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Find video protocols related to scientific articles indexed in Pubmed.
Upregulation of store operated Ca channel Orai1, stimulation of Ca(2+) entry and triggering of cell membrane scrambling in platelets by mineralocorticoid DOCA.
Kidney Blood Press. Res.
PUBLISHED: 12-20-2014
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Mineralocorticoid excess leads to vascular injury, which is partially due to hypertension but in addition involves increased concentration of cytosolic Ca(2+) concentration in platelets, key players in the pathophysiology of occlusive vascular disease. Mineralocorticoids are in part effective by rapid nongenomic mechanisms including phosphatidylinositide-3-kinase (PI3K) signaling, which involves activation of the serum & glucocorticoid inducible kinase (SGK) isoforms. SGK1 has in turn been shown to participate in the regulation of the pore forming Ca(2+) channel protein Orai1 in platelets. Orai1 accomplishes entry of Ca(2+), which is in turn known to trigger cell membrane scrambling. Platelets lack nuclei but are able to express protein by translation, which is stimulated by PI3K signaling. The present study explored whether the mineralocorticoid desoxycorticosterone acetate (DOCA) influences platelet Orai1 protein abundance, cytosolic Ca(2+)-activity ([Ca(2+)]i), phosphatidylserine abundance at the cell surface and/or cell volume.
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Stimulation of Suicidal Erythrocyte Death by Ellipticine.
Basic Clin. Pharmacol. Toxicol.
PUBLISHED: 11-15-2014
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Ellipticine is a potent antineoplastic alkaloid effective in part by triggering apoptosis. Mechanisms involved in ellipticine-induced apoptosis include mitochondrial depolarization and DNA damage. Erythrocytes lack mitochondria and nuclei but may nevertheless enter suidical death or eryptosis, which is characterized by cell shrinkage and phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+) ]i ), ceramide formation and oxidative stress. The present study tested whether ellipticine stimulates eryptosis. Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca(2+) ]i from Fluo3-fluorescence, ceramide abundance from binding of specific antibodies and reactive oxygen species from 2',7'-dichlorodihydrofluorescein diacetate fluorescence. A 24-hr exposure of human erythrocytes to ellipticine (5 ?g/ml) significantly increased the percentage of annexin V binding cells, ceramide abundance and oxidative stress. Ellipticine did not significantly modify [Ca(2+) ]i and the stimulation of annexin V binding by ellipticine (5 ?g/ml) did not require presence of extracellular Ca(2+) . Ellipticine (5 ?g/ml) did not significantly modify forward scatter. Ionomycin (1 ?M, 1 hr) decreased forward scatter, an effect slightly but significantly blunted by ellipticine (5 ?g/ml). Ellipticine stimulates phosphatidylserine translocation in the erythrocyte cell membrane, an effect at least partially due to stimulation of oxidative stress and ceramide formation. This article is protected by copyright. All rights reserved.
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Impact of Aldosterone on Osteoinductive Signaling and Vascular Calcification.
Nephron Physiol
PUBLISHED: 11-06-2014
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Vascular calcification is frequently found already in early stages of chronic kidney disease (CKD) patients and is associated with high cardiovascular risk. The process of vascular calcification is not considered a passive phenomenon but involves, at least in part, phenotypical transformation of vascular smooth muscle cells (VSMCs). Following exposure to excessive extracellular phosphate concentrations, VSMCs undergo a reprogramming into osteo-/chondroblast-like cells. Such 'vascular osteoinduction' is characterized by expression of osteogenic transcription factors and triggered by increased phosphate concentrations. A key role in this process is assigned to cellular phosphate transporters, most notably the type III sodium-dependent phosphate transporter Pit1. Pit1 expression is stimulated by mineralocorticoid receptor activation. Therefore, aldosterone participates in the phenotypical transformation of VSMCs. In preclinical models, aldosterone antagonism reduces vascular osteoinduction. Patients with CKD suffer from hyperphosphatemia predisposing to vascular osteogenic transformation, potentially further fostered by concomitant hyperaldosteronism. Clearly, additional research is required to define the role of aldosterone in the regulation of osteogenic signaling and the consecutive vascular calcification in CKD, but more generally also other diseases associated with excessive vascular calcification and even in individuals without overt disease. © 2014 S. Karger AG, Basel.
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Mineralocorticoid and SGK1-Sensitive Inflammation and Tissue Fibrosis.
Nephron Physiol
PUBLISHED: 11-06-2014
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Effects of mineralocorticoids are not restricted to regulation of epithelial salt transport, extracellular volume and blood pressure; mineralocorticoids also influence a wide variety of seemingly unrelated functions such as inflammation and fibrosis. The present brief review addresses the role of mineralocorticoids in the orchestration of these latter processes. Mineralocorticoids foster inflammation as well as vascular, cardiac, renal and peritoneal fibrosis. Mechanisms involved in mineralocorticoid-sensitive inflammation and fibrosis include the serum- and glucocorticoid-inducible kinase 1 (SGK1), which is genomically upregulated by mineralocorticoids and transforming growth factor ? (TGF-?), and stimulated by mineralocorticoid-sensitive phosphatidylinositide 3-kinase. SGK1 upregulates the inflammatory transcription factor nuclear factor-?B, which in turn stimulates the expression of diverse inflammatory mediators including connective tissue growth factor. Moreover, SGK1 inhibits the degradation of the TGF-?-dependent transcription factors Smad2/3. Mineralocorticoids foster the development of TH17 cells, which is compromised following SGK1 deletion. Excessive SGK1 expression is observed in a wide variety of fibrosing diseases including lung fibrosis, diabetic nephropathy, glomerulonephritis, obstructive kidney disease, experimental nephrotic syndrome, obstructive nephropathy, liver cirrhosis, fibrosing pancreatitis, peritoneal fibrosis, Crohn's disease and celiac disease. The untoward inflammatory and fibrosing effects of mineralocorticoids could be blunted or even reversed by mineralocorticoid receptor blockers, which may thus be considered in the treatment of inflammatory and/or fibrosing disease. © 2014 S. Karger AG, Basel.
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On the Pleotropic Actions of Mineralocorticoids.
Nephron Physiol
PUBLISHED: 11-05-2014
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Classical effects of mineralocorticoids include stimulation of Na(+) reabsorption and K(+) secretion in the kidney and other epithelia including colon and several glands. Moreover, mineralocorticoids enhance the excretion of Mg(2+) and renal tubular H(+) secretion. The renal salt retention following mineralocorticoid excess leads to extracellular volume expansion and hypertension. The increase of blood pressure following mineralocorticoid excess is, however, not only the result of volume expansion but may result from stiff endothelial cell syndrome impairing the release of vasodilating nitric oxide. Beyond that, mineralocorticoids are involved in the regulation of a wide variety of further functions, including cardiac fibrosis, platelet activation, neuronal function and survival, inflammation as well as vascular and tissue fibrosis and calcification. Those functions are briefly discussed in this short introduction to the special issue. Beyond that, further contributions of this special issue amplify on mineralocorticoid-induced sodium appetite and renal salt retention, the role of mineralocorticoids in the regulation of acid-base balance, the involvement of aldosterone and its receptors in major depression, the mineralocorticoid stimulation of inflammation and tissue fibrosis and the effect of aldosterone on osteoinductive signaling and vascular calcification. Clearly, still much is to be learned about the various ramifications of mineralocorticoid-sensitive physiology and pathophysiology. © 2014 S. Karger AG, Basel.
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Macrophage Migration Inhibitory Factor Limits Activation-Induced Apoptosis of Platelets via CXCR7-Dependent Akt Signaling.
Circ. Res.
PUBLISHED: 09-29-2014
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Macrophage migration inhibitory factor (MIF) is released on platelet activation. Circulating MIF could potentially regulate platelets and thereby platelet-mediated inflammatory and regenerative mechanisms. However, the effect of MIF on platelets is unknown.
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Enhanced Eryptosis following Juglone Exposure.
Basic Clin. Pharmacol. Toxicol.
PUBLISHED: 09-17-2014
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Juglone, a quinone isolated from Juglans mandshurica Maxim, has previously been shown to be effective against malignancy. The effect is at least partially due to stimulation of suicidal death or apoptosis of tumour cells. On the other hand, juglone has been shown to counteract apoptosis, e.g. of neurons. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and breakdown of phosphatidylserine asymmetry of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+) ]i ). The present study explored whether juglone stimulates eryptosis. To this end, erythrocyte volume was estimated from forward scatter, phosphatidylserine exposure at the erythrocyte surface from FITC annexin V binding, ceramide abundance from binding of fluorescent antibodies in flow cytometry, and cytosolic ATP with a luciferin-luciferase-based assay. As a result, a 24-hr exposure of human erythrocytes to juglone (5 ?M) significantly decreased erythrocyte forward scatter. Juglone (1-5 ?M) significantly increased the percentage of annexin V binding cells. Juglone (5 ?M) significantly increased ceramide abundance at the erythrocyte surface and decreased erythrocyte ATP concentration. The effect of juglone (10 ?M) on annexin V binding was slightly but significantly blunted by removal of extracellular Ca(2+) and by addition of protein kinase C (PKC) inhibitor staurosporine (1 ?M). In conclusion, juglone stimulates suicidal erythrocyte death or eryptosis at least in part by up-regulation of ceramide abundance, energy depletion and activation of PKC. This article is protected by copyright. All rights reserved.
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Sphingosine kinase 1 (Sphk1) negatively regulates platelet activation and thrombus formation.
Am. J. Physiol., Cell Physiol.
PUBLISHED: 09-17-2014
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Sphingosine 1-phosphate (S1P) is a powerful regulator of platelet formation. Enzymes generating S1P include sphingosine kinase 1. The present study thus explored the role of sphingosine kinase 1 in platelet formation and function. Activation-dependent platelet integrin ?IIb?3 activation and secretion of platelets lacking functional sphingosine kinase 1 (sphk1(-/-)) and of wild-type platelets (sphk1(+/+)) were determined utilizing flow cytometry and chronolume luciferin assay. Cytosolic Ca(2+) activity ([Ca(2+)]i) and aggregation were measured using fura-2 fluorescence and aggregometry, respectively. In vitro platelet adhesion and thrombus formation were evaluated using a flow chamber with shear rates of 1,700 s(-1). Activation-dependent increase of [Ca(2+)]i, degranulation (release of alpha and dense granules), integrin ?IIb?3 activation, and aggregation were all significantly increased in sphk1(-/-) platelets compared with sphk1(+/+) platelets. Moreover, while platelet adhesion and thrombus formation under arterial shear rates were significantly augmented in Sphk1-deficient platelets, bleeding time and blood count were unaffected in sphk1(-/-) mice. In conclusion, sphingosine kinase 1 is a powerful negative regulator of platelet function counteracting degranulation, aggregation, and thrombus formation.
