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Background/Aims: A high surface-to-volume ratio and a spectrin membrane-skeleton (MS) confer to the mammalian red blood cells (RBCs) their characteristic deformability, mechanical strength and structural stability. During their 120 days of circulatory life in humans, RBCs decrease in size, while remaining biconcave disks, owing to a coordinated decrease in membrane surface area and cell water. It is generally believed that part of the membrane is lost with the shedding of spectrin-free vesicles of the same type that can be obtained in vitro by different treatments. If this were true, an excess of MS would arise in old RBCs, with respect to the lipid bilayer. Aim of this paper was to investigate this aspect. Methods: Quantification of spectrin by electrophoretic methods was carried out in RBCs of different age. Results: Spectrin decreases, on a per cell basis, with RBC ageing. On the other hand, the membrane raft protein marker flotillin-2, while decreasing in the membrane of old cells, was found to be strongly depleted in the membrane of in vitro-induced vesicles. Conclusion: Part of the membrane-skeleton is probably lost together with part of the lipid bilayer in a balanced way. These findings point to a mechanism for the in vivo release of membrane that is different from that which is known to occur in vitro. [ABSTRACT FROM AUTHOR]

Several types of K+ channels have been identified in epithelial cells. Among them high conductance Ca2+-activated K+ channels (BK channels) are of relevant importance for their involvement in regulatory volume decrease (RVD) response following hypotonic stress. The aim of the present work was to investigate the functional and molecular expression of BK in the eel intestine, which is a useful experimental model for cell volume regulation research. In the present paper using rat BK channel-specific primer, a RT-PCR signal of 696 pb cDNA was detected in eel intestine, whole nucleotide sequence showed high similarity (83%) to the alpha subunit of BK channel family. BK channel protein expression was verified by immunoblotting and confocal microscopy, while the functional role of BK channels in epithelial ion transport mechanisms and cell volume regulation was examined by electrophysiological and morphometric analysis on the intact tissue. BKCa channels appeared to be localized along all the plasma membrane of the enterocytes; the apical part of the villi showed the most intense immunostaining. These channels were silent in basal condition, but were activated on both membranes (apical and basolateral) by increasing intracellular Ca2+ concentration with the Ca2+ ionophore ionomycin (1μM). BKCa channels were also activated on both membranes by hypotonic swelling of the epithelium and their inhibition by 100 nM iberiotoxin (specific BKCa inhibitor) abolished the Regulatory Volume Decrease (RVD) of the intestinal cells after hypotonic swelling. In conclusion, our results demonstrated the molecular and functional expression of high conductance Ca2+ -activated K+ channels in eel intestine; the physiological role of these channels is mainly related to the RVD response of the epithelial cells following hypotonic swelling. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Serum- and glucocorticoid-inducible kinase-1 (SGK1) increases CFTR Cl currents in Xenopus oocytes by an unknown mechanism. Because SGK increases the plasma membrane expression of other ion channels, the goal of this paper was to test the hypothesis that SGK1 stimulates CFTR Cl currents by increasing the number of CFTR Cl channels in the plasma membrane. Methods: CFTR Cl currents were measured in Xenopus oocytes by the two-electrode voltage clamp technique, and CFTR in the plasma membrane was determined by laser scanning confocal microscopy. Results: wt-SGK1 stimulated CFTR Cl currents by 42% and increased the amount of CFTR in the plasma membrane by 35%. A kinase-dead SGK mutant (K127N) had a dominant-negative effect on CFTR, reducing CFTR Cl currents by 38%. In addition, deletion of the C-terminal PDZ-interacting motif (SGK1-ΔSFL) increased CFTR Cl currents by 108%. Thus, SGK1-ΔSFL was more effective than wt-SGK1 in stimulating CFTR Cl currents. Neither wt-SGK nor the K127N mutant had any effect on Cl currents in oocytes when expressed alone in the absence of CFTR. Conclusion: SGK1 stimulates CFTR Cl currents in Xenopus oocytes by increasing the number of channels in the plasma membrane. Moreover, the effect of SGK may be mediated by protein-protein interactions involving the PDZ interacting motif. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: In thyroid cells, the intracellular pH plays a key role in the control of the iodide uptake, since iodide accumulation is associated with an intracellular acidification. In the present paper we studied the kinetic proprieties of the Na+/H+ antiporter (NHE) and the molecular expression of different NHE isoforms in rat thyroid PC-Cl3 cells. In addition the intracellular buffer capacity was also evaluated. Methods: pHi was measured using the pH sensitive fluorescent dye BCECF-AM. Amiloride, 5-(N,NDimethyl) hydrochloride was used to inhibit NHE activity. RT-PCR and western blot analyses were used to study the expression of NHE mRNA and protein isoforms. Results: PC-Cl3 cells shown a resting pHi, in the absence of CO2/HCO3 -, of 6.94 ± 0.1; after an acid load PC-Cl3 cells recovered toward resting pHi value, using a Na-dependent H+ extrusion mechanisms which was amiloride sensitive (Ki = 23 μM). The kinetic parameters were K(Na)app = 10 ± 2 mM and Vmax = 0.23 ± 0.02 ΔpH/min x 105 cells. NHE1, NHE2 and NHE3 were expressed at the mRNA level as well as at the protein level. Conclusion: PC-Cl3 cells express a functional Na/H exchange activity and different isoforms (NHE1, NHE2 and NHE3) are expressed in the plasma membrane. [ABSTRACT FROM AUTHOR]

Background/Aims: The taurine derivative Taurolidine is effective against diverse bacteria and tumor growth. In the treatment of cancer, the substance is effective in part by triggering suicidal death or apoptosis of tumor cells. The Taurolidine-induced apoptosis involves mitochondria. Erythrocytes lack mitochondria but are nevertheless able to enter suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explores, whether Taurolidine induces eryptosis and, if so, which cellular mechanisms are involved. Methods: Phosphatidylserine exposure at the cell surface was estimated using annexin-V-binding, cell volume using forward scatter, [Ca2+]i using Fluo3-fuorescence, reactive oxygen species (ROS) formation using 2',7'-dichlorodihydrofuorescein (DCF)-dependent fluorescence, and ceramide abundance using specific antibodies. Results: A 48 hours exposure of human erythrocytes to Taurolidine (60 µg/ml) significantly enhanced the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly increased Fluo3-fluorescence and ceramide abundance, but not DCF-fluorescence. The effect of Taurolidine on annexin-V-binding was virtually abrogated by removal of extracellular Ca2+. Conclusion: Taurolidine triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry and paralleled by increase of ceramide abundance [ABSTRACT FROM AUTHOR]

Background/Aims: Although baicalein has been shown to increase insulin sensitivity in liver of mice, there is no literature available about the effect of baicalein on glucose transporter 4 (GLUT4) translocation from intracellular membrane pools to plasma membranes in adipocytes of diet-induced obese mice. Methods: In the present study, the obese model was induced in mice fed a high fat diet (20% carbohydrates, 21% protein and 59% fat) for 16 weeks. The diet-induced obese mice were given 20mg/kg baicalein intraperitoneally (i.p.) once a day for 21 days. The plasma insulin was measured by enzyme-linked immunosorbent assay. Fasting blood glucose and insulin resistance indexes were measured by glucose tolerance test (GTT). The expression levels of PGC-1α, UCP1, GLUT4, PPARy, pP38MAPK, pERK and pAKT in adipocytes were determined by quantitative real-time polymerase chain reaction and western blotting. Results: The present findings showed that administration of baicalein decreased pP38MAPK, pERK and PPARy levels, but enhanced pAKT, PGC-1α and UCP1 contents as well as GLUT4 expression in adipocytes, and reversed high fat diet-induced glucose intolerance, hyperglycemia and insulin resistance in diet-induced obese mice. Moreover, baicalein treatment increased GLUT4 concentration in plasma membranes of adipocytes, i.e. baicalein may prevent insulin resistance through the GLUT4 translocation from intracellular membrane compartments to plasma membranes in adipocytes. Conclusion: These results suggest that baicalein is a powerful and promising agent for treatment of obesity and insulin resistance via Akt/GLUT4 pathway. [ABSTRACT FROM AUTHOR]

