These results show that CRMP2 is required for PDGF-directed cell migration in vitro.The Platelet Derived Growth Factor (PDGF) family of ligands have well established functions in the induction of cell proliferation and migration during development, tissue homeostasis and interactions between tumours and stroma. However, the mechanisms by which these actions are executed are incompletely understood. Here we report a differential phosphoproteomics study, using a SILAC approach, of PDGF-stimulated mouse embryonic fibroblasts (MEFs). 116 phospho-sites were identified as up-regulated and 45 down-regulated in response to PDGF stimulation. These encompass proteins involved in cell adhesion, cytoskeleton regulation and vesicle-mediated transport, significantly expanding the range of proteins implicated in PDGF signalling pathways. Included in the down-regulated class was the microtubule bundling protein Collapsin Response Mediator Protein 2 (CRMP2). In response to stimulation with PDGF, CRMP2 was dephosphorylated on Thr514, an event known to increase CRMP2 activity. This was reversed in the presence of micromolar concentrations of the protein phosphatase inhibitor okadaic acid, implicating PDGF-induced activation of protein phosphatase 1 (PP1) in CRMP2 regulation. Depletion of CRMP2 resulted in impairment of PDGF-mediated cell migration in an  The PDGF family of ligands comprises four related gene products: PDGFA, PDGFB, PDGFC and PDGFD which can form both homodimers and heterodimers providing a repertoire of PDGF isoforms2. The PDGF family of ligands elicit their biological effects by interacting with two structurally related high affinity receptors of the tyrosine kinase family: PDGFR alpha and PDGFR beta3. PDGF-AA, AB, BB and CC induce PDGFR alpha homo-dimerisation; PDGF-BB and DD induce formation of PDGFR beta homodimers, and PDGF-AB, BB, CC and DD induce formation of PDGFR alpha/PDGFR beta heterodimers2. PDGF-BB homodimers are therefore universal PDGFR ligands capable of eliciting the full repertoire of PDGFR-mediated biological responses.Platelet Derived Growth Factor (PDGF) was first identified in platelet extracts as a potent mitogen for fibroblasts and endothelial cells4. This pattern is seen in multiple organs and tissues such as gut, kidney, gonads, somites and nervous system5. Extensive genetic studies of PDGF and PDGFR function in the mouse confirms that PDGF signalling from epithelial cells via PDGFRs is absolutely required for the expansion and migration of specific mesenchymal cell populations6. For example, in the kidney, PDGF signalling is required both for the multiplication of mesangial cell precursors and their migration into the glomerular space5. In the lung, PDGF is required for the expansion of myofibroblasts and their migration along the alveolar epithelium during lung morphogenesis7. In the central nervous system (CNS) oligodendrocyte precursor (O2A) cells require PDGF signalling both for population expansion and migration through the spinal cord8.PDGF receptors are expressed by cells of mesenchymal origin and the cognate PDGF ligands are expressed by overlying epithelial cells indicative of paracrine signalling from epithelium to the underlying mesenchyme. For example, in the lung, PDGFR alpha is expressed in the lung mesenchyme and PDGFA is expressed in the epitheliumin vitro9 and has been studied for its ability to promote wound healing through promoting the migration, proliferation and extracellular matrix deposition by fibroblasts10. Ectopic expression of PDGFB in the kidney glomerulus induces expansion of bone marrow derived mesenchymal cells to form a severe mesanagial glomerulopathy and fibrosis11. PDGF-BB is a mitogen for vascular smooth muscle cells and activation of PDGF signalling is required for pulmonary arterial hypertension (PAH) and pulmonary arterial remodelling12.PDGF signalling also has significant functions in adult tissue homeostasis and regeneration, acting on cells of mesenchymal origin. PDGF is an established mitogen and chemoattractant for mesenchymal derived cells 14. PDGF-BB produced by tumours of epithelial origin acts upon adjacent connective tissue cells, pericytes and vascular smooth muscle cells to regulate tumour angiogenesis and tumour-associated fibrosis15. PDGF-activated perivascular cells also promote metastasis via tumour-associated macrophages and act on tumour cells that have undergone epithelial-mesenchymal transition (EMT) to promote migration through the extracellular matrix17.The PDGF signalling axis is also implicated in regulation of tumour/stromal cell interactions2. To gain further insight into how PDGFs mediate their biological effects we analysed a differential phosphoproteomics dataset of PDGF-stimulated mouse embryo fibroblasts (MEFs)18. This shows that, in MEFs, PDGFR activation elicits a broad range of phosphorylation events mediated by downstream Ser/Thr kinases which impact on mediators of cytoskeletal function and cell motility. In particular, we show that Collapsin response mediator protein-2 (CRMP2) is dephosphorylated in response to PDGF and is required for PDGF-induced cell migration. CRMP2  is highly expressed in the nervous system and mediates cellular responses downstream of ephrins, neurotrophins and semaphorins, such as neuronal polarity, growth and axonal guidance20.Despite the biological significance of these activities, little is known about the downstream intracellular pathways that mediate these responses beyond canonical RTK signalling pathways such as PLC, AKT and MAPK/ERKCollectively these results significantly expand the range of proteins and phosphorylation events implicated in PDGF signalling pathways and reveal potential new targets for therapeutic intervention in PDGF signalling. They also reveal a role for CRMP2 in PDGF-induced cell migration which has broad implications for tissue repair and tumour development.21). As part of our analysis of LAR function WT MEFs were stimulated with PDGF, and here we have reanalysed this part of the dataset to identify novel PDGF-dependent phosphorylation events. Full details of the experimental setup and procedures used to obtain the phosphoproteomics and proteomics datasets have been previously described18. Methodology relevant to the PDGF-dependent dataset are summarised in Fig. 23. Being a tyrosine kinase, activation of PDGFR will initiate cell signalling via tyrosine phosphorylation. PDGF-induced tyrosine phosphorylation events have been previously studied using enrichment strategies to increase the relative abundance of tyrosine phosphorylated peptides25. As our aim was to gain an understanding of the global signalling landscape regulated by PDGF our enrichment strategy was not selective for either serine, threonine or tyrosine, enabling novel insight into the breadth of biological processes regulated by PDGF. From the 989 phosphopeptides identified, 161 showed a significant change in phosphorylation status upon PDGF stimulation   due to the absence of the intracellular phosphatase domain of Leukocyte Common Antigen-related (LAR) protein tyrosine phosphatase (PRIDE Accession PXD002545ge) Fig. . A total28. In agreement with these studies, we found increased phosphorylation of ERK1 on Tyr205  and Thr203 , and ERK2 on Tyr185 . The phosphorylation of these sites on ERK1/2 is essential for kinase activity. Phosphorylated ERK1/2 mediates diverse physiological functions such as cell growth, survival, cell adhesion, migration and differentiation28. We have also identified an increase in the phosphorylation of Bcl2-associated agonist of cell death (BAD) at Ser136 upon PDGF stimulation  increased upon PDGF stimulation  that have not previously been reported as PDGF-regulated sites (PhosphoSitePlus)34. Phosphorylation of Rab7a on Ser72 inactivates the protein, thereby impairing the recruitment to endosomal membranes and delaying the transport of EGFR from early to late endosomes35. This suggests that PDGFR activates an as yet unidentified kinase which phosphorylates Ser72 on Rab7a indicating an intersection between two major RTKs, PDGFR and EGFR in the regulation of endocytosis. In addition, we observed increased phosphorylation of Slit-Robo GTPase-activating protein 3 (srGAP3) on Ser858 (Supplementary Table 37. This site is phosphorylated by protein kinase A (PKA), promoting GAP activity towards Rac1 and inhibition of actin reorganisation in neurons38.After ERK1/2, the next most significant increase in phosphorylation was observed for Ser72 of Ras-related protein Rab7a , Neurofibromin , CLIP-associated protein 1 , Parva (Ser14), collapsin response mediator protein 2  and collapsin response mediator protein 4  Figs . These p46 was phosphorylated on Ser105 in response to PDGF stimulation  analysis of the phosphoproteins regulated by PDGF revealed a number of enriched GO biological processes Fig. . The dom56. In addition, KEGG analysis of the dataset also identified neurotrophin signalling and axon guidance as PDGF-regulated processes   and c-Jun N-terminal kinase (JNK1)  Fig. . RSK2 prK1) Fig. . The act67. Consistent with this, GSK3β has been shown to phosphorylate CRMP2 at Thr509, Thr514 and Ser518 thereby inhibiting CRMP2 activity leading to microtubule depolymerization and destabilization69. Within our dataset a doubly phosphorylated peptide from CRMP2  was significantly downregulated following PDGF stimulation  as an example of a novel PDGF-induced dephosphorylation target found within our dataset. CRMP2 regulates cytoskeletal remodelling, neurotransmission and axon guidance via its ability to promote microtubule assemblyion Fig. . Westernion Fig. . These d71. We therefore tested the effect of okadaic acid on CRMP2 phosphorylation at Thr514 following PDGF stimulation in MEFs. PP2A is inhibited at low concentrations (<1 μM) of okadaic acid, whereas higher concentrations (>1 μM) inhibit PP173. Western blot analysis showed no significant changes in PDGF-regulated CRMP2 dephosphorylation when okadaic acid was added at low (0.01 μM or 1 μM) concentrations . We showed that micromolar concentrations of okadaic acid, a serine/threonine phosphatase inhibitor, restored the phosphorylation of CRMP2 at Thr514. This is most likely due to preferential inhibition of protein phosphatase 1 (PP1)73. CRMP2 is not however dephosphorylated in non-stimulated cells showing that PDGF pathway activation is essential for the CRMP2 directed activity of this phosphatase. Given the intersection of CRMP2 in neuronal cells and MEFs we were interested to learn if CRMP2 was required for PDGF induced cell migration. Depletion of CRMP2 resulted in impairment of cell migration in a scratch wound assay in the presence of PDGF showing that it is required for PDGF induced cell migration in MEFs.We also report the identification of 45 phosphopeptides that are significantly down-regulated in response to PDGF stimulation. These include proteins such as CLASP1 and CRMP2. CRMP2 is of interest as an effector of PDGF-mediated cell migration as it has a well- established role as mediator of axon guidance, growth cone collapse and cell migration in neuronsOur quantitative phosphoproteomics study has significantly expanded the known repertoire of PDGF effectors and provided a resource for further experimental studies. The novelty of our findings is highlighted by the fact that we have identified a role for CRMP2 in PDGF-regulated cell migration, a fundamental biological process closely linked to developmental processes and progression of disease.18. Within the MaxQuant output, phosphorylation sites were considered to be localised correctly if the localisation probability was at least 0.75 (75%) and