Effects Of Thapsigargin Research Articles

Calcium dysregulation potentiates wild-type myocilin misfolding: implications for glaucoma pathogenesis.

Myocilin is secreted from trabecular meshwork cells to an eponymous extracellular matrix that is critical for maintaining intraocular pressure. Missense mutations found in the myocilin olfactomedin domain (OLF) lead to intracellular myocilin misfolding and are causative for the heritable form of early-onset glaucoma. The OLF domain contains a unique internal, hetero-dinuclear calcium site. Here, we tested the hypothesis that calcium dysregulation causes wild-type (WT) myocilin misfolding reminiscent of that observed for disease variants. Using two cellular models expressing WT myocilin, we show that the Ca2+ ATPase channel blocker thapsigargin inhibits WT myocilin secretion. Intracellular WT myocilin is at least partly insoluble and aggregated in the endoplasmic reticulum (ER), and stains positively with an amyloid dye. By comparing the effect ofthapsigargin on WT myocilin to that ona de novo secretion-competent Ca2+-free variant D478S, we discern that non-secretion of WT myocilin is due initially to calcium dysregulation, and is potentiated further by resultant ER stress. In E. coli, depletion of calcium leads to recombinantexpression of misfolded isolated WT OLF but the D478S variant is still produced as a folded monomer. Treatment of cells expressing a double mutant composed of D478S and either disease variants P370L or Y437H with thapsigargin promotes its misfolding and aggregation, demonstrating the limits of D478S to correct secretion defects. Taken together, the heterodinuclear calcium site is a liability for proper folding of myocilin. Our study suggests a molecular mechanism by which WT myocilin misfolding may contribute broadly to glaucoma-associated ER stress. This study explores the effect of calcium depletion on myocilin olfactomedin domain folding.

Small Molecule-mediated Insulin Hypersecretion Induces Transient ER Stress Response and Loss of Beta Cell Function.

Pancreatic islet beta cells require a fine-tuned endoplasmic reticulum (ER) stress response for normal function; abnormal ER stress contributes to diabetes pathogenesis. Here, we identified a small molecule, SW016789, with time-dependent effects on beta cell ER stress and function. Acute treatment with SW016789 potentiated nutrient-induced calcium influx and insulin secretion, while chronic exposure to SW016789 transiently induced ER stress and shut down secretory function in a reversible manner. Distinct from the effects of thapsigargin, SW016789 did not affect beta cell viability or apoptosis, potentially due to a rapid induction of adaptive genes, weak signaling through the eIF2α kinase PERK, and lack of oxidative stress gene Txnip induction. We determined that SW016789 acted upstream of voltage-dependent calcium channels (VDCCs) and potentiated nutrient- but not KCl-stimulated calcium influx. Measurements of metabolomics, oxygen consumption rate, and G protein-coupled receptor signaling did not explain the potentiating effects of SW016789. In chemical cotreatment experiments, we discovered synergy between SW016789 and activators of protein kinase C and VDCCs, suggesting involvement of these pathways in the mechanism of action. Finally, chronically elevated calcium influx was required for the inhibitory impact of SW016789, as blockade of VDCCs protected human islets and MIN6 beta cells from hypersecretion-induced dysfunction. We conclude that beta cells undergoing this type of pharmacological hypersecretion have the capacity to suppress their function to mitigate ER stress and avoid apoptosis. These results have the potential to uncover beta cell ER stress mitigation factors and add support to beta cell rest strategies to preserve function.

