Although the physiological relevance of this mechanism is under debate, emerging evidence suggests that there are critical factors in addition to Ca2+ that are required for gliotransmitters to be released from astrocytes. Interestingly, these factors include activated microglia and the proinflammatory cytokine Tumor Necrosis Factor α (TNFα), chemotactic cytokine Stromal cell-Derived Factor-1α (SDF-1α), and inflammatory mediator prostaglandin E2 (PGE2). Of note, microglial activation and release of inflammatory molecules from activated microglia and reactive astrocytes can occur within minutes of a triggering stimulus. Therefore, activation of astrocytes by inflammatory molecules combined with Ca2+ elevations may lead to gliotransmitter release, and be an important step in the early sequence of events contributing to hyperexcitability, excitotoxicity, and neurodegeneration in the damaged or diseased brain. In this review, we will first examine evidence questioning Ca2+-dependent gliotransmitter release from astrocytes in healthy brain tissue, followed by a close examination of recent work suggesting that Ca2+-dependent gliotransmitter release occurs as an early event in the development of neurological disorders and neuroinflammatory and neurodegenerative diseases.A prominent area of neuroscience research over the past 20 years has been the acute modulation of neuronal synaptic activity by Ca We will then review evidence suggesting that activated microglia and release of inflammatory mediators transform astrocytes into cells capable of releasing gliotransmitters in a Ca2+-dependent manner in the damaged or diseased brain.In this review, we will first examine evidence questioning Ca2+-dependent exocytosis of transmitters resulting in stimulation of synaptic receptors and acute synaptic modulation.The process of gliotransmission has been defined as analogous to neurotransmission, except that the source of the transmitters is glia  rather than neurons -expressing glial cells that express glutamate transporters, are coupled by gap junctions, and whose processes envelop synapses and form endfeet on the cerebrovasculature.It is first important to point out that there is no firm definition of an astrocyte. If the definition is exclusively morphological, i.e., highly ramified star-shaped cells, then the so-called “complex” glia could be included as an astrocyte subtype -sensitive intracellular stores, involving the canonical phospholipase C (PLC)/IP3 pathway. Upon Gq GPCR activation, PLC hydrolyzes the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to generate diacylglycerol (DAG) and IP3, leading to IP3R activation and Ca2+ release from the endoplasmic reticulum (ER). The lines of evidence for Gq GPCR-mediated Ca2+ sources in astrocytes are multiple. First, there is strong evidence that astrocytes express Gq GPCRs for endogenous neurotransmitters . Nevertheless, taken together the above evidence indicates that both evoked and spontaneous Ca2+ elevations in astrocytes are driven almost exclusively by Gq GPCR-coupled release from intracellular stores.The source of astrocyte Ca2+ was “necessary and sufficient” for this process  on both astrocytes and neurons as well as other cell types including microglia . These findings suggested that astrocyte Ca2+ elevations are not sufficient for gliotransmission in acute hippocampal slices. The results of these studies have already been well-documented as they have led to considerable debate as to whether the mechanisms of gliotransmission, as they have been conceived, are physiologically relevant  surrounding synapses, the relevant Ca2+ elevations involved in gliotransmission may be too small, too fast, and too local to detect using available Ca2+ indicators and two-photon imaging methods . In summary, the propagating intercellular Ca2+ wave is clear evidence for astrocytic release of gliotransmitters, evidence which is lacking in astrocytes from healthy tissue in situ or in vivo. However, as will be discussed below, in the diseased or damaged brain propagating astrocytic Ca2+ waves are evident, suggesting that changes occurring in reactive astrocytes are permissive for gliotransmitter release.Additional evidence questioning Ca2+ chelators BAPTA and/or EGTA in an effort to block astrocytic Gq GPCR Ca2+ elevations. These manipulations have produced clear effects on neuronal activity, suggesting that activity-induced astrocyte Ca2+ elevations modulate neuronal activity by gliotransmission. It has been difficult to reconcile these findings with those using IP3R2 knockout mice, in which astrocytic Gq GPCR Ca2+ elevations are also abolished but without a corresponding effect on hippocampal excitatory synaptic activity or NMDA receptor-dependent LTP , spotty Ca2+ signals were observed in cultured astrocytes that were not dependent on Ca2+ release from internal stores. Rather, these signals seemed to be dependent on extracellular influx of Ca2+ through transient receptor potential A1 (TRPA1) channels on the astrocyte membrane. These fast, focal Ca2+ elevations were shown to contribute to, and set resting Ca2+ levels in astrocytes. Could these channels represent the Ca2+ source required for gliotransmission? It cannot be ruled out; however, they seemed to open in an activity-independent manner and affect synaptic activity in hippocampal interneurons by a mechanism other than gliotransmission, i.e., by regulating the functional expression of astrocytic GABA transporters at the plasma membrane. It will be interesting in future work to determine if activity-independent Ca2+ influx setting basal Ca2+ levels is important for tonic release or uptake of gliotransmitters to set ambient concentrations of ATP/adenosine, d-serine, or glutamate  is 8 s  in the astrocyte membrane. Next, PLC mediates the conversion of inositol bisphosphate (PIP2) to IP3 and diacylglycerol (DAG). IP3 builds up in the astrocytic cytosol and binds its target receptor, IP3R2, on the ER. Activation of the IP3R2 results in channel opening and Ca2+ efflux from the ER into the cytosol, detected by the fluorescence indicator a few seconds after neuronal afferent stimulation. The locally elevated Ca2+ propagates into adjacent astrocytic compartments including the 20- to 30-nm sheet-like processes frequently surrounding synapses at a rate of 5–15 μm/s, where it encounters along the way small and diffuse synaptic-like microvesicles which are triggered to exocytose and release d-serine into the extrasynaptic space. The extracellular d-serine presumably reaches a sufficient concentration to percolate back into the synapse to bind synaptic NMDA receptors, permitting NMDA receptor channels to open to pass current and elevate postsynaptic Ca2+.Activity-dependent release of d-serine as a necessary co-agonist at the NMDA receptor occurred a 1000 times earlier in relative synaptic time  and oxygen free radicals  release. All of these molecules may act in concert through complex autocrine/paracrine or astrocyte-microglial signaling pathways to generate and amplify propagating intercellular astrocyte Ca2+ waves and increase neuronal excitability  in situ and in vivo, affecting the frequency, duration, and amplitude of astrocytic Ca2+ transients .As discussed above, evidence for intercellularly propagating Ca2+. A new report supports this hypothesis by showing that activated microglia recruit astrocytes to modulate neuronal activity early in the inflammatory process , trimeric TNF receptors (TNFR), or Gi/Gs GPCRs [prostaglandin E (EP) receptors; Bezzi et al., 2+-dependent exocytosis of glutamate from astrocytes. This assumption is based mainly on a study suggesting that CXCR4, TNFR, and EPs participate in the same sequence of events that ultimately leads to Ca2+-dependent exocytosis of glutamate, a response otherwise blocked by intracellular Ca2+ chelators or inhibitors of exocytosis  GPCRs, but also Gi/o (CXCR4) or Gi/Gs (EP) GPCRs. Additionally, the ligands of these receptors  and the ligand for TNFR (TNFα) appear to be mainly of microglial and astrocytic origin  exocytosis of cytokine vesicular carriers which could decrease the secretion of inflammatory mediators  in combination with neuronal electrophysiological readouts, which may lead to misinterpretation of how astrocytes actually control extracellular glutamate levels in a Ca2+-dependent manner.Interestingly, in the inflammatory process involving activated microglia and reactive astrocytes, glutamate release from astrocytes may be mediated by Ca2+-dependent glutamate release from astrocytes in acute hippocampal slices from otherwise healthy mice. Therefore, constitutive levels of inflammatory mediators could also regulate to some extent astrocytic glutamate release in brain tissue from healthy mice. However, it is worth noting that astrocytic inflammatory mediator-dependent glutamate release has been obtained in cultured astrocytes, mixed microglia/astrocyte co-cultures or acute hippocampal slices . Second, new research suggests that basal or resting astrocyte Ca2+ levels need to be carefully considered when Ca2+ chelators are infused into the astrocyte syncytium, which will block not only synaptically induced astrocyte Gq GPCR Ca2+ elevations, but also resting astrocyte Ca2+ levels. Manipulation of resting astrocyte Ca2+ may affect neuronal activity via mechanisms including changes in neurotransmitter uptake or ambient neurotransmitter concentrations. Third, propagating intercellular Ca2+ waves between astrocytes indicating release of the gliotransmitter ATP have not been substantiated in intact brain tissue from healthy subjects, arguing against gliotransmitter release in normal physiology. Last, because the endogenous means by which synaptically induced astrocyte Ca2+ elevations occur is through activation of Gq GPCRs, the kinetics of astrocyte Ca2+ elevations seem too slow to modulate neuronal synaptic activity occurring on a millisecond timescale  influence synaptic transmission affects the interpretation of a broad range of findings in neurophysiology. Thus, we propose that synaptically induced Gq GPCR Ca2+-dependent release of gliotransmitters  be rebranded as gliomodulation.A “calcium-centric” view has pervaded astrocyte research ever since the discovery of propagating astrocyte intercellular Cae Figure . It only2+-dependent release of gliotransmitters (glutamate and ATP) is also dependent on activated microglia, reactive astrocytes, and inflammatory molecules (Figure 2, mediators that are rapidly upregulated and secreted by activated microglia and reactive astrocytes. This new information is exciting for the following reasons: First, it provides a possible explanation for the disparate findings among research groups regarding the Ca2+-dependence of gliotransmitter release from astrocytes (Agulhon et al., 2+-dependent release of glutamate by astrocytes depends on glial mediators, as well as the second-to-minute time course of this response, argues that gliomodulation but not gliotransmission