Increase In Cytosolic Ca2 Research Articles

Effect of Bexarotene on Platelet Activation and Apoptosis

Background/Aims: The retinoid X receptor (RXRs) stimulator Bexarotene ((4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl] benzoic acid) is used for the treatment of several malignancies. Bexarotene is at least in part effective by stimulation of apoptosis of tumor cells. Moreover, Bexarotene triggers eryptosis, the suicidal death of erythrocytes. Similar to erythrocytes, blood platelets lack nuclei but are nevertheless able to enter an apoptosis-like phenotype, characterized by caspase activation, cell shrinkage and cell membrane scrambling with phospha-tidylserine translocation to the cell surface. Platelet apoptosis is triggered by increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i), which further leads to degranulation and integrin activation. Platelet activation and apoptosis could be elicited by thrombin or collagen related peptide (CRP). The present study explored whether treatment of platelets with bexarotene modifies platelet activation and apoptosis following exposure to thrombin or CRP. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to bexarotene (6 µg/ml) without or with an additional treatment with thrombin (0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca²⁺-activity ([Ca²⁺]_i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from α_IIbβ₃ integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, and relative platelet volume from forward scatter. Results: In the absence of thrombin or CRP, the administration of bexarotene slightly but significantly increased [Ca²⁺]_i, but did not significantly modify P-selectin abundance, activated α_IIbβ₃ integrin, annexin-V-binding, cell volume, or caspase activity. Exposure of platelets to thrombin or CRP was followed by significant increase of [Ca²⁺]_i, P-selectin abundance, active α_IIbβ₃ integrin, annexin-V-binding, and caspase activity. The effects of thrombin on [Ca²⁺]_i, annexin-V-binding, cell volume, and caspase activity as well as the effects of CRP on [Ca²⁺]_i, P-selectin abundance, activated α_IIbβ₃ integrin, annexin-V-binding, cell volume, and caspase activity were significantly augmented in the presence of bexarotene. Conclusions: Bexarotene sensitizes blood platelets for thrombin and/or CRP induced activation and apoptosis.

Stimulation of Phospholipid Scrambling of the Erythrocyte Membrane by 9-Cis-Retinoic Acid

Background/Aims: The endogenous retinoid 9-cis-retinoic acid has previously been shown to trigger apoptosis in a wide variety of cells including several tumor cells and has thus been suggested for the treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms participating in the accomplishment of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i) and formation of ceramide. The present study explored, whether 9-cis-retinoic acid induces eryptosis and whether the effect involves Ca²⁺ and/or ceramide. Methods: Flow cytometry was employed to estimate erythrocyte volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca²⁺]_i from Fluo3-fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to 9-cis-retinoic acid (≥ 0.5 µg/ml) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Exposure to 9-cis-retinoic acid (≥ 0.5 µg/ml) significantly increased Fluo3-fluorescence, and the effect of 9-cis-retinoic acid on annexin-V-binding was significantly blunted by removal of extracellular Ca²⁺. Exposure to 9-cis-retinoic acid (1 µg/ml) further significantly increased the ceramide abundance at the erythrocyte surface and significantly increased hemolysis. Conclusions: 9-cis-retinoic acid triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part downstream of Ca²⁺ and ceramide.

Simvastatin, a Novel Stimulator of Eryptosis, the Suicidal Erythrocyte Death

Background/Aims: The 3-hydroxy-3-methyl-glutaryl-Coenzyme A (HMG-CoA) reductase inhibitor simvastatin has been shown to trigger apoptosis of several cell types. The substance has thus been proposed as an additional treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the extracellular face of the erythrocyte cell membrane. Signaling contributing to stimulation of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), induction of oxidative stress, increase of ceramide abundance, and activation of SB203580-sensitive p38 kinase. The present study explored, whether simvastatin induces eryptosis and aimed to shed light on cellular mechanisms involved. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. Results: A 48 h exposure of human erythrocytes to simvastatin (1 µg/ml) significantly decreased the forward scatter, significantly augmented the percentage of annexin-V-binding cells, significantly increased Fluo3-fluorescence, and significantly enhanced DCFDA fluorescence. Simvastatin tended to increase ceramide abundance, an effect, however, escaping statistical significance. The effect of simvastatin on annexin-V-binding was significantly blunted by removal of extracellular Ca²⁺ and by addition of SB203580 (2 µM). Conclusions: Simvastatin stimulates eryptosis, an effect at least in part due to Ca²⁺ entry, oxidative stress, and p38 kinase.

