We have identified human ArhGAP9 as a novel MAP kinase docking protein that interacts with Erk2 and p38α through complementarily charged residues in the WW domain of ArhGAP9 and the CD domains of Erk2 and p38α. This interaction sequesters the MAP kinases in their inactive states through displacement of MAP kinase kinases targeting the same sites. While over-expression of wild type ArhGAP9 caused MAP kinase activation by the epidermal growth factor receptor (EGFR) to be suppressed and preserved the actin stress fibres in quiescent Swiss 3T3 fibroblasts, over-expression of an ArhGAP9 mutant defective in MAP kinase binding restored EGFR-induced MAP kinase activation and resulted in significant disruption of the stress fibres, consistent with the role of Erk activation in disassembly of actin stress fibres. The interaction between ArhGAP9 and the MAP kinases represents a novel mechanism of cross-talk between Rho GTPase and MAP kinase signaling. The extensive study of the molecular mechanisms of intracellular signal transduction over the past two decades have elucidated major pathways that are important in controlling cell functions and have provided important insights in drug discovery. Two of the most well-studied pathways include the Mitogen-activated protein (MAP) kinase cascade and signaling by small-GTP binding GTPases of the Ras superfamily. These pathways are essential in normal development and aberrant functions have resulted in a wide range of pathophysiological conditions.The MAP kinase cascades play important roles in a wide variety of cellular functions and are evolutionarily conserved. In mammals there are four subfamily members, namely, Erk1/2, p38, Jnk and Erk5. Each MAP kinase has its own activators, substrates and inactivators. The MAP kinase cascade is tightly regulated in order to achieve cellular functions that are spatially and temporally precise. Recent studies have revealed two mechanisms regulating the MAP kinase signaling, namely the docking and scaffolding interactions. MAP kinases possess a common docking (CD) domain that is localized to the C-terminal region of the kinase domain for interaction with docking proteins including upstream activating kinases MAP kinase kinases (MAPKKs), MAP kinases activating protein kinases (MAPKAPs), MAP kinase-specific phosphatases (MKPs) and MAP kinase substrates such as Elk , followed by centrifugation 13,000 g for 15 min at 4°C and collecting the supernatant. For in vitro pulldown assays, GST fusion proteins immobilized on glutathione beads were incubated with the lysates for 1 hour at 4°C with rotation, followed by washing of the beads with specifically bound proteins with cell lysis buffer for 5 times, each time for 5 min with rotation at 4°C. The protein complexes were then eluted with Laemmli Buffer and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by blotting onto PVDF membranes and detection with specific antibodies. For immuno-precipitation, antibodies were added to the lysates and incubated for 1 hour at 4°C with rotation, followed by addition of Protein A PLUS G beads  to capture the immuno-complexes. The immuno-complexes were washed and then eluted with Laemmli Buffer and resolved by SDS-PAGE, followed by western blotting with specific antibodies.Lysates for binding assays and immuno-precipitation were prepared by lysis of cells in the cell lysis buffer [20 mM HEPES (pH 7.5), 137 mM NaCl, 1% Triton X-100, 10% glycerol, 1.5 mM MgClPeptides for Erk2: LEQYYDPSDEPIAE, p38α: FAQYHDPDEPVAD and Jnk1: INVWYDPSEAEAPP were custom synthesized by NeoSystems . CD experiments were performed with a Jasco J-715 spectropolarimeter equipped with a Peltier cell holder and a PTC-348WI temperature controller. Far-UV CD spectra were measured in a 1-mm rectangular quartz cell. The buffer used was 20 mM Tris-HCl (pH 7.5), 500 mM NaCl and 1 mM DTT. Protein concentration of approximately 0.5 mg/ml determined by the Bradford assay method was used for wavelength scans. Wavelength scans were made at a scan rate of 10 nm/min and the average value of 3 scans of the same solution were obtained. Data were collected at 25°C over a wavelength range 190–260 nm with a bandwidth of 1 nm. To determine the conformational changes in WW domain upon peptide binding, the ellipticity of the corresponding peptides at equimolar concentration with the solvent were subtracted from the ellipticity of the WW domain-peptide complex and similarly the solvent spectrum were subtracted from the spectrum of WW domain before analysis. The far UV CD spectra were analyzed by using the secondary structure analysis program CDNN, version 2.1 .4 per glass bottom dish and grown overnight at 37°C in DMEM with 10% Cosmic calf serum. DNA was injected at 0.5 μg/ml into the nuclei using a custom setup microinjector and Olympus microscope (Olympus IMT-2) and the cells were returned to incubation at 37°C overnight to allow for protein expression. For DIC and fluorescence time-lapse analysis, cells were incubated on a heated stage at 37°C and imaged with a monochromator on a Zeiss Axiovert 200 M microscope enclosed in an incubator with CoolSNAP CCD camera.Swiss 3T3 fibroblasts were plated at 5 × 10The author(s) declare that they have no competing interests.


Dictyostelium discoideum lacks a Rho or Cdc42 ortholog but has several Rac related GTPases. Compared to our understanding of the downstream effects of Racs our understanding of upstream mechanisms that activate Rac GTPases is relatively poor.Rho subfamily GTPases are implicated in a large number of actin-related processes. They shuttle from an inactive GDP-bound form to an active GTP-bound form. This reaction is catalysed by Guanine nucleotide exchange factor (GEFs). GTPase activating proteins (GAPs) help the GTPase return to the inactive GDP-bound form. The social amoeba Guanine exchange factor for Rac GTPases), a Dictyostelium RacGEF. GxcDD is a 180-kDa multidomain protein containing a type 3 CH domain, two IQ motifs, three PH domains, a RhoGEF domain and an ArfGAP domain. Inactivation of the gene results in defective streaming during development under different conditions and a delay in developmental timing. The characterization of single domains revealed that the CH domain of GxcDD functions as a membrane association domain, the RhoGEF domain can physically interact with a subset of Rac GTPases, and the ArfGAP-PH tandem accumulates in cortical regions of the cell and on phagosomes. Our results also suggest that a conformational change may be required for activation of GxcDD, which would be important for its downstream signaling.We report on GxcDD (Dictyostelium, but is also involved in integrating different signals. We provide evidence for a Calponin Homology domain acting as a membrane association domain. GxcDD can bind to several Rac GTPases, but its function as a nucleotide exchange factor needs to be studied further.The data indicate that GxcDD is involved in proper streaming and development. We propose that GxcDD is not only a component of the Rac signaling pathway in  Rho GTPases are small monomeric GTPases of the Ras superfamily. Like any other GTPase Rho GTPases act as binary molecular switches cycling between a GTP-bound active and a GDP-bound inactive form. Guanine nucleotide exchange factors (GEFs) catalyze the activation reaction, and GTPase activating proteins (GAPs) convert the active to an inactive form. Further regulators, guanine nucleotide dissociation inhibitors (GDIs), block spontaneous activation and regulate cycling between membrane and cytosol. When activated, Rho GTPases undergo a conformational change enabling them to interact with their effector molecules and transduce signals for downstream events. Rho GTPases have been implicated in a large number of actin-related processes like motility, adhesion, morphogenesis, membrane trafficking and cytokinesis ,2.The human genome codes for 21 Rho GTPases and functions of most of them are only poorly understood. Of these, three, namely RhoA, Rac1 and Cdc42 are more extensively studied. RhoA generates myosin-based contractility and formation of adhesion complexes; Rac1 and Cdc42 are primarily involved in formation of protrusive structures, Rac1 regulates formation of lamellipodia and Cdc42 regulates filopodia formation and establishment of cell polarity [Dictyostelium discoideum revealed the presence of 18 Rac related GTPases, whereas a typical Rho or Cdc42 were absent  [Sequencing of the genome of the social amoeba  absent  ,4. Only  absent  ,6 and Ra absent  , Rac1A w absent   and RacC absent   and play absent  . RacG is absent   and RacH absent  .Dictyostelium at least 45 proteins contain a RhoGEF-PH module and most of them have a unique domain composition. The RhoGEF-PH (or diffuse B-cell lymphoma homology DH/pleckstrin homology PH) module is the structural feature that mediates the nucleotide exchange activity on Rho GTPases. Five of these RacGEFs have been studied in some detail. DdRacGap1 (DRG) containing both RhoGEF and Rho-GAP domains acts as a GEF for Rac1 and simultaneously acts as a GAP for RacE and Rab GTPases [C. elegans. Dictyostelium has 8 members of this family, but the functions of them remain to be elucidated.Compared to our understanding of the downstream effects of Rac GTPases less is known about the mechanisms that activate Rac GTPases controlled by GEFs, GAPs or GDIs. In  GTPases . RacGEF1 GTPases . The tai GTPases . More re GTPases . Finally GTPases ), has be GTPases . Nucleot GTPases . PresentDictyostelium development. The RacGEF domain of GxcDD can physically interact with several Rac  GTPases. Characterization of the individual domains revealed that the CH domain can act as a membrane anchor and the ArfGAP-PH tandem accumulates at cortical regions and on phagosomes. Our data also suggest that a conformational change is possibly required to activate GxcDD.In this study we focus on GxcDD, a novel multidomain RacGEF that contains a calponin homology (CH) domain, two IQ motifs, a DH domain, three PH domains and an ArfGAP domain. We show that, though dispensable for growth and development, GxcDD is required for proper streaming early in Guanine eXchange factor for raC) is located on chromosome 3. GxcDD is 1619 residues long with a calculated molecular mass of 179.652. It is a multidomain protein containing a CH domain, two IQ motifs, three PH domains, a RhoGEF domain and an ArfGAP domain  domain and this tandem DH-PH module is the signature motif of the Dbl family of guanine nucleotide exchange factors (GEFs). Similarly, in GxcDD, a DH domain responsible for the catalytic RacGEF activity resides between residues 464–637. There are three PH domains in GxcDD, a comparatively large and less defined PH domain between residues 664–910 and two more between residues 941–1038 and 1520–1618. An ArfGAP domain, which shows highest homology to centaurin-α is placed between residues 1258–1376. Centaurins are ArfGAPs with functions in intracellular trafficking and contain a PH domain. Centaurins act downstream of PI 3 kinases and are targets for PtdInsP3 [The gene coding for the RacGEF GxcDD  in E. coli, bound the protein to glutathione-sepharose beads and used them for incubation with lysates derived from Dictyostelium cells expressing different Rac GTPases as GFP fusion proteins. Rac GTPases interacting with GST-DH were identified by immunoblotting with GFP monoclonal antibodies P2, but highest binding was observed with PtdInsP3, the product of PI3K  and mediate the recruitment of proteins to membranes. When we tested whether the PH domain in the ArfGAP-PH domain could bind to PtdIns using a dot blot assay we found that the protein bound to PtdInsPI3K Fig. . The PH-To gain knowledge about the physiological role of GxcDD we disrupted the gxcDD gene by homologous recombination in wild type AX2 cells Fig. . The disgxcDD- cells did not show severely altered phenotypes when examined for growth on a bacterial lawn or in suspension, cytokinesis or actin organization in the cortex (not shown). The enrichment of the ArfGAP-PH domain at macropinosomes  had its peak expression at t8 in wild type and mutant , which induce a multitude of signaling events. Activation of cARs leads to dissociation of the heterotrimeric G-proteins and membrane localization of CRAC (cytosolic regulator of adenylyl cyclase), a PH domain containing protein, followed by activation of adenylyl cyclase (ACA), cAMP production and secretion [gxcDD- cells.To understand the physiological role of GxcDD, we generated ecretion . Activatecretion . The neiecretion . ACA is GxcDD is an unusual RacGEF containing CH domain, two IQ motifs, a RacGEF-PH tandem and an ArfGAP-PH tandem. It is present in cytosolic as well as membranous fractions. The CH