Inhibitor Of Endoplasmic Reticulum Ca2 Research Articles

Endoplasmic reticulum: Reduced and oxidized glutathione revisited

The reducing power of glutathione, expressed by its reduction potential EGSH, is an accepted measure for redox conditions in a given cell compartment. In the endoplasmic reticulum (ER), EGSH is less reducing than elsewhere in the cell. However, attempts to determine EGSH(ER) have been inconsistent and based on ineligible assumptions. Using a codon-optimized and evidently glutathione-specific glutaredoxin-coupled redox-sensitive green fluorescent protein (roGFP) variant, we determined EGSH(ER) in HeLa cells as -208±4 mV (at pH 7.0). At variance with existing models, this is not oxidizing enough to maintain the known redox state of protein disulfide isomerase family enzymes. Live-cell microscopy confirmed ER hypo-oxidation upon inhibition of ER Ca(2+) import. Conversely, stressing the ER with a glycosylation inhibitor did not lead to more reducing conditions, as reported for yeast. These results, which for the first time establish the oxidative capacity of glutathione in the ER, illustrate a context-dependent interplay between ER stress and EGSH(ER). The reported development of ER-localized EGSH sensors will enable more targeted in vivo redox analyses in ER-related disorders.

Inhibition of mitochondrial cytochrome c oxidase potentiates Aβ-induced ER stress and cell death in cortical neurons

Previously we reported that amyloid-β (Aβ) leads to endoplasmic reticulum (ER) stress in cultured cortical neurons and that ER-mitochondria Ca(2+) transfer is involved in Aβ-induced apoptotic neuronal cell death. In cybrid cells which recreate the defect in mitochondrial cytochrome c oxidase (COX) activity observed in platelets from Alzheimer's disease (AD) patients, we have shown that mitochondrial dysfunction affects the ER stress response triggered by Aβ. Here, we further investigated the impact of COX inhibition on Aβ-induced ER dysfunction using a neuronal model. Primary cultures of cortical neurons were challenged with toxic concentrations of Aβ upon chemical inhibition of COX with potassium cyanide (KCN). ER Ca(2+) homeostasis was evaluated under these conditions, together with the levels of ER stress markers, namely the chaperone GRP78 and XBP-1, a mediator of the ER unfolded protein response (UPR). We demonstrated that COX inhibition potentiates the Aβ-induced depletion of ER Ca(2+) content. KCN pre-treatment was also shown to enhance the rise of cytosolic Ca(2+) levels triggered by Aβ and thapsigargin, a widely used ER stressor. This effect was reverted in the presence of dantrolene, an inhibitor of ER Ca(2+) release through ryanodine receptors. Similarly, the increase in GRP78 and XBP-1 protein levels was shown to be higher in neurons treated with Aβ or thapsigargin in the presence of KCN in comparison with levels determined in neurons treated with the neurotoxins alone. Although the decrease in cell survival, the activation of caspase-9- and -3-mediated apoptotic cell death observed in Aβ- and thapsigargin-treated neurons were also potentiated by KCN, this effect is less pronounced than that observed in Ca(2+) signalling and UPR. Furthermore, in neurons treated with Aβ, the potentiating effect of the COX inhibitor in cell survival and death was not prevented by dantrolene. These results show that inhibition of mitochondrial COX activity potentiates Aβ-induced ER dysfunction and, to a less extent, neuronal cell death. Furthermore, data supports that the effect of impaired COX on Aβ-induced cell death occurs independently of Ca(2+) release through ER ryanodine receptors. Together, our data demonstrate that mitochondria dysfunction in AD enhances the neuronal susceptibility to toxic insults, namely to Aβ-induced ER stress, and strongly suggest that the close communication between ER and mitochondria can be a valuable future therapeutic target in AD.