Inhibition of Suicidal Erythrocyte Death by Reversine

Background/Aims: The A3 adenosine receptor antagonist reversine (2-(4-morpholinoanilino)-6-cyclohexylaminopurine) influences cellular differentiation, inhibits cell proliferation, induces cell-cycle arrest, triggers apoptosis, causes cell swelling with polyploidy and stimulates autophagy. The effect on apoptosis involves mitochondria and caspases. Erythrocytes are lacking mitochondria but express caspases and are, similar to apoptosis of nucleated cells, able to enter suicidal erythrocyte death or eryptosis. Stimulators of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), energy depletion and oxidative stress. The present study explored, whether reversine influences eryptosis. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding and cell volume from forward scatter. Measurements were made without or with energy depletion (glucose deprivation for 48 hours), Ca²⁺ loading (30 minutes treatment with 1 µM Ca²⁺ ionophore ionomycin), or oxidative stress (15 min exposure to 0.3 mM tert-butylhydroperoxide). Results: A 48 hours exposure of human erythrocytes to reversine (1-10 µM) did not significantly modify the percentage of annexin-V-binding cells and forward scatter. Energy depletion, Ca²⁺ loading, and oxidative stress were each followed by profound and significant increase of the percentage annexin-V-binding erythrocytes and a significant decrease of forward scatter. The effects of each, Ca²⁺ loading, energy depletion and oxidative stress on annexin-V-binding were significantly blunted in the presence of reversine (1-10 µM). The effect of ionomycin, but not the effects of energy depletion and oxidative stress on forward scatter were again significantly blunted in the presence of reversine (≥1 µM]. Conclusions: Reversine is a powerful inhibitor of cell membrane scrambling following energy depletion, Ca²⁺ loading and oxidative stress.

Camalexin-Induced Cell Membrane Scrambling and Cell Shrinkage in Human Erythrocytes

Background/Aims: The thaliana phytoalexin Camalexin has been proposed for the treatment of malignancy. Camalexin counteracts tumor growth in part by stimulation of suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms contributing to the complex machinery executing eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress, ceramide, protein kinase C and caspases. The present study explored, whether Camalexin induces eryptosis and, if so, to shed light on mechanisms involved. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo-3 fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to Camalexin significantly increased the percentage of annexin-V-binding cells (≥ 10 µg/ml), significantly decreased forward scatter (≥ 5 µg/ml) and significantly increased Fluo-3-fluorescence (≥ 10 µg/ml), but did not significantly modify DCFDA fluorescence or ceramide abundance. The effect of Camalexin on annexin-V-binding was significantly blunted by removal of extracellular Ca²⁺, by kinase inhibitors staurosporine (1 µM) and chelerythrine (10 µM), as well as by caspase inhibitors zVAD (10 µM) and zIETD-fmk (50 µM). Conclusions: Camalexin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part depending on Ca²⁺ entry, as well as staurosporine and chelerythrine sensitive kinase(s) as well as zVAD and zIETD-fmk sensitive caspase(s).

P38 Kinase, SGK1 and NF-κB Dependent Up-Regulation of Na+/Ca2+ Exchanger Expression and Activity Following TGFß1 Treatment of Megakaryocytes