Background/Aims: Intracellular Ca2+ signaling plays an important role in the regulation of autophagy. However, very little is known about the role of Ca2+ influx, which is induced by plasma membrane Ca2+ channels. Our previous study showed that transient receptor potential canonical channel-6 (TRPC6), a major Ca2+ influx pathway in podocytes, was activated by hypoxia. Here, we investigated whether TRPC6 is involved in hypoxia-induced autophagy in cultured human podocytes. Methods: In the present study, an immortalized human podocyte cell line was used. Fluo-3 fluorescence was utilized to determine intracellular Ca2+ concentration ([Ca2+]i), and western blotting was used to measure autophagy and protein expression. Results: We found that blockade TRPC6 by using either TRPC6 siRNA or a TRPC6 blocker attenuated hypoxia-induced autophagy, while enhancement of TRPC6 activity with a TRPC6 activator enhanced hypoxia-induced autophagy. Furthermore, TRPC6-dependent Ca2+ signaling is responsible for hypoxia-induced autophagy since both an intracellular and extracellular Ca2+ chelator abolished hypoxia-induced autophagy. Moreover, we found that blockade of TRPC6 by using either TRPC6 siRNA or a TRPC6 blocker decreased the expression of adenosine monophosphate-activated protein kinase (AMPK), an important signaling molecule in Ca2+-dependent autophagy activation, which is activated under hypoxic conditions. These data suggest that the involvement of TRPC6 in hypoxia-induced autophagy is associated with AMPK signaling. Conclusion: TRPC6 is essential for hypoxia-induced autophagy in podocytes. [ABSTRACT FROM AUTHOR]

Background/Aims: The anaplastic lymphoma (tyrosine) kinase (ALK) inhibitor ceritinib triggers apoptosis of tumor cells and eryptosis of erythrocytes. Blood platelets may similarly enter a state resembling apoptosis, which could be triggered by activation with collagen related peptide (CRP). CRP-induced platelet apoptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the platelet surface and cell shrinkage, preceded by externalization of Ca2+ channel Orai1, increase of cytosolic Ca2+-activity ([Ca2+]i), formation of reactive oxygen species (ROS), and caspase activation. The present study explored whether ceritinib triggers platelet apoptosis and/or modifies the CRP induced apoptosis. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to ceritinib (1.5 μg/ml) without or with 2.5 - 15 min pretreatment with CRP (2 μg/ml or 5 μg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, ROS abundance from 2',7'-dichlorodihydrofluorescein diacetate fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: In the absence of CRP, ceritinib slightly, but significantly decreased [Ca2+]i without significantly modifying the other measured parameters. CRP significantly increased [Ca2+]i, ROS abundance, P-selectin abundance, activated αIIbβ3 integrin, annexin-V-binding, caspase activity as well as aggregation and decreased cell volume, all effects significantly blunted in the presence of ceritinib. Conclusions: The present observations uncover a novel, unexpected effect of ceritinib, i.e. inhibition of CRP-induced platelet activation and apoptosis. [ABSTRACT FROM AUTHOR]

Background: Eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine-translocation, is triggered by fever and inflammation. Signaling includes increased cytosolic Ca2+-activity ([Ca2++]i), caspase activation, and ceramide. Inflammation is associated with increased plasma concentration of C-reactive protein (CRP). The present study explored whether CRP triggers eryptosis. Methods: Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ceramide abundance and caspase-3-activity utilizing FITC-conjugated antibodies. Moreover, blood was drawn from patients with acute appendicitis (9♀,11♂) and healthy volunteers (10♀,10♂) for determination of CRP, blood count and phosphatidylserine. Results: A 48h CRP treatment significantly increased the percentage of annexin-V-binding cells (⩾5μg/ml), [Ca2+]i (⩾5μg/ml), ceramide (20μg/ml) and caspase-activity (20μg/ml). Annexin- V-binding was significantly blunted by caspase inhibitor zVAD (10μM). The percentage of phosphatidylserine-exposing erythrocytes in freshly drawn blood was significantly higher in appendicitis patients (1.83±0.21%) than healthy volunteers (0.81±0.09%), and significantly higher following a 24h incubation of erythrocytes from healthy volunteers to patient plasma than to plasma from healthy volunteers. The percentage of phosphatidylserine-exposing erythrocytes correlated with CRP plasma concentration. Conclusion: C-reactive protein triggers eryptosis, an effect at least partially due to increase of [Ca2+]i, increase of ceramide abundance and caspase activation. [ABSTRACT FROM AUTHOR]

Background/Aims: The analkaloid drug quinine is utilized mainly for the chemoprophylaxis of malaria. The multiple side effects of quinine include hemolytic anemia and hemolytic uremic syndrome, disorders involving suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling contributing to stimulation of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide and D4476 sensitive casein kinase. The present study explored the putative effect of quinine on eryptosis and elucidated cellular mechanisms involved. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to quinine (≥ 50 µM) significantly increased the percentage of annexin-V-binding cells without significantly affecting forward scatter. Quinine significantly increased Fluo3-fluorescence, DCF fluorescence and ceramide abundance. The effect of quinine on annexin-V-binding was significantly blunted by removal of extracellular Ca2+ and by addition of D4476 (10 µM). Conclusions: Quinine triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, ceramide and D4476 sensitive casein kinase. [ABSTRACT FROM AUTHOR]

Background/Aims: Across the mitochondrial membrane an exceptionally intense exchange of O2 and CO2 occurs. We have asked, 1) whether the CO2 permeability, PM,CO2, of this membrane is also exceptionally high, and 2) whether the mitochondrial membrane is sufficiently permeable to HCO3- to make passage of this ion an alternative pathway for exit of metabolically produced CO2. Methods: The two permeabilities were measured using the previously published mass spectrometric 18O exchange technique to study suspensions of mitochondria freshly isolated from rat livers. The mitochondria were functionally and morphologically in excellent condition. Results: The intramitochondrial CA activity was exclusively localized in the matrix. PM,CO2 of the inner mitochondrial membrane was 0.33 (SD ± 0.03) cm/s, which is the highest value reported for any biological membrane, even two times higher than PM,CO2 of the red cell membrane. PM,HCO3- was 2·10-6 (SD ± 2·10-6) cm/s and thus extremely low, almost 3 orders of magnitude lower than PM,HCO3- of the red cell membrane. Conclusion: The inner mitochondrial membrane is almost impermeable to HCO3- but extremely permeable to CO2. Since gas channels are absent, this membrane constitutes a unique example of a membrane of very high gas permeability due to its extremely low content of cholesterol. [ABSTRACT FROM AUTHOR]

Background/Aims: The activation of acid sphingomyelinase by cellular stress or receptors or the de novo synthesis lead to the formation of ceramide (N-acylsphingosine), which in turn modifies the biophysical properties of cellular membrane and greatly amplifies the intensity of the initial signal. Ceramide, which acts by re-organizing a given signalosome rather than being a second messenger, has many functions in infection biology, cancer, cardiovascular syndromes, and immune regulation. Experimental studies on the infection of human cells with different bacterial agents demonstrated the activation of the acid sphingomyelinase/ ceramide system. Moreover, the release of ceramide was found to be a requisite for the uptake of the pathogen. Considering the particular importance of the cellular role of ceramide, it was necessary to develop sensitive and accurate methods for its quantification. Methods: Here, we describe a method quantifying ceramide in dendritic cells and defining the different fatty acids (FA) bound to sphingosine. The main steps of the method include extraction of total lipids, separation of the ceramide by thin-layer chromatography, derivatization of ceramide-fatty acids (Cer-FA), and quantitation of these acids in their methyl form by gas chromatography on polar capillary columns. The identification of FA was achieved by means of known standards and confirmed by mass spectrometry. Results: FA ranging between C10 and C24 could be detected and quantified. The concentration of the sum of Cer-FA amounted to 14.88 ± 8.98 nmol/106 cells (n=10). Oleic acid, which accounted for approximately half of Cer-FA (7.73 ± 6.52 nmol/106 cells) was the predominant fatty acid followed by palmitic acid (3.47 ± 1.54 nmol/106 cells). Conclusion: This highly sensitive method allows the quantification of different molecular species of ceramides. [ABSTRACT FROM AUTHOR]

