Both phosphoproteins accumulated at the plasma membrane and Anx-A1 was subsequently externalised thereby inhibiting thromboxane (Tx) B2 generation. When administered alone, cromoglycate or nedocromil had little effect on this pathway however, in the presence of a fixed sub-maximal concentration of GCs, increasing amounts of the cromoglycate-like drugs caused a striking concentration-dependent enhancement of Anx-A1 and PKCα/β phosphorylation, membrane recruitment and Anx-A1 release from cells resulting in greatly enhanced inhibition of TxB2 generation. GCs also stimulated phosphatase accumulation at the plasma membrane of U937 cells. Both cromoglycate and nedocromil inhibited this enzymatic activity as well as that of a highly purified PP2A phosphatase preparation. We conclude that stimulation by the cromoglycate-like drugs of intracellular Anx-A1 trafficking and release (hence inhibition of eicosanoid release) is secondary to inhibition of a phosphatase PP2A , which probably forms part of a control loop to limit Anx-A1 release. These experiments provide a basis for a novel mechanism of action for the cromolyns, a group of drugs that have long puzzled investigators.Using biochemical, epifluorescence and electron microscopic techniques in a U937 model system, we investigated the effect of anti-allergic drugs di-sodium cromoglycate and sodium nedocromil on the trafficking and release of the anti-inflammatory protein Annexin-A1 (Anx-A1) when this was triggered by glucocorticoid (GC) treatment. GCs alone produced a rapid (within 5 min) concentration-dependent activation of PKCα/β  and phosphorylation of Anx-A1 on Ser Anx-A1 is both induced and secreted from cells under the influence of GCs 1 antagonists such as ketotifen, azelastine, pemirolast and olopatidine, many of which appear to share a similar pharmacology (or exhibit cross-tachyphylaxis) with cromoglycate The ‘cromoglycate-like’ anti-allergic drugs (cromolyns) are a group of compounds of which sodium cromoglycate and sodium nedocromil are the exemplars. The family also embraces lodoxamide, traxanol and amlexanox as well as some HContemporary reviewers are unanimous in attributing the anti-asthmatic activity of the cromoglycate-like drugs to their anti-inflammatory properties  in 5% CO2 atmosphere. Proliferating monocytic cells were removed from the flask when 70–80% confluence was achieved, transferred to 12-well plates at a density of 106 cells/well and pre-incubated with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) for 24 h to promote monocytic differentiation after that period they acquire sensitivity to GCs U937 cells were obtained from the American Type Culture Collection and cultured in RPMI 1640 supplemented with 10% FCS, 1% g) for 5 min. The supernatant was removed and the resultant pellet resuspended in 500 μl of lysis buffer containing 1 mM EDTA (which removes Anx-A1 attached to cell membranes), 200 mM NaCl, 20 mM Tris–HCl (pH 8.0), 1 mM protease and 1 mM phosphatase inhibitors  and 0.1% Triton-X.For assessment of drug effects, GCs were tested alone or in combination with the anti-allergic drugs for different times. Three different procedures were used to prepare samples for analysis. To analyse proteins of interest, U937 cells were normally lysed at the end of the treatment period and the total protein content was used for further analysis. To do this U937 cells in suspension following drug treatment, were decanted into 1.5 ml Eppendorf tubes and gently centrifuged  according to the manufacturer's instructions.2.2U937 cells were stably transfected with a mouse Anx-A1 cDNA open reading frame-GFP as previously reported 6/well in DMEM. Transient transfection was performed the following day, using a Dharmafect reagent 2 , a liposoluble agent that fuses with the membrane, according to the manufacturer's protocol. These cells grow in suspension and after a period of 2 weeks in G418 selection, cells were sorted by FACS to enrich the GFP positive clones followed by a serial dilution in order to facilitate a clonal expansion.For transfection, the cells were plated in 6-well plates at a concentration of 10® 594 wheat germ agglutin  to visualise the plasma membrane of live Anx-A1–GFP transfected cells.To visualise export of Anx-A1–GFP cells were stimulated with GCs and/or cromoglycate-like drugs for 5–10 min, fixed in 4% paraformaldehyde (PFA), and stained with red fluorescent Alexa FluorThe slides were mounted in Mowiol . Fluorescence micrographs were obtained with a Cool-Snap-Pro colour camera  and image processing software (Image Pro Plus 4.5) linked to a Nikon Eclipse E800 microscope. The filter used to detect GFP-fluorescence was an excitation band-pass filter (450–490 nM), a dichroic mirror (510 nM), and an emission band filter (515–560 nM). Z-stack images at 0.5 μm separation were collected on an inverted epifluorescence TE2000 U Nikon microscope, and were subjected to nearest neighbour deconvolution using Openlab 5.5 software .2.327  and α-tubulin . A horseradish peroxidase-conjugated secondary antibody  detected bands related to the proteins of interest and these were revealed using ECL reagents and quantitated using the Image J densitometry program. All data were normalised to α-tubulin and expressed as percentage of control (differentiated cells stimulated with 10 ng/mL PMA for 24 h).The total cellular protein was determined and the supernatant analysed by conventional Western blotting techniques. Immunodetection was accomplished using different antibodies recognizing either the full-length Anx-A1 protein , Anx-A1 phosphorylated on Ser2.4Anx-A1 protein levels in conditioned medium were determined by ELISA as reported by Goulding et al. 2.5After drug treatment as described above, U937 cells were fixed with a mixture of freshly prepared 3% (w/v) paraformaldehyde and 0.5% (v/v) glutaraldehyde in PBS (pH 7.2) for 4 h at 4 °C, washed briefly in PBS, and transferred to a solution of 2.3 M sucrose (in PBS) at 4 °C overnight. The cyroprotected cells were slam-frozen , freeze-substituted at −80 °C in methanol for 48 h, and embedded at −20 °C in LRGold acrylic resin  in a Reichert freeze-substitution system. Ultrathin sections (50–80 nm) were prepared using a Reichert Ultracut-S ultratome and incubated at room temperature for 2 h with a well-characterised in-house polyclonal sheep anti-Anx-A1 antibody (dilution 1:200) followed by a second antibody labelled with immunogold . The serum and antiserum were diluted in 0.1 M phosphate buffer containing 0.1% egg albumin. After immunolabelling, sections were lightly counterstained with uranyl acetate and lead citrate  and examined with a JEOL 1010 transmission electron microscope .2 by dividing the total number of gold particles counted by the cell area. For measurement of cell area micrographs of each cell were taken at a magnification of 4000×. The cell areas were analysed from scanned micrographs using Axiovision software, version 3.4 . In all cases the analyst was blind to the sample code.The number of cytoplasmic and membrane 15 nm gold particles were counted in 30 cells and calculated as particles/μm2.6g for 1 h. An enzyme immunoassay was established to detect and quantify TxB2− released in the supernatant . The method was conducted following the manufacturer protocols. A standard curve ranging from 0.5 to 64 pg/well was prepared using the reagent provided and the optical density was then read at 450 nm in a microplate reader, within 30 min.Supernatants (2 ml) from individual samples were concentrated (10×) using Centricon centrifugal filter devices  with Ultracel YM-10 membrane centrifuged at 5000 × 2.72SO4 and the colour was measured on a microplate reader at 450 nm. The kinase activity in the cell lysate was calculated as a ratio between the average of absorbance in each sample (subtracted by the absorbance in the blank) and the amount of protein loaded per assay. A recombinant active protein kinase C was used as a positive control.The assay was performed using the PKC Kinase Activity Assay Kit  as described in the manufacturer's protocol: each sample was loaded on to a pre-coated plate with a substrate peptide for PKC and the reaction initiated by adding ATP. The phosphospecific substrate antibody  was added and detected by an HRP-conjugated anti-rabbit IgG and the colour developed with a TMB substrate in proportion to PKC phosphotransferase activity. The reaction was stopped with 100 μl of 1 M H2.8To detect protein phosphatase  activity in the samples, we used the colorimetric Sensolyte pNPP Protein Phosphatase kit . Membrane or recombinant PP2A samples were prepared according to the protocols suggested by the manufacturer. The colorimetric substrate p-nitrophenyl phosphate was used to assess the activity of generic phosphatase activity in our samples, yielding a yellow colour that can be quantified at 405 nm from which the substrate hydrolysis calculated from the molar extinction coefficient supplied. For a kinetic reading, the absorbance was measured every 5 min for 30 min. Samples containing drug alone without enzyme were monitored to check that they had no influence on the colour reaction.2.92SO4, methanol, NaCl, NaF, Na3VO4, paraformaldehyde, PMA, sucrose, Tris–HCl and 0.1% Triton-X) and drugs , MAP kinase inhibitor (PD98059), mifepristone (RU 486), okadaic acid and di-sodium cromoglycate) were purchased from Sigma–Aldrich, Poole, Dorset, UK. Highly purified (>90%) bovine PP2A 1800.0 U/mg was obtained from Calbiochem . Sodium nedocromil was a generous gift from Sanofi-Aventis.The following chemicals .2.10n = 30 cells per group. Preliminary analysis confirmed that the data were normally distributed. Subsequent analysis was undertaken by one-way analysis of variance (ANOVA) with post hoc analysis performed using Scheffe's test.For electron microscopy, all values for immunogold particles counted represent the mean ± S.E.M.: Elsewhere, all data are presented as mean ± S.E.M. and were tested for normality prior to analysis. Statistical differences between groups were established using one-way ANOVA followed by Bonferroni post hoc test.P < 0.05.In all cases differences were considered significant if 33.127 phosphorylated species within 5 min and this was sustained for up to 30 min. There were no changes in the total mass of Anx-A1 protein in cells prior to the 60 min time point after which steadily increasing amounts accumulated during the following 24 h. We therefore chose 5 min as the optimal time point for most of our subsequent assays as strong non-genomic effects of the GC