Knockdown of stromal interaction molecule 1 (STIM1) suppresses store-operated calcium entry, cell proliferation and tumorigenicity in human epidermoid carcinoma A431 cells

Store-operated calcium (Ca2+) entry (SOCE) is important for cellular activities such as gene transcription, cell cycle progression and proliferation in most non-excitable cells. Stromal interaction molecule 1 (STIM1), a newly identified Ca2+-sensing protein, monitors the depletion of endoplasmic reticulum (ER) Ca2+ stores and activates store-operated Ca2+ channels at the plasma membrane to induce SOCE. To investigate the possible roles of STIM1 in tumor growth in relation to SOCE, we established STIM1 knockdown (KD) clones of human epidermoid carcinoma A431 cells by RNA interference. Thapsigargin, an inhibitor of ER Ca2+-ATPase, -induced and phospholipase C-coupled receptor agonist-induced SOCEs were reduced in two STIM1 KD clones compared to a negative control clone. Re-expression of a KD-resistant full-length STIM1, but not a Ca2+ release-activated Ca2+ channel activation domain (CAD)-deleted STIM1 mutant, in the KD clone restored the amplitude of SOCE, suggesting the specificity of the STIM1 knockdown. The cell growth of the STIM1 KD clones was slower than that of the negative control clone. DNA synthesis assessed by BrdU incorporation, as well as EGF-stimulated EGF receptor activation, decreased in the STIM1 KD clones. Xenograft growth of the STIM1 KD clones was significantly retarded compared with that of the negative control. Cell migration was attenuated in the STIM1 KD clone and the STIM1 silencing effect was reversed by transient re-expression of the full-length STIM1 but not CAD-deletion mutant. These results indicate that STIM1 plays an important role in SOCE, cell-growth and tumorigenicity in human epidermoid carcinoma A431cells, suggesting the potential use of STIM1-targeting agents for treating epidermoid carcinoma.

Oxysterols modulate calcium signalling in the A7r5 aortic smooth muscle cell-line

Prolonged exposure to oxidized low density lipoprotein (oxLDL) can alter various aspects of cell biology, including modification of vasomotor responses and downregulation of calcium channel proteins in aortic smooth muscle cells. However, the components of oxLDL responsible for these effects have not been fully elucidated. The study reported here aimed at examining the consequences of extended exposure to oxysterols, cholesterol oxidation products whose levels are elevated in oxLDL as compared to unmodified LDL, on calcium signalling mechanisms in A7r5 cells, a model aortic smooth muscle cell-line. Within 24h of exposure, all three oxysterol congeners tested caused an elevation in the resting cytoplasmic Ca(2+) concentration. These oxysterols also inhibited Ca(2+) transients in response to arginine vasopressin and bradykinin, and some but not all congeners ablated Ca(2+) signals triggered by platelet activating factor, the ryanodine receptor calcium channel agonist 4-choloro-meta-cresol, or thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+) uptake. The effects of long-term exposure to the oxysterol congener 7β-hydroxycholesterol on arginine vasopressin stimulated Ca(2+) signals were mainly at the level of Ca(2+) release from intracellular stores rather than on Ca(2+) influx mechanisms. Of the calcium signalling proteins tested, only the type 1 ryanodine receptor and the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) were significantly downregulated by 24h exposure to oxysterols. Decreases in IP(3)R1 protein triggered by 7β-hydroxycholesterol were both time and concentration dependent, occurring over a concentration range encountered within atherosclerotic lesions. IP(3)R1 downregulation by certain oxysterols is mediated by proteasomal proteolysis, since it can be abolished by co-incubation with epoxomicin. Overall, these data demonstrate that major oxysterol components of oxLDL cause long-term alterations in Ca(2+) signalling in a model aortic smooth muscle cell. Such effects could contribute to the pathology of atherosclerotic disease.

Inhibition of endoplasmic reticulum Ca2+ ATPase in preBötzinger complex of neonatal rat does not affect respiratory rhythm generation

PreBötzinger complex (preBötC) neurons in the brainstem underlie respiratory rhythm generation in vitro. As a result of network interactions, preBötC neurons burst synchronously to produce rhythmic premotor inspiratory activity. Each inspiratory neuron has a characteristic 10–20mV, 0.3–0.8 s synchronous depolarization known as the inspiratory drive potential or inspiratory envelope, topped by action potentials (APs). Mechanisms involving Ca2+ fluxes have been proposed to underlie the initiation of the inspiratory drive potential. An important source of intracellular Ca2+ is the endoplasmic reticulum (ER) in which active Ca2+ sequestration is mediated by a class of transporters termed sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs). We aim to test the hypothesis that disruption of Ca2+ sequestration into the ER affects respiratory rhythm generation. We examined the effect of inhibiting SERCA on respiratory rhythm generation in an in vitro slice preparation. Bath application of the potent SERCA inhibitors thapsigargin or cyclopiazonic acid (CPA) for up to 90min did not significantly affect the period or amplitude of respiratory-related motor output or integral and duration of inspiratory drive in preBötC neurons. We promoted the depletion of intracellular Ca2+ stores by a transient bath application of 30mM K+ (high K+) in the continuous presence of thapsigargin or CPA. After washing out the high K+, respiratory rhythm period and amplitude returned to baseline values. These results show that after inhibition of SERCA and depletion of intracellular Ca2+ stores, respiratory rhythm remains substantially the same, suggesting that this source of Ca2+ does not significantly contribute to rhythm generation in the preBötC in vitro.

Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum

Most intracellular Ca(2+) signals result from opening of Ca(2+) channels in the plasma membrane or endoplasmic reticulum (ER), and they are reversed by active transport across these membranes or by shuttling Ca(2+) into mitochondria. Ca(2+) channels in lysosomes contribute to endo-lysosomal trafficking and Ca(2+) signalling, but the role of lysosomal Ca(2+) uptake in Ca(2+) signalling is unexplored. Inhibition of lysosomal Ca(2+) uptake by dissipating the H(+) gradient (using bafilomycin A1), perforating lysosomal membranes (using glycyl-L-phenylalanine 2-naphthylamide) or lysosome fusion (using vacuolin) increased the Ca(2+) signals evoked by receptors that stimulate inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] formation. Bafilomycin A1 amplified the Ca(2+) signals evoked by photolysis of caged Ins(1,4,5)P(3) or by inhibition of ER Ca(2+) pumps, and it slowed recovery from them. Ca(2+) signals evoked by store-operated Ca(2+) entry were unaffected by bafilomycin A1. Video-imaging with total internal reflection fluorescence microscopy revealed that lysosomes were motile and remained intimately associated with the ER. Close association of lysosomes with the ER allows them selectively to accumulate Ca(2+) released by Ins(1,4,5)P(3) receptors.

Abstract 9327: Depletion of Intracellular Ca 2+ Stores Induces Phosphorylation of Stromal Interaction Molecular 1 (STIM1), Which Contributes to the Sustained Phase of Store-operated Ca 2+ Influx in Vascular Endothelial Cells

Store-operated Ca 2+ influx (SOC) is a major Ca 2+ entry pathway in vascular endothelial cells, thus contributing to the regulation of endothelial barrier function, nitric oxide production, and angiogenesis. The basic molecular mechanisms underlying SOC have recently been elucidated. Namely, STIM1 serves as a store site Ca 2+ sensor and thereby activates SOC channels. However, the mechanisms regulating SOC remains largely unknown. STIM1 has been previously reported to be a phosphoprotein with unknown function. The present study thus investigated the role of STIM1 phosphorylation in the regulation of SOC. STIM1 phosphorylation was evaluated with a new SDS-PAGE technique utilizing a Phos-tag TM compound, which specifically binds to a phosphate group, thus allowing the separation of phosphoproteins from the unphosphorylated form. Thapsigargin, an inhibitor of endoplasmic reticulum Ca 2+ pump, induced a depletion of the Ca 2+ stores in porcine aortic endothelial cells (PAEC) and HeLa cells in the absence of extracellular Ca 2+ . The following repletion of the external Ca 2+ induced a sustained Ca 2+ influx. STIM1 knockdown abolished this influx. Thapsigargin induced >95% phosphorylation of STIM1 in the absence of extracellular Ca 2+ , which persisted irreversibly even after the repletion of external Ca 2+ . Thrombin induced Ca 2+ depletion and subsequent activation of SOC in PAEC, at a lower level than that seen with thapsigargin. Thrombin also induced a lower level of STIM1 phosphorylation in PAEC. However, it returned to the pre-stimulation level after Ca 2+ repletion. Myosin light chain kinase inhibitors, ML-9 and wortmannin, inhibited thapsigargin-induced STIM1 phosphorylation, while Rho kinase inhibitors, Y27632 and H1152, or PKC inhibitor GF109203X had no effect. The effect of the kinase inhibitors were correlated with the effect on the sustained phase of SOC. Analysis of various STIM1 deletion mutants revealed that the C-terminal region is the site of phosphorylation. These results provide the first evidence that STIM1 is phosphorylated following depletion of Ca 2+ stores and this phosphorylation contributes to the maintenance of Ca 2+ influx via SOC. STIM1 phosphorylation may provide a new therapeutic target to maintain normal endothelial functions.

