calcium entry. In addition, H(2)O(2) enhanced the extrusion of calcium from cells stimulated with thapsigargin and this effect was abolished in cells with down-regulated PKC and after treatment of the cells with the reducing agent beta-mercaptoethanol. In conclusion H(2)O(2) attenuates an increase in [Ca(2+)](i). As H(2)O(2) is produced in thyroid cells in a calcium-dependent manner, our results suggest that H(2)O(2) may participate in the regulation of [Ca(2+)](i) in these cells via a negative-feedback mechanism involving activation of PKC.

Redox modulation of intracellular free calcium concentration in thyroid FRTL-5 cells: evidence for an enhanced extrusion of calcium

Redox modulation is involved in the regulation of the intracellular free calcium concentration ([Ca2+]i) in several cell types. In thyroid cells, including thyroid FRTL-5 cells, changes in [Ca2+]i regulate important functions. In the present study we investigated the effects of the oxidizing compounds thimerosal and t-butyl hydroperoxide on [Ca2+]i in thyroid FRTL-5 cells. Thimerosal mobilized sequestered calcium, and evoked modest store-dependent calcium entry. Both compounds potently attenuated the increase in [Ca2+]i when store-operated calcium entry was evoked with thapsigargin. The entry of barium was not attenuated. Experiments performed with high extracellular pH, in sodium-free buffer and in the presence of vanadate suggested that thimerosal decreased [Ca2+]i by activating a calcium extrusion mechanism, probably a plasma membrane Ca2+-ATPase. All the observed effects were abrogated by the reducing agent beta-mercaptoethanol. The mechanism of action was apparently mediated via activation of protein kinase C, as thimerosal potently stimulated binding of [3H]phorbol 12, 13-dibutyrate, and was without effect on store-operated calcium entry in cells treated with staurosporine or in cells with down-regulated protein kinase C. Thimerosal did not depolarize the membrane potential, as evaluated using patch-clamp in the whole-cell mode. In immunoprecipitates obtained with an antibody against plasma membrane Ca2+-ATPase, we observed several phosphorylated bands in cells stimulated with thimerosal. In conclusion, we have shown that thimerosal attenuates an increase in [Ca2+]i, probably by activating a plasma membrane Ca2+-ATPase.