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Membrane Androgen Receptor Down-Regulates c-Src-Activity and Beta-Catenin Transcription and Triggers GSK-3beta-Phosphorylation in Colon Tumor Cells.
Cell. Physiol. Biochem.
PUBLISHED: 09-15-2014
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Functional membrane androgen receptors (mARs) have recently been described in colon tumor cells and tissues. Their activation by specific testosterone albumin conjugates (TAC) down-regulates the PI-3K/Akt pro-survival signaling and triggers potent pro-apoptotic responses both, in vitro and in vivo. The present study explored the mAR-induced regulation of gene products implicated in the tumorigenic activity of Caco2 colon cancer cells.
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Effect of TGF? on Na+/K+ ATPase activity in megakaryocytes.
Biochem. Biophys. Res. Commun.
PUBLISHED: 08-27-2014
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The Na(+)/K(+) ATPase generates the Na(+) and K(+) concentration gradients across the plasma membrane and is thus essential for cellular electrolyte homeostasis, cell membrane potential and cell volume maintenance. A powerful regulator of Na(+)/K(+) ATPase is the serum- and glucocorticoid-inducible kinase 1 (SGK1). The most powerful known regulator of SGK1 expression is TGFß1, which is pivotal in the regulation of megakaryocyte maturation and platelet formation. Signaling involved in the upregulation of SGK1 by TGFß1 includes p38 mitogen activated protein (MAP) kinase. SGK1 in turn phosphorylates the I?B kinase (IKK?/?), which phosphorylates the inhibitor protein I?B? thus triggering nuclear translocation of nuclear factor kappa B (NF-?B). The present study explored whether TGF? influences Na(+)/K(+) ATPase activity in megakaryocytes, and if so, whether the effect of TGß1 requires p38 MAP kinase, SGK1 and/or NF-?B. To this end, murine megakaryocytes were treated with TGFß1 and Na(+)/K(+) ATPase activity determined from K(+) induced current utilizing whole cell patch clamp. The pump current (Ipump) was determined in the absence and presence of Na(+)/K(+) ATPase inhibitor ouabain (100?M). TGFß1 (60ng/ml) was added in the absence or presence of p38 MAP kinase inhibitor skepinone-L (1?M), SGK1 inhibitor EMD638683 (50?M) or NF-?B inhibitor wogonin (50nM). As a result, the Ipump was significantly increased by pretreatment of the megakaryocytes with TGFß1, an effect reaching statistical significance within 16 and 24h and virtually abrogated in the presence of skepinone-L, EMD638683 or wogonin. In conclusion, TGFß1 is a powerful regulator of megakaryocytic Na(+)/K(+) ATPase activity. Signaling mediating the effect of TGFß1 on Na(+)/K(+) ATPase activity involves p38 MAP kinase, SGK1 and NF-?B.
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SPAK-Sensitive Regulation of Glucose Transporter SGLT1.
J. Membr. Biol.
PUBLISHED: 08-27-2014
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The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase SPAK is a powerful regulator of ion transport. The study explored whether SPAK similarly regulates nutrient transporters, such as the Na(+)-coupled glucose transporter SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of wild-type SPAK, constitutively active (T233E)SPAK, WNK-insensitive (T233A)SPAK or catalytically inactive (D212A)SPAK, and electrogenic glucose transport determined by dual-electrode voltage-clamp experiments. Moreover, Ussing chamber was employed to determine the electrogenic glucose transport in intestine from wild-type mice (spak (wt/wt)) and from gene-targeted mice carrying WNK-insensitive SPAK (spak (tg/tg)). In SGLT1-expressing oocytes, but not in water-injected oocytes, the glucose-dependent current (I g) was significantly decreased following coexpression of wild-type SPAK and (T233E)SPAK, but not by coexpression of (T233A)SPAK or (D212A)SPAK. Kinetic analysis revealed that SPAK decreased maximal I g without significantly modifying the glucose concentration required for halfmaximal I g (K m). According to the chemiluminescence experiments, wild-type SPAK but not (D212A)SPAK decreased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5 ?M) resulted in a decline of I g, which was similar in the absence and presence of SPAK, suggesting that SPAK did not accelerate the retrieval of SGLT1 protein from the cell membrane but rather down-regulated carrier insertion into the cell membrane. Intestinal electrogenic glucose transport was significantly lower in spak (wt/wt) than in spak (tg/tg) mice. In conclusion, SPAK is a powerful negative regulator of SGLT1 protein abundance in the cell membrane and thus of electrogenic glucose transport.
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SPAK and OSR1 Sensitivity of Voltage-Gated K(+) Channel Kv1.5.
J. Membr. Biol.
PUBLISHED: 08-26-2014
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SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1) are potent regulators of several transporters and ion channels. The kinases are under regulation of with-no-K(Lys) (WNK) kinases. The present study explored whether SPAK and/or OSR1 modify the expression and/or activity of the voltage-gated K(+) channel Kv1.5, which participates in the regulation of diverse functions including atrial cardiac action potential and tumor cell proliferation. cRNA encoding Kv1.5 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, wild-type OSR1, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1, and catalytically inactive (D164A)OSR1. Voltage-gated K(+) channel activity was quantified utilizing dual electrode voltage clamp and Kv1.5 channel protein abundance in the cell membrane utilizing chemiluminescence of Kv1.5 containing an extracellular hemagglutinin epitope (Kv1.5-HA). Kv1.5 activity and Kv1.5-HA protein abundance were significantly decreased by wild-type SPAK and (T233E)SPAK, but not by (T233A)SPAK and (D212A)SPAK. Similarly, Kv1.5 activity and Kv1.5-HA protein abundance were significantly down-regulated by wild-type OSR1 and (T185E)OSR1, but not by (T185A)OSR1 and (D164A)OSR1. Both, SPAK and OSR1 decrease cell membrane Kv1.5 protein abundance and activity.
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SPAK Dependent Regulation of Peptide Transporters PEPT1 and PEPT2.
Kidney Blood Press. Res.
PUBLISHED: 08-25-2014
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SPAK (STE20-related proline/alanine-rich kinase) is a powerful regulator of renal tubular ion transport and blood pressure. Moreover, SPAK contributes to the regulation of cell volume. Little is known, however, about a role of SPAK in the regulation or organic solutes. The present study thus addressed the influence of SPAK on the peptide transporters PEPT1 and PEPT2.
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Maternal vitamin d deficiency and fetal programming - lessons learned from humans and mice.
Kidney Blood Press. Res.
PUBLISHED: 08-19-2014
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Cardiovascular disease partially originates from poor environmental and nutritional conditions in early life. Lack of micronutrients like 25 hydroxy vitamin D3 (25OHD) during pregnancy may be an important treatable causal factor. The present study explored the effect of maternal 25OHD deficiency on the offspring.
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Stimulation of Suicidal Erythrocyte Death by Sulforaphane.
Basic Clin. Pharmacol. Toxicol.
PUBLISHED: 08-18-2014
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Sulforaphane, an isothiocyanate from cruciferous vegetable, counteracts malignancy. The effect is at least in part due to the stimulation of suicidal death or apoptosis of tumour cells. Mechanisms invoked in sulforaphane-induced apoptosis include mitochondrial depolarization and altered gene expression. Despite the lack of mitochondria and nuclei, erythrocytes may, similar to apoptosis of nucleated cells, enter eryptosis, a suicidal cell death characterized by cell shrinkage and phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca(2+) -activity ([Ca(2+) ]i ). This study explored whether sulforaphane stimulates eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure at the cell surface from annexin V binding and [Ca(2+) ]i from Fluo-3 fluorescence. A 48-hr treatment of human erythrocytes with sulforaphane (50-100 ?M) significantly decreased forward scatter, significantly increased the percentage of annexin V binding cells and significantly increased [Ca(2+) ]i . The effect of sulforaphane (100 ?M) on annexin V binding was significantly blunted but not abrogated by the removal of extracellular Ca(2+) . Sulforaphane (100 ?M) significantly increased ceramide formation. In conclusion, sulforaphane stimulates suicidal erythrocyte death or eryptosis, an effect at least partially, but not exclusively, due to the stimulation of Ca(2+) entry and ceramide formation.
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Glucocorticoids induce gastroparesis in mice through depletion of l-arginine.
Endocrinology
PUBLISHED: 07-24-2014
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Glucocorticoids (GCs) constitute a highly pleiotropic class of drugs predominantly employed in the treatment of inflammatory diseases. In our search for new mechanisms of action, we identified a hitherto unknown effect of GCs in the gastrointestinal tract. We found that oral administration of dexamethasone (Dex) to mice caused an enlargement of the stomach due to the induction of gastroparesis and that this effect was abolished in GR(dim) mice carrying the A458T mutation in the GC receptor (GR). Gastroparesis was unrelated to the enhanced gastric acid secretion observed after Dex treatment, although both effects were mediated by the same molecular mechanism of the GR. Using conditional GR-knockout mice, we could further rule out that GC effects on enterocytes or myeloid cells were involved in the induction of gastroparesis. In contrast, we found that Dex upregulated arginase 2 (Arg2) in the stomach both at the mRNA and protein level. This suggests that GC treatment leads to a depletion of l-arginine thereby impeding the production of nitric oxide (NO), which is required for gastric motility. We tested this hypothesis by supplementing the drinking water of the mice with exogenous l-arginine to compensate for the presumed shortage of this major substrate of NO synthases. Importantly, this measure completely prevented both the enlargement of the stomach and the induction of gastroparesis after Dex treatment. Our findings raise considerations of combining orally applied GCs with l-arginine to improve tolerability of GC treatment and provide a possible explanation for the antiemetic effects of GCs widely exploited in chemotherapy.
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Regulation of ClC-2 Activity by SPAK and OSR1.
Kidney Blood Press. Res.
PUBLISHED: 07-23-2014
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SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) are powerful regulators of diverse transport processes. Both kinases are activated by cell shrinkage and participate in stimulation of regulatory cell volume increase (RVI). Execution of RVI involves inhibition of Cl(-) channels. The present study explored whether SPAK and/or OSR1 regulate the activity of the Cl(-) channel ClC-2.
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Serum- and glucocorticoid-inducible kinase 1 sensitive NF-?B signaling in dendritic cells.
Cell. Physiol. Biochem.
PUBLISHED: 07-22-2014
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Dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity, are required for initiation of specific T cell-driven immune responses. Phosphoinositide-3-kinase (PI3K) suppresses proinflammatory cytokine production in DCs, which limits T helper (Th1) polarization. PI3K is in part effective by downregulation of transcription factor NF-?B. Downstream signaling elements of PI3K include serum- and glucocorticoid-inducible kinase 1 (SGK1) and its phosphorylation target N-myc downstream regulated gene 1 (NDRG1). The present study explored whether SGK1 and NDRG1 play a role in the regulation of NF-?B and DC-maturation.
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Na+/K+ ATPase inhibitors in cancer.
