Inhibitor Of Sarco-endoplasmic Reticulum Ca2 Research Articles

RPGR protein complex regulates proteasome activity and mediates store-operated calcium entry.

Ciliopathies are a group of genetically heterogeneous disorders, characterized by defects in cilia genesis or maintenance. Mutations in the RPGR gene and its interacting partners, RPGRIP1 and RPGRIP1L, cause ciliopathies, but the function of their proteins remains unclear. Here we show that knockdown (KD) of RPGR, RPGRIP1 or RPGRIP1L in hTERT-RPE1 cells results in abnormal actin cytoskeleton organization. The actin cytoskeleton rearrangement is regulated by the small GTPase RhoA via the planar cell polarity (PCP) pathway. RhoA activity was upregulated in the absence of RPGR, RPGRIP1 or RPGRIP1L proteins. In RPGR, RPGRIP1 or RPGRIP1L KD cells, we observed increased levels of DVl2 and DVl3 proteins, the core components of the PCP pathway, due to impaired proteasomal activity. RPGR, RPGRIP1 or RPGRIP1L KD cells treated with thapsigargin (TG), an inhibitor of sarcoendoplasmic reticulum Ca2+- ATPases, showed impaired store-operated Ca2+ entry (SOCE), which is mediated by STIM1 and Orai1 proteins. STIM1 was not localized to the ER-PM junction upon ER store depletion in RPGR, RPGRIP1 or RPGRIP1L KD cells. Our results demonstrate that the RPGR protein complex is required for regulating proteasomal activity and for modulating SOCE, which may contribute to the ciliopathy phenotype.

Reactive Oxygen Species Produced by a Photodynamic Effect Induced Calcium Signal in Neurons and Astrocytes.

Photodynamic therapy (PDT) leads to production of reactive oxygen species (ROS) and cell destruction due to oxidative stress. We used photodynamic effect of photosensitizer radachlorin to unravel the effect of photo-induced oxidative stress on the calcium signal and lipid peroxidation in primary culture of cortical neurons and astrocytes using live cell imaging. We have found that irradiation in presence of 200nM of radachlorin induces calcium signal in primary neurons and astrocytes. Photo-induced neuronal calcium signal depends on internal calcium stores as it was still observed in calcium-free medium and could be blocked by depletion of endoplasmic reticulum (ER) stores with inhibitor of sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) thapsigargin. Both inhibitors of phospholipase C activity U73122 and water-soluble analogue of vitamin E Trolox suppressed calcium response activated by PDT. We have also observed that the photodynamic effect of radachlorin induces lipid peroxidation in neurons and astrocytes. This data demonstrate that lipid peroxidation induced by PDT in neurons and astrocytes leads to activation of phospholipase C that results in production of inositol 1,4,5-trisphosphate (IP3).

Urokinase-type plasminogen activator (uPA) modulates monocyte-to-macrophage differentiation and prevents Ox-LDL-induced macrophage apoptosis

ObjectiveMonocyte-to-macrophage differentiation and macrophage death play a pivotal role in atherogenesis. uPA and its receptor uPAR are expressed in atherosclerotic lesion macrophages and contribute to atherosclerosis progression. In the present study we investigated the effect and mechanisms of action of uPA on monocyte-to-macrophage differentiation and on macrophage apoptotic death. Methods and resultsThe number of mouse peritoneal macrophages (MPM) harvested from uPAR-deficient (uPAR−/−) mice was significantly lower by 30% in comparison to control C57BL/6 mice. In vitro, uPA intensified PMA-induced THP-1 monocyte differentiation, as determined by increased expression of the macrophage marker CD36. This effect was mediated via G1 arrest, downregulation of G2/S phase and inhibition of PMA-induced cell death. uPA attenuated MonoMac6 (MM6) macrophage-like cell line apoptosis induced by oxidized LDL (Ox-LDL) and by thapsigargin (inhibitor of sarco-endoplasmic reticulum Ca2+-ATPase), but not by staurosporine (protein kinase inhibitor), suggesting that uPA antiapoptotic activity is Ca2+-independent, but involves a kinase activation. The antiapoptotic activity of uPA was dependent on the presence of uPAR, and it involved ERK1/2 activation-dependent downregulation of the proapoptotic protein Bim in macrophages stimulated with Ox-LDL. ConclusionsThe present study demonstrates, for the first time, that uPA stimulates the differentiation of monocytes into macrophages and attenuates Ox-LDL-induced macrophage apoptotic death via ERK1/2 activation-dependent Bim downregulation. These processes may result in prolonged macrophage survival in the lesion, increased lesion cellularity, and eventually necrosis, which accelerates lesion development.

Thapsigargin and trilobolide – sesquiterpene lactones with immunostimulatory properties

Thapsigargin (TG) and trilobolide (TB) are sesquiterpene lactones of guaianolide type isolated from Thapsia garganica L. and Laser trilobum (L.) Borkh., respectively. TG is widely used experimentally as an inhibitor of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) leading to rapid elevation of intracellular calcium.

