Signal crosstalk between distinct G protein-coupled receptors (GPCRs) is one mechanism that underlies pleiotropic signalling. Such crosstalk is also pertinent for GPCRs activated by gonadotrophic hormones; follicle-stimulating hormone (FSH) and luteinising hormone (LH), with specific relevance to female reproduction. Here, we demonstrate that gonadotrophin receptor crosstalk alters LH-induced Gαq/11-calcium profiles. LH-induced calcium signals in both heterologous and primary human granulosa cells were prolonged by FSHR coexpression via influx of extracellular calcium in a receptor specific manner. LHR/FSHR crosstalk involves Gαq/11 activation as a Gαq/11 inhibitor abolished calcium responses. Interestingly, the enhanced LH-mediated calcium signalling induced by FSHR co-expression was dependent on intracellular calcium store release and involved Gβγ. Biophysical analysis of receptor and Gαq interactions indicated that ligand-dependent association between LHR and Gαq was rearranged in the presence of FSHR, enabling FSHR to closely associate with Gαq following LHR activation. This suggests that crosstalk may occur via close associations as heteromers. Super-resolution imaging revealed that LHR and FSHR formed constitutive heteromers at the plasma membrane. Intriguingly, the ratio of LHR:FSHR in heterotetramers was specifically altered following LH treatment. We propose that functionally significant FSHR/LHR crosstalk reprograms LH-mediated calcium signalling at the interface of receptor-G protein via formation of asymmetric complexes. Anatomical ‘atlases’ of GPCR expression in diverse tissues suggest more than 100 different GPCRs are coexpressed in any one cell type, and that systems have distinct GPCR expression signatures4. Furthermore, one mechanism contributing to this pleiotropy in cell signalling is crosstalk of GPCR signalling, and that such crosstalk can occur via GPCR heteromerisation. The latter is defined as a complex of at least two different receptor protomers, with distinct biochemical properties from its individual components or homomers5. These GPCR heteromeric complexes can alter receptor function from monomeric or homo-di/oligomeric complexes, at multiple levels from ligand binding, G protein activation, receptor trafficking, and may possibly play a role in biased signalling9.How individual cells integrate and decode multiple signals from an array of distinct receptors is a fundamental question in cell biology. This is especially relevant for the superfamily of G protein-coupled receptors (GPCRs) that represent the largest family of signalling receptors with abundant and widespread expression in all physiological systems12. These hormones and their receptors play a central role in the regulation of sex steroid production, development of ovarian follicles, ovulation, and maintenance of corpus luteum and corpus luteum of pregnancy13. Thus, when coexpressed, heteromerisation of LHR FSHR is possible, but very little is known about their potential functional crosstalk.Crosstalk is highly pertinent for the GPCRs that play key roles in female reproduction and pregnancy. Upon ovarian follicular maturation, the follicle-stimulating hormone receptor (FSHR) and luteinising hormone receptor (LHR) are expressed in granulosa cells sequentially, starting with FSHR expression in early follicles, progressing to a stage of FSHR and LHR coexpression in preovulatory and luteinising follicles, and subsequently to LHR being primarily expressed in luteinised follicles2+) from intracellular stores; physiological relevant conditions that occur in the mural granulosa cells of the ovulatory follicle and the LH surge leading to ovulation15. We and others have demonstrated that LHR and FSHR are able to form constitutive homomers and heteromers21. Furthermore, we have demonstrated that the molecular composition of LHR protomers in lower order oligomers may modulate G protein activity19, highlighting the potential significance of gonadotrophin receptor organization in regulating signal crosstalk. LHR/FSHR crosstalk has been shown to negatively cross-modulate Gαs-cAMP signalling20. This inhibition of the primary G protein-signalling pathway by each receptor raises an interesting question in how such crosstalk may impact Gαq/11 signalling that is also key to follicular function.The primary G protein signalling pathway that the gonadotrophin receptors are coupled to is Gαs, which activates adenylyl cyclase and increases intracellular levels of cAMP. However, for the LHR, under conditions of high hormone concentrations and high receptor expression levels, this receptor also couples to Gαq/11 to activate phospholipase C which increases levels of diacyl glycerol and inositol phosphates that trigger release of calcium , and Cambridge BioScience, respectively. Coelenterazine h was purchased from Promega (UK), for BRET assays, and for Ca2+ imaging, Fluo-4AM direct was obtained from Invitrogen. Pharmacological inhibitors nifedipine and 2-aminoethyl diphenylborinate (2-APB) was purchased from Sigma Aldrich (UK), thapsigargin from Abcam (UK) and gallein from Calbiochem (UK). The Gαq/11 inhibitor FR900359 (UBO-QIC) was purchased from the Institute of Pharmaceutical Biology, University of Bonn.Recombinant LH and FSH was from the National Peptides and Hormones Program . CAGE 500 and 552 22. FLAG-tagged β2-adrenergic receptor was kindly provided by Prof. M. von Zastrow . Renilla luciferase 8 (Rluc8) and mVenus was provided by S. Gambhir  and A. Miyawaki , respectively. To generate C-terminally mVenus-tagged or Rluc8 FSHR and LHR, PCR was conducted to remove the stop codon, and sub-cloned into pcDNA3.1 plasmid containing Rluc8 or mVenus. Plasmid integrity was confirmed via sequencing. Plasmid DNA encoding Gαq and Gαs mVenus-tagged constructs and the untagged Gβ1 and Gγ2 subunits provided courtesy of J. Javitch 23.N-terminally FLAG-tagged human (h) LHR, and HA-tagged hFSHR were generated as described previously19. Cell lines stably expressing FLAG-hLHR or HA-FSHR were utilised for all functional studies. Stable cell lines were generated via Lipofectamine 2000® (Invitrogen)-based transfection of either FLAG-LHR or HA-FSHR plasmid DNAs, followed by G418 selection and assessment of cell surface LHR and FSHR expression levels by flow cytometry . Transient transfections were also conducted using Lipofectamine 2000® (as per manufacturer’s protocol) and experiments performed 48 h after transfection.HEK 293 cells were maintained and cultured as previously described24.Human granulosa-lutein cells were harvested from follicular aspirates obtained from women undergoing controlled ovarian hyperstimulation for IVF-embryo transfer under the approved study by the National Health Service National Research Ethics-Hammersmith and Queen Charlotte’s and Chelsea Research Ethics Committee (08/H0707/152). Written informed consent was obtained from all participants in accordance with the guidelines in The Declaration of Helsinki 2000. Subjects with idiopathic or male-factor infertility were recruited from the Infertility Clinic at Hammersmith Hospital, Imperial College London NHS Trust. Cells were isolated via Percoll density gradient as previously described1 was utilised to determine ligand-mediated receptor-Gα protein interactions, as described previously19. Briefly, cells were rinsed and harvested into PBS, and transferred into 96-well plates at a density of ∼200,000 cells/well. Following addition of Coelenterazine h substrate, the baseline BRET ratio (475 nm/535 nm) was recorded for 1 min, before addition of ligand and further recording for an additional 1 min. To calculate BRET signals, the values at 535 nm were divided by that omitted at 475 nm. For assessing constitutive receptor-G protein or receptor-receptor associations, net BRET values were calculated by subtracting the basal BRET ratio of receptor-tagged Rluc8 alone from all readings. The effect of ligand addition were assessed by subtracting basal net BRET readings from ligand-stimulated values, followed by subtracting any changes observed with control, PBS-treated wells. Constitutive receptor-G protein association readings were carried out in duplicate and ligand-induced BRET readings in triplicate.BRET2+ levels, Fluo4-AM Direct (Invitrogen) labelling was conducted, following the manufacturer’s protocol. Cells were incubated with Fluo4-AM Direct at 37 °C, for 30 min, followed by incubation at room temperature for an additional 30 min. Following incubation, agonist-dependent Ca2+ mobilization was assessed using a TCS-SP5 confocal microscope (Leica), with × 20 dry objective, capturing 1 frame per 1.2 s. Baseline Ca2+ was assessed for ~1 min prior to agonist treatment, and the effects of agonist assessed for a further 10 min. The inbuilt Leica LASAF analytical software was used to analyse the resulting time-lapse series. Intracellular cAMP concentrations were assessed by EIA as previously described25, with determined cAMP concentrations controlled for any variation in protein levels.To assess changes in intracellular Ca19. Briefly, FLAG and HA.11 primary antibodies were labelled with CAGE 500 and 552 photoswitchable dyes, respectively, following manufacturer’s protocol (Abberior). The degree of labelling was determined to be 1.0 ± 0.2 dye molecules per antibody for FLAG-CAGE 500 and 1.3 ± 0.1 dye molecules per antibody for HA.11-CAGE 552. Cells were plated onto 8-well 1.5 borosilicate coverglass chamber slides (Labtek) and incubated with HA.11-CAGE 552/FLAG-CAGE 500 antibodies for direct labelling of FSHR and LHR, respectively, for 30 min at 37 °C prior to ligand treatment. Subsequently, cells were washed with PBS and fixed for 30 min in 4% paraformaldehyde/0.2% glutaraldehyde. Cells were washed in PBS, and maintained in PBS for imaging. Images were acquired using an inverted Axiovert 200 manual wide-field fluorescent microscope  fitted with a commercial TIRF condenser , and a ×100 oil immersion, 1.45 numerical aperture objective. Image acquisition was as previously described19. Bright field images of each series were also acquired and grid images used post-acquisition to align CAGE 500 and 552 channels, using Fiji software.PD-PALM studies were conducted as previously described19. Data tables containing the x-y coordinates of localized receptors in 491- and 561-nm channels generated.Fluorescent intensity images of cropped non-overlapping 7 × 7 μm areas, within cell-cell boundaries, from 491- and 561-nm channels were analysed for localized receptors by QuickPALM Fiji plugin as previously describedx-y particle localization data tables were analysed using a custom Java application (PD-Interpreter), as previously described26, and coordinates from 491 and 561 nm fields plotted as an image. To determine the percentage and nature of FSH/LHR heteromerisation, a second order Getis Franklin neighbourhood analysis was conducted, using a search radius of 50 nm19. Outputted data were represented as a co-localization plot with differential colours distinguishing 491 and 561 channels, and heat maps generated to depict and represent the different numbers of associated receptor protomers observed.The number of associated receptor protomers derived from the 25. The fluorescence intensity of 10,000 cells was collected per sample, and technical replicates performed in triplicate.To quantify the cell surface expression levels of epitope-tagged receptors in stable cell lines and transient transfections, flow cytometry was carried out using a FACSCalibur flow cytometer (BD Biosciences), as previously described2+ data, baseline fluorescence was subtracted and area under the curve assessed for 20 cell areas for n = 3–5. To prevent the introduction of possible bias into the analysis, the presence of an LH-induced calcium response was visually confirmed (via change in fluorescence), and subsequent cell areas randomly selected and individual cell responses analysed. Area under the curve measurements were compared by One-way ANOVA with Dunnett’s post-hoc multiple comparison test. For cAMP analysis, ligand-induced cAMP levels were expressed as fold change over basal levels, and all conditions carried out in triplicate and compared by One-way ANOVA with Dunnett’s post-hoc multiple comparison test comparing different levels of FSHR transfection on LH-dependent