Background: TGFβ1, a decisive regulator of megakaryocyte maturation and platelet formation, has previously been shown to up-regulate both, store operated Ca²⁺ entry (SOCE) and Ca²⁺ extrusion by Na⁺/Ca²⁺ exchange. The growth factor thus augments the increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i) following release of Ca²⁺ from intracellular stores and accelerates the subsequent decline of [Ca²⁺]_i. The effect on SOCE is dependent on a signaling cascade including p38 kinase, serum & glucocorticoid inducible kinase SGK1, and nuclear factor NFκB. The specific Na⁺/Ca²⁺ exchanger isoforms involved and the signalling regulating the Na⁺/Ca²⁺ exchangers remained, however elusive. The present study explored, whether TGFβ1 influences the expression and function of K⁺ insensitive (NCX) and K⁺ sensitive (NCKX) Na⁺/Ca²⁺ exchangers, and aimed to shed light on the signalling involved. Methods: In human megakaryocytic cells (MEG01) RT-PCR was performed to quantify NCX/NCKX isoform transcript levels, [Ca²⁺]_i was determined by Fura-2 fluorescence, and Na⁺/Ca²⁺ exchanger activity was estimated from the increase of [Ca²⁺]_i following switch from an extracellular solution with 130 or 90 mM Na⁺ and 0 mM Ca²⁺ to an extracellular solution with 0 Na⁺ and 2 mM Ca²⁺. K⁺ concentration was 0 mM for analysis of NCX and 40 mM for analysis of NCKX. Results: TGFβ1 (60 ng/ml, 24 h) significantly increased the transcript levels of NCX1, NCKX1, NCKX2 and NCKX5. Moreover, TGFβ1 (60 ng/ml, 24 h) significantly increased the activity of both, NCX and NCKX. The effect of TGFβ1 on NCX and NCKX transcript levels and activity was significantly blunted by p38 kinase inhibitor Skepinone-L (1 µM), the effect on NCX and NCKX activity further by SGK1 inhibitor GSK-650394 (10 µM) and NFκB inhibitor Wogonin (100 µM). Conclusions: TGFβ1 markedly up-regulates transcription of NCX1, NCKX1, NCKX2, and NCKX5 and thus Na⁺/Ca²⁺ exchanger activity, an effect requiring p38 kinase, SGK1 and NFκB.

Inhibition of Erythrocyte Cell Membrane Scrambling by ASP3026

Background/Aims: The anaplastic lymphoma kinase (ALK) inhibitor ASP3026 is in clinical development for the treatment of ALK expressing non-small cell lung carcinoma (NSCLC). ASP3026 is in part effective by inducing apoptosis of tumor cells. Erythrocytes lack mitochondria and nuclei, key organelles in the execution of apoptosis, but are nevertheless able to enter suicidal death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Eryptosis is triggered by cell stress, such as energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i). The present study explored, whether ASP3026 impacts on eryptosis. Methods: Human erythrocytes have been exposed to energy depletion (glucose withdrawal for 48 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca²⁺ loading with Ca²⁺ ionophore ionomycin (1 µM for 60 min) in absence and presence of ASP3026 (1-4 µg/ml). Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, and cell volume from forward scatter. Results: Treatment with ASP3026 alone did not significantly modify annexin-V-binding or forward scatter. Energy depletion, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. ASP3026 significantly blunted the effect of energy depletion and oxidative stress, but not of ionomycin on annexin-V-binding. ASP3026 did not significantly influence the effect of any maneuver on forward scatter. Conclusions: ASP3026 is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion and oxidative stress.

Stimulation of Erythrocyte Cell Membrane Scrambling by Adarotene

Background/Aims: The atypical retinoid E23-(40-hydroxyl-30-adamantylbiphenyl-4-yl) acrylic acid (ST1926, adarotene) is used in the treatment of malignancy. The effect of ST1926 is at least in part due to stimulation of apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal death of erythrocytes. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca²⁺ activity [Ca²⁺]<Sub>i</Sub>, oxidative stress and ceramide. The present study explored, whether adarotene induces eryptosis and, if so, to test for the involvement of Ca²⁺ entry, oxidative stress and ceramide. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]<Sub>i</Sub> from Fluo3-fluorescence, reactive oxygen species (ROS) formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to adarotene (9 µM) significantly increased the percentage of annexin-V-binding cells, an effect paralleled by significant decrease of forward scatter, as well as significant increase of Fluo3-fluorescence, DCFDA fluorescence, and ceramide abundance. The effect of adarotene (9 µM) on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca²⁺. Conclusions: Adarotene stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by and at least in part due to Ca²⁺ entry, oxidative stress and ceramide.

