Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca EGF controls key cellular processes, such as proliferation, survival, differentiation during development, tissue homeostasis, and tumorigenesis  induce a distinctive robust oscillatory CaCercopithecus aethiops were cultured in Dulbecco's modified Eagle's medium  containing 10% (V/V) FBS (Sigma-Aldrich) at 37°C under an atmosphere of 5% CO2 and plated at a density of about 1.3 104 cells/cm2 on LabTek #1 borosilicate chambered slides (Thermo Fisher Scientific) previously coated with fibronectin (BD Biosciences) at a density of 1µg/cm2 for 1 hour and washed with Ca2+-free HBSS (Life Technologies). Cells were grown for 48–72 hours and deprived of serum 18 hours before imaging in DMEM-f12 medium without phenol red (Life Technologies). We ensured that cells were healthy by culturing low-passage cell lines, by systematically evaluating cell proliferation rate from growth curve data and by verifying cellular morphology before EGF stimulation experiments.COS-7 cells (American Type Culture Collection) derived from the kidney of the African Green Monkey, 2+ in the extracellular medium, 3 mM Ca2+o); 125 mM NaCl, 2.5 mM KCl, 1.1 mM NaH2PO4, 4 mM NaHCO3, 2 mM MgCl2, 3 mM CaCl2, 10 mM glucose, 10 mM Hepes, 0.2% (W/V) BSA or in Ca2+ free buffer  solution; 125 mM NaCl, 2.5 mM KCl, 1.1 mM NaH2PO4, 4 mM NaHCO3, 2 mM MgCl2, 10 mM glucose, 10 mM Hepes, 0.2% (W/V) BSA, 1 mM EGTA (Sigma). EGF solutions of 40 pM and 4 nM  were kept on ice at all times and warmed up to 30°C just before stimulation to avoid degradation of EGF.Mouse EGF (Life Technologies) was diluted in fresh imaging buffer solution , a blocker of Ca2+ indicator in view of the following considerations: i) high quantum efficiency upon binding Ca2+  excitation by light in the blue spectral range, which reduces cell phototoxicity, iii) low affinity for Ca2+  was chosen as CaCells were loaded with 3.5 µM fluo5F-AM in 0.5PBX Signal Enhancer (Becton Dickinson Biosciences) and 0.5 DMEM-f12 medium without phenol red for 20 min at 37°C and 20 min at room temperature. Loaded cells were washed and equilibrated with fresh imaging buffer solution at 30°C. Video time-lapse was started and 25 s after, EGF was applied volume to volume, leading to final concentrations of 20 pM and 2 nM.2+ indicator 2+ transients consisted in acquiring a sequence of 300 images with an integration time of 68 ms, and a 1s interval between consecutive images.Digital fluorescence images were obtained using an inverted Axio Observer.Z1 Zeiss microscope  equipped with an image acquisition system , a metal halide HXP-120 lamp for fluorescence excitation and a GFP fluorescence cube . To reduce photodamage, neutral density filters were introduced in the light path to illuminate samples with 380 µW as measured at the back aperture of the objective. Images were acquired with a CCD camera,  reaching a final pixel size of 322 nm after ×40 magnification and a 2× binning to obtain a good signal to noise ratio with minimal illumination of the cells. Temperature was maintained at 30°C to slow down loss of cytoplasmic CacellF) within a “region of interest” (ROI) covering the cell area (bkgF) in three regions of similar size to the cellular ROIs, which were located in the periphery of the recorded field  = F(t) – F(0)) were calculated (Sigma Plot software) where F(t) is the fluorescence at any time and F(0) the average of the 25 images preceding the addition of EGF. To correct for non-uniformities in dye concentration F(t) was divided by the pre-stimulus fluorescence. Thus, the ratio was: ΔF(t)/F(0) = (F(t) – F(0))/F(0).The changes in fluorescence /F(0)) with a single exponential function (F = 0.78+0.24et) values were normalized with the exponential function F(t)bleach to produce the ΔF/F traces (2+ (Ca2+i).Control experiments were performed for time-dependent dye bleaching over the course of the experiment. In 41 control cells, EGF-free buffer was added . FluoresF traces . This rath) which was defined from the distribution of normalized fluorescence intensity values from the 32 non-responsive control cells subjected to buffer in the absence of EGF: a mean (t0) of 0.047 and a standard deviation (SD) of 0.06 were extracted from a Gaussian fit curve of the values and th was set as t0+3SD  cells responded to 2 nM EGF and 49% (137/281 cells) to 20 pM EGF. Signals described as oscillating in Cell responsiveness