responses. Basal and ligand-dependent PD-PALM data are represented as the mean ± S.E.M. for 10–12 cells, from 3 independent experiments. Differences in basal versus LH induced LHR/FSHR heteromerisation were assessed by one-way ANOVA with Dunnett’s multiple comparison post-hoc test, comparing different time points to basal. For assessment of LH-dependent effects on individual LHR/FSHR dimer and oligomeric complexes, data were expressed as a percentage of each form, and a two-way ANOVA with Bonferroni’s multiple comparison post hoc test conducted, analysing the effect of LH on the associated receptor forms. Receptor trimeric and tetrameric ratios versus the composition of oligomeric complexes were analysed using a one-way ANOVA with Dunnett’s post hoc test.For analysis of Cap < 0.05. The data that support the findings of this study, and materials used, are available from the corresponding authors upon request. PD-Interpreter software is freely available to download at www.superimaging.org.Statistical analyses were carried out using GraphPad Prism Version 7 , and statistical significance determined as 2+14,15. Therefore, we assessed the impact of LHR/FSHR coexpression on LH-induced Ca2+ signalling in a heterologous cell system that enabled analysis of the contribution of each receptor in a controlled manner.It is established that one of the key physiological G protein pathways activated by LH/LHR, but not by FSH/FSHR, is Gαq/11, leading to activation of PLC and increase in inositol phosphates and intracellular Ca2+ indicator dye, Fluo4-AM, and imaged live via confocal microscopy. LHR induces Gαq/11-Ca2+ signalling only under high receptor and high hormone concentrations19. This cell line expressed at the plasma membrane between 4000–10, 000 receptors/cell (as assessed by PD-PALM), which is consistent with physiological levels reported for the ovary27. Stimulation of LHR-expressing cells with 100 nM LH induced a rapid increase in intracellular Ca2+ levels that declines within 20 sec of the maximal fluorescent response . LHR/FSHR co-expressing cells exhibited a more pronounced inhibition in the LH-induced sustained response (~60%), reverting the profile from a sustained to transient response  or 2-APB, a well characterized inhibitor of store-operated, or voltage-independent, Ca2+ channels in an IP3 receptor-independent manner32. Thapsigargin dramatically inhibited LH-induced increases in intracellular Ca2+ in cells expressing either LHR, or both LHR and FSHR  Canus Fig. . Surprisnus Fig. . This denus Fig. . Overall2+ signalling and altered G protein coupling may occur via formation of LHR/FSHR heteromers. These receptors are known to interact via BRET and fluorescence correlation spectroscopy approaches21 and confirmed in this study also via BRET  of this technique, we are able to identify and quantify dimers and low order oligomers, i.e. trimers and tetramers, that have different protomer compositions at the cell surface26. As LH-mediated Ca2+ responses and Gαq-associations are altered by FSHR, we employed PD-PALM for its ability to detect rearrangements in heteromeric makeup following ligand-dependent activation of LHR.The BRET data suggest that LH-dependent sustained Ca19  are notent Fig. . Interesent Fig. . Followient Fig. . Overall9. Crosstalk is highly pertinent for the GPCRs playing multiple key roles in both reproduction and pregnancy, which includes the gonadotrophin hormone receptors LHR and FSHR. Here we describe a mechanism for GPCR crosstalk resulting in positive modulation of Ca2+ signalling and formation of functional asymmetry at the interface of receptor-receptor and receptor-G protein organization.GPCRs exhibit remarkable signal pleiotropy given the limited set of heterotrimeric G protein signalling pathways they couple to. One key mechanism that has emerged underlying this signal diversity is receptor crosstalk, with an increasing number of reports identifying that this occurs at the receptor level via GPCR heteromerisation2+ signalling profile of the ligand-activated LHR. That an unliganded FSHR can alter LH/LHR Ca2+ signalling may suggest that this crosstalk is mediated by direct associations of FSHR with LHR that could impose a conformational change in LHR, effectively acting as an allosteric modulator. Additionally, as the glycoprotein hormone receptors exhibit strong negative cooperativity in binding41, LHR/FSHR crosstalk may favour LH binding, given that LHR, when expressed with FSHR, can increase dissociation of FSH from its receptor20. LHR and FSHR have been shown to exhibit cross-inhibitory effects on Gαs-cAMP signalling20, and confirmed in this study. Thus, in combination with our data demonstrating altered LH-dependent Ca2+ signalling, suggests that FSHR may positively modulate LH/LHR signalling, perhaps specifically to Gαq/11-βγ-Ca2+ signalling, while reciprocally LHR may negatively regulate FSHR-mediated cAMP signalling20.In this study, we demonstrate that the unliganded FSHR can dramatically alter the Gαq/11-dependent Ca2+ signalling in primary human granulosa-lutein cells supports these findings. Attempts to manipulate LHR/FSHR levels in these primary human cultures to directly demonstrate a requirement for both receptors in the LH-induced Ca2+ profile, however, have been unsuccessful.In the context of granulosa cells that co-express these receptors, one could speculate that gonadotrophin receptor crosstalk alters LH signalling during the switch from FSH/FSHR to LH/LHR dominance at a functional level, towards defined pathways required for ovulation and subsequent granulosa cell luteinisation. That we observe a similar profile of LH-mediated Ca2+ signalling by LHR/FSHR crosstalk is via Gαq/11, as a specific Gαq/11 inhibitor inhibited both the transient and sustained Ca2+ responses. A release of intracellular Ca2+ can lead to activation of ligand-gated ion channels, receptor-operated, or store-operated Ca2+ channels. However, the Ca2+ response in cells expressing LHR alone was insensitive to the Ca2+ channel blockers nifedipine and 2-APB, yet partially affected by removal of extracellular Ca2+, suggesting that the source of Ca2+ flux is primarily, but not completely, from intracellular stores. In contrast, in cells expressing LHR/FSHR the sustained response is sensitive to both the removal of extracellular Ca2+ and these Ca2+ channel blockers. This suggests there may be a differential mode for LH-dependent extracellular Ca2+ influx due to crosstalk with FSHR. Nifedipine is conventionally described as an L-type Ca2+ channel antagonist, however, HEK293 cells are not thought to express L-type Ca2+ channels, although inhibition of endogenous Ca2+ influx by dihydropyridines such as nifedipine have been previously described in this cell type28. Interestingly, nifedipine has also been shown by several groups to inhibit store-operated, or voltage-independent, Ca2+ channels37, suggesting this inhibitor has broader actions across distinct channel types. As we also observed that intracellular Ca2+ store depletion by thapsigargin inhibits transient and sustained Ca2+ suggests intracellular Ca2+ release is a prerequisite for the influx of extracellular Ca2+. Although human granulosa-lutein cells have been previously shown to express functional L-type Ca2+ channels42, the broader actions of nifedipine could also suggest involvement of non-voltage dependent Ca2+ channels. In the presence of FSHR, sustained Ca2+ signalling by LH is also Gαq/11-dependent suggesting that there are perhaps alterations in LHR signalling via FSHR crosstalk downstream of Gαq/11. Interestingly, rather than downstream of Gαq/11, mechanistically the LHR/FSHR crosstalk appears to be at the G protein level because BRET demonstrated that LHR/Gαq complexes are reorganized in the presence of FSHR to favour FSHR/ Gαq couplings specifically activated by LH. Furthermore, pharmacological inhibition of Gβγ partly inhibited the Ca2+ response by LHR/FSHR. This inhibition was only partial as it was likely counteracted by the unexpected sustained Ca2+ profile observed in LHR only expressing cells, as the LHR/FSHR-mediated Ca2+ response would represent a combination of LHR and LHR/FSHR responses, even within an individual cell. Given the potential differential modes of extracellular Ca2+ influx between LHR and LHR/FSHR expressing cells, and differential sensitivity to gallein, one possibility is that Gβγ may regulate the activity of specific Ca2+ channels activated by LHR and LHR/FSHR in an opposing manner. This is consistent with the concept that there may be differential cellular pools of Gβγ, composed of distinct Gβγ subunit combinations, with distinct cellular signalling functions40. Overall, our data clearly indicates how FSHR, in the absence of agonist activation, potentially reorganizes the signal machinery to reprogram LHR activation.The underlying mechanism of sustained Ca2+ signalling suggests this may be occurring at a direct receptor-receptor level via heteromerisation. The gonadotrophin receptors have been previously demonstrated to form homomers and heteromers in heterologous cells, the latter being demonstrated via BRET and fluorescence correlation spectroscopy approaches21. These approaches have their distinct advantages and disadvantages; however, super-resolution microscopy enables direct visualization of each receptor molecule at the plasma membrane with <10 nm resolution, enabling quantitation of each receptor form. Thus, we have previously demonstrated with PD-PALM that LHR exists as monomers, dimers and various oligomeric forms19. Employing LHR mutants that undergo functional complementation we were able to demonstrate that oligomers with functional asymmetry could modulate LHR signalling19. In the current study, super-resolution imaging of LHR/FSHR heteromers, which are functional asymmetric for LH-mediated Gαq/Ca2+ signalling, revealed that basally these receptors not only formed heterodimers but also distinct hetero-oligomers. Our prior studies clearly indicate that oligomers with >6 receptors are density dependent19, thus, we focused on the lower order oligomeric forms of trimers and tetramers. Under conditions of LHR activation where we observe a sustained Ca2+ signal profile by FSHR, only the heterotetramers increased in number. This was surprising as our prior studies have demonstrated that LHR homomers detected via PD-PALM were not altered via ligand activation19. Another advantage of PD-PALM over other techniques to study receptor di/oligomer formation is that it enables identification of the ratio of each protomer within distinct oligomeric forms. Further analysis of LHR/FSHR heterotetramers revealed that the predominant pre-formed complex consists of tetramers with 3LHRs to 1FSHR. However, ligand activation reorganizes these specific heterotetramers so there is an equal distribution of all 3 possible heterotetramers. Although we did not directly demonstrate a specific role of heterotetramers in mediating LHR/FSHR crosstalk, it is tempting to speculate that >1 FSHR protomer within a heterotetramer is required to achieve the modulations in Ca2+ signalling via LHR. This is potentially consistent with previous reports suggesting the active form of FSHR is a trimer43. Furthermore, the known negative cooperativity of LHR41 would suggest only one LHR protomer in a complex (homomer or heteromer) is sufficient for binding ligand. Other reported GPCR heterotetramers are formed by dimers of two distinct GPCRs that can enable coupling to two distinct G proteins46. However, our study suggests that the protomers in a GPCR heterotetramer complex can also be asymmetric. Collectively this work illustrates that GPCRs can not only form as monomers, dimers and oligomers, but even within a single oligomeric form, e.g. tetramers, there exist multiple possible active forms that could have distinct functional properties. We and others have demonstrated that gonadotrophin receptor homomer interaction interfaces are highly complex, involving both the extracellular domain and multiple possible transmembrane domains19. We have previously proposed that such complexity enables formation of these different forms , but also could enable diversity in associated forms within the context of a heteromer. The ability to identify complex-specific interacting sites for both gonadotrophin receptor homomers and heteromers remains to be determined.The ability of FSHR to induce these alterations in LHR/Gαq/Ca2+ signalling by LHR/FSHR is modulated at the G protein level. Furthermore, these receptors form specific hetero-tetramers in an LH-dependent manner. The distinct properties of this receptor crosstalk, possibly via highly specific heteromers, presents potential modalities to exploit this complexity and improve the specificity of current therapeutics, which is potentially applicable for many GPCRs in this superfamily.To conclude, we have demonstrated that GPCRs for the gonadotrophic hormones can alter signalling in a highly asymmetric manner and that this reprogramming of CaSupplementary information