Background/Aims: The increase of the intracellular Ca2+ content as well as the exposure of phosphatidylserine (PS) on the outer cell membrane surface after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA) has been investigated by a variety of research groups. Carrying out experiments, which we described in several previous publications, we observed some discrepancies when comparing data obtained by different investigators within our research group and also between batches of LPA. In addition, we found differences comparing the results of double and single labelling experiments (for Ca2+ and PS). Furthermore, the results of PS exposure depended on the fluorescent dye used (annexin V-FITC versus annexin V alexa fluor® 647). Therefore, it seems necessary to investigate these methodological approaches in more detail to be able to quantify results and to compare data obtained by different research groups. Methods: The intracellular Ca2+ content and the PS exposure of RBCs separated from whole blood have been investigated after treatment with LPA (2.5 μM) obtained from three different companies (Sigma-Aldrich, Cayman Chemical Company, and Santa Cruz Biotechnology Inc.). Fluo-4 and x-rhod-1 have been used to detect intracellular Ca2+ content, annexin V alexa fluor® 647 and annexin V-FITC have been used for PS exposure measurements. Both parameters (Ca2+ content, PS exposure) were studied using flow cytometry and fluorescence microscopy. Results: The percentage of RBCs showing increased intracellular Ca2+ content as well as PS exposure changes significantly between different LPA manufacturers as well as on the condition of mixing of LPA with the RBC suspension. Furthermore, the percentage of RBCs showing PS exposure is reduced in double labelling compared to single labelling experiments and depends also on the fluorescent dye used. Finally, data on Ca2+ content are slightly affected whereas PS exposure data are not affected significantly by the measuring method (flow cytometry, fluorescence microscopy). Conclusion: The LPA batch used and the mixing procedure of LPA and the RBC suspension has to be taken into consideration when comparing results of intracellular Ca2+ content and PS exposure of RBCs after LPA activation. In addition, one should consider that the results of single and double labelling experiments might be different depending on the fluorescent dyes used. [ABSTRACT FROM AUTHOR]

Background/Aims: Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. Methods: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Results: Treatment of RBCs with 4-bromo-A23187 (positive control), lysophosphatidic acid (LPA), or phorbol-12 myristate-13 acetate (PMA) in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS) and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 mV depended on the solutions and buffers used. Conclusion: An increase of intracellular Ca2+ or an activation of protein kinase C leads to the formation and release of MVs in human RBCs. [ABSTRACT FROM AUTHOR]

Background/Aims: Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. Methods: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Results: Treatment of RBCs with 4-bromo-A23187 (positive control), lysophosphatidic acid (LPA), or phorbol-12 myristate-13 acetate (PMA) in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS) and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potentialof MVs was determined in the range from - 40 mV to - 10 mV depended on the solutions and buffers used. Conclusion: An increase of intracellular Ca2+ or an activation of protein kinase C leads to the formation and release of MVs in human RBCs. [ABSTRACT FROM AUTHOR]