is involved. Definitive proof for Ca2+-dependent astrocyte glutamate release to occur via exocytosis awaits further evidence, and mechanisms other than exocytosis might also be at work including release through volume-regulated anion channels, pore-forming P2X7 channels, connexin hemichannels, or reversal of glutamate uptake (Haskew-Layton et al., 3R2 KO mice will be particularly useful for dissociating astrocyte Ca2+ specifically from other astrocyte transmitter release mechanisms. Future experiments that can measure and manipulate secretion and levels of inflammatory mediators vivo may also provide more compelling information regarding the conditions required for inflammatory mediator/Ca2+-dependent release of gliotransmitters by astrocytes. Overall, these findings provide promising new glial targets for therapeutical intervention to treat a wide range of central nervous system pathologies.Emerging research is suggesting that Cas Figure . These mThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Accurate chromosome segregation depends on coordination between cohesion resolution and kinetochore-microtubule interactions (K-fibers), a process regulated by the spindle assembly checkpoint (SAC). How these diverse processes are coordinated remains unclear. We show that in mammalian oocytes Shugoshin-like protein 2 (Sgol2) in addition to protecting cohesin, plays an important role in turning off the SAC, in promoting the congression and bi-orientation of bivalents on meiosis I spindles, in facilitating formation of K-fibers and in limiting bivalent stretching. Sgol2’s ability to protect cohesin depends on its interaction with PP2A, as is its ability to silence the SAC, with the latter being mediated by direct binding to Mad2. In contrast, its effect on bivalent stretching and K-fiber formation is independent of PP2A and mediated by recruitment of MCAK and inhibition of Aurora C kinase activity respectively. By virtue of its multiple interactions, Sgol2 links many of the processes essential for faithful chromosome segregation.DOI:http://dx.doi.org/10.7554/eLife.01133.001 Human reproductive cells—eggs and sperm—are produced through a process called meiosis. This means a ‘parent’ cell in the ovaries or testes undergoes two stages of cell division: it first divides into two cells, which then divide again to produce four ‘daughter’ cells. A crucial part of meiosis is ensuring that each daughter cell has half the number of chromosomes that the parent cell did.Before the first round of meiosis, the chromosomes in the parent cell are copied to produce enough chromosomes for the four daughter cells. The distribution of these chromosomes between the daughter cells is determined by the opposing forces acting on them. The pairs of identical chromosomes produced during the copying process are held together by a proteinaceous glue, while microtubules attached to a structure called the spindle—which has poles at opposite ends of the cell—try to pull these pairs of chromosomes apart.Rattani et al. now show that a protein called shugoshin-like 2 (Sgol2), which is involved in holding pairs of identical chromosomes together after the first round of division, has a bigger role than was previously realised. Sgol2 performs three other functions: it helps to align the chromosomes prior to division by, it is thought, facilitating the formation of the K-fibers that attach the microtubules to the chromosomes; it turns off the checkpoint that monitors the alignment of the chromosomes and the attachment of the microtubules; and it regulates a number of the enzymes involved in the process. The specific interactions that allow Sgol2 to perform these diverse functions in meiosis were also identified. Thus, Rattani et al. show that in linking so many essential processes throughout meiosis, Sgol2 appears to have a key, if not unique, role in determining the fate of chromosomes as cells divide.DOI:http://dx.doi.org/10.7554/eLife.01133.002 The production of haploid gametes from diploid germ cells depends on two rounds of chromosome segregation (meiosis I and II) without an intervening round of DNA replication. Defects during the first or second meiotic division in oocytes lead to formation of aneuploid eggs, which in humans occurs with a frequency between 10 and 30% and is a major cause of fetal miscarriages . UnderstAt the heart of this process are two opposing forces. The pulling forces produced by kinetochore-microtubules attachments (K-fibers) and resisting forces generated by sister chromatid cohesion, which counteracts K-fiber forces if and when kinetochores attach to microtubules with different polarities. During meiosis I, cohesion along chromosome arms holds bivalent chromosomes together following the creation of chiasmata produced by reciprocal recombination between homologous non-sister chromatids. This cohesion must persist during the attachment of maternal and paternal kinetochores to microtubules from opposite poles (bi-orientation) and resist the resulting spindle forces. The degree of traction exerted by meiotic spindles must create sufficient tension to facilitate bi-orientation . During Cdc20 can direct destruction of securin and cyclin B and thereby activate separase. Another pre-condition for cleavage, at least in yeast, is phosphorylation of cohesin’s kleisin subunit by a pair of protein kinases, namely CK1δ/ε and DDK  whose destruction of securin and cyclin B activates a thiol protease called separase that cleaves the kleisin subunit of the cohesin complex holding sister chromatids together . This pr and DDK . During Drosophila MEI-S332 protein, called shugoshins. Members of this family contain a conserved C-terminal basic region and an N-terminal homodimeric parallel coiled coil, which provides a docking site for protein phosphatase 2A’s (PP2A) C and B’ subunits , and Shugoshin-like protein 2 (Sgol2). The former prevents centromeric cohesin from a process called the ‘prophase pathway’ that removes cohesin from chromosomes by a non-proteolytic mechanism soon after cells enter mitosis . Sgol2, Cdc20 activation, suggesting that it was required to shut off the SAC. Sgol2 helps to turn off the SAC by binding directly both PP2A and the MCC protein Mad2, it moderates chromosome stretching by recruiting to kinetochores the kinesin MCAK, and likely promotes formation of K-fibers by down-regulating the activity of Aurora B/C kinase specifically at kinetochores. Meiotic chromosome segregation is a highly complex process dependent on an array of different biochemical processes. Our findings imply that through its multi-domain structure, Sgol2 has an important if not unique role in coordinating many of the key processes within this array.We show here that Sgol2 defective in PP2A binding fails to protect centromeric cohesin, as found for Sgo1 in yeast . HoweverAn essential feature of shugoshins is a conserved N-terminal homodimeric coiled coil that binds PP2A . Unlike sgol2Δ/Δ oocytes (n = 26). Importantly, microinjection of wild type Sgol2 encoding mRNA into sgol2Δ/Δ oocytes restored accumulation at kinetochores of both Sgol2 and PP2A at metaphase I. In contrast, microinjection of mRNAs carrying N54A, N55I, or 3A mutations restored Sgol2 but not PP2A , but also advanced activation so that it took place about 2 hr earlier than in wild type    providedif (MIM)  and far-if (MIM) . StrikinC/CCdc20 . They alC/CCdc20 . ImportaC/CCdc20  or MCAK C/CCdc20  to this C/CCdc20 . Interestivation  without tivation . We concsgol2Δ/Δ oocytes . In other words, bivalents were stretched more and possibly the stretching of distal parts of bivalent chromosomes resulted in increased separation of homologous pairs on chromosome spreads. Importantly, increased kinetochore separation in sgol2Δ/Δ oocytes was invariably observed on bivalents that had bi-oriented (t test p<0.0001), without altering spindle length (t test p<0.0001), regulation of bivalent stretching by Sgol2 does not depend on its ability to bind PP2A or Mad2. Thus, the effect cannot be due to changes either in arm cohesion protection or duration of metaphase I.We noticed that chiasmata appeared terminalized in a large fraction of bivalent chromosomes from  oocytes . Because oocytes , it is uoriented , but wasoriented . Interesoriented , but alse length . Becausesgol2Δ/Δ oocytes and more abundant (even than wild type) in mutant cells microinjected with Sgol2 mRNA (M.m Sgol2) by Aurora B has been implicated in the association of MCAK with Sgol2 in mitotic cells (sgol2Δ/Δ oocytes failed to restore accumulation of MCAK at kinetochores (t test p=0.027). Interestingly, neither the Aurora B/C inhibitor nor T521A T600A prevented MCAK’s recruitment to chromosome arms . We conclude that Sgol2 moderates the pulling force exerted on bivalents by recruiting MCAK to kinetochores, a process dependent on phosphorylation of Sgol2 by Aurora B/C kinases.These observations suggest that bivalent stretching might be inhibited by recruitment of MCAK to kinetochores by Sgol2. If so, increased inter-kinetochore distances in The period between Germinal Vesicle Breakdown (GVBD) and anaphase can be divided according to the state of spindles and chromosomes into four distinct phases . During sgol2Δ/Δ oocytes. Though GFP-MCAK co-localizes with MTOCs and microtubules, it never subsequently accumulates at kinetochores , raising the possibility that Sgol2 facilitates bi-orientation by a mechanism either independent of or in addition to MCAK’s recruitment to kinetochores.Crucially, the precocious bi-orientation caused by Sgol2 mRNA injection was unaffected by N54A or by H150E mutations but abrogated by T521A T600A , which asgol2Δ/Δ oocytes at 6 hr post-GVBD, when most kinetochores in wild type have relinquished lateral and formed end on attachments and chromosomes have bi-oriented. Kinetochore associated microtubules bundles (K-fibers) were thinner and more sparse, lateral attachments were greatly increased in frequency, and kinetochores (as detected by CREST antibodies) were frequently highly stretched in mutant oocytes  of eliminating a single function. Sgol2 possesses several different domains that regulate fundamentally different processes Rec8- Myc+ were crossed back to sgol2Δ/+ mice to generate sgol2Δ/+ (Tg)Rec8-Myc+ (controls) and sgol2Δ/Δ (Tg)Rec8-Myc+ female.Generation of the escribed . To dete2.Prophase-arrested oocytes were harvested from ovaries excised from 8–12 week old female mice. Antral follicles were isolated using sterile insulin needles in M2 medium  supplemented with 200 µM IBMX (Sigma-Aldrich). For intracellular microinjections, oocytes were placed in droplets of M2 medium with IBMX. For time-lapse confocal imaging and in vitro culture prior to preparation of chromosome spreads, oocytes were transferred into IBMX-free M16 medium and cultured at 37°C and 5% COCapped mRNA constructs with a poly-A tail were transcribed using T3 or T7 Ultra mMESSAGE kit  from plasmid cDNA encoding H2B-mCherry, wild type Sgol2 , and various mutants