Triggering of Suicidal Erythrocyte Death by Gefitinib

Background/Aims: The epidermal growth factor receptor-tyrosine kinase inhibitor gefitinib is effective against several malignancies and is mainly utilized in the treatment of epidermal growth factor receptor mutation positive non-small cell lung cancer. The anti-cancer effect of the drug involves stimulation of apoptosis. Side effects of gefitinib include anemia. At least in theory, the development of anemia during gefitinib treatment could result from triggering of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i) and generation of oxidative stress. The present study explored, whether gefitinib stimulates eryptosis and, if so, whether its effect involves Ca²⁺ entry and/or oxidative stress. Methods: Flow cytometry was employed to quantify cell volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca²⁺]_i from Fluo3-fluorescence, and reactive oxygen species (ROS) abundance from 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence. Results: A 48 hours exposure of human erythrocytes to gefitinib (≥ 2 µg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Gefitinib did not significantly increase Fluo3-fluorescence but the effect of gefitinib on annexin-V-binding was significantly blunted by removal of extracellular Ca²⁺. Gefitinib further significantly increased DCFDA fluorescence. Conclusions: Gefitinib triggers erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part dependent on extracellular Ca²⁺ and paralleled by oxidative stress.

Triggering of Eryptosis, the Suicidal Erythrocyte Death, by Perifosine

Background/Aims: The alkylphospholipid perifosine is used for the treatment of malignancy. The substance is effective by triggering suicidal tumor cell death or apoptosis. Side effects of perifosine include anemia. At least in theory, perifosine-induced anemia could result from stimulation of suicidal erythrocyte death or eryptosis. Hallmarks of eryptosis are cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms participating in the orchestration of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress, increase of ceramide abundance, as well as activation of staurosporine sensitive protein kinase C and/or of SB203580 sensitive p38 kinase. The present study explored, whether perifosine induces eryptosis and, if so, whether its effect involves and/or requires Ca²⁺ entry, oxidative stress, ceramide and kinase activation. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. Results: A 24 hours exposure of human erythrocytes to perifosine (2.5 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased average forward scatter, significantly increased the percentage of shrunken erythrocytes, and significantly decreased the percentage of swollen erythrocytes. Perifosine significantly increased the percentage of hemolytic erythrocytes. Perifosine significantly increased Fluo3-fluorescence, but decreased DCFDA fluorescence and ceramide abundance. The effect of perifosine on annexin-V-binding was significantly blunted by removal of extracellular Ca²⁺ and by addition of staurosporine (1 µM), but not by addition of SB203580 (2 µM). Conclusions: Perifosine triggers eryptosis, an effect at least in part due to Ca²⁺ entry and activation of staurosporine sensitive kinases.

Triggering of Eryptosis, the Suicidal Erythrocyte Death by Mammalian Target of Rapamycin (mTOR) inhibitor Temsirolimus

Background/Aims: The mammalian target of rapamycin (mTOR) inhibitor temsirolimus is utilized for the treatment of malignancy. Temsirolimus is at least in part effective by triggering suicidal tumor cell death. The most common side effect of temsirolimus treatment is anemia. At least in theory, the anemia following temsirolimus treatment could result from stimulation of eryptosis, the suicidal erythrocyte death. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the orchestration of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress, ceramide, as well as activation of staurosporine and chelerythrine sensitive protein kinase C, SB203580 sensitive p38 kinase, D4476 sensitive casein kinase 1, and zVAD sensitive caspases. The purpose of the present study was to test whether temsirolimus influences eryptosis and, if so, to shed light on the signaling involved. Methods: Flow cytometry was employed to estimate cell volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca²⁺]_i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was determined from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to temsirolimus (5 – 20 µg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Temsirolimus significantly increased Fluo3-fluorescence, DCFDA fluorescence and ceramide abundance at the erythrocyte surface. The effect of temsirolimus on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca²⁺ and by addition of staurosporine (1 µM) or chelerythrine (10 µM) but not significantly modified by addition of SB203580 (2 µM), D4476 (10 µM), or zVAD (10 µM). Chelerythrine (10 µM) further significantly blunted the effect of temsirolimus on DCFDA fluorescence but not ceramide formation. Removal of extracellular Ca²⁺ had no effect on temsirolimus induced ROS formation or ceramide abundance. Conclusions: Temsirolimus triggers eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca²⁺ entry, oxidative stress, ceramide and activation of staurosporine/Chelerythrine sensitive kinase(s).