could be easily observed without detectable genomic actions occurring. We tested dexamethasone, hydrocortisone, prednisolone, methylprednisolone and betamethasone in this system finding that all stimulated Anx-A1 Ser27 phosphorylation in a qualitatively comparable fashion, however prednisolone, methylprednisolone and betamethasone were not examined further in our assay systems.GCs increase both the synthesis (genomic) as well as the release (non-genomic) of Anx-A1 from cells. As most previous studies have focussed upon the former we determined, in a series of pilot studies, the time course of GC action on these separate processes in U937 cells (data not shown). Treatment with 1 μM dexamethasone strongly stimulated the production of the Ser27 phosphorylation. We examined the concentration–response curves of dexamethasone, prednisolone and hydrocortisone in detail and determined the relative EC50 concentrations to be 0.2, 0.4 and 1 nM respectively. In this paper we report data obtained mainly using dexamethasone but we observed that hydrocortisone exhibited qualitatively identical behaviour in terms of its interaction with the cromolyns.We then established the concentration dependency of GC-induced Anx-A1 phosphorylation in U937 cell lysates at 5 min. 27 phospho Anx-A1.Previous studies have implicated the enzyme PKC in this GC-induced phosphorylation of Anx-A1. We tested this in the present system using a pan-specific anti-phospho PKC antibody, finding that the abundance of phosphorylated PKC was increased in a similar concentration-dependent fashion . It is noteworthy that the background Anx-A1 phosphorylation sometimes observed in untreated cells was itself reduced by RU 486 (P < 0.05) perhaps implying that residual GCs in the medium may drive a low level phosphorylation of the protein.To confirm that the GC effect was dependent upon GR occupation we tested the effect of the GR antagonist RU 486 on the ability of dexamethasone to stimulate phosphorylation of Anx-A1. P < 0.001) to reverse the dexamethasone-induced increase in Anx-A1 phosphorylation. The MW of the phosphorylated PKC enzyme detected by Western blotting using the pan-specific PKC antibody was in the range 76–82 kDa suggesting the relevant isoform was either PKC δ (78 kDa), PKC θ (76 kDa) or PKCα/β (80–82 kDa). When we probed our blots with a panel of isoform specific anti-phospho PKC antisera, no PKC δ (Ser643) or PKC θ Thr538 was detectable. Some PKC δ Thr505 were detected but with a lower MW (78 kDa) than the GC stimulated species seen in our blots. However, the specific anti-phospho-PKCα/β Thr638/641 antibody showed good reactivity with a band that increased with GC treatment over a period of 5–30 min treatment  supernatant to the particulate fraction of the U937 cells. We refined this analysis by using differential centrifugation to prepare some 100,000 × g membranes from cells treated with drug combinations. P < 0.05) the catalytic activity of the enzyme in this compartment.PKCα/β is enriched at the plasma membrane when activated and Anx-A1 is likewise recruited to the membrane upon phosphorylation at Ser3.42, a prominent eicosanoid spontaneously released from U937 cells.The ability of GCs to arrest eicosanoid synthesis depends on the release of Anx-A1 from target cells in many systems. As the cromoglycate-like drugs are also reported to inhibit eicosanoid formation, we next determined whether these drugs alone, or in combination with GCs, enhanced Anx-A1 release thereby producing a concomitant decrease in the generation of TxB2 release it greatly potentiated (P < 0.001) the inhibitory effect of 2 nM dexamethasone (P < 0.01) alone. This correlated with a potentiation of Anx-A1 release into the medium as assessed by the ELISA assay (P < 0.001).2 release at concentrations of 20 nM nedocromil.2 generation that was correlated exactly with the increasing amounts (7–8-fold) of Anx-A1 released onto the outside of the cell (see blot insert).2 was caused entirely by increased Anx-A1 in this system we tested the effect of a neutralising anti-Anx-A1 monoclonal (mab 1A) and isotype-matched irrelevant monoclonal antibody. P < 0.001) the inhibitory effect of the dexamethasone–nedocromil combination on TxB2 release.To confirm that the drug-induced inhibition of TxB3.5To investigate the effect of the drugs on the intracellular movement of Anx-A1 at a fine level of detail, we utilised U937 cells that had been transfected with an Anx-A1–GFP construct and analysed the effects of drug treatment by confocal microscopy.The Western blots in ® 594 wheat germ agglutinin (WGA) labelling reagent that specifically stains membrane phospholipids. The Anx-A1–GFP in vehicle treated cells is localised in the cytoplasm (row a). Not much change is seen with nedocromil (5 nM) treatment alone (row b), however, treatment with dexamethasone (2 nM) alone induces some degree of Anx-A1 redistribution, as shown by a partial co-localisation of Anx-A1 with the membrane . When dexamethasone is combined with 5 nM nedocromil, however a striking degree of co-localisation of Anx-A1 with the membrane is observed (row d) and evidence of membrane ‘bubbling’ (possible related to secretory activity) may be inferred from the presence of GFP–Anx-A1 outside the cell membrane.Although we observed that the non-genomic actions of these drugs were evident at 5 min, we were curious about events that might take place over a shorter time-span. To investigate this we used a slightly different experimental design in which time-lapse video-microscopy was used. GFP-tagged Anx-A1 transfected U937 cells were filmed for 3 min under various experimental conditions, and the frames examined for changes that might reflect the release of Anx-A1 in response to drugs.3.6P < 0.01). In the presence of both GCs and nedocromil however (panel 6D), there was a significant (P < 0.01) increase of membrane-associated Anx-A1 and a corresponding decrease in cytosolic and vesicular associated material.The experiments with the transfected U937 cells could be compromised by the fact that Anx-A1 was conjugated to GFP and therefore that its intracellular trafficking might be atypical. To address this we used conventional electron microscopic and immuno-chemistry techniques to follow the fate of Anx-A1 in response to various drug treatments. 3.727 under the influence of activated PKCα/β, was released from the cell to exert an extracellular inhibitory effect on eicosanoid synthesis and that this process was somehow potentiated by the cromoglycate-like drugs.All the data generated so far pointed to a mechanism whereby Anx-A1, translocated to the membrane after phosphorylation on Ser27 phospho Anx-A1. Since okadaic acid is a well-characterised inhibitor of PP2A (and PP1 in higher concentrations), we investigated its action in the U937 system. P < 0.001) share the ability of cromoglycate to potentiate Anx-A1 release in this system.The catalytic activity of PKCα/β is limited by membrane phosphatases, in particular PP2A, and we hypothesised that the cromoglycate-like drugs inhibited the activity of these enzymes thus prolonging the catalytic action of PKCα/β and indirectly potentiating the generation and release of Serg membranes. In the experiment depicted in g membranes from U937 dexamethasone-treated cells (as described above) and then pre-incubated them for 5 min with either 5 nM nedocromil or 1 μM okadaic acid before assessing their phosphatase activity at 10 min . Strikingly, phosphatase activity was almost completely inhibited in the presence of either nedocromil or okadaic acid.We next measured the net phosphatase activity in U937 100,000 × 50s of approximately 0.65 and ∼1.7 nM respectively. As expected, okadaic acid was also strongly inhibitory (IC50 ≪ 1 μM).Finally, we tested the effect of cromoglycate and nedocromil on a highly purified PP2A preparation from bovine kidney. 4The Anx-A1 system in undifferentiated myelomonocytic U937 cells does not respond to glucocorticoids and it is necessary to pre-treat the cells with low concentrations of PMA to render them responsive 27 phosphorylation of Anx-A1 prior to membrane translocation and export and established that the kinase most likely to drive this process is the PKCα/β isoform of PKC.Concerning the former, we have confirmed and extended earlier reports  modulatory/targeting sub-unit. The nature of the latter determines the targeting and substrate specificity of the assembled enzyme complex. C-terminal carboxymethylation of the catalytic sub-unit at LeuThe degree of Anx-A1 phosphorylation, and hence the amount exported, will therefore depend upon the net catalytic activity of the PKCα–PP2A complex which, in turn will result from the reciprocal interactions between the two enzymes. The cumulative evidence from biochemical, immunocytochemical and imaging studies presented here all support the notion that whilst the cromoglycate-like drugs alone have only weak activity they are able greatly to accelerate the phosphorylation and release of Anx-A1 when this is primed (in this case) by GCs, which promote PKC activation and some degree of Anx-A1 phosphorylation and membrane localisation. Our analysis points to a direct inhibitory action of these drugs on PP2A enzyme activity as the most likely explanation for this synergism although we cannot entirely rule out the participation of PP1, another phosphatase, or the possibility that which ever phosphatase is involved, it acts directly to dephosphorylate Anx-A1 itself rather than PKC. Supporting the notion that PP2A is the actual target however is our observation that the PP2A inhibitor okadaic acid Experiments using anti-Anx-A1 neutralising antibodies Although there are inflammatory models in which Anx-A1 is not efficacious Whilst our hypothesis is novel, there have been several previous attempts to link cromolyn action to activation of signalling pathways and modification of potential down-stream molecular targets. Treatment of mast cells with cromoglycate results in the phosphorylation of (at least) four intracellular protein substrates including the erythrocyte band 4.1 group protein moesin There have also been previous reports One practical implication of this work arises from the observation that GCs and cromoglycate-like drugs exhibit a strong synergistic action that might be applicable to anti-inflammatory therapy. The notion that these drugs can inhibit PP2A may also suggest other uses for these agents in other conditions where PKC activation plays a significant role.The authors have no conflicting financial interests.