Objectives: Human mesenchymal stem cells (MSCs) are efficacious in various cellular therapeutic applications and have been isolated from several tissues. Recent studies have reported that human tonsil tissue contains a new source of progenitor cells, potentially applicable for cell-based therapies. Information about the effects of donor age, long-term passage and cryopreservation are essential for clinical applications and cell-based therapies. Therefore, the authors investigated how the morphology, cell-surface markers, proliferation potential and differentiation capacity of tonsil-derived MSCs (T-MSCs) were affected by donor age, long-term passage, and cryopreservation. Materials and Methods: T-MSCs were isolated from tonsillar tissue of 20 patients undergoing tonsillectomy. Authors evaluated the effects of donor-age, long-term passage, and cryopreservation on the morphology, surface markers, proliferation potential and differentiation capacities of T-MSCs. Results: T-MSCs exhibited a fibroblast-like, spindle-shaped appearance. There were no significant morphological differences according to donor age, long-term passage or cryopreservation. T-MSCs isolated from donors of various ages were positive for markers CD90, CD44, and CD73, but negative for CD45, CD31, and HLA-DR. There were no significant differences in the expression of positive and negative surface markers as a function of donor age, long-term passage and cryopreservation. T-MSCs from different donor age groups showed similar proliferation potentials after passage 2. After long-term passage and cryopreservation, there were no significant morphological differences. Cryopreservation did not affect the proliferation potential of T-MSCs, but there was a significant decrease in the proliferation potential in long-term passage T-MSCs (passage 15). The effect of donor age, long-term passage and cryopreservation on the in vitro adipogenic, osteogenic, and chondrogenic differentiation potential of T-MSCs was not significant. Conclusion: The effect of donor age, long-term passage culture, and cryopreservation on T-MSC properties are negligible, except for the proliferation capacity of long-term cultured T-MSCs. Therefore, T-MSCs are considered to be promising MSCs that can be used as future alternative sources for autologous or allogenic MSCs. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: Thiazolidinediones are highly beneficial in the treatment of type II diabetes. However, they are also associated with edema and increased risk of congestive heart failure. Several studies demonstrated that rosiglitazone (RGZ) increases the abundance of aquaporin-2 (AQP2) at the plasma membrane of renal cells. The aim of this study was to investigate whether RGZ might activate a transduction pathway facilitating AQP2 membrane accumulation in renal cells. Methods: We analyzed the effect of RGZ on renal AQP2 intracellular trafficking in MCD4 renal cells by confocal microscopy and apical surface biotinylation. Cytosolic Ca2+ dynamics were measured by a video-imaging approach in single cell. Transient Receptor Potential (TRP) channels expression was determined by RT-PCR. Results: We showed that in MCD4 cells, short-term exposure to RGZ dramatically increases the amount of apically expressed AQP2 independently on cAMP production, PKA activation and AQP2 phosphorylation. RGZ elicited a cytosolic Ca2+ transient due to Ca2+ influx prevented by ruthenium red, suggesting the involvement of TRP plasma membrane channels. We identified TRPV6 as the possible candidate mediating this effect. Conclusions: Taken together these results provide a possible molecular mechanism explaining the increased AQP2 membrane expression under RGZ treatment: in renal cells RGZ elicits Ca2+ transients facilitating AQP2 exposure at the apical plasma membrane, thus increasing collecting duct water permeability. Importantly, this effect suggests an unexplored application of RGZ in the treatment of pathological states characterized by impaired AQP2 trafficking at the plasma membrane. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: CD40 is an important costimulatory molecule in both celluar and humoral immune responses, involved in the pathogenic processes of chronic inflammatory diseases. Few studies were performed on the association of CD40 single nucleotide polymorphism (SNP) with chronic hepatitis B virus (HBV) infection. In this study, we studied whether the CD40-1C/T polymorphism had any effect on the progression of chronic HBV infection in Chinese population. Methods: CD40 -1C/T polymorphism in the 5′-untranslated region was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 453 chronic HBV carriers, who were divided into asymptomatic HBV carriers (ASC), moderate chronic hepatitis B group (MCHB) and severe chronic hepatitis B group (SCHB). 202 healthy individuals in the same region were enrolled in this study as the controls. The CD40 expression on B lymphocytes was detected by flow cytometry. The concentrations of soluble CD40 (sCD40) in sera were assayed by a commercial ELISA kit. Results: Our results showed the frequencies of TT genotype and T allele of CD40-1C/T polymorphism were higher significantly in ASC than those in controls (P< 0.05), while this result was not found in either MCHB or SCHB. On the surface of B lymphocytes, the CD40 expression levels in the individuals with TT genotype were significantly lower than those with CC and CT genotypes in either ASC group or healthy controls (P<0.001). The sCD40 levels in the sera of ASC, MCHB and SCHB groups were significantly higher than the controls (P<0.001). Conclusions: The CD40 -1C/T polymorphism may contribute to the susceptibility of asymptomatic HBV carriers through its effect on cell-surface CD40 expression, which indicated CD40 signaling was involved in immune tolerance of chronic HBV infection. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: Many vital processes in animal cells depend on monovalent ion transport across the plasma membrane via specific pathways. Their operation is described by a set of nonlinear and transcendental equations that cannot be solved analytically. Previous computations had been optimized for certain cell types and included parameters whose experimental determination can be challenging. Methods: We have developed a simpler and a more universal computational approach by using fewer kinetic parameters derived from the data related to cell balanced state. A file is provided for calculating unidirectional Na+, K+, and Cl- fluxes via all major pathways (i.e. the Na/K pump, Na+, K+, Cl- channels, and NKCC, KC and NC cotransporters) under a balanced state and during transient processes. Results: The data on the Na+, K+, and Cl- distribution and the pump flux of K+ (Rb+) are obtained on U937 cells before and after inhibiting the pump with ouabain. There was a good match between the results of calculations and the experimentally measured dynamics of ion redistribution caused by blocking the pump. Conclusion: The presented approach can serve as an effective tool for analyzing monovalent ion transport in the whole cell, determination of the rate coefficients for ion transfer via major pathways and studying their alteration under various conditions. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: 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. Methods: In Caco2 human colon cancer cells transcript levels were determined by RT-PCR, protein abundance and phosphorylation by Western blotting and confocal microscopy, as well as cytoskeletal architecture by confocal microscopy. Results: We report time dependent significant decrease in Tyr-416 phosphorylation of c-Src upon mAR activation. In line with the reported late down-regulation of the PI-3K/Akt pathway in testosterone-treated colon tumors, GSK-3beta was phosphorylated at Tyr-216 after long term stimulation of the cells with TAC, a finding supporting the role of this kinase to promote apoptosis. PCR analysis revealed significant decrease of beta-catenin and cyclin D1 transcript levels following TAC treatment. Moreover, confocal laser scanning microscopic analysis disclosed co-localization of beta-catenin with actin cytoskeleton. It is thus conceivable that beta-catenin may participate in the reported modulation of cytoskeletal dynamics in mAR stimulated Caco2 cells. Conclusions: Our results provide strong evidence that mAR activation regulates late expression and/or activity of the tumorigenic gene products c-Src, GSK-3beta, and beta-catenin thus facilitating the pro-apoptotic response in colon tumor cells. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: ClC-3, a member of the ClC family, is predicted to have six isoforms, ClC-3a to -3f, with distinct N- and C-terminal amino acid sequences. There have been conflicting reports on the properties of ClC-3a (also known as the N-terminal short form of ClC-3) and ClC-3b (the N-terminal long form of ClC-3) as plasmalemmal Cl- channels. Meanwhile, little is known about other isoforms. The amino acid sequence of ClC-3d (a C-terminal variant of the short form) listed in the NCBI database was derived from the genomic sequence, but there has been no experimental evidence for the mRNA. Methods: PCR-cloning was made to obtain the full coding region of ClC-3d from mouse liver. Its molecular expression on the plasma membrane was microscopically examined in HEK293T cells transfected with GFP-tagged ClC-3d. Its functional plasmalemmal expression and the properties of currents were studies by whole-cell recordings in the cells transfected with ClC-3d. Results: The cloned ClC-3d was found to be the only isoform which has an N-terminal amino acid sequence identical to ClC-3a. When introduced into HEK293T cells, a minor fraction of exogenous ClC-3d proteins was detected at the plasma membrane, and activation of anion currents was observed at neutral pH under normotonic conditions. The properties of ClC-3d currents were found to be shared by ClC-3a-mediated currents. Also, both ClC-3d and -3a currents were found to be sensitive to Cd2+. ClC-3d overexpression never affected the endogenous activity of acid- or swelling-activated anion channels. Conclusion: We thus conclude that plasmalemmal ClC-3d, like ClC-3a, mediates Cd2+-sensitive outwardly rectifying anion currents and that ClC-3d is distinct from the molecular entities of acid- and volume-sensitive anion channels. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Human pendrin (SLC26A4, PDS) is an integral membrane protein acting as an electroneutral anion exchanger. Loss of function mutations in pendrin protein cause Pendred syndrome, a disorder characterized by sensorineural deafness and a partial iodide organification defect that may lead to thyroid goiter. Additionally, pendrin up-regulation could play a role in the pathogenesis of several diseases including bronchial asthma and chronic obstructive pulmonary disease (COPD). Therefore, monitoring the plasma membrane abundance and trafficking of pendrin in the context of a living cell is crucially important. Methods: Trafficking of pendrin to the plasma membrane was monitored by fluorescence resonance energy transfer (FRET), a physical phenomenon occurring between two fluorophores (the FRET donor and acceptor) located in close spatial proximity. Because the efficiency of the energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, FRET is extremely sensitive to small changes in distance between the donor and acceptor and is therefore a powerful tool to determine protein-protein interactions. Results: FRET studies revealed that forskolin-induced cAMP production is associated with a significant increase of pendrin expression at plasma membrane, which is paralleled by a decrease in intracellular pH. Pendrin transposition to the membrane is accompanied with a partial depolymerization of actin cytoskeleton via Rho-GTPase inhibition. Conclusion: Trafficking to the plasma membrane is critical in the regulation of pendrin activity. Therefore, reliable tools for monitoring and quantifying this phenomenon are highly desirable. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: The quaternary benzo-phenanthridine alkaloid (QBA) chelerythrine (CET) is a pro-apoptotic drug and Na+/K+ pump (NKP) inhibitor in human lens epithelial cells (HLECs). In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS). Methods: Silver nanoparticles (AgNPs) prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 μM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm-1 marker band as a function of CET concentration. Results: SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. Conclusion: Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET+) accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Backgroud/Aims: Skeletal muscle (SM) constitutes more than 40% of the body weight in adulthood. Transports dietary glucose mainly through the insulin-dependent glucose transporter (Glut-4) located in the Transverse tubule membrane system (TT). The TT development ends shortly after birth. The TT membrane hosts the proteins involved in excitation-contraction coupling and glucose uptake. Glycaemic regulation through movement is a key function of fully developed skeletal muscle. In this study, we aimed to characterize the effect of gestational undernourishment (GUN) in rats GLUT-4 expression and on the protein/lipid content of the TT membranes. We also examined the effect of GUN on the mechanical properties of muscles as an indication of the metabolic condition of the SM at birth. Methods: Isolated TT membrane from SM of GUN rats were used to study lipid/protein content and protein stability by differential scanning calorimetry. The effect of GUN on the SM mechanical properties was determined in isolated Extensor Digitorum Longus (EDL) muscle. Results: We demonstrate that compared to control, GUN in the new-born produces; i) decreases body weight; ii) diminution in SM mass; iii) decreases the formation of TT membranes; iv) expresses TT membrane proteins with higher thermal stability. The TT membrane expression of GLUT-4 in GUN offspring was twice that of controls. The isolated EDL of GUN offspring was 20% stronger as measured by contractile force and more resistant to fatigue relative to controls. Conclusion; These results provide the first evidence of adaptive changes of the SM in new-borns exposed to severe gestational food restriction. The effects of GUN on muscle at birth are the first step toward detrimental SM metabolic function, contributing to the physiopathology of metabolic diseases in adulthood. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: The objective of this study is to examine the current signals in response to large hyperpolarizations with the aid of principal component analysis (PCA) to search for or even predict current fluctuations related to membrane electroporation-induced current (IMEP). Methods: The characteristics of principal eigenvalues generated for IMEP and the current signals at 10 sec prior to the start of initial IMEP (IPre) were examined. As membrane hyperpolarizations were applied at 0.1 Hz, the appearance of IMEP coincided with the higher principal eigenvalues extracted in PCA. Results: Subsequent addition of LaCl3 (100 µM) greatly reduced IMEP and associated principal eigenvalues. In real-time analysis for a single frame (i.e, 300 msec), in response to large hyperpolarization, multiple runs of heralded minuscule inward currents (Imin) occurring before large rise in current amplitudes were detected. With PCA, such heralded Imin was noted to coincide with the extreme principal eigenvalues. The duration of Imin together with large principal eigenvalues was influenced by different levels of membrane hyperpolarization. In GH3 cells, palmitoyl-L-carnitine (PALCAR), a long-chain acylcarnitine, effectively increased the IMEP amplitude with an EC50 value of 2.4 µM. However, in PALCAR-treated cells, the Imin together with higher principal eigenvalues disappeared, while in isoflurane-treated cells, Imin occurring before large rise of current amplitude remained intact. Similarly, the PCA analysis from IPre in RAW 264.6 macrophages showed the presence of herald Imin accompanied by the extreme principal eigenvalues. Conclusion: It is clear from this study that these large principal eigenvalues are representative of MEP-associated formation of electropores. Therefore, different compositions around the surface membrane of cells may alter the appearance of Imin followed by IMEP emergence. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: Janus-activated kinase-2 JAK2 is activated by energy depletion and hyperosmotic shock and modifies the activity of several Na+ coupled transporters. The Na+ coupled osmolyte transporter SMIT (myoinositol transporter) is upregulated by osmotic shock and downregulated by energy depletion. The present study thus explored whether JAK2 contributes to the regulation of SMIT activity. Methods: To this end, cRNA encoding SMIT was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, constitutively active V617FJAK2 or inactive K882EJAK2. Inositol-induced current (ISMIT) was determined by dual electrode voltage clamp and taken as measure for electrogenic inositol transport. Results: No appreciable ISMIT was observed in water injected oocytes. In SMIT expressing oocytes ISMIT was significantly decreased by additional coexpression of JAK2 or V617FJAK2, but not by coexpression of K882EJAK2. According to kinetic analysis coexpression of JAK2 decreased maximal ISMIT without significantly modifying the concentration required for halfmaximal ISMIT (KM). In oocytes expressing both, SMIT and JAK2, ISMIT was gradually increased by JAK2 inhibitor AG490 (40 μM). Disruption of carrier insertion with brefeldin A (5 μM) was followed by a decline of ISMIT to a similar extent in Xenopus oocytes expressing SMIT with JAK2 and in Xenopus oocytes expressing SMIT alone, suggesting that JAK2 did not affect carrier stability in the cell membrane. Conclusion: JAK2 contributes to the regulation of the inositol transporter SMIT. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Recent studies demonstrate that rapid translocation of the acid sphingomyelinase (ASM), a lysosomal hydrolase, to the outer leaflet of the cell membrane and concomitant release of ceramide constitute a common cellular signaling cascade to various stimuli including CD95 ligation, UV-irradiation, bacterial and viral infections. Reactive oxygen species (ROS) were shown to play a crucial role in regulating this signaling cascade at least for some bacterial infections and UV-irradiation. However, the precise role of ROS for regulation of ASM is unknown. Here, by confocal microscopy and flow cytometry analysis, we demonstrate that hydrogen peroxide (H2O2), a primary form of ROS in mammalian cells, induces very rapid translocation of ASM and formation of ceramide-enriched membrane platforms in the plasma membrane of Jurkat T cells. In parallel, H2O2 triggers lysosome trafficking and fusion with the plasma membrane, i.e. lysosome exocytosis, as detected by exposure of a lysosome-associated protein, LAMP1. Depletion of intracellular Ca2+ by cell permeable EGTA-AM inhibits H2O2-induced lysosome exocytosis, ASM translocation and formation of ceramide-enriched platforms. Pharmacological inhibition or genetic deficiency of ASM did not affect H2O2-induced lysosome exocytosis. These results indicate that ROS-induced membrane translocation of ASM is mediated by exocytosis of lysosomes, which is dependent on intracellular Ca2+ release. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