the score difference at least 5. Significance testing was performed in the Perseus software environment, which is part of MaxQuant , using a Student’s t-test on log 2 transformed ratios and controlled with a Benjamini-Hochberg FDR threshold of 0.05. Peptides quantified in three or more experimental repeats were deemed significantly changed and regulated by PDGF if they had a p-value of <0.05 and a ratio of <0.667 or >1.5 (at least a 1.5-fold change in abundance). The COMPARTMENTS database81 was used to assign proteins to subcellular localisations. Protein network visualization was performed using Cytoscape (version 3.3.0)82. DAVID 83 was used to identify over-represented KEGG pathways56  and REVIGO84 was used for identification and visualisation of over-represented GO terms. Kinases upstream of the identified PDGF-regulated phosphopeptides were predicted using Kinase Enrichment Analysis (KEA)57.Details of the experimental setup for the quantitative phosphoproteomics experiments have been previously described85. Goat anti-mouse-IgG IRDye-conjugated antibody (925-68070) and goat anti-rabbit-IgG (925-68071) HRP-conjugated antibodies were from LI-COR Biosciences . Recombinant human PDGF-BB (#8912) was purchased from Cell Signaling Technology and okadaic acid was purchased from Calbiochem .Antibodies against CRMP2 (#9393), CRMP2 Thr514 (#9397), and phospho-threonine (#9381) were purchased from Cell Signaling Technology . Antibodies against ERK1/2 (sc-514302) and ERK Thr202/Tyr204 (sc-136521) were purchased from Santa Cruz Biotechnology , and against tubulin (T6199) from Sigma-Aldrich Company Ltd (U.K.). The anti-Alix antibody was a gift from Carl Hendrik Heldin Mouse embryonic fibroblasts (MEFs) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 10 mg/mL streptomycin, and 250 μg/mL amphotericin B. For growth factor stimulation experiments, cells were grown to 80% confluence and starved for 16 h in serum-free DMEM before being treated with 20 ng/mL recombinant PDGF-BB at 37 °C. In some experiments, cells were incubated with either 0.01 μM, 1 μM or 1.5 μM okadaic acid.18. Immunoblots were visualised using fluorescence detection on the Odyssey Infrared Imaging System . Densitometric analysis was carried out using ImageJ86.Cell lysates were prepared, separated by SDS-PAGE, transferred to nitrocellulose membranes and immunoblotted using previously described protocols87. Nonsilencing control (NSC) siRNA was used as a control for each experiment.Cells were transiently transfected with 80 pmol of SMARTpool or individual ONTARGETplus siRNA for murine CRMP2  using Lipofectamine RNAiMAX  as described elsewhereCells were plated in 96-well cell culture plates and cultured to confluency. Cells were serum starved overnight. Scratch wounds were made using an automated wound maker , cells were stimulated with 20 ng/ml PDGF, and images collected at hourly intervals post-wounding using an IncuCyte ZOOM imaging system .88, used in this analysis have been previously deposited on the Proteome Xchange Consortium via the PRIDE partner repository with the dataset identifier PXD00254521.The mass spectrometry proteomics data, including the output from MaxQuantSupplementary InformationSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5


Dang  commenteOne classification with which Dang takes issue is the forward span task, arguing that it should be classified as working memory rather than inhibition. Performance on forward span tasks is indeed underpinned in part by cognitive processes associated with performance on working memory tasks, such as storage processes  in our analyses.Dang also contends that the sustained attention to response task (SART) is a response inhibition task, not a cognitive inhibition task. The reason for this contention is that the SART has a similar structure to the go/no-go  tasks but not with classic response inhibition measures  differed from studies that used response inhibition tasks (m = 6) or from studies that used other cognitive inhibition tasks (m = 13). In these analyses, the effect of stress on SART performance (g+ = −0.290) was significantly different from the effect of stress on response inhibition performance (g+ = 0.295), B = −0.585, t(6.4) = −3.57, p = 0.011, but the effect of stress on SART performance did not significantly differ from the effect of stress on other cognitive inhibition tasks (g+ = −0.182), B = −0.108, t(5.1) = −0.67, p = 0.531. Thus, the effects of stress on the SART were quite similar to the effects of stress on other cognitive inhibition tasks but quite different from the effects of stress on response inhibition tasks, corroborating the findings of prior research indicating that the SART is a cognitive inhibition task.Additional evidence for classifying the SART as cognitive inhibition comes from a reanalysis of the data from the meta-analysis itself. In particular, it is possible to test whether studies that used the SART (A final point contested by Dang was classifying certain newly developed tasks and complex emotional interference tasks as cognitive inhibition tasks. Dang states that because Sänger et al.  used a nDang's conclusion is that stress effects on inhibition are still pending. I agree. The meta-analysis included many heterogeneous paradigms, and the number of studies that examined response inhibition in it was small. Nonetheless, despite these limitations, the task classifications—and therefore, inferences—we made were determined by and are in agreement with prior research, whereas the classifications suggested by Dang were and are not. Future work should therefore be careful to follow established classifications for executive function tasks (e.g., Kornblum et al., The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A semi-quantitative analysis of atomic column compositions in the ordered L12 γ′ structure is provided. Co atoms were found to occupy the {1/2, 1/2, 0} face-center positions whereas Al, W, Ti and Ta atoms prefer to occupy the {0, 0, 0} cube corner positions in the L12 γ phase. These findings agree well with predictions from first principles simulations in the literature.Using state-of-the-art atomic scale super energy dispersive X-ray spectroscopy and high angle annular dark field imaging this study reveals the elemental partitioning preference between the γ′ and γ phases in a Co-Al-W-Ti-Ta superalloy and the site preference of its alloying elements in the ordered L1 The γ′ phase has an ordered L12 structure which is more resistant to dislocation movement than its parent disordered FCC phase, thus exhibiting a higher strength. The L12 structure was first identified in Ni3Al4 alloy in 1937 and later discovered in other alloys such as Cu3Au5 alloy. The Ni-based and Co-based superalloys are typical L12 containing alloys and they derive their high temperature strengths largely from their L12 precipitates. The Ni-based superalloys have been used in turbine engines6. Ti and Ta are often added in these alloys to further improve the structural stability of γ/γ′ two-phase microstructures by increasing the γ′ volume fraction11. It has been shown that the γ′ strengthened Co-Al-W-Ti-Ta superalloys display improved structural stability, exceeding both conventional Co-based superalloys and the first-generation Ni-base single-crystal superalloys in creep life at around 1000 °C12.Co-Al-W-based superalloys derive their high temperature strength largely from a γ′-Co2 γ′ precipitate. Due to the ordered structure, the Burger’s vector of a full dislocation in the L12 γ′ phase is twice the magnitude of that in the FCC γ phase, i.e., bγ = a/2 <110> full dislocation in the FCC γ phase functions only as a partial dislocation in the γ′ phase and that dislocations in the γ′ phase always move in pairs of different m <110> and n <112> combinations. The passing of these partial dislocation pairs produces different forms of stacking faults (SFs) and anti-phase boundaries (APBs)12. Therefore, stacking fault energy and anti-phase boundary energy directly influence the activation and slipping of these dislocations and thus the mechanical strength of the γ′ precipitates15.The superior creep resistance of Co-Al-W-Ti-Ta superalloys derives from the intrinsic high resistance to dislocation shear of the L13B type alloys containing other alloying elements, for example the addition of Ti and Ta in the Co-Al-W alloy. Therefore, knowledge of lattice site occupancy of alloying elements in γ′-Co-Al-W-Ti-Ta is important for developing high strength and creep resistant Co-based superalloys.Stacking fault energy and anti-phase boundary energy are in turn determined by the elemental structure of the atomic plane in question, particularly for A18. It is generally predicted that Ti has equal preference for the face-centered {1/2, 1/2, 0} and cube-corner {0, 0, 0} sites in the L12 structure17 and that Ta has a preference for the {0, 0, 0} sites18.Much effort has been made in theoretical analysis, mostly by means of first principles simulation, to predict lattice site preference of alloying elements in the γ′ phase20. Resolving atomic site preferences of elements in a thin region of the Co-Al-W-Ti-Ta alloy is challenging because of the weak signals and spatial resolution of conventional TEM equipment and energy-dispersive X-ray spectroscopy (EDS) detectors. These limitations can be overcome by using a spherical aberration (Cs) corrected probe and super energy-dispersive X-ray spectroscopy (super-EDS) detector22. In the present work, we investigated the elemental partitioning between the atomic columns in the γ′ and γ phases in a Co-Al-W-Ti-Ta alloy and the site preferences of Co, Al, W, Ti and Ta in the L12 γ′ structure using high resolution high angle annular dark field (HR-HAADF) and atomic super-EDS techniques.However, to date there has not been direct experimental evidence to support or verify such predictions. One possible experimental technique to use is atomically resolved chemical analysis using transmission electron microscopy (TEM)Figure γ′/γ, (defined as the ratio of the concentration in γ′ to that in γ) of the elements between the two phases. It is seen that Co has a lower concentration in the γ′ phase, apparently to meet the stoichiometry requirement for the L12 structure (75 at.%). This gives a Kγ′/γ value of 0.9 for Co. Al shows little preference between the two phases with a small partitioning coefficient of 1.06. By contrast, W, Ti and Ta show clear preference for the γ′ phase with partitioning coefficients of 1.81, 1.59 and 1.85, respectively. Given that the Co content in the γ′ phase is ~75 at.% of that expected in its theoretical form of Co3, i.e. the stoichiometry of the L12 structure, the {1/2, 1/2, 0} site is expected to contain 100 at.