CBL-INTERACTING PROTEIN KINASE 6 (GhCIPK6) gene.Potassium maintains cotton fiber cell elongation under conditions of calcium deficiency, through the  2+) is necessary for fiber cell development in cotton (Gossypium hirsutum), both as a cell wall structural component and for environmental signaling responses. It is also known that potassium (K+) plays a critical role in cotton fiber cell elongation. However, it is unclear whether Ca2+ integrates its activities with K+ to regulate fiber elongation. Here, we report the novel discovery that Ca2+ deficiency, when integrated with K+ signaling, promotes fiber elongation. Using inductively coupled plasma–mass spectrometry (ICP-MS), we determined dynamic profiles of the ionome in ovules and fibers at different developmental stages, and found that a high accumulation of macro-elements, but not Ca2+, was associated with longer fibers. Using an in vitro ovule culture system, we found that under Ca2+-deficient conditions, sufficient K+ (52 mM) rapidly induced ovule and fiber browning, while reduced K+ (2 or 27 mM) not only suppressed tissue browning but also altered fiber elongation. Reduced K+ also enhanced reactive oxygen species scavenging ability and maintained abscisic acid and jasmonic acid levels, which in turn compensated for Ca2+ deficiency. Ca2+ deficiency combined with reduced K+ (0 mM Ca2+ and 27 mM K+) produced longer fibers in cultured ovules, due to cell wall loosening by phytosulfokine (PSK), expansin (EXP), and xyloglucan endotransglycosylase/hydrolase (XTH), and an increase of the K+ content of fiber cells. Using transgenic cotton, we showed that the CBL-INTERACTING PROTEIN KINASE 6 (GhCIPK6) gene mediates the uptake of K+ under Ca2+-deficient conditions. This study establishes a new link between Ca2+, K+, and fiber elongation.Calcium (Ca Gossypium hirsutum) and provide the essential raw material for a significant part of the textile industry  and potassium (K+) also play critical roles in fiber elongation.Fiber cells initiate from the outer-layer cells of the ovule, which undergo 20 d of rapid elongation growth and secondary cell wall synthesis, and after a subsequent period of dehydration become a valuable crop. Fiber cells can extend to 15–40 mm, and also represent an excellent model for studying single-cell elongation . Cell wa2+ has an indispensable role in plant development, both as a structural component in lipid membranes and cell walls, and also as a secondary messenger responding to environmental signaling   and K+ transporters (GhKT1 and GhKT2) to promote fiber elongation . Using an ovule culture system, we found that CaGossypium hirsutum) plants TM-1, Xuzhou142, the Xuzhou142 lintless-fuzzless mutant (xu142-fl), YZ1, and Gossypium barbadense 3-79 were grown in the fields of Huazhong Agricultural University, Wuhan, Hubei province, China, under standard farming conditions. In the experimental plot, each cotton variety was grown in a subplot that contained 300 plants in 30 rows. The soil was yellow-brown loam . N fertilization (240 kg N ha−1), P fertilization (120 kg P2O5 ha−1), K fertilization (150 kg K2O ha−1), and irrigation were performed in accordance with standard agricultural practices. Bolls were tagged at the day of flowering as 0 d post-anthesis (DPA). At the required development stage, bolls were harvested and frozen in liquid nitrogen, and stored at –70 °C until analysis.Cotton (Gossypium hirsutum TM-1 and YZ1. Ovule culture was performed as described previously (JXB online. Flowers were collected on the afternoon of the day of flowering (0.5 DPA). After the flower organs had been peeled off, ovaries were sterilized with 0.1% HgCl2 for 15 min, washed with double-distilled water and cut off. Sterile ovules were stripped off and suspended on the ovule culture medium, which was supplemented with 0.5 µM GA3 and 5 µM IAA.To identify the effect of each mineral element on fiber development, ovule culture was carried out with two cotton varieties, 3, 2 mM KH2PO4, 3 mM CaCl2, 2 mM MgSO4, 100 µM H3BO3, 100 µM MnSO4, 30 µM FeSO4, 30 µM ZnSO4, 5 µM KI, 1 µM Na2MoO4, 0.1 µM CoCl2, 0.1µM CuSO4, vitamins , 1 mM inositol, and 120 mM glucose, at pH 5.0. Six concentrations were set up for each essential element , which were 0, 0.5-, 1-, 2-, 4-, and 8-fold the standard (1-fold) concentration in the medium. For the series concentration for K+, KNO3 was replaced by NH4NO3 in medium with an equivalent molarity of nitrogen: K0 indicates that 50 mM KNO3 was replaced by 25 mM NH4NO3, and K0.5 indicates that 50 mM KNO3 was replaced by 25 mM KNO3 and 12.5 mM NH4NO3. In the calcium and potassium interaction experiment, Ca0, Ca0.5 Ca1, Ca2, Ca4, and Ca8 represent concentrations of CaCl2 at 0, 1.5, 3, 6, 12 and 24 mM, respectively. For other ions , only the concentration of the corresponding compound was changed in the medium. Three replicates were performed for each treatment. At least six ovules of one replicate were collected for fiber length measurement after 5 or 10 d culture.The concentration of each element in the ovule culture medium was set according to the BT medium of Fiber-bearing ovules were collected from cultured ovules treated with different ions and used for fiber length measurement according to a previous method (G. hirsutum (Gh) TM-1, Xuzhou142, xu142-fl, and G. barbadense (Gb) 3–79. At 0 DPA, ovules from 200 flowers were collected for each sample. For samples at 3 and 5 DPA, fiber-bearing ovules from 100 bolls were collected. At 10, 15, 20, and 25 DPA, at least 20 bolls were collected from the same position on the plant, with each boll being collected from a different plant. For samples from 5 to 25 DPA, fibers were gently knocked off the ovules with a pestle in liquid nitrogen, and fibers and ovules were separately ground into powder. To analyse changes in ion contents in fiber-bearing ovules treated with different levels of K+ or Ca2+, fiber-bearing ovules cultured for 10 d with K0 (2 mM K+), K1 (27 mM K+), and K2 (52 mM K+), and fiber-bearing ovules cultured for 5 d with Ca0-K0.5 (0 mM Ca2+ and 27 mM K+), Ca1-K0.5 (3 mM Ca2+ and 27 mM K+), and Ca8-K0.5 (24 mM Ca2+ and 27 mM K+) were collected and washed with double-distilled water. After removing excess water with filter paper, samples were ground into powder with liquid nitrogen. Powder samples were dried in a vacuum freeze-drier  until constant weight was achieved and analysed for ion contents according to the method described previously by To quantify the content of each ion in ovules and fibers at different stages, samples were collected at 0 to 25 DPA from four cotton varieties grown in the field, + or Ca2+ for 5 or 10 d, all samples were collected and the fibers were gently removed with a pestle in liquid nitrogen. The fibers and ovules were then separately ground into powder, and 0.1 g samples were used for total RNA extraction according to the method described previously by GhUB7 (DQ116411) was used as the internal control to normalize gene expression levels.After fiber-bearing ovules had been cultured with different levels of exogenous K+  and fibers cultured for 5 d with three different levels of Ca2+  were used for total RNA extraction. Each treatment included two biological replicates, and each biological replicate included six repeats. Twelve samples of total RNA were sent to the BGI Company  for RNA-Seq quantification analysis using an Illumina HiSeqTM2000. The coding sequence (CDS) and genome sequences (BioProject ID: PRJNA248163) of G. hirsutum TM-1 were used as the reference. For each sample there were 12 million total reads. Clean RNA-Seq reads were mapped to the G. hirsutum genome and the uniquely mapped reads were extracted for estimation of the expression levels of genes using Cufflinks (Fibers cultured for 10 d with three different levels of K−1 2H6-ABA and 10 ng ml−1 dihydrojasmonic acid (dh-JA) as internal standards, and shaken on a shaker overnight at 4 °C in the dark. After centrifugation at 12 000 g for 15 min at 4 °C, the supernatant was transferred to a new 2-ml centrifuge tube. The precipitate was suspended in 450 µl 80% (v/v) methanol, shaken at 4 °C for 4 h, and tubes were centrifuged to ensure the extracted sample mixed with the remaining supernatant. After the mixed supernatant had been air-dried, the residues were dissolved in 300 µl 10% (v/v) methanol. The solution was filtered through a 0.22-µm nylon membrane  and the hormone was detected using LC-GC/MS according to the method reported previously by Fiber-bearing ovules cultured for 5 d under Ca0-K0.5, Ca1-K0.5, Ca8-K0.5, Ca0-K1, Ca1-K1, and Ca8-K1 treatments were used for the measurement of abscisic acid (ABA) and jasmonic acid (JA) contents. Fibers or ovules were ground into powder in liquid nitrogen. Samples of 0.1 g were extracted with 750 µl cold extraction buffer  supplemented with 10 ng ml+ or Ca2+ for 1, 2, 3, or 4 d were incubated in 0.01 M PBS buffer  containing 10 µM 2’,7’-DCFDA . The 2’,7’-DCFDA was dissolved in dimethyl sulfoxide (DMSO) for 30 min in the dark at 30 °C in advance (Ovules cultured in medium with different levels of KG. barbadense (Gb) 3–79, and G. hirsutum (Gh) TM-1, Xuzhou 142, and Xuzhou 142-fl; To capture the dynamic changes in the ionome of cotton ovules and fibers during different development stages, four cotton varieties [Gb are more desirable than those from Gh as they are much longer, finer, and stronger  and a minimum in the ovules (K+: 17.76 ± 0.67 mg g−1 DW to 12.47 ± 0.90 mg g−1 DW). After 10 DPA, contents of K, P, and Mg began to decrease in fiber cells and increase in ovules  accumulation in Gb than in Gh  for each of 11 essential elements  was set up. After 5 or 10 d culture, fiber phenotypes and lengths were determined  in the ovule culture system, Ca2+ deficiency induced ovule and fiber browning (a sign of tissue damage)  produced longer fibers (13.65 ± 0.22 mm) than the normal Ca2+ concentration  (11.53 ± 0.14 mm)  conditions, the length of fibers cultured under Ca2+ deficiency  was longer than when treated with standard Ca2+ and half-K+ , i.e. 8.13 ± 0.05 mm compared to 6.13 ± 0.27 mm (5 d culture) and 13.96 ± 0.03 mm compared to 12.22 ± 0.36 mm (10 d culture)  started to show browning by the 10th day  , while a0.14 mm) . After 5culture) , C. Fibe10th day . The dif2+ deficiency-promoted fiber elongation was found to depend on the concentration of K+ in the medium. Under Ca2+ deficiency, a sufficient concentration of K+  did not enhance fiber elongation, but instead resulted in a brown ovule with suppressed fiber development; however, reduced K+ (27 mM) alleviated Ca2+ deficiency-induced stress (2+ and 52 mM K+), which became more obvious after 5 d culture (2+ and 27 mM K+) did not brown, and the fibers were longer than in Ca1-K0.5 (3 mM Ca2+ and 52 mM K+) , the brown phenotype in the ovules was delayed, but fiber elongation was inhibited (+ in the growth medium compensates for Ca2+ deficiency-induced stress that results in browning.Cad stress . To furt culture . Ovules 2 mM K+) . If the nhibited . These r2+ deficiency under sufficient K+ conditions was caused by ROS-induced cell death (2+ and 52 mM K+) than in Ca0-K0.5 (0 mM Ca2+ and 27 mM K+) or Ca1-K1 (3 mM Ca2+ and 52 mM K+) at the first day (2+ and 52 mM K+) and were lower in this treatment than in Ca0-K0.5 (0 mM Ca2+ and 27 mM K+) at the fourth day (+ in Ca2+-deficient medium (0 mM Ca2+ and 52 mM K+) may derive from the uncontrolled accumulation of ROS.We hypothesized that the browning induced by Call death . ROS levirst day . As the urth day . When 2 urth day . This su+ (2 or 27 mM K+) in the growth medium delayed Ca2+ deficiency-induced browning. To test whether this might be due to increased ROS-scavenging ability, we performed RNA-seq analysis to screen genes that respond to Ca2+ deficiency or to reduced K+ (+) fibers revealed that oxidoreductase-related processes are significantly enriched (+ or Ca2+ deficiency were clustered (+), including the anti-oxidative genes LAC, POD, GST, APX, GPX, and MT  genes in K0 fibers, 37 were the Tau-type GST (2+ deficiency (63) was less than the number in fibers treated with reduced K+ (187)  . These r+ supply may activate other signaling systems to protect against damage by Ca2+ deficiency. Therefore, endogenous hormones were quantified in ovules and fibers cultured with different levels of K+ or Ca2+. After 5 d culture, the level of ABA was decreased and JA was increased in ovules and fibers cultured in Ca0-K0.5 (0 mM Ca2+ and 27 mM K+), compared to Ca1-K0.5 (3 mM Ca2+ and 27 mM K+)  ovules (2+ and 52 mM K+) or Ca0-K0.5 ovules  , B. ABA ) ovules , and thi5 ovules . These r2+ deficiency-induced browning in ovules and fibers  for RNA-seq analysis and identified genes involved in the Ca2+-deficiency response. A total of 3599 differentially expressed genes (DEGs) were identified  among the different treatments , EXP (expansin), and XTH (xyloglucan endotransglycosylases/hydrolases)  . Accordi qRT-PCR .