This study characterizes the complex mechanisms of acute regulation of organic cation (OC) transport across the basolateral membrane of isolated mouse proximal tubules. The fluorescent substrate ASP+, 4-(-4-(dimethylamino) styryl-N-methylpyridinium, was used to quantify OC transport using a microtiter plate based fluorescence reader method. Inhibition of phosphatidylinositol-3-kinase, of p56 tyrosine kinase, stimulation of PKC and inhibition of PKA reduced ASP+-uptake. ASP+-kinetic and Dixon plot analyses revealed effects on transporter trafficking as explanation for the inhibition of ASP+-uptake by these pathways. Angiotensin n (AII) via stimulation of Ca2+/calmodulin increased ASP+-uptake. This effect aroused from an altered substrate affinity. Bafilomycin, an inhibitor of the vacuolar H+-ATPase and thus endosomal and lysosomal function, reduced ASP+-uptake, but did not prevent the All effect on ASP+-uptake. Bafilomycin seemed to diminish the recycling rate of OCTs and hence to reduce the amount of transporters in the membrane. AII via Ca2+/calmodulin increased the substrate affinity of the remaining OCTs. The involvement of the cytoskeleton in acute regulation of OCTs became obvious as colchicine induced inhibition of microtubule polymerisation reduced ASP+-uptake. Acute regulation of mouse OCTs mostly involves changes in trafficking from and to the plasma membrane and only in the case of AII/CaM changes in substrate affinity. [ABSTRACT FROM AUTHOR]

Background/Aims: We have earlier found that the linoleic acid derivative DCP-LA could ameliorate learning and memory impairment by targeting &agr;7 ACh receptor The present study aimed at understanding DCP-LA-regulated &agr;7 ACh receptortrafficking. MeiAiods: We monitored currents through &agr;7 ACh receptors expressed in Xenopus oocytes and assayed the receptor mobilizations using fractions with a sucrose density gradient centrifugation, synaptosome preparation, and acutely dissociated neurons from rat hippocampal slices. Results: DCP-LA persistently potentiated &agr;7 ACh receptor currents, and the effect was inhibited by a protein kinase C (PKC) inhibitor or vesicular transport inhibitors. DCP-LA did not induce PKC phosphorylation of &agr;7 ACh receptors. DCP-LA stimulated translocation of &agr;7 ACh receptors from the cytosol toward the plasma membrane or from extra-synaptosomes into synaptosomes and accumulated the receptors at the presynaptic site in hippocampal neurons. Conclusion: The results of the present study demonstrate that DCP-LA increases surface localization of &agr;7 ACh receptors in hippocampal neurons, specifically at presynaptic terminals, by stimulating vesicular transport of the receptors, resulting in potentiation of &agr;7 ACh receptor responses, regardless of PKC phosphorylation of the receptors. [ABSTRACT FROM AUTHOR]

Background: ICln is a multifunctional protein involved in the generation of chloride currents activated during regulatory volume decrease (RVD) after cell swelling (IClswell). Growth factor receptors play a key role in different cellular processes and epidermal growth factor (EGF) regulates swelling-activated chloride permeability. Aim: We set out to investigate if the EGF-induced upregulation of IClswell could be explained by a rearrangement of ICln subcellular distribution and interaction with its molecular partners. Methods: NIH-3T3 fibroblasts were serum-deprived for 24 hours and stimulated with EGF (40 ng/ml) for 30 minutes. IClswell activation, ICln distribution and interaction with its molecular partner HSPC038 were assessed by whole cell patch clamp and fluorescence resonance energy transfer (FRET). Results: EGF treatment significantly enhanced the direct molecular interaction between ICln and HSPC038 and also resulted in an increase of ICln and HSPC038 association with the plasma membrane. Importantly, these events are associated with a significant increase of IClswell. Conclusions: The present data indicate that EGF might exert its role in the modulation of volume-sensitive chloride currents in part through activation and translocation of ICln and HSPC038 to the plasma membrane. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