of Sgol2, GFP-Sgol2, Securin-eGFP, GFP-MCAK, CENPB-eGFP, CENPB-eGFP-MCAK, and GFP-EB3.2.Fully-grown oocytes were injected with 5–10 pl mRNA at a final concentration of 0.1 mg/ml in RNase-free water (Ambion) using a Pneumatic PicoPump (World Precision Instruments). To allow for protein expression, following microinjection of mRNA, GV oocytes were cultured for 12 hr in M16 supplemented with IBMX. Oocytes were then washed in inhibitor-free M16 and thereafter cultured at 37°C and 5% CODuring time-lapse confocal microscopy experiments, oocytes were cultured in a PeCon environmental microscope incubator at 37°C and 5% CO2. A Zeiss LSM510 META confocal microscope equipped with PC-Apochromat 63x/1.2 NA water immersion and PC-Apochromat 20x/0.8 NA objective lenses was used for image acquisition. For detection of GFP and mCherry, 488-nm and 561-nm excitation wavelengths and BP 505–550 and LP 575 filters were used. During live-cell imaging, chromosomes labeled with H2B-mCherry were tracked with an EMBL-developed tracking macro  adapted 2O (Kodak) and PBS for 5 min, at room temperature. Slides were then processed for immunostaining. In this study, we used CREST , rabbit anti-Sgol2 serum , anti-p Barbero ), anti-M Barbero ), anti-p Barbero ), anti-p Barbero ), antibo2+ buffer  at 37°C and then fixed in 1% formaldehyde prepared in Ca2+ buffer for 30 min at room temperature. Following fixation, oocytes were washed and extracted overnight in PBT (PBS supplemented with 0.1% Triton X-100) at 4°C. Cells were then blocked in 3% BSA with PBT (blocking solution) for 1 hr at room temperature. For immuno-labeling oocytes were incubated overnight at 4°C with β-tubulin  and CREST  antibodies, prepared in blocking solution. After four washes in PBT, oocytes were incubated for 1 hr at room temperature with Alexa 488 and 568 conjugated secondary antibodies  prepared in blocking solution. Oocytes were briefly stained with Hoechst 33,342 (20 mg/ml) before confocal imaging.For analysis of K-fibers, oocytes were processed as previously described . BrieflyEscherichia coli BL21(DE3) RIPL  carrying corresponding expression cassettes in pMAL-c2x- (Flag-tagged subunit) and pET28b-based backbones (His-tagged subunit), grown at 37°C in 2xTY media supplemented with 25 μg/ml chloramphenicol and 100 μg/ml ampicillin for pMAL-c2x or 50 μg/ml kanamycin for pET28b. At OD600 = 0.4, MBP-Sgol2cc-His or -Flag synthesis was induced at 30°C by the addition of 0.1 mM IPTG. After 4 hr, the cells were harvested by centrifugation, frozen in dry ice, and stored at −80°C. The cell paste was re-suspended in 40 ml of lysis buffer  containing Roche complete EDTA free protease inhibitor, 1 mM PMSF and, for the His-tagged subunit 5 mM. Each aliquot was sonicated four times for 30 s on ice. Insoluble material was removed by centrifugation at 25,000 rpm for 1 hr at 4°C in a Beckman Coulter Avanti-J26XP JLA8.1000 rotor. The supernatant was incubated for 2 hr at 4°C on a 5 ml Talon column  or ANTI-FLAG M2 Affinity Gel (Sigma A2220). The M2 Flag beads were washed four times with 50 ml of lysis buffer and the Talon beads were washed two times with 50 ml of lysis buffer containing 20 mM imidazole and two times with 50 ml of lysis buffer containing 45 mM imidazole. MBP-Sgol2cc-Flag was eluted with four times 1 ml of lysis buffer containing 3 × FLAG peptide (150 ng/μl). MBP-Sgol2cc-His was eluted with 10 ml of lysis buffer containing 1 M of imidazole. The concentration of MBP-Sgol2cc-His and MBP-Sgol2cc-Flag were determined by nanodrop against the lysis solution.Recombinant MBP-Sgol2cc-His and MBP-Sgol2cc-Flag were purified from 1l of For all binding assays, highly-purified PP2A subunits (purified as previously described ) and MBPE.coli BL21(DE3)RIPL for 4 hr at 30°C with 0.1 mM of IPTG. Cells were spun at 25,000 rpm for 1 hr at 4°C in a Beckman Coulter Avanti-J26XP JLA8.1000 rotor. The pellet was re-suspended into lysis buffer . 10 mg of extract were incubated with 100 μl of Talon beads for 2 hr at 4°C. The beads were wash five times with the lysis buffer containing 23 mM of imidazole. MBP-Sgol2cc-His was eluted with 10 ml of lysis buffer containing 1 M of imidazole. Affinity purified material and input were resolved by SDS-PAGE. The abundance of His and Flag-tagged MBP-Sgol2cc were analyzed by western blot using anti-Flag  and anti-His  antibodies.His and Flag-tagged mSgol2cc from wild type and 3A were co-expressed in  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  There was some discussion among the reviewers as to whether it would be important to show the localization of a component of the SAC – is Mad2 remaining at kinetochores in Sgol2 knockout oocyte? On balance we decided that it was not essential, but if you have the data it would strengthen the message.2) All the reviewers felt you could improve the citation of previous work in the Introduction. In particular, you should provide a citation for the statement that stretching the bivalents causes terminalisation of chiasmata. Furthermore, you should discuss the claim that stretching bivalents causes terminalisation without any apparent loss of cohesin. 1) There was some discussion among the reviewers as to whether it would be important to show the localization of a component of the SAC – is Mad2 remaining at kinetochores in Sgol2 knockout oocyte? On balance we decided that it was not essential, but if you have the data it would strengthen the message.We tried to check if Mad2 residence is affected in Sgol2 knockout oocytes. We performed chromosome spreads at multiple time points, but unfortunately none of the three different antibodies we tested gave us a reliable signal that we could quantify. In short, at this time we do not have data for Mad2 levels in Sgol2 knockout oocytes.2) All the reviewers felt you could improve the citation of previous work in the Introduction. In particular, you should provide a citation for the statement that stretching the bivalents causes terminalisation of chiasmata. Furthermore, you should discuss the claim that stretching bivalents causes terminalisation without any apparent loss of cohesin.We do agree that this phrase could create confusion. Therefore in the revised version, we have removed the statement that stretching of the bivalents causes terminalisation of chiasmata from the Introduction and in the Results we have also modified the phrasing. We have described in the Results that though the bivalent in Sgol2 knockout oocytes appeared terminalised, they still retained cohesion between homologous chromosomes. Furthermore, we have added that this abnormal shape of bivalent chromosomes on chromosome spreads could possibly be explained by increased inter-kinetochore stretching on the proximal end of the chromosomes in Sgol2 knockout oocytes, but these bivalent chromosomes are still connected at distal ends by cohesin molecules.


In this study, we characterized the cellular mechanisms underlying Ca2+ mobilization induced by (RS)-3,5-dihydroxyphenylglycine  in the somata of acutely dissociated rat hippocampal neurons using microfluorometry. We found that DHPG activates mGluR5 to mobilize intracellular Ca2+ from ryanodine-sensitive stores via cyclic adenosine diphosphate ribose (cADPR), while the PLC/IP3 signaling pathway was not involved in Ca2+ mobilization. The application of glutamate, which depolarized the membrane potential by 28.5±4.9 mV (n = 4), led to transient Ca2+ mobilization by mGluR5 and Ca2+ influx through L-type Ca2+ channels. We found no evidence that mGluR5-mediated Ca2+ release and Ca2+ influx through L-type Ca2+ channels interact to generate supralinear Ca2+ transients. Our study provides novel insights into the mechanisms of intracellular Ca2+ mobilization by mGluR5 in the somata of hippocampal neurons.Group I metabotropic glutamate receptors  exert diverse effects on neuronal and synaptic functions, many of which are regulated by intracellular Ca Enhancement of neuronal excitability 2+-dependent proteins such as PICK1 and NCS-1 in mGluR-LTD has recently been demonstrated 2+2+ that contributes to these diverse effects have not yet been clearly elucidated.The group I metabotropic glutamate receptors (mGluRs), which include mGluR1 and mGluR5, play important roles in regulating intrinsic excitability and synaptic plasticity 2+ from intracellular stores in hippocampal neurons 2+ mobilization may involve the phospholipase C (PLC)/inositol-3-triphosphate (IP3) signaling pathways 2+ release by group I mGluR stimulation paired with backpropagating action potential (AP) was shown to be from IP3 receptor (IP3R)-sensitive intracellular stores in apical dendrites of CA1 hippocampus 2+ mobilization by group I mGluRs required cyclic ADPR ribose (cADPR)/ryanodine receptors (RyRs) as well as IP3/IP3Rs 2+ signaling is supported by the study showing that the glutamate-induced stimulation of ADP-ribosyl cyclase occurs preferentially in NG108-15 neuroblastoma/glioma hybrid cells over-expressing mGluR1, 3, 5, and 6 2+ mobilization by group I mGluR may be mediated by signal pathways other than PLC/IP3Rs should be tested in hippocampal neurons.It is well known that group I mGluRs mobilize Ca2+ signaling in neurons is highly compartmentalized, with Ca2+ having distinctive roles in each section 2+ signaling have been extensively studied due to their importance in the regulation of neurotransmitter release and synaptic plasticity 2+ signals also play important roles in regulating cellular excitability, synaptic plasticity and gene expression 2+ signals are not well studied. As different neuronal compartment may have distinct Ca2+ signaling machinery, results obtained from dendrites or axons may not extend to the somatic Ca2+ signals. Therefore, separate studies of somatic Ca2+ signals are warranted.Ca2+ mobilization by group I mGluRs. Using microfluorometric Ca2+ measurements in the somata of acutely dissociated rat hippocampal neurons loaded with Fura 2-AM, we discovered that stimulation of group I mGluRs induces the cADPR-dependent Ca2+ mobilization from ryanodine-sensitive stores. Our results represent a novel mechanism for Ca2+ mobilization by group I mGluRs in hippocampal neurons.In the current study, we directly investigated the signaling pathways underlying somatic Ca2+ increase by group I mGluR stimulation, acutely dissociated hippocampal CA1 neurons were loaded with 2 µM Fura 2-AM for microfluorometry experiments. The application of 50 µM (RS)-3,5-dihydroxyphenylglycine (DHPG), a specific group I mGluR agonist, to these cells rapidly increased intracellular Ca2+ concentrations in the somata. The amplitude of DHPG-induced Ca2+ increase (CaDHPG) was highly variable among cells, ranging from ∼20 nM to ∼500 nM , but CaDHPG values obtained from the same cell upon repetitive application of DHPG at 2 min intervals yielded consistent data . To investigate the mechanism of DHPG-induced Ca2+ increase, we regarded CaDHPG,1 as the control and applied various experimental conditions prior to the second application