During restenosis, vascular smooth muscle cells (VSMCs) migrate from the vascular media to the developing neointima. Preventing VSMC migration is therefore a therapeutic target for restenosis. Drugs, such as prostacyclin analogues, that increase the intracellular concentration of cyclic adenosine monophosphate (cAMP) can inhibit VSMC migration, but the mechanisms via which this occurs are unknown. Two main downstream mediators of cAMP are protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). This study has examined the effects of the prostacyclin analogue beraprost on VSMC migration and investigated the intracellular pathways involved.ex vivo serum-treated human veins. Using fluorescence resonance energy transfer, we demonstrated that beraprost activated Epac but not PKA. The mechanisms of this Epac-mediated effect involved activation of Rap1 with subsequent inhibition of RhoA. Cytoskeletal rearrangement at the leading edge of the cell was consequently inhibited. Interestingly, Epac1 was localized to the leading edge of migrating VSMC.In a chemotaxis chamber, human saphenous vein VSMC migrated towards a platelet-derived growth-factor-BB (PDGF) chemogradient. Incubation with therapeutically relevant concentrations of cAMP-producing agonist beraprost significantly decreased PDGF-induced migration. Direct activation of either PKA or Epac inhibited migration whereas inhibition of PKA did not prevent the anti-migratory effect of beraprost. Direct activation of Epac also prevented hyperplasia in These results indicate that therapeutically relevant concentrations of beraprost can inhibit VSMC migration via a previously unknown mechanism involving the cAMP mediator Epac. This may provide a novel target that could blunt neointimal formation. At a cellular level, VSMC migration is initiated by growth factors such as platelet-derived growth-factor-BB (PDGF), cytokines (for example tumour necrosis factor-α), and extracellular matrix components.4 These mediators stimulate migration in a spatially and temporally organized process. Initially, a remodelling of the actin cytoskeleton occurs throughout the cell resulting in protrusion of a leading edge.5 This leading edge extends towards a chemotactic stimulus , assisted by the actions of matrix metalloproteinases which degrade the extracellular matrix.6 Movement of the cell occurs via the actomyosin motors which generate force.5 The complex guidance cues and intracellular pathways which regulate this migration process in VSMC are still unclear. It is known that monomeric G-proteins have an integral and interdependent role in migration.7 In particular, the role of RhoA has been characterized in many cell types including VSMC.9 RhoA regulates actin polymerization via actin-binding proteins involved in focal contact formation with integrins as well as regulating the activity of myosin II.10 The actions of RhoA are mediated by Rho effectors such as p160ROCK (Rho-kinase) which can phosphorylate multiple target proteins.7The most common clinical interventions in coronary artery disease are balloon angioplasty followed by stent emplacement or coronary artery bypass graft. Both these treatments are associated with neointimal hyperplasia resulting in restenosis and vessel reocclusion.11 Although currently untested, this approach could potentially indicate that cAMP-activating drugs may be useful in drug-eluting stents to decrease neointimal hyperplasia. Indeed, prostacyclin and its analogues have been recognized for some time to inhibit neointimal hyperplasia in animal models via a variety of mechanisms, including elevation of intracellular cAMP concentrations.15 The mechanisms involved in cAMP-mediated inhibition of VSMC migration are not clear. Until recently the downstream effects of increased cAMP were assumed to be predominantly via the well-described mediator, protein kinase A (PKA). However, cloning of the cAMP-binding protein, exchange protein directly activated by cAMP (Epac), has offered additional cAMP-dependent mechanisms with different downstream consequences.17 Epac has 2 known isoforms, Epac1 and Epac2, with little functional difference ascribed to their effects. Epac is one of a family of guanine nucleotide exchange factors for the monomeric G-protein Rap1.17 Rap1 has several different functions dependent upon cell type and is frequently associated with regulation of the cell cytoskeleton.18 The Epac-Rap1 pathway is intimately involved in regulating cell migration and cell-cell interactions in a cell type dependent manner.20 Both Epac/Rap1 and PKA may be involved in relaxation of smooth muscle and could inhibit VSMC proliferation, although their role in VSMC migration is still unknown.23In addition to mechanisms which activate VSMC migration, there are endogenous mechanisms which maintain quiescence, thereby preventing migration. At the level of intracellular signalling, it is clear that increases in cyclic adenosine monophosphate (cAMP) can inhibit growth-factor-induced VSMC migration.In the current study, we have examined VSMC migration and the effects of increasing cAMP using the prostacyclin analogue beraprost. We now demonstrate that at therapeutically relevant concentrations of beraprost, VSMC migration is inhibited via a divergent mechanism dependent on cAMP and Epac, but does not involve PKA. These findings indicate that cAMP-producing agonists such as beraprost may be useful in decreasing restenosis by preventing VSMC migration. The study also indicates Epac/Rap1 as a potential therapeutic target.2.Supplementary material.An expanded Methods section is available in 2.1ex vivo organ culture, veins were incubated in 30% bovine calf serum for 14 days and medium changed every 48 h.Saphenous veins were obtained from patients undergoing coronary artery bypass graft surgery after obtaining written consent under procedures approved by the local ethical committee (reference 06/S0802/26). These procedures conform to the principles outlined in the Declaration of Helsinki. VSMC were cultured from vascular explants and maintained as previously described. All cells used were between passages 3–8. For 2.2Cells were kept in serum-free medium for 24 h and seeded into a 48 multi-well chemotaxis chamber as per the manufacturer's instructions. Migrated cells bound to the underside of the membrane were mounted onto a microscope slide and the cells counted using ImageJ. All samples were completed in duplicate.2.3Cells were plated into 96-well plates in 0.1% FCS DMEM for 24 h. Treatment medium was added along with the bromodeoxyuridine (BrdU) label for a further 24 h. The BrdU assay was carried out as per manufacturer's instructions and repeated in triplicate.2.424Protein extracts were prepared and subjected to SDS–PAGE followed by immunoblotting as previously described.2.524 Cells were imaged using a Zeiss 710 scanning confocal microscope with a 40× objective. During confocal imaging comparing different cell treatments, settings on the microscope were not changed . Experiments were repeated in triplicate.Cells were serum starved for 24 h and, following treatment, were fixed in 3% paraformaldehyde as previously described.2.624 Cells were transfected with either 37.5 nmol/L scrambled siRNA (Dharmacon) or 18.75 nmol/L of each PKAα and -β catalytic subunit siRNA  or 18.75 nmol/L Rap1A and Rap1B SMARTpool siRNA  using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. For sequence data, see Table S1Supplementary material online, . The cells were serum starved the following day for 24 h and then the protein was extracted for immunoblotting or the cells were used in the chemotaxis chamber.Knockdown of specific proteins was carried out as previously described for human VSMC.2.725 Images of 378 ms duration were captured every minute with a Roper Coolsnap 1392 × 1040 pixel camera with 4 × 4 binning. The Leica 11504119 Fret EX filter separated the fluorescent channels: a CFP donor channel (excitation at wavelength 427 ± 5 nm and emission at 482 ± 15 nm), an FRET channel (excitation at wavelength 427 ± 5 nm and emission at 542 ± 13 nm), and a YFP channel (excitation at wavelength 500 ± 6 nm and emission at 542 ± 13 nm). These images were used to create a pseudocolour sensitized FRET image for each time point using specific algorithms.25 Regions of interest were traced using the Leica Advanced Fluorescence software and FRET values recorded. The FRET data are displayed as percentage change from the initial control readings before treatments were added at 5 min, with values >0% reflecting protein activation and values <0% reflecting protein inactivation. All experiments were performed in duplicate.A human aortic smooth muscle cell nucleofector kit cc-2571 from Lonza was used to introduce the cDNA into the VSMC. For experiments investigating the Epac protein, 8 µg CFP-Epac-YFP cDNA was used . The PKA cDNA consists of a regulatory subunit construct and a catalytic subunit construct . The ratio of the two PKA cDNA was optimised and 2 µg of the donor and 3 µg of the acceptor were added. Briefly cells were passaged and transferred to an electroporation cuvette with the cDNA and nucleofector solution and transfected according to manufacturer's instructions. The cells were plated in single glass-bottom dishes and following stimulation time-lapse videos of transfected VSMC were captured using a Leica AF6000LX imaging system which captures simultaneously in cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and FRET configured channels. Correction factors were entered into the software to correct for channel bleed-through and background fluorescence. The correction factors have previously been generated using control experiments described.2.826 Cells were plated in 75 cm2 flasks and grown to 80–90% confluent. The cells were serum starved for 24 h. After this time period, treatment medium was added to the flasks for 5–10 min for the Rap1 experiments and 30 min for the Rho experiments. After incubation the Rap1 or Rho pull-down assay was carried out as per manufacturer's instruction (Thermo Scientific). Briefly, the cells were scraped and centrifuged at 16 000 g for 15 min at 4°C. Two controls were used including the GTPγs (positive control) and GDP (negative control) throughout the assay. The lysates were placed into spin cups with a GST–fusion protein of the Rap1-binding domain from human RalGDS to pull down the active Rap1 protein or the Rhotekin-binding domain along with glutathione agarose resin to pull down the active Rho protein. Immunoblotting was then carried out with either an anti-Rap1 or anti-Rho antibodies for the detection of the GTP-bound active form of the protein.Pull-down assays in human VSMC were performed as previously described.2.9All experiments were performed with either three or more individual patient vein explants. Data are expressed as the mean ± SEM. For experiments that were performed in duplicate or triplicate, replicates were averaged and average values used to calculate the mean.t-test was performed for the Rap1 RT-PCR  and Figure 6D, where the same cells at the same passage were used with different treatments. A two-way ANOVA was performed with Dunnett's post hoc test for the FRET data to