Extracts of Emmer Wheatgrass Grown with Distilled Water, Salinity or Selenium Differently Affect Germination and Cytosolic Ca2+ of Maize Pollen

In this work, the biological activity of emmer (Triticum turgidum L. spp. dicoccum (Schrank ex Shubler) Thell.) wheatgrass extracts obtained from grains sprouted with distilled water, or salinity (50 mM) or selenium (45 mg L−1 of Na2SeO3), was tested through an experimental biological model based on the germination and cytosolic Ca2+ homeostasis of maize pollen grains. The effects of thapsigargin (TG) and of four phenolic acids (PAs: ferulic, coumaric, salicylic and 3-HO benzoic) on maize pollen were also tested as controls. Wheatgrass extracts influenced both pollen cytosolic Ca2+ and germination. The Ca2+ agonist activity of emmer wheatgrass was transient, different from that of TG, which caused a depletion of the stored Ca2+ and a permanent alteration of Ca2+ homeostasis. The results obtained with extracts compared to those obtained with pure PAs suggest that PAs in unconjugated forms, which are known to be well represent in emmer wheatgrass, contribute to the biological activity of extracts. The extent of the biological response of emmer wheatgrass extracts was influenced by emmer sprouting conditions (i.e., distilled water, or salinity or selenium). Maize pollen treated with Se-enriched wheatgrass extracts showed a less perturbed cytosolic Ca2+ and a higher germination rate.

Testing Cellular Proliferation Responses to Oxidative, Glucocorticoid and Metabolic Challenges in the Baboon

Aging is associated with progressive loss of cellular homeostasis which results from intrinsic and extrinsic challenges. Testing how such challenges relate to the aging process is often limited by the available model systems. We use primary cells (fibroblasts) isolated from baboons as a non-human primate cellular model to address how stressful challenges affect resilience. Using a real-time live-cell imaging system, we characterized a protocol for testing the effects of pro-oxidant compounds (e.g hydrogen peroxide (H2O2), paraquat and thapsigargin), dexamethasone and low glucose environment on cellular proliferation in fibroblasts derived from baboons across the life-course. The inhibitory effect of H2O2 (50 and 100µM), an oxidative stress, on cell proliferation was dose-dependent, with a higher impact in old males (age 14.52-14.80 years; average life span 21 years) compared to young males (6.35-6.39 years). Exposure to a different oxidative stress, paraquat (100 and 200µM) tended to reduce cell proliferation rate with age in males but not females. Inhibitory effects of thapsigargin, an endoplasmic reticulum stress inducer, on cell proliferation were dependent on challenge duration (2 vs 24h), concentration (0.1 and 1µM) and donor age, with greater resilience in young males than young females (4.33-6.70 years). Dexamethasone (100 and 500µM), a glucocorticoid, reduced cell proliferation dose-dependently, with older males exhibiting more resilience than females. In response to low glucose (1mM), cell proliferation reduced with age. Donor’s chronological age and sex are important variables in cellular response to challenge compounds faced during aging, which will guide our on-going studies on the cellular transcriptome and proteome.

Inhibition of interleukin-6 on matrix protein production by glomerular mesangial cells and the pathway involved.

Activation of immunological pathways and disturbances of extracellular matrix (ECM) dynamics are important contributors to the pathogenesis of chronic kidney diseases. Glomerular mesangial cells (MCs) are critical for homeostasis of glomerular ECM dynamics. Interleukin-6 (IL-6) can act as a pro/anti-inflammatory agent relative to cell types and conditions. This study investigated whether IL-6 influences ECM protein production by MCs and the regulatory pathways involved. Experiments were carried out in cultured human MCs (HMCs) and in mice. We found that overexpression of IL-6 and its receptor decreased the abundance of fibronectin and collagen type IV in MCs. ELISA and immunoblot analysis demonstrated that thapsigargin [an activator of store-operated Ca2+ entry (SOCE)], but not the endoplasmic reticulum stress inducer tunicamycin, significantly increased IL-6 content. This thapsigargin effect was abolished by GSK-7975A, a selective inhibitor of SOCE, and by silencing Orai1 (the channel protein mediating SOCE). Furthermore, inhibition of NF-κB pharmacologically and genetically significantly reduced SOCE-induced IL-6 production. Thapsigargin also stimulated nuclear translocation of the p65 subunit of NF-κB. Moreover, MCs overexpressing IL-6 and its receptor in HMCs increased the content of the glucagon-like peptide-1 receptor (GLP-1R), and IL-6 inhibition of fibronectin was attenuated by the GLP-1R antagonist exendin 9-39. In agreement with the HMC data, specific knockdown of Orai1 in MCs using the targeted nanoparticle delivery system in mice significantly reduced glomerular GLP-1R levels. Taken together, our results suggest a novel SOCE/NF-κB/IL-6/GLP-1R signaling pathway that inhibits ECM protein production by MCs.