to EGF was determined by selecting cells that displayed calcium signals rising above an amplitude threshold  were regarded as noise oscillations around threshold and discarded.To quantify oscillatory properties in single cells  D–G, resKinetics of signals from responsive cells  and 3B w2+ on EGF responses , Gaussian fitting procedure , with p <0.001 considered to be statistically significant. Figures were prepared using SigmaPlot, Igor Pro, and Adobe Illustrator CS5. Unless otherwise stated, data distributions are presented as median values. Box and whisker plots are represented with the first and third quartiles at the ends of the box, the median is indicated by a horizontal line in the box, the 52+i) dynamics induced by EGF in COS-7 cells were studied by measuring changes in fluorescence intensity of the low affinity Ca2+ indicator fluo5F, which faithfully reports kinetics in single living cells 2+i variations were quantified as illustrated in 2+ levels subsequently reaching a plateau  displayed an oscillatory Ca2+ signal  were determined. While the average fluorescence intensity values steadily increased after irrelevant antibodies were added (n = 32 cells), possibly related to an application effect that was also seen immediately after buffer , no noticeable differences were found in the kinetics of the averaged Ca2+ signal , although statistically significant .A statistical comparison of the Caerformed . While aerformed  respondeth . Peak duration and area were evaluated on the first peak elicited after EGF addition to avoid measurement errors on peaks exceeding the recording time frame. Peak durations . Peak areas . However, the medians of the interspike intervals . Overall, quantifications suggest that 2 nM EGF elicit a modestly but significantly larger increase in Ca2+ load than 20 pM EGF, while Ca2+ signal kinetics are not significantly different.To analyze the properties of the oscillatory responses observed in response to 2 nM and 20 pM EGF, we defined a peak as a signal that rises and falls through the intensity threshold th , calculath . Then stth  of the rurations  obtainedak areas  were alsntervals  were not2+ signaling in response to nanomolar EGF has two components: Ca2+ release from the intracellular stores sequentially due to phospholipase (PLC) γ activation, inositol 1,4,5-trisphosphate (IP3) synthesis and IP3 receptor activation, and a net Ca2+ influx from the outer medium due to store-operated channels (SOC) 2+ was omitted from the extracellular medium and 1 mM EGTA was added , most cells still responded to 2 nM EGF (2+o vs 64% n =  28 in 0 mM Ca2+o). Also, single-cell responses appeared very similar whether Ca2+ was present or not in the extracellular medium. Average fluorescence curves . In contrast, responses to 20 pM EGF were totally abolished in the absence of extracellular Ca2+  of the cells responded in 0 mM Ca2+o  channel activity that results in a prolonged membrane potential hyperpolarization + channel activity and of intracellular Ca2+ have been found 2+ oscillation 2+ entry and Ca2+ activation of KCa3.1 channels. Based on these observations, we tested whether Ca2+-activated K+ channels could be involved in the oscillatory Ca2+ signal observed in response to 20 pM and 2 nM EGF. The application of charybdotoxin (chx), a high affinity blocker of Ca2+-activated K+ channels, revealed a clear difference (+ channel blocker vs 73% in the absence of chx (Fisher exact's test p = 0.006).Since oscillating responses represented more than 70% of the responses observed after application of 20 pM EGF , we invefference  I–J in tfference  in the afference  in the p2+ imaging, we characterized Ca2+signals elicited through high affinity EGFRs. These signals were specific for EGFR activation as i) when buffer was applied instead of EGF, negligible Ca2+i variations were seen (ii) the increase in average Ca2+i induced by EGF was inhibited by EGFR-specific neutralizing monoclonal M225 IgGs (Using sensitive Caere seen  and ii) 225 IgGs  [12].2+ signals, we were able to demonstrate the oscillating nature of the EGF-dependent Ca2+i transients  Ca3.1 channel activation in VMSC 2+ activate calmodulin and gate KCa3.1 channels, which are also regulated by class II phosphoinositide-3 kinase  signaling cascade, the central driver of cell proliferation in a PI3K-dependent mode, in this cell type