compare each timepoint with different treatments. All other experiments were analysed with a one-way ANOVA with Dunnett's post hoc test. A P value of <0.05 was taken as statistically significant.A paired 3.3.1Figure ). Similar results were obtained with another prostacyclin analogue iloprost (not shown). To ensure that changes in migration were not due to the actions of beraprost on the mitogenic effects of PDGF, BrdU incorporation was assessed . At 4 h, BrdU incorporation was not significantly increased by PDGF (not shown). Furthermore, at 24 h, although VSMC incubated with PDGF had significantly increased BrdU incorporation, the addition of beraprost did not significantly alter this effect.To determine whether beraprost can inhibit human VSMC migration, primary cultured VSMC were seeded onto membranes in a modified Boyden chemotaxis chamber and incubated with beraprost. After 4 h in the chamber, cells had significantly migrated towards a chemogradient established by 10 ng/mL PDGF. Beraprost  significantly inhibited VSMC migration  (selective PKA agonist) or 8-(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate (8-Me-cAMP) (selective Epac agonist). All of these cAMP analogues inhibited PDGF-induced VSMC migration . The effects of 6-Phe-cAMP and 8-Me-cAMP were not additive  despite the concentrations used being submaximal . Differences in the total migration achieved with PDGF can be observed in Figure  compared with Figure . This is due to variation within the patient samples obtained.Since beraprost inhibits PDGF-induced VSMC migration, we investigated the downstream mediators involved. VSMC were incubated with cAMP analogues that preferentially activate either PKA or Epac and PDGF-induced migration was determined. VSMC were incubated with either 8-bromoadenosine 3′,5′-cyclic monophosphorothioate Sp-isomer (Sp-cAMPS) (Epac and PKA agonist), 3.327 This peak plasma concentration does not increase with multiple doses. To determine whether beraprost activates either Epac or PKA at this therapeutically relevant concentration, we transfected VSMC with fluorescence resonance energy transfer (FRET) sensors for Epac or PKA. Single cells were stimulated with either 1, 10, or 100 nmol/L beraprost and FRET activity for each probe was measured and expressed as relative FRET activity of the whole cell. 1 nmol/L beraprost induced a significant decrease in FRET activity (increase in sensor activation) of the Epac probe 1 min after incubation compared with vehicle-treated cells (drug added after 5 min of baseline measurement). This reached a peak between 2 and 5 min after beraprost addition and decreased thereafter . However, 1 nmol/L beraprost did not change the activity of the PKA FRET probe in VSMC over 15 min, indicating that PKA is not activated at this concentration . Additional experiments carried out over longer periods of up to 60 min duration also did not reveal any significant change in PKA activity as assessed by both the % change in FRET activity (not shown) or area under the curve . Similarly, incubation with 10 nmol/L beraprost did not result in a change in PKA FRET probe activity compared with untreated cells over a 60 min timecourse . Epac activation, however, was significantly increased over a 60 min timecourse by incubation with 10 nmol/L beraprost . Notably, higher concentrations of beraprost (100 nmol/L) produced a significant increase in both Epac and PKA FRET activity in cells transfected with the respective probes . To ensure that the observed differences between activity of the Epac and PKA FRET probes to beraprost were not due to different sensitivities of the different sensors, VSMC were incubated with increasing concentrations of forskolin, an adenylate cyclase activator and FRET activity was measured. Changes in the magnitude and timecourse of FRET activity were not significantly different comparing Epac and PKA probes in response to 1 and 10 µmol/L forskolin . These data suggest that Epac, but not PKA, is activated by a therapeutically relevant concentration of beraprost to inhibit VSMC migration.Pharmacokinetic studies of beraprost in humans have determined that the peak plasma concentration of beraprost which can be achieved is up to 345 ng/mL (∼1 nmol/L).ex vivo model of hyperplasia to determine whether Epac could prevent hyperplasia in blood vessels. Four sequential human saphenous vein segments (from the same vein) were either; (i) fixed immediately (control), (ii) cultured for 14 days in 30% FBS-containing medium, (iii) cultured for 14 days in serum-free medium with 50 µmol/L Epac analogue 8-Me-cAMP, or (iv) cultured for 14 days in 30% FBS-containing medium and 50 µmol/L Epac analogue 8-Me-cAMP. Subsequent analysis of saphenous vein paraffin-embedded sections subjected to H&E staining revealed that the serum-induced increase in vessel wall thickness was significantly reversed by incubation with the Epac analogue .In addition, we examined an 3.4Supplementary material online, Methods). Alternatively we targeted Rap1 expression. VSMC were incubated with siRNA-specific for Rap1A and Rap1B. Rap1 (both Rap1A and Rap1B) protein expression was significantly decreased by 81 ± 17% compared with cells incubated with scrambled siRNA . This decrease in Rap1 protein expression had no significant effect on PDGF-induced VSMC migration as assessed using a modified Boyden chamber . However, inhibition of migration by 1 nmol/L beraprost was significantly reversed in these cells compared with cells incubated with scrambled siRNA or control (untransfected) cells indicating the involvement of the Rap1 pathway in the anti-migratory effects of beraprost. To determine the potential involvement of PKA, VSMC were transfected with siRNA which resulted in 74 ± 11% decrease in PKA catalytic subunit protein expression . This knockdown in PKA protein expression did not produce any change in either PDGF-induced migration or the inhibitory effect of 1 nmol/L beraprost . To ensure that the level of decrease in PKA protein expression was sufficient to induce a functional change, we examined phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a known downstream target protein of PKA. In cells transfected with scrambled siRNA, 100 nmol/L beraprost incubation  resulted in an increase in VASP phosphorylation within 60 min . This increase was significantly inhibited in cells transfected with PKA siRNA.An siRNA-mediated decrease in the expression of Epac1 and Epac2 was not possible in human saphenous vein VSMC . Beraprost also resulted in Rap1 activation by ∼2-fold compared with control after 5 min incubation. This remained elevated at 10 min but was not significant .The above results suggest that beraprost inhibits migration via activation of the Epac/Rap1 pathway. To ensure that beraprost activated Rap1 in human saphenous vein VSMC, we used a pull-down assay specific for GTP-bound Rap1. Forskolin induced an increase in Rap1 activation as determined by elevated GTP binding within 5 min of incubation . VSMC on coverslips were subjected to a scratch and left for 24 h to induce migration. Cells migrating towards the scratch centre with a clearly visible leading edge were analysed. Epac1 was located predominantly at the leading edge . Epac2 had a more even distribution throughout the cell although it was also present at the leading edge. PKA, however, was found almost exclusively in the perinuclear region. Incubation with PDGF did not alter the localization of Epac1, Epac2 or PKA . Beraprost treatment also did not change the distribution of these proteins .To determine which isoforms of Epac are expressed in human saphenous vein VSMC, we performed immunocytochemistry using specific antibodies for Epac1 and Epac2. Immunoblots and real-time PCR determined that Epac1 and Epac2 are expressed in these cells (3.621 We firstly examined the role of RhoA in migration of human saphenous vein VSMC. The Rho-kinase inhibitor Y27632 (1 µmol/L) had a similar inhibitory effect on PDGF-induced migration compared with 8-Me-cAMP . This effect obtained at 1 µmol/L Y27632 was submaximal . This suggests that activation of the RhoA/Rho-kinase pathway is required for PDGF-induced migration. The effects of Y27632 and 8-Me-cAMP combined were not additive. Using Rhotekin pull-down assays specific for GTP-bound RhoA, we observed that PDGF activated RhoA in human VMSC over a 30 min timecourse . Co-incubation of the cells with 1 nmol/L beraprost significantly inhibited RhoA activation. In addition, 8-Me-cAMP inhibited PDGF-induced RhoA activation at the same concentration used to inhibit VSMC migration . As a comparison, the effect of the PKA agonist 6-Phe-cAMP on PDGF-induced RhoA activation was also examined . Direct PKA stimulation also inhibited RhoA activation, although this effect will not be involved in beraprost-induced inhibition of migration . As RhoA is an important regulator of the actin cytoskeleton, we determined whether beraprost might prevent PDGF-induced effects on F-actin. F-actin filaments observed at the leading edge in PDGF-treated cells, as assessed using immunolocalization, were significantly decreased following co-incubation with 1 nmol/L beraprost .As therapeutic concentrations of beraprost activated the Epac /Rap1 pathway, we examined potential downstream mechanisms which might contribute to the inhibitory effect on migration. Rap1 has previously been shown to inhibit RhoA in several cell types including some smooth muscle cell types.4.in vitro to determine the potential of cAMP-producing drugs to inhibit VSMC migration. Beraprost, a prostacyclin analogue, was studied as this has already been indicated as potentially useful in some cardiovascular disease states and has therefore direct clinical relevance.27 This was assessed at therapeutically relevant concentrations comparable with those achievable in patient plasma.27 We reveal that beraprost can inhibit growth-factor-induced VSMC migration at these therapeutic concentrations. The mechanisms involved in this inhibitory effect occur via activation of the cAMP-activated binding protein Epac but do not involve the well characterized and more typical cAMP effector PKA. In our experiments, PKA was not activated by beraprost at clinical concentrations and PKA siRNA did not inhibit the beraprost-induced anti-migratory response. These results demonstrate that cAMP-producing agonists such as beraprost may help slow restenosis following angioplasty by inhibiting VSMC migration through Epac activation.Preventing migration of VSMC after angioplasty/stenting or vein grafting following bypass surgery is a promising but untested therapeutic target. Drugs which target VSMC migration would decrease the growth of neointima and thereby reduce restenosis. In the current study, human saphenous vein VSMC was examined 23 Activation of Epac via either a direct Epac activator or a cAMP-producing membrane receptor agonist, such as isoproterenol, can produce a vasodilatory effect. This occurs via an increase in Rap1 activity and subsequent inhibition of RhoA resulting in calcium desensitization of myosin II phosphorylation.21 In addition, Epac can also activate calcium-sensitive potassium channels in arteries leading to hyperpolarization