Curr Drug Targets
PUBLISHED: 07-22-2014
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Sodium potassium pump (Na(+)/K(+) ATPase) is a transmembrane protein complex found in all higher eukaryotes acting as a key energy-consuming pump maintaining ionic and osmotic balance in cells. Recently recognized as an important transducer and/or integrator of various signals as well as a protein-protein interaction scaffold forming receptor complexes with signaling properties, the most prominent pharmacological role of Na(+)/K(+) ATPase inhibitors is the increase of myocardial contractility in pathologic conditions such as congestive heart failure. Consequently, modulators of Na(+)/K(+) ATPase such as digoxin have been approved by regulatory authorities and are widely used in the treatment of cardiac failure since 1975. Initiating from early observations of reduction of cancer incidence in cardiac patients taking digoxin, recent epidemiological and other studies have consolidated the anti-cancer potential of Na(+)/K(+) ATPase inhibitors in indications such as prostate, breast, lung cancer or leukemia. More importantly, a new series of pharmacologically optimized Na(+)/K(+) ATPase inhibitors has recently shown strong anti-cancer activities in multiple preclinical assays and have reached early clinical trials. Altogether, these results suggest that Na(+)/K(+) ATPase is an emerging cancer target that merits further investigation. In this review, we summarize key functional properties of the enzyme that are highly relevant for cancer cell selectivity, review the most prominent chemical classes of Na(+)/K(+) ATPase inhibitors and analyze their downstream effectors. Moreover, we discuss overall development prospects of these candidate drugs on their way to becoming new effective treatments of cancer in patients.
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SPAK and OSR1 Dependent Down-Regulation of Murine Renal Outer Medullary K Channel ROMK1.
Kidney Blood Press. Res.
PUBLISHED: 07-17-2014
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The kinases SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) participate in the regulation of the NaCl cotransporter NCC and the Na(+),K(+),2Cl(-) cotransporter NKCC2. The kinases are regulated by WNK (with-no-K[Lys]) kinases. Mutations of genes encoding WNK kinases underly Gordon's syndrome, a monogenic disease leading to hypertension and hyperkalemia. WNK kinases further regulate the renal outer medullary K(+) channel ROMK1. The present study explored, whether SPAK and/or OSR1 have similarly the potential to modify the activity of ROMK1.
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Enhanced Orai1 and STIM1 expression as well as store operated Ca2+ entry in therapy resistant ovary carcinoma cells.
Oncotarget
PUBLISHED: 07-13-2014
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Mechanisms underlying therapy resistance of tumor cells include protein kinase Akt. Putative Akt targets include store-operated Ca(2+)-entry (SOCE) accomplished by pore forming ion channel unit Orai1 and its regulator STIM1. We explored whether therapy resistant (A2780cis) differ from therapy sensitive (A2780) ovary carcinoma cells in Akt, Orai1, and STIM1 expression, Ca(2+)-signaling and cell survival following cisplatin (100 µM) treatment. Transcript levels were quantified with RT-PCR, protein abundance with Western blotting, cytosolic Ca(2+)-activity ([Ca(2+)]i) with Fura-2-fluorescence, SOCE from increase of [Ca(2+)]i following Ca(2+)-readdition after Ca(2+)-store depletion, and apoptosis utilizing flow cytometry. Transcript levels of Orai1 and STIM1, protein expression of Orai1, STIM1, and phosphorylated Akt, as well as SOCE were significantly higher in A2780cis than A2780 cells. SOCE was decreased by Akt inhibitor III (SH-6, 10 µM) in A2780cis but not A2780 cells and decreased in both cell lines by Orai1 inhibitor 2-aminoethoxydiphenyl borate (2-ABP, 50 µM). Phosphatidylserine exposure and late apoptosis following cisplatin treatment were significantly lower in A2780cis than A2780 cells, a difference virtually abolished by SH-6 or 2-ABP. In conclusion, Orai1/STIM1 expression and function are increased in therapy resistant ovary carcinoma cells, a property at least in part due to enhanced Akt activity and contributing to therapy resistance in those cells.
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Piperlongumine-induced phosphatidylserine translocation in the erythrocyte membrane.
Toxins (Basel)
PUBLISHED: 07-03-2014
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Piperlongumine, a component of Piper longum fruit, is considered as a treatment for malignancy. It is effective by inducing apoptosis. Mechanisms involved in the apoptotic action of piperlongumine include oxidative stress and activation of p38 kinase. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in eryptosis include increase of cytosolic Ca²?-activity ([Ca²?]i), formation of ceramide, oxidative stress and activation of p38 kinase.
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Stimulation of eryptosis by cryptotanshinone.
Cell. Physiol. Biochem.
PUBLISHED: 07-03-2014
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Cryptotanshinone, a component of Salvia miltiorrhiza Bunge roots, may trigger suicidal death or apoptosis of tumor cells and has thus been recommended for the prevention and treatment of malignancy. On the other hand, Cryptotanshinone has been shown to counteract apoptosis of neurons and hepatocytes. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). The present study explored whether Cryptotanshinone stimulates eryptosis.
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Down-regulation of K? channels by human parvovirus B19 capsid protein VP1.
Biochem. Biophys. Res. Commun.
PUBLISHED: 06-24-2014
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Parvovirus B19 (B19V) can cause inflammatory cardiomyopathy and endothelial dysfunction. Pathophysiological mechanisms involved include lysophosphatidylcholine producing phospholipase A2 (PLA2) activity of the B19V capsid protein VP1. Most recently, VP1 and lysophosphatidylcholine have been shown to inhibit Na(+)/K(+) ATPase. The present study explored whether VP1 modifies the activity of Kv1.3 and Kv1.5 K(+) channels. cRNA encoding Kv1.3 or Kv1.5 was injected into Xenopus oocytes without or with cRNA encoding VP1 isolated from a patient suffering from fatal B19V-induced myocarditis. K(+) channel activity was determined by dual electrode voltage clamp. Injection of cRNA encoding Kv1.3 or Kv1.5 into Xenopus oocytes was followed by appearance of Kv K(+) channel activity, which was significantly decreased by additional injection of cRNA encoding VP1, but not by additional injection of cRNA encoding PLA2-negative VP1 mutant (H153A). The effect of VP1 on Kv current was not significantly modified by transcription inhibitor actinomycin (10 ?M for 36 h) but was mimicked by lysophosphatidylcholine (1 ?g/ml). The B19V capsid protein VP1 inhibits host cell Kv channels, an effect at least partially due to phospholipase A2 (PLA) dependent formation of lysophosphatidylcholine.
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Checkpoint kinase Chk2 controls renal Cyp27b1 expression, calcitriol formation, and calcium-phosphate metabolism.
Pflugers Arch.
PUBLISHED: 06-06-2014
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Checkpoint kinase 2 (Chk2) is the main effector kinase of ataxia telangiectasia mutated (ATM) and responsible for cell cycle regulation. ATM signaling has been shown to upregulate interferon-regulating factor-1 (IRF-1), a transcription factor also expressed in the kidney. Calcitriol (1,25 (OH)2D3), a major regulator of mineral metabolism, is generated by 25-hydroxyvitamin D 1?-hydroxylase in the kidney. Since 25-hydroxyvitamin D 1?-hydroxylase expression is enhanced by IRF-1, the present study explored the role of Chk2 for calcitriol formation and mineral metabolism. Chk2-deficient mice (chk2 (-/-)) were compared to wild-type mice (chk2 (+/+)). Transcript levels of renal 25-hydroxyvitamin D 1?-hydroxylase, Chk2, and IRF-1 were determined by RT-PCR; Klotho expression by Western blotting; bone density by ?CT analysis; serum or plasma 1,25 (OH)2D3, PTH, and C-terminal FGF23 concentrations by immunoassays; and serum, fecal, and urinary calcium and phosphate concentrations by photometry. The renal expression of IRF-1 and 25-hydroxyvitamin D 1?-hydroxylase as well as serum 1,25 (OH)2D3 and FGF23 levels were significantly lower in chk2 (-/-) mice compared to chk2 (+/+) mice. Plasma PTH was not different between the genotypes. Renal calcium and phosphate excretion were significantly higher in chk2 (-/-) mice than in chk2 (+/+) mice despite hypophosphatemia and normocalcemia. Bone density was not different between the genotypes. We conclude that Chk2 regulates renal 25-hydroxyvitamin D 1?-hydroxylase expression thereby impacting on calcium and phosphate metabolism.
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High-frequency ultrasound-guided disruption of glycoprotein VI-targeted microbubbles targets atheroprogressison in mice.
Biomaterials
PUBLISHED: 06-06-2014
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Targeted contrast-enhanced ultrasound (CEU) using microbubble agents is a promising non-invasive imaging technique to evaluate atherosclerotic lesions. In this study, we decipher the diagnostic and therapeutic potential of targeted-CEU with soluble glycoprotein (GP)-VI in vivo. Microbubbles were conjugated with the recombinant fusion protein GPVI-Fc (MBGPVI) that binds with high affinity to atherosclerotic lesions. MBGPVI or control microbubbles (MBC) were intravenously administered into ApoE(-/-) or wild type mice and binding of the microbubbles to the vessel wall was visualized by high-resolution CEU. CEU molecular imaging signals of MBGPVI were substantially enhanced in the aortic arch and in the truncus brachiocephalicus in ApoE(-/-) as compared to wild type mice. High-frequency ultrasound (HFU)-guided disruption of MBGPVI enhanced accumulation of GPVI in the atherosclerotic lesions, which may interfere with atheroprogression. Thus, we establish targeted-CEU with soluble GPVI as a novel non-invasive molecular imaging method for atherosclerosis. Further, HFU-guided disruption of GPVI-targeted microbubbles is an innovate therapeutic approach that potentially prevents progression of atherosclerotic disease.
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Enigmatic Cassandra: renal FGF23 formation in polycystic kidney disease.
Kidney Int.
PUBLISHED: 05-31-2014
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Fibroblast growth factor 23 (FGF23) counteracts phosphate excess and tissue calcification. Phosphate intake, Ca(2+), parathyroid hormone, and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) stimulate FGF23 release from bone. FGF23 inhibits renal 1,25(OH)2D3 formation and phosphate reabsorption. Spichtig and colleagues demonstrate that FGF23 is generated in rodent polycystic kidneys, leading to an increase in plasma FGF23 concentration before reduction in kidney function. FGF23 fails to appreciably downregulate renal phosphate transporter and 1?-25OH-vitamin D hydroxylase activities. Unraveling underlying mechanisms may open diagnostic and therapeutic opportunities.
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Effect of saponin on erythrocytes.
Int. J. Hematol.