of DHPG. The relative amplitude of CaDHPG,2 compared with CaDHPG,1  was obtained to study the contribution of each variable to the DHPG-induced Ca2+ increase.To investigate the mechanisms underlying Ca2+ transient was significantly suppressed by the selective mGluR5 antagonist, MPEP (25 µM), but not by the mGluR1 antagonist LY367385 (100 µM), indicating that mGluR5 is responsible for the DHPG-induced Ca2+ increases in hippocampal CA1 neurons , but they were markedly suppressed when cells were pretreated with the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin (2 µM) for 5 min, indicating that DHPG mobilizes Ca2+ from its intracellular stores  were completely inhibited by U73122 -containing pipette with or without heparin (1 mg/ml), and re-applied DHPG to elicit a second Ca2+ transient. We confirmed that the amplitudes of the second Ca2+ transients, which were measured at a holding potential of −60 mV, did not differ from those of the first Ca2+ transients    by ADP-ribosyl cyclases 2+ from intracellular stores. The involvement of ADP-ribosyl cyclase and/or cADPR in agonist-induced intracellular Ca2+ mobilization has been described in a variety of cell types 2+ release from midbrain dopaminergic neurons was shown to be mediated by both IP3 and cADPR 2+ mobilization.Alternatively, cADPR, which is metabolized from nicotinamide adenine dinucleotide (NAD2+ transients (2+ in neurons loaded with 8-NH2-cADPR (100 µM), a competitive antagonist of cADPR 2+ transients by DHPG  for 5 min to inhibit ADP-ribosyl cyclase ansients . Similar by DHPG . Importa neurons .2+ release in the somata of hippocampal neurons. In the brain, glutamate is the natural neurotransmitter that stimulates mGluRs; glutamate can activate both mGluRs and ionotropic glutamate receptors (iGluRs), which include AMPA and NMDA receptors. NMDA receptors and the Ca2+-permeable AMPA receptors are possible sources of Ca2+ entry into the cell. In addition, glutamate-induced membrane depolarization should activate voltage-gated Ca2+ channels (VGCCs) to cause Ca2+ influx. Indeed, we confirmed that glutamate depolarizes the membrane potential by 29.0±4.6 mV (n = 4). As shown in 2+ transients (CaGlu) was significantly greater than CaDHPG. The average CaDHPG was 92.8±10.3 nM from 74 cells, whereas CaGlu was 284.3±23.6 nM from the same population .Our results demonstrated a predominant role for the cADPR/RyR signaling pathway in mGluR5-induced Capulation , p<0.01.2+ entry when cells were stimulated with glutamate. Repetitive application of glutamate at 2 min intervals yielded reproducible CaGlu values . When the bath solution was replaced with a Ca2+-free solution prior to the second application of glutamate, the CaGlu,2 was significantly decreased so that the CaGlu,2/CaGlu,1 ratio was 50.4±9.6% , a specific blocker of the Ca2+-permeable AMPA receptor, had no significant effect on glutamate-induced Ca2+ transients  in the presence of CNQX, which is significantly less than that observed in the control .Subsequently, experiments were performed to determine the source of Ca% n = 6, . Unexpec% n = 7, . In cont% n = 8, , suggestansients . These r2+ channel blocker, significantly reduced glutamate-induced Ca2+ transients (Glu was not significantly affected by ω-conotoxin GVIA (1 µM), ω-agatoxin IVA (200 nM), and NiCl2 (100 µM), indicating that N-type, P/Q type, T-type and R-type Ca2+ channels are not involved , an L-type Caansients . In continvolved . These d2+ signaling, it is necessary to examine the interaction between mGluR-induced Ca2+ release and glutamate-induced Ca2+ influx. It has been shown that Ca2+ entry through VGCCs interacts synergistically with IP3 to enhance mGluR-mediated Ca2+ release in apical dendrites of hippocampal CA1 neurons 2+ release by DHPG along with either membrane potential depolarization or NMDA receptor activation was also demonstrated in primary cultured hippocampal neurons 2+ channels are enhanced by mGluR5-induced Ca2+ mobilization from ryanodine-sensitive stores in the GABAergic interneurons of the hippocampus 2+ channels induced by both orthograde and retrograde pathways in NG108-15 cells 2+ transients by DHPG and glutamate, under the assumption that CaGlu represents the sum of Ca2+ influx (CaInflux), Ca2+ mobilization by mGluR5 (CaDHPG) and the supralinear Ca2+ transients (CaSUP) by synergistic effects of mGluR5 and Ca2+ influx (CaGlu = CaInflux+CaDHPG+CaSUP).To understand the complexity of mGluR-mediated CaSUP by subtracting the sum of CaDHPG . In this series of experiments, CaDHPG was 34.3±10.2% of CaGlu , and found that the mechanisms were consistent with those found in acutely dissociated immature neurons. Therefore, the mechanisms of mGluR5-induced somatic Ca2+ mobilization found in the current study may be extended into at least young adult neurons.In this study, the experiments were performed using acutely dissociated neurons, as dissociated neurons have several advantages in studying signaling mechanisms in the somata. In this preparation, indirect effects or presynaptic components can be excluded. Furthermore, rapid application and wash-out of drugs are guaranteed. It is very difficult to obtain healthy cells by enzymatic dissociation method from rats over 2 weeks old, and therefore we used immature rats (P7-P14). However, glutamate signaling is still developing at this age, and the Ca2+ channels, but not NMDA receptors, are responsible for the Ca2+ influx by glutamate. It was shown previously that NMDA-dependent Ca2+ entry evokes Ca2+ increases primarily in spines, which are more concentrated with oblique dendrites 2+ influx by NMDA receptors exclusively at oblique dendrites, whereas backpropagating APs evoke Ca2+ increase at all dendritic locations. Thus, in acutely dissociated neurons that usually lack oblique dendrites the role of NMDA receptors in Ca2+ influx should be limited and membrane potential depolarization by AMPA receptors and the opening of L-type Ca2+ channels may be responsible for Ca2+ influx instead. Another notable finding is that the contribution of Ca2+ influx to CaGlu was larger than that of Ca2+ release . Animal handling was conducted in accordance with national and international guidelines. The number of animals used was minimized, and all necessary precautions were taken to mitigate pain or suffering.see below) saturated with 95% O2 and 5% CO2. Transverse hippocampal slices (400 µm thick) were prepared using a vibratome . After a 30 min recovery period at 32°C, the slices were treated with protease type XIV  for 30–60 min, and with protease type X  for 10–15 min at 32°C. The slices were allowed to recover during a 1-hour incubation period at room temperature. The CA1 region was identified and punched out under a binocular microscope , placed in a recording chamber containing normal Tyrode (NT) solution (see below) and mechanically dissociated using a Pasteur pipette to release individual neurons. The dissociated neurons were allowed to adhere to the bottom of the recording chamber for 10–20 min. Cells were identified as pyramidal neurons by their typical large pyramidal-shaped cell body with a thick apical dendritic stump of ∼50 µm under an inverted microscope . The isolation of hippocampal CA1 neurons from PLCβ1 or PLCβ4 knockout mice : NaCl 150, KCl 5, CaCl2 2, MgCl2 1, glucose 10, Hepes 10, and was adjusted to pH 7.4 with Tris-OH. To make Ca2+-free NT solutions, CaCl2 was replaced with equimolar MgCl2 and 0.1 mM EGTA. Pipette solutions used for electrophysiology studies contained (in mM) K-gluconate 110, KCl 30, Hepes 20, Mg-ATP 4, Na-vitamin C 4, Na-GTP 0.3, EGTA 0.1 titrated to pH 7.3 with KOH.ACSF contained (in mM): NaCl 125, NaHCORS)-3,5-DHPG, LY367385, MPEP, SKF96365, CNQX, AP-5, ryanodine, TTX were purchased from Tocris. U73122 was purchased from Biomol. Fura 2, Fura 2-AM and 8-NH2-cADPR were obtained from Molecular Probes, and ω-conotoxin-GVIA and ω-agatoxin-IVA were from Anygen. All other drugs were purchased from Sigma. Stock solutions of drugs were made by dissolving in deionized water or DMSO according to manufacturer's specifications and were stored at −20°C. On the day of the experiment one aliquot was thawed and used. The final concentration of DMSO in solutions was maintained below 0.1%., which was coupled to the epi-illumination port of an inverted microscope  via a quartz light guide and a UV condenser. Microfluorometry was performed with a 40× water immersion objective  and a photodiode (TILL-Photonics).Keff) was calculated from Keff = [Ca2+](Rmax−Rint)/(Rint−Rmin), where [Ca2+] was entered as 231 nM (assuming a dissociation constant (Kd) of BAPTA of 222 nM at pH 7.2). The estimated Rmin, Rmax and Keff (µM) measured using an inverted microscope were typically 0.27, 3.95 and 0.93, respectively. A standard two-wavelength protocol was used for fluorescence measurement of cells. Fluorescence intensity was measured at 1 Hz with double wavelength excitation at 340 nm (F340) and 380 nm (F380). The ratio R = F340/F380 was converted to [Ca2+] values using the equation [Ca2+] = Keff (R−Rmin)/(Rmax−R).Calibration parameters were determined using in vivo calibration as described in Current clamp recordings of membrane potential were performed using an EPC-10 amplifier (HEKA Elektronik) at room temperature. Membrane potentials were recorded from acutely dissociated hippocampal CA1 neurons in a conventional whole cell configuration at a sampling rate of 10 kHz filtered at 1 kHz. Data were acquired using an IBM-compatible computer running Pulse software v8.67 (HEKA Elektronik). The patch pipettes were pulled from borosilicate capillaries (Hilgenberg-GmbH) using a Narishige puller . The patch pipettes had a resistance of 3–5 megaohms when filled with above-mentioned K-based pipette solutions.2-cADPR was performed by single cell electroporation. Micropipettes were pulled as described above and were filled at their tips with NT solutions containing heparin (20 mg/ml) or 8-NH2-cADPR (100 µM) plus Alexa Fluor-488 . Micropipettes were controlled by a micromanipulator (Burleigh) to reach cells, and square electric pulses generated with an electroporator  were applied to transfer the mixture into the cells.The loading of heparin and 8-NHn represents the number of cells studied. The significance of differences between the peaks was evaluated using a Student's t-test with confidence levels of p<0.01 (**) and p<0.05 (*).Data were analyzed using IgorPro  and Origin  software. Statistical data are expressed as the mean ± S.E., where