% Co atoms and the {0, 0, 0} site contains all the other elements of Al, W, Ti and Ta. The partitioning of W, Ti and Ta in the {0, 0, 0} sites of the L12 structure implies that the bonding energies of Co-W, Co-Ta and Co-Ti bonds are higher than that of Co-Co bond, as predicted by Saal and Wolverton in their density functional theory (DFT) calculations23.Quantitative EDS analysis of the composition of the two phases was conducted. Figure 2 unit cell. The {1/2, 1/2, 0} positions are labelled as the A sites  and {0, 0, 0} positions are denoted the B sites . Figure 1.6–1.7, where Z is the atomic number24, or in this case the average atomic number of the atomic column in the direction of the electron beam. In this image, the bright spots are the {0, 0, 0} sites (site B) and the dark spots which are three times more populous than the bright spots are the {1/2, 1/2, 0} sites (site A). The intensity ratio of two adjacent atoms in a HAADF image gives a qualitative comparative measure of the aggregated atomic weights of the columns without distinction between specific elements. By comparison, the EDS mapping provided by the Super-EDS technique gives a semi-quantitative description of the actual elements and their partitions between the columns.Figure Figure a sites, which are surrounded by four other A sites and the Ab sites, which are surrounded by two A sites and two B sites. It needs to be noted that the Aa and Ab sites are totally equivalent in the 3-dimentional environment in the lattice. They are different here only with respect to the viewing direction, in which the composition analysis was conducted. Figure While the HAADF image technique can detect the difference in average atomic number between different atomic columns, it is unable to detect which individual element may have contributed to the average atomic number in an atomic column. For this, atomically resolved EDS mapping analysis was carried out for the alloying elements in the γ′ phase in the Co-7Al-8W-4Ta-Ti alloy, as presented in Fig. a, Ab) and the B sites. For the analysis, six site Aa atomic columns, twelve site Ab atomic columns and six site B atomic columns were analyzed, and the data shown in Fig. B/Aa on Al, W, Ti and Ta are higher than values of KB/Ab, i.e., the Ab site exhibits less partitioning than the Aa site relative to the B site. This is attributed to the higher level of signal contamination of the Ab site due to its proximity to the B site.Figure a site and 80.8 at.% at the Ab site. The lower concentration at the Ab site is apparently due to the signal contamination from the B site and thus is considered a less accurate representation of the A site . Therefore, the Aa site composition is considered a more accurate measure of the composition of the A site, though still not a perfect representation due to the inevitable signal contamination of the technique used. However, a significant amount of Co is also detected at the B site, with a detected concentration of 61.0 at.%. In an ideal Co3X L12 structure the A[001] column is expected to be 100% Co and the B[001] column is expected to have no Co. The Co content being lower than purity at the A site is due to signal contamination from the surrounding columns caused by the spread of the electron beam as it penetrates the sample25. Similarly, the detection of Co at the B site is also due to column signal contamination. Considering that a B<001> column is always surrounded by four A<001> columns, the level of contamination is expected to be higher than that of the A<001> columns, thus the high Co content measured.Co is found to have a preference for the A site. Its concentration is measured to be 85.8 at.% at the Aa and site Ab were measured separately, as presented in Fig. 2 structure, the two sites show clear differences in element concentrations, with lower Co concentrations and higher concentrations of other elements at the Ab site than Aa site. This is apparently related to the closer neighboring relationship of Ab to the B sites.To verify this, column compositions of site A26, sample alignment and thickness27 limit the spatial resolution of composition quantification. In our experiment, the beam convergence angle is about 30 mrad and the beam spot size is about 2 Å. As seen in Fig. b columns is closer to that of the B site columns due to the influence of beam-spread induced signals from the adjacent B columns. In this regard, the measured composition of the Aa site is considered less contaminated and thus a more reliable measurement.A number of factors including beam spread, probe channeling, image delocalization, thermal scatteringet al. conducted DFT calculations and suggested that Co3Al and Co3W are less stable L12 phases23. In this regard, the actual γ′ phase in the Co-7Al-8W-4Ti-1Ta alloy can be described as Co3, i.e., an ordered L12 structure between the A and B sites with a disordered solid solution of W, Al, Ti and Ta at the B site. Neglecting the Co content measured, the B site composition may be estimated to be 14.5 at.%W, 11.6 at.%Al, 14.2 at.%Ti and 1.7 at.%Ta. This gives a weighted average atomic number of 32.26 for the B site. For the A site, the measurement from the Aa site is used, because of its relatively lower signal contamination, and the composition is estimated to be 5.7 at.%W, 5.2 at.%Al, 2.9 at.%Ti and 0.4 at.%Ta. This gives a weighted average atomic number of 28.99 for the A site. In this regard, the average atomic number ratio between the B site and the A site is estimated to be 32.26/28.99 = 1.11, according to the Super-EDX measurements.Al, W, Ti and Ta all show preferences for the B site. Saal a site is 2.2, as determined in the centre of a γ′ phase region in a HAADF image. It has been suggested that the HAADF intensity (I) of an atomic column and its weighted average atomic number (Z) follow the relationship of I ∝ Z1.6–1.7. Using this relationship, the average atomic number ratio between the B site and the Aa site is estimated to be 1.59–1.64. The ratio is closer to a nominal Co74.9Al7.1W9.1Ti6.9Ta1.9 obtained from γ′ phase and it has an ideal atomic number ratio of 1.56. It is seen that the two values determined by the two methods are different, but both are larger than 1, indicating the segregation of heavy elements to the B sites. It can be conclude that the average atomic numbers obtained from the EDS atomic scale mapping method are less accurate than those obtained from a HAADF image.On the other hand, the average atomic number ratio may also be estimated from HAADF measurement. The HAADF intensity ratio of the B site to the A29.Atom Probe Tomography (APT) is another material analysis technique offering extensive capabilities for 3D imaging and chemical composition measurements at the atomic scale. This technique has spatial resolutions of typically 1–3 Å in depth and 3–5 Å in lateral directions. The super-EDS technique differs from APT in several aspects. First, the super-EDS in TEM technique used in this study has a higher lateral spatial resolution of ~0.7 Å when used in a Cs-corrected TEM. This is more suited for composition analysis of atomic columns such as those which are separated by 1.8 Å in our alloy. Second, the 3D tomography needs a reconstruction process that introduces secondary errors, whereas the Super-EDS in TEM technique quickly generates direct elemental mapping in real time. Third, the APT technique is more advantageous than the Super-EDS method by not suffering from signal contamination caused by beam spread. The APT method conducts composition analysis layer by layer by progressively ablating the sample surface with laser or high voltage pulsed ion beam, thus avoiding the problem of beam spreading and minimizing signal contamination from adjacent atomic columns2 unit cell. These findings are consistent with the findings of first principles investigations reported in the literature and this study provided direct experimental evidence for the first time.Using atomic scale super energy dispersive X-ray spectroscopy and high angle annular dark field imaging we studied elemental partitioning in the γ′/γ structure and atomic site preference in a Co-Al-W-Ti-Ta superalloy. The Super-EDS technique provides a semi-quantitative analysis of elemental partitioning. Co was found to have a site preference for the {1/2, 1/2, 0} sites whereas Al, W, Ti and Ta have preferences for the {0, 0, 0} positions of the L111. The alloy was fabricated into single crystal bars of 15 mm in diameter and 150 mm in length by means of directional solidification using the conventional Bridgman method. The single-crystal ingot was solution treated at 1270 °C for 24 h in a flowing Ar atmosphere, and then aged at 900 °C for 50 h to encourage γ′ phase formation followed by air cooling. TEM samples were prepared by means of a twin-jet electrochemical polishing method. The alloy we used is a highly crystalline single crystal with the cubic shape γ′ phase about 200 nm and we chose the center in a 200 nm γ′ phase to get EDS mapping results. The thickness measured in our experiments is about 50–70 nm by the two beam CBED method. The region we measured in the γ′ phase did not contain γ′ phase lying beneath the ordered phase. TEM, HAADF and EDS mapping experiments were carried out using an FEI Titan G2 60–300 microscope operated at 300 kV. Quantification was performed from average data of 6 Aa, 12 Ab and 6 B columns in one image. For quantitative EDS analysis, the power law method was applied to remove the background. The K series peaks were chosen for Al, Ti and Co and the L series peaks were chosen for W and Ta. Phase partitioning coefficient Kγ′/γ and site preference partitioning coefficient KB/A were defined as the concentration ratios of an element in γ′ to γ phase at sites A and B, respectively.The experimental alloy has a nominal composition of Co-7Al-8W-4Ti-1Ta (at.%)


In the bladder neck, a marked phosphodiesterase type 4 (PDE4) expression has also been described and PDE4 inhibitors, as rolipram, produce smooth muscle relaxation. This study investigates the role of PDE4 isoenzyme in bladder neck gaseous inhibitory neurotransmission. We used Western blot and double immunohistochemical staining for the detection of NPP4 (PDE4) and PDE4A and organ baths for isometric force recording to roflumilast and tadalafil, PDE4 and PDE5, respectively, inhibitors in pig and human samples. Endogenous H2S production measurement and electrical field stimulation (EFS) were also performed. A rich PDE4 and PDE4A expression was observed mainly limited to nerve fibers of the smooth muscle layer of both species. Moreover, roflumilast produced a much more potent smooth muscle relaxation than that induced by tadalafil. In porcine samples, H2S generation was diminished by H2S and NO synthase inhibition and augmented by roflumilast. Relaxations elicited by EFS were potentiated by roflumilast. These results suggest that PDE4, mainly PDE4A, is mostly located within nerve fibers of the pig and human bladder neck, where roflumilast produces a powerful smooth muscle relaxation. In pig, the fact that roflumilast increases endogenous H2S production and EFS-induced relaxations suggests a modulation of PDE4 on NO- and H2S-mediated inhibitory neurotransmission.Nitric oxide (NO) and hydrogen sulfide (H While the cAMP pathway seems to play a key role in detrusor relaxation so that PDE4 inhibitors have been suggested for the treatment of bladder overactivity, the NO/cGMP signaling would be involved in regulating urethral contractility. In fact PDE5 inhibitors, such as tadalafil, are useful for the treatment of lower urinary tract symptoms (LUTS) associated with benign prostatic enlargement (LUTS/BPE)19.The cAMP pathway is crucial in the control of urinary bladder smooth muscle tension. cAMP, via PKA activation, regulates smooth muscle function by targeting the activity of several K22 and hydrogen sulfide (H2S)24 play a pivotal role in nerve-mediated relaxation. Along with these mediators, other non-gaseous molecules, such as adenosine 5′-triphosphate (ATP)25, serotonin26 and peptides, such as pituitary adenylate cyclase-activating polypeptide 3828, are also implicated in the bladder neck smooth muscle relaxation. Our lab has previously described a marked PDE4 expression in pig and human bladder outflow region, where the selective PDE4 inhibitor rolipram causes a powerful smooth muscle relaxation29. The study of the underlying mechanisms regulating the bladder neck smooth muscle tension is necessary to provide drugs causing bladder outlet region relaxation during the voiding in obstructive