domain of GxcDD localizes to the plasma membrane and may recruit GxcDD to the membrane and possibly regulate function and localization of the protein by binding to the ArfGAP-PH tandem. This is to the best of our knowledge the first evidence of membrane targeting of a CH, which would reveal an additional function for this functionally diverse domain. Our studies show that the RacGEF domain has the ability to physically interact with several Rac GTPases. To get a better insight to GxcDD function we need to perform nucleotide exchange assays to find the actual substrates for GxcDD. The ArfGAP-PH domain is associated with the leading edge of the cell and is able to associate with phosphatidyl inositides. It is also present in phagosomes and in Triton X-100 insoluble fractions. Deletion of the gene did not result in major phenotypic changes associated with growth, endocytic processes, actin organization, cytokinesis and cell motility, but shows subtle defects in formation of streams during aggregation and a delay in development.D. discoideum cells of strain AX2 were grown either with Klebsiella aerogenes on SM agar plates or axenically in liquid nutrient medium [gxcDD- cells were cultivated in axenic medium containing 3.5 μg/ml blasticidin . To analyse development, cells were grown axenically to a density of 2–3 × 106/ml, washed twice in Soerensen phosphate buffer  and 5 × 107 cells were plated on phosphate agar plates. The streaming pattern was studied by allowing 1 ml of 5 × 106 cells/ml to settle on a well of a NUNC six-well plate and observed at 3 min intervals with a Leica DM-IL inverse microscope.t medium  in shakiGxcDD gene in AX2 cells, a GxcDD gene replacement vector was constructed using the plasmid pBSBsrΔBam [For disruption of the SBsrΔBam . A 1.1-kE. coli. The C-terminal 1-kb fragment encoding residues 1269–1619 containing the ArfGAP-PH tandem was amplified and cloned into pBsr-GFP for expression in AX2 cells and into pGEX-4T3 for expression in E. coli. Generation of strains expressing RacF1, RacG, RacH fused to GFP has been described elsewhere [For expression of the CH domain of GxcDD fused to green fluorescent protein (GFP) at the N-terminus, a 0.4-kb fragment encoding the first 134 residues of GxcDD was amplified from AX2 cDNA and cloned into pBsr-GFP ,38. A 1-lsewhere . For explsewhere  and the Polyclonal antibodies specific for GxcDD were obtained by immunising female white New Zealand rabbits with GST-ArfGAP-PH , followed by two boosts of 100 μg each at two weeks intervals. The antiserum was affinity purified by incubating with GST-ArfGAP-PH bound glutathione-sepharose beads.8 cells/ml in MES buffer ). Cells were lysed by sonication and membrane and cytosolic fractions separated by centrifugation at 100,000 g for 30 min at 4°C.For separating membrane and cytosolic fractions cells were washed in Soerensen buffer and resuspended at a density of 1 × 107 cells/ml and 300 μl of cell suspension were lysed using an equal volume of TIC buffer  and incubated on ice for 10 min followed by incubation at RT for 10 min. The insoluble cytoskeleton fraction was separated by centrifugation at 10,000 g for 4 min. Supernatant and pellet fractions were subjected to western blot analysis.Triton X-100 was used for preparing cytoskeletal fractions. Cells were washed as before and resuspended in Soerensen buffer at a density of 5 × 10E. coli and bound to glutathione-sepharose beads. For interaction of GxcDD with Rac proteins, 4 × 107 AX2 cells expressing Dictyostelium Racs as GFP fusions were lysed in lysis buffer ) and incubated with equal amounts of GST-RacGEF bound beads for 3 hrs at 4°C. Beads were washed with wash buffer . The eluate of the pulldown was immunoblotted and the Rac protein detected using a GFP specific monoclonal antibody [GST-RacGEF and GST-ArfGAP-PH were expressed in antibody . Cells eImmunoflorescence was done by fixing cells using cold methanol for 10 min followed by staining for actin using actin-specific monoclonal antibody Act1-7  and Cy3 Monoclonal antibodies recognizing α-actinin , contact7 cells/ml were stimulated with 1 μM cAMP and 50 μl samples were taken at various time points. Samples were immediately lysed in lysis buffer  and stained for 1 hr and centrifuged at 10,000 g for 5 min. Pellets were extracted with 1 ml methanol overnight and fluorescence (540/565) measured in a fluorimeter.F-actin levels upon cAMP stimulation were determined as described . BrieflyFor crosslinking experiments GST-ArfGAP-PH was thrombin cleaved on glutathione-sepharose beads and the purified protein subjected to dialysis against PBS, pH 7.4, for 6 hrs at 4°C. To equal amount of protein increasing amount of glutaraldehyde (0–0.1% v/v) was added. The final reaction volume was 40 μl. Crosslinking was carried out at 4°C for 30 min. The reaction was stopped by addition of 5 μl 1 M glycine. Samples were subjected to imunoblotting.2, PtdInsP2, PtdInsP3) were spotted on a PVDF membrane and incubated with ArfGAP-PH domain. Protein bound to lipids was detected using polyclonal GxcDD antibodies.Phosphoinositides binding was performed by a dot blot assay. 200 pmoles of each phophoinositide PgxcDD- and AX2 cells were allowed to starve in Soerensen buffer at a density of 1 × 107 cells/ml and samples were withdrawn at the indicated times. The percentage of aggregated cells was determined by measuring the OD600.For cell aggregation in suspension For northern blot analysis total RNA was isolated from AX2 cells of different developmental stages as described previously and separated in agarose gels containing 6% formaldehyde ,50. The SM and DN were involved in carrying out all the experiments. SM drafted the manuscript. FR and AAN supervised the work and revised the manuscript.


In this study, we examined thepossibility that the interaction of olopatadine with S100A12inhibits the proinflammatory effects of S100A12. Pretreatment ofolopatadine with S100A12 reduced migration of THP-1, a monocytecell line, induced by S100A12 alone, but did not affectrecombinant human regulated upon activation, normal T cellexpressed and secreted (RANTES)-induced migration. Amlexanox,which also binds to S100A12, inhibited the THP-1 migration inducedby S100A12. However, ketotifen, another histamine 1Hreceptor antagonist, had little effect on the activity of S100A12.These results suggest that olopatadine has a new mechanism ofaction, that is, suppression of the function of S100A12, inaddition to histamine 1H receptor antagonistic activity.Olopatadine hydrochloride (olopatadine) is an antiallergic drugwith histamine  Olopataultiform . Olopataultiform . Olopataultiform , 18. HowRecently, Okada et al have found that olopatadinebinds specifically to S100A12 and S100A13 in a calcium-dependentmanner as amlexanox and cromolyn do . In the ® tablets purchased from Takeda ChemicalIndustries Inc . These drugs were dissolved in DMSO.Recombinant human regulated upon activation, normal T cellexpressed and secreted (RANTES) was purchased from R&D SYSTEMS.Olopatadine hydrochloride (olopatadine) wassynthesized in Yokkaichi Plant, Kyowa Yuka Co, Ltd .Ketotifen fumarate (ketotifen) was purchased from Sigma Chemical. Amlexanox was extracted fromSOLFAHCl (pH 7.5) containing 1 mmol/Lethylenediaminetetraacetic acid, and then centrifuged at10 000 g for 60 min. The supernatant was dialyzed against20 mmol/L Tris-HCl (pH 7.5) (buffer A), and loadedonto a HiPrep 16/10 Q XL column  equilibrated with buffer A. The protein was eluted with alinear gradient from 0 to 250 mmol/L NaCl. Fractionscontaining the protein were pooled and loaded onto a HiLoad 16/60Superdex 75 pg (Amersham Biosciences) equilibrated withbuffer A. Fractions containing the protein were pooled and furtherpurified using a Mono Q HR 5/5 column (Amersham Biosciences)equilibrated with buffer A. The protein was eluted with a lineargradient from 0 to 200 mmol/L NaCl, and stored at−80°C. Purity was confirmed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. In S100- A12preparations used for this study, endotoxin levels were less than0.1 pg/μg of S100A12 as measured by the limulusamebocyte assay .Human S100A12 cDNA was a gift from Dr Ryoji Kobayashi. Recombinant protein wasexpressed in E coli strain BL21 (DE3) and purified as previouslydescribed by Yamashita et al , with so2CO in air.Human monocytic THP-1 cells  were cultured in RPMI 1640supplemented with 10 vol% heat-inactivated fetal bovine serum(FBS) , 100 U/mL penicillinand 100 μg/mL streptomycin  at 37°C 5% 5 cells) was added to the upperchamber and incubated for 3 h at 37°C in 5%2CO. Cells passing through the membrane were collectedfrom the lower well and counted with the EPICS XL/MCL flowcytometry system  followedby mixing of a predetermined number of flow-count fluorospheres(Beckman Coulter). The total number of cells that had migrated tothe lower wells was determined by the ratio of cells tofluorospheres using the following formula: cells per microliter =[(cells counted)/(fluorospheres counted)] × fluorospheres/microliter. Each sample was assayed in triplicate.The migration of THP-1 monocytes was studied using aChemotaxicell 24-well disposable chamber with 5 μm pores. Cultured cells wereharvested and washed three times with Hank's balanced saltsolution and then re-suspended in assay medium piperazine-N'-(4-butanesulfonic acid), pH7.4). Each drug and stimulant were premixed in the assay mediumand incubated at 37°C for 30 min. Then 0.05 mL ofthe mixture was placed into the lower chamber contained0.45 mL/well of the assay medium. Then 0.2 mL of cellsuspension .Data were presented as means ± SE. The Student The migration of THP-1 monocytes was studied in thepresence of S100A12 or RANTES. S100A12 induced the migration ofTHP-1 cells at concentrations between 0.75 and 2 μmol/L(data not shown). Therefore, we examined the effect of olopatadineon the activity of S100A12 at 1 μmol/L, a submaximalconcentration. After pretreatment of S100A12 for 30 min, 26095± 1044 cells/well migrated to the bottom chamber at 1 μmol/L of S100A12,indicating a 3.4-fold increase in comparisonwith the control value of 7699 ± 693 cells/well. On the Amlexanox is also reported to bind to S100A12, 14. Acc1H receptorantagonistic activity of olopatadine is related inhibition of theactivity of S100A12, ketotifen, another histamine 1Hreceptor antagonist, was tested. 1Hreceptor antagonistic activity than olopatadine [1Hreceptor antagonistic activity.Next, to determine whether histamine patadine ,22.TheS100A12 has been identified as a binding protein to antiallergicdrugs, such as olopatadine, amlexanox, and cromolyn, usingdrug-affinity columns . S100A12Previous studies showed that S100A12 induced chemotactic responsesat much lower concentrations (0.01–1 nmol/L), 10, whe1H receptorantagonistic activity provide high degree of efficacy in thetreatment of allergic diseases.Ketotifen in contrast to olopatadine did not suppress the THP-1migration induced by S100A12, suggesting that olopatadine may havethe distinctive feature of direct inhibition of the function ofS100A12. This new mechanism of action of olopatadine may inaddition to its potent histamine