Chlorogenic acid against palmitic acid in endoplasmic reticulum stress-mediated apoptosis resulting in protective effect of primary rat hepatocytes

BackgroundWe demonstrated growing evidence supports a protective role of chlorogenic acid of rat hepatocytes elicited by two compounds, i.e. thapsigargin and palmitic acid. Nevertheless, little is known about the mechanisms of palmitic acid induced endoplasmic reticulum (ER) stress and cell death.MethodsThe proliferation of primary rat hepatocytes was detected by MTT assay. The expression of GRP78, CHOP and GRP94 was detected by Western blot analyses.Caspase-3 activity was detected by a Caspase-3 substrate kit. Cell apoptosis was detected by Hoechst 33342 staining.ResultsWe demonstrated that incubation of hepatocytes for 16 h with palmitic acid elevated cell death. Moreover, Western blot analyses demonstrated increased levels of the endoplasmic reticulum stress markers — glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and glucose regulated protein 94 (GRP94). Chlorogenic acid could inhibit ER stress induced cell death and levels of indicators of ER stress caused by palmitic acid. The effect of thapsigargin, which evokes ER stress were reversed by chlorogenic acid.ConclusionsAltogether, our data indicate that in primary rat hepatocytes, chlorogenic acid prevents ER stress-mediated apoptosis of palmitic acid.

The effect of thapsigargin on the expression of bile salt export pump: A pathologic link between ER stress and cholestasis

The effect of thapsigargin on the expression of bile salt export pump: A pathologic link between ER stress and cholestasis

Store-operated calcium entry suppressed the TGF-β1/Smad3 signaling pathway in glomerular mesangial cells.

Our previous study demonstrated that the abundance of extracellular matrix proteins was suppressed by store-operated Ca2+ entry (SOCE) in mesangial cells (MCs). The present study was conducted to investigate the underlying mechanism focused on the transforming growth factor-β1 (TGF-β1)/Smad3 pathway, a critical pathway for ECM expansion in diabetic kidneys. We hypothesized that SOCE suppressed ECM protein expression by inhibiting this pathway in MCs. In cultured human MCs, we observed that TGF-β1 (5 ng/ml for 15 h) significantly increased Smad3 phosphorylation, as evaluated by immunoblot. However, this response was markedly inhibited by thapsigargin (1 µM), a classical activator of store-operated Ca2+ channels. Consistently, both immunocytochemistry and immunoblot showed that TGF-β1 significantly increased nuclear translocation of Smad3, which was prevented by pretreatment with thapsigargin. Importantly, the thapsigargin effect was reversed by lanthanum (La3+; 5 µM) and GSK-7975A (10 µM), both of which are selective blockers of store-operated Ca2+ channels. Furthermore, knockdown of Orai1, the pore-forming subunit of the store-operated Ca2+ channels, significantly augmented TGF-β1-induced Smad3 phosphorylation. Overexpression of Orai1 augmented the inhibitory effect of thapsigargin on TGF-β1-induced phosphorylation of Smad3. In agreement with the data from cultured MCs, in vivo knockdown of Orai1 specific to MCs using a targeted nanoparticle small interfering RNA delivery system resulted in a marked increase in abundance of phosphorylated Smad3 and in nuclear translocation of Smad3 in the glomerulus of mice. Taken together, our results indicate that SOCE in MCs negatively regulates the TGF-β1/Smad3 signaling pathway.

Sodium-Calcium Exchanger in Pig Coronary Artery.