p38 protein kinase is activated by hyperosmotic shock, participates in the regulation of cell volume sensitive transport and metabolism and is involved in the regulation of various physiological functions including cell proliferation and apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo suicidal death or eryptosis, which is paralleled by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include hyperosmotic shock, which increases cytosolic Ca2+ activity and ceramide formation. The present study explored whether p38 kinase is expressed in human erythrocytes, is activated by hyperosmotic shock and participates in the regulation of eryptosis. Western blotting was utilized to determine phosphorylation of p38 kinase, forward scatter to estimate cell volume, annexin V binding to depict phosphatidylserine exposure and Fluo3 fluorescence to estimate cytosolic Ca2+ activity. As a result, erythrocytes express p38 kinase, which is phosphorylated upon osmotic shock (+550 mM sucrose). Osmotic shock decreased forward scatter, increased annexin V binding and increased Fluo3 fluorescence, all effects significantly blunted by the p38 kinase inhibitors SB203580 (2 μM) and p38 Inh III (1 μM). In conclusion, p38 kinase is expressed in erythrocytes and participates in the machinery triggering eryptosis following hyperosmotic shock. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: FKBP8 is a multifunctional protein involved in many distinct processes like formation of central nervous system, viral RNA replication and inhibition of apoptosis. Fkbp8 expression was reported in different tissues, various cell lines and malignancies, in the latter displaying changes during carcinogenesis. Loss of Fkbp8 leads to substantial neurodegenerations during regular mouse development, thus hearing onset in mice could also potentially depend on Fkbp8 expression. Since Fkbp8 is crucial for patterning of neuronal function, we studied its expression during maturation of the rodent auditory function. Methods: Fkbp8 gene expression in rodent cochlear samples was studied by RT-PCR, qPCR, and western blot. Localization of Fkbp8 transcripts and protein was analyzed by in-situ hybridization and immunohistochemistry. Results: Studies of auditory organ demonstrate that Fkbp8 gene activity is increasing just before hearing onset and gradually decreasing after onset of hearing. Western blot analysis suggests substantial levels of Fkbp8 protein before hearing onset, and slow degradation after onset of hearing. The Fkbp8 mRNA is localized in spiral ganglion of cochlea but its distribution changes over time to the stria vascularis, a finding supported by immunohistochemistry staining. Additionally, in pre-hearing time Fkbp8-specific signal was also observed in the tectorial membrane, whose α- and β-Tectorin components show similar time-dependent expression of mRNA as Fkbp8. Conclusion: These results indicate a temporal shift in expression of Fkbp8 which correlates with cochlear maturation, strongly suggesting a contribution of Fkbp8 to the onset of the rodent hearing processes. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The exposure of phosphatidylserine (PS) on the outer membrane leaflet of red blood cells (RBCs) serves as a signal for eryptosis, a mechanism for the RBC clearance from blood circulation. The process of PS exposure was investigated as function of the intracellular Ca2+ content and the activation of PKCα in human and sheep RBCs. Cells were treated with lysophosphatidic acid (LPA), 4-bromo-A23187, or phorbol-12 myristate-13 acetate (PMA) and analysed by flow cytometry, single cell fluorescence video imaging, or confocal microscopy. For human RBCs, no clear correlation existed between the number of cells with an elevated Ca2+ content and PS exposure. Results are explained by three different mechanisms responsible for the PS exposure in human RBCs: (i) Ca2+-stimulated scramblase activation (and flippase inhibition) by LPA, 4-bromo-A23187, and PMA; (ii) PKC activation by LPA and PMA; and (iii) enhanced lipid flop caused by LPA. In sheep RBCs, only the latter mechanism occurs suggesting absence of scramblase activity. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Dicoumarol, a widely used anticoagulant, may cause anemia, which may result from enhanced erythrocyte loss due to bleeding or due to accelerated erythrocyte death. Erythrocytes may undergo suicidal death or eryptosis, characterized by cell shrinkage and phospholipid scrambling of the cell membrane. Eryptosis may be triggered by increase of cytosolic 2+-activity ([Ca2+]i). The present study explored, whether dicoumarol induces eryptosis. [Ca2+]i was estimated from Fluo3-fluorescence, cation channel activity utilizing whole cell patch clamp, cell volume from forward scatter, phospholipid scrambling from annexin-V-binding, and hemolysis from haemoglobin release. Exposure of erythrocytes for 48 hours to dicoumarol (=10 μM) significantly increased [Ca2+]i, enhanced cation channel activity, decreased forward scatter, triggered annexin-V-binding and elicited hemolysis. Following exposure to 30 μM dicoumarol, annexin-V-binding affected approximately 15%, and hemolysis 2% of treated erythrocytes. The stimulation of annexin-V-binding by dicoumarol was abrogated in the nominal absence of 2+. In conclusion, dicoumarol stimulates suicidal death of erythrocytes by stimulating Ca2+ entry and subsequent triggering of 2+ dependent cell membrane scrambling. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The effects of (2-hydroxypropyl)- β-cyclodextrin (HPβCD), a cyclic oligomer, on membrane electroporation-induced inward current (IMEP) in pituitary tumor (GH3) cells were experimentally and analytically characterized. Depletion of membrane cholesterol by exposing cells to HPβCD (2 mM) increased the activation time constant of delayed rectifier K+ current. Such maneuver resulted in a significant reduction of IMEP density. 2,2'-Azo-bis(2-amidinopropane) dihydrochloride (AAPH), an initiator of free radicals, increased the magnitude of IMEP. AAPH-stimulated IMEP was not reversed by the blockers of Ca2+-activated K+ channels, but by LaCl3 or MnCl2. However, in HPβCD-treated cells, the ability of AAPH to enhance IMEP was abolished. Under such maneuver, the gating charge of IMEP activation was increased by 2 fold, along with a hyperpolarized shift of the activation curve by 30 mV. No change in single-channel conductance of MEP-induced channels during cell exposure to HPβCD was demonstrated. The energy change of IMEP in untreated and HPβCD-treated cells was estimated to be -17.7 and -44.8 kJ/mol, respectively, and the perturbation of free energy following HPβCD treatment was -27.1 kJ/mol. Based on an MEP model, cell exposure to HPβCD increased the edge energy of the electropore size. By use of a two barrier-one site barrier model, HPβCD treatment can increase both the peak height and well depth of the barrier profile. Taken together, depletion of membrane cholesterol by HPβCD can elevate the edge energy of pore formation, thereby decreasing the IMEP magnitude. The channel-suppressing properties during membrane cholesterol depletion with HPβCD might thus contribute to the underlying mechanisms by which such maneuver alters neuronal or neuroendocrine function. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Voltage gated calcium channels (VGCCs) regulate cellular activity in response to membrane depolarization by altering calcium homeostasis. Because calcium is the most versatile second messenger, regulation of the amount of VGCCs at the plasma membrane is highly critical for several essential cellular processes. Among the different types of VGCCs, the Cav2.3 calcium channel and its regulation mechanisms are least understood due to Cav2.3's resistance to most pharmacological agents. Methods: In order to study regulation and surface expression of Cav2.3, a yeast two hybrid (Y2H) screen with the II-III loop of human Cav2.3 as bait, was performed. APLP1, a member of the APP gene family and Rab5A, an endocytotic catalyst were identified as putative interaction partners. The interaction were confirmed by immunoprecipitation. To study the functional importance of the interaction, patch-clamp recordings in Cav2.3 stably transfected HEK-293 cells (2C6) and surface biotin endocytosis assays were performed. Results: We are able to show that the II-III loop of the Cav2.3 calcium channel binds APLP1 and that this binding promotes internalization of the channel. In addition, Rab5A also binds to the same loop of the channel and exerts an inhibitory effect on APLP1 mediated channel internalization. Conclusions: This study identifies a regulation mechanism of Cav2.3's surface expression, which implicates APLP1 as a regulator of calcium homeostasis. Thus APLP1 may play a crucial role in neuropathological mechanisms, which involve modulation of surface expression of voltage-gated Ca2+ channels. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims. It has been shown that MDCK cells, a cell line derived from canine renal tubules, develop cell domes due to fluid pumped under cell monolayer and focal detachment from the adhesion surface. In vitro studies have shown that primary cilia of kidney tubular epithelial cells act as mechanosensors, increasing intracellular calcium within seconds upon changes in fluid shear stress (SS) on cell membrane. We then studied the effect of prolonged SS exposure on cell dome formation in confluent MDCK cell monolayers. Methods. A parallel plate flow chamber was used to apply laminar SS at 2 dynes/cm2 to confluent cell monolayers for 6 hours. Control MDCK cell monolayers were maintained in static condition. The effects of Ca2+ blockade and cell deciliation on SS exposure were also investigated. Results. Seven days after reaching confluence, static cultures developed liquid filled domes, elevating from culture plate. Exposure to SS induced almost complete disappearance of cell domes (0.4±0.8 vs. 11.4±2.8 domes/mm2, p < 0.01, n=14). SS induced dome disappearance took place within minutes to hours, as shown by time-lapse videomicroscopy. Exposure to SS importantly affected cell cytoskeleton altering actin stress fibers expression and organization, and the distribution of tight junction protein ZO-1. Dome disappearance induced by flow was completely prevented in the presence of EGTA or after cell deciliation. Conclusions. These data indicate that kidney tubular cells are sensitive to apical flow and that these effects are mediated by primary cilia by regulation of Ca2+ entry in to the cell. SS induced Ca2+ entry provokes contraction of cortical actin ring that tenses cell-cell borders and decreases basal stress fibers. These processes may increase paracellular permeability and decrease basal adhesion making dome disappear. Elucidation of the effects of apical fluid flow on tubular cell function may open new insights on the pathophysiology of kidney diseases associated with cilia dysfunction. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The Janus-activated kinase-2 JAK2 is involved in the signaling of leptin and erythropoietin receptors and mediates neuroprotective effects of the hormones. In theory, JAK2 could be effective through modulation of the glutamate transporters, carriers accounting for the clearance of glutamate released during neurotransmission. The present study thus elucidated the effect of JAK2 on the glutamate transporters EAAT1, EAAT2, EAAT3 and EAAT4. To this end, cRNA encoding the carriers was injected into Xenopus oocytes with or without cRNA encoding JAK2 and glutamate transport was estimated from glutamate induced current (Iglu). Iglu was observed in Xenopus oocytes expressing EAAT1 or EAAT2 or EAAT3 or EAAT4, but not in water injected oocytes. Coexpression of JAK2 resulted in an increase of Iglu by 83% (EAAT1), 67% (EAAT2), 42% (EAAT3) and 126% (EAAT4). As shown for EAAT4 expressing Xenopus oocytes, the effect of JAK2 was mimicked by gain of function mutation V617FJAK2 but not by the inactive mutant K882EJAK2. Incubation with JAK2 inhibitor AG490 (40 μM) resulted in a gradual decrease of Iglu by 53%, 79% and 92% within 3, 6 and 24 hours. Confocal microscopy and chemiluminescence analysis revealed that JAK2 coexpression increased EAAT4 protein abundance in the cell membrane. Disruption of transcription did not appreciably modify the up-regulation of Iglu in EAAT4 expressing oocytes. The decay of Iglu following inhibition of carrier insertion with brefeldin A was similar in oocytes expressing EAAT4 + JAK2 and oocytes expressing EAAT4 alone, indicating that JAK2 did not appreciably affect carrier retrieval from the membrane. In conclusion, JAK2 is a novel powerful regulator of glutamate transporters and thus participates in the protection against excitotoxicity. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Uroguanylin (UGN) has been proposed as a key regulator of salt and water intestinal transport. Uroguanylin activates cell-surface guanylate cyclase C receptor (GC-C) and modulates cellular function via cyclic GMP (cGMP), thus increasing electrolyte and net water secretion. It has been suggested that the action of UGN could involve the Na+/H+ exchanger, but the actual contribution of this transporter still remains unclear. The objective of our study was to investigate the putative effects of UGN on some members of the Na+/H+ exchanger family (NHEs), as well as to clarify its consequences on transepithelial fluid flow in T84 cells. In order to do so, transepithelial fluid flow (Jv) was studied by optic techniques and intracellular pH (pHi) was measured with a fluorescence method. Results showed that NHE2 is found at the apical membrane and has a major role in Na+ absorption; NHE1 and NHE4 are localized at the basolateral membrane with a house-keeping role in steady state pHi. In the assayed conditions, cell exposure to apical UGN increases net secretory Jv, without changing short-circuit currents nor transepithelial resistance, and reduces NHE2 activity. Therefore, at physiological pH, the effect on net Jv was produced mainly by a reduction in normal Na+ absorption through NHE2, rather than by the stimulation of electrolyte secretion. Our study shows that the effect of UGN on pHi is GC-C/cGMP-mediated and enhanced by sildenafil, thus involving PDE5 enzyme. Additionally, cell exposure to apical UGN results in intracellular alkalinization, probably due to indirect effects on basolateral NHE1 and NHE4, which have a major role in pHi regulation. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in X. oocytes and is required for the expression of amiloride sensitive sodium channels (ENaC). Oocytes were injected with α, β, and γ mENaC and xShroom1 sense or antisense oligonucleotides. We used voltage clamp techniques to study the amiloride-sensitive Na+ currents (INa(amil)). We observed a marked reduction in INa(amil) in oocytes co-injected with xShroom1 antisense. Oocytes expressing a DEG mutant β-mENaC subunit (β-S518K) with an open probability of 1 had enhanced INa(amil) although these currents were also reduced when co-injected with xShroom1 antisense. Addition of low concentration (20 ng/ml) of trypsin which activates the membrane-resident ENaC channels led to a slow increase in INa(amil) in oocytes with xShroom1 sense but had no effect on the currents in oocytes coinjected with ENaC and xShroom1 antisense. The same results were obtained with higher concentrations of trypsin (2 μg/ml) exposed during 2.5 min. In addition, fluorescence positive staining of plasma membrane in the oocytes expressing α, β and γ mENaC and xShroom1 sense were observed but not in oocytes coinjected with ENaC and xShroom1 antisense oligonucleotides. On this basis, we suggest that xShroom1-dependent ENaC inhibition may be through the number of channels inserted in the membrane. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