Coronavirus contains about 25 species of coronaviruses (CoVs), which are important pathogens causing highly prevalent diseases and often severe or fatal in humans and animals. No licensed specific drugs are available to prevent their infection. Different host receptors for cellular entry, poorly conserved structural proteins (antigens), and the high mutation and recombination rates of CoVs pose a significant problem in the development of wide-spectrum anti-CoV drugs and vaccines. CoV main proteases (Mpros), which are key enzymes in viral gene expression and replication, were revealed to share a highly conservative substrate-recognition pocket by comparison of four crystal structures and a homology model representing all three genetic clusters of the genus Coronavirus. This conclusion was further supported by enzyme activity assays. Mechanism-based irreversible inhibitors were designed, based on this conserved structural region, and a uniform inhibition mechanism was elucidated from the structures of Mpro-inhibitor complexes from severe acute respiratory syndrome-CoV and porcine transmissible gastroenteritis virus. A structure-assisted optimization program has yielded compounds with fast in vitro inactivation of multiple CoV Mpros, potent antiviral activity, and extremely low cellular toxicity in cell-based assays. Further modification could rapidly lead to the discovery of a single agent with clinical potential against existing and possible future emerging CoV-related diseases.The genus  pros may rapidly lead to an antiviral agent against Coronavirus-associated diseases.Structure-assisted optimization of compounds capable of inactivating CoV M Coronavirus belongs to the plus-strand RNA virus family of the Coronaviridae and currently contains about 25 species that are classified into three groups according to their genetic and serological relationships  was utilized to evaluate their inhibition as an approximation of the pseudo second-order rate constant (k3/iK) if very rapid inactivation occurs. The iK of N1 ranges from approximately 1.11–10.7 μM and k3 ranges from approximately 4.1–50 × 10−3s−1; the iK of N9 ranges from approximately 0.9–6.7 μM, and k3 ranges from approximately 2.6–19.5 × 10−3s−1. Compared with N1 and N9, N3 demonstrated more potent inhibition on TGEV, FIPV, MHV, and IBV Mpros with obsk/[I] ranging from approximately 4,700–47,000 M−1•s−1. We therefore solved the crystal structure of SARS-CoV and TGEV Mpros individually complexed with N1, which revealed a common mechanism of inhibition among CoV Mpros.Based on the complex structure of I2, we entered into a second round of optimization focusing on the P2 and P4 recognition sites. For the P2 subsite, the phenyl group was substituted by a more flexible Leu side chain. In order to enhance the binding affinity, a series of residues were utilized as substituents at P4, followed by a heterocycle that should increase the Van der Waals contacts with residues flanking at either side. From this round of modification, two inhibitors designated as N1 and N9, and a more efficacious derivative named N3, were identified with fast in vitro inactivation of all CoV Msays see . These iN1 binds to protomers A and B of SARS-CoV in an identical and normal manner. On binding N1, the S1 subsite in protomer B adopts an active conformation compared with the partially collapsed S1 pocket of protomer B in the native structure , which cpro complexed with N1 and the native enzyme show that certain residues constituting the S1 and S2 subsites undergo large conformational changes on inhibitor binding  toward the interior of the protein compared with the native enzyme. The Michael acceptor remains in a plane following the Michael addition since it is stabilized by a water molecule. This ordered water molecule donates a long 3.3-Å hydrogen bond to the carboxylate oxygen of the ester and then accepts a 2.8-Å hydrogen bond from the backbone NH of 143-Gly and a 3.0-Å hydrogen bond from the carboxamide nitrogen of 142-Asn. The position of Sγ in 145-Cys implies that it undergoes nucleophilic attack on Cβ by approaching the π-electron cloud from above. The carbonyl oxygen occupies the oxyanion hole and is close to backbone NHs of 143-Cys and 145-Cys, mimicking the tetrahedral oxyanion intermediate formed during Ser protease cleavage. However, the standard hydrogen bonds are not formed. The ethyl ester portion extends into the S1′ site, with sufficient size, and in an extended conformation, to interact with the alkyl portions of 25-Thr and 27-Leu by van der Waals interaction.Clear electron density showed that the Sα of Leu at the P2 site in N1 moves into the S2 subsite by approximately 1 Å relative to the corresponding carbon atom in I2, and Cβ–Cγ of Leu forms an angle of approximately 40° to the phenyl at P2 in the I2 complex, inserting deeply into the S2 subsite. Another notable difference between N1 and I2 is the insertion of an Ala between P3 and P4 in I2, the latter of which was replaced by an isoxazole to block the N-terminal. As expected, the side chain of Ala at the current P4 position readily enters into the S4 subsite. Simultaneously, the backbone NH of Ala donates a hydrogen bond to the carbonyl oxygen of 190-Thr. The isoxazole at P5 makes Van der Waals contacts with 168-Pro and the backbone of residues 190–191.The lactam at P1 inserts favorably into the S1 subsite and forms two stable 2.6-Å hydrogen bonds: one between the lactam oxygen and NE2 of 163-His, and another between the lactam NH and a water molecule at the bottom of this subsite. The Cpro complexed with N9 . The rate of chemical inactivation presumably depends on how the reactive vinyl group is oriented and on the extent to which the transition-state intermediate can be stabilized by proteases  was used as an approximation of the pseudo second-order rate constant to evaluate the inhibitors and was measured at approximately 2–4 different inhibitor concentrations. The error associated with this determination (obsk/[I]) is less than 20% of a given value.In the experiment, the 2.Murine DBT cells (generously provided by Dr. Lishan Su of University of North Carolina) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37 °C in 5% CODBT cells were suspended in growth medium in triplicate wells in 6-well plates and preincubated with appropriate concentrations of the inhibitor. The next day, the medium was aspirated, and MHV-A59 was added to each well at a titer of 100 PFU/well. After incubation for 1 h, the virus inoculum was aspirated, and 2 ml of a media-agar overlay with appropriate concentrations of inhibitor was added to each well. The plates were further incubated for 24 h and stained with neutral red to visualize plaques.DBT cells were suspended in growth medium in 96-well plates. The next day, appropriate concentrations of the inhibitor were added to the medium. Two days later, the relative numbers of surviving cells were measured by MTT  assay in accordance with the manufacturer's instructions.Felis catus whole fetus (macrophage) cells , and DBT cells were cultured in minimal essential medium (MEM) supplemented with 25 mM HEPES, Glutamax I, nonessential amino acids, 10% FBS, and antibiotics at 37 °C in 5% CO2. Nearly confluent monolayers of MRC-5 (incubated at 33 °C following infection), FCWF, and DBT cells, which were grown in 6-well plates, were infected with HCoV-229E, FIPV (strain 79–1146), and MHV-A59, respectively, at a multiplicity of infection of 3 TCID50 per cell. After 60 min of virus adsorption, the virus inoculum was replaced with cell culture medium containing varying concentrations of N3 or in the absence of inhibitor. At 14 h postinfection, the virus titers in the cell culture supernatants were determined using standard procedures. All experiments were performed in triplicate and mean values were determined.Human embryonic lung fibroblast cells , Figure S1The N9 inhibitor is shown in gold and covered by an omit map contoured at 1.0 σ. Residues forming the substrate-binding pocket are shown in silver. Two water molecules (in red) form hydrogen bonds with N9.(425 KB PDF).Click here for additional data file.Figure S2pro solution with 10 μM substrate.Activity profile curves were displayed at two different inhibitor concentrations for (A–F). (A) 0.1 μM HCoV 229E Mpro solution with 20 μM substrate.(B) 0.1 μM TGEV Mpro solution with 10 μM substrate.(C) 0.05 μM FIPV Mpro solution with 20 μM substrate.(D) 0.6 μM MHV Mpro solution with 20 μM substrate.(E) 0.8 μM IBV Mpro solution with 20 μM substrate.(F) 1 μM SARS-CoV Mpro of a newly identified CoV (HCoV-HKU1). Curve A represents the activity curve of 1 μM Mpro of HCoV-HKU1 in cleaving 20 μM substrate with time; curves B and C individually represent the decrease in enzyme activity when N3 was added with 2-fold and 4-fold molar of protease.(G) The preliminary inhibitory assay of N3 on Mpro of a recently identified CoV (HCoV-NL63). Curve A represents the activity curve of 0.5 μM Mpro of HCoV-NL63 in cleaving 10 μM substrate with time; curves B and C individually represent the decrease in enzyme activity when N3 was added with 2-fold and 4-fold molar of protease.(H) The preliminary inhibitory assay of N3 on M(1.2 MB PDF).Click here for additional data file.Figure S3(124 KB PDF).Click here for additional data file.Table S1(106 KB PDF).Click here for additional data file.Table S2(128 KB PDF).Click here for additional data file.Table S3(121 KB PDF).Click here for additional data file.Protocol S1(137 KB PDF).Click here for additional data file.http://www.rcsb.org/pdb/) accession numbers for the structures of SARS Mpro individually complexed with I2, N1, N3, and, N9, and TEGV Mpro in complex with N1 are 1WNQ, 1WOF, 2AMQ, 2AMD, and 2AMP, respectively. The GenBank (http://www.ncbi.nih.gov/Genbank/) accession number for IBV Mpro is DQ157446.The Protien Data Bank (


III) and methylarsonous acid (MAsIII) inhibit insulin-stimulated glucose uptake (ISGU) in 3T3-L1 adipocytes by suppressing the insulin-dependent phosphorylation of protein kinase B (PKB/Akt).Increased prevalences of diabetes mellitus have been reported among individuals chronically exposed to inorganic arsenic (iAs). However, the mechanisms underlying the diabetogenic effects of iAs have not been characterized. We have previously shown that trivalent metabolites of iAs, arsenite , which synthesizes phosphatidylinositol-3,4,5-triphosphate (PIP3), or on phosphorylation of PTEN (phosphatase and tensin homolog deleted on chromosome ten), a PIP3 phosphatase. Neither iAsIII nor MAsIII interfered with the phosphorylation of 3-phosphoinositide-dependent kinase-1 (PDK-1) located downstream from PI-3K. However, PDK-1 activity was inhibited by both iAsIII and MAsIII. Consistent with these findings, PDK-1-catalyzed phosphorylation of PKB/Akt(Thr308) and PKB/Akt activity were suppressed in exposed cells. In addition, PKB/Akt(Ser473) phosphorylation, which is catalyzed by a putative PDK-2, was also suppressed. Notably, expression of constitutively active PKB/Akt restored the normal ISGU pattern in adipocytes treated with either iAsIII or MAsIII.Subtoxic concentrations of iAsIII or MAsIII, and possibly for impaired glucose tolerance associated with human exposures to iAs.These results suggest that inhibition of the PDK-1/PKB/Akt-mediated transduction step is the key mechanism for the inhibition of ISGU in adipocytes exposed to iAs Arsenic (As) is a naturally occurring toxic metalloid and a potent human carcinogen . The can2) at the plasma membrane to phosphatidylinositol-3,4,5-triphosphate (PIP3) (3 facilitates 3-phosphoinositide-dependent kinase-1/2 (PDK-1/2) dependent phosphorylation/activation of protein kinase B (PKB/Akt) and two atypical enzymes of the protein kinase C (PKC) family, PKCλ and ζ  upon binding of insulin to the α-subunit of the receptor (IRα), the subsequent tyrosine phosphorylation of insulin receptor substrate 1 or 2 (IRS-1 or -2), and the binding of a phosphorylated IRS (p-IRS) to the regulatory (p85) subunit of the class IA phosphatidylinositol 3-kinase (PI-3K) that leads to the activation of its catalytic (p110) subunit. The activated PI-3K catalyzes the phosphorylation of phosphatidylinositol-4,5-bisphosphate (PIPe (PIP3) . PIP3 faCλ and ζ . The phoCλ and ζ . In addiCλ and ζ . The mecCλ and ζ  and actiCλ and ζ  contribuCλ and ζ  and to iCλ and ζ .in vitro studies have consistently shown significant increases in basal (insulin-independent) glucose uptake by various types of cells or dissected tissues exposed to cytotoxic concentrations of a trivalent iAs, arsenite (iAsIII), or an aromatic derivative of AsIII, phenylarsine oxide (PAO) , in drinking water , and dimethylarsinous acid (DMAsIII)  . Consistng water , 2005. Ong water , 1995 anng water . PAO didng water . The effng water . We haveDMAsIII) , inhibitIII and MAsIII, focusing mainly on the components of the insulin-activated signal transduction pathway that regulate PKB/Akt phosphorylation in adipocytes. Results of this work show that iAsIII and MAsIII inhibit PDK-1 activity, thus suppressing PDK-1–catalyzed phosphorylation of PKB/Akt and p-PKB/Akt–mediated translocation of GLUT4 transporters to the plasma membrane. Notably, MAsIII was an order of magnitude more potent than iAsIII as an inhibitor of the PDK-1/PKB/Akt signal transduction step and of glucose uptake in insulin-stimulated adipocytes. Thus, the formation of MAsIII in the methylation pathway for iAs may play a critical role in determining the extent of the diabetogenic effects associated with chronic exposures to iAs.In the present study we examined the molecular mechanisms of ISGU inhibition by iAs–3T3-L1 preadipocytes expressing constitutively active PKB/Akt lacking the pleckstrin homology (PH) domain were provided by S. Summers . Addition of the src myristoylation sequence promotes constitutive membrane association and activation of PKB/Akt  with high glucose, 10% fetal bovine serum , penicillin, and streptomycin . Cells were cultured at 37°C in a humidified incubator in a 90% air and 10% CO2 atmosphere. To induce differentiation, postconfluent cells were treated with a mixture of 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1 μg/mL insulin  for 48 hr and cultured in insulin-containing medium for an additional 48 hr  or methylarsine oxide . Identity and purity of