which decreases calcium entry via voltage-gated calcium channels and also results in vasodilation.22 It is not clear whether this effect on potassium channels is dependent on Rap1 signalling. Additionally, Epac can inhibit proliferation in rat and human VSMC.28 This effect, at least in rat VSMC, is synergistic with PKA as Epac activation alone was not sufficient to inhibit the proliferative effect.22 In the present study we did not detect any inhibition of human VSMC proliferation, even at relatively high concentrations of the cAMP-producing agonist beraprost, which can activate both PKA and Epac. However, our current study now indicates that Epac activation (in the absence of PKA activation) is sufficient to inhibit human VSMC migration. A recent contradictory study has indicated that a synthetic Epac activator enhances rat VSMC migration, although therapeutically relevant concentrations of cAMP, such as those generated by cAMP-producing agonists, were not assessed.29 This is in contrast to most studies which indicate a protective role for cAMP signalling in the vasculature. We have also examined migration in rat VSMC and although no enhancement was observed with a synthetic Epac agonist, we also could not reproduce our results with regard to the inhibitory effects on human VSMC migration (results not shown). This suggests that there are clear species differences which will be important in determining translational applicability. Regardless, we provide evidence here that Epac is a novel drug target which could potentially be exploited to limit restenosis.Until very recently, the functional roles of Epac in VSMC were unknown. Studies using Epac selective activators have now indicated that Epac may be involved in the vasculoprotective effects of cAMP, such as vasorelaxation of VSMC and an inhibition of VSMC proliferation.in vivo), only Epac is activated while at higher beraprost concentrations both Epac and PKA are activated. This indicates that lower levels of cAMP production lead to Epac activation alone, although this was not directly measured in our study. Such data correspond with that previously observed in cardiac fibroblasts and provides further evidence for the relevant physiological cAMP affinities for Epac vs. PKA.19 Other than these potential differences in cAMP affinity, we have also uncovered further evidence which indicates divergent functions of Epac and PKA. Our study demonstrates that Epac1 is localized predominantly at the leading edge of migrating VSMC cells and is therefore situated at the main site for directional migration. This is in contrast to PKA which has a perinuclear localization relatively distant from both the leading and trailing edge. These distinct localizations may indicate a compartmentalization of signalling with regard to these mediators. Both the potentially lower cAMP affinity together with differences in localization indicate that Epac1 may be more likely (compared with PKA) to be part of the regulation of migration, acting as a brake on leading edge progression.An important finding of this study is that Epac and PKA are differentially regulated in VSMC by varying the concentration of a cAMP-producing agonist. At lower beraprost concentrations . This likely occurs via Rap1-mediated inhibition of RhoA. Evidence for an important role of Rap1 in VSMC function has been recently demonstrated using Rap1b−/− mice. These mice develop hypertension in part via functional changes to VSMC and this indicates an essential role of Rap1 (and possibly Epac) in maintaining normal vascular physiology.30 With regard to the Rap1-induced inhibition of RhoA, other studies have previously demonstrated this link although not in relation to VSMC migration.21 We now provide evidence of the importance of this Rap1/RhoA mechanism in a pathologically relevant process and this is summarized in Figure 7.Although studies have demonstrated the potential importance of Epac in VSMC function, the downstream signalling of Epac is highly relevant to fully understand the mechanisms involved. We reveal that the inhibition of migration induced by Epac is dependent on the subsequent activation of Rap1 which in turn inhibits RhoA activation. The RhoA/Rho-kinase pathway is essential for PDGF-induced migration in human VSMC and is a well-described regulator of the cell cytoskeleton.27 It must be acknowledged that, in addition to the short half-life, beraprost also has side-effects that could preclude this drug from becoming a routine option following angioplasty.31 However, recent developments in technology, including drug-eluting stents and encapsulation of beraprost in slow release nanoparticles, now provide a route of administration which results in more targeted action with lower systemic doses required.32 Such improvements in drug delivery would decrease side-effects and increase its therapeutic potential. Beraprost is generally cardioprotective via its vasodilatory and anti-platelet effects. It is a prostacyclin analogue which binds to prostanoid receptors leading to activation of adenylate cyclase via the heterotrimeric G-protein, Gs. It has a relatively short half-life in vivo and therefore the maximum concentration which can be achieved in plasma is limited to ∼1 nmol/L.27 We now demonstrate in human VSMC that this therapeutically relevant concentration of beraprost activates Epac. This suggests that many of the clinically beneficial effects may in fact arise from Epac activation. Our results also indicate that beraprost activation of Epac/Rap1 may be useful in preventing restenosis following angioplasty by blocking VSMC migration. Indeed a recent study has examined the effects of beraprost in an in vivo model of in-stent restenosis.12 These authors have demonstrated that intravenous beraprost treatment during stent emplacement surgery significantly limited neointimal thickening after 4 weeks although the intracellular mechanisms were not assessed. Our results now suggest that an inhibition of migration may explain the observed beneficial effects of prostacyclin analogues in vivo.12 Together these data provide evidence of a potential therapeutic benefit of this drug target in restenosis.An important part of our study has demonstrated that the clinically approved drug beraprost activates Epac at therapeutically relevant concentrations. Orally-active beraprost is currently used in the treatment of pulmonary hypertension in some countries and may have clinical benefits in peripheral vascular disease.In conclusion, we have revealed, for the first time, that therapeutically relevant concentrations of the prostacyclin analogue beraprost can inhibit VSMC migration via activation of the cAMP effector Epac. The mechanism of this Epac-mediated inhibition is via activation of Rap1 and subsequent inhibition of RhoA preventing actin cytoskeletal changes. This indicates that cAMP-producing agonists such as beraprost may be beneficial following angioplasty to prevent neointimal hyperplasia.Cardiovascular Research online.Supplementary material is available at Conflict of interest: none declared.British Heart foundation (grant FS/11/23/28730). J.S.M. was funded by a British Heart Foundation PhD studentship. Funding to pay the Open Access publication charges for this article was provided by the Charities Open Access Fund (UK).This work was supported by the


Fasting is characterised by profound changes in energy metabolism including progressive loss of body proteins. The underlying mechanisms are however unknown and we therefore determined the effects of a 72-hour-fast on human skeletal muscle protein metabolism and activation of mammalian target of rapamycin (mTOR), a key regulator of cell growth.Eight healthy male volunteers were studied twice: in the postabsorptive state and following 72 hours of fasting. Regional muscle amino acid kinetics was measured in the forearm using amino acid tracers. Signaling to protein synthesis and breakdown were assessed in skeletal muscle biopsies obtained during non-insulin and insulin stimulated conditions on both examination days.Fasting significantly increased forearm net phenylalanine release and tended to decrease phenylalanine rate of disappearance. mTOR phosphorylation was decreased by ∼50% following fasting, together with reduced downstream phosphorylation of 4EBP1, ULK1 and rpS6. In addition, the insulin stimulated increase in mTOR and rpS6 phosphorylation was significantly reduced after fasting indicating insulin resistance in this part of the signaling pathway. Autophagy initiation is in part regulated by mTOR through ULK1 and fasting increased expression of the autophagic marker LC3B-II by ∼30%. p62 is degraded during autophagy but was increased by ∼10% during fasting making interpretation of autophagic flux problematic. MAFbx and MURF1 ubiquitin ligases remained unaltered after fasting indicating no change in protesomal protein degradation.Our results show that during fasting increased net phenylalanine release in skeletal muscle is associated to reduced mTOR activation and concomitant decreased downstream signaling to cell growth. The ethical committee of central region Denmark approved the study.Data from the present study on growth hormone (GH) signaling and insulin sensitivity have been published previously 2. The subjects did not take prescription medicine.Eight healthy men with no family history of diabetes participated. The average age was 26±4 years, body weight 82.9±8.8 kg and body mass index 23.8±1.6 kg/mThe protocol is visualized in Intravenous catheters  were placed for infusions and blood sampling. One was placed in a dorsal hand vein for sampling, and the hand was placed in a 65°C heated box for arterialization of the blood. In the same arm, a catheter was placed in an antecubital vein for infusions. Furthermore, a third catheter was placed in the contralateral antecubital vein in a retrograde direction for deep venous samples. Before deep venous sampling the wrist cuff was inflated to a supra systolic pressure.15N]phenylalanine, [2H4]tyrosine, [15N]tyrosine and [13C]urea  were used as amino acid tracers. The only metabolic fate of phenylalnine and tyrosine in muscle is protein synthesis and degradation 15N]phenylalanine (0.7 mg/kg), [15N]tyrosine (0.3 mg/kg), [2H4]tyrosine (0.5 mg/kg) and [13C]urea (309.6 mg), continuous infusions of [15N]phenylalanine (0.7 mg/(kg*h)), [2H4]tyrosine (0.5 mg/(kg*h)) and [13C]urea (42 mg/h) were maintained for 4 hours. After 210 min. with continuous infusions, steady state was accomplished and blood samples were taken in triplicate during the last 30 min. of the period. Solutions were prepared under sterile conditions and were tested free of bacteria and pyrogens before use. Venous occlusion plethysmography was used to determine forearm blood flow.Subjects were studied for 6 h from 0800 to 1400 h (t = 0 to 360 min.). During the first 4 hour-basal period (t = 0–240), phenylalanine, [2H4]tyrosine, [15N]tyrosine and [13C]urea enrichment were measured using gas chromatography-mass spectrometry (GC-MS) and concentrations of phenylalanine and tyrosine were measured  using l-[2H8]phenylalanine and l-[13C6]tyrosine as internal standards [Qphe) and tyrosine