+ in the vacuole provides cells turgor, and PSK can slow down the efflux of K+ to promote fiber cell elongation , Gh_D13G1983 (CIPK6), Gh_D01G1760 (HAK5), and Gh_D05G0538 (encoding a BTB/POZ domain with WD40/YVTN repeat-like protein) (CBL-INTERACTING PROTEIN KINASE6 (CIPK6) interacting with AKT2 (a Shaker-type K+ channel) controls K+ uptake under K+-deficient conditions in Arabidopsis  . It has G. hirsutum, there were eight CIPK6 genes that showed high similarity to AtCIPK6 and they were divided into four clades (GhCIPK6.3a(Gh_A07G0210) and GhCIPK6.3d(Gh_D07G0265) expression were undetectable in ovules and fibers, but three other clades of CIPK6 genes were induced by reduced K+  or Ca2+ deficiency (GhCIPK6 genes were significantly up-regulated after 6 h treatment in reduced K+ (K0) (GhCIPK6 in K+ uptake.In r clades . GhCIPK6ficiency . Three G K+ (K0) , consistGhCIPK6.2a(Gh_A06G0873) (line OE24) or silencing GhCIPK6 by RNAi (line Ri16) (GhCIPK6 genes were detected in 0 DPA ovules of transgenic and wild-type cotton. Only GhCIPK6.2a(Gh_A06G0873) was up-regulated in line OE24 and three GhCIPK6 genes  were down-regulated in line Ri16  (+ contents were significantly lower in the GhCIPK6-suppressed line (Ri16) than in the wild-type under Ca2+-deficient conditions  in the Ri16 line under reduced K+ (K0) conditions is consistent with this view  after 5 or 10 d culture, displaying an increased sensitivity to Ca2+ deficiency (GhCIPK6-suppressed cotton line (Ri16) on the other hand showed a higher tolerance to Ca2+ deficiency than the wild-type  . The traine Ri16 . To confent (K0) . Ionome nditions , confirmhis view . In ovulficiency . The GhCild-type . These r2+ in fiber cells can promote elongation. Ionome analysis during fiber developmental stages revealed a low content of Ca2+ and a high content of K+, which suggests that cotton might behave as a calcifuge during fiber elongation  has a greater effect in promoting fiber elongation than a more normal higher Ca2+ concentration (3 mM) (2+ pool release channel blocker 2-aminoethoxydiphenyl borate (2-APB) inhibits Ca2+ influx into fiber cells, which suppresses elongation. Moreover, it also found that Ca2+ starvation (0 or 1 mM) promotes early fiber elongation, and excess Ca2+ (50 mM) inhibits elongation  induced ovule and fiber browning due to the effects of ROS; however, half-K+ (27 mM) not only suppressed tissue browning but also promoted fiber elongation, whilst very low K+ (2 mM) inhibited both tissue browning and fiber elongation  tetraacetic acid (EGTA) to the ovules treated with different levels of Ca2+ or K+. After 3 d culture, ovules and fibers treated with 1 mM EGTA showed the same phenotypes as those that were not treated  or sufficient K+, while GhCIPK6-silencing resulted in a higher tolerance to Ca2+ deficiency  proteins and the response of GhCIPK6 to reduced KAnalysis of K+ content in the GhCIPK6-suppressed line (Ri16) and wild-type cotton under Ca2+ deficiency.Fig. S8. 2+ chelator EGTA on fiber development under different Ca2+ or K+ conditions after 3 d culture.Fig. S9. Effect of the CaTable S1. List of primers used for qRT-PCR in this study.Table S2. Fiber quality parameters of three cotton varieties.G. hirsutum TM-1.Table S3. List of essential mineral element concentrations in the third leaf from the top of Table S4. List of enriched GO terms for DEGs that were up-regulated in fibers in the K0 treatment compared with K1.+ or Ca2+ deficiency, based on the RPKM value from RNA-seq analysis.Table S5. List of ROS generation and scavenging genes expressed in response to reduced KTable S6. List of enriched GO terms for 1262 up-regulated genes in the Ca0K0.5 treatment compared with Ca1K0.5.Table S7. List of enriched GO terms for 1132 up-regulated genes in the Ca0K0.5 treatment compared with Ca8K0.5.Table S8. List of enriched GO terms for 774 genes up-regulated in the Ca0K0.5 treatment compared with both Ca1K0.5 or Ca8K0.5.+ and Ca2+ concentrations determined by ICP-MS in Ca2+-deficient growth medium treated with the Ca2+ chelator EGTA for 3 d.Table S9. KSupplementary Figures S1-S9Click here for additional data file.Supplementary Tables S1-S8Click here for additional data file.


UAEC) express P2Y2 receptors and at high cell density show sustained and synchronous [Ca2+]i burst responses in response to ATP. Bursts in turn require coupling of transient receptor potential canonical type3 channel (TRPC3) and inositol 1,4,5‐triphosphate receptor type 2 (IP3R2), which is upregulated in P‐UAEC in a manner dependent on connexin 43 (Cx43) gap junctions. While there is no known direct interaction of TRPC3 with Cx43, early descriptions of TRPC3 function showed it may also be influenced by altered membrane potential (Vm). Herein, we ask if enhanced TRPC3 Ca2+ bursting due to enhanced Cx43 coupling may be coupled via dynamic alterations in Vm in P‐UAEC, as reported in some (HUVEC) but not all endothelial cells. Following basic electrical characterization of UAEC, we employed a high sensitivity cell imaging system to simultaneously monitor cell Vm and [Ca2+]i in real time in continuous monolayers of UAEC. Our findings show that while acute and sustained phase [Ca2+]i bursting occur dose‐dependently in response to ATP, Vm is not coregulated with any periodicity related to [Ca2+]i bursting. Only a small but significant progressive change in Vm is seen, and this is more closely related to overall mobilization of Ca2+. Surprisingly, this is also most apparent in NP‐UAEC > P‐UAEC. In contrast [Ca2+]i bursting is more synchronous in P‐UAEC and even achieves [Ca2+]i waves passing through the P‐UAEC monolayer. The relevance of these findings to mechanisms of pregnancy adaptation and its failure in hypertensive pregnancy are discussed.Pregnancy‐derived uterine artery endothelial cells (P‐ To that end, we herein report our investigations of parallel changes in ATP‐stimulated Ca2+ and observed Vm responses in UAEC from nonpregnant and pregnant ewes. Specifically, we have for the first time characterized the basic electrical properties of individual P‐UAEC and NP‐UAEC using electrophysiology, which can now be used as baseline references for any future studies involving channels that may alter Vm or ion flow in these cells. Secondly, acknowledging the importance of cell–cell communication in UAEC, we have investigated responses from cells in confluent monolayers using dual‐fluorescence microscopy to observe real‐time changes of [Ca2+]i and corresponding Vm in an ATP dose‐dependent manner. The outcome of our studies suggest that while acute and sustained phases of [Ca2+]i signaling are each regulated dose‐ and time‐dependently by ATP, Vm is not strongly or dynamically coregulated in either P‐UAEC or NP‐UAEC. Our data further suggest that Vm most clearly associates with overall Ca2+ mobilization rather than the [Ca2+]i being tightly controlled by more dynamic shift in Vm. Despite the observation of only minor changes in Vm, we have also observed P‐UAEC are capable of mounting Ca2+ waves across cell monolayers, while these waves are far less apparent or even completely lacking in monolayers of NP‐UAEC. These findings considerably further our understanding of the mechanisms underlying endothelial cell adaptation of Ca2+ signaling that is necessary for enhanced vasodilator production in pregnancy, and may also provide mechanistic insight into how this may fail in preeclampsia and other hypertensive disorders involving endothelial vasodilatory dysfunction.One possibility, of course, is that Cx43 mediates the transfer of Ca2+ or IP3 between cells and there is no need for other explanations. However, published studies on other endothelial cell types have shown agonist‐stimulated responses that include reciprocal periodicity of both [Ca2+]i and 2 was from Calbiochem ; 35‐mm dishes with glass‐bottom coverslip windows were from MatTek Corp. . Electrophysiology pipettes were made from 1.65‐mm‐OD glass capillary tubing .Minimum essential medium (MEM), Fura‐2 AM, Pluronic Acid, DIBAC4(3), and round 13‐mm glass cover slips were obtained from Thermo Fisher Scientific . ATP , electrophysiology reagents, and all other chemicals were from Sigma‐Aldrich  unless noted otherwise. CaCl2 (2 mmol/L), KH2PO4 (1 mmol/L), and MgSO4 (1 mmol/L) at pH 7.4. ATP stock solutions were freshly made at 100× concentration and diluted to final concentration upon addition.Kreb's solution used in imaging and electrophysiology (bath solution) studies comprised of KCl (5 mmol/L), NaCl (125 mmol/L), HEPES (25 mmol/L), glucose (6 mmol/L), CaClμg/mL gentamycin (growth medium) to passage 3 before trypsinization and storage in liquid nitrogen in the same growth medium containing 10% dimethylsulfoxide (DMSO) from Sigma‐Aldrich . For all subsequent imaging experiments, a pool of cells from four animals each was created (NP and P pools). Passage 3 cells from four individual animals were thawed and grown in growth medium to near confluence, and then combined and split 1:35 for freezing at passage 4 in growth medium with 10% DMSO. These cell pool aliquots were then used for all subsequent cell imaging studies as required.Animal handling protocols for experimental procedures were approved by the University of Wisconsin‐Madison Research Animal Care Committees of the School of Medicine and Public Health and of the College of Agriculture and Life Sciences and followed the guidelines for humane treatment and euthanasia of laboratory farm animals as recommended by the American Veterinary Medicine Association. Uterine artery endothelial cells were obtained from pregnant ewes (120–130 days gestation) and nonpregnant ewes  of Polypay and mixed Western breed during nonsurvival surgery as previously described , and MgCl2 (4 mmol/L) at pH 7.2. Backfill solution included 120 μg/mL amphotericin B. Standard pipette solution contained KCl (30 mmol/L), K‐gluconate (83 mmol/L), K2HEPES (10 mmol/L), K2EGTA (5.5 mmol/L), CaCl2 (0.5 mmol/L), MgCl2 (4 mmol/L), K2ATP (4 mmol/L), and Na2GTP (0.1 mmol/L) at pH 7.2. High K+ solution contained KCl (100 mmol/L), NaCl (30 mmol/L), HEPES (25 mmol/L), glucose (6 mmol/L), CaCl2 (2 mmol/L), KH2PO4 (1 mmol/L), and MgSO4 (1 mmol/L) at pH 7.4. Recordings were carried out on single, voltage‐clamped cells using an Axopatch 200B amplifier system, Digidata1440A digitizer, and pCLAMP 9.0 data acquisition software . Voltage steps or ramp protocols changed the membrane potential from −150 to +50 mV over 1.5 sec, with in between holding potential of −60 mV. Additional stock solutions of 100 μmol/L ATP and 100 mmol/L K+ were diluted with Kreb's just prior to perfusion. Curve fitting and statistical analysis was completed using Microsoft Excel and SigmaPlot 12  with a voltage correction of −9 mV applied to all values due to the existence of a liquid junction potential between the bath solution and pipette tip causing a flowing saturated KCl bridge.Whole‐cell configuration of patch clamp electrophysiology was performed to determine the basic electrical properties of P‐UAEC and NP‐UAEC. Briefly, cells were cultured in 20% FBS‐MEM based growth medium on round glass coverslips or in glass‐bottom dishes and then mounted to the microscope recording platform. Cells were perfused with Krebs' solution at room temperature using a gravity fed multi‐line perfusion system controlled by electronic shut‐off valves at a rate of ~1 mL/min. Healthy individual, isolated cells were chosen for patch‐clamping through visual confirmation at 60× magnification. Fire polishing of pipette tips resulted in an impedance of 2–8 mol/L·Ω. Perforated patch pipette solution contained KCl (30 mmol/L), K‐gluconate (83 mmol/L), K2HEPES (10 mmol/L), K2EGTA (5.5 mmol/L), CaClImaging control and image acquisition was achieved with MetaFluor 7.7.9 software . Calibration of [Ca2+]i for Fura‐2 studies was achieved by establishing a standard curve using buffer solutions of known free [Ca2+]  and Fura‐2 free acid.