methylarsine oxide was confirmed by 1H-NMR and mass spectrometry. In aqueous solutions, methylarsine oxide is hydrolyzed to form MAsIII  were prepared before each experiment to minimize the oxidation of iAsIII to iAsV or MAsIII to methylarsonic acid (MAsV). Adipocytes were incubated with arsenicals or vehicle in a cell culture incubator for 4 hr.We obtained 3T3-L1 preadipocytes from Y. Patel . Myr-PKB/Akt PKB/Akt , 1996b. al 48 hr . All expm MAsIII . Fresh s14C]-deoxy-d-glucose (0.1 μCi/well) . To measure basal (insulin-independent) glucose uptake, we incubated cells with radiolabeled glucose without pretreatment with insulin. After the incubation, cells were washed twice with PBS (0°C), and lysed in a solution of 0.5 N NaOH and 10% SDS. Radioactivity in cell lysates was measured, using a Wallac 1409 liquid scintillation counter .The glucose uptake assay followed the previously described procedures . Brieflyl-lysine  and treated with arsenicals. Cells were then fixed in 4% buffered-paraformaldehyde and permeabilized in a solution of 0.1% Triton X-100 and 0.1% sodium citrate . DNA strand breaks were enzymatically labeled on 3′-OH termini with fluorescein-linked nucleotides, using the In Situ Cell Death Detection Kit . Nuclei of both normal and apoptotic cells were stained with 100 nM 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) . Labeled cells were visualized using a Nikon Microphot FXA fluorescent microscope .To determine cell viability, we used the MTT assay, which measures the conversion of 3--2,5-diphenyltetrazolium bromide (MTT) to purple formazan by mitochondrial dehydrogenases of viable cells , as prevd-glucose . After fixation with 4% buffered-paraformaldehyde, adipocytes were rinsed with ice-cold PBS and incubated with poly-l-lysine (0.5 mg/mL) for 1 min. Cells were then treated with a hypotonic buffer  and pulse sonicated for 5 sec in a sonication buffer , using a Fisher Model 100 Sonic Dismembrator equipped with a 12.7 × 1.3 × 0.3-cm probe . Plasma membrane sheets attached to the coverslip were washed twice with the sonication buffer, incubated with an anti-GLUT4 antibody  and labeled with Alexa-fluor 594 (Molecular Probes). Fluorescent images were captured, using a Zeiss LSM 110 fluorescent microscope .Adipocyte cultures on glass cover slips were treated with arsenicals, activated with insulin, and incubated with III or MAsIII using hydride generation atomic absorption spectrometry (HG-AAS) as previously described , total methylarsenic (MAs = MAsIII + MAsV), and total dimethylarsenic (DMAs = DMAsIII + DMAsV) species were determined. We confirmed the identities of arsenicals in spectral peaks using aliquots of samples spiked with standards. Calibration curves for each of the arsenicals  were generated to quantify results of the analyses.We analyzed arsenicals in cell cultures exposed to iAsescribed . Cells ad-glucose, using a 25-mM HEPES (pH 7.4) lysis buffer containing 1% NP 40, 100 mM NaCl, 2% glycerol, 5 mM sodium fluoride, 1 mM EDTA, 1 mM sodium orthovanadate (Na3VO4), 1 mM sodium pyrophosphate, 1 mM PMSF, 40 μg/mL aprotinin, 20 μg/mL leupeptin, and 20 μg/mL pepstatin . Protein extracts were separated by 10% SDS-PAGE, electroblotted to Immobilon-P membranes , and probed using the following antibodies: anti-p38 MAPK (mitogen-activated protein kinase), anti-p-p38 MAPK, anti-PKB/Akt, anti-p-PKB/Akt(Ser473) and anti-p-PKB/Akt(Thr308), anti-PTEN (phosphatase and tensin homolog deleted on chromosome ten), anti-p-PTEN(Ser380), anti-p-PDK-1(Ser241) ; anti-PI-3K(p85) ; and anti-β-actin . The antigen-antibody complexes on immunoblots were treated with horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized using autoradiography or the Gene Gnome imaging system .Protein extracts were prepared from cells treated with arsenicals, activated with insulin, and incubated with We measured PI-3K, PKB/Akt, and PDK-1 activities in cell lysates from adipocytes treated with arsenicals and activated with insulin after immunoprecipitation with specific antibodies bound to protein G agarose beads (Santa Cruz Biotechnology). For a single assay we used the immunoprecipitate from cells cultured in one 10-cm plate. The assay conditions were as follows:2, 200 μM adenosine, 40 μM adenosine 5′-triphosphate (ATP) , 20 μCi [γ-32P]-ATP , and l-α-phosphatidylinositol (PI)  as a substrate (2). To simplify the analysis, a 10-min incubation that yielded only PIP was used throughout this study. Radiolabeled phospholipids were extracted in chloroform:methanol (CHCl3:CH3OH) (1:1). The organic phase was washed with CH3OH:HCl (1:1), evaporated under nitrogen, and the residue was dissolved in CHCl3:CH3OH (2:1). The extract was separated by thin-layer chromatography (TLC) on glass silica plates pretreated with 1% potassium oxalate, using an n-propanol:2 N acetic acid (65:35) solvent system . Standards were visualized on developed TLC plates by treatment with 50% sulfuric acid at 100°C for 1 hr.PI-3K was immunoprecipitated from control (untreated) adipocytes or from adipocytes exposed to arsenicals with an anti-phosphotyrosine (PY20) antibody. PI-3K immunoprecipitated from insulin-activated adipocytes pretreated with 1 nM wortmannin (a specific inhibitor of PI-3K) was used as a negative control. The enzyme activity was measured in a 50-μL assay mixture containing the immunoprecipitated PI-3K, 20 mM HEPES (pH 7.4), 50 mM MgClubstrate . The reat system . The dis3VO4, 1 mM dithiothreitol, 10 mM protein kinase A (PKA) inhibitor peptide (Upstate Biotechnology), 19 mM MgCl2, 125 μM ATP, 5 μCi [γ-32P]-ATP, and Akt/SGK peptide as a substrate. Incubation was carried out at 30°C for 10 min with continuous shaking. The radiolabeled peptide was blotted on P81 phosphocellulose and quantified by liquid scintillation.We measured PKB/Akt activity using an Akt1/PKBα Immunoprecipitation-Kinase Assay Kit (Upstate Biotechnology), following the manufacturer’s protocol. PKB/Akt was immunoprecipitated with an antibody raised against the pleckstrin homology domain of PKB/Akt. The assay mixture contained 20 mM morpholine-propanesulfonic acid (pH 7.2), 25 mM β-glycerophosphate, 5 mM EGTA, 1 mM Na2, 100 μM ATP and 5 μCi [γ-32P]-ATP. The assay was performed at 30°C in two incubation steps. In the first 30-min step, PDK-1 phosphorylates (activates) recombinant serum and glucocorticoid-induced protein kinase-1 (SGK1). In the second 10-min step, the activated SGK1 phosphorylates a synthetic peptide (RPRAATF) using [γ-32P]-ATP as a phosphate donor. The radiolabeled peptide is blotted on P81 phosphocellulose and quantified by liquid scintillation.PDK-1 activity was measured using a PDK-1 Kinase Assay Kit (Upstate Biotechnology). PDK-1 was immunoprecipitated with an anti-p-PDK-1(Ser241) antibody . The assay mixture contained 50 mM Tris–HCl (pH 7.5), 0.1 mM EGTA, 0.1 mM EDTA, 1 mM Tris(2-carboxyethyl) phosphine, 25 μM PKA peptide inhibitor (Upstate Biotechnology), 1 μM microcystin-LR, 10 mM magnesium acetate, 15 mM MgClp < 0.05 were considered statistically significant.All experiments were replicated to ensure the reproducibility of results. Representative findings are shown. Results of the cell viability, glucose uptake, and protein kinase activity assays were evaluated by analysis of variance with Tukey multiple comparison posttest using a GraphPad Instat statistical software package . Differences among means with III or MAsIII at a wide range of concentrations. Consistent with our previous report . The estimated IC50 (concentration that results in the inhibition of ISGU by 50%) values for the inhibition of ISGU were 25 μM for iAsIII and 4 μM for MAsIII. In comparison, the LC50 (concentration that results in a decrease of cell viability by 50%) values characterizing the cytotoxic effects were 11 mM for iAsIII and 15 μM for MAsIII. Thus, the inhibition of ISGU by iAsIII and MAsIII at or below IC50 values was not due to impaired adipocyte viability. However, both iAsIII and MAsIII can induce cell apoptosis , a cell-permeable caspase-3 inhibitor, prevented caspase-3 activation by both arsenicals and by hydrogen peroxide. However, pre-treatment with AC-DEVD-CHO did not prevent the decrease in ISGU in cells treated with either iAsIII or MAsIII . Therefore, the inhibition of ISGU is not associated with stress and is likely due to specific effects of these arsenicals on mediators of insulin signaling or on the cellular components involved in glucose transport. Based on these results, 4-hr exposures to 50 μM iAsIII and 2 μM MAsIII were used in further experiments to examine the effects of iAsIII or MAsIII on components of the insulin-activated signal transduction pathway in 3T3-L1 adipocytes.Our previous work has shown that trivalent arsenicals inhibit ISGU by 3T3-L1 adipocytes. However, the possible association between the inhibition of ISGU and a general loss of cell functions due to the cytotoxicity of arsenicals has not been thoroughly examined. In this study we examined ISGU and cell viability in adipocytes exposed for 4 hr to iAss report , stimulaM MAsIII . In contpoptosis . At earlpoptosis . Adipocyr MAsIII , suggestM MAsIII . AdipocyIII or MAsIII on mediators of insulin signaling would ultimately depend on the intracellular concentrations and metabolic conversion of these arsenicals. We examined the distribution of As species in adipocytes after a 4-hr exposure to iAsIII or MAsIII, using HG-AAS. Cells exposed to 50 μM iAsIII retained about 3 times more As than cells exposed to 2 μM MAsIII  cells that were or were not stimulated with insulin , immunoprecipitated with an anti-phosphotyrosine (PY20) antibody, which reacts with phosphorylated tyrosine residues of IRS in the insulin-activated PI-3K complex . Effects of MAsIII on PI-3K activity were further analyzed in an in vitro assay mixture containing PI-3K immunoprecipitated from control insulin-stimulated adipocytes. Addition of MAsIII into this mixture at concentrations up to 50 μM did not inhibit PI-3K activity (data not shown). PTEN, a PIP3 phosphatase, is involved in the regulation of PIP3 levels in adipocytes. PTEN activity is regulated by a casein kinase 2–catalyzed phosphorylation on its C-terminal non-catalytic regulatory domain, which includes Ser380   . No chanr MAsIII .III or 2 μM MAsIII had no significant effects on the level of Ser241-phosphorylated PDK-1 in insulin-stimulated adipocytes. However, PDK-1 activity was significantly lower in cells exposed to either iAsIII or MAsIII, 47% and 57% of that in control cells, respectively  phosphorylation, which is essential for PDK-1 activity. These results suggest that iAsIII and MAsIII inhibit PDK-1 activity through direct interactions with the enzyme. Sulfhydryl groups of vicinal or closely spaced cysteines are typical high-affinity targets for trivalent arsenicals in protein structures  appear to be compatible with this type of exposure. Thus, taken together, this work provides a mechanistic basis for the diabetogenic effects of chronic environmental and occupational exposures to iAs.In summary, subtoxic concentrations of iAs PKB/Akt . The inh