PUBLISHED: 05-22-2014
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Saponins, naturally occurring glycosides and triterpene glycosides in plants, are considered useful in the prophylaxis and treatment of several disorders, including malignancy. The effect of these substances is partly attributable to induction of both apoptosis and necrosis. Saponin has previously been shown to trigger hemolysis. Erythrocytes may avoid hemolysis by entering programmed cell death or eryptosis, characterized by cell shrinkage and cell membrane scrambling, leading to phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by increase of cytosolic Ca(2+) activity ([Ca(2+)](i)). The present study explored, whether exposure of human erythrocytes to saponin modifies [Ca(2+)](i), ceramide formation, hemolysis, and eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release, [Ca(2+)](i) from Fluo3-fluorescence, and ceramide utilizing specific antibodies. A 24 h exposure to saponin (15 µg/ml) resulted in a significant increase of annexin V binding and a significant stimulation of hemolysis. Saponin (15 µg/ml) further increased [Ca(2+)](i) and ceramide formation. Annexin V binding was significantly blunted but not abrogated in the nominal absence of extracellular Ca(2+). Saponin thus triggers cell membrane scrambling, an effect partially due to entry of extracellular Ca(2+) and ceramide formation.
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Niflumic acid affects store-operated Ca(2+)-permeable (SOC) and Ca (2+)-dependent K (+) and Cl (-) ion channels and induces apoptosis in K562 cells.
J. Membr. Biol.
PUBLISHED: 05-10-2014
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Non-steroidal anti-inflammatory drugs (NSAIDs) are known to induce apoptosis in a variety of cancer cells. However, the precise mechanisms by which NSAIDs facilitate apoptosis in tumor cells are not clear. In the present study, we show that niflumic acid (NA), a member of the fenamates group of NSAIDs and Cl(-) and Ca(2+)-activated Cl(-) (CAC) channels blocker, induced apoptosis (by ~8 %, 24 h treatment) and potentiated (by 8-10 %) apoptotic effect of endoplasmic reticulum Ca(2+) mobilizer thapsigargin (Tg) in human erythroleukemic K562 cell line. The whole-cell patch clamp and Fluo-3 flow cytometric experiments confirmed an inhibitory effect of NA (100 and 300 µM) on store-operated (SOC) channels. We also found that NA-blocked CAC channels were activated by acute application of Tg (2 µM) in K562 cells. NA blockage of CAC channels was accompanied by activation of Ca(2+)-activated K(+) (SK4) channels. The observed effects of NA were not connected with COX-2 inhibition since 100-nM NA (IC50 for COX-2 inhibition) did not induce either apoptosis or affect the channels activity. We conclude that inhibition of SOC channels plays a major role in NA-induced apoptosis. Increased apoptotic levels in Tg-treated K562 cells in the presence of NA may be due to the blockage of CAC and stimulation of SK4 channels in addition to SOC channels inhibition.
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In vitro sensitization of erythrocytes to programmed cell death following baicalein treatment.
Toxins (Basel)
PUBLISHED: 05-09-2014
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The polyphenolic flavonoid Baicalein has been shown to trigger suicidal death or apoptosis of tumor cells and is thus considered for the prevention and treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and ceramide. The present study explored whether Baicalein stimulates eryptosis. To this end, forward scatter was taken for measurement of cell volume, annexin-V-binding for phosphatidylserine-exposure, Fluo3 fluorescence for [Ca2+]i and fluorescent antibodies for ceramide abundance. As a result, a 48 h exposure of human erythrocytes to Baicalein was followed by significant decrease of forward scatter (?10 µM), significant increase of the percentage of annexin-V-binding cells (?25 µM), significant increase of [Ca2+]i (50 µM) and significant increase of ceramide abundance (50 µM). The effect of Baicalein (50 µM) on annexin-V-binding was significantly blunted but not abrogated by removal of extracellular Ca2+. In conclusion, at the concentrations employed, Baicalein stimulates suicidal erythrocyte death or eryptosis, an effect at least in part due to the combined effects of Ca2+ entry and ceramide formation.
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Stimulation of erythrocyte cell membrane scrambling by gedunin.
Cell. Physiol. Biochem.
PUBLISHED: 05-08-2014
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Gedunin, an inhibitor of heat shock protein HSP90, triggers apoptosis of tumor cells and is thus effective against malignancy. Moreover, the drug has antimalarial potency. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). The present study explored whether gedunin stimulates eryptosis.
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Pre-activated blood platelets and a pro-thrombotic phenotype in APP23 mice modeling Alzheimer's disease.
Cell. Signal.
PUBLISHED: 05-06-2014
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Platelet activation and thrombus formation play a critical role in primary hemostasis but also represent a pathophysiological mechanism leading to acute thrombotic vascular occlusions. Besides, platelets modulate cellular processes including inflammation, angiogenesis and neurodegeneration. On the other hand, platelet activation and thrombus formation are altered in different diseases leading to either bleeding complications or pathological thrombus formation. For many years platelets have been considered to play a role in neuroinflammatory diseases such as Alzheimer's disease (AD). AD is characterized by deposits of amyloid-? (A?) and strongly related to vascular diseases with platelets playing a critical role in the progression of AD because exposure of platelets to A? induces platelet activation, platelet A? release, and enhanced platelet adhesion to collagen in vitro and at the injured carotid artery in vivo. However, the molecular mechanisms and the relation between vascular pathology and amyloid-? plaque formation in the pathogenesis of AD are not fully understood. Compelling evidence is suggestive for altered platelet activity in AD patients. Thus we analyzed platelet activation and thrombus formation in aged AD transgenic mice (APP23) known to develop amyloid-? deposits in the brain parenchyma and cerebral vessels. As a result, platelets are in a pre-activated state in blood of APP23 mice and showed strongly enhanced integrin activation, degranulation and spreading kinetics on fibrinogen surfaces upon stimulation. This enhanced platelet signaling translated into almost unlimited thrombus formation on collagen under flow conditions in vitro and accelerated vessel occlusion in vivo suggesting that these mice are at high risk of arterial thrombosis leading to cerebrovascular and unexpectedly to cardiovascular complications that might be also relevant in AD patients.
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Up-regulation of hepatic alpha-2-HS-glycoprotein transcription by testosterone via androgen receptor activation.
Cell. Physiol. Biochem.
PUBLISHED: 05-05-2014
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Fetuin-A (alpha-2-HS-glycoprotein, AHSG), a liver borne plasma protein, contributes to the prevention of soft tissue calcification, modulates inflammation, reduces insulin sensitivity and fosters weight gain following high fat diet or ageing. In polycystic ovary syndrome, fetuin-A levels correlate with free androgen levels, an observation pointing to androgen sensitivity of fetuin-A expression. The present study thus explored whether the expression of hepatic fetuin-A is modified by testosterone.
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Conjugated bilirubin triggers anemia by inducing erythrocyte death.
Hepatology
PUBLISHED: 04-13-2014
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Hepatic failure is commonly associated with anemia, which may result from gastrointestinal bleeding, vitamin deficiency and liver-damaging diseases, such as infection and alcohol intoxication. At least in theory, anemia during hepatic failure may result from accelerated clearance of circulating erythrocytes. Here we show that bile duct ligation (BDL) in mice leads to severe anemia despite increased reticulocyte numbers. Bilirubin stimulated suicidal erythrocyte death of human erythrocytes. Mechanistically, bilirubin triggered rapid Ca(2+) influx, sphingomyelinase activation, formation of ceramide and subsequent translocation of phosphatidylserine to the erythrocyte surface. Consistent with our in vitro and in vivo findings, incubation of erythrocytes in serum from patients with liver disease induced suicidal death of erythrocytes in relation to their plasma bilirubin concentration. Consistently, in patients with hepatic disease bilirubin plasma levels correlated negatively with erythrocyte count and positively with reticulocyte count. In conclusion, bilirubin triggers suicidal erythrocyte death thus contributing to anemia during liver disease. (Hepatology 2014;).
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A steroidal Na+/K+ ATPase inhibitor triggers pro-apoptotic signaling and induces apoptosis in prostate and lung tumor cells.
Anticancer Agents Med Chem
PUBLISHED: 04-11-2014
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Recently we have reported potent anti-cancer actions of various steroidal Na(+)/K(+) ATPase inhibitors in multiple cell lines. Furthermore, the most powerful compound identified in this study, the 3-[(R)-3-pyrrolidinyl]oxime derivative (3-R-POD), was highly effective in various tumor cell lines in vitro, and exhibited significant tumor growth inhibition in prostate and lung xenografts in vivo. In the present study we have addressed the molecular mechanisms implicated in the anti-cancer actions of 3-R-POD. We report here that 3-R-POD induces strong apoptotic responses in A549 lung- and in DU145 prostate- cancer cells. These effects are accompanied by significant upregulation of caspase-3 activity. Focussing on A549 cells, we further demonstrate late downregulation of BCL-2- and upregulation of c-Fos- gene transcription. In addition, the steroidal Na(+)/K(+) ATPase inhibitor induced late de-phosphorylation of Focal Adhesion Kinase (FAK) and activation of p38 MAPK. Our findings suggest that the steroidal Na(+)/K(+) ATPase inhibitor 3-R-POD induces apoptosis, paralleled by altered BCL-2 and c-Fos gene transcription, inhibition of the pro-survival FAK signalling, up-regulation of the pro-apoptotic p38 MAPK pathway and stimulation of caspase-3 activity.
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Upregulation of excitatory amino acid transporters by coexpression of Janus kinase 3.
J. Membr. Biol.
PUBLISHED: 04-03-2014
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Janus kinase 3 (JAK3) contributes to cytokine receptor signaling, confers cell survival and stimulates cell proliferation. The gain of function mutation JAK3(A572V) is found in acute megakaryoplastic leukemia. Replacement of ATP coordinating lysine by alanine yields inactive JAK3(K855A). Most recent observations revealed the capacity of JAK3 to regulate ion transport. This study thus explored whether JAK3 regulates glutamate transporters EAAT1-4, carriers accomplishing transport of glutamate and aspartate in a variety of cells including intestinal cells, renal cells, glial cells, and neurons. To this end, EAAT1, 2, 3, or 4 were expressed in Xenopus oocytes with or without additional expression of mouse wild-type JAK3, constitutively active JAK3(A568V) or inactive JAK3(K851A), and electrogenic glutamate transport was determined by dual electrode voltage clamp. Moreover, Ussing chamber was employed to determine electrogenic glutamate transport in intestine from mice lacking functional JAK3 (jak3(-/-)) and from corresponding wild-type mice (jak3(+/+)). As a result, in EAAT1, 2, 3, or 4 expressing oocytes, but not in oocytes injected with water, addition of glutamate to extracellular bath generated an inward current (Ig), which was significantly increased following coexpression of JAK3. Ig in oocytes expressing EAAT3 was further increased by JAK3(A568V) but not by JAK3(K851A). Ig in EAAT3 + JAK3 expressing oocytes was significantly decreased by JAK3 inhibitor WHI-P154 (22 µM). Kinetic analysis revealed that JAK3 increased maximal Ig and significantly reduced the glutamate concentration required for half maximal Ig (Km). Intestinal electrogenic glutamate transport was significantly lower in jak3(-/-) than in jak3(+/+) mice. In conclusion, JAK3 is a powerful regulator of excitatory amino acid transporter isoforms.