Incubation of Caco-2 cell monolayers (15 min) with the dietary phosphodiesterase inhibitors caffeine and theophylline inhibited Gly–Sar uptake across the apical membrane. Pentoxifylline, a phosphodiesterase inhibitor given orally to treat intermittent claudication, also decreased Gly–Sar uptake through a reduction in capacity (Vmax) without any effect on affinity (Km). The reduction in dipeptide transport was dependent upon both extracellular Na+ and apical pH but was not observed in the presence of the selective Na+/H+ exchanger NHE3 (SLC9A3) inhibitor S1611. Measurement of intracellular pH confirmed that caffeine was not directly inhibiting hPepT1 but rather having an indirect effect through inhibition of NHE3 activity. NHE3 maintains the H+-electrochemical gradient which, in turn, acts as the driving force for H+-coupled solute transport. Uptake of β-alanine, a substrate for the H+-coupled amino acid transporter hPAT1 (SLC36A1), was also inhibited by caffeine. The regulation of NHE3 by non-nutrient components of diet or orally-delivered drugs may alter the function of any solute carrier dependent upon the H+-electrochemical gradient and may, therefore, be a site for both nutrient–drug and drug–drug interactions in the small intestine.The H Indeed, dipeptides can stimulate Na+ absorption in the human small intestine and Na+ uptake in human intestinal Caco-2 cell monolayers +-coupled di/tripeptide transport and Na+ absorption + influx activated an apical Na+/H+ exchanger and thus Na+ absorption. This model also explains the partial Na+-dependence of di/tripeptide uptake observed in mammalian small intestine and Caco-2 cell monolayers +/H+ exchange by removal of extracellular Na+, prevents the enterocyte from maintaining intracellular pH (pHi) during H+/solute influx and, therefore, abolishes the driving force  for further transport. The apical Na+/H+ exchanger specifically activated by di/tripeptide uptake is NHE3 (SLC9A3) which plays a significant role in Na+ absorption in the small intestine When expressed heterologously, hPepT1 functions as a Naii by preventing breakdown), affect hPepT1 function. The dietary phosphodiesterase inhibitors caffeine and theophylline and the orally-delivered drug pentoxifylline all inhibit dipeptide uptake across the brush-border membrane of human intestinal epithelial Caco-2 cell monolayers. This inhibition is not a direct effect on hPepT1 but rather is indirect through inhibition of NHE3.As hPepT1 is involved in both protein assimilation and drug delivery it is important to elucidate how altering hPepT1 function may affect nutritional status and oral bioavailability. Progress has been made in understanding how intestinal di/tripeptide transport is regulated by hormonal and neural signals as well as by disease states and surgical intervention (for review see 22.114C]sarcosine (specific activity 57 mCi mmol− 1)] was obtained from Cambridge Research Biochemicals . [3-3H]β-Alanine (specific activity 50 Ci mmol− 1)] was obtained from American Radiolabeled Chemicals . PACAP (pituitary adenylate cyclase-activating polypeptide) was from Bachem . Biotrak cAMP enzyme immunoassay was from GE Healthcare . 2′,7′-Bis(2-carboxyethyl-5(6)-carboxyfluorescein) acetoxymethyl ester (BCECF-AM) was from Invitrogen . Transwell polycarbonate filters were from Corning . S1611 was obtained from H.J. Lang . Forskolin, phosphodiesterase inhibitors, cell culture media and supplements were from Sigma-Aldrich . All other chemicals were from Sigma-Aldrich or VWR  and were of the highest quality available.GlycylGly–Sar  or [3H]β-alanine  uptake across the apical membrane of Caco-2 cell monolayers was measured, essentially as described previously 4, 0.99; KH2PO4, 0.34; NaH2PO4, 0.3; CaCl2, 2.8; Glucose, 10) or Na+-free solution . Uptake was measured at apical pH 5.5 or pH 6.5  with the basolateral solution being pH 7.4  in all experiments. Uptake was measured over 15 min (37 °C) at the end of which the monolayers were rinsed thoroughly . Various compounds were added to the buffers for the duration of uptake (see figure legends for details). Cell monolayer-associated radioactivity was determined by scintillation counting.2.6t-test or one-way analysis of variance (ANOVA) (using the Tukey–Kramer or Bonferroni's multiple comparisons post-test) as appropriate. Significance was assumed if p < 0.05. Curves were fitted using FigP software.Data are expressed as mean ± SEM unless stated otherwise. Statistical comparisons of mean values were made using paired two-tailed Student's 33.1− 2 [15 min]− 1, n = 17–18, p < 0.001, + reduces Gly–Sar uptake .Uptake of the dipeptide Gly–Sar (glycylsarcosine) across the apical membrane of Caco-2 cell monolayers was measured over 15 min at an apical pH (pH 6.5) representative of that found at the surface of the proximal small intestine. Incubation of Caco-2 cell monolayers with the dietary phosphodiesterase inhibitor caffeine (5 mM) at both the apical and basolateral surfaces (for the duration of the 15 min uptake period) reduced Gly–Sar uptake by around 50% . This concentration of S1611 is sufficient to inhibit NHE3 completely but would not inhibit NHE2 or NHE1 +, S1611 has no effect on Gly–Sar uptake into Caco-2 cells Xenopus laevis oocytes, dipeptide uptake is not inhibited by S1611 p > 0.05) suggesting that caffeine and S1611 inhibit hPepT1 activity through a common mechanism (i.e. NHE3 inhibition). Gly–Sar uptake increases with decreasing apical pH, consistent with hPepT1 being a H+/dipeptide symporter. However, NHE3 activity decreases with acidification of the apical solution and is inactive by pH 5.5 due to the unfavourable transmembrane pH gradient +p > 0.05) suggesting again that caffeine is inhibiting Gly–Sar uptake through inhibition of NHE3.Previously we have shown that the Nainactive B. At api3.2+ (p < 0.001) but not in Na+-free conditions (p > 0.05). Previously, we have shown that the enteric neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) and the related neuropeptide VIP  reduce dipeptide uptake across the apical membrane of Caco-2 cell monolayers by inhibition of NHE3 1 receptor which is coupled to adenylate cyclase and, therefore, increases [cAMP]i. Phosphodiesterase inhibitors structurally-related to caffeine and either found in diet (theophylline), or used as orally-delivered therapeutics (pentoxifylline) or used as laboratory tools  were tested for inhibition of hPepT1 . Theophyi in Caco-2 cell monolayers was measured. Monolayers were incubated with caffeine (5 mM), theophylline (5 mM), pentoxifylline (5 mM) or the adenylate cyclase activator forskolin (10 μM) which can also inhibit dipeptide uptake i measured by enzyme immunoassay. Caffeine, theophylline and pentoxifylline caused [cAMP]i to more than double from control levels of 950 ± 230 fmol cm− 2  to 2200 ± 830, 2780 ±300 and 2330 ± 150 fmol cm− 2 , respectively. However, the change in cAMP was modest compared to [cAMP]i detected in the presence of forskolin .Phosphodiesterase inhibitors increase the levels of cAMP within cells by preventing cAMP breakdown. However, caffeine can also elicit cell signalling effects such as antagonism of adenosine receptors and activation of calcium release from intracellular stores +-dependent (NHE3-dependent) component of Gly–Sar uptake by phosphodiesterase inhibitors was concentration-dependent  for Gly–Sar uptake was significantly reduced from 8246 ± 913 to 5369 ± 117 pmol cm− 2 [15 min]− 1  by pentoxifylline but the affinity (Km) was not affected . Reduction in the capacity for dipeptide uptake is consistent with a reduction in the driving force  for uptake through inhibition of NHE3. Previously, we have shown that S1611 and forskolin similarly decrease the Vmax for dipeptide uptake across the apical membrane of Caco-2 cell monolayers Gly–Sar uptake across the apical membrane was measured at apical pH 6.5 at various concentrations of Gly–Sar . In the 3.4+/dipeptide symport in Caco-2 cell monolayers +, or factors that increase [cAMP]i, such as forskolin and VIP, all inhibit the ability of the cell to regulate pHi after H+/solute-induced intracellular acidification i was measured using the pH-sensitive dye BCECF  under conditions where hPepT1 activity would be maximal but where there would be no concurrent NHE3 activity . After 5 min, Gly–Sar had induced a large intracellular acidification. When Gly–Sar was removed and the apical solution returned to a Na+-containing, pH 7.4 solution, pHi recovered rapidly back to baseline levels. The same monolayers were then incubated with caffeine (5 mM) in both the apical and basolateral solutions for 10 min. Gly–Sar was then reintroduced for 5 min in the continued presence of caffeine. The degree and rate of acidification caused by Gly–Sar was not altered by caffeine suggesting that hPepT1 is not directly regulated by phosphodiesterase inhibitors. However, the ability of the cell to recover pHi after Gly–Sar-induced acidification was attenuated after the 15 min exposure to caffeine .Previous work demonstrated that NHE3 (but not NHE1 or NHE2) is selectively activated by the intracellular acidification associated with Hye BCECF . Caco-2 caffeine A. The derecovery B. In theXenopus laevis oocytes, as described previously To confirm that phosphodiesterase inhibitors do not have any direct inhibitory effect on dipeptide transport, hPepT1 was expressed in 3.5+-electrochemical gradient will similarly be regulated by these compounds. The H+-coupled amino acid transporter hPAT1 (SLC36A1) has been isolated from Caco-2 cell monolayers +-independent but hPAT1-mediated amino acid uptake into Caco-2 cells is partially Na+-dependent +/H+ exchange by NHE3 +-coupled dipeptide uptake, H+-coupled amino acid uptake into Caco-2 cells is inhibited by forskolin, S1611 and VIP in a Na+ and pH-dependent manner via inhibition of NHE3 p < 0.001) but not the absence of extracellular Na+ (p > 0.05) suggesting that H+-coupled amino acid uptake via hPAT1 is also modulated indirectly through regulation of NHE3.The indirect nature of the inhibition of hPepT1 by phosphodiesterase inhibitors suggests that any apical solute transporter dependent upon the transmembrane H4The di/tripeptide transporter hPepT1 acts as a high-capacity route for solutes across the first barrier to oral-bioavailability, the brush-border membrane of the small intestine. Many, orally-active peptidomimetics and amino acid-conjugated pro-drugs have been identified as hPepT1 substrates +- and pH-dependent (+ or by addition of S1611) the cells are no longer able to maintain pHi during solute-induced acidification and, therefore, the driving force  for further dipeptide uptake is reduced. Previously, we have shown that hPepT1 can be inhibited by other factors which are known to increase cAMP in intestinal epithelial cells such as the enteric neuropeptides VIP and PACAP i. The increase is relatively small compared to that produced by forskolin. However, this could be due to the cAMP signal being compartmentalised (as demonstrated in cardiomyocytes i (data not shown). Lastly, the concentrations of caffeine, theophylline and pentoxifylline required to produce inhibition of hPepT1  and release of intracellular calcium (EC50≈ 5–20 mM) 2 receptors by luminal adenosine (5 mM) in mouse small intestine in vivo 2 receptors are positively coupled to adenylate cyclase so antagonism of the receptor should cause a decrease in [cAMP]i and not the increase noted here The data presented here show that the inhibition of Gly–Sar uptake by phosphodiesterase inhibitors is both Naependent  suggestiCaffeine (and to a lesser extent theophylline) is found in high levels in many beverages either naturally or by addition. For example, the average cup of coffee contains 85 mg of caffeine Since the initial reports of phosphodiesterase activity it has become evident that there are many types of phosphodiesterase. Currently there are 11 different PDE families each of which can contain several isoforms and splice variants Caffeine, theophylline, IBMX and pentoxifylline generally inhibit all PDE isoforms with roughly equal efficacy +-coupled dipeptide transport. Functional cooperativity between hPepT1 and NHE3 results not only in the absorption of di/tripeptides but also stimulation of Na+ absorption +-coupled amino acid transporter hPAT1 (SLC36A1) is regulated by caffeine in a similar, indirect manner to hPepT1 (+ electrochemical gradient for function may be regulated in a similar manner.In conclusion, we have shown that non-nutrient components of diet and orally-delivered therapeutic agents both regulate activity of the nutrient and drug transporter hPepT1. This regulation is an indirect effect through inhibition of apical NHE3 function, which acts as a pH homeostatic mechanism during Ho hPepT1 . Thus, t


Xenopus laevis embryo undergo extensive remodeling beginning at the midblastula transition (MBT) of early development. Cell divisions 2–12 consist of rapid cleavages without gap phases or cell cycle checkpoints. Some remodeling events depend upon a critical nucleo-cytoplasmic ratio, whereas others rely on a maternal timer controlled by cyclin E/Cdk2 activity. One key event that occurs at the MBT is the degradation of maternal Wee1, a negative regulator of cyclin-dependent kinase (Cdk) activity.The cell cycles of the In order to assess the effect of Wee1 on embryonic cell cycle remodeling, Wee1 mRNA was injected into one-cell stage embryos. Overexpression of Wee1 caused cell cycle delay and tyrosine phosphorylation of Cdks prior to the MBT. Furthermore, overexpression of Wee1 disrupted key developmental events that normally occur at the MBT such as the degradation of Cdc25A, cyclin E, and Wee1. Overexpression of Wee1 also resulted in post-MBT apoptosis, tyrosine phosphorylation of Cdks and persistence of cyclin E/Cdk2 activity. To determine whether Cdk2 was required specifically for the survival of the embryo, the cyclin E/Cdk2 inhibitor, Δ34-Xic1, was injected in embryos and also shown to induce apoptosis.X. laevis embryos.Taken together, these data suggest that Wee1 triggers apoptosis through the disruption of the cyclin E/Cdk2 timer. In contrast to Wee1 and Δ34-Xic1, altering Cdks by expression of Chk1 and Chk2 kinases blocks rather than promotes apoptosis and causes premature degradation of Cdc25A. Collectively, these data implicate Cdc25A as a key player in the developmentally regulated program of apoptosis in  Xenopus laevis embryo provides a rich context in which to investigate cell cycle regulation and the interplay between the cell cycle and development. The first twelve cleavage cycles following fertilization consist of rapid oscillations between S and M phase without intervening gap phases. These cell cycles do not engage checkpoints in response to damaged or unreplicated DNA ATP [2 cpm/fmol]). Reactions were terminated with 25 μl of 2× gel loading buffer containing 25% β-mercaptoethanol, heated at 95°C for 2 min, and resolved by PAGE. The gels were stained with Coomassie Blue and dried. Incorporation of [γ-32P]ATP was determined by Cerenkov scintillation counting of the excised histone H1 band.For kinase assays, immunoprecipitates were washed twice in kinase buffer , and then incubated 20 min at 27°C with 25 μl of kinase buffer containing 0.2 mg/ml bovine serum albumin, 0.5 mg/ml histone H1, 200 μM [γ-2, 10 mM β-mercaptoethanol) and stored at -80°C.Rosetta cells (Novagen) were transformed with pGEX vector encoding the GST-p27Xic1CK- containing 4 mutations in p27Xic1 . Bacterial pellets were resuspended in buffer ×  containing 1 tablet of Complete protease inhibitors (Roche) and 1 mM DTT. Cell suspension was incubated at room temperature with 100 μg/mL lysozyme for 20 min with stirring then for an additional five minutes with 1/20 volume 10% Triton ×-100. Cell suspension was then sonicated 2 × 30 seconds and centrifuged at 17,510 × g for 1 hr at 4°C. The supernatant fraction was loaded onto a column of glutathione-agarose beads (Sigma) previously equilibrated with 5 volumes of extraction buffer . Supernatant was passed through the column twice. The column was then washed with 10 volumes of buffer A  and 10 volumes of buffer B . p27Xic1CK- protein was eluted with 5 mL buffer × containing 100 mM glutathione , pH 8.0. Purified p27Xic1CK- was then concentrated by buffer exchange using Amicon Ultra Ultracel low binding regenerated cellulose tubes , resuspended in buffer  declare that there are no competing interests.BNW designed and carried out the experiments and drafted the manuscript. CVF helped design and perform the Xic experiments and helped draft the manuscript. JCS participated in the project and experimental design and data interpretation, performed the nuclear morphology and kinase assays and drafted the manuscript. All authors have read and approved the final version of the manuscript.