Second harmonic generation (SHG) microscopy is a recently developed technique which allows examination of fibrillar collagen structures with a high degree of specificity and resolution. The goal of this study was to examine the potential utility of SHG and multiphoton excitation fluorescence (MPEF) microscopy in understanding the relation between tenocytes and their surrounding collagenous matrix in early tendon overuse lesions.Tenocytes have been implicated in the development of tendinosis, a chronic condition commonly seen in musculoskeletal overuse syndromes. However, the relation between abnormal tenocyte morphology and early changes in the fibrillar collagen matrix has not been closely examined Histological preparations of tendon were prepared from adult male Sprague-Dawley rats subjected to an Achilles tendon loading protocol for 12 weeks (Rat-A-PED), or from sedentary age-matched cage controls. Second harmonic generation and multiphoton excitation fluorescence were performed simultaneously on these tissue sections in at least three different areas.SHG microscopy revealed an association between abnormal tenocyte morphology and morphological changes in the fibrillar collagen matrix of mechanically loaded Achilles tendons. Collagen density and organization was significantly reduced in focal micro-regions of mechanically loaded tendons. These pathological changes occurred specifically in association with altered tenocyte morphology. Normal tendons displayed a regular distribution of fibre bundles, and the average size of these bundles as determined by Gaussian analysis was 0.47 μm ± 0.02. In comparison, fibre bundle measures from tendon regions in the vicinity of abnormal tenocytes could not be quantified due to a reduction in their regularity of distribution and orientation.SHG microscopy allowed high resolution detection of focal tendon abnormalities affecting the fibrillar collagen matrix. With ongoing repetitive loading, these tenocyte-associated focal collagen defects could predispose to the progression of overuse pathology. SHGI) scales as SHG I≈ N2 Here N is the effective number of radiating molecules. Likewise, isotropic macromolecules generate MPEF signal due to their endogenous fluorescence. Multiphoton excitation fluorescence intensity (MPEFI) emitted by these molecules undergoing multiphoton excitation can be expressed as, MPEF I≈ N. While SHG signal intensity scales as a square of number of collagen molecules, the MPEF intensity scales linearly with the number of other endogenously fluorescent molecules. Since MPEF and SHG involve different principles and contrast mechanisms, SHG and MPEF can be captured simultaneously to visualize structural changes of biological macromolecules. Particularly, SHG signals originating from the collagen structures can be used to generate high resolution images of individual collagen fibre bundles and quantify collagen density in a given tissue. Hence, SHG provides the potential to investigate precise spatial relations between collagen organization and underlying tenocyte biology.Multiphoton and associated microscopy methods have been widely used for imaging dynamic interactions in cells and tissues with submicron resolution -3. AmongTendinosis is a histological entitity characterized by collagen remodeling and angiofibroblastic hyperplasia. Tendons which rupture often demonstrate evidence of pre-existing tendinosis . HoweverResearch into the early pathogenesis of tendinosis has suggested that tenocytes may initiate degradation and remodelling of the load-bearing collagen matrix in response to mechanical loading. This model runs counter to a traditional "wear and tear" hypothesis of collagen microtrauma . Dudhia in vivo model of mechanical overload. We hypothesized that collagen density and fibrillar organization would be significantly reduced in highly localized areas associated with abnormal tenocyte morphology.The goal of the current study was to determine whether SHG microscopy could be used to examine a potential association between abnormal tenocyte morphology and local reductions of collagen density in a relevant Twelve male Sprague-Dawley rats aged 4 months, weight 448.7 ± 10 grams were obtained locally and housed singly in standard cage conditions. Following a 48 hour acclimatization period, rats were divided into controls , or runners , providing 12 tendons per group. Ethic approval for the study was obtained from the UBC animal care committee (A07-0274).Runners were subjected to a previously published mechanical loading protocol (Rat-A-PED) using a dedicated rat treadmill to induce Achilles tendinosis  . Rats we2, and whole bilateral Achilles tendons were removed with the muscle still attached, oriented longitudinally in Tissue Tek embedding medium , then frozen in isopentane chilled in liquid nitrogen. Tendons were cryosectioned onto charged glass slides at 5 micron thickness and processed for H&E , Alcian blue / fast nuclear red (stains sulphated glycosaminoglycans and cell nuclei), or Picrosirius red (enhances birefringence of collagen when viewed under polarized light).Rats were euthanized with COxyR) values follow: 20X/0.7 NA (xyR≈0.50 μm); 63X/1.2 NA (xyR≈0.292 μm). Upon entering the Leica microscope system, the laser beam was directed to the scanning mirrors, then through a 670 nm long pass dichroic mirror  and subsequently focused on the specimens through the objective lens. The backscattered emission from the sample was collected through the objective lens. Leica Confocal Software TCS SP2 was used for the image acquisition. Non-descanned detectors and spectral scanning mode both in the reflection geometry were used for capturing the MPEF images as well as for the spectral signal characterizations respectively. In the non-descanned PMT detectors , a 700 nm short pass filter  was used to prevent the scattered IR laser radiation from reaching the detector and a 455 long pass dichroic beam splitter  was used to separate SHG signal from the MPEF signal. SHG signal in forward direction was captured using a non-descanned detector in the transmission geometry equipped with a 440/20 nm band pass filter  and high NA condenser. All SHG and MPEF spectral data were generated using the de-scanned PMT detector  located inside the scan head where the emission signals were delivered through the AOBS detection system with the maximum confocal pinhole setting at 600 μm via the spectral dispersion prism. The width of the slits in front of each PMT could be software adjusted such that each PMT could detect spectral regions spanning from a 5 nm bandwidth up to the overall spectral capacity of the system (400-800 nm). With this instrument configuration, a series of individual images were collected using a narrow detection window with a width of ~5 nm, and each image detected at this specific emission wavelength band (i.e. ~ 5 nm) provided a data point in the spectral graph. The gain and the offset of the PMTs were adjusted for optimized detection using the color gradient to avoid pixel intensity saturation and background. Images (8 bit) acquired at slow scan speed i.e. 10 sec per 512 X 512 pixels.The microscope system used in our present experiments is the same as described previously . SpecifiLateral line profiles of collagen fibers were generated using Leica comprehensive image processing software from 2D spectrally clean SHG images. The sizes of the collagen fibers and their size distribution were plotted by fitting the lateral line profiles to a Gaussian curve using Origin Lab Software.A Mann-Whitney U test was used to detect differences in collagen density and blood flow. Values are expressed as means ± SE. Results were considered statistically significant if α was less than 0.05.In order to determine the microregional properties of collagen surrounding abnormal tenocytes in mechanically loaded tendon, we assessed standard histological preparations using multiphoton and second harmonic generation (SHG) microscopy. Initially, normal tendon was analyzed. Representative images originating from spectrally clean SHG signal originating from collagen (1A) and the MPEF signal originating from endogenously fluorescent tissue components Figure  are showAfter confirming the specificity of collagenous structures visualized with SHG in normal tendons, we next examined the collagen organization in sedentary and mechanically loaded tendons. All Achilles tendons analyzed from animals subjected to the mechanical loading protocol demonstrated microregions of abnormal tenocyte morphology, typified by the appearance of tenocytes with prominent cytoplasm and rounded morphology Figure . These mSHG signals emitted from the Achilles tendon proper region in normal tendon vs tendinosis lesions were next characterized by spectral analysis. The wavelength scans of both normal tendon from sedentary rats, and of tendon surrounding abnormal tenocytes from rats subjected to the mechanical loading protocol, showed a highly specific SHG signal manifested by a narrow peak at 440 nm Figure . The res2) to yield a mean collagen density value. The collagen density from micro-regions corresponding to abnormal tenocytes in rats subjected to the overuse protocol was 729 ± 90.3 AU, compared to the value of 1135 ± 60.1 in normal tendon (p < 0.05).We then applied a noise removal filter to SHG images to define the boundary between foreground and background, and the lower threshold in the histogram was set to 10% of the highest pixel intensity value. The total SHG signal intensity values thus generated were normalized by the cropped collagen area (μmSecond harmonic generation (SHG) resulted in high resolution, spectrally clean signals originating from fibrillar collagen. The collagen matrix surrounding abnormal tenocytes demonstrated an abnormal organization and a reduced density compared to sedentary cage controls. This reduction in SHG signal occurred specifically in association with the appearance of abnormal tencoytes in the tendons subjected to mechanical loading. These results provide support to an emerging model of tendinosis where degradation and/or remodelling of the load-bearing collagen matrix in response to exercise is mediated by the local activity of tenocytes, rather than being due to a passive accumulation of fatigue damage ,14.increased collagen staining using this overuse tendinopathy model based on assessment of H&E preparations [reduced SHG signal observed in the current study accurately reflects a loss of collagen density in the tendinosis samples. Indeed, H&E evaluation of tendinosis samples may be misleading as tendinosis lesions can be highly eosinophilic, but we have found these eosinophilic regions to generate very weak or absent SHG signals indicating that they may comprise provisional matrix with a higher component of non-collagenous elements. We are therefore able here to refine and further validate the description of early pathology which occurs in this model.Glazebrook et al. recently reported arations . HoweverIn the current study, SHG was used to estimate the collagen fibre bundle diameter with submicron resolution in normal tendons, but not in tendinosis samples. The longitudinal alignment of collagen fibres was reduced in the tendinosis tendons, in association with a reduction of SHG signal intensity. The technique of measuring fibre bundle diameter relied on the fibres demonstrating an orderly, longitudinal orientation, which was disrupted in areas of tendinosis.in vivo are accompanied by highly localized foci of reduced collagen density which may predispose to the progression of overuse tendinopathy.Second harmonic generation analysis of mechanically loaded tendon provides the ability to examine collagen organization in standard histological preparations, providing insight into mechanisms of early load-induced tendon changes. Tenocyte responses to mechanical loading The authors declare that they have no competing interests.AS and TA conceived of the study. AS supervised the tendon mechanical loading protocol and carried out the imaging and histological examination. GF assisted with imaging analysis. TA and AS conducted the image analysis. TA designed and oversaw the SHG and MPEF microscopy. All authors read and approved of the final manuscript.Alex Scott is a physical therapist engaged in full time research and teaching. Gloria Fong is a Research Assistant with a BSc in Medical and Laboratory Sciences. Thomas Abraham is a specialist in optical physics engaged in full time research.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/26/prepubFigure 1S Glycosaminoglycan distribution in mechanically loaded and sedentary tendon. Alcian blue staining in tendinosis (A) and normal healthy (B) tendon. Tendinosis tendon demonstrates increased sulphated glycosaminoglycan intra- and peri-cellularly, particularly in chains of abnormal-appearing tenocytes. Mast cells also stain blue with this technique in the paratendon regions (not shown). 20× objective lens.Click here for file