LUTS20.In the bladder outflow region, nitric oxide (NO)33. As the PKA signaling pathway might represent a valuable therapeutic target for patients suffering LUTS/BPE, the present study was designed to investigate the role played by PDE4 isoenzyme and PDE4 inhibitors, such as roflumilast, in bladder neck gaseous inhibitory neurotransmission.Roflumilast is an orally active PDE4 inhibitor with anti-inflammatory and bronchodilator effects approved for the treatment of severe chronic obstructive pulmonary disease (COPD)Neuronal NPP4 (PDE4) and PDE4A expression in pig (n = 5 pigs) and human (n = 4 persons) bladder neck samples were investigated by double staining immunohistochemistry using NPP4 and PDE4A selective antibodies together with pan-neuronal marker protein gene product PGP) 9.5. Similar NPP4 (PDE4) and PDE4A immunoreactivities were observed mostly to co-localize with PGP 9.5, within nerve fibers running parallel to the smooth muscle bundles, both in the pig Figs  and 2A–D1P 9.5. Si23123132S level produced by pig bladder neck samples (0.96 ± 0.13 nM.min-1.g-1) was diminished as consequence of CSE and NO synthase inhibition with, respectively, PPG (1 mM)24 and L-NOARG (100 µM)24, and recovered by administration of increasing concentrations of roflumilast (0.1–10 µM) (Table The HM) Table .Table 1E29 produced a sustained tone of 2.4 ± 0.1 g (n = 36) and 2.3 ± 0.1 g (n = 4) in pig and human, respectively, strips.Strips of pig (n = 36 pigs) and human  bladder neck were equilibrated to a tension of 2.1 ± 0.1 g and 1.9 ± 0.1 g, respectively. PhE (1 μM)2 and Emax values of 8.0 ± 0.1 and 100 ± 2% and 6.0 ± 0.1* and 100 ± 8%, for roflumilast and tadalafil, respectively, *P < 0.05 versus roflumilast value, unpaired t–test, n = 8)   and tadalafil (0.1 nM–10 μM) produced relaxations of pig (pD 8) Fig.  and huma 4) Fig.  bladder 2 and Emax values of 9.1 ± 0.1 and 87 ± 9% and 8.2 ± 0.1* and 76 ± 5%, in the absence or presence, respectively, of PPG, *P < 0.05 versus control value, unpaired t–test, n = 5) and L-NOARG (100 µM) , blockers of H2S synthase CSE and NO synthase, respectively, reduced roflumilast responses.PPG (1 mM)    roflumilast concentrations  Roflumilast relaxation was also diminished by H2S and NO synthesis blockade. (4) EFS- and isoproterenol-induced relaxations were enhanced in the presence of roflumilast threshold concentrations.The main finding of the present study is that neuronal PDE4, essentially PDE4A isoform, plays a crucial role in regulating contractility of the pig and human bladder outflow region. In fact, PDE4 inhibition and subsequent increased neuronal [cAMP] produces a potent smooth muscle relaxation due to its facilitatory action on nerve-mediated response. The above conclusion is maintained by the following data: (1) NPP4 and PDE4A are mainly detected within nerve fibers distributed along the smooth muscle layer of the pig and human bladder neck, where roflumilast exerts a much more potent relaxation compared to that induced by the PDE5 inhibitor tadalafil. (2) In pig bladder neck membranes, endogenous H36. In the present study, in the pig and human bladder neck samples, PDE4 (NPP4) and PDE4A expression were consistently detected. In fact, western blot analysis in the pig and human bladder neck samples showed a consistent PDE4 (NPP4) and PDE4A protein expression. Moreover, double-labeling immunofluorescence assays revealed IR for NPP4 and PDE4A as nerve fibers running alongside the smooth muscle layer. These results agree with those obtained in the human seminal vesicles, where immunosignals specific for PDE4 co-localized with vasoactive intestinal peptide were found in nerve fibers and nerve endings37. In our study, the presence of NPP4 and PDE4A within nerve fibers also suggested a modulatory role for PDE4 in the inhibitory efferent neurotransmission of the bladder neck. Selective PDE5 inhibitors change the endogenous production and/or effect of neurotransmitters in the lower urinary tract. In fact, in human detrusor, sildenafil causes smooth muscle relaxation and increases the H2S generation, thus indicating that PDE5 inhibitor relaxation on the human bladder involves the H2S signaling pathway38. Moreover, tadalafil enhances the inhibitory effect of the α1A-adrenergic receptor antagonist tamsulosin on EFS-elicited neurogenic contraction of human bladder neck39. In the pig bladder neck, NO and H2S play a pivotal role in the NANC inhibitory neurotransmission. NO, whose release from intramural nerves is modulated by pre-junctional α2-adrenoceptor and voltage-gated K+ channel activation, relaxes smooth muscle through a mechanism dependent on the activation of guanylyl cyclase, in which post-junctional K+ channels do not seem to be involved21. In addition, H2S, synthesized by CSE and released from nerves, produces bladder neck smooth muscle relaxation through opening of ATP-dependent K+ channels and by intracellular Ca2+ desensitization-dependent mechanisms. H2S also promotes the release of neuropeptides and cyclooxygenase-1 pathway-derived prostanoids from capsaicin sensitive primary afferents, via transient receptor potential A1, transient receptor potential vanilloid 1 and/or related ion channel activation24. In the present study, in the pig bladder neck, H2S synthesis enzyme CSE and NO synthase blockade diminished the endogenous H2S production, thus indicating that H2S is generated through activation of CSE, also involving a NO synthetic pathway. H2S production was, nervetheless, completely restored and significantly increased, in a dose-dependent manner, above control sample levels, as a consequence of stimulation with increasing roflumilast concentrations, thus indicating that PDE4 isoenzyme, present in the nerves, would be involved in regulating production and/or release of gaseous inhibitory neurotransmitter/s. Since PDE4 inhibition enhanced H2S generation, these results suggest a facilitatory role for cAMP on neurotransmitter release in the bladder neck.Several PDE isoenzymes, such as PDE1A, PDE4A, PDE4B and PDE5A have been identified in the human urethra and prostatic smooth muscle, where selective inhibitors of PDE4 and PDE5 cause relaxation, thus indicating that, in addition to the NO/cGMP/PKG pathway, the cAMP/PKA-dependent signaling might be a valuable target to treat symptoms of LUTS/BPE35. Since NO21 and H2S24 are the major NANC inhibitory neurotransmitters in pig bladder neck, the samples in this study were treated with NO synthase and H2S synthase CSE inhibitors, which reduced the roflumilast response, thus suggesting that PDE4 inhibition and the subsequent increase in neuronal cAMP levels may favour the release of NO and H2S. This fact, together with the potentiation of EFS-elicited neurogenic relaxation observed in presence of a threshold roflumilast concentration and the marked expression of PDE4, especially PDE4A, within nerve fibers, suggests that pig bladder neck roflumilast relaxation is produced via neuronal H2S- and NO-dependent signaling pathways. These results agree with those described in human detrusor, where the L-cysteine/H2S pathway is involved in the relaxation produced by PDE5 inhibitors. In fact, not only stable analogs of cGMP but also cAMP elevate the H2S levels in the human bladder, thus supporting the role of cAMP/PKA signaling pathway in the H2S-mediated relaxation38.The fact that roflumilast relaxation was much more potent than that induced by tadalafil agrees with that described in human urethra, where PDE4 inhibitors exhibit a higher rank order of efficacy versus that shown by the PDE5 inhibitors sildenafil, vardenafil and tadalafil41. In the current study, a roflumilast threshold concentration evoked a leftward displacement of the relaxation concentration-response curve to the β-adrenoceptor agonist isoproterenol, thus suggesting that PDE4 inhibitors, in addition to cause a powerful smooth muscle relaxation per se, enhance the relaxant effect produced via β-adrenoceptor stimulation in the bladder outlet region.In airways smooth muscle, an interaction between PDE4 inhibitors and beta-adrenoceptor agonists has been documented. In fact, PDE4 inhibitors, such as rolipram and quercetin, cause a potent smooth muscle relaxation of human bronchus and mice trachea, respectively, and potentiate β-adrenoceptor stimulation relaxation45. Therefore, PDE4 inhibitory activity might provide, in addition to relaxant action, an anti-inflammatory therapy for bladder urine outflow region. The fact that roflumilast, an orally active anti-inflammatory drug used as an add-on treatment to long-acting bronchodilators has been approved for the treatment of severe COPD33, suggests the possible usefulness of roflumilast and/or its active metabolite N-oxide, alone or in combination with the PDE5 inhibitors or α1A-adrenoceptor antagonists for smooth muscle relaxation of lower urinary tract outflow region in patients suffering LUTS/BPE.Selective PDE4 inhibitors also attenuate the development of cyclophosphamide-induced cystitis improving the bladder symptoms, as voiding behaviour and histological damage by suppressing cytokine production and inducible NO synthase induction, and causing a significant decrease on the amplitude and frequency of spontaneous contractions without changing cystometric parameters, thus suggesting that PDE4 inhibitors might be an attractive strategy for the treatment of chemically-induced bladder overactivity2S production and EFS relaxation suggests that PDE4 inhibition and subsequent increased neuronal [cAMP] facilitates NO- and H2S-mediated bladder neck inhibitory neurotransmission. To our knowledge this is the first time that it is suggested a modulatory role for neuronal PDE4 in the gaseous inhibitory transmission.In conclusion, current results suggest that PDE4, predominantly PDE4A, is mostly expressed within nerve fibers of the pig and human bladder neck, where roflumilast promotes a much more powerful smooth muscle relaxation in comparison with that produced by tadalafil. In pig, the fact that roflumilast increases endogenous HUrinary bladders from adult male pigs were provided from the Matadero Madrid Norte slaughterhouse. Human bladder samples were taken from 4 men. One of them was 53 years old and died as consequence of a cerebrovascular injury, and another 43 years old who died in a traffic accident. Human samples were also obtained from two other patients, 64- and 56-year old, who had undergone cystectomy for bladder cancer. All methods were carried out in accordance with relevant guidelines and regulations. The study protocol with human samples was approved by the Ethics Committees from Hospital Universitario Puerta de Hierro-Majadahonda and Hospital Universitario Ramón y Cajal de Madrid , with informed consent obtained from all subjects or families of the organ donors. Bladders were maintained in physiological saline solution at 4 °C.Proteins extracted from pig and human bladder neck (50 μg of protein) were separated by 10% polyacrylamide gel (SDS-PAGE), after which the transfer membranes were exposed to specific antibodies against NNP4 and PDE4A  at a 1:50 dilution. Mouse anti-β-actin antibody was used as a loading control . Membranes were incubated with the secondary antibody (1:5000 dilution); the blots were detected with an ECL solution , using an Image-Quant LAS 500 system chemiluminescence imaging system .Pig and human bladder neck segments were placed in an solution of 4% paraformaldehyde in phosphate buffer , and subsequently placed in 30% sucrose in PB for cryoprotection. The tissue was sliced into transversal sections 5 μm thickness and preincubated in 10% normal donkey serum in PB containing 