Extracellular ATP-induced nuclear Ca2+ transient is mediated by inositol 1,4,5-trisphosphate receptors in mouse pancreatic β-cells

Extracellular ATP (eATP) induces an intracellular Ca2+ transient by activating phospholipase C (PLC)-associated P2X4 purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent Ca2+ release from intracellular stores in mouse pancreatic β-cells. Using laser scanning confocal microscopy, Ca2+ indicator fluo-4 AM, and the cell permeable nuclear indicator Hoechst 33342, we examined the properties of eATP-induced Ca2+ release in pancreatic β-cell nuclei. eATP induced a higher nuclear Ca2+ transient in pancreatic β-cell nuclei than in the cytosol. After pretreatment with thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pumps, the amplitude of eATP-induced Ca2+ transients in the nucleus was still much higher than those in the cytosol. This effect of eATP was not altered by inhibition of either the plasma membrane Ca2+-ATPase (PMCA) or the plasma membrane Na+/Ca2+ exchanger (NCX) by LaCl3 or by replacement of Na+ with N-Methyl-Glucosamine. eATP-induced nuclear Ca2+ transients were abolished by a cell-permeable IP3R inhibitor, 2-aminoethoxydiphenyl borate (2-APB), but were not blocked by the ryanodine receptor (RyR) antagonist ryanodine. Immunofluorescence studies showed that IP3Rs are expressed on the nuclear envelope of pancreatic β-cells. These results indicate that eATP triggers nuclear Ca2+ transients by mobilizing a nuclear Ca2+ store via nuclear IP3Rs.

Thapsigargin, a selective inhibitor of sarco-endoplasmic reticulum Ca2+-ATPases, modulates nitric oxide production and cell death of primary rat hepatocytes in culture

Increased cytosolic calcium ([Ca2+]i) and nitric oxide (NO) are suggested to be associated with apoptosis that is a main feature of many liver diseases and is characterized by biochemical and morphological features. We sought to investigate the events of increase in [Ca2+]i and endoplasmic reticulum (ER) calcium depletion by thapsigargin (TG), a selective inhibitor of sarco-ER-Ca2+ -ATPases, in relation to NO production and apoptotic and necrotic markers of cell death in primary rat hepatocyte culture. Cultured hepatocytes were treated with TG (1 and 5 micromol/L) for 0-24 or 24-48 h. NO production and inducible NO synthase (iNOS) expression were determined as nitrite levels and by iNOS-specific antibody, respectively. Hepatocyte apoptosis was estimated by caspase-3 activity, cytosolic cytochrome c content and DNA fragmentation, and morphologically using Annexin-V/propidium iodide staining. Hepatocyte viability and mitochondrial activity were evaluated by ALT leakage and MTT test. Increasing basal [Ca2+]i by TG, NO production and apoptotic/necrotic parameters were altered in different ways, depending on TG concentration and incubation time. During 0-24 h, TG dose-dependently decreased iNOS-mediated spontaneous NO production and simultaneously enhanced hepatocyte apoptosis. In addition, TG 5 micromol/L produced secondary necrosis. During 24-48 h, TG dose-dependently enhanced basal NO production and rate of necrosis. TG 5 micromol/L further promoted mitochondrial damage as demonstrated by cytochrome c release. A selective iNOS inhibitor, aminoguanidine, suppressed TG-stimulated NO production and ALT leakage from hepatocytes after 24-48 h. Our data suggest that the extent of the [Ca2+]i increase and the modulation of NO production due to TG treatment contribute to hepatocyte apoptotic and/or necrotic events.

Apoptosis in insulin-secreting cells. Evidence for the role of intracellular Ca2+ stores and arachidonic acid metabolism.

This study investigated the role of intracellular free Ca2+ concentration ([Ca2+]i) in apoptosis in MIN6 cells, an insulin secreting cell line, and in mouse islets. Thapsigargin, an inhibitor of sarcoendoplasmic reticulum Ca2+-ATPases (SERCA), caused a time- and concentration-dependent decrease in the viability of MIN6 cells and an increase in DNA fragmentation and nuclear chromatin staining changes characteristic of apoptosis. Two structurally distinct SERCA inhibitors, cyclopiazonic acid and 2,5-di-[t-butyl]-1,4-hydroquinone also caused apoptosis, but agents that increased [Ca2+]i by other mechanisms did not induce apoptosis in MIN6 cells. Carbachol- or ionomycin-releasible intracellular Ca2+ stores were completely depleted in cells treated by SERCA inhibitors, but not by other agents that increase [Ca2+]i. The ability of thapsigargin to induce cell death was not affected by blocking Ca2+ influx or by clamping [Ca2+]i with a cytosolic Ca2+ buffer suggesting that the process did not depend on changes in [Ca2+]i per se. However, application of the lipoxygenase inhibitors 5,8,11-eicosatrienoic acid and nordihydroguaiaretic acid partially prevented MIN6 cell apoptosis, while exposure of cells to the product of lipoxygenase, 12-hydroxy-[5,8,10,14]-eicosatetraenoic acid, caused apoptosis. In contrast, inhibition of cyclooxygenase with indomethacin did not abolish thapsigargin-induced apoptosis in MIN6 cells. Our findings indicate that thapsigargin causes apoptosis in MIN6 cells by depleting intracellular Ca2+ stores and leading to release of intermediate metabolites of arachidonic acid metabolism.

A tool coming of age: thapsigargin as an inhibitor of sarco-endoplasmic reticulum Ca(2+)-ATPases.

Thapsigargin is the most widely used inhibitor of the ubiquitous sarco-endoplasmic reticulum Ca(2+)-ATPases in mammalian cells. Over the past ten years, this guaianolide compound of plant origin has become a popular tool in a host of studies directed at elucidating the mechanisms of intracellular Ca2+ signalling. Its remarkable potency and selectivity have been instrumental in widening our view of the function of intracellular Ca2+ stores to include such key aspects as store-operated Ca2+ entry or the involvement of the stores in protein synthesis or cell growth. In this article Marek Treiman, Casper Caspersen and Søren Brøgger Christensen review the key pharmacological features of thapsigargin action; they also discuss some of the ways in which its unique properties have shown to be important for obtaining new insights into the biology of Ca2+ stores, and how these properties might encompass a therapeutic potential. In parallel, attention is drawn to some of the limitations and pitfalls encountered when working with thapsigargin.