Genetic studies have demonstrated that GJA8 is involved in maintaining lens opacity and proper lens development. In this study, a cataract model with GJA8 gene knockout was developed via co-injection of Cas9/sgRNA mRNA into rabbit zygotes. Our results showed that gene mutation efficiency in the GJA8 locus reached 98.7% in embryos and 100% in pups, demonstrating that the Cas9/sgRNA system is a highly efficient tool for gene editing in rabbits. In agreement with other studies, our genetic and histology results showed that impaired GJA8 function caused microphthalmia, small lens size and cataracts. In summary, our novel rabbit model of cataracts will be an important drug-screening tool for cataract prevention and treatment.Cataracts are the leading cause of vision loss in the world, although surgical treatment can restore vision in cataract patients. Until now, there have been no adequate animal models for  GJA1, GJA3 and GJA8 play critical roles in transmitting information from highly metabolically active cells to less metabolically active cells. Consequently, these are critical genes in development and function of vertebrate lenses4678GJA8 encodes connexin50 (Cx50) and has abundant expression in the lens, which is necessary for lens growth and maturation of lens fiber cells10Congenital cataracts, characterized by lens opacity, are the leading cause of blindness in childhood2In terms of anatomy and physiology, rabbits are more similar to humans than mice or rats, and they have a lower cost maintenance and shorter gestation period than pigs or monkeys13in vitro transcribed mRNA and CRISPR/Cas9 have been successfully used for genome modifications in several mammalian embryos17Recently, the CRISPR/Cas9 system has been extensively used for gene editing in various organisms, such as mice, rabbits, sheep and pigs151617GJA8 in humans67GJA8 knockout mice displayed recessively inherited cataractsGJA8 inheritance patterns between humans and animals. Because rabbits and humans are similar, the objective of this study was to create a novel animal model that recapitulated human congenital cataracts by using CRISPR/Cas9-mediated GJA8 gene knockout in rabbits. To evaluate this cataract model, we examined the gene editing efficiency of CRISPR/Cas9, the phenotypes and mutant gene heritability.Previous studies have reported that cataracts are commonly caused by dominant gene mutations of GJA8 in rabbits, two sgRNAs targeting the CDS of GJA8 were designed , revealing that CRISPR/Cas9 generated mosaicism was commonly detected in F0 animals  were transferred into the oviducts of two surrogate rabbits . After 3eletions . This re animals . Since tGJA8 (+/−) rabbits when compared to WT counterparts. Photographs showed eight of 11 F0 GJA8 mutated rabbits appeared to develop cataracts with lens opacities  were determined by PCR and T-cloning Sanger sequencing. As shown in T rabbit . Taken tGJA8 (+/−) rabbits showed opacity, smaller lens and obvious microphthalmia  and WT rabbits. According to immunolabeling results, weak fluorescent signals of GJA8 protein were observed in the outermost fiber cells of F1-23 (WT/-52) rabbit, but stronger punctate signals were seen in WT rabbits  mice appeared to have normal eyes and lenses, while only GJA8 (−/−) mice developed microphthalmia with smaller lenses compared to WT miceGJA8 (+/−) rabbits developed cataracts had microphthalmia and smaller lenses. These data were consisted with the clinical data of human GJA8 cataract phenotypes, due to the dominant gene mutations56GJA8 (+/−) rabbits have dominant cataracts, which was more similar to human cataracts than the mouse in inheritance pattern. Yet, not all these rabbit pups in the F0 or F1 generations exhibited the cataract phenotype. The different lengths of deletion fragments could possibly cause these different lens phenotypes. According to the genotype analysis, cataracts were not observed in rabbits with deletion fragment lengths that were multiples of 3, such as F0-4 (WT/-57bp), F0-6 (WT/-51bp) and F0-10 (WT/-51bp)  rabbits in the F0 generation. These results suggested that GJA8 transcription was not terminated but produced a mutated protein. The 3D structure indicating that these mutated protein structures of GJA8 were unstable in the rabbits  and purified using an RNeasy Mini Kit (Qiagen).3x FLAG-NLS-SpCas9-NLS was synthesized and cloned into the Pcs2+ vector (Addgene ID 48137). The recombinant Cas9 expression vector was linearized with