This review focuses on the sodium-calcium exchangers (NCX) in the left anterior descending coronary artery smooth muscle. Bathing tissues in Na+-substituted solutions caused them to contract. In cultured smooth muscle cells, it increased the cytosolic Ca2+ concentration and extracellular entry of 45Ca2+. All three activities were attributed to NCX since they were inhibited by NCX inhibitors. The tissues also expressed the sarco/endoplasmic reticulum (SER) Ca2+ pump SERCA2b whose activity was much greater than that of NCX. Inhibiting SERCA2b with thapsigargin decreased the NCX-mediated 45Ca2+ accumulation by the cells. The decrease was not observed in cells loaded with the Ca2+-chelator BAPTA. The results are consistent with a limited diffusional space model with a proximity between NCX and SERCA2b. NCX molecules appear to be colocalized with the subsarcolemmal SERCA2b based on studies on membrane flotation experiments and microscopic fluorescence imaging of antibody-labeled cells. Thapsigargin inhibition of SERCA2b moved NCX even closer to SER. This provides a model for the NCX-mediated Ca2+ refilling of SER in the arterial smooth muscle. The model for the NCX-mediated refilling of the depleted SER proposed for smooth muscle did not apply to endothelium in which NCX levels were greater and SERCA levels were lower than in smooth muscle. The effect of thapsigargin on the NCX-mediated Ca2+ accumulation which was observed in smooth muscle was absent in the endothelium. We propose that the coupling between NCX and smooth muscle may be tissue dependent.

Effects of Thapsigargin on the Proliferation and Survival of Human Rheumatoid Arthritis Synovial Cells

A series of experiments have been carried out to investigate the effects of different concentrations of thapsigargin (0, 0.001, 0.1, and 1 μM) on the proliferation and survival of human rheumatoid arthritis synovial cells (MH7A). The results showed that thapsigargin can block the cell proliferation in human rheumatoid arthritis synovial cells in a time- and dose-dependent manner. Results of Hoechst staining suggested that thapsigargin may induce cell apoptosis in MH7A cells in a time- and dose-dependent manner, and the percentages of cell death reached 44.6% at thapsigargin concentration of 1 μM treated for 4 days compared to the control. The protein and mRNA levels of cyclin D1 decreased gradually with the increasing of thapsigargin concentration and treatment times. Moreover, the protein levels of mTORC1 downstream indicators pS6K and p4EBP-1 were reduced by thapsigargin treatment at different concentrations and times, which should be responsible for the reduced cyclin D1 expressions. Our results revealed that thapsigargin may effectively impair the cell proliferation and survival of MH7A cells. The present findings will help to understand the molecular mechanism of fibroblast-like synoviocytes proliferations and suggest that thapsigargin is of potential for the clinical treatment of rheumatoid arthritis.

Small Molecule Inhibition of PAX3-FOXO1 through AKT Activation Suppresses Malignant Phenotypes of Alveolar Rhabdomyosarcoma

Alveolar rhabdomyosarcoma comprises a rare highly malignant tumor presumed to be associated with skeletal muscle lineage in children. The hallmark of the majority of alveolar rhabdomyosarcoma is a chromosomal translocation that generates the PAX3-FOXO1 fusion protein, which is an oncogenic transcription factor responsible for the development of the malignant phenotype of this tumor. Alveolar rhabdomyosarcoma cells are dependent on the oncogenic activity of PAX3-FOXO1, and its expression status in alveolar rhabdomyosarcoma tumors correlates with worst patient outcome, suggesting that blocking this activity of PAX3-FOXO1 may be an attractive therapeutic strategy against this fusion-positive disease. In this study, we screened small molecule chemical libraries for inhibitors of PAX3-FOXO1 transcriptional activity using a cell-based readout system. We identified the Sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCA) inhibitor thapsigargin as an effective inhibitor of PAX3-FOXO1. Subsequent experiments in alveolar rhabdomyosarcoma cells showed that activation of AKT by thapsigargin inhibited PAX3-FOXO1 activity via phosphorylation. Moreover, this AKT activation appears to be associated with the effects of thapsigargin on intracellular calcium levels. Furthermore, thapsigargin inhibited the binding of PAX3-FOXO1 to target genes and subsequently promoted its proteasomal degradation. In addition, thapsigargin treatment decreases the growth and invasive capacity of alveolar rhabdomyosarcoma cells while inducing apoptosis in vitro. Finally, thapsigargin can suppress the growth of an alveolar rhabdomyosarcoma xenograft tumor in vivo. These data reveal that thapsigargin-induced activation of AKT is an effective mechanism to inhibit PAX3-FOXO1 and a potential agent for targeted therapy against alveolar rhabdomyosarcoma.

