Venkanna Pasham

Stimulation of suicidal erythrocyte death by benzethonium.

Benzethonium, an antimicrobial surfactant widely used as preservative of pharmaceuticals, topical wound care product and oral disinfectant, triggers apoptosis of several cell types. The apoptosis is preceded and possibly triggered by mitochondrial depolarization. Even though lacking mitochondria, erythrocytes may similarly undergo suicidal cell death or eryptosis. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis may be triggered by energy depletion, which leads to increase of cytosolic Ca2+-activity with subsequent Ca2+-sensitive cell shrinkage and cell membrane scrambling. Ca2+-sensitivity is enhanced by ceramide. The present study explored the effect of benzethonium on eryptosis. Cell membrane scrambling was estimated from binding of fluorescent annexin V to phosphatidylserine, cell volume from forward scatter in FACS analysis, cytosolic Ca2+-concentration from Fluo3-fluorescence, hemolysis from hemoglobin release, lactate formation by colorimetry and ceramide utilizing fluorescent antibodies. A 48 hours exposure to benzethonium (=5µM) significantly increased cytosolic Ca2+-concentration, decreased forward scatter and triggered annexin V-binding affecting some 30% of the erythrocytes at 5 µM benzethonium. Only 5% of treated erythrocytes were hemolytic. The effects of benzethonium on annexin V binding were blunted in the nominal absence of Ca2+ and in the presence of amiloride (1 mM) but not in the presence of the pancaspase inhibitor zVAD (10 µM). Benzethonium further significantly enhanced the effect of glucose depletion on cytosolic Ca2+-concentration and annexin V-binding, but significantly blunted the effect of glucose depletion on forward scatter. Benzethonium (5 µM) significantly enhanced lactic acid formation but not ceramide abundance. The present observations disclose a novel effect of benzethonium, i.e. triggering of suicidal death of erythrocytes.

Enhanced erythrocyte membrane exposure of phosphatidylserine following sorafenib treatment: an in vivo and in vitro study.

Background: Sorafenib (Nexavar®), a polytyrosine kinase inhibitor, stimulates apoptosis and is thus widely used for chemotherapy in hepatocellular carcinoma (HCC). Hematological side effects of Nexavar® chemotherapy include anemia. Erythrocytes may undergo apoptosis-like suicidal death or eryptosis, which is characterized by cell shrinkage and phosphatidylserine-exposure at the cell surface. Signaling leading to eryptosis include increase in cytosolic Ca2+activity ([Ca2+]i), formation of ceramide, ATP-depletion and oxidative stress. The present study explored, whether sorafenib triggers eryptosis in vitro and in vivo. Methods: [Ca2+]i was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, ceramide with antibody binding-dependent fluorescence, cytosolic ATP with a luciferin–luciferase-based assay, and oxidative stress from 2’,7’ dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Results: A 48 h exposure of erythrocytes to sorafenib (≥0.5 µM) significantly increased Fluo 3 fluorescence, decreased forward scatter, increased annexin-V-binding and triggered slight hemolysis (≥5 µM), but did not significantly modify ceramide abundance and cytosolic ATP. Sorafenib treatment significantly enhanced DCFDA-fluorescence and the reducing agents N-acetyl-L-cysteine and tiron significantly blunted sorafenib-induced phosphatidylserine exposure. Nexavar® chemotherapy in HCC patients significantly enhanced the number of phosphatidylserine-exposing erythrocytes. Conclusions: The present observations disclose novel effects of sorafenib, i.e. stimulation of suicidal erythrocyte death or eryptosis, which may contribute to the pathogenesis of anemia in Nexavar®-based chemotherapy.