http://crispr.mit.edu/. Two complementary DNA oligonucleotides were annealed at 95 °C for 5 min to generate double-stranded DNA. Then, DNA was cloned into the BbsI-linearized pUC57-T7 vector (Addgene ID 51306). The sgRNAs oligonucleotides sequences targeting GJA8 were listed in The sgRNAs were designed according to the following website: 3 , 0.750 g Hepes , 0.05 g penicillin, 0.06 g streptomycin, 1.755 g NaCl, 3.0 g BSA, and 1 L Milli Q H2O. A mixture of Cas9 and sgRNA mRNA  was microinjected into the embryo cytoplasm to knock out the GJA8 gene. The injected embryos were transferred into EBSS medium for short-term culture at 38.5 °C, 5% carbon dioxide and humidity conditions. 30–50 injected embryos were transferred into the oviduct of a recipient rabbit.The female New Zealand White rabbits (6–8 months old) were superovulated with FSH (60 IU) at intervals of 12 h for 6 times. After the last injection, female rabbits were mated with the male rabbits. Females then received a 100 IU human chorinonic gonadotrophin (HCG) injection. At 18 h post HCG injection, female rabbits were euthanized, and oviducts were flushed with 5 ml DPBS-BSA for zygotes collection. Rabbit embryos at the pronuclear stage (around 18–20 h post-mated) were collected and transferred into oocyte manipulation medium, which contained 9.5 g TCM-199, 0.05 g NaHCOGJA8 knockout (KO) rabbit pups was isolated using the TIANamp Genomic DNA Kit . The DNA was amplified with 2×Taq Plus MasterMix (TIANGEN) and PCR primers used to detect mutation were as follows: F- 5′ CGAGAATGTCTGCTACGATGAG- 3′, and R- 5′ CCGGAAACCATACAGGAAGTAG- 3′. The PCR products were purified and cloned into the pGM-T vector , and then analyzed by Sanger sequencing. The colonies were picked and sequenced to confirm exact gene mutations.The injected embryos were collected at the blastocyst stage. Genomic DNA was extracted with an embryo lysis buffer (1% NP40) at 50 °C for 20 minutes and 90 °C for 5 minutes in a BIO-RAD PCR Amplifier. Genomic DNA from wild type (WT) and http://crispr.mit.edu/). The PCR products of the POTS were sequenced and confirmed by T7E1 enzyme digestion, as previously describedSeven potential off-target sites (POTS) for each sgRNA were predicted to analyze site-specific cleavage by the CRISPR/Cas9 system according to an online design tool  and viewed under a Nikon ts100 microscope.Eyes tissues from WT and GJA8 polyclonal antibody  was used to measure protein levels. Anti-Beta actin monoclonal antibody  was used as an internal control.Imediately after rabbits were euthanized, the eyes were ground under liquid nitrogen. The powder of eye tissues was lysed in a protein lysis buffer on ice for 30 min. Protein concentrations were measured using the BCA Protein Assay Kit (Beyotime). Anti-GJA8 mutant proteins were built from their amino acid sequences according to the web site: http://swissmodel.expasy.org/ 3 D structure models of the WT and GJA8 KO rabbits were fixed in 4% paraformaldehyde for 48 h, embedded in paraffin wax and sectioned for slides. Thin sections were de-waxed with dimethylbenzene and dehydrated using an ethanol gradient . The slides were placed in 0.1% Triton solution at 4 °C for 1 h. Slides were blocked with 5% goat serum for half an hour at 37 °C and rinsed with PBS 3 times. Slides were incubated with primary antibody  at 4 °C for overnight. Slides were then incubated with and anti-rabbit secondary antibody  in 5% goat serum for 1 h at room temperature in the dark. Finally, cell nuclei were counter stained with hochest 33342 for 7 min in the dark. Confocal laser microscopy was used to examine the GJA8 fluorescence.Eye lenses from WT and GJA8 KO eye lenses were cut into small pieces, and fixed in 0.1 M cacodylate buffer  for 2–4 hours at room temperature. As previous study described4, tannic acid, and uranyl acetate. Then, they were embedded in Epon. Ultrathin sections were cut with a diamond knife, mounted on copper grids and viewed under an electron microscope (Hitachi H-7650) at 80 Kv.For morphological analysis by electron microscopy, WT and in vitro embryo development in the 2-cell, morular and blastocyte groups was compared by chi-square test. A value of p < 0.05 was considered statistically significant.Percentage for How to cite this article: Yuan, L. et al. CRISPR/Cas9-mediated GJA8 knockout in rabbit recapitulates human congenital cataract. Sci. Rep.6, 22024; doi: 10.1038/srep22024 (2016).