Su1719 Effectiveness and Safety of Baclofen Treatment in Alcohol-Dependent Patients With or Without Liver Cirrhosis: Results of an Open Study in True Life

Background and Aims: The endoplasmic reticulum (ER) is a multifunctional organelle involved in synthesis, processing and folding of proteins, lipid biosynthesis, and regulation of calcium and redox homeostasis. Altered ER-function due to accumulation of misfolded/unfolded proteins, oxidative stress or depletion of ER calcium, lead to a condition called “ER-stress”, particularly important in cells with a high burden of protein synthesis as hepatocytes. Efavirenz, a non-nucleoside analog reverse transcriptase inhibitor, is a cornerstone in the combined therapy of HIV1 infection. Despite being considered generally safe, it has been related to development of adverse events such as hepatic toxicity. Here we report the presence of specific ER-stress in human hepatic cells. Methods: Hep3B cells and primary human hepatocytes were exposed to short-term treatment (24h) with clinically relevant concentrations of Efavirenz. General cell biology methods were employed (RNA interference, RT-PCR, Western blot and fluorescence microscopy). Results: ER-stress markers indicative of several ER-stress pathways were up-regulated concentration-dependently by Efavirenz, including the expression of CHOP and GRP78, at protein and mRNA level, and the phosphorylated form of eIF-2a. Furthermore, we also detected an increase in the transcription of ATF4 and XBP-1, and presence of the spliced form s-XBP-1. Efavirenz also induced morphological changes in ER with enhanced ER-content and dilatation of its cisternae, and led to an increase in the cytosolic calcium level. All these changes were similar to those produced by the standard pharmacological ER-stressor, Thapsigargin. On the contrary, while Thapsigargin induced an increment in the mitochondrial calcium level, Efavirenz provoked a decrease. A differential effect was also observed with transient silencing of CHOP. The stimulating effect of Thapsigargin on the generation of reactive oxygen species and the increment in the ER-content was more pronounced in CHOP-depleted cells, but this exacerbated action was not observed when cells were treated with Efavirenz. Conclusions: Clinical concentrations of Efavirenz induce ER-stress in hepatic cells in a compound-specific manner. Given the lifelong and widespread use of Efavirenz in the multidrug therapy of HIV, this novel mechanism of cellular response to drug-induced stress could help to understand the frequent hepatic toxicities that accompany the treatment of HIV1 infection.

Abstract 989: Mild ER stress increases human ABC transporter expression and drug transport function in polarized cell monolayers.

Abstract Expression of ATP-binding cassette (ABC) transporter genes such as MDR1 (ABCB1/P-gp) is associated with poor outcome in chemotherapy and a multidrug resistant phenotype in cancer. Endoplasmic reticulum (ER) stress has been implicated in tumor growth but its role in drug resistance remains unclear. Here, we hypothesize that ER stress could increase expression of drug efflux transporters. To test this hypothesis, we developed a transient expression system in which human ABC transporters were ectopically expressed in a cell line monolayer by the BacMam baculovirus. The baculovirus is able to infect several mammalian cell lines including LLC-PK1, T-84, and MA104 without affecting the tightness of the host cell monolayers. In LLC-PK1 cells infected by BacMam vectors, induction of mild ER stress by pre-incubation (pre-conditioning) with thapsigargin (0.5 μg/ml) or tunicamycin (1 μg/ml), which are well-characterized ER-stress inducers, increased ABCB1 (P-gp) and ABCG2 (BCRP) levels. Drug transport assays showed that P-gp expressing cells pre-conditioned with thapsigargin showed increased directional transport of [3H] paclitaxel across the cell monolayer. The effect of thapsigargin was inhibited by the presence of the Erk inhibitor PD098059 but not the P38(MAPK) inhibitor SB203580, or the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002, suggesting that expression of ABC transporter is influenced by mild ER stress response and, at least in part, by the ERK/MAPK signaling pathway. Although pre-conditioning with tunicamycin can increase P-gp expression, it also inhibits N-glycosylation of P-gp. Collectively, our findings indicate that mild ER stress responses up-regulate drug transporters introduced into cells by BacMam vectors. Citation Format: King Leung Fung, Jessica Pixley, Darrell J. Talbert, Khyati Kapoor, Suresh Ambudkar, Michael M. Gottesman. Mild ER stress increases human ABC transporter expression and drug transport function in polarized cell monolayers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 989. doi:10.1158/1538-7445.AM2013-989