Inhibition of Suicidal Erythrocyte Death by Volasertib

Background/Aims: The Polo-like kinase 1 (Plk1) inhibitor volasertib is used in the treatment of malignancy. Volasertib is partially effective by triggering suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal cell death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Stimulators of eryptosis include energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i). The present study explored, whether volasertib impacts on eryptosis. Methods: Human erythrocytes have been exposed to energy depletion (glucose withdrawal for 48 hours), hyperosmotic shock (addition of 550 mM sucrose for 6 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca²⁺ ionophore ionomycin (1 µM for 60 min) in absence and presence of volasertib (0.5-1.5 µg/ml) and flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3 fluorescence, reactive oxygen species from 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence and ceramide abundance utilizing antibodies. For comparison, annexin-V-binding and forward scatter were determined following a 48 hours exposure of human leukemic K562 cells in RPMI-1640 medium to volasertib. Results: Treatment with volasertib alone did not significantly modify annexin-V-binding or forward scatter in mature erythrocytes. Energy depletion, hyperosmotic shock, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. Volasertib significantly blunted the effect of energy depletion and hyperosmotic shock, but not of oxidative stress and ionomycin on annexin-V-binding. Volasertib did not significantly influence the effect of any maneuver on forward scatter. In K562 cells, volasertib enhanced annexin-V-binding and decreased the forward scatter. Conclusions: Volasertib is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion and hyperosmotic shock, effects contrasting the stimulation of K562 cell apoptosis.

Effects of Antimalarial Tafenoquine on Blood Platelet Activity and Survival

Background/Aims: The 8-aminoquinoline tafenoquine has been shown to be effective against Plasmodia, Leishmania and Trypanosoma. The substance is at least in part effective by triggering apoptosis of the parasites. Moreover, tafenoquine has been shown to trigger eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. The effect of tafenoquine on eryptosis is in part due to stimulation of Ca²⁺ entry and oxidative stress. Ca²⁺ entry is a critical event in the activation of blood platelets by thrombin and collagen related peptide (CRP). The present study explored, whether tafenoquine influences Ca²⁺ entry, activation and apoptosis of blood platelets. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to tafenoquine (2.5 µg/ml) without or with an additional treatment with thrombin (0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca²⁺-activity ([Ca²⁺]<Sub>i</Sub>) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from α<Sub>IIb</Sub>β<Sub>3</Sub> integrin abundance, phosphatidylserine abundance from annexin-V-binding, relative platelet volume from forward scatter, reactive oxygen species (ROS) from DCF fluorescence, caspase 3 activity with an active caspase-3 Staining kit, and aggregation utilizing staining with CD9-APC and CD9-PE. Results: Both, thrombin (0.01 U/ml) and CRP (2 µg/ml or 5 µg/ml), significantly increased [Ca²⁺]<Sub>i</Sub>, P-selectin abundance, active α<Sub>IIb</Sub>β<Sub>3</Sub> integrin, and annexin-V-binding, and both significantly decreased platelet volume, activated caspase 3 and stimulated aggregation. Administration of tafenoquine (2.5 µg/ml, 30 min) significantly decreased [Ca²⁺]<Sub>i</Sub> both, in the absence and presence of thrombin and CRP. Tafenoquine significantly blunted the effect of thrombin and CRP on [Ca²⁺]<Sub>i</Sub>, P-selectin abundance, and active α<Sub>IIb</Sub>β<Sub>3</Sub> integrin, but significantly increased ROS and annexin-V-binding, significantly augmented the effect of thrombin on caspase 3 activity and platelet volume and significantly enhanced platelet aggregation. Conclusions: Tafenoquine counteracts thrombin and CRP induced increase of cytosolic Ca²⁺ activity and platelet activation, but enhances platelet apoptosis and platelet aggregation.