Plasmodium parasites in human erythrocytes. Especially, the development into the mature forms (trophozoite and schizont) of Plasmodium falciparum  causes severe malaria symptoms due to a distinctive property, sequestration which is not shared by any other human malaria. Ca2+ is well known to be a highly versatile intracellular messenger that regulates many different cellular processes. Cytosolic Ca2+ increases evoked by extracellular stimuli are often observed in the form of oscillating Ca2+ spikes (Ca2+ oscillation) in eukaryotic cells. However, in lower eukaryotic and plant cells the physiological roles and the molecular mechanisms of Ca2+ oscillation are poorly understood. Here, we showed the observation of the inositol 1,4,5-trisphospate (IP3)-dependent spontaneous Ca2+ oscillation in P. falciparum without any exogenous extracellular stimulation by using live cell fluorescence Ca2+ imaging. Intraerythrocytic P. falciparum exhibited stage-specific Ca2+ oscillations in ring form and trophozoite stages which were blocked by IP3 receptor inhibitor, 2-aminoethyl diphenylborinate (2-APB). Analyses of parasitaemia and parasite size and electron micrograph of 2-APB-treated P. falciparum revealed that 2-APB severely obstructed the intraerythrocytic maturation, resulting in cell death of the parasites. Furthermore, we confirmed the similar lethal effect of 2-APB on the chloroquine-resistant strain of P. falciparum. To our best knowledge, we for the first time showed the existence of the spontaneous Ca2+ oscillation in Plasmodium species and clearly demonstrated that IP3-dependent spontaneous Ca2+ oscillation in P. falciparum is critical for the development of the blood stage of the parasites. Our results provide a novel concept that IP3/Ca2+ signaling pathway in the intraerythrocytic malaria parasites is a promising target for antimalarial drug development.Malaria remains one of the world’s most important infectious diseases and is responsible for enormous mortality and morbidity. Resistance to antimalarial drugs is a challenging problem in malaria control. Clinical malaria is associated with the proliferation and development of  Plasmodium species is complex . Each of these asexual merozoites invades an erythrocyte and enters into another phase of asexual reproduction, and then bursts the cell, releasing 8–32 more merozoites to invade more erythrocytes (blood stage). In infected erythrocytes, development of the parasites is accompanied by morphological changes such as ring form, trophozoite and schizont stages. P falciparum is responsible for the lethal form of human malaria. The mature forms of the intraerythrocytic parasite (trophozoite and schizont) remodel the cytoskeleton and plasma membrane to create cytoadherence knobs as well as nutrient permeation pathways and alter the mechanical stability of the erythrocytes, causing them to stick to blood vessels Malaria continues to be a worldwide public health problem causing significant morbidity and mortality and its resistance to existing antimalarial drugs is a growing problem  complex . Infecti2+) oscillations are ubiquitous intracellular signals responsible for numerous cellular processes, including excitation–contraction coupling, fertilization, cell growth, transformation, secretion and neuronal signalling Plasmodium, Toxoplasma and Cryptosporidium, Ca2+-mediated signalling controls various vital functions such as protein secretion, motility, cell invasion and differentiation 2+ signalling in Plasmodium species, numerous studies have focused on calcium-dependent protein kinases, which are activated downstream of Ca2+ release from intracellular Ca2+ stores, as an important therapeutic target for antimalarial drug development 2+ has been considered to be a key regulator of the parasite egress and invasion of erythrocytes 2+ signalling in intraerythrocytic development of Plasmodium species.Calcium  receptor inhibitor 2-aminoethyl diphenylborinate (2-APB) occur in the ring form and trophozoite. Examination of the effects of 2-APB on the in vitro intraerythrocytic parasite development and electron microscopic observations revealed that blockage of Ca2+ oscillations caused severe degeneration and breakdown of successive asexual reproduction in the intraerythrocytic parasites, resulting in death of them. Furthermore, 2-APB showed a similar effect against the chloroquine-resistant K1 strain of P. falciparum.In this study, we observed the intracellular dynamics of Ca2+ images of each intraerythrocytic developmental stage of the FCR-3 strain: early ring forms , late ring forms , early trophozoites , late trophozoites , schizonts  and merozoites (M). Ca2+ imaging of parasites was performed in culture chambers at 37°C in an atmosphere of 5% O2 and 5% CO2, conditions identical to those in conventional in vitro parasite culture. The Fluo-4 fluorescence in a parasite cytoplasm (F) was calculated by subtraction of the background fluorescence and normalized to the minimum fluorescence during the imaging period (Fmin). In early ring forms (ERf) and early trophozoites (ET), spontaneous Ca2+ oscillations were observed   . This re2+ oscillations by 2-APB in early ring forms and early trophozoites strongly suggests that the observed Ca2+ oscillations are regulated by a putative IP3 receptor/Ca2+ channel that is activated by IP3 binding. In Plasmodium the phospholipase C (PLC) pathway is known to be involved in the release of Ca2+ from the intracellular Ca2+ store, and the effectiveness of U73122, a commonly used PLC inhibitor, has been demonstrated 3. As shown in 2+ oscillations in early ring forms  and early trophozoites  were almost completely inhibited. In apicomplexan parasites, two intracellular Ca2+ stores are known to be involved in Ca2+ release: the endoplasmic reticulum (ER) and acidocalcisomes including the food vacuole 2+ oscillations, we depleted Ca2+ stores before imaging by using thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic reticulum Ca2+–ATPase, and concanamycin A (CMA), a specific inhibitor of vacuolar-type H+-ATPase. The effects of these inhibitors have been demonstrated in Plasmodium species 2+ by Ca2+ imaging in early ring forms and early trophozoites using perfusion experiments  and early trophozoites . In contrast, Ca2+ depletion by pre-treatment with 100 nM CMA had no effect on Ca2+ oscillations in early ring forms  and early trophozoites   . In thesThe effects of 2-APB on intraerythrocytic development were investigated using synchronized parasite cultures in the ring form stage, with initial parasitaemia of approximately 1%. P. falciparum using synchronized parasite cultures in the ring form stage with initial parasitaemia of approximately 2%. At 24 h of the assay a tendency towards decreasing trophozoite parasitaemia was reproducibly observed in parasites cultured with 100 µM 2-APB  of P. falciparum2+ signalling. Dilatation of the nuclear envelope and ER is possibly among the features of cell degeneration, but it may also compensate for disrupted Ca2+ signalling, since ER is a multifunctional and highly dynamic organelle, undergoing constant movement and reorganization depending on cellular conditions Ultrastructural changes induced by 2-APB were observed by transmission electron microscopy. As shown in 2+ signalling and accumulation of aberrant proteins is known to cause ER stress, a hallmark of cell death that is associated with many neurodegenerative diseases 2+ signaling systems in unicellular organisms. In this study, we clearly showed the stage-specific spontaneous Ca2+ oscillations in the intraerythrocytic stages of P. falciparum, a unicellular eukaryote, without any exogenous extracellular stimulation. We further demonstrated that the blockage of these Ca2+ oscillations by 2-APB caused severe cellular degeneration resulting to the death of the parasites. The half maximal inhibitory concentration (IC50) value of 2-APB for inhibition of IP3-induced Ca2+ release in mammalian cells was ∼40 µM 2+ oscillations of the early trophozoites. First, the normal development of the parasites was observed in 2-APB-pretreated erythrocytes  was diluted 10-fold with BSA(−) medium for Ca2+ imaging  supplemented with 25 mM HEPES, 24 mM sodium bicarbonate, 0.5 g/L L-glutamine and 50 mg/L hypoxanthine. Cultures of erythrocytes (1 mL) were collected by centrifugation (1000 g for 5 min at room temperature) and resuspended in 350 µL BSA(−) medium. Loading Solution was prepared for staining nuclei with Hoechst 33342 and loading of Fluo-4-AM . A suspension of erythrocytes (350 µL) was mixed with 150 µL of Loading Solution and shaken at 200 rpm for 30–60 min at 37°C. Erythrocytes were then washed once with 10 mL BSA(−) medium (1000 g for 5 min at room temperature) and resuspended in 600 µL of BSA(+) medium [BSA(−) medium supplemented with 0.5% Albumax I and 25 µg/mL gentamicin (Sigma)]. A suspension of erythrocytes (100 µL) was inoculated in a 35 mm glass-bottomed dish (MatTek) that had been coated with 1 mg/mL poly-L-lysine before use. After 30 min of incubation in an O2/CO2 incubator, suspended erythrocytes were removed by gentle washing with BSA(+) medium. The glass-bottomed dish was then placed in the culture chamber in which O2, CO2, temperature and humidity were maintained under conditions used for conventional in vitro parasite culture.A culture of infected erythrocytes .Sequential time-lapse imaging of Hoechst 33342, Fluo-4-AM and transparent images was performed using the Leica confocal microscope system  with a 63× (N.A.1.42) oil immersion objective lens and excitation at 410 nm (diode laser) for Hoechst 33342 and 488 nm (Argon laser) for Fluo-4-AM and transparent images. Emissions were collected using the true spectral detection method developed by Leica Microsystems. Images were captured every 5–15 s for 300–600 s. Specific Fluo-4 fluorescence in a parasite (F) was calculated by subtraction of background fluorescence and normalized to minimum fluorescence during the imaging period (FThe 2-APB concentration used was determined by preliminary experiments with a variety of concentrations ranging from 1 to 150 µM (data not shown). Effects of 2-APB on the intraerythrocytic parasite development were assayed using parasite cultures in the ring form stage with initial parasitaemia of approximately 1%–2%. Cultures (500 µl) were placed in each well of a tissue culture plate . 