Bortezomib induction of C/EBPβ mediates Epstein-Barr virus lytic activation in Burkitt lymphoma

AbstractEpstein-Barr virus (EBV) is associated with a variety of lymphoid malignancies. Bortezomib activates EBV lytic gene expression. Bortezomib, a proteasome inhibitor, leads to increased levels of CCAAT/enhancer-binding proteinβ (C/EBPβ) in a variety of tumor cell lines. C/EBPβ activates the promoter of the EBV lytic switch gene ZTA. Bortezomib treatment leads to increased binding of C/EBP to previously recognized binding sites in the ZTA promoter. Knockdown of C/EBPβ inhibits bortezomib activation of EBV lytic gene expression. Bortezomib also induces the unfolded protein response (UPR), as evidenced by increases in ATF4, CHOP10, and XBP1s and cleavage of ATF6. Thapsigargin, an inducer of the UPR that does not interfere with proteasome function, also induces EBV lytic gene expression. The effects of thapsigargin on EBV lytic gene expression are also inhibited by C/EBPβ knock-down. Therefore, C/EBPβ mediates the activation of EBV lytic gene expression associated with bortezomib and another UPR inducer.

Effect of thapsigargin on Ca2+ fluxes and viability in human prostate cancer cells

Effect of the carcinogen thapsigargin on human prostate cancer cells is unclear. This study examined if thapsigargin altered basal [Ca2+]i levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca2+-sensitive fluorescent probe. Thapsigargin at concentrations between 10 nM and 10 µM increased [Ca2+]i in a concentration-dependent fashion. The Ca2+ signal was reduced partly by removing extracellular Ca2+ indicating that Ca2+ entry and release both contributed to the [Ca2+]i rise. This Ca2+ influx was inhibited by suppression of phospholipase A2, but not by inhibition of store-operated Ca2+ channels or by modulation of protein kinase C activity. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished thapsigargin-induced Ca2+ release. Conversely, pretreatment with thapsigargin greatly reduced BHQ-induced [Ca2+]i rise, suggesting that thapsigargin released Ca2+ from the endoplasmic reticulum. Inhibition of phospholipase C did not change thapsigargin-induced [Ca2+]i rise. At concentrations of 1-10 µM, thapsigargin induced cell death that was partly reversed by chelation of Ca2+ with BAPTA/AM. Annexin V/propidium iodide staining data suggest that apoptosis was partly responsible for thapsigargin-induced cell death. Together, in PC3 human prostate cancer cells, thapsigargin induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive Ca2+ channels. Thapsigargin also induced cell death via Ca2+-dependent pathways and Ca2+-independent apoptotic pathways.

Effect of thapsigargin on P-glycoprotein-negative and P-glycoprotein-positive L1210 mouse leukaemia cells