We compared 10 healthy controls with 12 patients with SSc, first after interruption of treatment with DTCCA and second after 2 weeks of treatment with nifedipine (60 mg/day). O2•- production by monocytes stimulated with phorbol myristate acetate (PMA) was quantified by the cytochrome c reduction method. We also investigated the effects in vitro of DTCCA on O2•- production and protein phosphorylation in healthy monocytes and on protein kinase C (PKC) activity using recombinant PKC. After DTCCA had been washed out, monocytes from patients with SSc produced more O2•- than those from controls. Nifedipine treatment considerably decreased O2•- production by PMA-stimulated monocytes. Treatment of healthy monocytes with nifedipine in vitro inhibited PMA-induced O2•- production and protein phosphorylation in a dose-dependent manner. Finally, nifedipine strongly inhibited the activity of recombinant PKC in vitro. Thus, the oxidative stress damage observed in SSc is consistent with O2•- overproduction by primed monocytes. This was decreased by nifedipine treatment both in vivo and in vitro. This beneficial property of nifedipine seems to be mediated by its cellular action and by the inhibition of PKC activity. This supports the hypothesis that this drug could be useful for the treatment of diseases associated with oxidative stress.We have reported previously that dihydropyridine-type calcium-channel antagonists (DTCCA) such as nifedipine decrease plasma markers of oxidative stress damage in systemic sclerosis (SSc). To clarify the cellular basis of these beneficial effects, we investigated the effects  Systemic sclerosis (SSc) is a connective tissue disease, characterised by vascular involvement with generalised microangiopathy culminating in systemic fibrosis. Several lines of evidence suggest that the generation of oxygen free radicals is of major importance in the pathogenesis of SSc . Frequen2•-) than those from healthy subjects and patients with primary Raynaud's phenomenon [Histological studies of cutaneous SSc lesions have revealed early mononuclear cell infiltration of perivascular spaces around small vessels ,4, and menomenon .2•- production ex vivo by monocytes from patients with SSc after a wash-out and during nifedipine treatment. We also investigated the effects of nifedipine in vitro on O2•- release from human monocytes, on protein phosphorylation with phorbol myristate acetate (PMA) as stimulator and on protein kinase C (PKC) activity.Dihydropyridine-type calcium-channel antagonists (DTCCA) are an essential treatment in SSc because they decrease vasospastic propensity. These drugs are suspected to have anti-oxidant properties in other diseases ,9. We reReagents, except when specified, were provided by Sigma .Twelve non-smoking patients with SSc were included (three men and nine women); their mean age was 56 ±10 SD) years and the mean disease duration was 8 ± 5 years. Each of the patients had been hospitalised for systematic follow-up of the disease. SSc was classified as limited cutaneous or diffuse cutaneous according to the criteria of LeRoy and colleagues . The cli0 SD yearThe initial evaluation included biological tests performed on the morning of admission, after 1 hour of rest at room temperature; patients stopped taking calcium-channel blockers 3 days before admission . This wag for 25 min at room temperature to separate mononuclear cells from red blood cells and polymorphonuclear neutrophils. Mononuclear cells collected at the interface were washed twice in RPMI 1640 solution and separated into lymphocytes and monocytes by gradient centrifugation with 47% Percoll  at 150 g for 25 min. The purity of the monocyte population (more than 90%) was assessed by May–Grunwald–Giemsa staining. Cells were diluted in RPMI and used immediately.Blood samples (20 ml) were collected on EDTA. Whole blood was diluted 1:2 in RPMI 1640 solution , layered on 15 ml of Ficoll-Hypaque  and centrifuged at 150 2•- was quantified by the cytochrome c reduction assay at 550 nm [® spectrophotometer equipped with a thermostat-controlled cuvette holder and a magnetic stirrer. In brief, 840 µl of monocytes (2 × 106 cells) were incubated at 37°C with 80 µM cytochome c in 1 ml of PBS with stirring before stimulation with 10 µl of PMA (100 nM). Absorbance was read against a reference blank curve containing PBS and cytochrome c at 5, 10 and 15 min. Results are expressed in nmoles of O2•- per 106 cells using 21.2 mM/cm as the extinction coefficient. For in vitro evaluation of the effects of DTCCAs, control monocytes were preincubated for 30 min with the desired concentration of drugs or vehicle  before stimulation with PMA.Ot 550 nm  with a UThe respiratory burst of control and nifedipine-treated monocytes induced by formyl-Met-Leu-Phe (fMLP) was measured by a sensitive fluorimetric assay for hydrogen peroxide under standard conditions  using thc. The reaction was started by adding 0.1 U/ml xanthine oxidase. Absorbance was read at 550 nm.The scavenging effect of nifedipine was studied in an assay system containing nifedipine at the desired concentration with 0.15 mM hypoxanthine, 1.5 mM EDTA and 0.025 mM cytochrome 3VO4 and 1 µg/ml each of aprotinin, pepstatin and leupeptin). Cells were then disrupted by sonication (three times 5 s) and proteins were delipidated and desalted before being subjected to SDS gel electrophoresis. Protein phosphorylation was also studied after treatment of a cytosolic fraction of resting monocytes with PMA for 10 min. Protein samples (150 µg of a 1 mg/ml protein sample) were treated in vitro with nifedipine for 30 min before stimulation of PKC with a mixture of diacylglycerol and calcium. This was done with the PKC Biotrak® enzyme assay  in accordance with the manufacturer's instructions. Proteins (50 µg) were separated by SDS gel electrophoresis. Proteins were fluorescently stained by fixing the gels overnight in 50% methanol and 10% trichloracetic acid. The gels were washed with deionised water for 10–20 min, stained with Pro-Q-Diamond for 180 min and destained by three washes in 4% acetonitrile in 50 mM sodium acetate, pH 4, for 2 hours. Gels were scanned with a fluorimager, a Typhoon 9400 laser scanner (Amersham Biosciences), with excitation at 532 nm and a 580 nm band pass emission filter for Pro-Q-diamond dye detection. Phosphorylated proteins were quantified densitometrically with the Image Quant software. Results represent the net phosphorylation induced by PMA, expressed as percentages of the control PMA lane (taken as 100%).Phosphorylated proteins were detected by using the fluorescent Pro-Q-Diamond dye (Molecular Probes), which can directly detect phosphate groups attached to tyrosine, serine or threonine residues in gels. In brief, monocytes from healthy donors were treated with or without nifedipine and stimulated for 10 min with 100 nM PMA (which induces the activation of PKC). Reactions were stopped with ice-cold buffer and cell lysates were prepared by incubating monocytes for 30 min in 0.5 ml of ice-cold extraction buffer  and a non-radioactive PKC kinase assay kit . The principle is based on an enzyme-linked immunosorbent assay method that uses a specific synthetic peptide as a substrate for PKC and a polyclonal antibody that recognised the phosphorylated form of the substrate. In brief, 1 µl of recombinant PKC (5 µg/ml) in 30 µl of kinase assay dilution buffer was incubated for 20 min at 37°C with the drug vehicle (control) or with nifedipine or two other PKC inhibitors . PKC was then activated by 10 µl of PMA (100 nM) for 10 min at 37°C and the reaction was blocked by incubating tubes in ice-cold water. PKC activity was then determined as recommended by the manufacturer. Results are expressed as percentages of the net activity induced by PMA (taken as 100%).PKC assays were performed The study was approved by local ethics committee, and all patients gave written informed consent.P < 0.05 was considered significant. All quantitative data are expressed as means ± SD.Data were compared with the nonparametric Mann–Whitney (unpaired data) and Wilcoxon (paired data) tests. n = 12) not treated by nifedipine produced a significantly greater amount of O2•- than controls (n = 10) after stimulation ex vivo with the PKC activator PMA for 15 min  but not when they were stimulated for 5 min . These observations suggest that the biochemical modifications affecting monocytes from patients with SSc might not affect the kinetics of NADPH oxidase but rather its late regulatory mechanisms.Monocytes from patients with SSc , 10 min  and 15 min  for 14 days produced a significantly smaller amount of Oin vitro with nifedipine for 30 min before stimulation with PMA. Nifedipine induced a concentration-dependent inhibition of O2•- production at concentrations of 10 and 50 µM (n = 5)  by approximately 3–5 µM nifedipine. This inhibitory effect of nifedipine was not due to any cytotoxic effect, as determined by the trypan blue exclusion test (cell death less than 5%). The effects of nifedipine were next studied on the monocyte respiratory burst induced by an inflammatory agonist such as fMLP. However, fMLP induced very weak monocyte O2•- production in standard conditions, so respiratory burst was measured with a fluorimetric assay. Treatment of monocytes for 30 min with 5 and 10 µM nifedipine significantly inhibited hydrogen peroxide production by 55 ± 7% and 90 ± 9%, respectively, relative to controls (n = 3).To determine whether monocytes were directly altered by nifedipine, monocytes from healthy donors were treated  5) Fig. . PMA ind2•- production of healthy monocytes (n = 5) .Next we studied whether other calcium-channel blockers alter the O 5) Fig. . A stron2•- by phagocytes is dependent on the activation of PKC, a family of isoenzymes that phosphorylate various proteins including some components of the NADPH oxidase. Because PMA directly activates various PKC isoforms, we studied the effect of nifedipine on PKC-dependent protein phosphorylation in healthy monocytes. Phosphorylated proteins were directly detected in polyacrylamide gels by using Pro-Q-Diamond, a fluorescent dye that binds to phosphate groups on proteins. In the absence of PMA, nifedipine inhibited the basal phosphorylation of various proteins in monocytes  strongly inhibits the ability of monocytes to produce O2•- ex vivo; and third, nifedipine inhibits O2•- production by healthy monocytes in vitro, a property associated with the inhibition of PKC-dependent protein phosphorylation and PKC activity.In this report we show that, first, 2•- than controls [ex vivo by PMA release about twice as much O2•- as controls, which is similar to previous findings [2•- suggests that circulating monocytes are in a primed state, which is consistent with their prior exposure to a substimulatory dose of agonists such as cytokines or immune complexes [Since the proposal that free radicals have a function in SSc , severalcontrols . This isfindings . This PMomplexes . These domplexes .2•- by monocytes and leads to the disappearance of the monocyte priming state. PMA was chosen because it mimics the activation of NADPH oxidase by PKC, a mechanism also induced by most of the physiological activators of monocytes. Mononuclear cells are thought to be important in SSc [The main finding of this study is that the treatment of patients with SSc with nifedipine (60 mg/day) strongly decreases the production of Ot in SSc -5 and int in SSc . The rest in SSc . Togethet in SSc , these rt in SSc ,21 but sin vitro, as shown by the strong decrease in PMA-induced protein phosphorylation in monocytes and in a cell-free system and the inhibition of a recombinant isoform of PKC. Analysis of the protein phosphorylation pattern showed that nifedipine also inhibits basal phosphorylation of proteins, indicating that other protein kinases might be altered. The molecular mechanism by which nifedipine inhibits PKC cannot be derived from our results and will require further experiments. However, nifedipine inhibits both basal and PMA-induced protein phosphorylation as well as PKC activity, which suggests that nifedipine might interact with both the regulatory and catalytic domains of PKC.Several reports have demonstrated the anti-oxidant properties of calcium-channel blockers. In hypertensive rats, dihydropyridine calcium-channel blockers decrease lipoprotein oxidation and prolong survival independently of modifications of blood pressure . In hype50 values were above 3–5 µM nifedipine for PMA-induced O2•- production in vitro  significantly inhibited Otrophils . The IC5o 100 pM  and the o 100 pM . We usedtrations . Thus, o2•- production by activated monocytes than other calcium-channel blockers evaluated; this was previously reported using human neutrophils [2•- production.We found that diltiazem had a weaker effect on Otrophils  and suggMost of the classical properties of nifedipine are exerted through the modulation of L-type voltage-gated calcium channels on smooth muscle cells. Such channels are also present on monocytes; we suspect that the molecular mechanism highlighted is independent of these channels, but this will require further investigations. Calcium channel-independent properties have previously been reported and because these are not represented in endothelial cells they cannot account for these cellular effects . It was ex vivo PKC-dependent production of O2•- relative to healthy monocytes. Treatment of patients with SSc or of healthy monocytes in vitro markedly decreased the ability of monocytes to generate O2•- . Biochemical analysis of protein phosphorylation in monocytes and cell-free systems suggested that nifedipine directly inhibits PKC activity. These results demonstrate the potential anti-oxidant effects of this drug, which might have important clinical implications for SSc and other oxidative stress-associated diseases.Monocytes from patients with SSc adopt a priming state resulting in increased 50 = concentration for 50% inhibition; PKC = protein kinase C; PMA = phorbol myristate acetate; SSc = systemic sclerosis.DTCCA = dihydropyridine-type calcium-channel antagonists; fMLP = formyl-Met-Leu-Phe; ICThe author(s) declare that they have no competing interests.YA, DB and AP devised the study. YA, HL and AP performed all the experiments. OGE and AK assisted in the writing of the report. All authors read and approved the final manuscript.