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Stimulation of Erythrocyte Cell Membrane Scrambling by Nystatin.
Basic Clin. Pharmacol. Toxicol.
PUBLISHED: 04-03-2014
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The antifungal ionophore nystatin dissipates the Na(+) and K(+) gradients across the cell membrane, leading to cellular gain of Na(+) and cellular loss of K(+) . The increase of cellular Na(+) concentration may result in Ca(2+) accumulation in exchange for Na(+) . Increase of cytosolic Ca(2+) activity ([Ca(2+) ]i ) and loss of cellular K(+) foster apoptosis-like suicidal erythrocyte death or eryptosis, which is characterised by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the erythrocyte surface. The present study explored whether nystatin stimulates eryptosis. Cell volume was estimated from forward scatter (FSC), phosphatidylserine exposure from annexin V binding and [Ca(2+) ]i from Fluo3-fluorescence in flow cytometry. A 48-hr exposure to nystatin (15 ?g/ml) was followed by a significant increase of [Ca(2+) ]i , a significant increase of annexin V binding and a significant decrease of FSC. The annexin V binding after nystatin treatment was significantly blunted in the nominal absence of extracellular Ca(2+) . Partial replacement of extracellular Na(+) with extracellular K(+) blunted the nystatin-induced erythrocyte shrinkage but increased [Ca(2+) ]i and annexin V binding. Nystatin triggers cell membrane scrambling, an effect at least partially due to entry of extracellular Ca(2+) .
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Stimulation of erythrocyte cell membrane scrambling by mitotane.
Cell. Physiol. Biochem.
PUBLISHED: 04-03-2014
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background: Mitotane (1,1-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane), a cytostatic drug used for the treatment of adrenocortical carcinomas, is effective by triggering tumor cell apoptosis. In analogy to apoptosis of nucleated cells, eryptosis is the suicidal death of erythrocytes, which is typically paralleled by cell shrinkage and breakdown of cell membrane phosphatidylserine asymmetry with subsequent phosphatidylserine exposure at the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca(2+) concentration ([Ca(2+)]i). The present study tested, whether treatment of human erythrocytes with mitotane is followed by eryptosis.
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Lack of the serum- and glucocorticoid-inducible kinase SGK1 improves muscle force characteristics and attenuates fibrosis in dystrophic mdx mouse muscle.
Pflugers Arch.
PUBLISHED: 04-01-2014
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Duchenne muscular dystrophy (DMD) is a human genetic disease characterized by fibrosis and severe muscle weakness. Currently, there is no effective treatment available to prevent progressive fibrosis in skeletal muscles. The serum- and glucocorticoid-inducible kinase SGK1 regulates a variety of physiological functions and participates in fibrosis stimulation. Here, we investigated whether SGK1 influences structure, function and/or fibrosis of the muscles from the mdx mouse, an animal model for DMD. As expected, mdx muscles showed the typical pathological features of muscular dystrophy including fiber size variations, central nuclei of muscle fibers, fibrosis in the diaphragm, and force reduction by 30-50 %. Muscles from sgk1 (-/-) mice were histologically overall intact and specific force was only slightly reduced compared to wild-type muscles. Surprisingly, soleus and diaphragm muscles of mdx/sgk1 (-/-) mice displayed forces close to wild-type levels. Most muscle fibers of the double mutants contained central nuclei, but fibrosis was not observed in any of the tested limb and diaphragm muscles. We conclude that the sole lack of SGK1 in mouse muscle does not lead to pronounced changes in muscle structure and function. However, dystrophin-deficient mdx muscle seems to benefit from SGK1 deficiency. SGK1 appears to be an important enzyme in the process of fibrotic remodeling and subsequent weakness of dystrophin-deficient mouse muscle.
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CXCL16 is a novel diagnostic marker and predictor of mortality in inflammatory cardiomyopathy and heart failure.
Int. J. Cardiol.
PUBLISHED: 03-25-2014
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Cardiac inflammation has been suggested to play a critical role in the pathogenesis of inflammatory cardiomyopathy as well as in progressive heart failure (HF). CXC motif ligand 16 (CXCL16) is a recently discovered chemokine produced by several inflammatory cells and representing an important pathogenic mediator in the development of HF. The present study evaluates the diagnostic and prognostic relevance of CXCL16 expression in endomyocardial biopsies of consecutive patients with congestive HF.
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Gremlin-1 inhibits macrophage migration inhibitory factor-dependent monocyte function and survival.
Int. J. Cardiol.
PUBLISHED: 03-17-2014
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Monocyte migration and their differentiation into macrophages critically regulate vascular inflammation and atherogenesis and are governed by macrophage migration inhibitory factor (MIF). Gremlin-1 binds to MIF. Current experimental evidences present Gremlin-1 as a potential physiological agent that might counter-regulate the inflammatory attributes of MIF.
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Influence of annexin A7 on insulin sensitivity of cellular glucose uptake.
Pflugers Arch.
PUBLISHED: 03-11-2014
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Insulin sensitivity is decreased by prostaglandin E2 (PGE2), a major product of cyclooxygenase (COX). As shown in erythrocytes, PGE2 formation is inhibited by annexin A7. The present study defined the role of annexin A7 in glucose metabolism. Gene-targeted mice lacking annexin A7 (annexin7 (-/-)) were compared to wild-type mice (annexin7 (+/+)). The serum 6-Keto-prostaglandin-F1? (6-Keto-PGF1?) concentration was measured by ELISA and hepatic COX activity determined by an enzyme assay. Expression of COX-1, COX-2, prostaglandin E synthase, GLUT-4, and insulin receptor was determined by Western blotting. Glucose and insulin serum concentrations were analyzed following an intraperitoneal glucose load and glucose serum levels after intraperitoneal injection of insulin. Experiments were done without and with pretreatment of the mice with COX-inhibitor aspirin. The serum 6-Keto-PGF1? level and hepatic COX activity were significantly higher in annexin7 (-/-) than in annexin7 (+/+) mice. Hepatic COX-1 expression was higher in annexin7 (-/-) mice. Glucose tolerance was decreased in annexin7 (-/-) mice. Intraperitoneal insulin injection decreased the serum glucose level in both genotypes, an effect significantly less pronounced in annexin7 (-/-) mice. Glucose-induced insulin secretion was higher in annexin7 (-/-) mice. GLUT-4 expression in skeletal muscle from annexin7 (-/-) mice was reduced. Aspirin pretreatment lowered the increase in insulin concentration following glucose injection in both genotypes and virtually abrogated the differences in serum insulin between the genotypes. Aspirin pretreatment improved glucose tolerance in annexin7 (-/-) mice. In conclusion, annexin A7 influences insulin sensitivity of cellular glucose uptake and thus glucose tolerance. These effects depend on COX activity.
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Stimulation of suicidal erythrocyte death by increased extracellular phosphate concentrations.
Kidney Blood Press. Res.
PUBLISHED: 02-10-2014
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Anemia in renal insufficiency results in part from impaired erythrocyte formation due to erythropoietin and iron deficiency. Beyond that, renal insufficiency enhances eryptosis, the suicidal erythrocyte death characterized by phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be stimulated by increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). Several uremic toxins have previously been shown to stimulate eryptosis. Renal insufficiency is further paralleled by increase of plasma phosphate concentration. The present study thus explored the effect of phosphate on erythrocyte death.
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mTOR target NDRG1 confers MGMT-dependent resistance to alkylating chemotherapy.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-23-2013
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A hypoxic microenvironment induces resistance to alkylating agents by activating targets in the mammalian target of rapamycin (mTOR) pathway. The molecular mechanisms involved in this mTOR-mediated hypoxia-induced chemoresistance, however, are unclear. Here we identify the mTOR target N-myc downstream regulated gene 1 (NDRG1) as a key determinant of resistance toward alkylating chemotherapy, driven by hypoxia but also by therapeutic measures such as irradiation, corticosteroids, and chronic exposure to alkylating agents via distinct molecular routes involving hypoxia-inducible factor (HIF)-1alpha, p53, and the mTOR complex 2 (mTORC2)/serum glucocorticoid-induced protein kinase 1 (SGK1) pathway. Resistance toward alkylating chemotherapy but not radiotherapy was dependent on NDRG1 expression and activity. In posttreatment tumor tissue of patients with malignant gliomas, NDRG1 was induced and predictive of poor response to alkylating chemotherapy. On a molecular level, NDRG1 bound and stabilized methyltransferases, chiefly O(6)-methylguanine-DNA methyltransferase (MGMT), a key enzyme for resistance to alkylating agents in glioblastoma patients. In patients with glioblastoma, MGMT promoter methylation in tumor tissue was not more predictive for response to alkylating chemotherapy in patients who received concomitant corticosteroids.
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Regulation of ion channels and transporters by AMP-activated kinase (AMPK).
Channels (Austin)
PUBLISHED: 12-23-2013
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The energy-sensing AMP-activated kinase AMPK ensures survival of energy-depleted cells by stimulating ATP production and limiting ATP utilization. Both energy production and energy consumption are profoundly influenced by transport processes across the cell membane including channels, carriers and pumps. Accordingly, AMPK is a powerful regulator of transport across the cell membrane. AMPK regulates diverse K(+) channels, Na(+) channels, Ca (2+) release activated Ca (2+) channels, Cl(-) channels, gap junctional channels, glucose carriers, Na(+)/H(+)-exchanger, monocarboxylate-, phosphate-, creatine-, amino acid-, peptide- and osmolyte-transporters, Na(+)/Ca (2+)-exchanger, H(+)-ATPase and Na(+)/K(+)-ATPase. AMPK activates ubiquitin ligase Nedd4-2, which labels several plasma membrane proteins for degradation. AMPK further regulates transport proteins by inhibition of Rab GTPase activating protein (GAP) TBC1D1. It stimulates phosphatidylinositol 3-phosphate 5-kinase PIKfyve and inhibits phosphatase and tensin homolog (PTEN) via glycogen synthase kinase 3? (GSK3?). Moreover, it stabilizes F-actin as well as downregulates transcription factor NF-?B. All those cellular effects serve to regulate transport proteins.
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Energy sensitive regulation of Na+/K+ ATPase by Janus kinase 2.
Am. J. Physiol., Cell Physiol.