flux (Qtyr) were calculated: i is the rate of tracer infusion (µmol/(kg*h)), Ei is enrichment of the tracer infused and Ep is the plasma enrichment of the tracer at isotopic plateau Whole body phenylalanine flux (Ipt) was calculated: 15N-Tyrei and 15N-Pheei are the isotopic enrichments of the respective tracers in plasma and Iphe is the infusion rate of 15N-phenylalanine (µmol/(kg*h))The rate of phenylalanine conversion by hydroxylation to tyrosine  was calculated: Ea and PheEv represent phenylalanine isotopic enrichment in arterialized and venous blood, respectively phe), which represents the muscle protein synthesis rate, was calculated as: In this study, net phenylalanine release was calculated as follows using Fick's principle: 4P2O7, 10 NaF, 2 EDTA, 1 MgCl2, 1 CaCl2, 2 Na3VO4, 1% (vol/vol) Nonidet P-40, 10% (vol/vol) glycerol, 2 µg/ml aprotinin, 5 µg/ml leupeptin, 0.5 µg/ml pepstatin, 10 µg/ml antipain, 1.5 mg/ml benzamidine, and 100 µM 4-(-2-aminoethyl)-benzenesulfonyl fluoride, hydrochloride (pH 7.4), and samples were rotated for 60 min at 4°C. Insoluble materials were removed by centrifugation at 16,000×g for 20 min at 4°C. Western blot analyses were used to assess protein and phosphorylation levels of various proteins. Primary antibodies against mTOR, p-mTOR(Ser2448), 4EBP1, non-p-4EBP1(Thr46), ULK1, p-ULK(Ser757), rpS6, p-rpS6(Ser235-236), TSC2, p-TSC2(Ser1387), eIF2α, p-eIF2α(Ser51), FOXO3a, p-FOXO3a(Ser318-321), light chain LC3B (LC3B) and β-actin were from Cell Signaling Technology . p62/SQSTM1/sequestosome-1, MAFbx and MURF1 antibodies were from Abcam .Muscle biopsies were homogenized in an ice cold buffer containing (in mM) 50 HEPES, 137 NaCl, 10 NaHorseradish peroxidase (HRP)-conjugated goat antirabbit or goat antimouse IgG secondary antibody or HRP-streptavidin was used, and proteins were visualized by BioWest enhanced chemiluminescence (Pierce) and quantified using UVP BioImaging System . Unless otherwise mentioned, quantifications of total protein are expressed as ratio of protein to β-actin and protein phosphorylation as a ratio of total protein expression measured on the same membranes. Tissue amount only allowed measurements of 4EBP1, ULK1, MAFbx, and MURF1 in tissue from 6 out of 8 participants.Results are expressed as means ± SE. Normal distribution was assessed by inspection of QQ-plots, and the Levene Median test was used to test for equal variance. Isolated comparisons between control and 72 hours of fasting were assessed by a paired t-test. Comparisons between the main effects and interaction of 72 hours of fasting and insulin stimulation were assessed by a two-way repeated measurements ANOVA. When the two-way repeated-measurements ANOVA revealed significant differences, post-hoc test multiple comparison procedures using Student-Newman-Keul's method were performed. Correlations were measured using pearson product-moment correlation. P<0.05 was considered significant.During fasting FFA concentration was increased by ∼100% and glucose concentration was decreased ∼25%. These changes in metabolic substrates were accompanied with a significant decrease in the circulating levels of insulin, c-peptide and triiodothyronine, and increased glucagon levels. Comparable levels of insulin and glucose were achieved 30 minutes into the hyperinsulinemic euglycemic clamp on both occasions, but glucagon and FFA were not normalized at this time-point .phe  with no change in Raphe   . Whole beakdown) .2448 on mTOR by ∼40% (p<0.05). Insulin stimulated mTOR phosphorylation on both examination days, but the increase during fasting was substantially reduced (46 (observed by increased non-phosphorylated 4EBP1) and ULK1Ser757 (235-236 phosphorylation expressed as ratio to β-actin was decreased (Fasting significantly reduced phosphorylation of the activating site Ser reduced . To testK1Ser757  and 4A. K1Ser757 . Howeverecreased .2448 phosphorylation correlated with changes in downstream phosphorylation (non-phospho-4EBP1(Thr46) Correlation Coefficient (CC) = −0.67, p = 0.005; ULK1(Ser757) CC = 0.58, p = 0.017; rpS6(Ser235-236)/β-actin CC = 0.51, p = 0.012). In addition, mTOR phosphorylation was correlated  with Ser473 phosphorylation on previously examined Akt Insulin increased phosphorylation on all examined target-sites downstream of mTORC1 , and 4A.1387 was not altered by fasting or insulin stimulation (51 phosphorylation showed significant interaction (p = 0.048) between fasting and insulin stimulation after the 72-hour-fast and post-hoc testing showed a trend (p = 0.059) toward decreased phosphorylation after 72 hours of fasting  phosphorylation at the AMP-activated protein kinase (AMPK) target site Sermulation . Eukaryo fasting . In addi757. Consequently, we examined markers of autophagy and fasting increased light chain 3 (LC3)B-II protein content by ∼30% compared to LC3B-I. Insulin stimulation reduced LC3B-II levels on both experimental days with no effects of fasting  in parallel with decreased mTOR Ser2448 phosphorylation. This phosphorylation is essential for mTORC1 downstream regulation of ribosomal mRNA translation 235-236 phosphorylation through ribosomal protein S6 kinase of 70 kDa (p70S6K), and this phosphorylation site was also decreased after 72 hours fasting when expressed as ratio of β-actin. The reduced phosphorylation on mTOR Ser2448 together with decreased phosphorylation on all examined downstream mTORC1 target sites clearly indicate reduced activity of this complex after fasting. The decreased mTORC1 activity is in accordance with the observed increase in amino acid release from the forearm and the trend towards reduced phenylalanine rate of disappearance, as well as previous studies describing mTORC1 and protein metabolism in other models The current study demonstrates that increased skeletal muscle net phenylalanine release following a 72-hour-fast is associated with decreased mTOR activity. The mTORC1 complex is a key regulator of cell growth and anabolism, and has been extensively studied in cell lines and during different conditions of nutrient availability 473, a positive correlation between mTOR Ser2448 and Akt Ser473 phosphorylation was observed. This positive correlation indicates a connection between the regulation of these two proteins during fasting and insulin stimulation. Our study does not define the hormonal mechanisms regulating the observed alterations of intramyocellular protein metabolic signaling. However, insulin inhibits muscle protein breakdown and activates Akt and mTOR phosphorylation 473 and mTOR Ser2448 phosphorylation was increased on both examination days. Interpretation of the hierarchical order of these phosphorylations is impossible because skeletal muscle biopsies only were obtained once during insulin stimulation.Even though no fasting effect was observed on the validated mTORC2 downstream target site, Akt Ser2448 and rpS6 Ser235-236 (expressed as ratio of β-actin) did not respond to the same extend after 72 hours fasting as after the overnight fast, indicating insulin resistance in this part of the insulin signaling pathway. Consequently, we examined modulators of the signal downstream of Akt. To elucidate whether the reduced phosphorylations could be explained by changes in AMP/ATP ratio, we also examined the AMPK target site, upstream of mTORC1, Ser1387 on TSC2 1387 phosphorylation. This is in accordance with the previously reported unaltered AMPK activity after 72 hours fasting 51 phosphorylation. If anything – we found a trend towards decreased phosphorylation of eIF2α after 72 hours fasting, indicating that the increased net phenylalanine release is not due to eIF2α repression of translation. It can be assumed that venous phenylalanine concentrations reaches equilibrium with intramyocellular concentrations across the capillary bed. The present unchanged venous phenylalanine concentrations and absence of increased eIF2α phosphorylation after 72 hours fasting suggests that the decreased mTORC1 activity is not due to decreased amino acid concentrations.Insulin stimulated phosphorylation on mTOR Ser757 phosphorylation. Phosphorylation of ULK1 at Ser757 has previously been reported as a direct mTORC1 phoshorylation site involved in mTORC1 mediated suppression of autophagy 757 phosphorylation as a mechanism by which autophagy is activated in response to fasting. AMPK has also been shown to increase autophagy by activating ULK1 through phosphorylation of distinct sites 757 during insulin stimulation, suggesting mTORC1 mediated phosphorylation of ULK1 as a potential mechanism by which insulin suppresses autophagy in human skeletal muscle. However, p62 levels remained unaltered during insulin stimulation, and we are therefore unable to determine whether or not autophagic flux is affected. In addition to enzymatic control, autophagy has also been shown to be transcriptionally regulated by forkhead box O (FOXO) 3a through a mechanism phosphorylating Ser318318 phosphorylation remained unaltered in response to fasting and insulin stimulation. This finding either suggests that the divergence between our LC3B-II and p62 findings reflects unaltered autophagic flux, or that the autophagic response to fasting and insulin are independent of FOXO3a Ser318 phosphorylation. Collectively, our data suggest that LC3B lipidation is controlled by a mechanism that involves mTORC1 mediated phosphorylation and dephosphorylation of ULK1 at Ser757 in response to fasting and insulin stimulation in human skeletal muscle. However, due to the conflicting nature of the data used to determine autophagic flux, we are unable to associate these findings to the increased skeletal muscle net phenylalanine release following a 72-hour-fast.Autophagy is a catabolic process that delivers proteins to the lysosomes for degradation and is regulated in response to changing nutrient and energetic conditions. Studies in mice indicate a pivotal role for autophagy in surviving short periods without nutrient supply phe  was observed after 72 hours fasting we measured MAFbx and MURF1 protein expression, because amino acids from increased protein breakdown could be used for incorporation in newly synthesized proteins and therefore escape detection by the tracer dilution method employed. However, in accordance with previously reported unchanged MAFbx and MURF1 mRNA levels in human skeletal muscle after fasting for 40 hours In parallel with autophagy being essential for mice to survive nutrient deprivation, fasted mice do also show upregulation of ubiquitin protein E3-ligase increased proteasome mediated protein degradation Arteriovenous amino acid kinetics across muscle beds assessing net muscle breakdown, rate of protein synthesis and protein breakdown have been used to define the muscle response to fasting but have produced conflicting results. It has been reported that net muscle amino acid release increases with fasting In conclusion, by use of a phenylalanine tracer we observed increased net skeletal muscle breakdown and a trend towards a decrease in human skeletal muscle protein synthesis after 72 hours fasting. This was associated with reduced mTOR activation and concomitant decreased downstream signaling to cell growth.