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. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Epithelial-to-Mesenchymal Transition (EMT) is an important pathogenic mechanism mediating glomerular injury or sclerosis in a variety of renal and systemic diseases such as hyperhomocysteinemia (hHcys). The present study was designed to test whether Hcys-induced EMT in podocytes is reversed by growth hormone (GH), a hormone regulating cell differentiation and growth and to explore the cellular and molecular mechanism mediating its action. It was found that Hcys induced significant EMT in podocytes, as shown by marked decreases in slit diaphragm-associated protein P-cadherin and zonula occludens-1 as epithelial markers and by dramatic increases in the expression of mesenchymal markers, fibroblast specific protein-1 and α-smooth muscle actin, which were detected by all examinations via immunocytochemistry, real time RT-PCR and Western blot analysis. When podocytes were treated with GH at 25 ng/mL, however, Hcys failed to induce podocyte EMT. Using electromagnetic spin resonance spectrometry, Hcys-induced superoxide (O2.-) production via NADPH oxidase was found to be significantly inhibited by GH (66%). Functionally, GH was shown to substantially inhibit Hcys-induced increases in the permeability of podocyte monolayers and to block the decrease in podocin expression in these cells. In addition, NADPH oxidase subunit, gp91phox and GH receptors aggregated in membrane raft clusters, which produced O2.- in response to Hcys and could be blocked by GH, membrane raft disruptors filipin and MCD or NADPH oxidase inhibitor, apocynin. It is concluded that Hcys-induced podocyte EMT is associated with transmembrane membrane raft-redox signaling and that GH reverses this Hcys-induced EMT protecting podocytes from functional disturbance. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Aquaporin-2 (AQP2) increases the water permeability of renal collecting ducts in response to vasopressin. Vasopressin stimulation is accompanied by a profound remodeling of actin cytoskeleton whose dynamics are regulated by crosstalk between intracellular and extracellular signals. Here, we report that AQP2 contains a conserved RGD domain in its external C-loop. Co-immunoprecipitation experiments demonstrated that AQP2 binds integrin β1 in renal tissue and in MCD4 cells. To investigate the role of this interaction on AQP2 trafficking, cells were exposed to synthetic RGD-containing peptides, GRGDNP or GRGDSP, able to bind certain integrins. Incubation with these peptides increased the membrane expression of AQP2 in the absence of hormonal stimulation as assessed by confocal analysis and cell surface biotinylation. To identify the signals underlying the effects of peptides on AQP2 trafficking, some possible intracellular messengers were evaluated. Exposure of MCD4 cells to GRGDNP increased intracellular cAMP as assessed by FRET studies while GRGDSP increased intracellular calcium concentration. Taken together, these data propose integrins as new players controlling the cellular localization of AQP2, via two distinct signal transduction pathways dependent on cAMP and calcium respectively. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Previous studies using bloodstream form Trypanosoma brucei have shown that glycerol transport in this parasite occurs via specific membrane proteins, namely a glycerol transporter and glycerol channels [1]. Later, we cloned, expressed and characterized the transport properties of all three aquaglyceroporins (AQP1-3) [2], which were found permeable for water, glycerol and other small uncharged solutes like dihydroxyacetone [3]. Here, we report on the cellular localization of TbAQP1 and TbAQP3 in bloodstream form trypanosomes. Indirect immunofluorescence analysis showed that TbAQP1 is exclusively localized in the flagellar membrane, whereas TbAQP3 was found in the plasma membrane.In addition, we analyzed the functions of all 3 AQPs, using an inducible inheritable double-stranded RNA interference methodology. All AQP knockdown cell lines were still able to survive hypo-osmotic stress conditions, except AQP2 knockdown parasites. Depleted TbAQP2 negatively impacted cell growth and the regulatory volume recovery, whereas AQP1 und 3 knockdown trypanosomes displayed phenotypes consistent with their localization in external membranes. A simultaneous knockdown of all 3 AQPs showed that the cells were able to substitute the missing glycerol uptake capability through a putative glycerol transporter. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: It has been widely accepted that chloride ions moving along chloride channels act to dissipate the electrical gradient established by the electrogenic transport of H+ ions performed by H+-ATPase into subcellular vesicles. Largely known in intracellular compartments, this mechanism is also important at the plasma membrane of cells from various tissues, including kidney. The present work was performed to study the modulation of plasma membrane H+-ATPase by chloride channels, in particular, CFTR and ClC-5 in kidney proximal tubule. Methods and Results: Using in vivo stationary microperfusion, it was observed that ATPase-mediated HCO3- reabsorption was significantly reduced in the presence of the Cl- channels inhibitor NPPB. This effect was confirmed in vitro by measuring the cell pH recovery rates after a NH4Cl pulse in immortalized rat renal proximal tubule cells, IRPTC. In these cells, even after abolishing the membrane potential with valinomycin, ATPase activity was seen to be still dependent on Cl-. siRNA-mediated CFTR channels and ClC-5 chloride-proton exchanger knockdown significantly reduced H+-ATPase activity and V-ATPase B2 subunit expression. Conclusion: These results indicate a role of chloride in modulating plasma membrane H+-ATPase activity in proximal tubule and suggest that both CFTR and ClC-5 modulate ATPase activity. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