Triggering of Suicidal Erythrocyte Death by Exemestane

Background/Aims: The steroidal aromatase inactivator exemestane blocks estrogen biosynthesis and is thus employed for the prevention and treatment of breast cancer. Exemestane is in part effective by stimulation of suicidal cell death or apoptosis. Side effects of exemestane treatment include anemia. At least in theory, exemestane induced anemia could be secondary to stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress, ceramide, several kinases and caspases. The present study explored, whether exemestane is able to trigger eryptosis and, if so, to shed some light on the signaling involved. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCF fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to exemestane (≥ 10 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying forward scatter. Exemestane significantly increased Fluo3-fluorescence (10 and 20, but not 40 µg/ml), DCF fluorescence (40 µg/ml), and ceramide abundance (40 µg/ml). The effect of exemestane (40 µg/ml) on annexin-V-binding was significantly blunted by antioxidant N-acetylcysteine (1mM), but was not significantly modified by removal or increase of extracellular Ca²⁺, by p38 kinase inhibitor SB203580 (2 µM), casein kinase inhibitor D4476 (10 µM) and caspase inhibitor zVAD (10 µM). Conclusions: Exemestane triggers phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by enhanced [Ca²⁺]_i, oxidative stress, and increased ceramide abundance.

Human salivary gland cells express bradykinin receptors that modulate the expression of proinflammatory cytokines.

Bradykinin is an important peptide modulator that affects the function of neurons and immune cells. However, there is no evidence of the bradykinin receptors and their functions in human salivary glands. Here we have identified and characterized bradykinin receptors on human submandibular gland cells. Both bradykinin B1 and B2 receptors are expressed on human submandibular gland cells, A253 cells, and HSG cells. Bradykinin increased the intracellular Ca2+ concentration ([Ca2+ ]i ) in a concentration-dependent manner. Interestingly, a specific agonist of the B1 receptor did not have any effect on [Ca2+ ]i in HSG cells, whereas specific agonists of the B2 receptor had a Ca2+ mobilizing effect. Furthermore, application of the B1 receptor antagonist, R715, did not alter the bradykinin-mediated increase in cytosolic Ca2+ , whereas the B2 receptor antagonist, HOE140, showed a strong inhibitory effect, which implies that bradykinin B2 receptors are functional in modulating the concentration of cytosolic Ca2+ . Bradykinin did not affect a carbachol-induced rise of [Ca2+ ]i and did not modulate translocation of aquaporin-5. However, bradykinin did promote the expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), implying the role of bradykinin in salivary gland inflammation. These data suggest that bradykinin receptors are involved in Ca2+ signaling in human submandibular gland cells and serve a unique role, which is separate from that of other salivary gland G protein-coupled receptors.

Structural perturbations induced by Asn131 and Asn171 glycosylation converge within the EFSAM core and enhance stromal interaction molecule-1 mediated store operated calcium entry

A major intracellular calcium (Ca2+) uptake pathway in both excitable and non-excitable eukaryotic cells is store-operated Ca2+ entry (SOCE). SOCE is the process by which endoplasmic reticulum (ER)-stored Ca2+ depletion leads to activation of plasma membrane Ca2+ channels to provide a sustained increase in cytosolic Ca2+ levels that mediate a plethora of physiological processes ranging from the immune response to platelet aggregation. Stromal interaction molecule-1 (STIM1) is the principal regulator of SOCE and responds to changes in ER stored Ca2+ through luminal sensing machinery composed of EF-hand and SAM domains (EFSAM). The EFSAM domain can undergo N-glycosylation at Asn131 and Asn171 sites; however, the precise role of EFSAM N-glycosylation in the Ca2+ sensing mechanism of STIM1 is unclear. By establishing a site-specific chemical approach to covalently linking glucose to EFSAM and examining α-helicity, thermal stability, three dimensional atomic-resolution structure, Ca2+ binding affinity and oligomerization, we show that N-glycosylation of the EFSAM domain enhances the properties that promote STIM1 activation. This augmentation occurs through changes in structure localized near the Asn131 and Asn171 sites that together permeate through the protein core and lead to decreased Ca2+ binding affinity, reduced stability and enhanced oligomerization. Congruently, Ca2+ influx via SOCE in HEK293 cells co-expressing Orai1 and STIM1 was diminished when N-glycosylation was blocked by introducing Asn131Gln and Asn171Gln mutations. Collectively, our data suggests that N-glycosylation enhances the EFSAM destabilization-coupled oligomerization in response to ER Ca2+ depletion thereby augmenting the role of STIM1 as a robust ON/OFF regulator of SOCE. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