Bradykinin may play a role in the autodigestive disease acute pancreatitis, but little is known about its pancreatic actions.2+ signal generation in normal mouse pancreatic lobules.In this study, we have investigated bradykinin‐elicited Ca2+ signalling between pancreatic acinar (PACs) and stellate cells (PSCs). Pathophysiologically relevant bradykinin concentrations consistently evoked Ca2+ signals, via B2 receptors, in PSCs but never in neighbouring PACs, whereas cholecystokinin, consistently evoking Ca2+ signals in PACs, never elicited Ca2+ signals in PSCs.We found complete separation of Ca2+ signals were due to initial Ca2+ release from inositol trisphosphate‐sensitive stores followed by Ca2+ entry through Ca2+ release‐activated channels (CRACs). The Ca2+ entry phase was effectively inhibited by a CRAC blocker.The bradykinin‐elicited CaB2 receptor blockade reduced the extent of PAC necrosis evoked by pancreatitis‐promoting agents and we therefore conclude that bradykinin plays a role in acute pancreatitis via specific actions on PSCs.2+ signals. We have compared Ca2+ signalling in PSCs and their better studied neighbouring acinar cells (PACs) and found complete separation of Ca2+ signalling in even closely neighbouring PACs and PSCs. Bradykinin (BK), at concentrations corresponding to the slightly elevated plasma BK levels that have been shown to occur in the auto‐digestive disease acute pancreatitis in vivo, consistently elicited substantial Ca2+ signals in PSCs, but never in neighbouring PACs, whereas the physiological PAC stimulant cholecystokinin failed to evoke Ca2+ signals in PSCs. The BK‐induced Ca2+ signals were mediated by B2 receptors and B2 receptor blockade protected against PAC necrosis evoked by agents causing acute pancreatitis. The initial Ca2+ rise in PSCs was due to inositol trisphosphate receptor‐mediated release from internal stores, whereas the sustained phase depended on external Ca2+ entry through Ca2+ release‐activated Ca2+ (CRAC) channels. CRAC channel inhibitors, which have been shown to protect PACs against damage caused by agents inducing pancreatitis, therefore also inhibit Ca2+ signal generation in PSCs and this may be helpful in treating acute pancreatitis.Normal pancreatic stellate cells (PSCs) are regarded as quiescent, only to become activated in chronic pancreatitis and pancreatic cancer. However, we now report that these cells in their normal microenvironment are far from quiescent, but are capable of generating substantial Ca Bradykinin may play a role in the autodigestive disease acute pancreatitis, but little is known about its pancreatic actions.2+ signal generation in normal mouse pancreatic lobules.In this study, we have investigated bradykinin‐elicited Ca2+ signalling between pancreatic acinar (PACs) and stellate cells (PSCs). Pathophysiologically relevant bradykinin concentrations consistently evoked Ca2+ signals, via B2 receptors, in PSCs but never in neighbouring PACs, whereas cholecystokinin, consistently evoking Ca2+ signals in PACs, never elicited Ca2+ signals in PSCs.We found complete separation of Ca2+ signals were due to initial Ca2+ release from inositol trisphosphate‐sensitive stores followed by Ca2+ entry through Ca2+ release‐activated channels (CRACs). The Ca2+ entry phase was effectively inhibited by a CRAC blocker.The bradykinin‐elicited CaB2 receptor blockade reduced the extent of PAC necrosis evoked by pancreatitis‐promoting agents and we therefore conclude that bradykinin plays a role in acute pancreatitis via specific actions on PSCs. This effect can be abolished by phospholipase C inhibition or blockade of IP3 receptors (IP3Rs). Following the initial intracellular Ca2+ release, there is opening of conventional CRAC channels : NaCl, 140; KCl, 4.7; Hepes (KOH), 10; MgCl2, 1; glucose 10; CaCl2 1; pH 7.2. Pancreatic lobules were then incubated with fluorescent dye following the manufacturer's description. All experiments were carried out with freshly prepared pancreatic lobules, attached to the coverslip of the perfusion