Our results suggest the prime binding site for fluoxetine on BC to be at or near tryptophan residues. Fluoxetine quenched the fluorescence by a static process, which specifically indicates the formation of a complex. Cystatins constitute a superfamily of homologous proteins. The major role of cystatins is to regulate the unwanted proteolysis and to protect the organism against endogenous proteases released from lysosomes, invading microorganisms and parasites that use cysteine proteases to enter the body. Imbalance in regulation of proteolytic activity may lead to a wide range of human diseases. An enormous progress has been made in understanding of protein degradation process under normal and pathological conditions; infact proteases are now clearly viewed as important drug targets. Fluoxetine a selective serotonin reuptake inhibitor (SSRI) is an antidepressant. It is used to treat major depressive disorders.In the present study binding of fluoxetine to cystatin was studied by UV and fluorescence quenching technique. Intrinsic fluorescence of fluoxetine complexed with purified buffalo brain cystatin (BC) was measured by selectively exciting the tryptophan residues. Gradual quenching was observed on complex formation. When cystatin was added to fluoxetine solutions at a molar ratio of 1 : 0.5, it not only quenched more than half of its fluorescence but also reduced the activity of cystatin. Stern-Volmer plots obtained from experiments carried out at 25 The most studied inhibitors of the papain family are the cystatins. They are present in mammals, birds, insects, plants, and protozoa. They function both intracellularly and extracellularly. Cystatins are competitive, reversible, and tight binding protein inhibitors which display structural and functional similarities. They serve a protective function by regulating the activities of endogenous proteinases, which if not regulated may cause uncontrolled proteolysis and damage to cells and tissues. On the basis of homology, inhibition of target enzymes and presence or absence of disulphide bonds, cystatin superfamily has been divided into three families. Family I also called as stefins include members of low molecular weight proteins (approximately 11 KDa), which lack disulphide bonds and carbohydrate content. This family includes cystatin A, cystatin B, stefin C, and stefin D. Family II known as cystatin family is represented by the inhibitors of a bit larger molecular weight proteins (approximately 13 KDa) compared to stefins and possesses disulphide bonds towards carboxyl terminal. This family comprises cystatins C, D, S, SN, E, F, and M. Family III or kininogens are higher molecular weight inhibitors containing both disulphide linkage and carbohydrate content. They are found only in blood plasma. There are three distinct types of kininogens designated as high molecular weight kininogen HK (MW120 KDa), low molecular weight kininogens LK (MW 50–70 KDa), and T-kininogen found only in rat plasma  can be used to determine K as well as n.When small molecules bind independently to set of equivalent sites on macromolecules, the equilibrium between free and bound molecules is given by the following equation , 22:(2)R is the gas constant (8.314(J/mol × K))8.314 Jmol × K, T is the temperature [K], and K is the equilibrium constant.The determination of the change of free enthalpy based on the Van't Hoff equation:μM while the drug concentration was varied for different drugs to different extent. Absorption spectra were recorded on a double beam Shimadzu UV-vis spectrophotometer UV-1700 using a cuvette of 1 cm path length.The UV measurement of brain cystatin in the presence and absence of antidepressants was made in the range of 200–300 nm and the inhibitor (cystatin) concentration was fixed at 1 μM) was incubated with increasing concentrations of drugs at 25°C for 30 min before the activity was measured. Activity of untreated BC was taken as 100%.The inhibitory activity of the purified inhibitor (BC) under native conditions was assessed by its ability to inhibit caseinolytic activity of papain by the method of Kunitz . The inhμM) in the presence of different concentrations of fluoxetine were recorded in the range of 300–400 nm upon excitation at 280 nm. The drug caused quenching of the intrinsic fluorescence of cystatin (μM of drug concentration leading to quenching up to 51%. These results indicated that there were interactions between fluoxetine and cystatin (BC); moreover the binding reactions resulted in nonfluorescent complex.In this study fluorescence spectra of Cystatin  of the interactions was calculated as described in Methods section. The value was found to be −38.2 KJ/mol showing the reaction to be spontaneous  in 50 mM sodium phosphate buffer pH 7.5 at room temperature for 30 min; its inhibitory activity was determined by caseinolytic assay of papain  with BSA.Fluorescence technique has been widely used for drug-protein interaction studies , 43. In han site . SimilarStudies on the binding mechanism between protein and small molecules provide useful information. For example, a detailed characterization of drug-protein binding properties was essential for understanding the function of drugs and hence interest in drug-protein interaction has attracted much attention and the development of drugs based on inhibition of cystatins has advanced into clinical testing with targeting compounds .The static type of quenching is indicative of a complex formation between the protein and the drug molecule.G0) is estimated for the interaction of fluoxetine with cystatin which is shown in (G0) support the assertion that the binding process is spontaneous.Stern-Volmer equation is used to study the interaction of cystatin with fluoxetine. The interaction forces between proteins and ligands may comprise hydrophobic, hydrogen bonds, van der Waals, and electrostatic interactions . The freshown in . The negμM fluoxetine , its inhibitory activity decreased in concentration dependent manner. On interaction with 0.5 μM fluoxetine 48% loss of cystatin activity was noticed (μM drug concentration 52% of inhibitor's activity was compromised. A drastic decline (90%) was noticed at 2 μM drug concentration.When 1  noticed . At 1 μMThis could be explained by the fact that fluoxetine is a competitive inhibitor of serotonin uptake which may interact with the same portion of the carrier protein responsible for the transport of serotonin; the structural features responsible for substrate-carrier protein recognition may be different from those responsible for inhibitor-carrier protein recognition . From th


PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear. We analyzed the effects of PDE10A inhibition in vivo by immunohistochemistry, and imaged cAMP, cAMP-dependent protein kinase A (PKA), and cGMP signals with biosensors in mouse brain slices. PDE10A inhibition in mouse striatal slices produced a steady-state increase in intracellular cAMP concentration in D1 and D2 MSNs, demonstrating that PDE10A regulates basal cAMP levels. Surprisingly, the PKA-dependent AKAR3 phosphorylation signal was strong in D2 MSNs, whereas D1 MSNs remained unresponsive. This effect was also observed in adult mice in vivo since PDE10A inhibition increased phospho-histone H3 immunoreactivity selectively in D2 MSNs in the dorsomedial striatum. The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition. These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs. This study shows that PDE10A inhibitors share with antipsychotic medications the property of activating preferentially PKA-dependent signaling in D2 MSNs.Type 10A phosphodiesterase (PDE10A) is highly expressed in the striatum, in striatonigral and striatopallidal medium-sized spiny neurons (MSNs), which express D Their activity is associated with either the initiation of movement or action suppression, respectively. Biosensor imaging revealed that pharmacological inhibition of type 10A phosphodiesterase increased cAMP levels in D1 and D2 neurons in the same manner, but only D2 neurons exhibited an increase in the protein kinase A-mediated phosphorylation level. This effect resulted from an asymmetrical regulation of phosphatases by DARPP-32. D2 neurons are thus more prone to respond to a tonic cAMP signal than D1 neurons, a property that may explain how phosphodiesterase 10A inhibitors produced antipsychotic-like behavioral effects. This D1/D2 imbalance may also be critical for reward-mediated learning and action selection.The striatum is mainly composed of medium-sized spiny neurons that express either dopamine D1 receptors (hereafter termed D1 MSNs); whereas, MSNs projecting to the external globus pallidus highly express adenosine A2A and dopamine D2 receptors  inhibitors were recently proposed as a treatment for schizophrenia ; howeverBesides PDE10A, striatal neurons express a number of specific signaling proteins that markedly differ from those in other brain regions , and tha2 receptors and thus, in the striatum, increase PKA-dependent phosphorylation selectively in D2 MSNs , controlled humidity (55 ± 10%), and food and water available ad libitum. Homozygous mice expressing DARPP-32 with the T34A or T75A mutation  were obtm): 125 NaCl, 0.4 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 25 glucose, and 1 kynurenic acid, saturated with 5% CO2 and 95% O2. The slices were incubated in this solution for 30 min and then placed on a Millicell-CM membrane (Millipore) in culture medium . We used the Sindbis virus as a vector to induce expression of the various biosensors after overnight incubation (H150 (5 particles per slice) was added, and slices were incubated overnight at 35°C under an atmosphere containing 5% CO2. Before the experiment, slices were incubated for 30 min in the recording solution . Recordings were performed with a continuous perfusion of the same solution at 32°C. MSNs constitute 95% of neurons in the striatum. Large neurons , presumably cholinergic interneurons, were excluded.Brain slices were prepared from male mice that were 8–12 days of age. Coronal brain slices were cut with a VT1200S microtome. Slices were prepared in an ice-cold solution of the following composition  tuned at 850 nm for CFP excitation. Galvanometric scanners  were used for raster scanning, and a piezo-driven objective scanner  was used for software . The micWide-field images were obtained with an Olympus BX50WI or BX51WI upright microscope with a 40× 0.8 NA water-immersion objective and an ORCA-AG Camera (Hamamatsu). Images were acquired with iVision (Biovision). The excitation and dichroic filters were D436/20 and 455dcxt. Signals were acquired by alternating the emission filters, HQ480/40 for CFP, and D535/40 for yellow fluorescent protein, with a filter wheel (Sutter Instruments). These filters were obtained from Chroma Technology.No correction for bleed-through or direct excitation of the acceptor was applied, since this correction, while increasing the absolute amplitude of ratio changes, also increases the noise in the measurement .The biosensor chromophores are sensitive to nonspecific environmental disturbances. We used a mutated version of AKAR3 in which the threonine residue of the PKA phosphorylation site was replaced with an alanine residue (T391A) as a control. This AKAR3 (T391A) control sensor reported no ratio change in response to PDE10A inhibition in MSNs.H150 sensors. The pseudocolor images display the ratio value coded in hue and the fluorescence of the preparation coded in intensity.Images were analyzed with custom routines written in the IGOR Pro environment (Wavemetrics). The emission ratio was calculated for each pixel, as follows: F535/F480 for AKAR2-NLS and AKAR3, and F480/F535 for cygnet2 and Epac-S1 and D2 MSNs, provided that the infection level was kept low and no fluorescence overlap between neighboring neurons was observed. The optical cross-contamination resulting from out-of-focus light was evaluated by the final response to CGS 21680 and SKF-38393, applied sequentially: cells were rejected from analysis if the cross-contamination was >30%. For cGMP imaging .H150 biosensor from the ratio measurement using the Hill equation, with a Kd of 4.4 µm and a Hill coefficient of 0.77, as determined from Rmax) was determined for each neuron at the end of the recording. This level was obtained by applying 13 µm forskolin (FSK), a dose known to be sufficient to maximally phosphorylate the highly sensitive probe AKAR3 in MSNs. For cAMP biosensors, this Rmax value was obtained with 200 µm IBMX and 13 µm forskolin.The free cAMP concentrations were estimated with the Epac-Sm SQ22536, which resulted in a ratio decrease, measured in wide-field microscopy, of −4.0% of the maximal ratio change. This decrease in baseline ratio was −4.9 ± 0.7 and −3.3 ± 0.7 , respectively, in D1 and D2 MSNs. Assuming that adenylyl cyclase inhibition effectively decreased cAMP levels down to a level sufficient to reach the minimal ratio level (Rmin), these values suggest a baseline cAMP concentration in a range of ∼100 nm.The baseline cAMP level in control conditions was evaluated by inhibiting adenylyl cyclases with 200 µm PBS, pH 7.5. Brains were post-fixed overnight in the same solution and stored at 4°C. The 30-μm-thick sections were cut with a vibratome and stored at −20°C in a solution containing 30% (v/v) ethylene glycol, 30% (v/v) glycerol and 0.1 m sodium phosphate buffer, until they were processed for immunofluorescence. Sections were processed as described in m was included in all buffers and incubation solutions. Histone H3 phosphorylation was revealed with a rabbit polyclonal antibody against phospho-Ser10-H3  the specificity of which was confirmed in a previous study (m Tris) before mounting in 1,4-diazabicyclo-[2. 