The effect of chronic alcohol consumption on mitochondrial calcium handling in hepatocytes.

The damage to liver mitochondria is universally observed in both humans and animal models after excessive alcohol consumption. Acute alcohol treatment has been shown to stimulate calcium (Ca2+) release from internal stores in hepatocytes. The resultant increase in cytosolic Ca2+ is expected to be accumulated by neighboring mitochondria, which could potentially lead to mitochondrial Ca2+ overload and injury. Our data indicate that total and free mitochondrial matrix Ca2+ levels are, indeed, elevated in hepatocytes isolated from alcohol-fed rats compared with their pair-fed control littermates. In permeabilized hepatocytes, the rates of mitochondrial Ca2+ uptake were substantially increased after chronic alcohol feeding, whereas those of mitochondrial Ca2+ efflux were decreased. The changes in mitochondrial Ca2+ handling could be explained by an up-regulation of the mitochondrial Ca2+ uniporter and loss of a cyclosporin A-sensitive Ca2+ transport pathway. In intact cells, hormone-induced increases in mitochondrial Ca2+ declined at slower rates leading to more prolonged elevations of matrix Ca2+ in the alcohol-fed group compared with controls. Moreover, treatment with submaximal concentrations of Ca2+-mobilizing hormones markedly increased the levels of mitochondrial reactive oxygen species (ROS) in hepatocytes from alcohol-fed rats, but did not affect ROS levels in controls. The changes in mitochondrial Ca2+ handling are expected to buffer and attenuate cytosolic Ca2+ increases induced by acute alcohol exposure or hormone stimulation. However, these alterations in mitochondrial Ca2+ handling may also lead to Ca2+ overload during cytosolic Ca2+ increases, which may stimulate the production of mitochondrial ROS, and thus contribute to alcohol-induced liver injury.

Molecular Mechanism of Dihydropyridine Ca2+ Channel Blockers in Pulmonary Hypertension.

Idiopathic pulmonary arterial hypertension (IPAH) is a progressive and fatal disease of unidentified pathogenesis. IPAH is pathologically characterized as sustained vasoconstriction and vascular remodeling of the pulmonary artery. In pulmonary arterial smooth muscle cells (PASMCs), an increase in cytosolic Ca2+ concentration ([Ca2+]cyt) triggers vasoconstriction and stimulates cell proliferation leading to vascular remodeling. However, dihydropyridine-type voltage-dependent Ca2+ channel blockers are only effective in very few patients with IPAH (<10%). It is unclear why dihydropyridine Ca2+ channel blockers are not therapeutically effective in a majority of IPAH patients. We have previously shown that extracellular Ca2+-sensing receptor (CaSR) is upregulated in PASMCs from IPAH patients, and it contributes to enhanced [Ca2+]cyt responses and augmented cell proliferation. In this study, the effects of dihydropyridine Ca2+ channel blockers on [Ca2+]cyt responses mediated by CaSR were examined in IPAH-PASMCs. Nifedipine (dihydropyridines) enhanced the CaSR-mediated increase in [Ca2+]cyt in IPAH-PASMCs, but not in PASMCs from normal subjects. Nicardipine (dihydropyridines) and Bay K 8644 (a dihydropyridine Ca2+ channel activator) also augmented the CaSR-mediated [Ca2+]cyt increase in IPAH-PASMCs. In contrast, non-dihydropyridine Ca2+ channel blockers such as diltiazem (benzothiazepines) and verapamil (phenylalkylamines) had no effect on the [Ca2+]cyt response in IPAH-PASMCs. Finally, in monocrotaline-induced pulmonary hypertensive rats, nifedipine caused further increase in right ventricular systolic pressure and thus right ventricular hypertrophy. In conclusion, dihydropyridine Ca2+ channel blockers could exacerbate symptoms of pulmonary hypertension in IPAH patients with upregulated CaSR in PASMCs.