μmol/L Fura‐2 AM + 0.05% pluronic acid and incubated at 32°C in the dark for 45 min. Cells were then rinsed twice with Kreb's solution containing 250 nmol/L DIBAC4(3) to remove excess Fura‐2 AM. After rinsing, 2 mL of 250 nmol/L DIBAC4(3) were added to the dish and cells were allowed 30 min at room temperature in the dark to complete Fura‐AM hydrolysis. At the end of loading, cells were placed on a heated microscope stage using a 35 mmol/L culture dish heater  and maintained at 23–26°C by a temperature controller box . A specific patch of tightly grouped cells was identified under 380 nm and/or 480 nm UV excitation using a Nikon Ti‐E inverted microscope equipped with a 20× phase/fluor objective and a 175W Lambda LS xenon arc lamp . Individual cells (59 cells) were selected and imaged at 2‐sec intervals via excitation filters alternating 340 nm and 380 nm (300 and 150 milliseconds per excitation) for Fura‐2 responses and additionally at 6‐sec intervals via 480 nm (100 milliseconds per excitation) for DIBAC4(3). Emission light passed through a 505 nm dichroic mirror, and was filtered at 520 nm via a second filter wheel . Responses were captured with a Cascade 512B high‐speed camera  with data acquisition controlled by Metafluor software. Baselines were observed for 5 min prior to stimulating UAEC with ATP . ATP stock was diluted to 10× in a vial using dish media, then returned to the dish, and immediately mixed to a final concentration by manual pipette suction/expulsion with stimulation responses being recorded for 30 min. Post hoc video analysis of the [Ca2+]i response was later performed on 340/380 ratio TIF image files recorded in MetaFluor . Images were merged into a single AVI video file using MetaFluor and viewed at 30.7 fps with ImageJ 1.50i software .Pooled cells frozen at passage 4 were thawed and grown on 35‐mm glass‐bottom dishes in standard growth medium to 90% or greater confluency. Cells were loaded in Kreb's solution (1 mL) containing 10 Vm data at either fixed time points, or as “area under the curve” (AUC), were compared using student's t‐test, or paired t‐test as appropriate. For regression analysis, where a dose response was expected, a Sigmoidal fit was used. Other datasets were analyzed using a linear or nonlinear regression based on the equation which best fit the data . Differences in P‐value <0.05 were considered significant.[Ca2+]i and n = 37) and NP‐UAEC (n = 7). We determined the average membrane potential (Vm) of P‐UAEC, which was −22.0 ± 2.25 mV and for NP‐UAEC, was −23.9 ± 2.20 mV with values corrected by −9 mV for liquid junction potential. The membrane capacitance (Cm) values were P‐UAEC (17.1 ± 2.33 pF) and NP‐UAEC (16.1 ± 1.89 pF), and values of membrane resistance (Rm) were P‐UAEC (3.96 ± 0.726 GΩ) and NP‐UAEC (2.78 ± 0.810 GΩ). None of these baseline electrical parameters were statistically different between P‐UAEC and NP‐UAEC at low cell density. Application of voltage ramp protocols . Additionally, the average inward current responses between P‐UAEC and NP‐UAEC were compared at 20 mV intervals with no significant differences noted .Whole‐cell patch clamp recordings were undertaken to establish baseline biophysical parameters for isolated P‐UAEC (μmol/L), and we used voltage ramp whole‐cell methods to monitor the current response. Figure Vm values completely reversed upon reintroduction of basal Kreb's buffer solution. Under our recording conditions, exposure to ATP  or to ATP , but hyperpolarization and depolarization events or periods of fluctuating Vm sometimes occurred . Additionally, exposure to ATP during perforated patch current clamp recordings did not consistently induce a change in Vm (1 of 3 cells), but when hyperpolarization resulted, the event lasted <60 sec as seen in Figure P‐UAEC combined dish average responses . ATP‐stimulated P‐UAEC clearly show a dose‐dependent [Ca2+]i response for both the initial [Ca2+]i peak and for the secondary sustained [Ca2+]i phase. ATP dose‐dependently increased both the initial [Ca2+]i peak's height and the duration of the peak's downslope, and [Ca2+]i failed to completely return to baseline even showing a secondary “bump” of [Ca2+]i at 200 sec at the higher ATP doses  were stimulated with ATP (30 or 100 μmol/L), we saw a dose‐dependent increase in the initial [Ca2+]i peak  were expressed as a fold of the averaged 100 μmol/L ATP AUC response and data were fit using a sigmoidal curve. A clear ATP dose response is seen for the initial [Ca2+]i peak  and had a very abrupt increase between 1 and 10 μmol/L ATP before the response plateaued. By this analysis at least, sustained phase Ca2+ seemed to occur as an “all or nothing” event triggered within a very narrow range of ATP stimulation. Nonetheless, when we examine dose dependency of synchronous bursting . DIBAC4(3) is capable of reporting membrane potential change in nonexcitable cells, and studies show that a 1% change in DIBAC4(3) fluorescence approximates a 1 mV change in membrane potential  which recovered to baseline by 5 min. The majority of individual P‐UAEC showed decreasing DIBAC4(3) fluorescence over 30 min (data not shown), and the overall dish‐averaged Vm response trended in the hyperpolarized direction. On agonist stimulation, UAEC did not show added oscillatory Vm behavior as has been reported in some types of endothelial cells . Overall, a “dose dependency” curve fit for this small response failed to achieve significance. NP‐UAEC Vm data are also plotted on the curve in Figure μmol/L ATP responses significantly different from control. The 100 μmol/L ATP‐stimulated net Vm change in P‐UAEC (−16 ± 3.5 mV) was not statistically different from NP‐UAEC (−20 ± 3.2 mV) at 30 min. Indeed, UAEC appear to tightly maintain Vm even under artificially challenged situations, and ATP only slightly changes Vm at the very highest dose of ATP.In establishing baseline p with Ca+i. We thye DIBAC4. DIBAC4. Nonetheless, Figure Vm values that spanned from depolarized to hyperpolarized shifts in Vm. Likewise, the significant correlation of AUC of the sustained [Ca2+]i phase to net Vm change  and more predictive in showing greater hyperpolarization as AUC increased for both the initial peak and sustained [Ca2+]i phases.Given that ATP could dose‐dependently alter [Ca2+]i, and only to a lesser extent magnify hyperpolarization in P‐UAEC, we next asked if [Ca2+]i was directly correlated with/could actually be directly responsible for the small nge Fig. B also stVm values that fell within the expected range of Vm values (−52 mV to −16 mV) reported in other endothelial isolated cell studies  which have a more cobblestone morphology have been reported to show slightly more negative Vm values at high density compared to low cell density (−24 mV vs. −16 mV) P3‐mediated release of Ca2+ from the ER as proposed previously  is simply unreliable as a monitor of Vm change in UAEC, or if progressive Vm hyperpolarization is indeed a property of UAEC that differs from observations in HUVEC or other endothelial cells. We found DIBAC4(3) responses are subject to mild photobleaching, so for prolonged recordings such as ours, DIBAC4(3) may not optimally report Vm change. Pipette media addition itself also resulted in a specific transient fluorescence response artifact. Nonetheless, both of these artifacts in the absence of ATP were constant across observations, and could be identified and corrected for. The observed Vm changes seen with added agonist were over and above these artifacts, and at 30 μmol/L and 100 μmol/L ATP became significantly greater than no agonist control responses. This leads us to conclude that the ATP‐stimulated Vm responses are small but real events in UAEC. Unlike the clear dose‐dependent changes in [Ca2+]i in the exact same cells, an analysis of ATP dose dependency on Vm failed to achieve a significant overall curve fit. Given such a weak relationship between ATP dose and change in Vm, the question then follows if Vm changes are not significantly related to receptor occupancy , then are they secondary to changes in [Ca2+]i? Regression analyses of the observed Vm change versus total AUC of [Ca2+]i shows clearly there is a significant correlation between the two variables for P‐UAEC, and even more so for NP‐UAEC. This stronger correlation again is the clearest evidence that it is changes in [Ca2+]i that drive Vm directly in UAEC, and this alters with pregnancy. Given the whole‐cell nature of our initial observations, and the limitations of DIBAC4(3), we cannot rule out the additional possibility that greater magnitude Vm changes could be occurring within subcellular regions, perhaps related to local gap junction function and/or the frequency of local [Ca2+]i bursting activity within a cell. Higher magnification and higher speed imaging studies beyond this study would be necessary to determine this absolutely.Given our failure to detect any substantial dynamic quantitative, but also qualitative in nature to coordinate individual burst responses in ways beyond a simple increase in [Ca2+]i. Of note, our averaged [Ca2+]i tracings (which are a composite of multiple dishes of cells) still clearly reflect defined periods of [Ca2+]i bursting. Such periodic [Ca2+]i elevations are not only consistent in timing across multiple cells, but average number of “bumps” per monolayer reported here coincide with the number of synchronous Ca2+ bursts previously reported in individual P‐UAEC by Yi et al.  that is key. Altered endothelial function in pregnancy extends to adapted monolayer function as cells shift to support communication beyond their own membrane boundaries and work together in harmony.The combination of analyses in this study shows that the enhanced nature of the sustained Ca2+]i phase response is not just i et al. . The faci et al. , 2011 an+i phase ion Fig.  despite Vm was not supported, there are still three main findings from this study of importance: (1) the basic electrical properties of both P‐UAEC and NP‐UAEC have been described for the first time. (2) imaging studies have determined while global Vm changes that dynamically follow [Ca2+]i bursting are not observed, overall mobilization of Ca2+ may be responsible for the small secondary changes in Vm that are observed (particularly in NP‐UAEC). (3) the most significant finding, while ATP stimulation can mobilize similar amounts of Ca2+ in the sustained phase in NP‐ and P‐UAEC, the qualitative nature of Ca2+ signaling in P‐UAEC is what differs between NP‐UAEC and P‐UAEC responses and specifically allows [Ca2+]i wave propagation in P‐UAEC. Given our finding that it is net Ca2+ mobilization that drives secondary changes in Vm rather than the reverse, further studies are now needed to understand what subtypes of KCa channels play a role in UAEC [Ca2+]i signaling and so vasodilator production, and how this may change in pregnancy. These studies are currently underway in isolated UAEC and in intact uterine artery vessels.In summary, while our initial proposed hypothesis that P‐UAEC enhance and better coordinate function via dynamic changes in The authors have no disclosures related to this study.Videos S1 and S2. Observation of a [Ca2+]i wave propagation in P‐UAEC versus an asynchronous [Ca2+]i response in NP‐UAEC. Video files from which the snapshots shown in Figure Click here for additional data file. Click here for additional data file.