2. 2]-octane (Sigma-Aldrich).Mice, 8-10 weeks old, were treated with the drug for 60 min and then rapidly anesthetized with pentobarbital  and were transcardially perfused with 4% (w/v) paraformaldehyde in 0.1 us study . GFP wasus study  and goatus study . SectionSingle-labeled images  were obtt test for comparing two groups. Paired data were analyzed with a Student’s t test. A p value of <0.05 was used as a cutoff for statistical significance. All error bars represent the SEM; n indicates the number of experiments , with at least four neurons of each type in each experiment. All experiments were performed on at least three different brain slices from at least two animals.Data were analyzed with SPSS statistical software version 22.0. Normality in variable distributions and homogeneity of variances across groups were assessed with the Shapiro–Wilk and Levene tests, respectively. Variables that failed any of these tests were analyzed with nonparametric statistics using the Kruskal–Wallis ANOVA on ranks followed by Mann–Whitney rank sum test with a Dunn–Sidak adjustment test for pairwise multiple comparisons. Variables that passed the normality test were analyzed with ANOVA followed by Bonferroni post hoc test for multiple comparisons or by Student’s SKF-38393 hydrobromide, CGS 21680 hydrochloride, 1-methyl-3-isobutylxanthine (IBMX), rolipram, papaverine, roscovitine, okadaic acid, cantharidin, gabazine, CNQX, APV, and forskolin were obtained from Tocris Cookson. TTX was from Latoxan. PQ-10, MP-10, and roflumilast were a gift from Janssen Pharmaceuticals. TP-10 was provided by Pfizer through the Compound Transfer Program.1 and D2 MSNs express high levels of PDE10A protein  and an agonist of dopamine D1 receptors  were applied sequentially, triggering a positive cAMP response in D2 and D1 MSNs, respectively, thereby functionally identifying MSN subtypes. The final application of the general adenylyl cyclase activator FSK (13 µm) together with the nonselective phosphodiesterase inhibitor IBMX (200 µm) produced the maximal ratio response used for normalization.Since both D protein , we usedm) increased cAMP levels in all MSNs (1 and D2 MSNs (m), cAMP responses reached ∼35% of the maximal response to FSK plus IBMX, which corresponds to a concentration of free cAMP of ∼2 µm . These experiments showed that, in the basal condition, cAMP is tonically produced in striatal slices and that PDE10A contributes significantly to its degradation.PDE10A inhibition with PQ-10  strongly increased the emission ratio of AKAR3 in the D2 MSNs ; whereas, in D1 MSNs, the ratio remained at a much lower level (6 ± 2%). These results indicated a significantly higher phosphorylation of AKAR3 in D2 than in D1 MSNs in response to PQ-10. The effect of PQ-10 on AKAR3 ratio in D2 MSNs was steeply dose dependent with a maximal effect reached at <100 nm  completely reversed the PQ-10-induced AKAR3 response in D2 MSNs, monitored with wide-field microscopy (2 MSNs with 11 ± 3% (n = 5) of the maximal FSK response in D2 MSNs compared with 9 ± 2% in D1 MSNs  and papaverine (1 µm) on AKAR3 ratio were similar . In contrast, PDE4 inhibitors  had no effect on basal AKAR3 ratio (data not shown).We then determined whether the effect selectivity for D similar C,D, ind2 MSNs express adenosine A2A receptors, we examined whether the tonic presence of extracellular adenosine in our brain slice preparation activated adenylyl cyclase and might be responsible for the positive response to PQ-10 recorded specifically in these MSNs. When A2A receptors were blocked with SCH 58261 (100 nm), PQ-10 still elicited positive responses in D2 MSNs (2A agonist CGS 21680 (data not shown). These results showed that tonic activation of A2A receptors was not required for the phosphorylation response to PQ-10 in D2 MSNs. We then examined whether the phosphorylation signal involved endogenous neuronal activity, or required the glutamate and GABA which may be present in the brain slice. This was not the case since, in the presence of blockers of voltage-gated sodium channels , calcium channels , non-NMDA receptors , NMDA receptors , and GABAA receptors , the selective effect of PQ-10 on D2 MSNs was still present .As D D2 MSNs E. In coin vivo (m) had no effect on the baseline cGMP levels . In addition, PQ-10 (1 µm) had no effect on the cGMP steady-state level obtained with the nitric oxide (NO) donor DEANO , a nonselective inhibitor of PP-1 and PP-2A, had no effect by itself on the AKAR3 emission ratio (m) was applied in the bath . Since these responses did not return to the baseline after drug washout, it was impossible to distinguish between D1 and D2 MSNs, and all MSNs were pooled (m), a selective inhibitor of PP-2A , whereas, as in wild-type mice, no effect was observed in D1 MSNs  had no effect on the D1/D2 imbalance in the response to PQ-10 , a substrate for several protein kinases including PKA , a PDE10A inhibitor known to produce clear behavioral effects (2 MSNs) in the dorsomedial striatum, whereas in lateral parts of the striatum both GFP-negative and GFP-positive D2 MSNs exhibited PH3 immunoreactivity  were similar, with a strong effect of TP-10 in D2 MSNs but not in D1 MSNs  or vehicle  and brain sections analyzed by immunofluorescence for PH3 60 min after injection. Whereas wild-type littermates strongly responded to TP-10, the effect of TP-10 was completely abolished in the DARPP-32 T34A mutant mice in all regions of the striatum (1 and D2 MSNs in both the dorsomedial and dorsolateral striatum in vivo.Our experiments in striatal slices showed that the inhibition of PP-1 by DARPP-32 phosphorylated on Thr34 was necessary for the selective responsiveness to PDE10A inhibition on AKAR phosphorylation. We then investigated whether the effects of PDE10A inhibition striatum A,B. The2 MSNs than in D1 MSNs. We provide evidence that this difference is present in all striatal regions in brain slices and in the dorsomedial striatum of adult mice in vivo. Moreover, we show that the regulation of PP-1 activity by DARPP-32 is required for these specific effects of PDE10A inhibitors. These observations provide novel insights into the regulation of the cAMP/PKA pathway in the two populations of MSNs and the possible antipsychotic action of PDE10A inhibitors.Our study shows that although PDE10A is expressed and functional in all types of MSNs, its inhibition in striatal slices produces a higher PKA-dependent protein phosphorylation response in D1 and D2 MSNs. MSNs thus contrast with pyramidal neurons of the prefrontal cortex in which basal cAMP is predominantly controlled by PDE4 (2 MSNs (PDE10A is one of the enzymes specifically enriched in the striatum , and the by PDE4 . Althoug (2 MSNs , biosensIn vivo, PDE10A inhibition was shown to increase cGMP levels and to affect synaptic transmission (in vivo, PDE10A inhibitors recruit nitric oxide synthase-positive striatal interneurons through a global increase in network activity.PDE10A inhibition showed no effect on basal or stimulated cGMP production. A similar lack of effect of PDE10A inhibitors on cGMP has already been reported in brain slice preparations . PDE10A smission , and why1 and D2 MSNs share a number of cellular features, more detailed studies revealed subtle differences (A receptors and DARPP-32, which were higher in D2 MSNs than in D1 MSNs (1/D2 differences, which lies at the level of DARPP-32-mediated PP-1 regulation. In D1 MSNs, the Thr34 of DARPP-32 is in a lower phosphorylation state than in D2 MSNs and, thus, the PP-1 activity reverts PKA target sites to the dephophorylated state (1 MSNs. Thr34 residue is efficiently dephosphorylated by both PP-2A and PP-2B (but not by PP-1; 1 and D2 MSNs. In contrast to tonic cAMP levels induced by PDE10A inhibition, cAMP signals elicited by D1 receptor stimulation lead to a phosphorylation of T34 and inhibition of PP-1 (1 MSNs, PDE10A inhibition only affects PKA-dependent modulation of synaptic transmission when cAMP production is stimulated (1 MSNs may improve the detection of powerful but brief events such as the phasic dopamine signal associated with reward and novelty (While Dferences , such asferences . Differe D1 MSNs . Our wored state . This hy of PP-1 , an effe of PP-1 . This isimulated . This no novelty , while f2 MSNs DARPP-32 is phosphorylated on the Thr34 residue, as previously demonstrated (2 MSNs fail to respond to PDE10A inhibition (In contrast, in Dnstrated . In thishibition .1 and D2 MSNs that may contribute to the higher level of DARPP-32 phosphorylation on Thr34.Further work is needed to analyze the possible differences in PP-2A and PP-2B activities between D1/D2 differences is the phosphorylation of DARPP-32 on Thr75, which is catalyzed by Cdk5 and is responsible for PKA inhibition (2 MSNs, since we found no alteration of these responses in DARPP-32 T75A knock-in mutant mice.Another potential player in the Dhibition . Since Thibition , it coul2 MSNs at the level of PKA signaling is opposed in vivo by the activity of D2 receptors: the simple blockade of these receptors by D2 antagonists strongly activates cAMP-dependent phosphorylation (2 MSNs, and not in D1 MSNs (2 MSNs is functionally important for some effects of antipsychotic drugs since conditional knockout of DARPP-32 in D2 MSNs leads to an increased locomotor activity and a strongly reduced catalepsy upon administration of D2 receptor inhibitors (2 receptors since both potentiate the PKA pathway selectively in D2 MSNs. The particular sensitivity of D2 MSNs includes DARPP-32 Thr34 phosphorylation, which is more increased by PDE10A inhibitors in this population than in D1 MSNs (2 MSNs to the blockade of either D2 receptors or PDE10A activity.The increased responsiveness of Drylation . Accordi D1 MSNs . This rohibitors . Our dat D1 MSNs . Thus, hin vivo (in vivo (The cellular effects of PDE10A inhibition affect MSNs neuronal properties, which are integrated through the basal ganglia network. For example, PDE10A inhibition was shown to potentiate D-amphetamine-dependent dopaminergic neuromodulation in vivo . In addi(in vivo , whereasIn vivo, the inhibition of PDE10A also selectively activates D2 MSNs in the medial striatum. Interestingly, this striatal subregion is innervated by prefrontal and cingulate cortices, which are involved in the limbic system, and dopamine neurons originating from the ventral tegmental area. D1 and D2 receptors exert contrasting roles selectively in the dorsomedial striatum during behavioral inhibition in the stop-signal task in rats ( in rats , and les in rats . This re in rats . Medial 1/D2 difference is present in brain slices in both medial and lateral striatum, and remains when network activity is blocked, showing that the D1/D2 difference is an intrinsic property of D1 and D2 MSNs. The in vivo effects of PDE10A inhibitors were totally abolished in T34-DARPP-32 mutant mice, confirming the role of the DARPP-32/PP-1 loop as the initial determinant of the positive PKA response obtained during PDE10A inhibition.The differences observed between medial and lateral striatum likely involve network effects and differences in synaptic plasticity reported between these two striatal subregions , and fur1 and D2 MSNs might be of interest to understand the pathophysiology of other diseases that affect neuromodulatory processes in basal ganglia such as Parkinson’s disease or Huntington’s disease (Further work is needed to determine whether the imbalance in PKA signal integration between D disease .