PUBLISHED: 12-04-2013
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Janus kinase 2 (JAK2) contributes to intracellular signaling of leptin and erythropoietin, hormones protecting cells during energy depletion. The present study explored, whether JAK2 is activated by energy depletion and regulates Na+/K+-ATPase, the major energy consuming pump. In Jurkat cells JAK2 activity was determined by radioactive kinase assay, phosphorylated JAK2 detected by western blotting, ATP levels measured by luciferase-assay, as well as Na+/K+-ATPase ?1-subunit transcript and protein abundance determined by real time PCR and western blotting, respectively. Ouabain-sensitive K+-induced currents (Ipump) were measured by whole-cell patch-clamp. Ipump was further determined by dual-electrode voltage-clamp in Xenopus oocytes injected with cRNA encoding JAK2, active V617FJAK2 or inactive K882EJAK2. As a result, in Jurkat T cells, JAK2 activity significantly increased following energy depletion by sodium azide (NaN3) or 2,4- dinitro phenol (DNP). DNP- and NaN3-induced decrease of cellular ATP was significantly augmented by JAK2 inhibitor AG490 and blunted by Na+/K+-ATPase inhibitor ouabain. DNP decreased and AG490 enhanced Ipump as well as Na+/K+-ATPase ?1-subunit transcript and protein abundance. The ?1-subunit transcript levels were also enhanced by STAT5-inhibitor CAS 285986-31-4. In Xenopus oocytes Ipump was significantly decreased by expression of JAK2 and V617FJAK2 but not of K882EJAK2, effects again reversed by AG490. In V617FJAK2-expressing Xenopus oocytes neither DNP nor NaN3 resulted in further decline of Ipump. In Xenopus oocytes the effect of V617FJAK2 on Ipump was not prevented by inhibition of transcription with actinomycin. In conclusion, JAK2 is a novel energy sensing kinase that curtails energy consumption by downregulating Na+/K+ ATPase expression and activity.
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Dynamic adhesion of eryptotic erythrocytes to immobilized platelets via platelet phosphatidylserine receptors.
Am. J. Physiol., Cell Physiol.
PUBLISHED: 11-27-2013
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Glucose depletion of erythrocytes triggers suicidal erythrocyte death or eryptosis which leads to cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes adhere to endothelial cells by a mechanism involving phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 at the endothelial cell membrane. Nothing is hitherto known about an interaction between eryptotic erythrocytes and platelets, the decisive cells in primary haemostasis and major players in thrombotic vascular occlusion. The present study thus explored whether and how glucose depleted erythrocytes adhere to platelets. To this end, adhesion of phosphatidylserine exposing erythrocytes to platelets under flow conditions was examined in a flow chamber model at arterial shear rates. Platelets were immobilized on collagen and further stimulated with adenosinediphosphate (ADP, 10 µM) or thrombin (0.1 U/ml). As a result, a 48 hours glucose depletion triggered phosphatidylserine translocation to the erythrocyte surface and augmented the adhesion of erythrocytes to immobilized platelets, an effect significantly increased upon platelet stimulation. Adherence of erythrocytes to platelets was blunted by coating of erythrocytic phophatidylserine by Annexin V or by neutralization of platelet phosphatidylserine receptors CXCL16 and CD36 with respective antibodies. In conclusion, glucose-depleted erythrocytes adhere to platelets. The adhesive properties of platelets are augmented by platelet activation. Erythrocyte adhesion to immobilized platelets requires phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 expression on platelets. Thus, platelet-mediated erythrocyte adhesion may foster thrombo-occlusive complications in diseases with stimulated phosphatidylserine exposure of erythrocytes.
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Annexin 7 in the Regulation of Gastric Acid Secretion.
Cell. Physiol. Biochem.
PUBLISHED: 11-25-2013
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Background/Aims: Glucocorticoids enhance gastric acid secretion and inhibit gastric cyclooxygenase, thus downregulating formation of PGE2, an inhibitor of gastric acid secretion. In erythrocytes, PGE2 formation is inhibited by annexin 7. The present study thus explored whether annexin 7 participates in the regulation of gastric acid secretion. Methods: Annexin 7 protein expression was determined by Western blotting, cytosolic pH (pHi) of parietal cells utilizing BCECF-fluorescence, and gastric acid secretion by determination of Na(+)-independent pHi recovery from an ammonium pulse (?pHi/min). Experiments were performed in isolated glands from gene targeted mice lacking annexin 7 (anx7(-/-)) and in respective wild type animals (anx7(+/+)). Results: Prior to treatment pHi and ?pHi/min were similar in isolated gastric glands from anx7(-/-) and from anx7(+/+) mice. Aspirin (100 µM added to the glands 1 hr prior to the experiment) significantly increased ?pHi/min to similar values in both genotypes. The administration of dexamethasone (10 µg/g BW subcutaneously for 4 consecutive days prior to the experiments) significantly increased ?pH/min in anx7(+/+) mice but not in anx7(-/-) mice. Following dexamethasone treatment, the luminal pH was significantly lower and the acid content significantly higher in anx7(+/+) mice than in anx7(-/-) mice. An increase of extracellular K(+) concentration to 35 mM (replacing Na(+)/NMDG(+)) significantly increased ?pHi/min in both genotypes. In neither genotype dexamethasone increased ?pH/min further in the presence of 35 mM K(+) or presence of aspirin. Conclusions: Annexin 7 is required for the stimulation of gastric acid secretion by glucocorticoids. © 2014 S. Karger AG, Basel.
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Geldanamycin-Induced Phosphatidylserine Translocation in the Erythrocyte Membrane.
Cell. Physiol. Biochem.
PUBLISHED: 11-19-2013
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Background/aims: Geldanamycin, a benzoquinone ansamycin antibiotic, and its analogues induce apoptosis of tumor cells and are thus considered for the treatment of cancer. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-concentration ([Ca(2+)]i) and formation of ceramide. The present study explored, whether geldanamycin modifies [Ca(2+)]i, ceramide formation, cell volume and phosphatidylserine abundance at the erythrocyte surface. Methods: Erythrocyte volume was estimated from forward scatter, phosphatidylserine-abundance from annexin V binding, hemolysis from hemoglobin release, ceramide formation from binding of fluorescent antibodies and [Ca(2+)]i from Fluo3-fluorescence. Results: A 48 hours exposure to geldanamycin significantly decreased forward scatter (? 5 µM), significantly increased annexin-V-binding (? 25 µM), but did not significantly modify Fluo3-fluorescence (up to 50 µM). The annexin-V-binding following geldanamycin treatment was not significantly modified by removal of extracellular Ca(2+) but was paralleled by significantly increased ceramide formation (50 µM). Conclusions: Geldanamycin stinulated eryptosis, an effect at least partially due to ceramide formation. © 2014 S. Karger AG, Basel.
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Estramustine-Induced Suicidal Erythrocyte Death.
Cell. Physiol. Biochem.
PUBLISHED: 11-14-2013
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Background: The nitrogen mustard derivative of estradiol-17?-phosphate estramustine is used for the treatment of prostate cancer. Estramustine may trigger suicidal death of cancer cells. Side effects of estramustine include anemia. At least in theory, estramustine could cause anemia by stimulation of eryptosis, the suicidal death of erythrocytes. Hallmarks of eryptosis include cell shrinkage, increased cytosolic Ca(2+) activity ([Ca(2+)]), ceramide formation and phosphatidylserine translocation to the outer leaflet of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Eryptosis is stimulated by increase of cytosolic Ca(2+) activity ([Ca(2+)]i). The present study explored whether estramustine triggers eryptosis. Methods: [Ca(2+)]i was estimated from Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, and hemolysis from hemoglobin release. Results: A 24 h exposure to estramustine (? 100 µM) significantly increased [Ca(2+)]i, increased annexin V binding and increased hemoglobin release. The effect of estramustine on annexin V binding was significantly blunted by removal of extracellular Ca(2+). Conclusions: Estramustine stimulates both, eryptosis and hemolysis. The estramustine induced translocation of phosphatidylserine to the cell surface is at least partially due to increase of cytosolic Ca(2+) activity. © 2013 S. Karger AG, Basel.
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Acid sphingomyelinase regulates platelet cell membrane scrambling, secretion, and thrombus formation.
Arterioscler. Thromb. Vasc. Biol.
PUBLISHED: 11-14-2013
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Platelet activation is essential for primary hemostasis and acute thrombotic vascular occlusions. On activation, platelets release their prothrombotic granules and expose phosphatidylserine, thus fostering thrombin generation and thrombus formation. In other cell types, both degranulation and phosphatidylserine exposure are modified by sphingomyelinase-dependent formation of ceramide. The present study thus explored whether acid sphingomyelinase participates in the regulation of platelet secretion, phosphatidylserine exposure, and thrombus formation.
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Triggering of Suicidal Erythrocyte Death by Penta-O-galloyl-?-d-glucose.
Toxins (Basel)
PUBLISHED: 10-23-2013
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The polyphenolic 1,2,3,4,6-penta-O-galloyl-beta-d-glucose from several medicinal herbs triggers apoptosis and has, thus, been proposed for treatment of malignancy. The substance is at least partially effective through caspase activation. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Eryptosis is triggered by increase of cytosolic Ca2+-activity ([Ca2+]i). The sensitivity to [Ca2+]i is enhanced by ceramide. The present study explored whether penta-O-galloyl-?-d-glucose stimulates eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin-release, [Ca2+]i from Fluo3-fluorescence and ceramide abundance from fluorescent antibodies. A 48-h exposure of human erythrocytes to penta-O-galloyl-?-d-glucose significantly decreased forward scatter (50 µM) and significantly increased annexin V binding (10 µM). Up to 50 µM penta-O-galloyl-?-d-glucose did not significantly modify [Ca2+]i. However, the effect of penta-O-galloyl-?-d-glucose (25 µM) induced annexin V binding was slightly, but significantly, blunted by removal of extracellular Ca2+, pointing to sensitization of erythrocytes to the scrambling effect of Ca2+. Penta-O-galloyl-?-d-glucose (25 µM) further increased ceramide formation. In conclusion, penta-O-galloyl-?-d-glucose stimulates suicidal erythrocyte death or eryptosis, an effect partially due to stimulation of ceramide formation with subsequent sensitization of erythrocytes to Ca2+.
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Effect of Nitazoxanide on Erythrocytes.
Basic Clin. Pharmacol. Toxicol.
PUBLISHED: 10-23-2013
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Nitazoxanide, a drug effective against a variety of pathogens, triggers apoptosis and is thus considered to be employed against malignancy. Similar to nucleated cells, erythrocytes may undergo an apoptosis-like suicidal cell death or eryptosis. Hallmarks of eryptosis include cell shrinkage and phospholipid scrambling of the cell membrane with translocation of phosphatidylserine to the erythrocyte surface. Stimulators of eryptosis include increase in cytosolic Ca(2+) -activity ([Ca(2+) ]i ). The Ca(2+) -sensitivity of eryptosis is increased by ceramide. This study explored whether nitazoxanide triggers eryptosis. [Ca(2+) ]i was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin-V-binding, ceramide abundance utilizing fluorescent antibodies and haemolysis from haemoglobin release. A 48-hr exposure to nitazoxanide (1-50 ?g/ml) did not significantly modify [Ca(2+) ]i but significantly increased ceramide formation, decreased forward scatter (?10 ?g/ml), increased the percentage of annexin-V-binding erythrocytes (?10 ?g/ml) and, at higher concentrations (?20 ?g/ml), stimulated haemolysis. The stimulation of annexin-V-binding was significantly blunted in the absence of calcium. Nitazoxanide thus stimulates eryptosis, an effect in part due to ceramide formation.