Deregulated activation of cyclin-dependent kinase-5 (Cdk5) is implicated in neurodegenerative disorders such as Alzheimer's disease. One of the restricting factors for developing specific Cdk5 inhibitors is the lack of reproducible and well-characterized cellular in vitro assay systems.HEK293 cells were transfected with Cdk5 and its activator p25 as a starting point for an assay to screen for Cdk5 kinase inhibitors. To identify suitable substrates for Cdk5 we utilized an antibody that recognizes phospho serine in a consensus motif for Cdk substrates.Western blot analysis of transfected cells detected a 200 kDa band that was identified, by mass spectrometry, as non-muscle myosin heavy chain, type B (NMHC-B). Phosphorylation of NMHC-B was evident only in cells that were double transfected with Cdk5/p25 and was dose-dependently inhibited by Roscovitine and other Cdk5 inhibitors. Cdk5 was found to phosphorylate NMHC-B also in the human neuroblastoma SH-SY5Y cell line.A novel Cdk5 substrate NMHC-B was identified in this study. A cellular assay for screening of Cdk5 inhibitors was established using NMHC-B phosphorylation as a read-out in Cdk5/p25 transfected HEK293 cells. A novel Cdk5 inhibitor was also pharmacologically characterized in this assay system. Cyclin-dependent kinase-5 (Cdk5) is a member of the cyclin-dependent kinase (Cdk) family of serine/threonine kinases . Unlike Although Cdk5 activity is necessary for many physiological functions and development of the nervous system, deregulated Cdk5 activity is neurotoxic and has been linked to neurodegenerative diseases such as Alzheimer's disease (AD). Conversion of p35 to p25 by the calcium activated protease calpain, is thought to cause deregulation of Cdk5 activity in AD brain ,7. The dTaken together, data supporting the role of Cdk5 in different pathways connected to pathological processes in the central nervous system is convincing thus making it a potentially important target for drug research. Furthermore, availability of specific and selective Cdk5 inhibitors would enable even more detailed studies on its pathological and biological roles. One of the restricting factors for identifying specific Cdk5 inhibitors is the lack of a reproducible and well-characterized cellular assay system. One of the major reasons is the almost exclusive localization of the active Cdk5/p35(p25) complex to cells of neuronal origin, which makes it difficult to find easy-to-handle cell lines for assay purposes.We previously investigated retinoic acid and brain-derived neurotrophic factor (RA-BDNF) differentiated SH-SY5Y cells in an attempt to establish a cellular system to study Cdk5 involvement in tau phosphorylation. However, in basal conditions the involvement of Cdk5 in tau phosphorylation is minor  and alsoWe report the establishment of a new cellular screening system, which enables pharmacological characterization of specific Cdk5 inhibitors. In the course of the study, we also identified non-muscle myosin heavy chain, type B (NMHC-B), as a substrate for Cdk5.5 cells/cm2 in 6-well culture dishes . Day 1 after plating, the cells were transfected with equal amount of p25 plasmid  and Cdk5 plasmid , 1.5 μg each. Lipofectamine™2000  was used as a transfection reagent. Lipofectamine™2000 (7.5 μl/transfection) was first diluted in cell culture medium without FCS and incubated for 5 min at RT. The plasmid DNA diluted in medium was then combined with Lipofectamine and incubated for further 20 min at RT. The complexes were added to the cells and the transfection was carried out for 24 hours. Treatment with Cdk5 inhibitors was carried out during the last 4 hours of transfection.Human embryonic kidney 293 (HEK293) cells were grown in Dulbecco's Modified Eagle Medium  with 4.5 g/l glucose, 2 mM glutamine and 110 mg/l sodium pyruvate. The medium was supplemented with 1% non-essential amino acids  and 10% heat-inactivated Fetal Calf Serum . For transfection experiments, the cells were plated at a density of 2.0 × 10The p25 and Cdk5 genes were cloned into mammalian expression vectors, pcDNA3 and pcDNA3.1(-) , respectively and the expression was under the control of CMV promoter. Cdk5 inhibitors used in this study were Roscovitine , 7-ethyl-4-[(4-fluorophenyl) amino]-3,5,7-triaza bicyclo [4.4.0.] deca-1,3,5,9-tetraen-8-one from Warner Lambert company (WL compound) ,19 and 44 cells/cm2 in 6-well culture dishes using cell medium with 10% FCS. For differentiation, the cells were treated 6 days with 10 μM all-trans-retinoic acid  in the medium containing 1% FCS. In some experiments the cells were left undifferentiated and these cells were cultured in medium containing 10% FCS. SH-SY5Y cells have high endogenous levels of Cdk5 and therefore only the p25 plasmid (pAPC227) was transfected to the cells. Transfections were carried out in the same manner as for HEK293 cells except that 10 μl of Lipofectamine™2000 was used for each transfection with 8 μg p25 plasmid.SH-SY5Y cells were grown in medium with equal amount of Minimum Essential Medium  and Nutrient Mixture Ham's F-12 , supplemented with 1% non-essential amino acids and with heat-inactivated FCS. Cells were plated at a density of 4.0 × 103O4V, 0.5% NP-40 and 1 complete protease inhibitor cocktail tablet /10 ml buffer. Lysis buffer for SH-SY5Y cells contained 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 1 mM Na3VO4,1% Triton X-100 and 1 complete protease inhibitor cocktail tablet/10 ml buffer. Cell lysates were centrifuged at 14 000 rpm  for 15 min. The protein content in the supernatants was measured using BCA Protein Assay kit . Samples containing 40–50 μg protein were resolved in 10% NuPage®Bis-Tris gels  and the proteins were transfered to Hybond nitrocellulose membranes . Membranes were blocked in PBS with 0.05% Tween 20 containing 5% nonfat dry milk for 1 hour at room temperature (RT). Primary antibodies were diluted in either 5% BSA or 5% milk and incubations were carried out at 4°C over night. Primary antibodies were used at the following dilutions: Cdk5  1:2000, p35  1:2000, pSer Cdk substrate  1:800 and non-muscle heavy chain myosin [3H2]  1:1000. Horseradish-peroxidase (HRP) conjugated secondary antibodies  were incubated 1 hour at RT in 5% milk at the dilution of 1:5000 for anti-rabbit and 1:10 000 for anti-mouse antibody. Blots were developed using the enhanced chemiluminescence (ECL) Western blotting detection system . When needed, membranes were stripped with Restore Western blot stripping buffer  for 30 min at 50°C. Average density of the bands was measured in Fluor-S™MultiImager  by using Quantity One software. The inhibition curves were analysed by non-linear regression using Graph Pad Prism.HEK293 cells were lysed in buffer containing 10 mM Tris-HCl pH 7.2, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 1 mM Na200 μg of protein in 100 μl lysis buffer was precleared for 1 hour at 4°C with 10 μl protein A/G Plus agarose beads . The samples were centrifuged at 14 000 rpm (Eppendorf 5417R) for 1 min and the supernatants were transferred to new tubes. 10 μl of pSer Cdk substrate antibody was added to the supernatants and incubated rotating over night at 4°C, 20 μl protein A/G Plus agarose beads was added and incubated further for 1 hour. The samples were centrifuged at 6000 rpm (Eppendorf 5417R) for 2 min. The supernatants were discarded and the pellets washed three times in 150 μl lysis buffer. The immunoprecipitates were then diluted in lysis buffer and processed for Western blot analysis.® -stained gel pieces were manually treaded as follows; wash with 100 μl ddH2O three times for 15 min, followed by wash with 25 mM ammoniumbicarbonate/acetonitril (1:1 v/v) three times for 15 min. Thereafter acetonitril was added to cover the gel pieces to shrink them down. The gel pieces were rehydrated by incubating with 50 μl 25 mM ammoniumbicarbonate for 5 min, adding acetonitril and incubating for another 15 min. Thereafter all liquid was removed and the gel pieces were dried completely in vacuo. The samples were placed on ice and a freshly prepared and chilled digestion solution containing 10 ng/μl of trypsin  in 25 mM ammoniumbicarbonate was added to cover the gel pieces. Incubation on ice for 10 min and in RT for 30 min was carried out and more buffer was added if necessary. The samples were then placed on a heater at 37°C over night. One-hour incubation with equal amount of 1% formic acid stopped the reaction. To concentrate and desalt the samples ZipTip  was used according to manufacturers descriptions. Samples were then washed twice with 1% formic acid and eluted with 2 μl of matrix solution α-cyano-4-hydroxycinmic acid  direct onto 96-position MALDI target plate  and crystals of matrix and peptide were formed. After MALDI-ToF  was used to attain the mass of each peptide and the resulting peaklist was imported into the search engineers Protein Lynx Global Server 2  and MASCOT . Several databases were selected as information sources.Protein spots of interest were excised from gels using a spot cutter robot  and transferred to 96-well plates. The gel plugs were transferred to eppendorf tubes approximately 10 pieces in each tube. Sypro Ruby33P]ATP , 2 μM unlabelled ATP and 10 mM MgCl2 in an final assay volume of 21 μl. After incubation for 40 min at RT, each reaction was terminated by the addition of 30 μl stop solution containing 24 mM EDTA, 2.2 mM ATP and 0.225 mg streptavidine coated SPA beads . The microtiter plates were centrifuged for 2 minutes at 200 g and the radioactivity was determined in a liquid scintillation counter . The inhibition curves were analysed by non-linear regression using Graph Pad Prism.The assay experiments were carried out in duplicate with 10 different concentrations of the inhibitors in clear-bottom 384-well microtiter plates . Recombinant human Cdk5/p25  was added at a final concentration of 3.3 nM in an assay buffer containing 23.3 mM HEPES, pH 7.35, 0.2 mM EDTA, 12.5 mM KCl, 10 mM β-glycerophosphate, 0.02% β-mercaptoethanol, 0.007% Brij 35, 0.8% glycerol and 0.05% BSA. After incubation for 15 min the reaction was initiated by the addition of 2.95 μM  of a biotinylated peptide substrate, Biotin-Ala-Lys-Lys-Pro-Lys-Thr-Pro-Lys-Lys-Ala-Lys-Lys-Leu-OH , 0.07 μCi [γTo identify novel Cdk5 substrates suitable for characterising enzyme inhibitors, we transfected HEK293 cells with Cdk5 and p25 to get an active Cdk5/p25 complex. Cell lysates from transfected cells were analyzed with Western blotting using an antibody that recognizes phospho serine in a consensus motif for Cdk substrates.The pSerCdk substrate antibody recognized several phosphoproteins. A band with molecular weight of approximately 200 kDa appeared in cells that were double transfected with Cdk5 and p25 but did not show up in non-transfected or single transfected (only Cdk5 or p25) cells Fig. . The 200In initial studies, the HEK293 cells were transfected with 1, 1.5 or 2 μg of Cdk5 and p25 plasmids. The setting where the cells were transfected with 1.5 μg of each plasmid, i.e. 3 μg total plasmid DNA, showed least effect on cell morphology and viability (data not shown) with sufficient phosphorylation of Cdk substrates Fig . This plP35580). The total number of mass values matched was 130 and the sequence coverage 57%. Verification that the 