The Ca2+ permeability is a crucial functional parameter to understand the physiological role of nAChRs, in particular considering their ability to modulate Ca2+-dependent processes such as neurotransmitter release. The rings of extracellular and intracellular charged amino acid residues adjacent to the pore-lining TM2 transmembrane segment have been shown to play a key role in the cation selectivity of these receptor channels, but to date a quantitative relationship between these structural determinants and the Ca2+ permeability of nAChRs is lacking. In the last years the Ca2+ permeability of several nAChR subtypes has been experimentally evaluated, in terms of fractional Ca2+ current . In the present study, the available Pf-values of nAChRs are used to build a simplified modular model describing the contribution of the charged residues in defined regions flanking TM2 to the selectivity filter controlling Ca2+ influx. This model allows to predict the currently unknown Pf-values of existing nAChRs, as well as the hypothetical Ca2+ permeability of subunit combinations not able to assemble into functional receptors. In particular, basing on the amino acid sequences, a Pf > 50% would be associated with homomeric nAChRs composed by different α subunits, excluding α7, α9, and α10. Furthermore, according to the model, human α7β2 receptors should have Pf-values ranging from 3.6% (4:1 ratio) to 0.1% (1:4 ratio), much lower than the 11.4% of homomeric α7 nAChR. These results help to understand the evolution and the function of the large diversity of the nicotinic receptor family.Nicotinic acetylcholine receptors (nAChRs) are cation-selective ligand-gated ion channels exhibiting variable Ca Furthermore, the constant ratio CNa/CCa has been substituted by a single constant const. Thus, Pf data have been fitted with the following equation:With a strong oversimplification, the term ΔR2-value has been reported, and the predicted Pf-values have been compared with the real ones. The const and ki-values obtained by fitting the charge distribution in the five out the eight regions were then used to predict the Pf-values of several pentameric subunit combination giving rise to unreal or existing nicotinic receptors. The natural unit of electrical charge (nuec) has been used as charge unit throughout the paper.For each fit the 2+ permeability of 16 different nAChRs  correlated with the local distribution of the electrical charges associated to the amino acid sequences of their subunits , in which the sigmoidal function described by Equation (5) was used to best fit the 16 known Pf-values, starting using as independent variable the sum of electrical charges in position {20′}, where it was observed the most significant correlation. Successive fit procedures used the electrical charges present in two or more positions as independent variables, progressively choosing the positions giving the higher R2-values. In Figure Pf-values are plotted using as abscissa the weighted sum of the charges in different receptor regions, where the ki constant were derived from the fitting procedure. For each fit the comparison between observed and predicted Pf-values is reported. As expected from the linear regression analysis, the best monodimensional fit was obtained using the position {20′} data as independent variable .The second aim of this study was to quantitatively estimate the contribution of the charges distributed in different receptor regions to the nAChR Cae Figure . However Figures , not reaki constants values, reported in Table Pf-value. In this way it was possible to predict the hypothetical Pf-values of several combinations of human subunits which are not able to assemble into functional nAChRs in living cells  for homomeric nAChRs constituted by α subunits (from α1 to α6), all lacking the five negative charges in position {25′ 29′} in the α7 subunit, which determine for this last receptor the reduction to 10.4%. Interestingly, if the charges in position {−1′} are removed, as for α7E237A, the Pf-value is 0.0, exactly as expected from the direct measure of Ca2+ permeability reported for this mutated nAChR  pentameric combination of nicotinic subunits. Relating known Pf-values to the charge spatial design of the corresponding nAChRs provided several observations.The main results of this study are represented by an improved picture of the functional relations between the distribution of electrical charges and Ca2+ permeability of these receptors does not depend on the total amount of charge present in the large extracellular or intracellular domains. All extracellular domains have an overall negative charge, with muscle nAChRs exhibiting the most negative values in this region. The extracellular overall negative charge may be functionally relevant for agonist binding, assembly, posttranslational modification, and interactions with extracellular molecular apparatus, in particular for muscle nAChRs which cooperate with a large number of molecular components at the endplate . These results confirm for nAChRs the observation that negatively charged sites may either facilitate Ca2+ flow, reducing the electrostatic energy profile for divalent cations, or counteract it, in particular if the negative residues represent a high affinity Ca2+ binding site in which Ca2+ itself may produce a long lasting electrical repulsion for other incoming divalent cations  only muscle nAChRs exhibit a slight less negative charge than all other receptors. In the intermediate ring a reduction of the negative charge is associated to reduced 2+ permeability to α5* nAChRs. A hypothetical α5 homomeric nAChR would exhibit, according to this model, the highest calculated Pf-value, and the replacement by an α5 subunit of any other α subunit in a heteromeric nAChR always enhance Ca2+ permeability. This finding is of relevant physiological interest, given the role of α5* nAChRs in modulating the self-administration of nicotine and the overall use of tobacco  an increased negativity in the immediate intra- and extra-cellular proximity of the channel pore , with a mechanism similar to the selectivity filter of voltage-gated Ca2+ channels 2(α7)2 vs. (α4β2)2α4 . In this view, the insertion in heteromeric neuronal nAChRs of β2 or β4 subunits together with α2, α3, α4, or α6 appears a protective process, adding positive lysine residues instead of negative ones in the extracellular ring (position {20′}). The same role of “Ca2+ limiter” is played by β2 subunit in heteromeric nAChRs containing α7 subunits: decreasing the ratio α7:β2 considerably lowers the Pf-values of the corresponding nAChRs, suggesting distinct physiological roles for different subunit combinations forming these recently described heteromeric receptors (Wu et al., The analysis of the predicted 2+ permeability depend also on uncharged residues (Di Castro et al., 2+ fluxes, in an extremely complex picture.The predictive model presented in this study is based on a strongly oversimplified interpretation of the distribution of charged amino acid residues in selected regions of the receptor-channels, and presents clear limitations. First, it is well-known that the nAChR Ca2+ fluxes through nAChRs, but a single acid residue in the extracellular domain has been shown to strongly decrease the Ca2+ permeability of α7 nAChR (rat aspartate 44, corresponding to human aspartate 42; Colón-Sáez and Yakel, Furthermore, in the present study the overall extracellular distribution of charged amino acids did not appear to affect CaPf-values, strongly indicating that the amount of negative charges in strategic intracellular and extracellular TM2-flanking regions represents the main determinant of nAChR Ca2+ permeability.Despite all these known limitations, the present simple approach led to a surprisingly good description of the relation between charge distribution and 2+ permeability of other receptor-channels, as for instance the highly relevant insect nAChRs, representing the major target for several insecticides (Dupuis et al., Pf. Furthermore, in the future this approach could be useful to analyze other classes of ligand-gated channels, such as ionotropic glutamate receptors, whose Ca2+ permeability play a major role in synaptic plasticity and in neurodegenerative processes. The knowledge of the molecular mechanisms regulating Ca2+ entry through ion channels represents the first step to understand how to modulate it, in particular when and where the excessive accumulation of intracellular Ca2+ endangers cell health and survival.This study might be useful as a starting point to build future more sophisticated models, to plan future experimental studies on poorly described heteromeric nAChRs (see Table SF designed, wrote and approved this study, and agrees to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.