Expression of drug-transporting P-glycoprotein (P-gp, an integral protein of the plasma membrane) in neoplastic cells confers multidrug resistance and also involves alteration of cell sensitivity to inhibitors of the sarco/endoplasmic reticulum calcium pump thapsigargin (Th). Mouse leukaemia L1210 cell sublines that overexpress P-gp due to selection with vincristine (R) or stable transfection with a gene encoding human P-gp (T) were less sensitive to Th than the parental cell line (S). Th at a concentration of 0.1 μmol/l did not induce alterations in the amount of P-gp mRNA in R or T cells (S cells did not contain any measurable amount of this transcript as assessed by RT-PCR) or in the amount of calnexin (CNX) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in all three cell sublines. However, when using a concentration of 10 μmol/l, Th decreases the amounts of CNX, GAPDH (in S, R and T cells) and P-gp (in R and T cells) mRNAs. In contrast to R and T cells (which contain abundant P-gp), S cells did not contain any P-gp detectable by the c219 antibody on a Western blot. Th at a concentration of 0.1 μmol/l induced a reduction in the amount of P-gp present in R and T cells, particularly in isoforms with higher molecular weights (i.e., mature fully glycosylated isoforms). Similar results were observed when Th was used at a concentration of 10 μmol/l. R and T cells contained lower levels of CNX than S cells. While Th at a lower concentration did not alter the levels of CNX in S, R or T cells, a higher concentration of this substance induced a measurable decrease in the amount of CNX. S, R and T cells did not differ with respect to GAPDH content, but Th induced a reduction in the amount of this protein in all cell sublines. More pronounced results were observed when Th was applied at a concentration of 10 μmol/l comparing with a concentration of 0.1 mmol/l. These changes may be involved together with the Th efflux activity of P-gp in Th-resistance associated with the P-gp-mediated multidrug resistance of R and T cells.

Characterization of Ca2+ signaling pathways in mouse adrenal medullary chromaffin cells

In the present study, we characterized the Ca2+ responses and secretions induced by various secretagogues in mouse chromaffin cells. Activation of the acetylcholine receptor (AChR) by carbachol induced a transient intracellular Ca2+ concentration ([Ca2+](i)) increase followed by two phases of [Ca2+](i) decay and a burst of exocytic events. The contribution of the subtypes of AChRs to carbachol-induced responses was examined. Based on the results obtained by stimulating the cells with the nicotinic receptor (nAChR) agonist, 1,1-dimethyl-4-phenylpiperazinium iodide, high K(+) and the effects of thapsigargin, it appears that activation of nAChRs induces an extracellular Ca2+ influx, which in turn activate Ca(2+)-induced Ca2+ release via the ryanodine receptors. Muscarine, a muscarinic receptor (mAChRs) agonist, was found to induce [Ca2+](i) oscillation and sustained catecholamine release, possibly by activation of both the receptor- and store-operated Ca2+ entry pathways. The RT-PCR results showed that mouse chromaffin cells are equipped with messages for multiple subtypes of AChRs, ryanodine receptors and all known components of the receptor- and store-operated Ca2+ entry. Furthermore, results obtained by directly monitoring endoplasmic reticulum (ER) and mitochondrial Ca2+ concentration and by disabling mitochondrial Ca2+ uptake suggest that the ER acts as a Ca2+ source, while the mitochondria acts as a Ca2+ sink. Our results show that both nAChRs and mAChRs contribute to the initial carbachol-induced [Ca2+](i) increase which is further enhanced by the Ca2+ released from the ER mediated by Ca(2+)-induced Ca2+ release and mAChR activation. This information on the Ca2+ signaling pathways should lay a good foundation for future studies using mouse chromaffin cells as a model system.

Siphonodictyal B1 from a Marine Sponge Increases Intracellular Calcium Levels Comparable to the Ca2+-ATPase (SERCA) Inhibitor Thapsigargin

Siphonodictyal B1 is a sesquiterpene-hydroquinone isolated from the Caribbean coral reef bioeroding sponge Siphonodictyon coralliphagum. Siphonodictyal B1 increased intracellular calcium levels in neuroendocrine cells (PC12) in the presence and absence of extracellular calcium using Fura-2 as a calcium-sensitive dye. The calcium rise was comparable in amplitude and timing to the application of the sarco-endoplasmic reticulum calcium-ATPase (SERCA) inhibitor thapsigargin from the terrestrial plant Thapsia garganica. The effects of thapsigargin and siphonodictyal B1 on intracellular calcium levels were not distinguishable in pharmacological experiments conducted with caffeine, ryanodine, muscarine, and thapsigargin in calcium-free and calcium-containing buffer, although thapsigargin was effective at lower concentrations. Thapsigargin is a sesquiterpene-lactone and has no structural similarities to siphonodictyal B1. We conclude that thapsigargin and siphonodictyal B1 share SERCAs as cellular targets. Siphonodictyal B1 may be involved in the process of bioeroding the calcium carbonate endoskeleton of the scleractinian corals attacked by S. coralliphagum.