200 kDa pSerCdk substrate band indeed was myosin was done by immunoprecipitation. When Cdk5/p25 transfected samples were immunoprecipitated with pSerCdk substrate antibody and then run on Western blot, the pSerCdk substrate antibody and NMHC-B antibody detected the same band  . This biochemical assay measures inhibition of the recombinant human Cdk5/p25 protein kinase by different compounds with a synthetic peptide as a substrate. Several compounds displayed a potency in the nanomolar range and were further tested in Cdk5/p25 transfected HEK293 cells. Figure tine Fig , 45 nM ftine Fig  and 355 tine Fig .To further investigate Cdk5 involvement in NMHC phosphorylation, we used the three pharmacological inhibitors of Cdk5 and analyzed NMHC phosphorylation by Western blot using the pSerCdk substrate antibody.50 of 1.18 μM, 2.55 μM and 4.36 μM, respectively. . A close analogue to it was co-crystallised with the Cdk5/p25 complex and the X-ray structure showed that the ligand is not directly bound to the backbone (Glu 81 and Cys83) in the ATP site of the kinase as is the usual case. Instead, a water molecule was found to form a bridging interaction between the ligand and the hinge backbone . The AZ compound is assumed to bind in a similar fashion, although X-ray crystallography would be required to confirm this.Phosphorylation of NMHC-B was evident only in Cdk5/p25 double transfected cells indicating that only Cdk5 phosphorylates this substrate in HEK293 cells. Cdk5 inhibitors concentration-dependently inhibited NMHC-B phosphorylation and at higher concentrations phosphorylation was totally blocked. Described assay with NMHC-B phosphorylation as a read-out is thus very specific and sensitive. However, a limitation of the system at present is its relatively low throughput caused by the Western blot based quantification of the phosphorylation signal. An attempt to solve this could for instance be use of Image analysis in a high content screening setting such as ImageXpress.A novel Cdk5 substrate, NMHC-B, was identified in this study. Using phosphorylation of this substrate as a read-out, we developed a specific and sensitive cellular assay that can be used for validation of compounds designed to inhibit Cdk5. A novel Cdk5 inhibitor was also pharmacologically characterized in this assay system.AD: Alzheimer's disease; Cdk5: cyclin-dependent kinase; HEK293: human embryonic kidney 293; NMHC-B: non-muscle myosin heavy chain, type B; RA: retinoic acid; SPA: Scintillation proximity assay.The authors declare that they have no competing interests.AJ carried out experiments in SH-SY5Y cells, participated in design of the study and drafted the manuscript. KA, ACH and AJ all participated in experiments with HEK293 cells. KA also participated in the design of the study and edited the manuscript. JO carried out the sequence analysis using mass spectrometry and protein identification using Mascot server. GH carried out SPA assays. JM was responsible for the design and synthesis of the novel Cdk5 kinase inhibitor used in the study. GL designed the plasmids used in transfection experiments and participated in the initial transfection experiments in HEK293 cells. MV organized the study, participated in study design and coordination and revised the manuscript. All authors read and approved the final manuscript.


P2, that has been implicated in various trafficking events associated with the endocytic pathway. We have now directly compared the effects of siRNA-mediated knockdown of PIKfyve in HeLa cells with a specific pharmacological inhibitor of enzyme activity. Both approaches induce changes in the distribution of CI-M6PR and trans-Golgi network (TGN)-46 proteins, which cycles between endosomes and TGN, leading to their accumulation in dispersed punctae, whilst the TGN marker golgin-245 retains a perinuclear disposition. Trafficking of CD8-CI-M6PR (retromer-dependent) and CD8-Furin (retromer-independent) chimeras from the cell surface to the TGN is delayed following drug administration, as is the transport of the Shiga toxin B-subunit. siRNA knockdown of PIKfyve produced no defect in epidermal growth factor receptor (EGFR) degradation, unless combined with knockdown of its activator molecule Vac14, suggesting that a low threshold of PtdInsP2 is necessary and sufficient for this pathway. Accordingly pharmacological inhibition of PIKfyve results in a profound block to the lysosomal degradation of activated epidermal growth factor (EGF) and Met receptors. Immunofluorescence revealed EGF receptors to be trapped in the interior of a swollen endosomal compartment. In cells starved of amino acids, PIKfyve inhibition leads to the accumulation of the lipidated form of GFP-LC3, a marker of autophagosomal structures, which can be visualized as fluorescent punctae. We suggest that PIKfyve inhibition may render the late endosome/lysosome compartment refractory to fusion with both autophagosomes and with EGFR-containing multivesicular bodies.The phosphoinositide 5-kinase (PIKfyve) is a critical enzyme for the synthesis of PtdIns P is generated by the class III PtdIns 3-kinase, hVps34, and is concentrated on early/sorting endosomes 3-phosphoinositides control the identity and dynamics of endocytic compartments, through the recruitment of multiple effector proteins containing specific recognition domains. PtdIns3P is also a precursor for the generation of PtdInsP2 by the FYVE domain containing PtdIns 5-kinase, PIKfyve P2 increase due to various stresses, osmotic shock in yeast PtdIns3bone fide effectors of PtdInsP2 has proven elusive P and PtdInsP2, and altering its expression levels leads to changes in the distribution of CI-M6PR Identification of Caenorhabditis elegans (C. elegans) mutant hypomorphic for the PIKfyve homologue was suggested to possess a defect in retrieval of membrane from mature lysosomes Drosophila PIKfyve yields cells with enlarged endosomes and a defect in degradation of Wingless and Notch, without any apparent signalling defects Various studies have examined the effects of manipulating PIKfyve and Vac14 levels. A Jefferies et al. have recently characterized a novel inhibitor of PIKfyve, YM201636, which provides the first opportunity for acute inhibition of the enzyme 50 of 23 nm, whereas an inactive analogue MF2 showed no activity even at 5 μm. Corresponding MF4 values for class I PtdIns 3-kinases which we determined are 0.25 μm (p110α), 1 μm (p110β), 0.9 μm (p110γ) and 0.8 μM (p110δ). Application of MF4 gives a vacuolar phenotype in all cells within 4 h. Electron microscopic analysis indicates that the large phase lucent vacuoles are inaccessible to internalized horseradish peroxidase (HRP), but they do become surrounded by a second class of smaller (but still swollen) HRP-containing vacuoles, positive for early (EEA1) or late endosomal markers [lysosome-associated membrane protein (LAMP-2)] ] , which wLAMP-2)] . MF4 doeVE probe .cis-Golgi marker GM130 retains its ribbon-like disposition  and YM210636 (33 nm) are similar towards PIKfyve, we obtain less selectivity over PtdIns 3-kinase p110α than that reported for YM210636 α and β were used. Both inhibitors caused a significant reduction in phosphorylation of AKT/PKB but had no effect on EGFR degradation (data not shown). These results in combination with the observation that MF4 does not cause a reduction in initial EGF-dependent phosphorylation of AKT/PKB, suggest that the effects on EGFR degradation are not due to a non-specific inhibition of class 1 PtdIns 3-kinases.PIKfyve siRNA studies of EGF-induced EGFR downregulation have not revealed any defect in EGFR downregulation P generated by the PtdIns 3-kinase Vps34 is required for initial steps of autophagosome formation, but the role of PtdInsP2 is less well established. The Atg18 gene in yeast binds both 3-phosphoinositides with a preference for PtdInsP2PtdIns3P2 particularly if the enzyme is controlled by a positive feedback loop, (ii) it is not clear if phenotypes observed are because of loss of enzyme activity or of some other function of the protein and (iii) depletion is very gradual, so that any phenotype may constitute a very indirect consequence of protein loss over a prolonged time period. Nevertheless, these studies and our current study, have shown that the distribution of proteins which traffic between the sorting endosome and the TGN is perturbed Previous studies in mammalian cells have assessed phenotypes arising from siRNA knockdown. These have the following drawbacks; (i) PIKfyve has proven hard to completely deplete, so that residual enzymatic activity could provide enough PtdInsAs well as the steady-state distribution, the kinetics of transport from the cell surface via the endosome to the TGN is delayed for several proteins , which may utilize retromer-dependent  and retromer-independent (CD8-Furin) sorting mechanisms from the endosome. Interestingly, retrieval to the TGN is delayed and not completely blocked following loss of PIKfyve activity, suggesting that either cargo may traffic on alternate, less efficient pathways or that enzymatic activity is rate-limiting for this process but not essential.P2 to a greater extent than PIKfyve alone. This condition revealed a modest inhibition of EGFR degradation  by recruitment of effector molecules with specific lipid binding domains and (ii) allosteric regulation of resident endocytic proteins, e.g. enzyme or channel activity. Although Svp1 in yeast is an authentic PtdInsP2 sensor it is less clear for its mammalian orthologues WIPI-1 and WIPI-2. WIPI-1, like Svp1 can bind both PtdIns3P and PtdInsP2in vitroA role for PtdIns3P2 are permissive for these pathways.In summary, juxtaposition of siRNA knockdown of PIKfyve with pharmacological inhibition links the observed siRNA phenotype of disrupted endosome to TGN trafficking, with enzymatic activity. However, pharmacological inhibition reveals further defects on the receptor degradation and autophagy pathway, suggesting that relatively low levels of PtdInsLimulus polyphemus haemocyanin (Covance) and affinity purified on peptide columns. Sheep anti-TGN46, sheep anti-GM130 antibodies and sheep anti-GFP were generous gifts of Vas Ponnambalam  Francis Barr and Ian Prior  respectively. Rabbit polyclonal anti-PIKfyve and anti-CI-M6PR antibodies were generous gifts from Lois Weisman  and Paul Luzio . Mouse monoclonal anti-Met (25H2) antibody, rabbit monoclonal phosphorylated AKT (pAkt) (Ser 473) and mouse monoclonal phosphorylated MAPK (pMAPK)  antibodies were from Cell Signalling. Monoclonal anti-EGFR R1 was from Cancer Research UK Laboratories (CRUK) and goat polyclonal anti-EGFR 1005 were from Santa Cruz. Mouse monoclonal anti-tubulin was from Sigma. Mouse monoclonal anti-golgin 245 (p230) antibody was from BD Biosciences. Rabbit polyclonal anti-EEA-1 was previously described Rabbit polyclonal human Vac14 antibodies were raised by immunizing rabbits with the N-terminal amino acids of Vac14 (HLEVRHQRSGRGDHLDRR), conjugated via a cysteine to 2Cl2 (20 mL) and cooled on ice. Triflic anhydride  was added drop-wise and the reaction was warmed to room temperature. Further CH2Cl2 was added (10 mL) and the reaction was allowed to proceed overnight. The solvent was removed in vacuo and the product was purified by silica gel flash chromatography