JAK2 mutation observed in these diseases (V617FJAK2) enforces Mcl-1 transcription via STAT3 signaling. Targeting this lesion with JAK inhibitor I (JAKi-I) attenuates STAT3 binding to the Mcl-1 promoter and suppresses Mcl-1 transcript and protein expression. The neutralization of Mcl-1 in V617FJAK2-harboring myelodyssplastic syndrome cell lines sensitizes them to apoptosis induced by the BH3-mimetic and Bcl-xL/Bcl-2 inhibitor, ABT-263. Moreover, simultaneously targeting JAK and Bcl-xL/-2 is synergistic in the presence of the V617FJAK2 mutation. These findings suggest that JAK/Bcl-xL/-2 inhibitor combination therapy may have applicability in a range of hematological disorders characterized by activating JAK2 mutations.Constitutive activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) axis is fundamental to the molecular pathogenesis of a host of hematological disorders, including acute leukemias and myeloproliferative neoplasms (MPN). We demonstrate here that the major  FLT3, KIT, JAK2, JAK3, and MPL. Though several oncogenic JAK2 variants have been described, JAK2 mutation manifests primarily as a single non-conservative substitution (V617F) in the JH2 pseudokinase domain. This lesion disables the auto-inhibitory interaction between pseudokinase domain and activation loop residues producing a constitutively active kinase. As JAK2 mutation is observed in nearly all cases of PV, JAK2 mutational status is now a major diagnostic criterion for this disease. Moreover, JAK2 or MPL mutation in ET and PMF is considered diagnostic of clonal hematopoeisis [Inappropriate STAT activation plays a central role in the molecular pathogenesis of a range of hematologic disorders including acute myeloid leukemia (AML) ,2, acuteitution V17F in thopoeisis . Severalopoeisis , and RuxJAK2V617F-positive leukemias.The STAT target genes Mcl-1 and Bcl-XL collaborate to oppose apoptosis mediated by pro-apoptotic BH3-only proteins ,11. We rJAK inhibitor I  was purchased from Calbiochem. SET-2, HEL, MV4;11, and K562 cells were obtained from ATCC and cultured as recommended. UKE-1 cells were purchased from Walter Fiedler (University of Hamburg). Cell lysates were either prepared using CHAPS lysis buffer  or cell extraction buffer containing 1% Triton X-100, 0.1% SDS, and 0.5% deoxycholate. All buffers were supplemented with protease and phosphatase inhibitor cocktails prior to use.For immunoprecipitation, lysates were prepared in CHAPS lysis buffer and 2 mg of cell lysate was mixed with at least 8 μg of immunoprecipitating antibody overnight at 4°C. The following day, 30 μl of a Protein A- or Protein G-agarose slurry was added for an additional 2 hr. Immunoprecipitates were washed three times in CHAPS lysis buffer, and heated in 1.5x loading buffer at 95°C for 5 min.Transfection of siRNAs was performed using Lipofectamine RNAiMAX according to the manufacturer’s recommendations. Cell viability was determined using the alamarBlue cell viability assay (Invitrogen) according to manufacturer’s suggested protocol after exposure to drug combinations for 72 hr. Caspase-3 activity was determined using the Caspase-GLO 3/7 Assay (Promega) in parallel with the CellTiter-GLO viability assay (Promega). The data are expressed as Caspase-3/7 activity divided by cell viability.Ki values of JAKi-I for individual kinases were determined by time-resolved fluorescence resonance energy transfer (TR-FRET) by displacement of proprietary Oregon Green-labelled probes with test compounds. ChIP using STAT3 antibodies was carried out using the EZ-ChIP assay kit (Millipore).Synergistic activities of JAKi-I and ABT-263 were determined using the Bliss additivity model  where thJAK2V617F mutation was STAT3/5 phosphorylation inhibited following treatment with JAKi-I  followed by ABT-263 and monitored the activity of caspase-3. Whereas neither JAKi-I nor ABT-263 alone induced caspase-3 activity, a synergistic induction was evident within four hours specifically in cell lines harboring JAK2V617F  with a JAK2 inhibitor (TG101209) has recently been shown to synergistically reduce the proliferation of JAK2V617F-positive cells. Also, the use of a JAK2 inhibitor in combination with suppression of the PI3K/Akt or mTOR pathways synergistically reduced the proliferation of JAK2V617F-positive cells [Combination of JAK2 inhibitors with other therapeutic agents has demonstrated beneficial effects on growth inhibition of JAK2ve cells . TherefoJAK2 mutant cell lines by demonstrating a key role of Mcl-1 regulation in this synergistic effect. Mcl-1 is apparently regulated by STAT3 as determined by CHIP analysis, which may also implicate STAT5 due to co-regulation by JAK. The biological properties of ABT-263, a potent, orally bioavailable, Bad-like, BH3 mimetic  have been reported previously [In this study, we expanded upon previous results ,23 that eviously . In vivoeviously . In celleviously ,26. Receeviously ,28.V617E mutation. As tyrosine phosphorylation of STAT proteins induces transcriptional activation through homodimerization, selective inhibition of STAT3/5 phosphorylation in JAK2V617F-harboring leukemia lines suggested that transcriptional targets of STAT3/5 may be silenced selectively in these lines. Mcl-1 is a STAT transcriptional target [JAK2V617F. This suggests that leukemias that express JAK2V617F may display a reduced threshold for apoptosis induced by ABT-263 in combination with JAKi-I. The presence of alternative STAT3/5 activating lesions in MV;411 (FLT3ITD) and K562 (BCR-ABL), renders STAT3/5 phosphorylation JAK-independent [JAK2V617F constitutively phosphorylates and activates STAT3/5, thus enforcing expression of the transcriptional targets Mcl-1 and Bcl-xL. Mcl-1 collaborates with Bcl-xL to oppose apoptosis and support viability. Inhibition of JAK2 in this context silences JAK/STAT-driven transcription of Mcl-1, leaving survival largely dependent upon remaining Bcl-xL. Neutralization of Bcl-xL with ABT-263 is then achieved at a lower dose and is sufficient to induce apoptosis (The JAK/STAT pathway is constitutively activated (phosphorylated) in cells harboring the JAKl target ,30,31 anl target ,12,13. Mependent ,33,34; tpoptosis . These fS1 DatasetIndividual Ki values are given in the table.(XLS)Click here for additional data file.S2 DatasetData represent means +/- standard deviation for two independent determinations each performed in triplicate (data in Summary tab). Individual experimental data in exp 051409 and repeat Mcl1 tabs.(XLS)Click here for additional data file.S3 Dataset(XLS)Click here for additional data file.S4 DatasetData are means of duplicate samples and are representative of two independent experiments.(XLS)Click here for additional data file.S5 Datasete.g., cell number will be reduced as cell die).In (XLS)Click here for additional data file.S6 DatasetData are means of duplicate determinations, and are representative of at least three independent experiments.(XLS)Click here for additional data file.


Non-specific lipid transfer proteins (nsLTPs) are important pollen and food allergens in the Mediterranean area. Peach LTP Pru p 3 is considered as primary sensitizer inducing LTP cross-reactive IgE antibodies. Despite the evidence on the importance of Pru p 3, it is unclear whether reactivity to multiple LTPs is due to antibody cross-reactivity or to independent sensitization events. Aim of the study was (1) to compare antigenicity of LTPs from pollen and food, and (2) to investigate whether primary sensitization to LTPs promotes secondary sensitization to LTPs from unrelated sources. IgE sensitization to peach LTP was compared in different mouse strains . IgE high responder mice (CBA/J) were used to compare the antigenicity and IgE cross-reactivity of LTPs from pollen  and foods . To investigate whether an established Th2 immune response to LTPs facilitates a secondary immune response and cross-reactivity to unrelated LTPs, CBA/J mice were immunized with (1) Pru p 3 followed by Art v 3, (2) Art v 3 followed by Pru p 3, and (3) a mixture of Pru p 3 and Art v 3. Induction of Pru p 3 and Art v 3 specific IgE and IgG titers, and cross-reactivity between the two LTPs were determined by ELISA. CBA and C3H mice showed strong IgE-responses to Pru p 3, whereas BALB/c mice did not. Remarkably, a species-specific immune response lacking any IgE- and IgG-cross-reactivity was observed for all tested LTPs except for Pru p 3 and Pru av 3. Although LTPs show high structural similarity, Pru p 3 and Art v 3 displayed different antigenicity. In comparison to Art v 3, Pru p 3 showed much lower IgE-sensitizing capacity. In contrast, IgE response to Pru p 3 was promoted in mice previously sensitized to Art v 3, which was even the case upon concurrent administration of both LTPs. Neither sequential immunization nor immunization with the mixture induced IgE antibodies to unrelated LTPs, e.g. Cor a 8. In summary, the immune response to LTPs in mice varied strongly between strains. The LTPs studied, displayed different immunogenic properties and did not induce cross-reactive IgE antibodies. Primary sensitization to the pollen LTP Art v 3 conditioned the animals for subsequent sensitization to the food LTP Pru p 3. The extent to which the findings are applicable to the manifestation of clinical cross-reactivity to LTPs in humans needs to be investigated.