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Sensitization of erythrocytes to suicidal erythrocyte death following water deprivation.
Kidney Blood Press. Res.
PUBLISHED: 10-16-2013
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Background/Aims : Klotho deficiency results in excessive formation of 1,25(OH)2D3, accelerated ageing and early death. Moreover, klotho deficiency enhances eryptosis, the suicidal erythrocyte death characterized by phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)]i), glucose depletion, hyperosmotic shock and oxidative stress. Klotho expression is decreased and 1,25(OH)2D3-formation enhanced by dehydration. The present study thus explored whether dehydration influences eryptosis. Methods : Blood was drawn from hydrated or 36h dehydrated mice. Plasma osmolarity was determined by vapour pressure method, plasma 1,25(OH)2D3 and aldosterone concentrations using ELISA, and plasma Ca(2+)-concentration utilizing photometry. Erythrocytes were exposed to Ca(2+)-ionophore ionomycin (1 µM, 30 min), energy depletion (12 h glucose removal), hyperosmotic shock (500 mM sucrose added, 2 h) and oxidative stress (100 µM tert-butyl-hydroperoxide, 30 min) and phosphatidylserine exposure at the erythrocyte surface estimated from annexin V binding. Results : Dehydration increased plasma osmolarity and plasma 1,25(OH)2D3 and aldosterone concentrations. Dehydration did not significantly modify phosphatidylserine-exposure of freshly drawn erythrocytes but significantly enhanced the increase of phosphatidylserine-exposure under control conditions and following treatment with ionomycin, glucose-deprivation, hyperosmolarity or tert-butyl-hydroperoxide. Conclusions : Dehydration sensitizes the erythrocytes to spontaneous eryptosis and to the triggering of eryptosis by excessive Ca(2+)-entry, energy depletion, hyperosmotic shock and oxidative stress. © 2013 S. Karger AG, Basel.
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Intestinal Na Loss and Volume Depletion in JAK3-Deficient Mice.
Kidney Blood Press. Res.
PUBLISHED: 10-14-2013
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Background/Aims : The Janus kinase 3 JAK3 participates in the signaling of immune cells. Lack of JAK3 triggers inflammatory bowel disease, which in turn has been shown to affect intestinal activity of the epithelial Na(+) channel ENaC and thus colonic sodium absorption. At least in theory, inflammatory bowel disease in JAK3-deficient mice could lead to intestinal salt loss compromizing extracellular volume maintenance and blood pressure regulation. The present study thus explored whether JAK3 deficiency impacts on colonic ENaC activity, fecal Na(+) exretion, blood pressure and extracellular fluid volume regulation. Methods : Experiments were performed in gene-targeted mice lacking functional JAK3 (jak3(-/-)) and in wild type mice (jak3(+/+)). Colonic ENaC activity was estimated from amiloride-sensitive current in Ussing chamber experiments, fecal, serum and urinary Na(+) concentration by flame photometry, blood pressure by the tail cuff method and serum aldosterone levels by immunoassay. Results : The amiloride (50 µM)-induced deflection of the transepithelial potential difference was significantly lower and fecal Na(+) excretion significantly higher in jak3(-/-) mice than in jak3(+/+) mice. Moreover, systolic arterial blood pressure was significantly lower and serum aldosterone concentration significantly higher in jak3(-/-) mice than in jak3(+/+) mice. Both, absolute and fractional renal Na(+) excretion were significantly lower in jak3(-/-) mice than in jak3(+/+) mice. Conclusions : JAK3 deficiency leads to impairment of colonic ENaC activity with intestinal Na(+) loss, decrease of blood pressure, increased aldosterone release and subsequent stimulation of renal tubular Na(+) reabsorption. © 2013 S. Karger AG, Basel.
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Differential cystine and dibasic amino acid handling after loss of function of the amino acid transporter b0,+AT (Slc7a9) in mice.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 10-09-2013
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Cystinuria is an autosomal recessive disease caused by mutations in SLC3A1 (rBAT) and SLC7A9 (b(0,+)AT). Gene targeting of the catalytic subunit (Slc7a9) in mice leads to excessive excretion of cystine, lysine, arginine, and ornithine. Here, we studied this non-type I cystinuria mouse model using gene expression analysis, Western blotting, clearance, and brush-border membrane vesicle (BBMV) uptake experiments to further characterize the renal and intestinal consequences of losing Slc7a9 function. The electrogenic and BBMV flux studies in the intestine suggested that arginine and ornithine are transported via other routes apart from system b(0,+). No remarkable gene expression changes were observed in other amino acid transporters and the peptide transporters in the intestine and kidney. Furthermore, the glomerular filtration rate (GFR) was reduced by 30% in knockout animals compared with wild-type animals. The fractional excretion of arginine was increased as expected (?100%), but fractional excretions of lysine (?35%), ornithine (?16%), and cystine (?11%) were less affected. Loss of function of b(0,+)AT reduced transport of cystine and arginine in renal BBMVs and completely abolished the exchanger activity of dibasic amino acids with neutral amino acids. In conclusion, loss of Slc7a9 function decreases the GFR and increases the excretion of several amino acids to a lesser extent than expected with no clear regulation at the mRNA and protein level of alternative transporters and no increased renal epithelial uptake. These observations indicate that transporters located in distal segments of the kidney and/or metabolic pathways may partially compensate for Slc7a9 loss of function.
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Tannic acid induced suicidal erythrocyte death.
Cell. Physiol. Biochem.
PUBLISHED: 09-26-2013
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The polyphenol tannic acid with antioxidant and antimicrobial potency may trigger suicidal death of nucleated cells or apoptosis and thus may counteract tumor growth. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, a suicidal death characterized by cell shrinkage and cell membrane scrambling with appearance of phosphatidylserine at the erythrocyte surface. A major trigger of eryptosis is increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). Erythrocytes could be sensitized to the eryptotic effect of cytosolic Ca(2+) by ceramide.
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Down-Regulation of the Na-Coupled Phosphate Transporter NaPi-IIa by AMP-Activated Protein Kinase.
Kidney Blood Press. Res.
PUBLISHED: 09-25-2013
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Background/Aims: The Na(+)-coupled phosphate transporter NaPi-IIa is the main carrier accomplishing renal tubular phosphate reabsorption. It is driven by the electrochemical Na(+) gradient across the apical cell membrane, which is maintained by Na(+) extrusion across the basolateral cell membrane through the Na(+)/K(+) ATPase. The operation of NaPi-IIa thus requires energy in order to avoid cellular Na(+) accumulation and K(+) loss with eventual decrease of cell membrane potential, Cl(-) entry and cell swelling. Upon energy depletion, early inhibition of Na(+)-coupled transport processes may delay cell swelling and thus foster cell survival. Energy depletion is sensed by the AMP-activated protein kinase (AMPK), a serine/threonine kinase stimulating several cellular mechanisms increasing energy production and limiting energy utilization. The present study explored whether AMPK influences the activity of NAPi-IIa. Methods: cRNA encoding NAPi-IIa was injected into Xenopus oocytes with or without additional expression of wild-type AMPK (AMPK(?1)-HA+AMPK(?1)-Flag+AMPK(?1)-HA), of inactive AMPK(?K45R) (AMPK(?1K45R)+AMPK(?1)-Flag+AMPK(?1)-HA) or of constitutively active AMPK(?R70Q) (AMPK(?1)-HA+AMPK(?1)-Flag+AMPK?1(R70Q)). NaPi-IIa activity was estimated from phosphate-induced current in dual electrode voltage clamp experiments. Results: In NaPi-IIa-expressing, but not in water-injected Xenopus oocytes, the addition of phosphate (1 mM) to the extracellular bath solution generated a current (Ip), which was significantly decreased by coexpression of wild-type AMPK and of AMPK(?R70Q) but not of AMPK(?K45R). The phosphate-induced current in NaPi-IIa- and AMPK-expressing Xenopus ooocytes was significantly increased by AMPK inhibitor Compound C (20 µM). Kinetic analysis revealed that AMPK significantly decreased the maximal transport rate. Conclusion: The AMP-activated protein kinase AMPK is a powerful regulator of NaPi-IIa and thus of renal tubular phosphate transport. © 2013 S. Karger AG, Basel.
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Effect of Carbon Monoxide Donor CORM-2 on Vitamin D3 Metabolism.
Kidney Blood Press. Res.
PUBLISHED: 09-17-2013
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Background/Aims: Carbon monoxide (CO) interferes with cytochrome-dependent cellular functions and acts as gaseous transmitter. CO is released from CO-releasing molecules (CORM) including tricarbonyl-dichlororuthenium (II) dimer (CORM-2), molecules considered for the treatment of several disorders including vascular dysfunction, inflammation, tissue ischemia and organ rejection. Cytochrome P450-sensitive function include formation of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) by renal 25-hydroxyvitamin D3 1-alpha-hydroxylase (Cyp27b1). The enzyme is regulated by PTH, FGF23 and klotho. 1,25(OH)2D3 regulates Ca(2+) and phosphate transport as well as klotho expression. The present study explored, whether CORM-2 influences 1,25(OH)2D3 formation and klotho expression. Methods: Mice were treated with intravenous CORM-2 (20 mg/kg body weight). Plasma 1,25(OH)2D3 and FGF23 concentrations were determined by ELISA, phosphate, calcium and creatinine concentrations by colorimetric methods, transcript levels by quantitative RT-PCR and protein expression by western blotting. Fgf23 mRNA transcript levels were further determined in rat osteosarcoma UMR106 cells without or with prior treatment for 24 hours with 20 µM CORM-2. Results: CORM-2 injection within 24 hours significantly increased FGF23 plasma levels and decreased 1,25(OH)2D3 plasma levels, renal Cyp27b1 gene expression as well as renal klotho protein abundance and transcript levels. Moreover, treatment of UMR106 cells with CORM-2 significantly increased Fgf23 transcript levels. Conclusion: CO-releasing molecule CORM-2 enhances FGF23 expression and release and decreases klotho expression and 1,25(OH)2D3 synthesis. © 2013 S. Karger AG, Basel.
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25-hydroxyvitamin d3 1-alpha-hydroxylase-dependent stimulation of renal klotho expression by spironolactone.
Kidney Blood Press. Res.