with 25% EtOAc:Hexanes to yield 67 mg (24% yield). Liquid chromatography–electrospray ionisation–mass spectrometry (LC-ESI-MS) [MH]+ m/z calculated for C20H15F3N4O5S 481.1, found 480.9. This product , cesium carbonate  and nicotinamide  were added to dioxane (4 mL). Xantphos  and Pd2(dba)3  were added and the reaction was heated to 100°C for 24 h 2Cl2, filtered and the solvent was removed in vacuo. The product was purified by silica gel chromatography using a gradient of 2–3% MeOH:CH2Cl2 and by reverse phase high performance liquid chromotography (HPLC) using a MeCN/H2O/0.1% trifluoroacetate (TFA) solvent system to give a white solid. LC-ESI-MS [MH]+ m/z calculated for C25H20N6O3453.2, found 453.0. MF2 was prepared similarly using PIK-112 + m/z calculated for C26 H22N6O2451.2, found 451.4.Synthesis of the PIKfyve inhibitor (hereafter designated MF4) and its inactive analogue, MF2 is described below. PI-103 m to 0.001 μm in a total reaction volume of 25 μL. The assay buffer contained 10 μm ATP, 2 μCi of (γ-32P) ATP, 100 μm PI-3P, 400 μm phosphatidyl-ethanolamine, 25 mm HEPES (pH 7.4), 120 mm NaCl, 1.5 mm MgCl2, 1 mm DTT, 0.5 mg/mL BSA and 2% dimethyl sulphoxide (DMSO). Reactions were terminated by spotting onto nitrocellulose membranes, which were then washed five or six times to remove unbound radioactivity and dried. Transferred radioactivity was quantitated using phosphorimaging, and IC50 values were calculated by fitting the data to a sigmoidal dose-response using Prism (GraphPad).Purified GST-PIKfyve bound to 5 μL of glutatione beads was incubated with inhibitors at fourfold dilutions over a concentration range of 5 μ2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum and 1% non-essential amino acids. To retain expression of stably transfected constructs, culture medium for CD8 cell lines was supplemented with 0.5 mg/mL G418. On-Target Plus siRNA oligos against PIKfyve and Vac14 were purchased from Dharmacon. A 2-hit protocol over 72 h was used. Briefly, HeLa cells were seeded in six-well plates at a density of 2.4 × 105 cells/well. Cells were transfected with 40 nm of the corresponding oligo using oligofectamine (Invitrogen), 24 h later. After a further 24 h, cells were reseeded onto six-well plates at the same density or onto cover-lips at a density of 0.12 × 106 cells/well and transfected again with 40 nM oligos for 24 h. Cells were then processed for western blotting and immunofluorescence as described below.HeLa and HEK293A cells were cultured in 5% COm Tris/Cl, pH 7.5, 100 mm NaCl). Lysates were cleared by centrifugation, resuspended in 5× SDS-sample buffer and resolved by SDS-PAGE followed by immunoblotting for detection with a Licor Odyssey.Where indicated, cells were serum-starved for 16 h and then stimulated with 100 ng/mL EGF or hepatocyte growth factor (HGF). Cells were washed twice in ice-cold phosphate buffered saline solution and lysed in Nonidet P-40 lysis buffer  in PBS, permeabilized with 0.2% Triton-X-100, blocked in 0.2% fish skin gelatin in PBS and incubated with antibodies in blocking buffer. Secondary antibodies were conjugated to either AlexaFluor 488, 594 or 633. Cover-slips were imaged using a Leica confocal SP2 AOBS (HCX PL APO CS 63.0 × 1.40 oil objective).m Tris-Cl pH 7.6, followed by incubation for 10 min in 0.1% diaminobenzidine  in Tris buffer and then for 10 min in 0.1% DAB and 0.01% H202 in Tris buffer at room temperature in the dark. The reaction was terminated, by washing 3 × 5 min in Tris buffer, and then 2 × 5 min in PBS. Cells were then post-fixed in the dark on ice with 1% OsO4 in 0.1 m phosphate buffer pH 7.4 for 1 h. Subsequently, the cells were washed 3 × 30 mi in PBS, followed by 2 × 30 min in distilled water and then incubated in 5% uranyl acetate in 30% ethanol for 1 h. Samples were dehydrated by sequential 10-min ethanol washes in 30%, 60%, 70%, 80% and then twice in 100% ethanol, then infiltrated with a 1:1 ratio of resin to 100% ethanol for 30 min. The infiltration resin was then replaced with 100% resin and the flat-embedded samples were polymerized at 60°C for 2 to 3 days.Cells were incubated in 10 mg/mL HRP in serum-free medium at 37°C for 10 min, 1 h or 4 h. Subsequently, cells were washed three times in ice-cold PBS and fixed in 0.5% glutaraldehyde for 30 min. Cells were washed three times at room temperature with 0.1 m Hepes and 0.2% fatty-acid free BSA) for 15 min. CD8-CIM6PR and CD8-Furin receptors at the cell surface were labelled by placing cover-slips, cell side down, upon a 100 μL drop of DHB containing 1 μg monoclonal anti-CD8 antibody at 4°C, for 1 h. Cover-slips were washed twice in ice-cold PBS, and then transferred to pre-warmed growth medium for the indicated time periods to allow uptake of the anti-CD8 antibody to occur. At the assay endpoint, cells were fixed and processed for immunofluorescence as described earlier.HeLaM CD8-CIM6PR or CD8-Furin cells were seeded onto cover-slips and treated with either siRNA or PIKfyve inhibitor. Cover-slips were then transferred to a well containing 3 mL ice-cold dihydroxybenzoate (DHB)  or cells were washed four times with Earl's buffered saline solution and then starved for 2 h in the final wash. Where indicated, Leupeptin was added to the starvation medium at a concentration of 0.25 mg/mL. Following starvation, cells were either fixed and processed for immunofluorescence or lysed in 1× SDS-sample buffer. After protein determination lysates were supplemented with 1 m


The progression from Barrett's metaplasia to adenocarcinoma is associated with the acquirement of an apoptosis-resistant phenotype. The bile acid deoxycholate (DCA) has been proposed to play an important role in the development of esophageal adenocarcinoma, but the precise molecular mechanisms remain undefined. The aim of this study was to investigate DCA-stimulated COX-2 signaling pathways and their possible contribution to deregulated cell survival and apoptosis in esophageal adenocarcinoma cells.Following exposure of SKGT-4 cells to DCA, protein levels of COX-2, MAPK and PARP were examined by immunoblotting. AP-1 activity was assessed by mobility shift assay. DCA-induced toxicity was assessed by DNA fragmentation and MTT assay.DCA induced persistent activation of the AP-1 transcription factor with Fra-1 and JunB identified as the predominant components of the DCA-induced AP-1 complex. DCA activated Fra-1 via the Erk1/2- and p38 MAPK while Erk1/2 is upstream of JunB. Moreover, DCA stimulation mediated inhibition of proliferation with concomitant low levels of caspase-3-dependent PARP cleavage and DNA fragmentation. Induction of the anti-apoptotic protein COX-2 by DCA, via MAPK/AP-1 pathway appeared to balance the DCA mediated activation of pro-apoptotic markers such as PARP cleavage and DNA fragmentation. Both of these markers were increased upon COX-2 suppression by aspirin pretreatment prior to DCA exposure.DCA regulates both apoptosis and COX-2-regulated cell survival in esophageal cells suggesting that the balance between these two opposing signals may determine the transformation potential of DCA as a component of the refluxate. Bile acids are normal constituents of the gastro-intestinal tract where they act as trophic factors for the gut epithelium and as detergents for the absorption of cholesterol and fat-soluble vitamins ,2. TypicWhile bile acids such as DCA cannot induce tumors, they are generally believed to be tumor promoters. The exact mechanism of their tumor promoting activity is uncertain but it is thought to involve alterations in cellular signaling cascades including activation of protein kinase C and gene expression systems . Bile acOver the past two decades there has been a significant increase in the incidence of Barrett's esophagus , a premain vivo -2 [4'-methylsulfinylphenyl]-5-[4'-pyridyl] imidazole), Z-VAD-FMK  and nuclear extracts were prepared as described previously . EMSA waμg/sample using a protein assay (Bio-Rad), according to the manufacturer's instructions. Equal protein content during affinity precipitation was assessed on acetone-precipitated supernatants.Affinity precipitation of DNA binding proteins was performed with the optimal binding sequence for AP-1 (5'-CGC TTG ATG AGT CAG CCG GAA-3') (Sigma genosys UK) as previously described  with the3VO4 and 1 mM PMSF and 10 μg leupeptin (Sigma) and incubating on ice for 20 minutes. Cell nuclei and debris were eliminated by centrifugation at 10,000 × g for 10 min. The total protein content per sample was standardized to 50–100 μg as above.SKGT4 cells were stimulated and total cell lysates obtained using 25 mM Tris-HCl, pH 7.9, 0.2% NP-40, 15 mM NaCl, 1 mM sodium fluoride, 5% glycerol (v/v), 0.05 mM EDTA, 1 mM NaEqual amounts of proteins were separated on a 10% SDS-polyacrylamide gel and transferred onto a PVDF membrane . Membranes were incubated with specific antibodies against Erk1/2, p38 and JNK (at a dilution of 1:1000)  or their corresponding phosphorylated forms Erk1/2 (p-Tyr204-Erk1/2), JNK (p-Thr-183/Tyr-185-JNK)  and p38 (p-Thr180/Tyr182-p38) (at a dilution of 1:50)  overnight at 4°C. Antibodies against COX-2  and PARP  (at a dilution of 1:100) were also used and incubated overnight at 4°C. Membranes were then incubated with corresponding secondary horseradish peroxidase-conjugated antibodies  (at a dilution of 1:2000) for 1 h at room temperature. Specific immunocomplexes were visualised using the ECL detection system . For sequential detection, membranes were stripped in 100 mM 2-Mercaptoethanol, 2% SDS, and 62.5 mM Tris pH 6.8 for 30–45 min at 50°C.4) were plated in a flat-bottomed micro-titre plate and incubated for 24 hr at 37°C and 5% CO2. Cells were incubated either with increasing concentrations of DCA (0 – 500 μM) or over a period of 1 – 24 hr with 300 μM DCA. Following stimulation, MTT   was added and cells were further incubated for 1 to 3 hr. Absorbance was measured at 490 nm. Viability is expressed as the percentage of cells remaining in cultures treated with bile acids relative to untreated controls.SKGT4 cells  according to the manufacturer's standard protocols. Absorbance was measured at 405 nm using an ELISA plate reader.DCA regulates gene transcription through AP-1 activation in colonic cells . We examAP-1 dimer composition is crucial in determining the induction of specific target genes and consequent cellular responses. EMSA was used to determine which Jun and Fos proteins form part of the DCA-induced AP-1 complex. Nuclear extracts from SKGT4 cells stimulated with 300 μM DCA, were incubated with antibodies against c-Fos, Fra-1 and c-Jun prior to incubation with the radiolabelled AP-1 probe. Blocking antibodies that prevent the binding of the corresponding transcription factor rather than causing a supershift were used . The speTo further characterize the composition of the DCA-induced AP-1 complex, total cell lysates were prepared from SKGT4 cells treated with 300 μM DCA for 1 – 6 hr and used for DNA affinity precipitation assays with the AP-1 consensus sequence (5'-CGC TTG ATG AGT CAG CCG GAA-3'). Only active forms of the Jun and Fos proteins are able to bind to this oligonucleotide and can be therefore affinity purified and detected by Western blot analysis with specific antibodies. For these assays, we concentrated on c-Jun and Fra-1 as suggested by EMSA and also included JunB and JunD, as they have been shown to respectively counteract or enhance c-Jun activity . These ede novo protein expression anthracene; GERD: gastroesophageal reflux disease; Erk: extracellular signal regulated kinase; MAPK: mitogen activated Protein Kinase; PdBu: phorbol 12, 13-dibutyrate; PMA: phorbol 12-myristate 13-acetate.ANIS: anisomycin; AP-1: activator protein-1; ASA: acetylsalicylic acid; COX-2: cyclooxygenase-2; DCA: deoxycholic acid; DMBA: 7,12-dimethylbenz [The authors declare that they have no competing interests.EL carried out the study and prepared the manuscript. MAL participated in the design of the study experimentation and edited the manuscript. VAM made contributions to the study methodology of MAPK and AP-1 analysis. SD contributed to the design of the study experimentation and drafting of the manuscript. AL coordinated the study and edited the manuscript. DK designed and supervised the study. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/190/prepub