Streptococcus pneumoniae Infection of Host Epithelial Cells via Polymeric Immunoglobulin Receptor Transiently Induces Calcium Release from Intracellular Stores

The pneumococcal surface protein C (PspC) is a major adhesin of Streptococcus pneumoniae (pneumococci) that interacts in a human-specific manner with the ectodomain of the human polymeric immunoglobulin receptor (pIgR) produced by respiratory epithelial cells. This interaction promotes bacterial colonization and bacterial internalization by initiating host signal transduction cascades. Here, we examined alterations of intracellular calcium ([Ca(2+)](i)) levels in epithelial cells during host cell infections with pneumococci via the PspC-hpIgR mechanism. The release of [Ca(2+)](i) from intracellular stores in host cells was significantly increased by wild-type pneumococci but not by PspC-deficient pneumococci. The increase in [Ca(2+)](i) was dependent on phospholipase C as pretreatment of cells with a phospholipase C-specific inhibitor U73122 abolished the increase in [Ca(2+)](i). In addition, we demonstrated the effect of [Ca(2+)](i) on pneumococcal internalization by epithelial cells. Uptake of pneumococci was significantly increased after pretreatment of epithelial cells with the cell-permeable calcium chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-tetraacetoxymethyl ester or use of EGTA as an extracellular Ca(2+)-chelating agent. In contrast, thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)ATPase, which increases [Ca(2+)](i) in a sustained fashion, significantly reduced pIgR-mediated pneumococcal invasion. Importantly, pneumococcal adherence to pIgR-expressing cells was not altered in the presence of inhibitors as demonstrated by immunofluorescence microscopy. In conclusion, these results demonstrate that pneumococcal infections induce mobilization of [Ca(2+)](i) from intracellular stores. This may constitute a defense response of host cells as the experimental reduction of intracellular calcium levels facilitates pneumococcal internalization by pIgR-expressing cells, whereas elevated calcium levels diminished bacterial internalization by host epithelial cells.

Calcium mobilization by the plant estrogen ferutinin does not induce blood platelet aggregation

Platelet activation is closely associated with an increase in intracellular Ca2+ concentration. Various compounds including Ca2+ ionophores are able to trigger platelet aggregation by increasing intracellular Ca2+ concentration in platelets. In the present study, we monitored the effect of the phytoestrogen ferutinin, which acts as a Ca2+ ionophore in human blood platelets; its ionophore-like properties include upregulation of [Ca2+]in, activation of fibrinogen receptors and increased fibrinogen binding. Using spectrofluorometry and triple-color flow cytometry, we demonstrate that ferutinin increases [Ca ] in both isolated platelets and platelets in whole blood from humans. This effect was almost completely blocked by the Ca2+ chelator EGTA and was not sensitive to either Gd3+ or econazole, which inhibit VOC and SOC channels, respectively. Nor was the effect sensitive to thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPases. Ferutinin stimulated the expression of the active form of the GPIIb-IIIa complex and whole blood platelet aggregation only weakly and had no statistically significant effect on the binding of fibrinogen. These results demonstrate apparently inconsistent effects of ferutinin, which raises intraplatelet Ca2+ concentration but fails to have an effect on spontaneous blood platelet aggregation. This pattern of responses may be caused by the combination of ferutinin’s Ca2+ ionophoric and estrogenic properties.

Intracellular calcium homeostasis in patients with early stagesof chronic kidney disease: effects of vitamin D3 supplementation