2-APB was dissolved in DMSO  at 10 mM. Stock solutions were diluted with PPMI medium and added to each well of the culture plate to give a defined concentration. DMSO diluted with medium served as control. A drop of cultured erythrocytes was smeared on a glass slide and stained with Giemsa. The number of parasite-infected erythrocytes in 2000 erythrocytes was counted and defined as the level of parasitaemia.Giemsa-stained smears were observed under the Nikon Eclipse 80i microscope (Nikon), photographed using the Nikon DXM 1200F camera and uploaded on a personal computer using digital photo manager software . To measure parasite size, we randomly selected 50 parasites and manually delineated areas containing parasites with lines on the screen. The area, perimeter and maximum diameter of the parasites were calculated by WinROOF software package Ver.5.8.1 .Transmission electron microscopy was performed as previously described 2+ imaging. ER-Tracker Red was then added at a final concentration of 0.5 µM to the erythrocyte suspension and shaken at 200 rpm for 30 min at 37°C. Erythrocytes were then washed once and resuspended in RPMI1640 medium without phenol red and observed under fluorescence microscopy (Leica DM 2500).The nucleus and ER of the parasites was stained with Hoechst 33342 and ER-Tracker Red (Invitrogen). Staining with Hoechst 33342 was performed as described above for fluorescence CaThe human erythrocytes stock for the parasite culture was provided by the Hokkaido Kushiro Red Cross Blood Centre under the ethical guidelines for the blood products and obtained according to their acquisition guidelines. Written informed consent from the donor of the human erythrocytes was obtained. This study was done without a regular review from ethics committees of the Obihiro University of Agriculture and Veterinary medicine, where the parasite culture was made, because the erythrocytes stock was provided as blood product from the Red Cross after completing their ethical review for the experiment.Figure S12+) dynamics during intraerythrocytic Cytosolic calcium . (A–D) Dimethyl sulfoxide (DMSO) was used as a solvent control. Each colour represents cytosolic Ca2+ dynamics acquired from individual parasites of late ring forms (LRf), late trophozoites (LT), schizonts (S) and merozoites (M) in the presence (right columns) or absence (left columns) of 100 µM 2-APB. Embedded images in left panels are representative images of Fluo-4-loaded P. falciparum during each intraerythrocytic stage (indicated by arrowheads). Scale bars, 5 µm.(TIF)Click here for additional data file.Figure S22+ distribution in late trophozoite treated with 100 µM 2-APB.Subcellular Ca The parasite Ca2+ and nucleus and were stained simultaneously with Fluo-4 (green) and Hoechst 33342 (blue). Merged images are shown in the right column (Merge). Scale bars, 5 µm.(TIF)Click here for additional data file.Figure S32+) fluctuations.Effects of 2-aminoethyl diphenylborinate (2-APB) on mean amplitude of periodic calcium  was detected only in merozoites. LRf, late ring forms; LT, late trophozoites; S, schizonts; M, merozoites.(TIF)Click here for additional data file.Figure S42+ oscillations in early ring forms (ERf) and early trophozoites (ET).Effects of U73122, thapsigargin (Tg) and concanamycin A (CMA) on Ca (A) Spontaneous Ca2+ oscillations observed in ERf (left) and ET (right) disappeared after 5 min of pre-treatment with 10 µM U73122.  Erythrocytes were prepared in a manner identical to that for Ca2+ imaging in the culture chamber. Ca2+ imaging was initiated under the same conditions as those used for perfusion with normal culture medium. Replacement of perfusion medium with medium containing 2 µM thapsigargin (Tg) (B) or 100 nM concanamycin A (CMA) (C) induced an increase in Ca2+ concentration in ERf (open circles) and ET (closed squares). Black horizontal bars represent the period of perfusion with test compounds. (D) Ca2+ depletion induced by 30 min of pre-treatment with 2 µM Tg significantly diminished Ca2+ oscillations in ERf (left) and ET (right). (E) Ca2+ depletion induced by 30 min of pre-treatment with 100 nM CMA had no effect on Ca2+ oscillations in ERf (left) and ET (right).(TIF)Click here for additional data file.Figure S5Pre-treatment of erythrocytes with 2-APB did not inhibit intraerythrocytic development of P. falciparum. To evaluate the effects of 2-APB on host cells, erythrocytes were pre-treated with 100 µM 2-APB for 1 h at 37°C, washed with RPMI medium and resuspended at a haematocrit of 5% in complete culture medium. Cultures of late schizonts  were diluted four times with pre-treated erythrocytes, and the culture was continued. Cultures  were terminated at 40 h of the assay, and thin smears of erythrocytes were prepared for parasite counting. Parasitaemia with ring forms did not differ significantly between dimethyl sulfoxide (DMSO)- and 2-APB-pre-treated groups . Parasitaemia is shown as mean + S.D. of three independent counts of three wells. Stages with parasitaemia of less than 0.1% are not shown.(TIF)Click here for additional data file.Figure S6Effect of 2-APB on the area, perimeter and maximum diameter of the chloroquine-resistant strain K1. No statistically significant difference was observed in the area, perimeter and maximum diameter of intraerythrocytic parasites between DMSO- and 100 µ M 2-APB-cultured groups after 24 h of the assay, but the 3 parameters showed a tendency to decrease. Error bars represent mean + S.D. (n = 50). P values are given in each panel (two-tailed unpaired t test).(TIF)Click here for additional data file.Figure S72+ dynamics and parasite development in the blood stage of Timetable of the effects of 2-APB on CaP. falciparum. Images show Giemsa-stained parasites. Blue bars above the parasite images indicate stages of the parasites in which spontaneous Ca2+ oscillations or small periodic Ca2+ fluctuations were observed. Red lines represent the period of 2-APB treatment. Red circles indicate the initiation of 2-APB treatment. Black, dark and light grey boxes show the extent of the effect of 2-APB during each sampling period: black, severe effect with developmental delay and abnormal morphology; dark grey, weaker effect with developmental delay and abnormal morphology than that when parasites were exposed to 2-APB for 40 h; light grey, slight effect with developmental delay or abnormal morphology compared to that when parasites were exposed to 2-APB for 40 h.(TIF)Click here for additional data file.Figure S8Electron micrographs of parasites in DMSO control culture. (A) At 30 h of the assay . (B) Higher magnification image of the part marked with an asterisk in (A) . (C) At 30 h of the assay . (D) Higher magnification image of the part marked with an asterisk in (C) . N, nucleus; NE, nuclear envelope; Ri, ribosome; Rh, rhoptry.(TIF)Click here for additional data file.Figure S9Effects of 2-APB on the number of merozoites in each schizont. (A) Box plot of the number of merozoites (M) formed in each schizont (S) after 40 h of culture with DMSO (white boxes) or 2-APB (light grey boxes). The central rectangle spans the first quartile to the third quartile. The segment inside the rectangle shows the median and whiskers above and below the box show the minimum and maximum. 2-APB significantly decreased the number of M per S (two-tailed unpaired t test with Welch’s correction). (B) Frequency distribution of the raw data shown in (A).(TIF)Click here for additional data file.


Ostericum koreanum Maximowicz has been used as a traditional medicine called “Kanghwal” in Korea(or “Qianghuo” in China). The purpose of this study was to investigate the vasorelaxant activity and mechanism of action of an ethanol extract of theO. koreanum root (EOK). We used isolated rat aortic rings to assess the effects of EOK on various vasorelaxant or vasoconstriction factors. EOK induced vasorelaxation in phenylephrine hydrochloride (PE) or KCl precontracted aortic rings in a concentration-dependent manner. However, the vasorelaxant effects of EOK on endothelium-intact aortic rings were reduced by pretreatment withL-NAME or methylene blue.In Ca2+-free Krebs-Henseleit solution, pretreatment with EOK (0.3 mg/mL) completely inhibited PE-induced constriction. In addition, EOK (0.3 mg/mL) also completely inhibited vasoconstriction induced by supplementalCa2+ in aortic rings that were precontracted with PE or KCl.Furthermore, the EOK-induced vasorelaxation in PE-contracted aortic rings was inhibited by preincubation with nifedipine.These results indicate that the vasorelaxant effects of EOK are responsible for the induction of NO formation fromL-Arg and NO-cGMP pathways, blockage of the extracellular Ca2+entry via the receptor-operative Ca2+ channel and voltage-dependent calcium channel, and blockage of sarcoplasmic reticulumCa2+ release via the inositol triphosphate pathway.The root of  Ostericum koreanum Maximowicz (Umbelliferae), is an example of this type of herbal medicine.Traditional Chinese medicine (TCM) was introduced to Korea in the 6th century . TherefoO. koreanum is a perennial herb widely distributed in Korea. The root of this plant has been used as a traditional medicine called “Kanghwal.” However, the pharmacopoeias of Korea, China, and Japan describe this plant's origin differently. The Chinese and Japanese pharmacopoeias list only Notopterygium incisum and Notopterygium forbesii as being of “Kanghwal” origin (“Qianghuo” in Chinese), while the Korean pharmacopoeia includes O. koreanum as also being of “Kanghwal” origin , maintained at 37°C, and aerated with a mixture of 95% O2 and 5% CO2. After careful removal of the connective tissue and fat, approximately 2 mm long aortic rings were cut and suspended in organ chambers containing 10 mL K-H solution at 37°C and aerated with a mixture of 95% O2 and 5% CO2. The aortic rings were placed between 2 tungsten stirrups and connected to an isometric force transducer . After incubation under no tension for 30 min, the vessel segments were allowed to equilibrate for 1 h at a resting tension of 1.0 g. During the