Brugia malayi and Trichinella spiralis to influence C5a-mediated chemotaxis of human peripheral blood granulocytes in vitro was examined. Secreted products from B. malayi microfilariae almost completely abolished chemotaxis. This inhibition was blocked by phenylmethylsulphonyl fluoride, indicating the presence of a serine protease, which was subsequently shown to cleave C5a. In contrast, secreted products from T. spiralis infective larvae showed modest inhibition of C5a-mediated granulocyte chemotaxis, and this was blocked by potato carboxypeptidase inhibitor, an inhibitor of several metallocarboxypeptidases. Adult and larval stages of both parasites were demonstrated to secrete carboxypeptidases which cleaved hippuryl-l-lysine and hippuryl-l-arginine, and the T. spiralis enzyme was partially characterised. The data are discussed with reference to inflammation in parasitic nematode infection.Given the importance of the complement anaphylatoxins in cellular recruitment during infection, the ability of secreted products from larval stages of  Inflammation does not always match that expected from secretion of potently antigenic material, for example in lymphatic filariasis, in which individuals frequently harbour high burdens of larval parasites in the circulatory system with no outward sign of infection . In contAncylostoma caninum also secretes Neutrophil Inhibitory Factor (NIF), which binds the β integrin CD18/CD11b and inhibits neutrophil adhesion to vascular endothelial cells and subsequent activation  and C3-deficient animals  had higher numbers of larvae in the lungs during primary infection, indicating a role for complement in parasite attrition during migration through somatic tissues  and cultured in serum-free RPMI 1640 for up to 5 days as previously described . All procedures involving animals were approved by the Imperial College Ethical Review Committee and performed under licence from the UK Home Office. Animal care and maintenance was performed by College staff as directed by the licence and institutional guidelines.escribed . Adults escribed . Culture2.22+ and Ca2+ supplemented with 0.1% BSA, 10 mM Hepes, pH 7.2, and 10 mM glucose). Chemotaxis plates were blocked with GAFS buffer supplemented with 1% BSA for 30 min at 37 °C in a humidified chamber prior to assay. Recombinant human C5a (Sigma) was used as the chemoattractant at a concentration of 10 nM, and pre-incubated with parasite secreted products in the presence or absence of protease inhibitors at 37 °C. Reactions were applied to the chemotaxis plate together with human granulocytes at a density of 4 × 106 cells ml−1, and incubated for 2 h at 37 °C, 5% CO2. Cells were quantified by flow cytometry, with the chemotactic index representing the number of cells that migrated towards the chemoattractant divided by the number of cells that migrated to GAFS buffer alone. Phenylmethylsulphonyl fluoride (PMSF), l-transepoxysuccinyl-leucylamido-[4-guanidino]butane (E64) and pepstatin were purchased from Amersham Pharmacia Biotech AB, 1,10-phenanthroline and potato carboxypeptidase inhibitor (PCI) were from Sigma, and guanidinoethylmercaptosuccinic acid (GEMSA) was from EMB Chemicals. Statistical analysis of data in which two groups were compared was by Student’s t-test assuming unequal variance.Chemotaxis assays were performed in 96-well chemotaxis chambers using membranes with a 5 μM pore diameter (Neuroprobe). Cells were isolated from venous blood of healthy donors, granulocytes isolated by standard procedures and suspended in Gated Autofluorescence/Forward Scatter (GAFS) buffer (HBSS without Mg2.3l-arginine or hippuryl-l-lysine (Sigma) in 25 mM Tris, pH 8.0, 1 mM CoCl2 in a total volume of 15 μl at 37 °C for 2 h. The reaction was stopped on ice, 100 μl 0.25 M phosphate buffer, pH 8.3 added, and further developed by the addition of 75 μl 3% cyanuric acid in 1,4 dioxane. After 2 min agitation, the plate was centrifuged at 3000g for 10 min, 100 μl of the supernatant was transferred to a fresh plate and absorbance at 405 nm determined. Carboxypeptidase activity was determined from hippuric acid standards, with porcine pancreatic carboxypeptidase B (Sigma) as a positive control. One unit of activity is equivalent to the hydrolysis of 1 μmol substrate at 37 °C per min.Carboxypeptidase activities were measured using a microplate colourimetric assay that detects the release of hippuric acid from hippuryl-conjugated substrates . Parasit2.4125I using the chloramine T method , whereas incubation of granulocytes with BmSP alone had minimal chemotactic effect  exists as an inactive zymogen in plasma, bound to plasminogen and activated by plasmin or thrombin . CPR is B. malayi and a carboxypeptidase from T. spiralis. Carboxypeptidases were secreted by adult and larval stages of both parasites but sufficient quantities of secreted proteins could only be recovered from T. spiralis cultures for characterisation. Mammalian plasma CPR is inhibited preferentially by PCI over GEMSA and is partially inhibited by the addition of cobalt. In contrast, CPN is activated by the addition of cobalt, inhibited by GEMSA but relatively insensitive to PCI  of mice with iliensis  and conts larvae . This ralishment . Onchoce pathway .T. spiralis and B. malayi. Mannose-binding lectin (MBL) binds oligosaccharides on the surface of T. spiralis muscle stage larvae, and binds to glycoproteins in their secreted products (B. malayi microfilariae with subsequent activation of C3 (−/− deficient mice than in matched wild-type animals, and this was accompanied by a profound defect in antigen-specific IgM production (B. malayi microfilariae (The lectin pathway has also been shown to be responsible for activation of complement on the surface of products . Murine on of C3 . Intriguoduction . This cofilariae . It was filariae  and, intfilariae .N. brasiliensis L3s are trapped in the skin of IL-5 transgenic mice for extended periods, indicative of susceptibility to eosinophil-mediated attack (T. spiralis and Toxocara canis appear unaffected (T. spiralis (T. spiralis by skewing cytokine production and thereby inhibiting nitric oxide synthesis (T. canis infective larvae inhibit the deposition of C3 and adherence of eosinophils to N. brasiliensis, suggesting that a proteolytic activity may degrade C3 (T. canis secreted products to the inoculum of N. brasiliensis L3s enhances the number of parasites which migrate to the lungs of IL-5 transgenic mice, indicating that the protective effect is also operative in vivo (Although d attack , other naffected , and eosspiralis . More reynthesis . Secretegrade C3 . Moreove in vivo .Necator americanus (T. spiralis and N. brasiliensis secrete metallo- and serine proteases which cleave eotaxin (Enzymes secreted by nematodes have been shown to degrade other pro-inflammatory chemotactic factors. For example, eotaxin is cleaved and inactivated by a secreted metalloprotease from ericanus , and it  eotaxin . A probl eotaxin . The mos eotaxin . Hopeful


Associations between cardiovascular diseases and mercury have been frequently described, but underlying mechanisms are poorly understood.We investigate the procoagulant activation of erythrocytes, an important contributor to thrombosis, by low-level mercury to explore the roles of erythrocytes in mercury-related cardiovascular diseases.ex vivo and in vivo thrombosis models in rats to investigate mercury-induced procoagulant activity.We used freshly isolated human erythrocytes and 2+; 0.25–5 μM for 1–48 hr) induced erythrocyte shape changes from discocytes to echinocytes to spherocytes, accompanied by microvesicle (MV) generation. These MVs and remnant erythrocytes expressed phosphatidylserine (PS), an important mediator of procoagulant activation. Hg2+ inhibited flippase, an enzyme that recovers PS into the inner leaflet of the cell membrane, and activated scramblase, an enzyme that alters lipid asymmetry in the cell membrane. Consistent with these activity changes, Hg2+ increased intracellular calcium and depleted ATP and protein thiol. A thiol supplement reversed Hg2+-induced MV generation and PS exposure and inhibited the increase in calcium ion (Ca2+) and depletion of ATP, indicating that free-thiol depletion was critical to Hg2+-mediated procoagulant activity. The procoagulant activity of Hg2+-treated erythrocytes was demonstrated by increased thrombin generation and endothelial cell adhesion. We further confirmed Hg2+-mediated procoagulant activation of erythrocytes in ex vivo and in vivo rat thrombosis models, where Hg2+ treatment (0.5–2.5 mg/kg) increased PS exposure and thrombus formation significantly.Prolonged exposure to low-dose mercuric ion (HgThis study demonstrated that mercury could provoke procoagulant activity in erythrocytes through protein-thiol depletion–mediated PS exposure and MV generation, ultimately leading to enhanced thrombosis. Mercury (Hg) is a heavy metal element widely distributed in the earth’s crust, seawater, freshwater, and air . Concomi2+) by catalase. Orally absorbed methylmercury is preferentially distributed into erythrocytes (~ 90%) and slowly turns into Hg2+ through demethylation in the spleen and liver. It has been demonstrated that 6–15% of total mercury in the blood of populations consuming high-fish diets exists in the form of inorganic mercury  translocation to the external surface of the erythrocyte cell membrane  through the modulation of a clotrimazole-sensitive potassium ion (K+) channel. These authors described the PS-exposing effects of Hg2+ in relation to the apoptosis of erythrocytes. However, the implications of PS-externalized erythrocytes in procoagulant activation and subsequent CVDs were not addressed. In another study, alterations of the erythrocyte membrane, including PS exposure and PS-bearing microvesicle (MV) formation, were reported to be able to render erythrocytes procoagulant, enabling the active participation of erythrocytes in thrombosis . Of note, Hg2+-mediated PS exposure and MV generation could enhance thrombin generation and adhesion to vascular endothelial cells, which is considered a direct marker for procoagulant activity. We examined the mechanism underlying, and in vivo relevancy of, Hg2+-induced procoagulant activation of erythrocytes in