Chronic renal failure has been referred to as a state of cellular calcium toxicity. The aim of this study was to investigate the status of free cytosolic calcium ([Ca(2+)](i)), intracellular calcium reserves and the capacitative calcium entry in peripheral blood mononuclear cells (PBMCs) of early-stage chronic kidney disease (CKD) patients, and to determine the effect of vitamin D(3) supplementation on these parameters. The study involved 44 patients with CKD stages 2-3; 27 of them were treated with cholecalciferol (5000 IU/week) for 12 months. [Ca(2+)](i) was measured using Fluo-3 AM fluorimetry. Intracellular calcium reserves were emptied by the application of thapsigargin (Tg), a specific inhibitor of endoplasmic reticulum Ca(2+)-ATPase. 2-Aminoethyl-diphenyl borate (2APB) was used to examine the capacitative calcium entry. [Ca(2+)](i) of CKD patients was substantially higher in comparison with healthy subjects: 123 (115-127) versus 102 (98-103) nmol/l, P < 0.001. The calcium concentration of Tg-sensitive stores and the capacitative calcium entry were also significantly increased in CKD patients. After the 12-month vitamin D(3) supplementation, there was a marked decrease in [Ca(2+)](i) [105 (103-112) nmol/l, P < 0.001 versus baseline], independently of the increase in 25(OH)D(3) or the decrease in PTH levels. No significant changes in intracellular calcium reserves and the capacitative calcium entry were found. Our results demonstrate that (1) [Ca(2+)](i), intracellular calcium stores and the capacitative calcium entry were significantly increased already in early stages of CKD; (2) long-term vitamin D(3) supplementation normalized [Ca(2+)](i) without any effect on intracellular calcium reserves or the capacitative calcium entry.

CGMP-independent anti-apoptotic effect of nitric oxide on thapsigargin-induced apoptosis in the pancreatic β-cell line INS-1

Aims Low concentrations of nitric oxide (NO) produced by constitutive NO synthase (cNOS) in pancreatic β-cells have been suggested to be a physiological regulator of insulin secretion. In contrast, excessive NO produced by inducible NO synthase is known to mediate β-cell apoptosis. The aim of the present study was to investigate the effect of low concentrations of NO on β-cell apoptosis. Main methods Apoptosis of the pancreatic β-cell line INS-1 was quantitatively determined by Annexin V flow cytometry. Key findings The 24-h incubation with 1 mM DETA/NO, a long half-life NO donor, induced β-cell apoptosis, which was insensitive to the soluble guanylate cyclase (sGC) inhibitor ODQ. In contrast, DETA/NO at lower concentrations until 300 μM concentration-dependently decreased the apoptosis induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca 2+-ATPase. ODQ did not affect the anti-apoptotic effect of DETA/NO. Moreover, neither the cGMP analogue 8-Br-cGMP nor the sGC activator YC-1 mimicked the anti-apoptotic effect of DETA/NO. Significance These results suggest that low levels of NO protect β-cells from thapsigargin-induced apoptosis in a cGMP-independent manner.

Neurotoxic effect of oligomeric and fibrillar species of amyloid-beta peptide 1-42: Involvement of endoplasmic reticulum calcium release in oligomer-induced cell death

The nature of the toxic form of amyloid-beta peptide (Aβ) involved in early Alzheimer's disease (AD) pathology and whether it is the fibrillar or the oligomeric peptide that is the most deleterious to neurons remain controversial. This work aimed to compare the neurotoxicity of different amyloid-beta peptide 1-42 (Aβ1-42) assemblies, using fresh and aged samples enriched in oligomeric and fibrillar species, respectively, and also isolated oligomers and fibrils. The results obtained with fresh and aged Aβ1-42 preparations suggested that oligomeric species are more toxic to cortical neurons in culture than fibrillar forms, which was confirmed by using isolated oligomers and fibrils. In order to further elucidate the mechanisms involved in soluble Aβ toxicity, the involvement of endoplasmic reticulum (ER) calcium (Ca 2+) release in oligomer-induced apoptosis was evaluated. We observed that oligomeric Aβ1-42 depletes ER Ca 2+ levels leading to intracellular Ca 2+ dyshomeostasis involving phospholipase C activation. Moreover, in the presence of dantrolene, an inhibitor of ER Ca 2+ release through ryanodine receptors, the oligomer-induced apoptosis was prevented demonstrating the involvement of ER Ca 2+ release.

Effects of nonylphenol on the calcium signal and catecholamine secretion coupled with nicotinic acetylcholine receptors in bovine adrenal chromaffin cells