chamber at room temperature (23°C). Penetration of various substances deep into the pancreatic lobule was highly dependent on the distance from the surface . It was therefore necessary, in several cases, to use relatively high concentrations  and relatively long pre‐incubation times.Pancreatic lobules and big clusters were isolated from the pancreas of adult C57Bl/6 male mice. Animals were killed according to UK Schedule 1 regulations. The pancreas was rapidly dissected, transferred to collagenase Na‐Hepes‐based solution  and incubated at 37°C for 5–6 min. After digestion, the tissue was kept in Na‐Hepes‐based extracellular media, containing (in mm Fluo‐4 acetoxymethyl ester (AM), for 10 min at room temperature. Cells were transferred to a flow chamber and perfused with the Na‐Hepes‐based extracellular solution as described above. Cells were visualized using a Leica SP5 MPII two‐photon confocal microscope, with a 63× 1.2 NA objective lens. Fluo‐4 was excited with a 488 nm Ar laser, at 1–4% power, and emitted light was collected at 510–580 nm. Generally, a series of images was recorded at 512×512 pixel resolution (at a speed of 0.3 frames s−1), and analysed using Leica Confocal Software . Fluorescence signals were plotted as F/F0 . Statistical analysis was performed using ANOVA or Student's t‐test.Intact cells were loaded with 5 μm ethanol, or 500 μm palmitoleic acid ethyl ester (POAEE) or 0.5% sodium choleate for 2 h in the presence or absence of 0.5 μm or 10 μm WIN 64338. Ethanol‐ or bile‐induced pancreatic necrosis was visualized by staining the lobules with propidium iodide (PI) and compared with the control necrosis level (without any treatment). Simultaneously lobules were stained with the nuclear dye Hoechst 33342 to enable cell counting. Both stainings were performed according to the manufacturer's protocols. Fluorescence of Hoechst 33342 and PI was recorded using confocal microscopy, with a 63× 1.2 NA objective lens .Pancreatic lobules were exposed to 350 met al.  and then with secondary (CFL) antibodies as described above.Immunocytochemistry in pancreatic lobules and big clusters was performed as described by Lur et al.  with somChemicals, unless otherwise indicated, were obtained from Sigma or Calbiochem . BK, R‐715 and WIN64338 were purchased from Tocris Biosciences . Fluo‐4 AM, PI and Hoechst 33342 were purchased from Invitrogen . Antibodies against CCK‐A (CCK1) receptor (sc‐16172), desmin antibody (sc‐7559) and donkey anti‐goat IgG‐CFL 594 (sc‐362275) were from Santa Cruz Biotechnology .et al. A–C). The presence of vitamin A in lipid droplets is another characteristic of PSCs .It has been shown that desmin is a good marker for PSCs (Apte C) took up the Ca2+‐sensitive fluorescent probe Fluo‐4 much more avidly than PACs . In large lobules stained with Fluo‐4 , it was often possible to observe PSCs as bright ‘strings’, whereas in smaller cell clusters, they typically appeared as individual bright cells at the periphery of acinar units .Cells situated in the position characteristic of desmin‐containing cells Fig. C took uACs Fig. A–C. In o‐4 Fig. A, B, itits Fig. Ca.2+ signals in response to a micromolar concentration of BK  with a sharp transient rise in [Ca2+]i, quickly followed by a longer lasting (plateau) phase of elevated [Ca2+]i . Neighbouring PACs never displayed any change in [Ca2+]i during the BK‐induced PSC Ca2+ signals (>100 experiments) , indicating lack of functional BK receptors on PACs and lack of direct communication between neighbouring PACs and PSCs. The typical, and physiologically important, PAC stimulants ACh and CCK evoked Ca2+ signals in PACs  . ATP (100 μm) elicited Ca2+ signals in a proportion of PSCs (41 of 107 cells), but failed to do so in many others . When ATP evoked a [Ca2+]i rise in a PSC, it was never transmitted to the neighbouring PAC .Cultured PSCs produce cytosolic Caet al.  and Ca2+ signals in PSCs consistently failed to be transmitted to neighbouring PACs . Furthermore, although a fluorescent antibody to CCK1 receptors clearly identified