an effort to gain insight into the CVDs associated with mercury exposure.In the present study, we discovered that both MV generation and PS exposure could be induced in human erythrocytes by low-dose Hg2), calcium chloride (CaCl2), EDTA, bovine serum albumin (BSA), KH2PO4, NaCl, Na2HPO4, KCl, Tris/HCl, MgCl2, NaH2PO4, dextrose, sodium citrate, Tris-base, NaHCO3, dimethyl sulfoxide, ethanol, Triton X-100, trichloroacetic acid, Tris-acetate, ATP bioluminescent assay kit, iodoacetic acid, adenosine, ethylene glycol tetraacetic acid (EGTA), acetic acid, purified human thrombin, and calcium ionophore A23187 from Sigma Chemical Co. . Other chemicals were of the highest grade available.We purchased mercury chloride  and once with Tris buffer  or Ringer solution . Washed erythrocytes were resuspended in Tris buffer or Ringer solution to a cell concentration of 5 × 107 cells/mL, and the final CaCl2 concentration was adjusted to 1 mM before use.This study was approved by the Institutional Review Board at the Seoul National University/Health Service Center, and all subjects provided written informed consent. On the day of each experiment, human blood was obtained from healthy male donors (18–25 years of age) into Vacutainers containing acid citrate dextrose . Platelet-rich plasma and buffy coat were removed by aspiration after centrifugation at 200 × 2+ and observed using confocal microscopy equipped with an argon laser . Excitation and emission filters were set at 488 nm and 550–600 nm, respectively.After fixation with 2% glutaraldehyde solution for 1 hr at 4°C, the erythrocytes were centrifuged and washed three times with PBS, followed by postfixation with 1% osmium tetroxide for 30 min at room temperature. After washing with PBS several times, the samples were dehydrated serially with 50%, 75%, 90%, and 100% ethanol. After drying and coating with gold, the images were observed on a scanning electron microscope . For confocal microscopy, 200 μL erythrocyte suspension was added and attached for 1 hr to an eight-chambered coverslip  that had been coated with 0.1 mg/mL poly-l-lysine. After washing the coverslip three times with Tris-buffered saline (TBS) containing 2% BSA, erythrocytes were stained with TBS buffer containing anti–glycophorin-A–FITC for 30 min and washed three times again. Erythrocytes were then incubated with vehicle (TBS) or Hg2. For detection of intracellular calcium increase, erythrocytes were loaded with 3 μM Fluo-4-AM  for 1 hr at 37°C in the dark. Subsequently, the cells were washed twice and then resuspended in Tris buffer to a final concentration of 5 × 107 cells/mL with 1 mM of CaCl2. For the confirmation of interference of Fluo-4 calcium signal by Hg2+, N,N,N′,N-tetrakis(2-pyridylmethyl)ethylenediamine  was added for 5 min to extract Fluo-4–bound Hg2+. To quench extracellular calcium, 3 mM EGTA was added to the erythrocyte suspension. Samples were analyzed on a FACScalibur flow cytometer (Becton Dickinson). Data from 10,000 events were collected and analyzed using CellQuest Pro software (Becton Dickinson).We used fluorescein isothiocyanate (FITC)-labeled annexin V  as a marker for PS positivity and phycoerythrin-labeled monoclonal antibody against human glycophorin A  to identify erythrocytes. Negative controls for annexin V binding were stained with annexin V-FITC in the presence of 2.5 mM EDTA instead of 2.5 mM CaCl7 cells/mL) were incubated with Hg2+ and then loaded with 0.5 μM 1-Palmitoyl-2-[6-[amino]hexanoyl]-sn-glycero-3-phospho-l-serine  for the flippase activity assay or 1-oleoyl-2-[6-[amino]hexanoyl]-sn-glycero-3-phosphocholine  for the scramblase activity assay. Aliquots of cell suspension were removed at the indicated time intervals, placed in cold Tris buffer, and incubated on ice for 10 min in the presence or absence of 1% BSA. The amount of internalized probe was determined by comparing the fluorescence intensity associated with the cells before and after back-extraction by BSA. Samples were analyzed on the FACScalibur flow cytometer.We measured phospholipid translocation according to the method described by 2+, erythrocytes were washed and resuspended in Tris buffer containing 1 mM CaCl2. The aliquot was mixed vigorously with 10% trichloroacetic acid solution and TAE buffer  and then cooled on ice for 20 min. The sample was centrifuged, and the aliquot of resultant supernatant was mixed with cold TAE buffer. Samples were adapted to the luciferin/luciferase assay in a Luminoskan microplate reader  using an ATP assay kit (Sigma). We calculated the ATP concentrations based on the ATP standard curve.After incubation with Hg2+, erythrocytes were centrifuged at 7,000 × g for 1 min, and the supernatant was removed. The pellet was resuspended with lysis buffer  and incubated on ice for 30 min. Total lysate was resuspended with 5% perchloric acid 2:5 and then centrifuged at 7,000 × g for 2 min. The pellet was solubilized in 1 mL Tris-EDTA buffer  containing 1% sodium dodecyl sulfate. 5,5′-Dithio-bis-(2-nitrobenzoic acid)  was added to the samples, and the change of absorbance was measured at 412 nm. The content of protein thiol was calculated on the basis of a glutathione calibration curve and divided by the protein content, which was measured by Bio-Rad protein assay kit .We determined protein thiol concentrations using a modified assay based on the colorimetric method described by 2+ for 4 hr, erythrocytes were incubated with 5 nM factor Xa and 10 nM factor Va  in Tyrode buffer  for 3 min at 37°C. Thrombin formation was initiated by adding 2 μM prothrombin. Exactly 3 min after addition of prothrombin, an aliquot of the suspension was transferred to a tube containing stop buffer . Thrombin activity was determined using the chromogenic substrate S2238 . We calculated the rate of thrombin formation from the change in absorbance at 405 nm using a calibration curve generated with active-site–titrated thrombin.After incubation with Hg2+-treated erythrocytes were added to plasma, and under gentle magnetic stirring, thrombin formation was initiated by adding human recombinant tissue factor  diluted  in Tris buffer containing 100 mM CaCl2 to the mixture. After 10 min, the aliquots were collected and transferred to Tris buffer containing 20 mM EDTA. The thrombin concentration was obtained as described above for the prothrombinase assay.We measured thrombin generation in plasma according to the method described by 2 incubator. Before the experiments, 1 × 105 cells were seeded into a T25 flask and grown for 5 days. Erythrocyte adherence to HUVEC was measured using a modification of the method described by 2+-treated erythrocytes were washed twice and resuspended in endothelial basal medium (EBM)-2 (Clonetics) to a cell concentration of 5 × 107 cells/mL. After HUVEC were washed twice with EBM-2 to remove media, the erythrocytes were layered onto a confluent HUVEC monolayer and incubated for 45 min at 37°C. After the incubation, the flask was rinsed three times with EBM-2 to remove nonadherent erythrocytes. The number of adherent erythrocytes was counted on a light microscope. The experiments were performed in triplicate, and 28 fields were selected randomly for counting erythrocytes.The HUVEC (three passages) were maintained in EGM kit  at 37°C in a 95% air/5% CO2  into a left femoral vein; 1 hr later blood was collected from the abdominal aorta using 3.8% trisodium citrate as anticoagulant. An aliquot of the blood sample was diluted 200-fold with buffer  and stained with annexin V-FITC for 15 min in the dark. PS exposure was measured as described above.We used male Sprague-Dawley rats  weighing 180–250 g for animal studies. For the measurement of PS exposure, animals were injected intravenously with saline (vehicle) or HgCl2  into a left femoral vein, 1,000-fold diluted thromboplastin was infused to induce thrombus formation. Stasis was initiated by tightening the two threads, first the proximal one, and 30 sec later the distal one. The abdominal cavity was provisionally closed, and blood stasis was maintained for 15 min. After the abdomen was reopened, the ligated venous segment was excised and opened longitudinally to remove the thrombus. The isolated thrombus was blotted of excess blood and immediately weighed.Venous thrombosis was induced by stasis combined with hypercoagulability. Rats (180–250 g body weight) were anesthetized with intraperitoneal urethane (1.25 g/kg), the abdomen was surgically opened, and the vena cava was exposed. Two loose cotton threads were prepared around the vena cava 16 mm apart, and all side branches were ligated tightly with cotton threads. One hour after intravenous injection of saline or HgClp < 0.05 indicated significance.We calculated mean ± SE for all treatment groups. The data were subjected to one-way analysis of variance followed by Duncan’s multiple range test to determine which means were significantly different from control. In all cases, 2 using SEM. As shown in 2+. It is well known that erythrocytes change into spherocytes by a substantial loss of membrane surface through vesiculation and MV generation. To visually identify MV generation, we treated erythrocytes attached to a poly-l-lysine–coated coverslip chamber with Hg2+ and observed them under confocal microscopy using erythrocyte-specific anti–glycophorin-A–FITC. Hg2+ treatment induced typical MV generation on erythrocyte membranes  or the antioxidant Trolox. As shown in 2+-induced PS exposure and MV generation, indicating a major role of thiol depletion. Indeed, Hg2+ treatment induced significant thiol depletion ] induced PS exposure on erythrocytes ex vivo  and promoted clot formation in a stasis- and hypercoagulability-induced venous thrombosis model after thromboplastin infusion , suggesting that Hg2+-induced procoagulant activity in erythrocytes could contribute to increased thrombosis in vivo.We confirmed prothrombotic effects of Hgn plasma . Moreoveon HUVEC . To evalhrocytes , which s2+ can induce shape changes, MV generation, and PS exposure on erythrocytes, important mediators of procoagulant activation of erythrocytes. Hg2+-mediated thiol depletion was responsible for calcium increase–mediated and ATP depletion–mediated flippase inhibition and scramblase activation, leading to increased thrombin generation, enhanced adhesion of erythrocytes to endothelial cells, and ultimately, accelerated thrombus formation  and ionic balance , can be readily modified by thiol-depleting agents, leading to a increased fragility of the erythrocyte membrane and disruption of ionic homeostasis. Moreover, depletion of free thiols in cells can cause depletion of ATP, inhibition of flippase (In our study, we determined thiol depletion to be a key mediator for calcium increase, ATP depletion, PS exposure, and MV generation in erythrocytes by Hglic Hg2+ . Intraceflippase , and incin vivo, mercury-induced procoagulant activation of erythrocytes might contribute to enhanced thrombosis in the population exposed to mercury, providing an important clue for the elucidation for the CVDs associated with mercury.We demonstrated that low-dose mercury can induce thrombogenic PS exposure and MV generation through thiol-depletion–mediated ATP depletion and calcium increase in erythrocytes. As shown by increased PS exposure and clot formation