Effects of thapsigargin and phenylephrine on calcineurin and protein kinase C signaling functions in cardiac myocytes

Neonatal rat cardiac myocytes were exposed to 10 nM thapsigargin (TG) or 20 muM phenylephrine (PE) to compare resulting alterations of Ca(2+) homeostasis. Either treatment results in resting cytosolic [Ca(2+)] rise and reduction of Ca(2+) signals in myocytes following electrical stimuli. In fact, ATP-dependent Ca(2+) transport is reduced due to catalytic inhibition of sarcoplasmic reticulum ATPase (SERCA2) by TG or reduction of SERCA2 protein expression by PE. A marked rise of nuclear factor of activated T cells (NFAT)-dependent expression of transfected luciferase cDNA is produced by TG or PE, which is dependent on increased NFAT dephosphorylation by activated calcineurin and reduced phosphorylation by inactivated glycogen synthase kinase 3beta. Expression of SERCA2 (inactivated) protein is increased following exposure to TG, whereas no hypertrophy is produced. On the contrary, SERCA2 expression is reduced, despite high CN activity, following protein kinase C (PKC) activation by PE (or phorbol 12-myristate 13-acetate) under conditions producing myocyte hypertrophy. Both effects of TG and PE are dependent on NFAT dephosphorylation by CN, as demonstrated by CN inhibition with cyclosporine (CsA). However, the hypertrophy program triggered by PKC activation bypasses SERCA2 transcription and expression due to competitive recruitment of NFAT and/or other transcriptional factors. A similar dependence on CN activation, but relative reduction under conditions of PKC activation, involves transcription and expression of the Na(+)/Ca(2+) exchanger-1. On the other hand, significant upregulation of transient receptor potential channel proteins is noted following PKC activation. The observed alterations of Ca(2+) homeostasis may contribute to development of contractile failure.

A New Conformation in Sarcoplasmic Reticulum Calcium Pump and Plasma Membrane Ca2+ Pumps Revealed by a Photoactivatable Phospholipidic Probe

The purpose of this work was to obtain structural information about conformational changes in the membrane region of the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca(2+) pumps. We have assessed changes in the overall exposure of these proteins to surrounding lipids by quantifying the extent of protein labeling by a photoactivatable phosphatidylcholine analog 1-palmitoyl-2-[9-[2'-[(125)I]iodo-4'-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine ([(125)I]TID-PC/16) under different conditions. We determined the following. 1) Incorporation of [(125)I]TID-PC/16 to SERCA decreases 25% when labeling is performed in the presence of Ca(2+). This decrease in labeling matches qualitatively the decrease in transmembrane surface exposed to the solvent calculated from crystallographic data for SERCA structures. 2) Labeling of PMCA incubated with Ca(2+) and calmodulin decreases by approximately the same amount. However, incubation with Ca(2+) alone increases labeling by more than 50%. Addition of C28, a peptide that prevents activation of PMCA by calmodulin, yields similar results. C28 has also been shown to inhibit ATPase SERCA activity. Interestingly, incubation of SERCA with C28 also increases [(125)I]TID-PC/16 incorporation to the protein. These results suggest that in both proteins there are two different E(1) conformations as follows: one that is auto-inhibited and is in contact with a higher amount of lipids (Ca(2+) + C28 for SERCA and Ca(2+) alone for PMCA), and one in which the enzyme is fully active (Ca(2+) for SERCA and Ca(2+)-calmodulin for PMCA) and that exhibits a more compact transmembrane arrangement. These results are the first evidence that there is an autoinhibited conformation in these P-type ATPases, which involves both the cytoplasmic regions and the transmembrane segments.