0.3% Triton-X-100, for 2–3 h. Followed the sections were incubated with either goat anti-PDE4  at a 1:50 dilution plus a mouse anti-protein gene product 9.5 (anti-PGP 9.5), diluted 1:100 and mouse anti PDE4A  at a 1:50 dilution plus a rabbit anti-protein gene product 9.5 (anti-PGP 9.5) diluted 1:50 during 48 h at 4 °C. After rinsing the sections with PB, fluorochrome-labeled secondary antibodies were applied for 3 h at room temperature  (Abcam). The slides were mounted with DAPI (Invitrogen), which stains all cell nuclei.On the original raw images, the intensity of immunostainining was measured using ImageJ free software . All microphotographs were obtained in the same conditions.2S was previously described46. Briefly, tissues were homogenized (1:10 w/v) in ice-cold 50 mM potassium phosphate, pH 6.8. The incubation solution (1 ml in each incubation chamber) contained of 10 mM L-cysteine, 2 mM pyridoxal 5′-phosphate, 100 mM potassium phosphate buffer, pH 7.4, and 50 mg of tissue homogenate. DL-propargylglycine , NG-nitro-L-arginine  or roflumilast  were added to the incubation solution in order to examine the role of an irreversible CSE inhibitor, a NO synthase inhibitor, and the selective PDE4 inhibitor, respectively. The reaction was initiated by transfer of the tubes from ice to a shaking water bath at 37 °C. After incubation for 30 min, 1% zinc acetate was injected to trap-generated H2S, and, then the reaction was stopped by applying 500% (w/v) trichloroacetic acid to denature the protein. Subsequently, N,N-dimethyl-p-phenylenediamine sulfate  in 7.2 M HCl was added, immediately followed by FeCl3  in 1.2 M HCl. The absorbance of the resulting solution at 670 nm was measured 20 min later by spectrophotometry. The H2S concentration was calculated against a calibration curve of NaHS standard. Results are expressed as nanomoles of H2S formed per mg of soluble protein per 20 min. Protein was determined with the Lowry assay .The method used for measuring endogenous production and release of H2 and 5% CO2 obtaining a final pH of 7.4. One end of the preparation was connected to an isometric force transducer (Grass FT03C) and the other one to a micrometer screw, with the signal continuously recorded on a polygraph (Graphtec Multicorder MC6621). The strips were allowed to equilibrate, under a passive tension of 2.0 g, for about 60 min in PSS and washed with (37 °C) PSS at 20 min intervals. The contractile capacity of the preparations was tested by stimulating them with 124 mM potassium-rich PSS (124 mM KPSS). In strips precontracted with 1 μM phenylephrine (PhE) concentration-response relaxation curves to roflumilast, tadalafil and isoproterenol, PDE4 and PDE5 inhibitors, and β-adrenergic receptor agonist, respectively, were constructed. Due to that relaxation curves to roflumilast were not reproducible, preparations from the same animal were run in parallel, using one of them as control, and the other one to assess the specific treatment for 30 min.Pig and human bladders were kept in chilled physiological saline solution (PSS) at 4 °C. Urothelium and suburothelium were removed by microdissection under the microscope. The absence of these layers was confirmed by histological study. Bladder neck strips (4–6 mm long and 2–3 mm wide) were suspended horizontally in 5 ml organ baths containing PSS at 37 °C and continuously gassed with 95% OIn electrical field stimulation (EFS) assays, pig bladder neck strips were pre-treated with guanethidine (10 μM) and atropine (1 μM) in order to block noradrenergic neurotransmission and muscarinic receptors, respectively, for 1 h, replacing the solution every 20 min, and these drugs were present throughout the experiment. Under these conditions and after 1 μM PhE-induced tone, relaxations to EFS in the absence or presence of threshold roflumilast concentrations were carried out in the same preparation. EFS was carried out using a stimulator , connected to two platinum electrodes placed on each side of the strip in parallel to its longitudinal axis, by delivering rectangular pulses , at 4 min intervals. Control EFS curves run parallel.G-nitro-L-arginine (L-NOARG), phenylephrine (PhE), DL-propargylglycine (PPG), roflumilast and tadalafil, all from Sigma (USA). PPG and roflumilast were dissolved in dimethylsulphoxide. Tadalafil was dissolved in acetonitrile. The other compounds were dissolved in double-distilled water. Preliminary experiments revealed no effects of the solvents used on bladder neck samples contractility.The drugs used were: Isoproterenol, N2 1.2, NaHCO3 24.9, glucose 11, CaCl2 1.5, KH2PO4 1.2, ethylenediamine tetraacetic acid (EDTA) 0.027. PSS was maintained at 37 °C and gassed with 95% O2 and 5% CO2, thus obtaining a final pH of 7.4 in the organ bath. To prepare K+-enriched PSS (KPSS), the NaCl in PSS was replaced with KCl on an equimolar basis.PSS composition (in mM)was: NaCl 119, KCl 4.6, MgCl2, where pD2 = −log EC50 and EC50 is the agonist concentration needed to produce half-maximal response. pD2 was estimated by computerized non-linear regression analysis . Relaxations are shown as a percent of the pre-contraction induced by PhE. Results are expressed as the mean ± s.e.m. of n (number of pigs or persons). Differences were analyzed using one-way analysis of variance (ANOVA) or paired or unpaired Student’s t-test when appropriate. Probability levels less than 5% were considered significant (P < 0.05). The calculations were carried out by using a standard software .Sensitivity to roflumilast, tadalafil and isoproterenol is expressed in terms of pDDataset 1


Although the incidence of pulmonary hypertension is higher in females, the severity and prognosis of pulmonary vascular disease in both neonates and adults have been shown to be worse in male subjects. Studies of sex differences in pulmonary hypertension have mainly focused on the role of sex hormones. However, the contribution of sex differences in terms of vascular signaling pathways regulating pulmonary vascular function remains incompletely understood. Consequently, we investigated pulmonary vascular function of male and female swine in vivo, both at rest and during exercise, and in isolated small pulmonary arteries in vitro, with a particular focus on the NO‐cGMP‐PDE5 pathway. Pulmonary hemodynamics at rest and during exercise were virtually identical in male and female swine. Moreover, NO synthase inhibition resulted in a similar degree of pulmonary vasoconstriction in male and female swine. However, NO synthase inhibition blunted bradykinin‐induced vasodilation in pulmonary small arteries to a greater extent in male than in female swine. PDE5 inhibition resulted in a similar degree of vasodilation in male and female swine at rest, while during exercise there was a trend towards a larger effect in male swine. In small pulmonary arteries, PDE5 inhibition failed to augment bradykinin‐induced vasodilation in either sex. Finally, in the presence of NO synthase inhibition, the pulmonary vasodilator effect of PDE5 inhibition was significantly larger in female swine both in vivo and in vitro. In conclusion, the present study demonstrated significant sex differences in the regulation of pulmonary vascular tone, which may contribute to understanding sex differences in incidence, treatment response, and prognosis of pulmonary vascular disease. Endothelial function is a key factor in vascular development as well as in maintenance of vascular structure and function throughout life. In the pulmonary vasculature, a healthy endothelium is essential for the transition from intrauterine to extrauterine life after birth, and endothelial dysfunction is an important factor in neonatal pulmonary vascular diseases such as bronchopulmonary dysplasia and neonatal pulmonary hypertension. Also later in life, endothelial dysfunction plays a critical role in the pathogenesis of adult pulmonary vascular disease, including pulmonary hypertension (PH). The pathogenesis of PH encompasses a combination of endothelial dysfunction, vasoconstriction, inflammation, structural remodeling of the pulmonary vasculature with formation of plexiform lesion and a high incidence of in situ thrombosis  pathway plays an important role in the pathogenesis of pulmonary hypertension. In patients with pulmonary hypertension, NO deficiency contributes to the increased pulmonary vascular tone and vascular remodeling  entered the study. Daily adaptation of animals to laboratory conditions started 1 week before surgery and continued during the first week after surgery.−1 i.m.) and midazolam (1 mg kg−1 i.m.), anesthetized with thiopental (10–15 mg kg−1 i.v.), intubated, and ventilated with a mixture of O2 and N2 (1:2) to which 0.2–1% (v/v) isoflurane was added. Anesthesia was maintained with midazolam (2 mg kg−1 + 1 mg kg−1 h−1 i.v.) and fentanyl (10 μg kg−1 h−1 i.v.), and the depth of anesthesia was checked regularly using a pain stimulus (toe‐pinch). Swine were instrumented under sterile conditions as previously described , and hemoglobin concentration  as well as for the infusion of drugs. A flow probe  was positioned around the ascending aorta for measurement of cardiac output. Catheters and electrical wires were tunneled subcutaneously to the back and the chest was closed in layers. Animals were allowed to recover, receiving analgesia (0.3 mg buprenorphine i.m.) for 2 days and antibiotic prophylaxis (25 mg kg−1 amoxicillin and 5 mg kg−1 gentamicin i.v.) for 5 days. The catheters were flushed daily with heparinized saline (1000–5000 IE mL−1). After completing all experimental protocols, animals were killed by an intravenous overdose of pentobarbitone sodium.Swine were sedated with ketamine  resting on the treadmill, resting hemodynamic measurements, consisting of heart rate, cardiac output, mean aortic pressure (MAP), mean pulmonary artery pressure (PAP), and mean left atrial pressure (LAP), were obtained. Rectal temperature was measured, and arterial and mixed venous blood samples were collected. Subsequently, a five‐stage treadmill exercise protocol was started (1–5 km h−1); each exercise stage lasted 3 min. Hemodynamic variables were continuously recorded and blood samples were collected during the last 45 sec of each exercise stage, at a time when hemodynamics had reached a steady state  was administered at a dose of 20 mg kg−1 i.v. in 24 male and 19 female swine. Ten minutes after completion of the infusion, resting measurements were obtained and the 5‐stage exercise protocol was repeated  the NO synthase inhibitor Nμg kg−1 min−1 i.v. in 7 male and 12 female swine. Ten minutes after starting the infusion, resting measurements were obtained and the 5‐stage exercise protocol was repeated  the phosphodiesterase‐5 inhibitor EMD360527  was infused continuously in a dose of 300 −1 i.v. in five male and five female swine. Ten minutes after completion of the infusion, resting measurements were obtained and the 5‐stage exercise protocol was repeated. Ninety minutes later, animals received the phosphodiesterase‐5 inhibitor EMD360527 (300 μg kg−1 min−1 i.v.). Ten minutes after starting the infusion, resting measurements were obtained and swine underwent a third exercise trial  the NO synthase inhibitor LNNA was administered in a dose of 20 mg kg2 (mmHg), paCO2 (mmHg), and pH, sO2 and hemoglobin (g/100 mL) were then immediately performed with a blood gas analyzer , and corrected for body temperature. Blood O2 content (μmol/mL) was computed as follows: (Hb × 0.621 × sO2) + (0.00131 × pO2). Body O2 consumption (BVO2) was calculated as the product of cardiac output and the difference in O2 content between arterial and mixed venous blood.Blood