Tanshinone IIA stimulates erythrocyte phosphatidylserine exposure

Tanshinone IIA, an antimicrobial, antioxidant, antianaphylactic, antifibrotic, vasodilating, antiatherosclerotic, organo-protective and antineoplastic component from the rhizome of Salvia miltiorrhiza, is known to trigger apoptosis of tumor cells. Tanshinone IIA is effective in part through mitochondrial depolarization and altered gene expression. Erythrocytes lack mitochondria and nuclei but may undergo eryptosis, an apoptosis-like suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis is triggered by increase of cytosolic Ca2+ activity, ATP depletion and ceramide formation. The present study explored, whether tanshinone IIA elicits eryptosis. Cytosolic Ca2+-concentration was determined from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from binding of fluorescent annexin V, hemolysis from hemoglobin concentration in the supernatant, ATP concentration utilizing luciferin–luciferase and ceramide formation utilizing fluorescent anticeramide antibodies. Clearance of circulating erythrocytes was estimated by CFSE-labeling. A 48 h exposure to tanshinone IIA (≥10 µM) significantly increased cytosolic Ca2+-concentration, decreased ATP concentration (25 µM), increased lactate concentration (25 µM), increased ceramide formation (25 µM), decreased forward scatter, increased annexin-V-binding and increased (albeit to a much smaller extent) hemolysis. The effect of 25 µM tanshinone IIA on annexin-V binding was partially reversed in the nominal absence of Ca2+. Labelled tanshinone IIA-treated erythrocytes were more rapidly cleared from the circulating blood in comparison to untreated erythrocytes. The present observations reveal a completely novel effect of tanshinone IIA, i.e. triggering of Ca2+ entry, ATP depletion and ceramide formation in erythrocytes, events eventually leading to eryptosis with cell shrinkage and cell membrane scrambling.

Sgk1 sensitivity of Na+/H+ exchanger activity and cardiac remodeling following pressure overload

Sustained increase of cardiac workload is known to trigger cardiac remodeling with eventual development of cardiac failure. Compelling evidence points to a critical role of enhanced cardiac Na+/H+ exchanger (NHE1) activity in the underlying pathophysiology. The signaling triggering up-regulation of NHE1 remained, however, ill defined. The present study explored the involvement of the serum- and glucocorticoid-inducible kinase Sgk1 in cardiac remodeling due to transverse aortic constriction (TAC). To this end, experiments were performed in gene targeted mice lacking functional Sgk1 (sgk1−/−) and their wild-type controls (sgk1+/+). Transcript levels have been determined by RT-PCR, cytosolic pH (pHi) utilizing 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, Na+/H+ exchanger activity by the Na+-dependent realkalinization after an ammonium pulse, ejection fraction (%) utilizing cardiac cine magnetic resonance imaging and cardiac glucose uptake by PET imaging. As a result, TAC increased the mRNA expression of Sgk1 in sgk1+/+ mice, paralleled by an increase in Nhe1 transcript levels as well as Na+/H+ exchanger activity, all effects virtually abrogated in sgk1−/− mice. In sgk1+/+ mice, TAC induced a decrease in Pgc1a mRNA expression, while Spp1 mRNA expression was increased, both effects diminished in the sgk1−/− mice. TAC was followed by a significant increase of heart and lung weight in sgk1+/+ mice, an effect significantly blunted in sgk1−/− mice. TAC increased the transcript levels of Anp and Bnp, effects again significantly blunted in sgk1−/− mice. TAC increased transcript levels of Collagen I and III as well as Ctgf mRNA and CTGF protein abundance, effects significantly blunted in sgk1−/− mice. TAC further decreased the ejection fraction in sgk1+/+ mice, an effect again attenuated in sgk1−/− mice. Also, cardiac FDG-glucose uptake was increased to a larger extent in sgk1+/+ mice than in sgk1−/− mice after TAC. These observations point to an important role for SGK1 in cardiac remodeling and development of heart failure following an excessive work load.

Effect of bacterial lipopolysaccharide on Na(+)/H(+) exchanger activity in dendritic cells.

The function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity, is stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS). In macrophages, ROS formation is paralleled by activation of the Na+/H+ exchanger, a carrier involved in the regulation of cytosolic pH and cell volume. The present study explored whether LPS influence Na+/H+ exchanger activity in DCs. The DCs were isolated from murine bone marrow, cell volume was estimated from forward scatter in FACS analysis, ROS production from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, apoptosis from annexin V binding, cytosolic pH (pHi) from 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence and Na+/H+ exchanger activity from the Na+ dependent realkalinization following an ammonium pulse. Exposure of DCs to LPS (1 µg/ml) led to a transient increase of Na+/H+ exchanger activity. Moreover, LPS increased forward scatter and ROS formation and decreased apoptosis. The NHE1 inhibitor cariporide (10 µM) virtually abrogated Na+/H+ exchanger activity, inhibited LPS-induced cell swelling, blunted LPS-induced ROS formation and reversed the antiapoptotic effect of LPS. Na+/H+ exchanger activity was stimulated by oxidative stress and LPS induced stimulation of NHE activity was abolished in presence of ROS chelators (Tempol, Tiron and Vitamin C). In conclusion, LPS treatment transiently upregulates the Na+/H+ exchanger in DCs, an effect required for the effects of LPS on DC survival, cell volume and ROS formation.