This study tested the hypotheses that the hypertonic environment of the renal medulla regulates the expression of cystic fibrosis transmembrane conductance regulator protein (CFTR) and its natural splice variant, TNR-CFTR. To accomplish this, Madin-Darby canine kidney (MDCK) stable cell lines expressing TNR-CFTR or CFTR were used. The cells were treated with hypertonic medium made with either NaCl or urea or sucrose (480 mOsm/kg or 560 mOsm/kg) to mimic the tonicity of the renal medulla environment. Western blot data showed that CFTR and TNR-CFTR total cell protein is increased by hypertonic medium, but using the surface biotinylation technique, only CFTR was found to be increased in cell plasma membrane. Confocal microscopy showed TNR-CFTR localization primarily at the endoplasmic reticulum and plasma membrane. In conclusion, CFTR and TNR-CFTR have different patterns of distribution in MDCK cells and they are modulated by a hypertonic environment, suggesting their physiological importance in renal medulla. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The serum- and glucocorticoid-regulated kinase (Sgk1) is essential for hormonal regulation of ENaC-mediated sodium transport and is involved in the transduction of growth-factor-dependent cell survival and proliferation. The identification of molecular partners for Sgk1 is crucial for the understanding of its mechanisms of action. We performed a yeast two-hybrid screening based on a human kidney cDNA library to identify molecular partners of Sgk1. As a result the screening revealed a specific interaction between Sgk1 and a 60 kDa Lysophospholipase (LysoLP). LysoLP is a poorly characterized enzyme that, based on sequence analysis, might possess lysophospholipase and asparaginase activities. We demonstrate that LysoLP has indeed a lysophospholipase activity and affects metabolic functions related to cell proliferation and regulation of membrane channels. Moreover we demonstrate in the Xenopus oocyte expression system that LysoLP downregulates basal and Sgk1-dependent ENaC activity. In conclusion LysoLP may represent a new player in the regulation of ENaC and Sgk1-dependent signaling. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Rosuvastatin (RSV) is a synthetic statin with favourable pharmacologic properties including minimal metabolism, hepatic selectivity and enhanced inhibition of HMG-CoA reductase. An induction of osteoblast differentiation has been reported in vitro with lipophilic statins but not with RSV, which, like pravastatin, is relatively hydrophilic compared with other statins. To mediate its action, an active transport mechanism via solute carrier (SLC) transporters from the SLC16, SLC21/SLCO and SLC22 gene family - specifically Slc16a1, Slco1a1, Slco2b1 and Slc22a8 - may be present to allow effective entry in osteoblastic cells. In this study, we demonstrate that RSV induced osteoblast differentiation, as measured by increased BMP-2 gene expression and secretion, and ALP activity in MC3T3-E1 osteoblast cells, without significantly affecting cell proliferation within the concentration range of 0.001-10 μM. Low concentrations of RSV (0.001-0.01 μM) were protective against cell death whereas higher concentrations (10-100 μM) showed cytotoxicity. Moreover, MC3T3-E1 osteoblasts expressed high levels of Slco1a1 and Slc16a1 mRNA and low levels of Slco2b1 and Slc22a8 mRNA, when compared with kidney and liver tissues from mice. Slco1a1 gene expression increased 12-fold during osteoblast differentiation and was further regulated after RSV treatment. In conclusion, as for other statins, RSV promotes osteoblast differentiation, and also, demonstrated for the first time, regulates the expression of Slco1a1, which may constitute the transport system for RSV across the cell membrane in mature osteoblasts. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Premature death of Plasmodium-infected erythrocytes is considered to favourably influence the clinical course of malaria. Amphotericin B has previously been shown to trigger suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling leading to phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing cells are rapidly cleared from circulating blood. The present study thus tested whether amphotericin B exerts a direct effect on Plasmodium falciparum and influences eryptosis of infected erythrocytes, parasitemia and host survival in murine malaria. To this end, human erythrocytes were infected in vitro with Plasmodium falciparum and mice were infected with Plasmodium berghei ANKA by in vivo intraperitoneal injection of parasitized murine erythrocytes (1x106). Half of the infected mice received amphotericin B (1.5 mg/kg b.w. i.v.) from the 8th day of infection. Amphotericin B (≥ 1 μM) compromised the intracellular development of the parasite in human erythrocytes as evident from in vitro growth and DNA amplification assays. Amphotericin B further augmented the eryptosis of infected human erythrocytes. The administration of amphotericin B to infected mice tended to delay the increase of parasitemia and significantly delayed host death. All nontreated mice died from malaria within 27 days. In contrast, some 50% of amphotericin B-treated mice survived for more than 27 days after infection. In conclusion, amphotericin B augmented the suicidal death of infected erythrocytes and delayed the lethal course of malaria in Plasmodium berghei infected mice. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Hyperosmotic stress causes cell death through activation of apoptotic pathways if the protective osmolyte response is impaired. In this study we attempt to elucidate the molecular mechanisms of hypertonicity-induced apoptosis and the effect of major organic osmolytes upon those. Methods: Hypertonicity-induced changes in Bcl2-family protein abundance and the presence of cytochrome c and apoptosis inducing factor (AIF) in the cytoplasm, were measured using western blot and immunofluorescence labeling. To determine dissipation of mitochondrial membrane potential (Δ &Psi) though the permeability transition pore (PTP), the lipophilic cationic carbocyanine fluorescence probe JC-1 and TMRM fluorescence probes were used. Results: Hypertonic culture conditions increase the abundance of proapoptotic Bax and the concentration of cytochrome c and apoptosis inducing factor (AIF) in the cytoplasm. These changes are associated with a dissipation of Δ &Psi and increased permeability of the PTP. We further show that organic osmolytes stabilize the Δ &Psi and decrease the concentration of cytochrome c and AIF in the cytoplasm. Conclusion: Our study shows that organic osmolytes prevent hypertonicity-induced apoptosis by preventing dissipation of Δ &Psi through stabilization of the PTP. These findings further support the important role of organic osmolytes in preventing hypertonicity-mediated cell death in medullary kidney cells. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]