samples were maintained in iced syringes until the conclusion of each exercise trial. Measurements of paO2 were indexed to body weight . Statistical significance was accepted at 2 and PVC, regression analysis was performed with sex, drug treatment, and BVO2, as well as their interactions as independent variables and animal as case label . Statistical analysis of the effect of drug intervention (vs. control) on PVC at rest and at maximal exercise was performed using unpaired t‐test to compare data from male and female swine .To test for the effects of sex and drug intervention on the relation between BVOn = 14 and female, n = 13) were obtained at a local slaughterhouse. Pulmonary small arteries (diameter ≈ 300 μm) were dissected out from the lower lung lobe and stored overnight in cold, oxygenated Krebs bicarbonate solution of the following composition (in mmol/L): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, and glucose 8.3; pH 7.4. The next day, pulmonary small arteries were cut into segments of ~2 mm length and mounted in microvascular myographs (Danish MyoTechnology) with separate 6 mL organ baths containing Krebs bicarbonate solution aerated with 95% O2 and 5% CO2, and maintained at 37°C. Changes in contractile force were recorded with a Harvard isometric transducer. Following a 30 min stabilization period, the internal diameter was set to a tension equivalent to 0.9 times the estimated diameter at 20 mmHg effective transmural pressure. Vessels were then exposed to 30 mmol/L KCl twice. Endothelial integrity of pulmonary arteries was verified by observing dilation to 10 nmol/L substance P after precontraction with 100 nmol/L of the stable thromboxane A2 analog pyridoxalphosphate‐6‐azophenyl‐2′4′‐disulfonic acid (U46619). Vessels were then subjected to 100 mmol/L KCl to determine the maximal vascular contraction. Thereafter, pulmonary arteries were allowed to equilibrate in fresh Krebs solution for 30 min before initiating different experimental protocols  or presence of NO synthase inhibition with Nω‐Nitro‐L‐arginine methyl ester hydrochloride , PDE5 inhibition with 10−8 mol/L sildenafil  or combined NO synthase‐ and PDE5 inhibition .Pulmonary small arteries of male and female swine were subjected to the endothelium‐dependent vasodilator bradykinin (BK) in incremental concentrations ranging from 10Vascular relaxation response to BK was expressed as percentage of contraction to U46619.P ≤ 0.05. Data are presented as mean ± SEM.Statistical analysis was performed using SPSS version 21.0 (IBM) and Prism version 5.0 . Statistical significance was accepted at Emax) and half maximal effective concentration (EC50) in each experiment was calculated using the GraphPad Prism version 5 for Windows . Statistical analysis of maximal relaxation and EC50 to bradykinin was performed using two‐way (sex and drug intervention) analysis of variance (ANOVA) for repeated measures using SPSS version 21.0 (IBM). Statistical analysis of the effect of drug intervention (vs. control) at baseline and at bradykinin‐induced maximal relaxation was performed using unpaired t‐test to compare data from male and female swine .The maximal relaxation (μm) were isolated from the same lungs as used in the myograph experiments, and snap frozen in liquid nitrogen. Small pieces of the frozen arteries (<30 mg) were homogenized by adding RLT lysisbuffer (Qiagen) using a homogenizer. After a prot K treatment at 55°C for 10 min, total RNA was isolated using RNeasy Fibrous Tissue Mini Kit (Qiagen). RNA was eluted in water and stored at −80°C. The concentration was determined by using a nanodrop and RNA integrity was confirmed by Bioanalyzer. cDNA was synthesized from 100 ng of total RNA with SensiFAST cDNA Synthesis Kit (Bioline). Quantitative real‐time PCR  was performed with SensiFAST SYBR & Fluorescein Kit (Bioline). Target gene mRNA levels were normalized against β‐actin, glyceraldehyde‐3‐phosphate dehydrogenase (GADPH), and Cyclophilin using the CFX manager software (Bio‐Rad). Primer sequences are shown in Table For detection of eNOS, soluble guanylyl cyclase (sGC) and PDE5A mRNA, pulmonary small arteries  significantly enhanced the precontraction to U46619 . In contrast to the comparable effect of NO synthase inhibition in both sexes in vivo, the BK‐induced relaxation after L‐NAME administration was significantly smaller in male swine as compared to female swine  were not statistically different between sexes.In vitro, addition of the NO synthase inhibitor L‐NAME 10−mol/L sigP = 0.077 for PDE5 inhibition x sex, Fig. PDE5 inhibition resulted in a significant increase in systemic vascular conductance at rest Fig.  that was−8mol/L) tended to attenuate the precontraction to U46619 .Although PDE5 inhibition by sildenafil . The change in PVC in response to combined PDE5 ‐and NO synthase inhibition was significantly smaller in male as compared to female swine at rest , this larger pulmonary vasodilator effect in female swine is unlikely to be explained by a higher presence of sGC.Altogether, these data suggest that there is a pathway different from the NO pathway that results in activation of sGC or pGC to produce cGMP. Furthermore, both in vivo and in vitro data indicate that in the presence of NO synthase inhibition, PDE5 inhibition has a larger pulmonary vasodilator effect in female, as compared to male swine. Since the mRNA expression levels of sGC did not differ between male and female swine  pulmonary hemodynamics at rest and during incremental exercise did not differ between male and female swine. (2) NO synthase inhibition with LNNA resulted in pulmonary vasoconstriction to a similar extent in male and female swine at rest and during exercise. (3) NO synthase inhibition with L‐NAME also reduces BK‐induced vasodilation in isolated pulmonary small arteries; however this reduction was slightly larger in male than in female swine. (4) PDE5 inhibition with EMD360527 resulted in pulmonary vasodilation to a similar extent in male and female swine at rest and during exercise, but PDE5 inhibition with sildenafil failed to augment the bradykinin‐induced vasodilation in small pulmonary arteries isolated from either male or female swine. (5) In the presence of NO synthase inhibition, the pulmonary vasodilator effect of PDE5 inhibition was significantly larger in female swine both in vivo (at rest) and in vitro. The implications of the present findings are discussed below.PVC is defined as cardiac output divided by mean PAP minus mean pulmonary backpressure. Pulmonary backpressure can be measured either by pulmonary wedge pressure or by left atrial pressure  are enzymes catalyzing the production of nitric oxide from L‐arginine. Two classes of NOSs exist; the constitutive isoforms endothelial NOS (eNOS) and neuronal NOS (nNOS), and the inducible isoform (iNOS). Both LNNA and L‐NAME demonstrate a 10‐ to 30‐fold selectivity for eNOS and nNOS over iNOS  produced similar hemodynamic responses compared to 20 mg kg−1  and nNOS (IC50 = 0.05 μmol/L)  compared with PDE6 (IC50 = 0.049 μmol/L), 41‐fold selectivity for PDE1 (IC50 = 0.35 μmol/L), 376‐fold selectivity for PDE4 (IC50 = 3.2 μmol/L), 447‐fold selectivity for PDE10 (IC50 = 3.8 μmol/L), and >1000‐fold selectivity for PDE2, PDE3, and PDE7 (IC50 > 10 μmol/L) (Bischoff 50 = 0.007 μmol/L) compared with PDE6 (IC50 = 0.32 μmol/L), 94‐fold selectivity for PDE1 (IC50 = 0.66 μmol/L), 137‐fold selectivity for PDE10 (IC50 = 0.96 μmol/L), and >1,400‐fold selectivity for PDE2, PDE3, PDE4, and PDE7 (IC50 > 10 μmol/L) . These findings are consistent with the comparable pulmonary vasoconstriction produced by NO synthase inhibition both at rest and during exercise in vivo between male and female swine. In vitro, NO synthase inhibition reduced BK‐induced vasorelaxation in pulmonary small arteries from male swine to a greater extent as compared to female swine. Although this apparent discrepancy between in vivo and in vitro findings is not readily explained, several possible explanations could be forwarded. First, eNOS activation by circulating estrogen in the pulmonary circulation , was observed suggesting a more active endogenous PDE5 in male swine. This is consistent with recent post hoc analyses of the PHIRST and SUPER trials  or even enhance  the pulmonary vasodilator responses to PDE5 in patients with pulmonary hypertension. Future studies are required to further investigate the mechanisms underlying these sex‐related differences in pulmonary vascular control mechanisms.The authors do not have any potential or actual personal, political, or financial interest in the material, information, or techniques described in this paper.


Cyclic AMP (cAMP) had no effect on ERK1/2 activity in human spermatozoa, but stimulated the MAPK in mouse pituitary LβT2 gonadotrope cells. cAMP via PKA attenuates PKC-dependent ERK1/2 activation only in the presence of proAKAP4. St-HT31, which disrupts PKA-regulatory subunit II (PKA-RII) binding to AKAP abrogates the inhibitory effect of cAMP in human spermatozoa and in HEK293T cells expressing proAKAP4. In transfected HEK293T cells, PMA relocated proAKAP4, but not proAKAP4-T265A to the Golgi in an ERK1/2-dependnet manner. Similarly, AKAP4 is localized to the spermatozoa principal piece and is relocated to the mid-piece and the postacrosomal region by PMA. Furthermore, using capacitated sperm we found that cAMP reduced PMA-induced ERK1/2 activation and acrosome reaction. Thus, the physiological role of the negative crosstalk between the cAMP/PKA/AKAP4 and the PKC/ERK1/2 pathways is to regulate capacitation and acrosome reaction.Mammalian spermatozoa undergo capacitation and acrosome reaction in order to fertilize the egg. The PKC-ERK1/2 pathway plays an important role in human spermatozoa motility, capacitation and the acrosome reaction. Here we demonstrate that ERK1/2 phosphorylates proAKAP4 on Thr265 in human spermatozoa  Capacitation ends in the process of acrosome reaction and fertilization of the egg23456789Mammalian spermatozoa acquire the ability to fertilize the egg during their path to the oocyte. Spermatozoa undergo a series of biochemical and physiological processes known as capacitationA-kinase anchoring proteins (AKAPs) are a family of scaffolding proteins that target PKA and other signaling enzymes to selective subcellular locations121314et al.Evidence for the involvements of AKAPs in the PKC-ERK1/2 pathway was presented by Su AKAP4  is a one of the major components (~50%) of the sperm fibrous sheath, a structure known to be involved in sperm motility. AKAP4 has a precursor termed proAKAP4 that is processed to mature AKAP4 during sperm differentiation in the human testes. Both proteins are highly homologous to mouse AKAP4. In contrast to mouse AKAP4, there is still a considerable portion of proAKAP4 that is not processed to AKAP4, and is localized to the entire sperm principal piece20We and others have previously reported that the PKC/ERK1/2 pathway plays an important role in human sperm motility, capacitation and acrosome reaction242526in vitro  lacking all the potential MAPK phosphorylation sites was not phosphorylated and the proAKAP4T265A mutant exhibited a faint phosphorylation band that was hardly detected , but not the A and B-Raf26We then investigated whether the inhibitory effect of cAMP on the PKC/ERK1/2 pathway is AKAP-dependent. Disruption of PKARII anchoring to AKAP can be achieved by using St-HT31, an anchoring inhibitor peptide derived from the RII binding domain of a human thyroid AKAPIn order to specifically assess the involvement of proAKAP4 in the inhibitory effect of cAMP on the PKC-ERK1/2 pathway we repeated the experiment in HEK293T