Several studies link hematological dysfunction to severity of sepsis. Previously we showed that platelet-derived microparticles from septic patients induce vascular cell apoptosis through the NADPH oxidase-dependent release of superoxide. We sought to further characterize the microparticle-dependent vascular injury pathway.During septic shock there is increased generation of thrombin, TNF-α and nitric oxide (NO). Human platelets were exposed for 1 hour to the NO donor diethylamine-NONOate (0.5 μM), lipopolysaccharide , TNF-α (40 ng/ml), or thrombin (5 IU/ml). Microparticles were recovered through filtration and ultracentrifugation and analyzed by electron microscopy, flow cytometry or Western blotting for protein identification. Redox activity was characterized by lucigenin (5 μM) or coelenterazine (5 μM) luminescence and by 4,5-diaminofluorescein (10 mM) and 2',7'-dichlorofluorescein (10 mM) fluorescence. Endothelial cell apoptosis was detected by phosphatidylserine exposure and by measurement of caspase-3 activity with an enzyme-linked immunoassay.phox, protein disulfide isomerase and Nox. Endothelial cells exposed to the exosomes underwent apoptosis and caspase-3 activation, which were inhibited by NO synthase inhibitors or by a superoxide dismutase mimetic and totally blocked by urate (1 mM), suggesting a role for the peroxynitrite radical. None of these redox properties and proapoptotic effects was evident in microparticles recovered from platelets exposed to thrombin or TNF-α.Size, morphology, high exposure of the tetraspanins CD9, CD63, and CD81, together with low phosphatidylserine, showed that platelets exposed to NONOate and LPS, but not to TNF-α or thrombin, generate microparticles similar to those recovered from septic patients, and characterize them as exosomes. Luminescence and fluorescence studies, and the use of specific inhibitors, revealed concomitant superoxide and NO generation. Western blots showed the presence of NO synthase II (but not isoforms I or III) and of the NADPH oxidase subunits p22We showed that, in sepsis, NO and bacterial elements are responsible for type-specific platelet-derived exosome generation. Those exosomes have an active role in vascular signaling as redox-active particles that can induce endothelial cell caspase-3 activation and apoptosis by generating superoxide, NO and peroxynitrite. Thus, exosomes must be considered for further developments in understanding and treating vascular dysfunction in sepsis. The concept of exosomes appeared with the description of the shedding process of the transferrin receptor by maturing reticulocytes . DivergiEndothelial activation is physiologically important in the context of the inflammatory response as well as pathophysiologically in ischemia/reperfusion, sepsis, and early atherosclerosis . In viewin vitro through a NADPH oxidase-dependent pathway [In previous work we have shown that, in sepsis, platelet-derived microparticles similar to exosomes can be recovered from plasma and that incubation of these microparticles with vascular cells induces apoptosis  pathway . Here weThe established endothelial cell line derived from rabbit aorta characterized by Venter and Buonassisi  was a giBlood samples (40 ml) were collected from 12 patients admitted to the intensive care unit of the Hospital Israelita Albert Einstein , with early (24 hours) diagnosis of septic shock, as defined in accordance with the criteria of the American College of Chest Physicians and the Society of Critical Care Medicine . Patientg for 10 minutes. Phenylmethanesulfonyl fluoride (3 mM), aprotinin (1 g/ml), and pepstatin (1 g/ml) as protease inhibitors were added to the supernatant, which was then sequentially filtered through 1.0, 0.45, and 0.22 μm nylon filters to remove platelets, cellular fragments, and apoptotic bodies. The remaining cell-free plasma was collected over ice and ultracentrifuged at 100,000 g for 90 minutes at 4°C. The pellet, containing exosomes, was first washed with PBS containing 0.1 mM EDTA to avoid contamination with plasma proteins, and then resuspended in 250 μl of PBS. The total exosome mass obtained was 9.6 ± 3.9 mg protein per sample. In previous work we have shown that this exosome population displayed almost exclusively platelet markers [Blood was collected in centrifuge tubes containing 10.5 mM trisodium citrate and was processed immediately. Initial procedures were performed at room temperature (between 20–25°C) to avoid artifactual platelet activation. Cells, platelets, and large debris were pelleted by centrifugation at 3,000  markers .g for 5 minutes at 20°C, and subsequently leukocytes were removed through a commercial filter system . Plasma-free platelet suspensions were obtained by centrifugation of platelet-rich plasma at 800 g for 15 minutes at 20°C, and the resultant pellet was resuspended in 5 ml of Krebs-HEPES buffer .Blood (40 to 50 ml) was collected from healthy volunteers who had not taken any medication known to interfere with platelet function within the previous 2 weeks. The blood was drawn into tubes containing acid citrate dextrose anti-coagulant . Platelet-rich plasma was first obtained by centrifugation at 800 g for 15 minutes) to obtain the platelet pellet fraction. The supernatant was further centrifuged  to obtain the microvesicle fraction, and the supernatant from that microvesicle fraction was filtered sequentially through 0.45 and 0.22 μm low-protein-binding nylon membranes. The filtered product was further centrifuged  to obtain the exosome pellet. All pellets were resuspended in 250 μl of PBS. The total exosome mass obtained was 10.6 ± 4.5 mg of protein per sample.Plasma-free platelet suspensions were incubated with agonist or with saline control (154 mM NaCl in water) for 1 hour as indicated, and the reaction was slowed down by placing samples on ice. Samples were centrifuged . Exosomes were resuspended in 100 μl of Krebs-HEPES buffer at a constant 100 μg/ml concentration. Luminescent or fluorescent probes were added 15 minutes before measurements started, and samples were equilibrated while being protected from light.8 platelets/ml and results were corrected to protein content. Luminescent counts are presented as relative luminescence units (RLU)/min per mg of protein.The luminescent probes lucigenin and coelenterazine were first used to detect the generation of ROS. The concentration of lucigenin and coelenterazine used (5 μM each) minimized the generation of artifactual readings, as shown previously . ReactioTo better characterize the generation of reactive species, 2',7'-dihydrodichlorofluorescein diacetate  for ROS  and 4,5-NG-methyl-L-arginine acetate; 5 mM), L-NAME  and D-NAME , urate (1 μM), the membrane-permeable superoxide dismutase mimetic Mn(III) tetrakis (4-benzoic acid) porphyrin chloride , and the specific NADPH oxidase inhibitory peptide gp91ds-tat (10 μM) [Further studies to characterize the source or type of reactive species were performed in the presence of specific inhibitors or quenchers such as L-NMA ( (10 μM) .For flow cytometry analysis, we used aliquots of exosome or apoptotic body suspensions with 200 μg of particle protein/ml. To identify specific epitopes, aliquots were incubated with fluorescein 5(6)-isothiocyanate (FITC) or R-phycoerythrin-conjugated antibodies directed to specific membrane antigens at 1 μg/ml final concentration , namely CD9, CD63, and CD81  ,8, and wBecause exosomes are, on average, too small for cytometry analysis, we believe that our data correspond to aggregates formed after ultracentrifugation. For this reason we did not attempt to perform any specific quantification.Pellets of exosomes obtained from platelets were fixed under 2.0% glutaraldehyde in 0.1 M sodium cacodylate for at least 2 hours and postfixed with 2% osmium tetroxide in 10.56% sucrose for 2 hours and finally incubated with 0.5% uranyl acetate and 10.56% sucrose overnight. Pellets were then dehydrated and embedded in Spurr resin. Ultrathin sections 70 to 80 nm thick were cut on an ultramicrotome , picked up on copper grids and stained for contrast with 1% uranyl acetate and 1% lead citrate. Specimens were examined with a transmission electron microscope , operated at 80 kV.6 cells/ml into Eppendorf vials. Annexin V-FITC was added to a final concentration of 100 ng/ml, and the cells were incubated in the dark for 10 minutes and then washed again with PBS. Propidium iodide (30 μl) was added before analysis. Cells were spread on clean slides, covered with glass coverslips, and immediately examined under fluorescence microscopy. From three high-power fields per sample, a minimum of 200 cells were counted. Cells were considered apoptotic when membrane-bound annexin-FITC fluorescence was positive and nuclear staining with propidium iodide (evidence of late apoptosis or necrosis) was negative. Results are expressed as apoptotic cells per 100 cells.Annexin V was used to detect apoptosis . In brie4P2O7 (10 mM), leupeptin (1 μg/ml), pepstatin (1 μg/ml), phenylmethylsulfonyl fluoride (3 mM), and Nonidet P40 (1% v/v), placed on ice for 10 minutes, and centrifuged at 10,000 g for 10 minutes. The activity of caspase-3 was measured at 405 nm with a Caspase-3 Colorimetric Detection Kit  in accordance with the manufacturer's instructions.Rabbit endothelial cells were cultured on six-well plates to 80 to 90% confluence as described. Cells were kept in 1% serum for 24 hours before use. A volume of microparticle suspension equivalent to 100 μg of protein was added to each well  and incubated for 30 minutes. Some experiments were performed after incubation with the membrane-permeable SOD mimetic (10 μM) or with L-NAME (1 μM). Exposure to TNF-α (50 ng/ml) was used as a positive control for caspase-3 activation. After incubation, plates were kept on ice. Cells were washed with ice-cold PBS and lysed with Nonidet lysis buffer containing Tris/HCl , NaCl (150 mM), Nab558 components p22phox, Nox1, and Nox2 (gp91phox)  or to inducible nitric oxide synthase (NOS), endothelial NOS or neuronal NOS  followed by horseradish peroxidase-conjugated secondary antibody  and developed with the Chemiluminescence-Phototope-HRP (horseradish peroxidase)-conjugated Detection Kit  as specified. Results are representative of at least three similar experiments.Exosome protein (40 μg), leukocyte and endothelial cell lysate (used as a positive control) were subjected to separation by SDS-PAGE and transferred to nitrocellulose. Equal separation and transference of the samples were confirmed by Ponceau staining during the preparation of membranes. Membranes were incubated with antibodies directed to the NADPH oxidase cytochrome P < 0.05 significance level.Data shown are means ± SD of three or more similar experiments. Comparisons between groups were performed by one-way analysis of variance followed by a Student–Newman–Keuls test at Exosomes are known to expose several different markers related to their cellular origin and putative functions. Phosphatidylserine is typically not exposed, differentiating exosomes from apoptotic bodies or cellular debris. In contrast, proteins of the tetraspan family are considered to be specifically sorted during exosome generation. As shown in Figure Exosomes in the former