Chenodeoxycholic acid requires activation of EGFR, EPAC, and Ca2+ to stimulate CFTR-dependent Cl- secretion in human colonic T84 cells.

Bile acids are known to initiate intricate signaling events in a variety of tissues, primarily in the liver and gastrointestinal tract. Of the known bile acids, only the 7α-dihydroxy species, deoxycholic acid and chenodeoxycholic acid (CDCA), and their conjugates, activate processes that stimulate epithelial Cl- secretion. We have previously published that CDCA acts in a rapid manner to stimulate colonic ion secretion via protein kinase A (PKA)-mediated activation of the dominant Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR) (Ao M, Sarathy J, Domingue J, Alrefai WA, and Rao MC. Am J Physiol Cell Physiol 305: C447-C456, 2013); however, PKA signaling did not account for the entire CDCA response. Here we show that in human colonic T84 cells, CDCA's induction of CFTR activity, measured as changes in short-circuit current (Isc), is dependent on epidermal growth factor receptor (EGFR) activation and does not involve the bile acid receptors TGR5 or farnesoid X receptor. CDCA activation of Cl- secretion does not require Src, mitogen-activated protein kinases, or phosphoinositide 3-kinase downstream of EGFR but does require an increase in cytosolic Ca2+ In addition to PKA signaling, we found that the CDCA response requires the novel involvement of the exchange protein directly activated by cAMP (EPAC). EPAC is a known hub for cAMP and Ca2+ cross talk. Downstream of EPAC, CDCA activates Rap2, and changes in free cytosolic Ca2+ were dependent on both EPAC and EGFR activation. This study establishes the complexity of CDCA signaling in the colonic epithelium and shows the contribution of EGFR, EPAC, and Ca2+ in CDCA-induced activation of CFTR-dependent Cl- secretion.

Hepatocyte growth factor inhibits hypoxia/reoxygenation-induced activation of xanthine oxidase in endothelial cells through the JAK2 signaling pathway.

Vascular endothelial cells (ECs) appear to be one of the primary targets of hypoxia/reoxygenation (H/R) injury. In our previous study, we demonstrated that hepatocyte growth factor (HGF) exhibited a protective effect in cardiac microvascular endothelial cells (CMECs) subjected to H/R by inhibiting xanthine oxidase (XO) by reducing the cytosolic Ca2+ concentration increased in response to H/R. The precise mechanisms through which HGF inhibits XO activation remain to be determined. In the present study, we examined the signaling pathway through which HGF regulates Ca2+ concentrations and the activation of XO during H/R in primary cultured rat CMECs. CMECs were exposed to 4 h of hypoxia and 1 h of reoxygenation. The protein expression of XO and the activation of the phosphoinositide 3-kinase (PI3K), janus kinase 2 (JAK2) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways were detected by western blot analysis. Cytosolic calcium (Ca2+) concentrations and reactive oxygen species (ROS) levels were measured by flow cytometry. The small interfering RNA (siRNA)-mediated knockdown of XO inhibited the increase in ROS production induced by H/R. LY294002 and AG490 inhibited the H/R-induced increase in the production and activation of XO. The PI3K and JAK2 signaling pathways were activated by H/R. The siRNA-mediated knockdown of PI3K and JAK2 also inhibited the increase in the production of XO protein. HGF inhibited JAK2 activation whereas it had no effect on PI3K activation. The siRNA-mediated knockdown of JAK2 prevented the increase in cytosolic Ca2+ induced by H/R. Taken together, these findings suggest that H/R induces the production and activation of XO through the JAK2 and PI3K signaling pathways. Furthermore, HGF prevents XO activation following H/R primarily by inhibiting the JAK2 signaling pathway and in turn, inhibiting the increase in cytosolic Ca2+.