Nonylphenol (NP) is the most critical metabolite of alkylphenol polyethoxylate detergents. NP is known as an endocrine disruptor with estrogenic activities and as an inhibitor of endoplasmic reticulum Ca 2+-ATPase. Estrogen has modulatory roles on ligand-gated ion channels, such as nicotinic acetylcholine receptors (nAChRs). Ca 2+-ATPase inhibitors can modulate the cytosolic calcium concentration ([Ca 2+] c]) and thus can affect the calcium signaling coupled with nAChRs. Therefore, NP is predicted to have complex effects on the Ca 2+ signaling and secretion coupled with nAChRs. This study investigated these effects using bovine adrenal chromaffin cells. The results show that NP suppressed the Ca 2+ signaling coupled with nAChRs and voltage-operated Ca 2+ channels in a dose-dependent manner, with IC 50s of 1 and 5.9 μM, respectively. Estradiol exhibits similar suppression but much lower inhibitory potencies. NP alone induced a transient rise in [Ca 2+] c in the presence or absence of extracellular calcium. Thapsigargin, an endoplasmic reticulum Ca 2+-ATPase inhibitor, partially suppressed the [Ca 2+] c rise induced by NP, but NP totally blocked the [Ca 2+] c rise induced by thapsigargin. This illustrates that NP can cause Ca 2+ release from thapsigargin-insensitive pools. Thapsigargin suppressed the Ca 2+ signaling coupled with nAChRs but increased that coupled with voltage-operated Ca 2+ channels. We propose that three routes are responsible for the effects of NP on nAChRs: named receptor channels, voltage-gated Ca 2+ channels, and Ca 2+-induced Ca 2+ release. Three routes are related to the characteristics of NP as steroid-like compounds and Ca 2+-ATPase inhibitor.

Modulation of Ca 2+ transients in cultured endothelial cells in response to fluid flow through αv integrin

In order to determine whether integrin dynamics is associated with intracellular Ca 2+ concentration ([Ca 2+] i) mobilization in ECs in response to hemodynamic forces, changes in [Ca 2+] i in fluo-4-loaded cultured bovine aortic endothelial cells (BAECs) under fluid flow conditions were visualized employing laser scanning confocal microscopy. Following the onset of flow stimulus, transient increases in [Ca 2+] i occurred several times in individual BAECs during the 30-min observation period. The frequency of these [Ca 2+] i transients was clearly reduced by the application of an integrin antagonist (GRGDSP peptide). Furthermore, treatment of cells with an integrin activator (Mn 2+) resulted in reduction of peak [Ca 2+] i levels and elevated frequency, which was markedly rescued upon GRGDSP administration. In contrast, an actin de-polymerizing agent (cytochalasin D) exerted no inhibitory effects; rather, cytochalasin D more likely facilitated [Ca 2+] i transients. Moreover, [Ca 2+] i transients, which were suppressed by short interference RNA-induced silencing of αv integrin, exhibited greater frequently in cells cultured on vitronectin substratum in comparison with those cultured on fibronectin or collagen substratum. Either removal of extracellular Ca 2+, application of an inhibitor of endoplasmic reticulum Ca 2+-ATPase (thapsigargin) or non-selective cation channel blocker (La 3+) inhibited the [Ca 2+] i transients. Additionally, [Ca 2+] i transients were attenuated by extracellular signal-regulated kinase (ERK) kinase inhibitor (U0126); in contrast, [Ca 2+] i transients were unaffected by tyrosine kinase inhibitor (genistein) or phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002). Therefore, our findings revealed that αv integrin dynamics modulates the frequency of flow-induced [Ca 2+] i transients in BAECs in an ERK-dependent fashion.

Dynamic Inositol Trisphosphate-mediated Calcium Signals within Astrocytic Endfeet Underlie Vasodilation of Cerebral Arterioles