cells expressing proAKAP4 . Indeed,In order to follow the fate of proAKAP4 after phosphorylation by ERK1/2, we first followed the cellular localization of proAKAP4 in transfected HEK293T cells. The cells were transfected with tGFP-proAKAP4 or tGFP-proAKAP4-T265A, point mutation in the ERK1/2 phosphorylation site, and then were pretreated with or without the MEK/ERK inhibitor U0126 for 20 min. Thereafter PMA was added for 5–30 min . PMA indWe then assessed whether AKAP4 also translocates in PMA-treated spermatozoa. To that aim we followed the localization of AKAP4 in capacitated spermatozoa under basal and PMA-activated cells. Indirect immunofluorescence microcopy showed that AKAP4 is localized to the principal piece under basal conditions and in the principal piece, mid-piece and the postacrosomal region under PMA stimulation . In ordeWe then investigated the biological significance of the negative crosstalk between the cAMP/PKA/AKAP4 and the PKC/ERK1/2 pathways. First, we examined the effect of cAMP upon PMA-stimulated motility. In noncapacitated cells as above  cAMP hadAKAPs are a wide family of proteins, known to bind multiple signaling molecules that were originally co-purified with PKA-R, but many of them have been shown to anchor PKC44AKAPs play an important role in spermatozoa compartmentalization by enabling the targeting of proteins and other signaling molecules to specific intracellular sites. A major protein component of the spermatozoa fibrous sheath is AKAP42347in vitro phosphorylation by ERK2, and mapped the phosphorylation site at Thr265. Indeed, Thr265 is a consensus sequence for ERK1/2 phosphorylation (PRTP). Furthermore, we could demonstrate the phosphorylation of proAKAP4 by MAPK in vivo in PMA-stimulated spermatozoa. Inhibition of the phosphorylation by the MEK inhibitor U0126 and the ERK1/2 inhibitor, GDC-0994, which prevents ERK1/2 activity through steric effects, without preventing MEK phosphorylation of ERK1/2, implicated ERK1/2 as the MAPK involved in proAKAP4 phosphorylation in vivo. It is well-recognized that phosphorylation of proteins on tyrosine residues is a marker of sperm capacitation39AKAP4 undergoes serine and tyrosine phosphorylation during capacitationWhen we examined the effect of cAMP, a vital molecule in sperm physiology33054553258596031We were then interested to know if proAKAP4 participates in the negative regulation of PMA-induced ERK1/2 activation by cAMP. Since ejaculated sperm cannot be transfected we resorted to HEK293T cells, in which cAMP had no effect on basal ERK1/2 levels as in sperm. Indeed, cAMP can decrease PMA-induced ERK1/2 activation only in HEK293T cells expressing proAKAP4. Further support that proAKAP4 participates in the cAMP inhibition of PMA-induced ERK1/2 activation was derived by the use of St-HT31. The peptide drug disrupts PKARII anchoring to AKAPs17Localization of AKAP4 in human spermatozoa may point to its role in sperm functions. We first focused our attention to transfected HEK293T cells, since mature sperm can’t be transfected. PMA treatment relocated proAKAP4 from the cytosol to the Golgi, as evident by colocalization with the Golgi marker, GRASP65. The effect of PMA was blocked by the MEK inhibitor, U0126 indicating that ERK1/2 was most likely involved by phosphorylation of proAKAP4. This was confirmed by showing that proAKAP4-T265A did not translocate to the Golgi by PMA. Importantly, during spermatogenesis the Golgi forms the acrosome vesicle2426We then investigated the biological significance of the negative crosstalk between the cAMP/PKA/AKAP4 and the PKC/ERK1/2 pathways. Although both cAMP and PMA stimulated forward motility on their own, their combination had no effect on motility (data not show). We hypothesize that the reduction in ERK1/2 observed in The cAMP/PKA and the PKC/ERK1/2 pathways play major roles in sperm biology32627303832P]ATP was obtained from PerkinElmer . Recombinant ERK2 was obtained from New England Biolabs . Mouse monoclonal anti-doubly phosphorylated (DP)-ERK1/2 antibodies and rabbit polyclonal antibodies to general ERK and rabbit anti-AKAP4 were obtained from Sigma-Aldrich . Rabbit anti-turboGFP was obtained from Evrogen . Mouse monoclonal anti-phospho-Ser/Thr-Pro (MPM2) antibody was from Upstate . Secondary horseradish peroxidase-conjugated goat anti mouse or goat anti rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories . Secondary anti-rabbit Alexa Fluor™ 488-labeled antibody was obtained from Thermo Fisher Scientific . Anti Raf-1 antibody was from Merck Millipore . tGFP-tagged ORF clone of human AKAP4 transcript variant 1 (NM_003886.2) was obtained from Origene . GRASP65-RFP construct was kindly provided by Dr. K. Hirschberg .Media, sera and antibiotics for cell cultures were obtained from Biological Industries . PMA, U0126, IBMX and 8-Br-cAMP were obtained from Sigma . GDC-0994 was obtained from Selleckchem . Myristoylated PKI 14–22 amide (PKI) was obtained from Tocris Bioscience . St-HT31 and St-HT31-P were obtained from promega . jetPRIME Transfection reagent was obtained from polyplus transfection . Proteins A/G agarose beads were obtained from Santa Cruz Biotechnology, Inc. .  ATP (4000 cpm/pmol) was added to the reaction and incubated for 10 min at 30 °C. The reaction was terminated by adding sample buffer, and the phosphorylated proteins were resolved on 10% SDS-PAGE, transferred to nitrocellulose membrane (1.5 h at 300 mA) and autoradiographed.HEK293T Cells were plated in 100-mm plates 24 h prior to transfection with tGFP-proAKAP4 and it mutants followed by transfection with 10 μg of plasmid DNA. 48 h later the cells were lysed with 100 mM NaCl, 20 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 15% glycerol, 0.2% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors mixture. The lysate was subjected to immunoprecipitation with an anti-turbo-green fluorescent protein antibody conjugated to protein A/G PLUS-agarose beads by gentle shaking overnight in 4 °C. The beads were washed once in RIPA buffer, followed by three washes with LiCl 0.5 M Tris 0.1 M pH = 7.4 and another wash in RIPA buffer in order to remove proteins that bound non-specifically to the beads and might have co-precipitated with tGFP-proAKAP4 or its mutants. Samples were frozen in −20 °C until their subjection to HEK293T cells were plated on 18 mm cover slips and transfected using the calcium phosphate method with 1 μg tGFP-proAKAP4, tGFP-proAKAP4-T265A or GRASP5. 24 h after transfection, cells were serum starved  and were pretreated with U0126 for 20 min and then PMA was added for various time points. Subsequently, the cells were washed with PBS, fixed in 4% paraformaldehyde (PFA) in PBS and then probed with DAPI. Cover slips were placed on glass slides with Vectashield mounting medium. Confocal fluorescent images were collected with a 63x magnification by Zeiss LSM-META confocal microscope.Sperm were washed and smeared on polylysine precovered slides and then allowed to air-dry. Cells were washed with PBS, permeabilized with Triton X-100 0.5% buffered in PBS. Nonspecific binding was blocked with 3% BSA buffered in PBS. Cells were probed first with monoclonal anti-AKAP4 (1:75), washed three times with PBS, and then probed with a secondary anti-rabbit Alexa Fluor™ 488-labeled antibody. Slides were viewed with Zeiss LSM-META confocal microscope.Results from two or more experiments were expressed as mean ± SEM. Where appropriate, data were subjected to statistical analysis by Student’s t test. Values of P < 0.05 were considered statistically significant.How to cite this article: Rahamim-Ben Navi, L. et al. A-Kinase Anchoring Protein 4 (AKAP4) is an ERK1/2 substrate and a switch molecule between cAMP/PKA and PKC/ERK1/2 in human spermatozoa. Sci. Rep.6, 37922; doi: 10.1038/srep37922 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Andrographis paniculata, “King of bitters” is a popularly known medicinal plant extensively used in many parts of the world for treatment of various diseases. Since recent past, anaphylactic/allergic type adverse events were reported upon A. paniculata usage, the study aimed to evaluate the anaphylactic and anaphylactoid potential of A. paniculata extract and andrographolide (a major phytoactive of A. paniculata). The anaphylactic potential was evaluated using active systemic anaphylaxis (ASA) assay in guinea pigs. Further, the release of allergic mediators was measured in immunoglobulin E (IgE) sensitized and non-IgE sensitized Rat Basophilic Leukemia (RBL-2H3) cell lines in-vitro. A. paniculata extract or andrographolide sensitized guinea pigs following the challenge antigen administration orally and intravenously did not demonstrate any clinical signs of anaphylaxis. IgE sensitized and non- IgE sensitized RBL-2H3 cells treated with A. paniculata extract did not induce release of allergic mediators. Whereas IgE sensitized and non- IgE sensitized RBL-2H3 cells treated with andrographolide demonstrated mild to moderate release of allergic mediators. A. paniculata extract has no anaphylactic and anaphylactoid potential in in-vivo and in-vitro studies. Whereas, andrographolide effects on allergic mediators in in-vitro studies needs to be scrutinized if they are of biologically important. F.) Nees belonging to family Acanthaceae is the most popular traditionally known medicinal plant used for the treatment of array of disease like viral fever, chicken pox, common cold, diarrhoea, dysentery, eczema, epidemic encephalitis B, hepatitis, herpes zoster, mumps, ulcer, neurodermatitis, inflammation, pharyngolaryngitis, pneumonia, respiratory infections A. paniculata is often used in combination with other herbs and health care products for treating patients suffering from various physical and mental disorders. It has been estimated that A. paniculata is used in more than 50% of herbal compositions commercialized in India for hepatic disorders In the Ayurvedic system of medicine, A. paniculata has been shown to possess wide spectrum of pharmacological properties viz., anti-microbial, anti-cancer, anti-inflammatory, anti-oxidant, immunostimulant, anti-diabetic, anti-infective, hepato-renal protective, anti-angiogenic, anti-allergic etc A. paniculata is rich in labdane diterpinoids. The pharmacological effects of A. paniculata have been attributed to the major bitter tasting secondary metabolite i.e, andrographolide, a labdane diterpinoid A. paniculata has immense potential for treating various diseases as per traditional and modern systems of medicine A. paniculata is vastly reported to possess safety and efficacy A. paniculata usage and reported an association between anaphylactic/allergic-type adverse drug reactions (ADRs) for products that contain A. paniculata as an ingredient. However, TGA could not conclude on particular type of extract or quantity of Andrographis to be causative for allergic/antigenic reactions associated with A. paniculata products A. paniculata extract and its major phytoactive andrographolide using in-vivo and in-vitro assays.The published systematic reviews indicate that A. paniculata extract and andrographolide in active systemic anaphylaxis (ASA) assay using guinea pigs. Further, the anaphylactic and anaphylactoid potential was investigated by measuring the release of allergic mediators such as histamine, β-hexosaminidase, leukotriene C4 (LTC4) and tryptase in IgE sensitized and non-IgE sensitized rat basophilic leukemia (RBL-2H3) cell line.The present study investigated the anaphylactic potential of 22.1A. paniculata (300 kg) were extracted with methanol under reflux. Thick paste obtained was dried under vacuum (≤65 °C), milled and sieved (#40) to get a uniform powdered extract of A. paniculata (18 kg). To the marc contained in the extractor, water was added and extracted under reflux. The concentrated liquid was then spray dried to get the water extract of A. paniculata (10 kg). The alcohol and water extracts were then analyzed for its active constituents and blended to get A. paniculata extract. The composition manufactured by Natural Remedies Private Limited known as KalmCold™/Ap-Bio™ adheres to the international quality requirements which include analysis of solvent residue, heavy metals residue, mycotoxin residue, pesticide residue evaluation and microbial contamination.Coarse ground leaves of 2.1.1A. paniculata extract on HPLC analysis was found to contain the following constituents, viz., andrographolide (>30.0% w/w), isoandrographolide (>0.3% w/w), neoandrographolide (>1.0% w/w), andrograpanin (>0.3% w/w), 14-deoxy-11,12-didehydroandrographolide (≤5.0% w/w), skull-capflavone I (>0.05% w/w) and 7-O-methylwogonin(>0.05% w/w).2.2The extract was subjected to liquid- liquid partitioning between ethyl acetate and water. The ethyl acetate layer was repeatedly chromatographed over silica gel using combinations of hexane: ethyl acetate and chloroform: methanol. Crystallisation of different chromatographic fractions yielded andrographolide. Identification of andrographolide was confirmed by comparing their 1H and 13C NMR data with literature. Purity of isolated compound was determined by HPLC 2.32.3.1Ovalbumin, Complete Freund’s Adjuvant (CFA) from Sigma Aldrich, USA; dimethyl sulfoxide (DMSO) and sodium chloride from Himedia Laboratories Pvt. Ltd., India were obtained. Mixture of ovalbumin and CFA was used as a positive control which was dissolved in normal saline, while 10% DMSO was used as a vehicle to dissolve the test substances.2.3.2ad libitum. All the animal procedures were approved by Institutional Animal Ethics Committee of Natural Remedies Pvt. Ltd., Bangalore (IAEC/NR-PCL/02/11.16).Male Dunkin-Hartley guinea pigs, 6–7 weeks of age were procured from Geniron Biolabs Pvt. Ltd., Bangalore. Guinea pigs were acclimatized for 7 days under optimal temperature of 25 ± 2 °C and 30–70% relative humidity before the initiation of the experimentation. Guinea pigs were allowed free access to feed pellets  and UV purified water 2.3.3A. paniculata extract (25 mg/kg), andrographolide (8 mg/kg) and DMSO (10 ml/kg) were orally administered to respective groups for 5 days. While for positive control, mixture of ovalbumin and CFA was administered subcutaneously weekly once for 3 weeks A. paniculata extract, andrographolide for sensitization corresponds to double the clinical dose of A. paniculata extract i.e., 200 mg/day.The study design including the sensitization and challenge schedule is briefed in A. paniculata extract or andrographolide, while the vehicle control and positive control groups were challenged with DMSO and ovalbumin respectively as presented in Two weeks following the final sensitization, the challenge antigen was either given orally or intravenously into the leg vein. Treatment groups were challenged with Clinical sign observations were performed 30 min to 3 h post challenge dose administration. The anaphylactic signs observed were scored according to Lee et al., Kouchi et al., Park et al. (−): No signs of anaphylaxis/Asymptomatic(±: Mild): Urination, evacuation, licking or rubbing the nose and ruffling the fur on occasion(+: Moderate): Cough, sneezing, weakness, restlessness and rales in addition to the above signs(++: Severe): Piloerection, nostril discharge, lacrimation, salivation, nasal bleeding, convulsion, dyspnoea, staggering gait, rhonchus, cyanosis, side position, flattening, prostration, retching, labored respiration in addition to the above signs(+++): Death2.42.4.12, 95% air).RBL-2H3 rat basophilic leukaemia (CRL-2256™) cell lines were obtained from American Type Culture Collection (ATCC) . RBL-2H3 cells was cultured in EMEM conditioned with 15% heat inactivated FBS (HI-FBS) at 37 °C in a humidified incubator (5% CO2.4.24 cells/well). After overnight culturing, cells were cultured with test items for 1 h at 37 °C RBL-2H3 cells were harvested and transferred into 96-well microplates  IgE for 24 h at 37 °C. IgE sensitized cells were treated with solvent control (DMSO 0.1%), or test substance at non-cytotoxic concentrations for 10 min. Cells were incubated with phosphatidylserine (10 μg/ml) for 5 min and followed by 0.5 μg/ml of 2,4-Dinitrophenyl hapten conjugated to bovine serum albumin (DNP-BSA) as antigen for 30 min. Supernatant was collected to quantify histamine, LTC4, β – hexosaminidase and tryptase. The quantification of histamine and LTC4 levels were carried out by means of homogenous time resolved fluorescence (HTRF) according to the procedure described by the kit manufacturer . Tryptase and β – hexosaminidase levels were measured using ELISA kits .RBL-2H3 cells were seeded at a density of 1 × 102.4.45 cells/well on a 24 – well microplate in 0.5 ml of complete medium/well and incubated overnight at 37° C, 5% CO2, 95% air. The cells were treated with treatment medium containing solvent control (DMSO 0.1%), or test substance at non-cytotoxic concentrations for 60 min. The supernatant was collected to quantify histamine, LTC4, tryptase and β – hexosaminidase levels.RBL-2H3 cells were seeded at a density of 1 × 102.5in-vitro studies. Statistical analysis was performed using one-way analysis of variance, followed by post hoc Dunnett’s test. P-value <0.05 was considered as statistically significant.The results were expressed as scores for ASA assay and mean ± standard deviation for 33.1viz., piloerection, flattening or side position, convulsions in 3 of the 5 animals. While, the remaining 2 animals died within 30 min to 3 h post ovalbumin administration. Hence, the positive control clearly demonstrated death as well as severe signs of anaphylaxis. In DMSO (vehicle control) sensitized group that served as vehicle control group, only urination and evacuation of faeces were observed following dosing with challenge antigen  and andrographolide (0.37–10 μg/ml). Hence the non-cytotoxic concentrations of A. paniculata and andrographolide were used in further experiments.The viability of RBL-2H3 cells upon treatment with 3.2.23.2.2.1A. paniculata extract at 0.2, 2 and 20 μg/ml dose levels did not induce histamine, β-hexosaminidase, LTC4 and tryptase release  demonstrated significant increase in tryptase levels at all the dose levels tested when compared to DMSO (Treatment of non-IgE sensitized RBL-2H3 with andrographolide (dose range 0.1–10 μg/ml) did not induce any significant increase in the histamine release when compared to DMSO. While treatment with andrographolide (dose range 0.1–10 μg/ml) except at higher dose (>1 μg/ml) tested, did not induce any significant increase in β-hexosaminidase and LTC to DMSO .Fig. 5Ef4A. paniculata has immense potential for treating various ailments such as liver disorders, respiratory tract problems and is widely used in many countries. The herb’s efficacy and safety were also profoundly researched A. paniculata extract as well as for andrographolide.Complementary and alternative medicines (CAM) are gaining popularity globally for various ailments and their usage continues to expand rapidly across the world A. paniculata extract and andrographolide in guinea pigs.Anaphylaxis affects one or more organ systems following the exposure to allergen that activates mast cells or basophils via IgE prompting degranulation and immediate release (5–30 min) of preformed mediators such as histamine, leukotrienes, tryptase etc. which are responsible for the occurrence of clinical signs and symptoms of anaphylaxis A. paniculata extract or andrographolide and subsequently challenged with A. paniculata extract or andrographolide orally/intravenously to respective groups did not demonstrate any signs of anaphylaxis. Although urination and defecation were observed in the treatment groups, as they were reported in the vehicle control group and were not accompanied by other moderate or severe anaphylactic signs, these signs do not indicate antigenicity. Thus, the study findings demonstrated no anaphylactic potential of A. paniculata extract and andrographolide in ASA assay.In the ASA assay, positive control sensitized with mixture of ovalbumin and Complete Freund’s Adjuvant followed by ovalbumin challenge demonstrated severe clinical signs of anaphylaxis including death in two animals indicating the reliability of this model for evaluating antigenicity of test substance A. paniculata extract and andrographolide RBL-2H3 cells are used to study comprehensive events on mast cells induced by allergens. The present study employed Anti-DNP IgE sensitized and DNP-BSA stimulated RBL-2H3 cells to investigate the allergic effects of A. paniculata extract/andrographolide were compared to DMSO treatment. There was no significant difference between the DMSO and A. paniculata extract treated groups. A. paniculata extract did not induce release of histamine, β-hexosaminidase, LTC4 and tryptase from IgE sensitized RBL-2H3 cells indicating that extract does not have any antigenic potential. However, andrographolide did not induce release of histamine and LTC4 from IgE sensitized RBL-2H3 cells at low doses. At higher dose tested andrographolide induced histamine and LTC4 release in IgE sensitized RBL-2H3 cells.The results of treatment of IgE sensitized RBL-2H3 cells with A. paniculata extract did not result in degranulation and release of allergic mediators from non-IgE sensitized RBL-2H3 cells. However, andrographolide treatment resulted in release of tryptase and β-hexosaminidase and LTC4 at higher doses tested through a non-IgE mediated pathway indicating it might induce anaphylactoid reactions. Hu et al. reported similar anaphylactoid potential for andrographolide in P815 mast cell degranulation model in vitroin-vitro studies, these effects were not observed in in-vivo studies hence, the biological importance of the effects needs to be scrutinized Anaphylactoid reactions are non-IgE mediated release of histamine, tryptase and other allergic mediators from mast cells and basophils via different trigger mechanisms A. paniculata extract that is standardized to various constituents has not demonstrated any anaphylactic potential. As reported by Suwankesawong et al. there is a possibility that hypersensitivity reaction might be related to product contamination and its lack of standardization across brands A. paniculata.The study findings indicate that 5A. paniculata extract did not induce clinical signs of anaphylaxis in guinea pigs and also did not induce allergic mediators release from IgE sensitized and non-IgE sensitized RBL-2H3 cells indicating that A. paniculata extract does not have anaphylactic and anaphylactoid potential under the conditions tested in the present study.In conclusion, treatment with While andrographolide although did not induce clinical signs of anaphylaxis in guinea pigs, it induced allergic mediators release from IgE sensitized and non-IgE sensitized RBL-2H3 cells at higher concentrations however conclusive remark can be made on the anaphylactic potential of andrographolide after scrutinizing biological significance.Study conception and design: Edwin, Bharathi, Deepak.Acquisition of data: Sasikumar, Edwin, Ramanaiah.Analysis and interpretation of data: Edwin, Sasikumar, Bharathi, Chandrasekaran.Drafting of manuscript: Bharathi, Edwin, Ramanaiah, Sasikumar.Critical revision: Bharathi, Chandrasekaran, Deepak.The author(s) confirm that this article content has no conflict of interest.