equilibration period, the K-H solution was replaced every 20 min. Changes in tension were recorded by isometric transducers connected to a data acquisition system . When required, the endothelium was removed by gentle rubbing of the vessel lumen with a thin cotton swab. The presence of functional endothelium was verified by the ability of Ach (10 μM) to induce more than 80% relaxation of rings precontracted by PE (1 μM). In endothelium-denuded rings, less than 10% relaxation due to Ach was seen. Ca2+-free K-H solution was prepared by omission of CaCl2 and addition of EGTA (1 mM).We used male Sprague-Dawley rats  to examine the vasorelaxant effect of EOK. All animal procedures were conducted according to the animal welfare guidelines issued by the Kyung Hee University Institutional Animal Care and Use Committee [KHUASP (SE)-09-006]. The rats were housed under controlled conditions , with food and water available μM) or KCl (60 mM). After the plateau was attained, EOK was added cumulatively (0.03–1.0 mg/mL). The vasorelaxant effect on the aortic rings was calculated as a percentage of contraction in response to PE or KCl. To determine whether the arterial endothelium pathways were involved in EOK-induced vasorelaxation, endothelium-intact aortic rings were preincubated with l-NAME (10 μM) or MB (10 μM) for 20 min before contraction by PE (1 μM) treatment. The relaxant effects of EOK on the aortic rings were compared with the control (not treated with l-NAME or MB). To investigate the effect of EOK on extracellular Ca2+-induced contraction, we investigated the contractile responses induced by CaCl2 (0.3–10 mM) addition on aortic rings pre-contracted by PE (1 μM) or KCl (60 mM) in Ca2+-free K-H solution in the absence (control) and presence of a 10 min pre-incubation of EOK (0.3 mg/mL). CaCl2-induced contractile responses were compared in the absence (control) and presence of EOK. To investigate the effect of EOK on L-type voltage-dependent calcium channel, K+ channels, and Rho-kinase pathway, aortic rings were preincubated with nifedipine, 4-AP, glibenclamide, TEA, or Y-27632 (1 μM) for 20 min before the addition of PE (1 μM). After the plateau was attained, EOK was added cumulatively (0.01–0.8 mg/mL). The vasorelaxant effects on the aortic rings were calculated as a percentage of contraction in response to PE. To investigate the effect of EOK on intracellular Ca2+ release from sarcoplasmic reticulum- (SR-) induced contraction, we investigated the contractile responses induced by PE (1 μM) or caffeine (5 mM) on endothelium-denuded aortic rings in Ca2+-free K-H solution in the absence (control) and presence of a 10 min pre-incubation of EOK (0.3 mg/mL). Contractile responses induced by PE or caffeine were compared in the absence (control) and presence of EOK.In standard K-H solution, endothelium-intact aortic rings were precontracted by PE . Isoimperatorin, imperatorin, and oxypeucedanin hydrate were used as standard materials for the qualitative analysis of EOK. They were serially diluted , and HPLC chromatograms were obtained. The relationship between the concentration and the peak area was measured using the minimum square method . The HPLC chromatograms were obtained from Gilson System equipped with a 234 autoinjector, a UV/VIS-155 detector, and a 321 HPLC Pump . A Luna 4.60 × 250 mm C18 reversed-phase column with 5 μm particles  was used. Chromatographic separation was carried out using an isocratic solvent with acetonitrile  water mixture in the ratio of 30 : 70 (v/v). The column eluent was monitored at UV 254 nm, following which all solvents were degassed with a micromembrane filter . Chromatography was performed at room temperature at a flow rate of 1.0 mL/min, and 10 μL was analyzed for 20 min. The quantity of the EOK standards was expressed as follows: the amount (mg) of standard material = the quantitative amount (mg) of standard materials × AT/AS/n . Accurately weighted EOK (100.0 mg) was dissolved in 10 mL of methanol . And then it was filtered through a syringe filter  followed by the Tukey's post hoc test. All statistical analyses were performed using SPSS v.13.0 statistical analysis software . P values less than 0.05 were considered statistically significant. Data were expressed as mean ± standard error of mean (SEM). Statistical comparisons were made using Student's μM) or KCl (60 mM) precontracted rings in a concentration-dependent manner. The maximal relaxant effects were up to 99.8 ± 1.7% and 102.4 ± 3.6% at the concentration of 1.0 mg/mL, respectively ( EOK (0.03–1.0 mg/mL) relaxed PE (1 ectively . l-NAME (10 μM) decreased EOK-  induced relaxation of endothelium-intact aortic rings precontracted by PE (1 μM) treatment (μM) also decreased EOK- (0.01–0.4 mg/mL) induced relaxation of endothelium-intact aortic rings  pathway, PE (1 μM) was applied to induce stable contraction. CaCl2 (0.3–10 mM) was then added cumulatively to induce a progressive increase in contraction of the aortic rings. EOK (0.3 mg/mL) preincubation significantly inhibited the contraction induced by extracellular CaCl2 (0.3–10 mM) compared with the control group. And the contraction was decreased to −0.05 ± 0.02 g, −0.07 ± 0.02 g, −0.05 ± 0.02 g, and −0.05 ± 0.02 g, respectively  (2-induced (0.1–10 mM) contraction compared with the control group. Contraction decreased to 0.00 ± 0.02 g, −0.03 ± 0.02 g, −0.06 ± 0.02 g, −0.07 ± 0.03 g, and −0.08 ±0.03 g, respectively  (μM) for 20 min inhibited EOK-induced vasorelaxation on PE-contracted aortic rings ( To investigate the effects of EOK on the receptor-operative Ca 0.14 g) . To inve 0.06 g) . In addiic rings . 2+-free K-H solution, preincubation with EOK (0.3 mg/mL) for 10 min completely inhibited PE-induced (1 μM) contraction. However, preincubation with EOK (0.3 mg/mL) for 10 min did not alter caffeine-induced (5 mM) contraction (In the Catraction .μM) for 20 min did not alter EOK-induced vasorelaxation on PE-contracted aortic rings (Preincubation with Y-27632 (1 ic rings . μM) precontracted aortic rings were not altered by preincubation of the rings with various K+ channel blockers, including 4-AP (1 mM), glibenclamide (10 μM), or TEA (5 mM) (The vasorelaxant effects of EOK on PE (1 A (5 mM) . R2) of oxypeucedanin hydrate was y = 201168.349x − 153935.071 (0.999) and exhibited good linearity. The oxypeucedanin hydrate content in EOK was 1.80 ± 0.92%. The regression equation  of imperatorin was y = 514722.569 + 153935.071 (0.999) and exhibited good linearity. The imperatorin content in EOK was 0.42 ± 0.02%. The regression equation  of isoimperatorin was y = 301715.046x + 32227.015 (0.998) and exhibited good linearity. The isoimperatorin content in EOK was 0.70 ± 0.02%. l-Arg and NO-cGMP pathways, blockage of the extracellular Ca2+ entry via the ROCC and VDCC, and blockage of SR Ca2+ release via the IP3 pathway. In the present study, we investigated the vasorelaxant effects of EOK on rat aortic rings and its related mechanisms. The vasorelaxant effects caused by EOK were both endothelium dependent and endothelium independent. And the vasorelaxant mechanisms of EOK were responsible for the induction of NO formation from l-NAME, an inhibitor of NOS. In addition, the vasorelaxant effect of EOK was reduced by pretreatment with MB, a soluble guanylate cyclase inhibitor. These results suggested that the vasorelaxant effect of EOK is related to the induction of NO formation from l-arginine and NO-cGMP pathways. Vascular endothelium plays an important role in vasorelaxation. NO is one of the potent vasodilators secreted from vascular endothelium. And vascular smooth muscle is relaxed via NO-cGMP pathway . In the 2+ influx via transmembrane Ca2+ channels and Ca2+ release from intracellular stores [2+ concentration in vascular cells is the most important factor of vascular contraction and relaxation. The factors that increase calcium in cells consist of stimulus by neurotransmitter, opened calcium channels by membrane potential, and calcium ion release from the SR. Ca2+ concentration in cells increases to combine with calmodulin, a protein that binds to calcium in cells. The bonded Ca2+-calmodulin activates inactive myosin light chain kinase, which then phosphorylates myosin light. The phosphorylated myosin light chain then makes a phosphorylated crossbridge with actin to produce a contraction [Vascular smooth muscle also plays an important role in vasorelaxation, which is regulated by extracellular Car stores . Ca2+ cotraction .α-adrenergic agonist) contracts smooth muscle cells via extracellular Ca2+ influx through ROCC and by internal calcium release from specific IP3 receptor (IP3R) channels in the SR membrane [2+-free K-H solution, PE induced the contraction via the IP3 pathway. In the present study, pretreatment with EOK (0.3 mg/mL) for 10 min completely inhibited PE-induced (1 μM) contraction. This result suggests that EOK could inhibit Ca2+-induced vasoconstriction from specific IP3R channels in the SR membrane. And EOK (0.3 mg/mL) completely inhibited vasoconstriction induced by Ca2+ supplementation in aortic rings that were precontracted with PE (1 μM). This result suggested that EOK could inhibit vasoconstriction induced by extracellular Ca2+ entry via the ROCC.PE (an membrane . In Ca2+2+ influx through depolarization of the cell membrane and subsequent opening of VDCC [2+ supplementation in aortic rings that were precontracted with KCl (60 mM). This result suggests that EOK could also inhibit vasoconstriction induced by extracellular Ca2+ entry via the VDCC. L-type VDCC is the major mechanism of VDCC. In the present study, EOK-induced vasorelaxation on PE-contracted aortic rings was inhibited by preincubation with nifedipine . This result suggested that EOK could inhibit extracellular Ca2+ entry via blocking L-type VDCC. KCl contracts smooth muscle cells mainly by extracellular Ca of VDCC . EOK  channels in the SR membrane . EOK  channels, Ca2+-activated K+ (BKCa) channels, and ATP-sensitive K+ (KATP) channels are well-known distinct types of K+ channels in vascular smooth muscle [V channel blocker), TEA (BKCa channel blocker), and glibenclamide (KATP channel blocker). These results indicated that the vasorelaxant effects of EOK on the rat aortic rings were not related to the opening of K+ channels.Increase of Krization . Thus, th muscle . In the 2+ sensitization of smooth muscle [Rho kinase has been identified as one of the effectors of the small GTP-binding protein Rho. And Rho/Rho-kinase-mediated pathway has been implicated in the regulation of vascular tone . Y-27632h muscle . In thisOsterici Radix has been reported to contain several active compounds such as oxypeucedanin hydrate , oxypeucin vivo studies, and chemical analyses may be necessary to establish the efficacy of Osterici Radix for treating hypertension.  In conclusions our findings suggest that Osterici Radix might be a useful herbal medicine for treating cardiovascular diseases such as hypertension. However, more detailed mechanism studies,