Active neurons communicate to intracerebral arterioles in part through an elevation of cytosolic Ca2+ concentration ([Ca2+]i) in astrocytes, leading to the generation of vasoactive signals involved in neurovascular coupling. In particular, [Ca2+]i increases in astrocytic processes (“endfeet”), which encase cerebral arterioles, have been shown to result in vasodilation of arterioles in vivo. However, the spatial and temporal properties of endfoot [Ca2+]i signals have not been characterized, and information regarding the mechanism by which these signals arise is lacking. [Ca2+]i signaling in astrocytic endfeet was measured with high spatiotemporal resolution in cortical brain slices, using a fluorescent Ca2+ indicator and confocal microscopy. Increases in endfoot [Ca2+]i preceded vasodilation of arterioles within cortical slices, as detected by simultaneous measurement of endfoot [Ca2+]i and vascular diameter. Neuronal activity–evoked elevation of endfoot [Ca2+]i was reduced by inhibition of inositol 1,4,5-trisphosphate (InsP3) receptor Ca2+ release channels and almost completely abolished by inhibition of endoplasmic reticulum Ca2+ uptake. To probe the Ca2+ release mechanisms present within endfeet, spatially restricted flash photolysis of caged InsP3 was utilized to liberate InsP3 directly within endfeet. This maneuver generated large amplitude [Ca2+]i increases within endfeet that were spatially restricted to this region of the astrocyte. These InsP3-induced [Ca2+]i increases were sensitive to depletion of the intracellular Ca2+ store, but not to ryanodine, suggesting that Ca2+-induced Ca2+ release from ryanodine receptors does not contribute to the generation of endfoot [Ca2+]i signals. Neuronally evoked increases in astrocytic [Ca2+]i propagated through perivascular astrocytic processes and endfeet as multiple, distinct [Ca2+]i waves and exhibited a high degree of spatial heterogeneity. Regenerative Ca2+ release processes within the endfeet were evident, as were localized regions of Ca2+ release, and treatment of slices with the vasoactive neuropeptides somatostatin and vasoactive intestinal peptide was capable of inducing endfoot [Ca2+]i increases, suggesting the potential for signaling between local interneurons and astrocytic endfeet in the cortex. Furthermore, photorelease of InsP3 within individual endfeet resulted in a local vasodilation of adjacent arterioles, supporting the concept that astrocytic endfeet function as local “vasoregulatory units” by translating information from active neurons into complex InsP3-mediated Ca2+ release signals that modulate arteriolar diameter.

Mitochondrial free cholesterol loading sensitizes to TNF- and Fas-mediated steatohepatitis

The etiology of progression from steatosis to steatohepatitis (SH) remains unknown. Using nutritional and genetic models of hepatic steatosis, we show that free cholesterol (FC) loading, but not free fatty acids or triglycerides, sensitizes to TNF- and Fas-induced SH. FC distribution in endoplasmic reticulum (ER) and plasma membrane did not cause ER stress or alter TNF signaling. Rather, mitochondrial FC loading accounted for the hepatocellular sensitivity to TNF due to mitochondrial glutathione (mGSH) depletion. Selective mGSH depletion in primary hepatocytes recapitulated the susceptibility to TNF and Fas seen in FC-loaded hepatocytes; its repletion rescued FC-loaded livers from TNF-mediated SH. Moreover, hepatocytes from mice lacking NPC1, a late endosomal cholesterol trafficking protein, or from obese ob/ob mice, exhibited mitochondrial FC accumulation, mGSH depletion, and susceptibility to TNF. Thus, we propose a critical role for mitochondrial FC loading in precipitating SH, by sensitizing hepatocytes to TNF and Fas through mGSH depletion.

A Novel Effect of Bifemelane, a Nootropic Drug, on Intracellular Ca2+ Levels in Rat Cerebral Astrocytes

We investigated the effects of bifemelane, a nootropic drug, on the intracellular calcium concentration ([Ca2+]i) in rat cerebral astrocytes using a Ca2+ imaging device. At concentrations of 10 - 30 microM, bifemelane induced a slow onset and small increase in the [Ca2+]i, while at higher concentrations (100 - 300 microM), it induced a rapid transient increase in the [Ca2+]i during administration and a second large increase was seen during drug washout. The first peak was observed in Ca2+-free medium, but its onset was significantly delayed, and no second peak was seen. Neither of these effects was seen in cells treated with thapsigargin, a specific inhibitor of endoplasmic reticulum Ca2+-ATPase, in Ca2+-free medium. When thapsigargin-treated astrocytes were returned to normal medium containing Ca2+ (1.8 mM), the [Ca2+]i increased significantly, and this effect was reversely inhibited by bifemelane. We conclude that bifemelane causes the first peak by stimulating release from intracellular Ca2+ stores and the second by capacitive entry through store-operated Ca2+ channels. Although the detail mechanisms of action of the drug are still unknown, bifemelane will be provided as a pharmacological tool for basic studies on astrocytes.