Recent advances in extracellular signaling suggest that extracellular protein phosphorylation is a regulatory mechanism outside the cell. The list of reported active extracellular protein kinases and phosphatases is growing, and phosphorylation of an increasing number of extracellular matrix molecules and extracellular domains of trans-membrane proteins is being documented. Here, we use public proteomic databases, collagens – the major components of the extracellular matrix, extracellular signaling molecules and proteolytic enzymes as examples to assess what the roles of extracellular protein phosphorylation may be in health and disease. We propose that novel tools be developed to help assess the role of extracellular protein phosphorylation and translate the findings for biomedical applications. Furthermore, we suggest that the phosphorylation state of extracellular matrix components as well as the presence of extracellular kinases be taken into account when designing translational medical applications.The online version of this article (doi:10.1186/s12967-015-0482-4) contains supplementary material, which is available to authorized users. Technical advances have significantly contributed to insights into the functional role of extracellular protein phosphorylation. Most recently, protein kinases such as VLK and FAM20C , have been shown to be secreted through the classical ER-Golgi secretory pathway ,2. In adSince VLK and FAM20C kinases are translated with a signal peptide it is easy to understand that they can be secreted from cells into the extracellular matrix. Other members of this protein family and closely related to FAM20C, such as FAM20A, FAM20B , FAM198,A sufficiently high ATP concentration is essential for extracellular phosphorylation to occur. ATP concentrations in human plasma were reported in a range of 30nM-11μM in different studies and under different conditions . FurtherLarge scale data mining has revealed that a large number of extracellular and cell surface proteins, and extracellular domains of trans-membrane proteins can exist in phosphorylated states . Among tProtein phosphorylation in the ECM may well be a mechanism to regulate protein processing and degradation. Phosphorylated sites close to a cleavage site may either promote or prevent proteolytic enzyme binding. Indeed, many in vitro studies support this possibility. Whether such events happen in vivo needs to be demonstrated. Phosphorylation of vitronectin at position S362 reduces its cleavage by plasmin at the position R361-S362 . PhosphoSimilarly, phosphorylation of other collagens, most notably fibril forming collagens, could be of physiological importance in the processing of procollagen to collagen by affecting cleavage of the N- and C-propeptides. Many phosphorylated sites are located close to the cleavage sites in procollagen α1 (I), α2 (I), α1 (II) and α1 (III)  is another important collagen binding partner. It has recently been discovered that the sequence IKGHRGFSGL in the C-terminal region of the human collagen α1(I) chain is a high affinity binding motif for PEDF. The motif overlaps with the heparin and heparan sulfate proteoglycan motif KGHRG(F/Y) . It is iRGDS). In addition, the Goodpasture antigen was isolated in phosphorylated and non-phosphorylated forms from human kidney, suggesting that the phosphorylation may have a role in the disease [SAVPGFRGD), human collagen α3(IV) at the residue S1435 (RGDS), mouse collagen α2(V) at the residue S137 (SQGPRGD), human collagen α3(VI) at residue S2048 (RGDRGPIGSIG), human collagen α2(XI) at residue T576 (RGDT) and human collagen α1(XXII) at residue S1038 (SRGD) . Collagen IV is a major component of basement membranes. A domain within collagen IV molecules, the Goodpasture antigen, plays an important role in the Goodpasture syndrome, a disease whereby autoimmune antibodies attack the lung and kidney. The antigen was shown to be phosphorylated in vitro at a serine residue next to an RGD sequence  ,24,84. PThe matrix metalloproteinase MMP2 was reported to be regulated by PKC phosphorylation . Human rSRSRR-AA) leads to its activation. While there are no phosphorylated residues close to this cleavage site in the sequence, a potential site that might be recognized by FAM20C is located directly in the recognition sequence [Recombinant human BMP15, as well as growth and differentiation factor (GDF)-9, has been reported to be phosphorylated . Furthersequence .Our insights into extracellular protein phosphorylation have grown during the last 40 years. With novel techniques and methods, the field has experienced a recent revival leading to astonishing and promising findings . As show


Astyanax gr. bimaculatus and Aequidens metae during rainy and dry season at three different sites of Ocoa River called Nacimiento , Centauros , Caño Seco . Also, fish from a reference site with little likelihood of contamination were sampled. Alterations such as decrease in erythrocyte count, lymphocytes, haemoglobin concentration and haematocrit percentage, and increased thrombocytes and neutrophils counts, together with nuclear abnormalities in erythrocytes such as increased frequency of micronuclei, lobed, blebbed and notched nuclei and binucleate cells were observed in fish from the site 3, followed by the site 2 in rainy season. These results shown that during rainy season, sites 2 and 3 receive domestic and industrial wastewater leading to genotoxic and haematological changes in the monitored organisms.The studies evaluating the impact of environmental pollution on aquatic organisms have had a growing concern in recent decades. The Ocoa River runs through Villavicencio city, receiving the wastewater produced in the city. To date studies reporting the effect and the consequences of Ocoa River pollution on aquatic organisms inhabiting this water body are unknown. Haematological and genotoxicity biomarkers were evaluated in peripheral blood of two native fish species The online version of this article (doi:10.1186/s40064-016-1753-0) contains supplementary material, which is available to authorized users. Aequidens and Astyanax are freshwater fish species used in biomonitoring of both lentic and lotic waters  and Aequidens metae (Pisces: Cichlidae), native fish of the Ocoa River, Villavicencio, Meta, Colombia.The use of physiological biomarkers to determine haematological variables or genotoxicity alterations estimating the occurrence of micronuclei in peripheral blood are frequently used as diagnostic techniques to establish the health status of the fish exposed to a complex mixture of available pollutants in water bodies. Studies have been performed showing the relevance of using in situ haematology studies as a tool to reveal the effects of exposure to the discharge of wastewater with high levels of toxic metals such as iron and mercury in the shad Monitoring sites at the Ocoa River, Villavicencio, Meta are shown in the Fig. Aequidens metae—ICN 18884 and Astyanax gr. bimaculatus—ICN 18885. Twelve fish of each species were caught at each monitoring site. Live fish were collected using conventional fishing net .The sampled fish were identified at the Institute of Natural Sciences of the National University of Colombia. The classification corresponded to The monitoring was done during dry season (February 2012 and March 2013) and rainy season (October 2012 and May 2013). During each sampling event, water samples were collected to determine the concentration of cadmium and mercury (atomic absorption spectrometry), surfactants (spectrophotometry) and total hydrocarbons (liquid–liquid extraction) in the Water Laboratory at the Industrial University of Santander, Colombia.3, mg/L) were determined with a HACH kit.Likewise, the physicochemical water characteristics were monitored at each site and each sampling season, including temperature (°C), dissolved oxygen (mg/L), pH, conductivity (mS/cm) and TDS (mg/L) using for this purpose a multiparameter probe  and a pH meter . Alkalinity (mg/L), hardness (mg/L) and ammonium (NH®) and the fork length measured. The blood was collected by puncture of the caudal vessels with a 1 ml syringe (using 25Gx5/8″ gauge needle) with heparin as anticoagulant . Afterwards, fish were weighed  and also allow for the Welfare Committee from the University of the Llanos.In each sample, a completed haemogram was made. For the blood cells total count , a Natt-Herrick solution was used Conroy  in a 1:2The haematocrit was quantified by centrifugation (7.000 g during 5 min). The haemoglobin concentration was determined by the cyanmethemoglobin method with an absorbance of 540 nm  using a commercial kit . The erythrocytes index such as mean corpuscular volume (MCV), mean haemoglobin corpuscular (MHC) and mean corpuscular haemoglobin concentration (MCHC) were determined following the methodology proposed by Conroy and Conroy .®) and observed at 100X magnification counting 100 cells per smear  previously filtered during 10 min. In each smear, 2000 cells with intact nuclear and cytoplasmic membranes were counted  and other nuclear abnormalities, two smears were done per fish. These smears were stained with Wright-Methanol (Merck®) and stored at −70 °C until analysis.Immediately after blood collection, fish were desensitized by medullary court immediately after blood collection. Liver (n = 6) of each species were sampled and a portion of each was fixed in 10 % buffered formaldehyde for histopathological analysis. The remaining tissue was aliquoted and transported in vapour nitrogen (Dry shipper Taylor-WhartonLipid peroxidation (LPO) was determined using the FOX assay (ferrous oxidation xylenol)  and data normal distribution (Kolmogorov–Smirnov) were done. A two-way ANOVA was done to evaluate the effect of the different sampling sites and the season on the variables followed by a Tukey test to detect differences between treatments. Transformation was done when data was abnormal and with heterogeneous variance, however, when data did not fulfil those tests, a ranking transformation was done. In all cases, a p value <0.05 was used to show statistical significant differences. The statistical procedures were done in SAS for Windows version 9.02  and in GraphPad v 5.0.The contaminant concentration evaluated, except for mercury, was within the levels accepted by the Colombian legislation in water bodies Act 1594/84). Temperature, dissolved oxygen, conductivity and ammonium had significant differences compared to the reference site  and length (4.6 cm) were observed in A. gr bimaculatus sampled at the site 3 which corresponds to the farthest site from the city. On the other hand, A. metae sampled at the site 2 had the lowest weight (10.83 g) and length (7.34 cm). In both species, the higher weight and length were observed in fish sampled at site 1 for A. gr. bimaculatus  and at the reference site for A. metae .In the first sampling site was not possible to find A. metae sampled at site 2 and site 3 (p < 0.05) when compared to the reference site (Table A. metae sampled at sites 2 and 3 (p < 0.05) when compared to the reference site  compared to the reference site  of the Ocoa River did not allow adaptation of the A. metae at Centauros (site 2) and Caño Seco (site 3) during the rainy season could be explained to the possible decrease in the haematopoiesis caused by intrasplenic and intrahepatic haemorrhage generated for the water contaminants such as heavy metals  and Centauros (site 3) in rainy season and the decrease in haemoglobin concentration in both sites in dry season could be due to the presence of high levels of ammonia and toxic metals such as mercury  and Caño Seco (site 3) when compared to the reference site can be associated with feedback responses of membranes erythrocytes structural damage as a result of the hemolysis and impaired haemoglobin synthesis and also to the erythrocytes release stress due to the hypoxia observed at sites 2 and 3. Similar results were reported by Kayode and Shamusideen (Oreochromis niloticus) to sublethal concentrations of diesel (23.4 mg/L) and drilling fluid (492 mg/L) for 28 days.The increase in the MCH and MCHC concentration observed in musideen  who expoSiganus rivulatus exposed to different concentrations of sea water nearby a sewage from the Egyptian Mediterranean coast and also to the study done by Elahee and Bhagwant  and cellular alterations  observed in this study  and Centauros (site 2) during both seasons, being these findings together with higher genotoxic damage evidenced by the presence of micronuclei, lobed nuclei, notched nuclei and binuclear erythrocytes. The relation between LPO and genotoxicity could be attributed to the fact that in the presence of damage in the lipid cell membrane, the genetic material is expose and can be trigger irreversible mutations due to a hydrogen deletion in the DNA, being this reaction higher than the capacity to restore the DNA. Supporting this finding, a study from Katsumiti et al.  and DNA damage expressed as MN presence (Ali et al. i et al.  evaluatei et al. , similarA. gr. bimaculatus is more sensitive to the formation of this type of nuclear abnormalities.It has been suggested that problems in segregating tangled and attached chromosomes or gene amplification via the Breakage–Fusion–Bridge cycle could cause lobed nuclei or blebbed nuclei during the elimination of amplified DNA from the nucleus Çavaş . LikewisLabeo bata and Oreochromis mossambica (Talapatra et al. Oreochromis niloticus, Oreochromis aureus and Tilapia zilli exposed in situ to domestic and industrial sewage discharged to the Nile (Ali et al. Finally, several studies have shown that the occurrence of binuclear erythrocytes is associated to the presence of wastewater from thermal power plants in In conclusion, the results obtained demonstrated haematological and genotoxic effects in fish caught at site 2 and site 3, areas in which wastewater from the city of Villavicencio are discharged. This effect could have a negative impact in the decline of fish populations inhabiting the Ocoa River. Finally, the information obtained through this study using bioindicators and sensitive biomarkers can be useful for the Colombian Government agencies to legislate on the sewage dumping.