the presence of CCK1 receptors on PACs, it failed to do so with regard to PSCs (four experiments) .Phillips et al.  have sugACs Fig. A. Furthts) Fig. B.2+ signals  could elicit PSC Ca2+ signals and that maximal responses were obtained at 1 nm . Overall, in the course of this investigation, BK responses to 1 nm have been observed in >100 experiments. At the low BK concentrations we used, there was no sign of desensitization . Within the time frame of our protocols, the [Ca2+]i elevation was maintained as long as the stimulus was maintained (n = 19) . It would therefore appear that normal PSCs are sufficiently sensitive to BK to be able to sense relatively small increases in the surrounding BK level.It seemed important to explore the levels of BK needed to evoke Caals Fig.  in relation Fig. A. Withi19) Fig. A. It woet al. n = 6)  or trypsin (n = 18)  in cells that responded to BK with a clear Ca2+ signal.Although the PSCs in our lobule preparation were much more sensitive to BK than those previously studied in culture  . In contrast, the B1 receptor blocker R‐715  . Furthermore, the specific B1 agonist Sar‐des‐Arg‐BK did not evoke any changes in [Ca2+]i of PSCs (n = 8) . It would appear that the plateau phase of the BK‐induced response to some extent depends on continued B2 receptor activation, because application of WIN64338 immediately after the initial BK‐elicited Ca2+ spike shortened the duration of the plateau phase .The BK receptor sub‐type responsible for generating Caion n = 2 , POAEE (500 μm) or bile acids .As a previous study showed that BK levels increase in acute pancreatitis and that blockade of B2 receptors attenuated the cellular changes underlying the development of acute pancreatitis evoked by obstruction of the pancreatico‐biliary duct in rats . Re‐admission of Ca2+ resulted in a transient increase in [Ca2+]i and enabled a subsequent BK application to evoke a normal response . We tested the ability of PSCs to generate store‐operated Ca2+ entry, by using the now ‘classical’ protocol for assessing this phenomenon , employing cyclopiazonic acid (CPA) to block the Ca2+ pumps in the endoplasmic reticulum (ER). As shown in Fig. B and C, re‐admission of external Ca2+ after a period of external Ca2+ deprivation, and BK stimulation, resulted in a transient rise in [Ca2+]i, but, if the Ca2+ re‐admission occurred after and during a period of continued ER Ca2+ pump inhibition, the [Ca2+]i elevation was sustained . In this situation, Ca2+ entering store‐operated Ca2+ channels cannot be taken up into the ER, but will only slowly be extruded by the plasma membrane Ca2+ pumps.As shown in Figs ase Fig. A, B. Rense Fig. A, B. Wenon Fig. C, emploned Fig. C. In th2+ entry pharmacologically. 2‐APB, a well‐known, but relatively unspecific, blocker of CRAC channels , which may be due to blockade of IP3Rs . GSK‐7975A also blocked the Ca2+ entry normally occurring when external Ca2+ was re‐admitted after a period of external Ca2+ deprivation (n = 14) .We tried to block the store‐operated Caion Fig. D, whichpike n = , we tested the hypothesis that this Ca2+ liberation occurs through IP3Rs. It is now well stablished that caffeine inhibits opening of IP3Rs . BK probably activates phospholipase C, as U73122  .Finally, we examined the mechanism underlying the initial Cares Fig. A, we teing n = 2. BK probet al. et al. 2+ signals in normal PSCs in their normal micro‐environment and therefore cast doubt on the hitherto prevailing concept of the quiescent PSC . Therefore, our results do not provide support for the hypothesis that CCK acting on PSCs will increase ACh secretion from these cells, which in turn would activate PACs . Given that B2 receptor blockade protects against pancreatitis‐like cellular changes .2+ signals in pancreatic stellate cells may influence negatively the outcome of acute pancreatitis. We tested the hypothesis that blockage of bradykinin type 2 (B2) receptors would offer protection against the pancreatic acinar cell necrosis caused by pancreatitis‐inducing agents, such as