Cyanea capillata (C. capillata) could cause a weak relaxation response mediated by nitric oxide (NO) using isolated aorta rings. However, the intracellular mechanisms of TE-induced vasodilation remain unclear. Thus, this study was conducted to examine the role of TE on Akt/eNOS/NO and Ca2+ signaling pathways in human umbilical vein endothelial cells (HUVECs). Our results showed that TE induced dose- and time-dependent increases of eNOS activity and NO production. And TE also induced Akt and eNOS phosphorylation in HUVECs. However, treatment with specific PI3-kinase inhibitor (Wortmannin) significantly inhibited the increases in NO production and Akt/eNOS phosphorylation. In addition, TE also stimulated an increase in the intracellular Ca2+ concentration ([Ca2+]i), which was significantly attenuated by either IP3 receptor blocker (Heparin) or PKC inhibitor (PKC 412). In contrast, extracellular Ca2+-free, L-type calcium channel blocker (Nifedipine), or PKA inhibitor (H89) had no influence on the [Ca2+]i elevation. Since calcium ions also play a critical role in stimulating eNOS activity, we next explored the role of Ca2+ in TE-induced Akt/eNOS activation. In consistent with the attenuation of [Ca2+]i elevation, we found that Akt/eNOS phosphorylation was also dramatically decreased by Heparin or PKC 412, but not affected by Nifedipine or H89. However, the phosphorylation level could also be decreased by the removal of extracellular calcium. Taken together, our findings indicated that TE-induced eNOS phosphorylation and activation were mainly through PI3K/Akt-dependent, PKC/IP3R-sensitive and Ca2+-dependent pathways.Our previous study demonstrated that tentacle extract (TE) from the jellyfish  Hypertension is the leading risk factor for cardiovascular diseases, causing almost 3.7% of total disability-adjusted life-years and 13% of all deaths , which hCurrently, many bioactive natural products, especially marine ones, have received considerable attentions. Compared with terrestrial counterparts, marine organisms evolved a stronger biological activity and a more complex structural diversity to adapt to the extreme marine environment, such as high salt, high pressure, low-nutrient and unstable temperature condition . TherefoCyanea capillata (C. capillata) could cause a weak relaxation response in isolated aorta rings, which might be mediated by nitric oxide (NO) (2+]i) (3Rs)  from the jellyfish ide (NO) . Howeveride (NO) . eNOS is) (2+]i) , which ii) (3Rs) . Besidesi) (3Rs) . Thus, tω-nitro-L-arginine methyl ester (L-NAME) and PI3-K inhibitor Wortmannin were purchased from Sigma-Aldrich . The antibodies against phospho-Akt (Ser473), Akt, phospho-eNOS (Ser1177) and eNOS were purchased from Cell Signaling Technology . The antibody against GAPDH was purchased from Abcam . HRP-conjugated anti-rabbit IgG and anti-mouse IgG were purchased from Beyotime . Fluo-4 AM was purchased from Invitrogen . The stock solution of 1 mM was prepared by adding dimethyl sulfoxide (DMSO) to solid powder. The working solution of 5 μM was prepared by adding serum free medium to the stock solution. 1 × HBSS  and 1 × HBSS  were purchased from Sangon Biotech . L-type calcium channel blocker Nifedipine, PKA inhibitor H89, IP3 receptor blocker Heparin and PKC inhibitor PKC 412 were purchased from Sigma-Aldrich . In the measurement of Ca2+ mobilization, Nifedipine solution of 100 μM was prepared by Ca2+-containing HBSS, whereas H89 solution of 10 μM, Heparin solution of 125 IU and PKC 412 solution of 10 μM were prepared by Ca2+-free HBSS.The HUVECs cell line was purchased from Zhongqiaoxinzhou Biotech . MTT assay kit and NOS assay kit were purchased from Beyotime . Human NO ELISA assay kit was purchased from Sangon Biotech . eNOS inhibitor NC. capillata were collected in June, 2014, in the Sanmen Bay, East China Sea, and identified by Professor Huixin Hong from the Fisheries College of Jimei University, Xiamen, China. The removed tentacles were preserved in plastic bags on dry ice and immediately shipped to Shanghai, where the samples were frozen at −70°C until use. TE was prepared following the method as described in previous reports  for over 8 h before use. The protein concentration in the preparations was determined using the method of Bradford.Specimens of  reports . Briefly2.Human umbilical vein endothelial cells (HUVECs) were cultured in high glucose DMEM  supplemented with 10% fetal bovine serum , 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified incubator with 95% air and 5% CO4 cells/ml. After incubation for 24 h, cell groups were respectively treated with various doses of TE (0–24 μg/ml) for 1 h or 6 h. Then, 20 μl MTT reagent (5 mg/ml) was added to each well. At 4 h later, the supernatants were removed and the formazan dye was subsequently dissolved in DMSO. The absorbance value at 490 nm was measured using a microplate reader .Cell viability was determined by the MTT assay. Cells were plated in 96-well culture plates at a density of 10stimulated − RFUinhibitor+stimulated)/(RFUunstimulated − RFUinhibitor+unstimulated).Cells were serum-starved overnight in 96-well culture plates before measurements. In the dose–effect experiments, cells were incubated with different concentrations of TE (0–4 μg/ml) at 37°C for 1 h. In the time-effect experiments, cells were incubated with TE (1 μg/ml) for different time durations (0–180 min) at 37°C. After treatments, eNOS activity was measured according to manufacturer’s instructions (NOS assay kit). Briefly, cells were exposed to 100 μl reaction buffer solutions and subsequent 100 μl reaction solutions for 2 h with/without eNOS inhibitor (L-NAME). Then the plates were observed on a microplate reader  at excitation/emission wavelengths of 495/515 nm. The activity of eNOS was calculated by the following equation: relative activity (eNOS) =  at 37°C for 1 h. In the time-effect experiments, cells were incubated with TE (1 μg/ml) for different time durations (0–180 min) at 37°C. In the third group, cells were incubated with eNOS inhibitor L-NAME or PI3-K inhibitor Wortmannin for 15 min before treatment with TE . After incubation, culture supernatants were collected, and NO concentration was measured using a microplate reader  in the absorbance value at 450 nm according to manufacturer’s instructions (Human NO ELISA assay kit).2+-containing, Nifedipine, extracellular Ca2+-free, H89, Heparin and PKC 412, respectively.In the time-effect group, HUVECs were treated with TE (1 μg/ml) for different time durations (0-60 min). In PI3K/Akt-dependent signaling pathways, HUVECs were treated with TE (1 μg/ml) for 30 min in the presence or absence of Wortmannin. In calcium-dependent signaling pathways, HUVECs were treated with TE (1 μg/ml) for 30 min in the presence of extracellular CaAfter treatments, cells were lysed on ice in RIPA buffer with protease inhibitor (1% PMSF). The protein content of the lysate was measured by the method of Bradford. Equal amounts of protein per sample were loaded on 10% SDS-PAGE gels and then transferred to the nitrocellulose membranes. The membranes were subsequently blocked with 5% non-fat dry milk in TBST  for 2 h at room temperature. Then, the samples were incubated overnight at 4°C with primary antibodies as follows: p-Akt , Akt , p-eNOS (1:500), eNOS (1:500) and GAPDH , with gentle shaking. The ECL method was used with secondary antibodies (HRP-conjugated anti-rabbit IgG and anti-mouse IgG) at a dilution of 1:5,000 for 2 h at room temperature. After that, membranes were exposed using a chemiluminescent detection system . Quantitative densitometric analyses of immunoblots were performed using an ImageJ software (Ver. 1.48), and the relative ratio was calculated.2+-free HBSS to remove excess extracellular dye.HUVECs were cultured in a confocal dish  and serum-starved overnight before use. Cells were loaded with Fluo-4 AM working solution (5 μM) at 37°C in the dark for 30 min, then washed three times with Ca2+-containing HBSS with/without Nifedipine (100 μM) for 15 min before the addition of TE . To examine the possible participation of intracellular calcium stores, the cells were pre-treated with Ca2+-free HBSS in the presence or absence of H89 (10 μM), Heparin (125 IU) or PKC 412 (10 μM) for 15 min before the addition of TE . All the concentrations of the inhibitors were screened out and proved effective in similar preparations of the preliminary experiments.To characterize the possible contribution of extracellular calcium, the cells were pre-treated with 1.26 mM Ca2+ mobilization in HUVECs were monitored using a laser scanning confocal microscope  with excitation and emission wavelengths of 488 nm and 526 nm, respectively. Fluorescence images were recorded as time-series mode per 10 s intervals . TE was added after the first five images were collected. Fluorescence intensities were obtained from the data set of images using FV10-ASW 3.1 .The fluorescence of Cat-test were used in statistical evaluation of the data as appropriate. P-values less than or equal to 0.05 were considered significant.In the experiments, the images shown were the representatives of at least three experiments performed on different experimental days. Data were presented as mean ± SEM. Analysis of variance (ANOVA) and Student’s 50 was 15.55 μg/ml. However, TE at 0.5–8 μg/ml, did not significantly influence the cell viability after 1 h treatment. Similarly, after treatment with TE for 6 h, the viability of cells was decreased at the concentrations of 4–24 μg/ml in a dose-dependent manner, and the IC50 was 14.88 μg/ml. But TE at 0.5–2 μg/ml did not significantly influence the cell viability, either. Therefore, the relatively safe dose of TE less than 8 μg/ml was selected for the next study.As shown in Treatments with various doses of TE (0–4 μg/ml) increased the activity of eNOS in a concentration-dependent manner within 1 h in HUVECs: a significant increase was induced at the concentration of 0.5 μg/ml and the peak at 4 μg/ml . SimilarWe next examined the phosphorylation of Akt and eNOS after the stimulation with TE (1 μg/ml). As shown in As shown in 2+ concentration in HUVECs, we incubated the cells with the Ca2+ indicator dye Fluo-4 AM, and then stimulated them with TE whilst the time-series images of intracellular Ca2+ levels were detected. As shown in 2+ concentration ([Ca2+]i), which peaked within 1 min and sustained for about 10 min. Secondly, to determine the involvement of extracellular calcium, we next used the L-type calcium blocker Nifedipine (in Ca2+-containing HBSS) and Ca2+-free HBSS instead of the normal HBSS for the examination of TE-induced [Ca2+]i changes. Our results showed that neither Nifedipine nor extracellular Ca2+-free had influences on the TE-induced [Ca2+]i rise, which indicated that TE-induced [Ca2+]i rise did not come from the extracellular Ca2+ influx, but mainly from the intracellular stored Ca2+ release. Therefore, to further explore the source of Ca2+, we next investigated the effects of PKA inhibitor H89, IP3 receptor blocker Heparin and PKC inhibitor PKC 412 on the [Ca2+]i elevation. Our results showed that the TE-induced [Ca2+]i rise was significantly attenuated either by Heparin or by PKC 412, but not affected by H89, suggesting that the IP3R and PKC signaling play major roles in the TE-induced [Ca2+]i elevation.To explore the effects of TE upon the Ca2+, we next explored the role of Ca2+ in the activation of Akt/eNOS pathway in response to TE. As shown in 2+ in the TE-induced Akt/eNOS activation. Firstly, we found that TE (in Ca2+-containing HBSS) could induce the phosphorylation of both Akt and eNOS, while the phosphorylation level was dramatically decreased by the removal of extracellular calcium, indicating that Ca2+ was essential to the TE-induced Akt/eNOS activation. Secondly, to further elucidate the contribution of extracellular and intracellular calcium to the effects of TE-induced Akt/eNOS activation, we similarly performed the Akt/eNOS activity assays in the presence of L-type calcium channel blocker Nifedipine (100 μM), PKA inhibitor H89 (10 μM), IP3 receptor blockers Heparin (125 IU) and PKC inhibitor PKC 412 (10 μM), respectively. As shown in 2+ release from the ER through IP3R and PKC pathways. This result was in consistent with that of the calcium fluorescence assay. However, the expressions of p-Akt/p-eNOS were not affected by Nifedipine or H89, which indicated that the Akt/eNOS activation was not due to the influx of extracellular Ca2+ via L-type calcium channel or through PKA-dependent Ca2+ signaling.Since eNOS is also activated by a rapid increase in the intracellular CaC. capillata. Our result suggested that TE may serve as a potential alternative of the nematocyst venoms with much richer source for isolating and purifying cardiovascular active proteins  jellyfish venoms are sticky, thermolabile and difficult to be separated ; and (2)proteins , becauseproteins . Besidesproteins , suggestIt is well-established that eNOS is tightly regulated not only at the transcriptional level but also by certain post-transcriptional mechanisms . In the 2+/calmodulin-dependent mechanism , we further explored the source of TE-evoked Ca2+ rise using Ca2+ signaling related inhibitors. Our results showed that neither Nifedipine nor extracellular Ca2+-free had a significant influence on the TE-induced [Ca2+]i rise, indicating that TE-induced [Ca2+]i rise did not come from the extracellular Ca2+ influx, but mainly from the intracellular stored Ca2+ release. In ER, the IP3R channel is capable of releasing a large quantity of Ca2+ to the cytosol, and is believed to play a primary role in Ca2+ mobilization  did induce the phosphorylation of both Akt and eNOS, while the phosphorylation level was dramatically decreased by the removal of extracellular calcium, indicating that extracellular Ca2+ was essential to TE-induced eNOS activation. Since we had confirmed that TE-induced Ca2+ mainly came from intracellular stored Ca2+ rather than extracellular Ca2+, we hypothesized that extracellular calcium might be necessary for Akt phosphorylation, and subsequently activate eNOS. In fact, it has been reported that calcium could phosphorylate three kinases  that are involved in the cell survival signalling in neuroblastoma  or PKC (PKC 412), but not affected by PKA inhibitor (H89), which was also in line with the results in fluorescence assay, indicating that TE-induced Akt/eNOS activation might be associated with the calcium release via IP3R, while IP3R was also modulated by PKC pathways rather than PKA pathways. Taken together, these findings confirmed that calcium pathway was also necessary in the activation of TE-induced Akt/eNOS signaling.On the other hand, since calcium ions play a crucial role in stimulating eNOS activity through a Caechanism , we nextlization . This st kinases . So we nblastoma . HoweverC. capillata could induce dose- and time-dependent activation of eNOS and NO production. Further investigation found that TE induced Ser1177 eNOS phosphorylation and activation mainly through PI3K/Akt-dependent, PKC/IP3R-sensitive and Ca2+-dependent pathway. Since dysfunction of endothelial NO production is one of the major predictors of cardiovascular events, these findings will contribute to a better understanding of the signaling mechanisms of TE in regulating the endothelial function. Although TE may require further purification and identification in the near future, the current study opens up the possibilities for the development of jellyfish-derived specific-acting drugs that can be used to treat and/or prevent cardiovascular diseases such as hypertension.In this study, we demonstrated that TE from 10.7717/peerj.3172/supp-1Data S1Click here for additional data file.10.7717/peerj.3172/supp-2Supplemental Information 1Click here for additional data file.