group, which are similar to those recovered from septic patients, exposed large amounts of the tetraspan family members CD9, CD63, and CD81 and exhibiting low binding of annexin V. Exosomes in the latter group were similar to the apoptotic bodies with lower tetraspan exposure and higher annexin V binding capability.Electron microscopy Figure  revealedPreliminary measurements of ROS-generating activity, performed with lucigenin, revealed that the redox activity of exosomes paralleled the surface characteristics disclosed by flow cytometry analysis. As seen in Figure To characterize the exosome redox profile better, measurements with the luminescent probe coelenterazine were also performed Figure . ResultsThe fluorescent probes DCHF and DAF were used for further clarification of the nature of ROS generated by the exosomes. DCHF is believed to react with hydrogen peroxide, whereas detection of superoxide radical with DCHF is still not clear. DCFH can also be oxidized by peroxinitrite . In contFigure Figure phox, Nox1, and Nox2 of the NADPH oxidase, as well as its regulatory protein protein disulfide isomerase (PDI). A non-specific protein staining can also be seen, which confirms equal gel loading between samples.Figure To verify a physiological or pathophysiological role for platelet-derived exosomes, we exposed cultured endothelial cells to the different types of exosome. As seen in Figure Caspase-3 is one central step in the apoptotic cascade, and it is well known to be redox sensitive -31. To vin vitro [A basic role for exosomes in intercellular communication implies that the cell of origin controls their content. In this respect, it has been suggested that different agents are able to induce the release of phenotypically distinguishable platelet microparticles in vitro . More rein vitro . In factin vitro . PDI, muin vitro . This leThe mechanisms regulating the secretory process of exosomes are as yet completely unknown. They emerge from an intracytoplasmic membrane complex known as multivesicular bodies, which can be understood as a processing compartment for internalized proteins, subjected to the influence of the trans-Golgi network. Regulation of specific protein sorting to the multivesicular bodies has been explored better and apparently depends on lipid signaling involving phosphadylinositol kinases and ubiquitination . In contIt must be pointed out that most of the studies concerning vascular signaling have been performed with a broader range of subcellular particles, known generically as microparticles. It is therefore difficult to perform comparisons and analysis of experimental results . DiffereAlthough the mechanisms of vascular damage are not fully understood, they have been related to the generation of ROS . In lineTo demonstrate that at least part of the proapoptotic activity of the exosomes could be related to the generation of ROS or RNS, we investigated the exosome-triggered SOD-mimetic, L-NAME, and urate inhibitable activation of caspase-3 in endothelial cells in culture. Caspase-3 activation and caspase-3-dependent apoptosis have been shown to be inhibited by S-nitrosation of a critical cysteine residue induced by exogenous NO donors . Other sin vitro may be a valuable model for the generation of exosomes involved in redox signaling.Taken together, our results confirm previous observations that exosome generation is a process subjected to specific regulatory pathways. In sepsis, both increased NO generation and the presence of LPS can trigger the release of platelet-derived exosomes, whereas thrombin or TNF-α induces the generation of phosphatidylserine-rich particles. Indicating an effective signaling role, septic-like platelet-derived exosomes induce caspase-3 activation and apoptosis of target endothelial cells through active ROS/RNS generation by NADPH oxidase and NO synthase type II. In addition, we propose that platelet exposure to LPS or NO Exosomes were first described in connection with the maturation of reticulocytes, and provide a method of sorting obsolete proteins, such as transferrin receptor, as the cells differentiate into erythrocytes. More recently, many other cell types have also been shown to secrete exosomes, such as antigen-presenting cells, which might use this mechanism to regulate the immune response. These findings prompted a reappraisal of the exosome's role from that of a 'garbage sack', releasing obsolete proteins, to a device involved in triggering intercellular communication. Here we propose that exosomes may have a major role in vascular redox signaling. In this context, exosomes could be a novel tool with which to further understand and possibly treat vascular dysfunction related to diabetes, hypertension, or sepsis.• In sepsis, increased NO generation, as well as the presence of LPS, can trigger the release of platelet-derived exosomes.• These exosomes induce caspase-3 activation and the apoptosis of endothelial cells through the generation of ROS and RNS.• The existence of both specific releasing stimuli and targets characterizes exosomes as vascular signaling effectors.• In sepsis, exosome generation may therefore contribute to endothelial dysfunction, leading to vascular failure.Nω-nitro-D-arginine methyl ester; ERK = extracellular signal-regulated kinase; FITC = fluorescein 5(6)-isothiocyanate; L-NAME = Nω-nitro-L-arginine methyl ester; L-NMA = NG-methyl-L-arginine acetate; LPS = lipopolysaccharide; NO = nitric oxide; NOS = NO synthase ; Nox1 and Nox2 = isoforms of membrane-bound subunits of NADPH oxidase; PBS = phosphate-buffered saline; PDI = protein disulfide isomerase; RNS = reactive nitrogen species; ROS = reactive oxygen species; SOD mimetic = membrane-permeable superoxide dismutase mimetic Mn(III) tetrakis (4-benzoic acid) porphyrin chloride; TNF = tumor necrosis factor.DAF = 4,5-diaminofluorescein diacetate; DCHF = 2',7'-dihydrodichlorofluorescein diacetate; DGK = diacylglycerol kinase; D-NAME = The authors declare that they have no competing interests.MHG performed Western blot and enzyme-linked immunosorbent assay studies and drafted the manuscript. AOC conducted the measurements of redox activity and apoptosis. LM participated in study design and performed all flow cytometry studies. SVF conducted Western blot studies as well as the electron microscopy. LRL participated in study design, coordination, and data analysis. MJ conceived of the study, participated in its design, coordination, and data analysis, and finished the manuscript. All authors read and approved the final manuscript.


This study aimed to examine whether ROS regulate α20), and myosin phosphatase stimulated by α1-adrenoceptor agonist phenylephrine were examined.Using endothelium-denuded rat tail artery (RTA) strips, effects of anti-oxidants on isometric force, ROS production, phosphorylation of the 20-kDa myosin light chain , and two NADPH oxidase inhibitors, apocynin and VAS2870, dose-dependently inhibited contraction activated by phenylephrine. Phenylephrine stimulated superoxide anion production that was diminished by the pretreatment of apocynin, VAS2870, superoxide scavenger tiron or mitochondria inhibitor rotenone, but not by xanthine oxidase inhibitor allopurinol or cyclooxygenase inhibitor indomethacin. Concurrently, NADPH oxidase activity in RTA homogenates increased within 1 min upon phenylephrine stimulation, sustained for 10 min, and was abolished by the co-treatment with apocynin, but not allopurinol or rotenone. Phenylephrine-induced MLC1-adrenoceptor-activated smooth muscle contraction by altering myosin phosphatase-mediated MLC20 phosphorylation through both RhoA/Rho kinase- and CPI-17-dependent pathways.ROS, probably derived from NADPH oxidase and mitochondria, partially regulate α However, ROS act as intracellular signaling molecules mediating various cellular functions including proliferation, apoptosis and survival . Emerginensin II ,4 and enensin II  or by deensin II . In addiensin II . How supIn the vasculature, the potential sources of ROS include NADPH oxidase, uncoupled endothelial nitric oxide synthase, xanthine oxidase, cyclooxygenase and the mitochondrial respiratory chain. Among these, NADPH oxidase is generally considered the major source of vascular ROS  and has 20) is a key determinant for smooth muscle contraction. The levels of MLC20 phosphorylation are determined by the activity ratio between myosin light chain kinase (MLCK) and myosin phosphatase. While MLCK activation depends on the cytoplasmic calcium concentration, myosin phosphatase activity is subject to the modulation by various signaling molecules [In vivo evidence showed that Rho kinase plays important roles in MYPT1 phosphorylation whereas protein kinase C catalyzes CPI-17 phosphorylation [Phosphorylation of the 20-kDa myosin light chains , and the experimental procedures were approved by the institutional Animal Care and Use Committee. Male Sprague-Dawley rats weighing 400 ~ 550 g were used in this study. After the animal was anesthetized with pentobarbital , the tail artery was removed and placed in oxygenated (95% O2 - 5% CO2) Krebs' physiological salt solution (PSS) with the following composition (in mM): 120 NaCl, 5.9 KCl, 25 NaHCO3, 1.2 NaH2PO4, 11.5 dextrose, 1.2 MgCl2 and 2.5 CaCl2 . The endothelium-denuded rat tail artery (RTA) strips were placed in tissue bathes with one end held in a muscle holder and the other end connected to a force transducer. After being stretched to the length that allows for maximal force production and being equilibrated at 37°C for at least 1 h, muscle strips were stimulated twice with 51 mM KCl-PSS (equimolar replacement of NaCl with KCl) to generate reproducible contraction. A dose response was generated with cumulative concentrations of α1-adrenoceptor agonist phenylephrine and the maximal force was used to normalize later contractile responses. To determine the involvement of ROS and NAD(P)H oxidase, tissues were incubated for 30 min in PSS containing vehicle, N-acetyl-L-cysteine (NAC), apocynin or VAS2870. A cumulative concentration-response for phenylephrine was then performed again. In all other experiments, 10 μM phenylephrine was used.This study conforms to the procedures described in the 4, 0.15 Na2HPO4, 0.4 KH2PO4, 5 NaHCO3, 5.5 glucose, 1.2 CaCl2, pH7.4) at 37°C for 30~60 min. After equilibration, RTA strips were incubated for 30 min in prewarmed PSS-HEPES buffer containing 10 mM DETC (Sigma) in the presence or absence of inhibitors for superoxide generation. DETC was used to inhibit the endogenous Cu2+/Zn2+ superoxide dismutase (SOD) activity. Following DETC treatment, RTA strips were placed in temperature-controlled (37 °C) sample dishes containing 2 ml HEPES buffer with lucigenin  plus or minus inhibitors and placed in the dark chamber of the Chemiluminescence Analyzing System . The background counts were measured for 5 min, phenylephrine in 100 μl HEPES buffer was then injected into the chamber and the chemiluminescence was measured for another 10 min. Lucigenin chemiluminescence was expressed as counts per min (cpm) per milligram tissue weight after subtracting background counts (without vessel).Superoxide production in RTA strips was measured by a lucigenin-enhanced chemiluminescence assay according to