Serotonin induces the increase in intracellular Ca2+ that enhances neurite outgrowth in PC12 cells via activation of 5-HT3 receptors and voltage-gated calcium channels

As a neurotransmitter and neuromodulator, serotonin (5-HT) influences neuronal outgrowth in the nervous systems of several species. In PC12 cells, 5-HT is known to have neuritogenic effects, although the signal transduction pathway responsible for these effects is not understood. In this study, we hypothesized that a 5-HT-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) could be involved in mediating the effects of 5-HT. Application of 5-HT to PC12 cells enhanced nerve growth factor (NGF)-induced neurite outgrowth in a dose-dependent manner, and the sensitivity of this neuritogenic effect was increased in differentiated PC12 cells. In accordance, an increase in [Ca(2+)](i) was observed following application of 5-HT in differentiated PC12 cells. This increase was amplified by further NGF treatment. 5-HT-induced increases in [Ca(2+)](i) were inhibited by MDL 72222, a selective 5-HT(3) receptor antagonist, and nifedipine, an L-type calcium channel blocker, but not by ketanserin, a 5-HT(2) receptor antagonist, or thapsigargin, a specific inhibitor of endoplasmic reticulum Ca(2+)-ATPase. These pharmacological tests indicated that 5-HT-induced increases in [Ca(2+)](i) are mediated by activation of voltage-gated calcium channels via 5-HT(3) receptors and that 5-HT-induced increases in [Ca(2+)](i) are likely to be independent of activation of 5-HT(2) receptors in PC12 cells. Furthermore, the neuritogenic effect of 5-HT was suppressed by MDL 72222, nifedipine, calmodulin (CaM) inhibitor, and calcineurin inhibitors. Taken together, our results indicate that 5-HT-induced increases in [Ca(2+)](i), which are mediated via 5-HT(3) receptors and L-type calcium channels in PC12 cells, and subsequent activation of CaM and calcineurin enhance NGF-induced neurite outgrowth.

Modulator of intracellular Ca&lt;sup&gt;2+&lt;/sup&gt;, Thapsigargin, interferes with in vitro secretion of cytokines and nitric oxide

Interference of thapsigargin (TG), an inhibitor of endoplasmic reticulum Ca(2+) ATPase, with immune reactivity of murine macrophages was investigated under conditions in vitro. The activation of cells with lipopolysaccharide (LPS), interferon-(gamma) (IFN-(gamma)), and with acyclic nucleoside phosphonate N(6)-isobutyl-9-[2-(phosphonomethoxy)ethyl]- 2,6-diaminopurine (N(6)-isobutyl-PMEDAP) resulted in enhanced production of cytokines TNF-alpha, IL-10, chemokines RANTES/CCL5 and MIP-1alpha/CCL3, as well as in substantially augmented production of nitric oxide (NO) triggered by IFN-(gamma). The effects were in a dual mode of action influenced by TG (1 microM). While TG upregulated secretion of TNF-alpha, it inhibited secretion of IL-10 and RANTES. The immune-stimulated secretion of MIP-1alpha remained virtually unaffected, though TG on its own activated expression of MIP-1alpha in macrophages. The high-output NO production induced by IFN-(gamma), high concentrations of LPS, or by combination of IFN-(gamma) plus LPS or N(6)-isobutyl-PMEDAP was inhibited by TG. On the other hand, production of NO which was marginally activated by low concentration of LPS was upregulated by TG.

Early events responsible for aluminum toxicity symptoms in suspension‐cultured tobacco cells

We investigated the aluminum (Al)-induced alterations in zeta potential, plasma membrane (PM) potential and intracellular calcium levels to elucidate their interaction with callose production induced by Al toxicity. A noninvasive confocal laser microscopy has been used to analyse the live tobacco (Nicotiana tabacum) cell events by means of fluorescent probes Fluo-3 acetoxymethyl ester (intracellular calcium) and DiBAC4 (PM potential) as well as to monitor callose accumulation. Log-phase cells showed no detectable changes in the PM potential during the first 30 min of Al treatment, but sustained large depolarization from 60 min onwards. Measurement of zeta potential confirmed the depolarization effect of Al, but the kinetics were different. The Al-treated cells showed a moderate increase in intracellular Ca2+ levels and callose production in 1 h, which coincided with the time course of PM depolarization. Compared with the Al treatment, cyclopiazonic acid, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase, facilitated a higher increase in intracellular Ca2+ levels, but resulted in accumulation of only moderate levels of callose. Calcium channel modulators and Al induced similar levels of callose in the initial 1 h of treatment. Callose production induced by Al toxicity is dependent on both depolarization of the PM and an increase in intracellular Ca2+ levels.