Curiously, the role of endothelial Ca2+ signaling in NVC has been rather neglected, although endothelial cells are known to control the vascular tone in a Ca2+-dependent manner throughout peripheral vasculature. Methods: We analyzed the literature in search of the most recent updates on the potential role of endothelial Ca2+ signaling in NVC. Results: We found that several neurotransmitters  and neuromodulators  can induce dilation of cerebral vessels by inducing an increase in endothelial Ca2+ concentration. This, in turn, results in nitric oxide or prostaglandin E2 release or activate intermediate and small-conductance Ca2+-activated K+ channels, which are responsible for endothelial-dependent hyperpolarization (EDH). In addition, brain endothelial cells express multiple transient receptor potential (TRP) channels , which induce vasodilation by activating EDH. Conclusions: It is possible to conclude that endothelial Ca2+ signaling is an emerging pathway in the control of NVC.Background: Neurovascular coupling (NVC) is the mechanism whereby an increase in neuronal activity (NA) leads to local elevation in cerebral blood flow (CBF) to match the metabolic requirements of firing neurons. Following synaptic activity, an increase in neuronal and/or astrocyte Ca Accordingly, the brain receives up to 20% of cardiac output and consumes ≈20% and ≈25% of total body’s O2 and glucose i) in brain microvascular endothelial cells.The brain comprises only 2% of the total body mass, yet it accounts for 20% of the overall energy metabolism . As the  glucose ,4. Brain glucose . Cerebra glucose . Through glucose ,8. NVC i glucose ,9. Synap glucose ,12,13,1420-HETE) . This is20-HETE) ,16,17. Eoxane A2 ,19,20,21dilation ,23. Heretunica intima), which is separated from an intermediate layer of smooth muscle cells (tunica media) by the internal elastic lamina. An outermost layer mainly comprised of collagen fibers and fibroblasts and enriched in perivascular nerves, known as tunica adventitia, is separated from the brain by the Virchow–Robin space, which is an extension of the sub-arachnoid space i could, in turn, drive the synthesis of Ca2+-dependent vasoactive mediators. At the same time, brain microvascular endothelial cells have the potential to regulate local CBF independently of NA by detecting changes in blood flow variations and in the concentration of blood-borne agonists through distinct patterns of intracellular Ca2+ elevation.The concept of NVU was first introduced during the first Stroke Progress Review Group meeting of the National Institute of Neurological Disorders and Stroke of the NIH July 2001) to highlight the relevance to CBF regulation of the intimate association between neurons and cerebral vessels . The NVU001 to hiid space ,6. Penetid space . While pid space ,6. Relevid space ,26,27,28id space . Finallyid space ,31. Astric waste ,33. Ther2+ concentration ([Ca2+]i) within the dendritic tree is the crucial signal that triggers the synthesis and release of vasoactive messengers in response to excitatory synaptic inputs i within perisynaptic astrocytic processes  1 and 5 , which stimulate phospholipase Cβ (PLCβ) through Gqα monomer and induce inositol-1,4,5-trisphosphate (InsP3)-dependent Ca2+ release from the endoplasmic reticulum (ER) i in NVC i and recruiting the Ca2+-dependent PLA2 .,4,9.2,4,c inputs . For insrneurons ,28,33,35rneurons ,35,36,37on VSMCs ,28. Intron VSMCs ,38. Longrocesses , which llum (ER) . Howevervent NVC ,39. Nevevent NVC ,40,41. Fchannels ,39. We ri in NVC ,30,39,42i in NVC ,44, whicent PLA2 . In addi vessels .2 supply to local cerebral demand i evoked by either chemical or mechanical inputs i delivers the crucial signal to induce the synthesis of multiple vasoactive mediators i) refill the ER in a sarco-endoplasmic reticulum Ca2+-ATPase (SERCA)-mediated fashion by releasing Ca2+ though the mitochondrial Na+/Ca2+ exchanger i in rat brain microvascular endothelial cells ER) and Orai1-2 channels, which provide the Ca2+-permeable channel-forming subunits on the plasma membrane [2+]ER, drives resting Ca2+ entry and maintains basal Ca2+ levels in endothelial cells , a, a2+ con2+ release  production , thereby inducing VSMC hyperpolarization and vessel dilation i both locally and ≈1200 μm upstream along the same vessel  i, which stimulates IKCa (KCa3.1) and SKCa (KCa2.3) channels to hyperpolarize the endothelial cell membrane  i induced by either mechanical or chemical stimuli may stimulate the Ca2+-dependent phospholipase A2 (PLA2), which cleaves AA from membrane phospholipids i engages PLA2 to liberate AA, thereby resulting in EET synthesis, either within astrocytes or adjacent smooth muscle cells I, which results in robust NO release i that activated KCa channels to induce rapidly-conducting vasodilation at distances >1 mm and travelled along the endothelium as an intercellular Ca2+ wave for 300–400 μm. As shown in previous studies i in vivo. It will also be important to decipher the role played by IKCa and SKCa channels in the hemodynamic response to NA. Earlier work showed that EDH is involved in local and retrograde vasodilation in parenchymal arterioles and middle cerebral arteries, but is absent in capillaries, where functional hyperemia is likely to initiate in response to sensory stimulation. The local vasodilation induced by NA increases the strength of laminar shear stress acting on vascular endothelium, a mechanism that leads to TRPV4-dependent NO release and EDH in peripheral circulation i in rat cerebral arteries endothelial cells. This mitochondrial-derived Ca2+ signal, in turn, induced eNOS activation, NO release and endothelium-dependent vasodilation [2+-dependent NO release by directly activating TRPV4 in several types of endothelial cells [2+ signals in human brain microvascular endothelial cells [2+ signals may also contribute to NVC.Neurovascular coupling is the crucial process to adjust local CBF to the metabolic requirements of active neurons and maintain brain function. Although functional magnetic resonance imaging is routinely employed to monitor the changes in neuronal spiking activity, BOLD signals actually reflect the increases in CBF induced by NA. Therefore, understanding the cellular and molecular bases of NVC is mandatory to interpret the complex relationship between neuronal firing, metabolism and blood flow in both physiological and pathological conditions ,7,8. Intal cells ,174. Theculation ,228. Theculation ,230,231.culation ,212. Forculation ,232,233. peptide , whose adilation . The phyal cells ,235. Somal cells . Given t


However, its role in the development of human cancers is poorly understood. By analyzing datasets from the Cancer Genome Atlas Research Network (TCGA) acute myeloid leukemia (AML) study, we discover that mutations and/or copy number variations of m6A regulatory genes are strongly associated with the presence of TP53 mutations in AML patients. Further, our analyses reveal that alterations in m6A regulatory genes confer a worse survival in AML. Our work indicates that genetic alterations of m6A regulatory genes may cooperate with TP53 and/or its regulator/downstream targets in the pathogenesis and/or maintenance of AML.Methylation of NThe online version of this article (doi:10.1186/s13045-017-0410-6) contains supplementary material, which is available to authorized users. Hence, there is a compelling reason to determine whether mutations, deletions, and amplifications of m6A regulatory genes are enriched in leukemia subtypes. Clinicopathological associations including patient survival have not previously been reported.Methylation of Nkaryotes . It is kkaryotes , 2. m6A 6A regulatory genes, METTL3, METTL14, YTHDF1, YTHDF2, FTO, and ALKBH5. Deep deletions are possibly homozygous deletions as measured using the Genomic Identification of Significant Targets in Cancer algorithm (GISTIC). Four distinct types of hematological malignancies were sequenced by the Cancer Genome Atlas Research (TCGA) Network: acute myeloid leukemia (AML), multiple myeloma (MM), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL), and genetic data has been made available via cBioPortal [6A regulatory genes were found in 2.6% (5/191) of AML, 2.4% (5/205) of MM, 1.0% (1/106) of ALL, and 0% (0/666) of CLL  and copy number gain (n = 13) of m6A regulatory genes . Notably, 4.7% (9/191) of AML patients had concomitant copy number gain or loss of more than one m6A regulatory gene .We determined whether mutations and copy number variations (CNVs) of mALKBH5 is associated with the clinicopathological and molecular features. Consistent with our findings in m6A regulatory genes overall, shallow/deep deletion of ALKBH5 was significantly associated with poorer cytogenetic risk and the presence of TP53 mutation in this AML cohort  and event-free survival (EFS) in patients with AML. As a group, patients with a mutation of any of the genes encoding m6A regulatory enzymes had a worse OS (P = 0.007) and EFS , unfavorable cytogenetic risk, and DNMT3A and TP53 mutations were significantly associated with inferior OS and/or EFS in univariate analyses . This group of patients had worse OS and EFS than patients who did not have any of these genetic alterations  of m6A regulatory gene(s)  and EFS (P < 0.042) compared to patients who had mutant FLT3 but no genetic alteration of m6A regulatory genes  when TP53 mutation was excluded from the model (Fig. 6A regulatory genes and TP53 mutation. The fact that one is confounding the other in predicting patients’ outcome suggests that both may be complementary in the pathogenesis and/or maintenance of AML.In a multivariate Cox proportional hazard model that includes variables associated with poorer survival, genetic alterations of m OS Fig. . Howeverdel Fig. . Similardel Fig. . Our res6A modification to RNA has broad physiological functions, its impairment may be associated with the development and progression of diverse cancers, including leukemia. The current WHO classification highlights epigenetic modifiers as being mutated early during the clonal evolution of AML [Identification of novel biomarkers and molecular targets to guide the development of anti-leukemic therapies remains a major challenge. Particularly for AML, the molecular markers to define subtypes and prognosis are under continuous refinement , 9. Give6A regulatory genes in AML. We found a striking association between genetic alterations of these genes as a group and TP53 mutations (Table 6A regulatory genes are associated with inferior outcome in AML patients, although this may be confounded by the adverse impact of TP53 mutations on survival [6A methyltransferase, METTL3, resulted in alternative splicing and gene expression changes of >20 genes involved in the TP53 signaling pathway including MDM2, MDM4, and P21 in a human liver cancer cell line [6A modifiers, TP53, and/or its regulator/downstream targets contribute in complementary pathways to the pathogenesis and/or maintenance of AML. Further studies in larger AML cohorts would assist in confirming our findings and spur future research into the functional role of m6A RNA modification in AML and its link to tumorigenesis pathways, especially TP53 signaling.Our present study is the first to determine the clinicopathological associations and impact of genetic alterations affecting mns Table . Importasurvival  (Additio