PI3 kinase and PDK1 in the regulation of the electrogenic intestinal dipeptide transport.

The phosphoinositol 3 kinase (PI3K) and the phosphoinositide dependent kinase (PDK1) stimulate the serum and glucocorticoid inducible kinase (SGK) and protein kinase B (PKB/Akt) isoforms, kinases stimulating a variety of transporters. Most recently, SGK1 was shown to stimulate the peptide transporters PepT1 and PepT2, and to mediate the glucocorticoid stimulation of PepT1. Basal electrogenic intestinal peptide transport was, however, not dependent on the presence of SGK1. The present study explored whether basal electrogenic intestinal peptide transport is dependent on PI3K or PDK1. To this end, peptide transport in intestinal segments was determined utilizing Ussing chamber analysis. Cytosolic pH (pHi) was determined by BCECF fluorescence. The luminal addition of 5 mM dipeptide gly-gly induced a current (Ip) across intestinal segments. Ip was significantly decreased in the presence of PI3 kinase inhibitors Wortmannin (1 µM) or LY294002 (50 µM). Exposure of isolated intestinal cells to 5 mM gly-gly was followed by cytosolic acidification (ΔpHi), which was significantly blunted by Wortmannin and by LY294002. Both, Ip and ΔpHi were significantly smaller in PDK1 hypomorphic mice (pdk1flfl) than in their wild type littermates (pdk1wt). In conclusion, PI3K and PDK1 participate in the regulation of basal peptide transport.

Upregulation of Na+/H+ exchanger by the AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is activated upon energy depletion and serves to restore energy balance by stimulating energy production and limiting energy utilization. Specifically, it enhances cellular glucose uptake by stimulating GLUT and SGLT1 and glucose utilization by stimulating glycolysis. During O(2) deficiency glycolytic degradation of glucose leads to formation of lactate and H(+), thus imposing an acid load to the energy-deficient cell. Cellular acidification inhibits glycolysis and thus impedes glucose utilization. Maintenance of glycolysis thus requires cellular H(+) export. The present study explored whether AMPK influences Na(+)/H(+) exchanger (NHE) activity and/or Na(+)-independent acid extrusion. NHE1 expression was determined by RT-PCR and Western blotting. Cytosolic pH (pH(i)) was estimated utilizing BCECF fluorescence and Na(+)/H(+) exchanger activity from the Na(+)-dependent re-alkalinization (DeltapH(i)) after an ammonium pulse. As a result, human embryonic kidney (HEK) cells express NHE1. The pH(i) and DeltapH(i) in those cells were significantly increased by treatment with AMPK stimulator AICAR (1mM) and significantly decreased by AMPK inhibitor compound C (10 microM). The effect of AICAR on pH(i) and DeltapH(i) was blunted in the presence of the Na(+)/H(+) exchanger inhibitor cariporide (10microM), but not by the H(+) ATPase inhibitor bafilomycin (10nM). AICAR significantly enhanced lactate formation, an effect significantly blunted in the presence of cariporide. These observations disclose a novel function of AMPK, i.e. regulation of cytosolic pH.

Influence of dexamethasone on na+/h+ exchanger activity in dendritic cells.

Glucocorticoids regulate the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. Glucocorticoids influence the function of other cell types by modulating the activity of the Na⁺/H⁺exchanger (NHE), a carrier involved in the regulation of cytosolic pH and cell volume. The present study explored whether dexamethasone influences Na⁺/H⁺ exchanger activity in DCs. The DCs were isolated from mouse bone marrow, cell volume was estimated from forward scatter in FACS analysis, cytosolic pH (pH_i) utilizing BCECF fluorescence and Na⁺/H⁺ exchanger activity from the Na⁺ dependent realkalinization after an ammonium pulse. Treatment with the glucocorticoid dexamethasone (100 nM; 1, 4, 16 and 24h) significantly decreased pH_i (≧4 h) and gradually increased Na⁺/H⁺ exchanger activity (=16 h). The stimulation of Na⁺/H⁺ exchanger activity by dexamethasone was virtually abrogated by glucocorticoid receptor blocker mefiprestone (1 µM) and NHE3 inhibitor dimethyl amiloride (5 µM), but not prevented by NHE1 inhibitor cariporide (10 µM). Dexamethasone treatment significantly increased SGK1 mRNA levels. Stimulation of Na⁺/H⁺ exchanger activity by dexamethasone was blunted in DCs lacking SGK1. Dexamethasone treatment did not significantly alter ROS formation but significantly decreased the forward scatter. Exposure of DCs to lipopolysacharide (LPS, 1 µg/ml) led to a transient increase followed by a decline of Na⁺/H⁺ exchanger activity and to enhanced forward scatter as well as ROS formation, all effects significantly blunted in the presence of dexamethasone (100 nM). In conclusion, glucocorticoid treatment decreased pH_i and cell volume, effects paralleled by upregulation of Na⁺/H⁺ exchanger activity in DCs. Moreover, glucocorticoids blunted the stimulation of Na⁺/H⁺ exchanger activity, cell swelling and ROS formation following LPS treatment.