a published method . RTA str2HPO4, pH 7.4, 10 μg ml-1 aprotinin, 10 μg ml-1 leupeptin, and 0.5 mM phenylmethylsulfonyl fluoride). Following 10-min centrifugation at 1,000 g, supernatant (30-40 μl) was mixed with reaction buffer containing (in mM) 37 NaCl, 2.7 KCl, 4.3 Na2HPO4, 1.5 KH2PO4, and 5 μM lucigenin as the detector. The reaction was started by the addition of NADPH  and photon emission was measured for 10 min at room temperature in a scintillation counter  in out-of-coincidence mode. NADPH oxidase activity was expressed as cpm per microgram protein. The specificity of lucigenin for ·O2- production was validated by adding a nonenzymatic scavenger of ·O2-, tiron , to quench the signal. To examine the effects of inhibitors, 1 mM apocynin, 10 μM VAS2870, 100 μM allopurinol, 10 μM rotenone or vehicle was added 30 min before RTA strips were stimulated with phenylephrine for 1 min.RTA strips were stimulated with phenylephrine, snap-frozen in liquid nitrogen at designated time points, and stored at - 80 °C until use. NAD(P)H oxidase activity was assayed according to a published method . Briefly20 phosphorylation was measured by two-dimensional polyacrylamide gel electrophoresis (PAGE) as previously described [20 over total (phosphorylated plus unphosphorylated) MLC20.MLCescribed . BrieflyThr697 or anti-MYPT1Thr855 antibody  or anti-CPI-17Thr38 antibody . The blots were washed and incubated with horseradish peroxidase-conjugated anti-rabbit IgG  for 1.5 h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence (PerkinElmer Life Sciences). Membranes were then stripped of bound antibodies by incubation in a buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 10 mM DTT for 30 min at 50°C with agitation. The blots were reprobed with anti-MYPT1  or anti-CPI-17  for loading control.Equal amounts of protein were resolved under reducing conditions on 8% SDS-PAGE (for MYPT1) or 12.5% SDS-PAGE (for CPI-17) and transferred electrically onto nitrocellulose membranes. Following 1.5 h blocking in a Tris-buffered saline (TBS) solution containing 3% nonfat dry milk at room temperature, membranes were incubated overnight at 4°C with phosphospecific anti-MYPT12, 2 EDTA, 10 μg ml-1 aprotinin and leupeptin, 1 phenylmethylsulfonyl fluoride, 50 sodium orthovanadate, 10 pyrophosphate, 10 NaF), the homogenates were centrifuged at 100,000 g, 4 °C for 30 min . The supernatant was collected as cytosolic fraction, and membrane proteins were extracted by incubating the pellet for 30 min in homogenization buffer containing 1% Triton X-100 and 0.25% sodium cholate. The extract was clarified at 800 g for 10 min, and the supernatant was collected as the particulate fraction. Protein concentrations were determined with a bicinchoninic acid kit (Pierce). Equal amounts of protein were electrophoresed on 12.5% SDS-PAGE followed by immunoblotting with a monoclonal anti-RhoA antibody . The result was expressed as the ratio of membrane RhoA/total RhoA (membrane RhoA + cytosolic RhoA).Translocation of RhoA from the cytosol to membrane fractions was determined according to a published method  with modt test was used for comparison between control and treatment group. P-values less than 0.05 were considered statistically significant.Data were expressed as means ± SEM. For multiple comparisons, ANOVA followed by Newman-Kuels or Dunnett's post-hoc test was used . An unpaired Student's 1-adrenoceptor-activated contraction was assessed by the effects of an antioxidant and two NAD(P)H oxidase inhibitors on phenylephrine-stimulated isometric force of RTA strips. As shown in Figure The involvement of ROS in α-1 vs. 464.3 ± 58.8 cpm mg tissue-1, p < 0.05). Pretreatment with a selective α1-adrenoceptor antagonist prazosin (1 μM) nearly abolished phenylephrine-stimulated superoxide production (672.0 ± 142.6 cpm mg tissue-1) and contractile response (data not shown). This result indicated that phenylephrine stimulates superoxide production in α1-adrenoceptor-dependent manner. To further unravel sources for phenylephrine-stimulated superoxide anions, we examined the effect of inhibitors for various ROS generation systems. Phenylephrine-induced ·O2- production was inhibited by the pretreatment with NAD(P)H oxidase inhibitors apocynin (1 mM) and VAS2870 (10 μM), a superoxide scavenger tiron (10 mM), and mitochondria respiratory chain inhibitor rotenone (10 μM). In contrast, xanthine oxidase inhibitor allopurinol (100 μM) and cyclooxygenase inhibitor indomethacin (10 μM) had no effect. These results suggested that NAD(P)H oxidase and mitochondria, but not xanthine oxidase or cyclooxygenase, may contribute to phenylephrine-stimulated superoxide production.We next examined whether phenylephrine stimulated ROS production in RTA strips using lucigenin-enhanced chemiluminescence assay. As shown in Figure -1 for control, 179 ± 5 and 217 ± 32 cpm μg protein-1 for 1-min and 10-min phenylephrine stimulation, respectively, p < 0.05 compared to control). As shown in Figure -1 for vehicle control, 86 ± 16 cpm μg protein-1 for apocynin; p < 0.05). In contrast, neither allopurinol (100 μM) nor rotenone (10 μM) exhibited significant inhibition. These results implied that NAD(P)H oxidase is involved in phenylephrine-stimulated superoxide anion production in RTA strips.The role of NAD(P)H oxidase in phenylephrine-induced superoxide production was further corroborated by activity assay in RTA homogenates. We tested both NADH and NADPH as the substrate for activity assay and found that 300 μM NADPH, but not NADH, elicited detectable signals. As shown in Figure 20 phosphorylation, we measured MLC20 phosphorylation levels in the presence and absence of apocynin. As depicted in Figure 20 phosphorylation increased markedly from 10 ± 1.1% to 43 ± 1.2% (p < 0.001), pretreatment of apocynin (1.5 or 3 mM) dose-dependently inhibited phenylephrine-stimulated MLC20 phosphorylation. Similarly, at 15-min phenylephrine stimulation, MLC20 phosphorylation was inhibited by apocynin in a dose-dependent manner . Pretreatment of RTA with apocynin abolished phenylephrine-induced RhoA translocation . Similar results were obtained at 15-min phenylephrine stimulation  production to regulate vasoconstriction. While inhibition of xanthine oxidase or cyclooxygenase did not affect ROS production in phenylephrine-stimulated RTA, two structurally distinct inhibitors of NAD(P)H oxidase abolished this response. Our results also indicated that phenylephrine-activated contractile response in RTA is only partially regulated by ROS because apocynin and VAS2870, at concentrations that attenuated 50~80% of ROS production, only decreased force by 30%~40%.Results of this study show that α1-adrenoceptor activation upon phenylephrine challenging increased ROS production. What is responsible for α1-adrenoceptor-mediated vascular ROS generation? Xanthine oxidase and cyclooxygenase can be excluded since inhibiton of either enzyme had no effect on phenylephrine-induced ROS formation. One probable source is NADPH oxidase. Our finding that phenylephrine-induced ROS production was attenuated by apocynin or VAS2870 pretreatment supports this notion. Furthermore, phenylephrine stimulation increased NADPH oxidase activity in RTA homogenate, which was abolished by apocynin. For more than a decade, apocynin has been used as a specific inhibitor to explore the role of NADPH oxidase in the field of vascular biology. However, Heumueller et al recently reported that apocynin is not an inhibitor of vascular NADPH oxidase and instead acts as an antioxidant, thereby reducing ROS bioavailability [1-adrenoceptor-activated vasoconstriction.Our results showed that αlability . These alability , inhibit1-adrenoceptor-induced ROS generation. An inhibitor of mitochondrial respiratory chain, rotenone, markedly decreased phenylephrine-induced ROS formation. This finding is in good agreement with a previous study showing that mitochondria are a major source of ROS in response to phenylephrine stimulation in rat mesenteric artery [1-adrenergic-dependent vasoconstriction [1-adrenergic-activated vasoconstriction.Mitochondria present another likely source for αc artery . Using mtriction . Anothertriction . Further20 phosphorylation and smooth muscle contraction [1-adrenoceptor stimulation activates NAD(P)H oxidase with concomitant increases in RhoA translocation, MYPT1Thr855 and MLC20 phosphorylation. Apocynin at concentrations that abolished increase in ROS production eliminated RhoA translocation and MYPT1Thr855 phosphorylation and decreased MLC20 phosphorylation. These results are consistent with the idea that ROS play upstream signaling roles in RhoA/Rho kinase activation and myosin phosphatase inhibition. At sustained phase, similar results were obtained with apocynin on RhoA translocation, MYPT1Thr855 phosphorylation and MLC20 phosphorylation. These results suggest that ROS may regulate α1-adrenoceptor-stimulated contraction through RhoA/Rho kinase-mediated myosin phosphatase inhibition and thereby increasing MLC20 phosphorylation. It was previously reported that exogenously generated ·O2- activates Rho/Rho kinase pathway to promote contraction [Thr855, resulting in enhanced MLC20 phosphorylation and contraction in rat pulmonary arteries [Myosin light chain phosphorylation levels are determined by the activity ratio between myosin light chain kinase and myosin phosphatase. Phosphorylation of MYPT1 inhibits myosin phosphatase activity, resulting in increased MLCtraction ,27. Resutraction . Knock earteries . Up to darteries . Resultsarteries , a redoxarteries .20 phosphorylation and smooth muscle contraction. The result that α1-adrenoceptor-induced CPI-17 phosphorylation was inhibited by apocynin suggested that CPI-17-mediated myosin phosphatase inhibition provides another mechanism accounting for ROS actions. Interestingly, apocynin was recently reported to inhibit Rho kinase without involving vascular NAD(P)H oxidase [Phosphorylation of CPI-17, a smooth muscle-specific protein inhibitor of myosin phosphatase, by PKC presents another important mechanism to inhibit myosin phosphatase activity ,33. It w oxidase . Our res2+ mobilization [2+ by inhibiting Ca2+-ATPase activity and by promoting inositol trisphosphate-induced Ca2+ release [2- locally to regulate ryanodine receptor activity and Ca2+ release from sarcoplasmic reticulum [20 phosphorylation via the activation of MLCK remains to be examined.In addition to modulating myosin phosphatase activity, ROS modulate intracellular Calization . It was  release . A receneticulum . Whether1-adrenoceptor-activated smooth muscle contraction is modulated by ROS generation. Alpha1 adrenoceptor-stimulated increases in ROS production, acting through RhoA/Rho kinase-mediated MYPT1Thr855 phosphorylation and Rho kinase-independent CPI-17Thr38 phosphorylation, lead to myosin phosphatase inhibition, MLC20 phosphorylation, and smooth muscle contraction.In summary, the present study showed that αThe authors declare that they have no competing interests.MHT designed and performed the experiments, analyzed the data, and drafted the manuscript; MJJ conceived of the study, designed the experiments, analyzed the data, and helped draft the manuscript. Both authors read and approved the final manuscript.