PUBLISHED: 09-13-2013
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Background: Klotho, a transmembrane protein, protease and hormone mainly expressed in kidney, is required for the suppression of 1,25(OH)2D3-generating 25-hydroxyvitamin D3 1-alpha-hydroxylase (Cyp27b1) by FGF23. Conversely, 1,25(OH)2D3 stimulates, by activating the vitamin D3 receptor (Vdr), the expression of klotho, thus establishing a negative feedback loop. Klotho protects against renal and vascular injury. Klotho deficiency accelerates aging and early death, effects at least partially due to excessive formation of 1,25(OH)2D3 and subsequent hyperphosphatemia. Klotho expression is inhibited by aldosterone. The present study explored the interaction of aldosterone and DOCA as well as the moderately selective mineralocorticoid receptor antagonist spironolactone on klotho expression. Methods: mRNA levels were determined utilizing quantitative RT-PCR in human embryonic kidney cells (HEK293) or in renal tissues from mice without or with prior mineralocorticoid (aldosterone or DOCA) and/or spironolactone treatment. In HEK293 cells, protein levels were determined by western blotting. The experiments in HEK293 cells were performed without or with silencing of CYP27B1, of vitamin D3 receptor (VDR) or of mineralocorticoid receptor (NR3C2). Results: In HEK293 cells aldosterone and in mice DOCA significantly decreased KLOTHO gene expression, effects opposed by spironolactone treatment. Spironolactone treatment alone significantly increased KLOTHO and CYP27B1 transcript levels in HEK293 cells (24 hours) and mice (8 hours or 5 days). Moreover, spironolactone significantly increased klotho and CYP27B1 protein levels in HEK293 cells (48 hours). Reduced NR3C2 expression following silencing did not significantly affect KLOTHO and CYP27B1 transcript levels in presence or absence of spironolactone. Silencing of CYP27B1 and VDR significantly blunted the stimulating effect of spironolactone on KLOTHO mRNA levels in HEK293 cells. Conclusion: Besides blocking the effects of aldosterone, spironolactone upregulates KLOTHO gene expression by upregulation of 25-hydroxyvitamin D3 1-alpha-hydroxylase with subsequent activation of the vitamin D3 receptor by 1,25(OH)2D3, an effect possibly independent from the mineralocorticoid receptor. © 2013 S. Karger AG, Basel.
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Effect of thioridazine on erythrocytes.
Toxins (Basel)
PUBLISHED: 09-04-2013
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Thioridazine, a neuroleptic phenothiazine with antimicrobial efficacy is known to trigger anemia. At least in theory, the anemia could result from stimulation of suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and by phospholipid scrambling of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca²?-concentration ([Ca²?](i)) and activation of p38 kinase. The present study explored, whether thioridazine elicits eryptosis.
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Inhibition of colonic tumor growth by the selective SGK inhibitor EMD638683.
Cell. Physiol. Biochem.
PUBLISHED: 09-03-2013
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The serum and glucocorticoid inducible kinase SGK1, which was originally cloned from mammary tumor cells, is highly expressed in some but not all tumors. SGK1 confers survival to several tumor cells. Along those lines, the number of colonic tumors following chemical carcinogenesis was decreased in SGK1 knockout mice. Recently, a highly selective SGK inhibitor (EMD638683) has been developed. The present study explored whether EMD638683 affects survival of colon carcinoma cells in vitro and impacts on development of colonic tumors in vivo.
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Age sensitivity of NF?B abundance and programmed cell death in erythrocytes induced by NF?B inhibitors.
Cell. Physiol. Biochem.
PUBLISHED: 09-03-2013
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Erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte outer membrane. Susceptibility to eryptosis is enhanced in aged erythrocytes and stimulated by NF?B-inhibitors Bay 11-7082 and parthenolide. Here we explored whether expression of NF?B and susceptibility to inhibitor-induced eryptosis is sensitive to erythrocyte age.
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Gremlin-1 is an inhibitor of macrophage migration inhibitory factor and attenuates atherosclerotic plaque growth in ApoE-/- Mice.
J. Biol. Chem.
PUBLISHED: 09-03-2013
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Monocyte infiltration and macrophage formation are pivotal steps in atherosclerosis and plaque vulnerability. Gremlin-1/Drm is crucial in embryo-/organogenesis and has been shown to be expressed in the adult organism at sites of arterial injury and to inhibit monocyte migration. The purpose of the present study was to evaluate and characterize the role of Gremlin-1 in atherosclerosis. Here we report that Gremlin-1 is highly expressed primarily by monocytes/macrophages in aortic atherosclerotic lesions of ApoE(-/-) mice and is secreted from activated monocytes and during macrophage development in vitro. Gremlin-1 reduces macrophage formation by inhibiting macrophage migration inhibitory factor (MIF), a cytokine critically involved in atherosclerotic plaque progression and vulnerability. Gremlin-1 binds with high affinity to MIF (KD = 54 nm), as evidenced by surface plasmon resonance analysis and co-immunoprecipitation, and reduces MIF-induced release of TNF-? from macrophages. Treatment of ApoE(-/-) mice with a dimeric recombinant fusion protein, mGremlin1-Fc, but not with equimolar control Fc or inactivated mGremlin1-Fc, reduced TNF-? expression, the content of monocytes/macrophages of atherosclerotic lesions, and attenuated atheroprogression. The present data disclose that Gremlin-1 is an endogenous antagonist of MIF and define a role for Gremlin-1/MIF interaction in atherosclerosis.
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Akt2 deficiency is associated with anxiety and depressive behavior in mice.
Cell. Physiol. Biochem.
PUBLISHED: 08-26-2013
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The economic burden associated with major depressive disorder and anxiety disorders render both disorders the most common and debilitating psychiatric illnesses. To date, the exact cellular and molecular mechanisms underlying the pathophysiology, successful treatment and prevention of these highly associated disorders have not been identified. Akt2 is a key protein in the phosphatidylinositide-3 (PI3K) / glycogen synthase 3 kinase (GSK3) signaling pathway, which in turn is involved in brain-derived neurotrophic factor (BDNF) effects on fear memory, mood stabilisation and action of several antidepressant drugs. The present study thus explored the impact of Akt2 on behaviour of mice.
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Chorein sensitivity of actin polymerization, cell shape and mechanical stiffness of vascular endothelial cells.
Cell. Physiol. Biochem.
PUBLISHED: 08-26-2013
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Endothelial cell stiffness plays a key role in endothelium-dependent control of vascular tone and arterial blood pressure. Actin polymerization and distribution of microfilaments is essential for mechanical cell stiffness. Chorein, a protein encoded by the VPS13A gene, defective in chorea-acanthocytosis (ChAc), is involved in neuronal cell survival as well as cortical actin polymerization of erythrocytes and blood platelets. Chorein is expressed in a wide variety of further cells, yet nothing is known about the impact of chorein on cells other than neurons, erythrocytes and platelets. The present study explored whether chorein is expressed in human umbilical vein endothelial cells (HUVECs) and addressed the putative role of chorein in the regulation of cytoskeletal architecture, stiffness and survival of those cells.
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AMPK?1-sensitivity of Orai1 and Ca(2+) entry in T - lymphocytes.
Cell. Physiol. Biochem.
PUBLISHED: 08-21-2013
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T-lymphocyte activation and function critically depends on Ca(2+) signaling, which is regulated by store operated Ca(2+) entry (SOCE). Human and mouse T lymphocytes express AMP activated kinase AMPK?1, which is rapidly activated following elevation of cytosolic Ca(2+) concentration ([Ca(2+)]i) by treatment of the cells with Ca(2+) ionophore or following inhibition of endosomal Ca(2+) ATPase with thapsigargin. AMPK is further activated by triggering of the T cell antigen receptor (TCR). The present study explored whether AMPK influences Ca(2+) entry and Ca(2+)-sensitive regulation of T-lymphocyte function.
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Upregulation of the Na(+)-Coupled Phosphate Cotransporters NaPi-IIa and NaPi-IIb by B-RAF.
J. Membr. Biol.
PUBLISHED: 08-20-2013
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B-RAF, a serine/threonine protein kinase, contributes to signaling of insulin-like growth factor IGF1. Effects of IGF1 include stimulation of proximal renal tubular phosphate transport, accomplished in large part by Na(+)-coupled phosphate cotransporter NaPi-IIa. The related Na(+)-coupled phosphate cotransporter NaPi-IIb accomplishes phosphate transport in intestine and tumor cells. The present study explored whether B-RAF influences protein abundance and/or activity of type II Na(+)-coupled phosphate cotransporters NaPi-IIa and NaPi-IIb. cRNA encoding wild-type NaPi-IIa and wild-type NaPi-IIb was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type B-RAF, and electrogenic phosphate transport determined by dual-electrode voltage clamp. NaPi-IIa protein abundance in Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified by chemiluminescence. Moreover, in HEK293 cells, the effect of B-RAF inhibitor PLX-4720 on NaPi-IIa cell surface protein abundance was quantified utilizing biotinylation of cell surface proteins and western blotting. In NaPi-IIa-expressing Xenopus oocytes, but not in oocytes injected with water, addition of phosphate to extracellular bath generated a current (I P), which was significantly increased following coexpression of B-RAF. According to kinetic analysis, coexpression of B-RAF enhanced the maximal IP. Coexpression of B-RAF further enhanced NaPi-IIa protein abundance in the Xenopus oocyte cell membrane. Treatment of HEK293 cells for 24 h with PLX-4720 significantly decreased NaPi-IIa cell membrane protein abundance. Coexpression of B-RAF, further significantly increased IP in NaPi-IIb-expressing Xenopus oocytes. Again, B-RAF coexpression enhanced the maximal IP. In conclusion, B-RAF is a powerful stimulator of the renal and intestinal type II Na(+)-coupled phosphate cotransporters NaPi-IIa and NaPi-IIb, respectively.
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Triggering of suicidal erythrocyte death by celecoxib.
Toxins (Basel)
PUBLISHED: 08-19-2013
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The selective cyclooxygenase-2 (COX-2) inhibitor celecoxib triggers apoptosis of tumor cells and is thus effective against malignancy. The substance is at least partially effective through mitochondrial depolarization. Even though lacking mitochondria, erythrocytes may enter apoptosis-like suicidal death or eryptosis, which is characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca2+-activity ([Ca2+]i). The present study explored whether celecoxib stimulates eryptosis. Forward scatter was determined to estimate cell volume, annexin V binding to identify phosphatidylserine-exposing erythrocytes, hemoglobin release to depict hemolysis, and Fluo3-fluorescence to quantify [Ca2+]i. A 48 h exposure of human erythrocytes to celecoxib was followed by significant increase of [Ca2+]i (15 µM), significant decrease of forward scatter (15 µM) and significant increase of annexin-V-binding (10 µM). Celecoxib (15 µM) induced annexin-V-binding was blunted but not abrogated by removal of extracellular Ca2+. In conclusion, celecoxib stimulates suicidal erythrocyte death or eryptosis, an effect partially due to stimulation of Ca2+ entry.
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