Rapamycin Sensitive ROS Formation and Na + /H + Exchanger Activity in Dendritic Cells

Rapamycin, a widely used immunosuppressive drug, has been shown to interfere with the function of dendritic cells (DCs), antigen-presenting cells contributing to the initiation of primary immune responses and the establishment of immunological memory. DC function is governed by the Na+/H+ exchanger (NHE), which is activated by bacterial lipopolysaccharides (LPS) and is required for LPS-induced cell swelling, reactive oxygen species (ROS) production and TNF-α release. The present study explored, whether rapamycin influences NHE activity and/or ROS formation in DCs. Mouse DCs were treated with LPS in the absence and presence of rapamycin (100 nM). ROS production was determined from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, cytosolic pH (pHi) from 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from the Na+-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, and TNF-α production utilizing ELISA. In the absence of LPS, rapamycin did not significantly modify cytosolic pH, NHE activity or cell volume but significantly decreased ROS formation. LPS stimulated NHE activity, enhanced forward scatter, increased ROS formation, and triggered TNF-α release, effects all blunted in the presence of rapamycin. NADPH oxidase inhibitor Vas-2870 (10 µM) mimicked the effect of rapamycin on LPS induced stimulation of NHE activity and TNF-α release. The effect of rapamycin on TNF-α release was also mimicked by the antioxidant ROS scavenger Tempol (30 µM) and partially reversed by additional application of tert-butylhydroperoxide (10 µM). In conclusion, in DCs rapamycin disrupts LPS induced ROS formation with subsequent inhibition of NHE activity, cell swelling and TNF-α release.

Effect of thymoquinone on cytosolic pH and Na+/H+ exchanger activity in mouse dendritic cells.

The anti-inflammatory Nigella sativa component thymoquinone compromises the function of dendritic cells (DCs), key players in the regulation of innate and adaptive immunity. DC function is regulated by the Na+/H+ exchanger (NHE), which is stimulated by lipopolysaccharides (LPS) and required for LPS-induced cell swelling, reactive oxygen species (ROS) production, TNF-α release and migration. Here we explored, whether thymoquinone influences NHE activity in DCs. To this end, bone marrow derived mouse DCs were treated with LPS in the absence and presence of thymoquinone (10 µM). Cytosolic pH (pHi) was determined from 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from the Na+-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNF-α production utilizing ELISA and DC migration with transwell migration assays. As a result, exposure of DCs to LPS (1 µg/ml) led within 4 hours to transient increase of NHE activity. Thymoquinone did not significantly modify cytosolic pH or cellular NHE activity in the absence of LPS, but abrogated the effect of LPS on NHE activity. Accordingly, in the presence of thymoquinone LPS-treatment resulted in cytosolic acidification. LPS further increased forward scatter and ROS formation, effects similarly abrogated by thymoquinone. Again, in the absence of LPS, thymoquinone did not significantly modify ROS formation and cell volume. LPS further triggered TNF-α release and migration, effects again blunted in the presence of thymoquinone. NHE1 inhibitor cariporide (10 µM) blunted LPS induced TNF-α release and migration. The effects of thymoquinone on NHE activity and migration were reversed upon treatment of the cells with t-butyl hydroperoxide (TBOOH, 5 µM). In conclusion, thymoquinone blunts LPS induced NHE activity, cell swelling, oxidative burst, cytokine release and migration of bone marrow derived murine dendritic cells. NHE inhibition may thus contribute to the antiinflammatory action of thymoquinone.