alcohol, fatty acid ethyl esters or bile acids. The results showed that specific B2 receptor blockade markedly reduced the extent of necrosis observed after treatment with ethanol, POAEE or a mixture of bile acids. This suggests that B2 receptor blockade in the early stage of acute pancreatitis may be helpful in reducing the severity of the disease. We also show that the bradykinin‐elicited sustained elevation of the cytosolic Ca2+ concentration in pancreatic stellate cells can be inhibited by a specific inhibitor of Ca2+ release – activated Ca2+ (CRAC) channels. We have recently shown that CRAC inhibition in PACs offers remarkable protection against necrosis induced by fatty acid ethyl esters. In the intact pancreas, CRAC inhibition would reduce excessive Ca2+ signal generation both in the acinar cells  and in the stellate cells (brought about by bradykinin). Both effects would be beneficial. Our work therefore indicates that combined treatment with a CRAC inhibitor and a B2 receptor – blocking agent should be tested in vivo as a potentially useful therapy against acute pancreatitis.Our work indicates that bradykinin‐elicited Ca


In vitro application of 100 nM lyso-Gb3 caused uptake of extracellular calcium in 10% of sensory neurons expressing nociceptor markers, rising to 40% of neurons at 1 μM, a concentration that may occur in Fabry disease patients. Peak current densities of voltage-dependent Ca2+ channels were substantially enhanced by application of 1 μM lyso-Gb3. These studies suggest a direct role for lyso-Gb3 in the sensitisation of peripheral nociceptive neurons that may provide an opportunity for therapeutic intervention in the treatment of Fabry disease-associated pain.Fabry disease is an X-linked lysosomal storage disorder characterised by accumulation of glycosphingolipids, and accompanied by clinical manifestations, such as cardiac disorders, renal failure, pain and peripheral neuropathy. Globotriaosylsphingosine (lyso-Gb3), a deacylated form of globotriaosylceramide (Gb3), has emerged as a marker of Fabry disease. We investigated the link between Gb3, lyso-Gb3 and pain. Plantar administration of lyso-Gb3 or Gb3 caused mechanical allodynia in healthy mice.  The noxious stimulus is detected by peripheral damage-sensing neurons (nociceptors). The depolarisation of the nociceptor is usually linked to an increase in cytosolic Ca22.1All experiments were approved by the UCL ethics board with prior approval by the UK home office. C57/BL6 mice  were treated in accordance with the UK Animal (Scientific Procedures) Act 1986.2.2Mechanical withdrawal thresholds were measured by applying von Frey hairs  to the plantar surface of the hindpaw. The 50% paw withdrawal threshold was calculated using the up-and-down method 2.3l-lysine and 0.02 mg/ml laminin pre-coated either 15 mm cover slips  or 35 mm petri dishes (for whole-cell patch clamp recording).DRG from all spinal levels were dissected from adult 7–8 week old mice and cultured as previously described 2.42+ recording buffer consisting of (in mM) 140 NaCl2, 4 KCl, 2CaCl2, 1 MgCl2, 10 HEPES, 10 glucose adjusted to pH 7.4 and osmolality 320 mOsm/l using NaOH and sucrose, respectively. Cells were incubated in recording buffer with 2.5 μM Fura-2AM  for 30 min at room temperature. In some experiments, cells were pre-treated with 5 μg/l IB4-FITC  in culture medium at 37 °C for 20 min before Fura-2AM loading. Cells were washed twice with Ca2+ recording buffer for 5 min each time at room temperature to remove excess Fura-2AM. A coverslip of cells was placed over the window of a SA-NIK stage adapter (Warner Instruments). The perfusing chamber was tightened onto the adapter, with vacuum grease silicone  to enhance the seal. 0.5 ml of Ca2+ recording buffer was loaded onto the perfusing chamber before the whole assembly was mounted onto the stage of a Nikon Eclipse TE300 microscope. The inlet tube of a perfusing system  was connected to the recording chamber, and the speed of superfusate was set to 250 μl/min. Images were obtained using either a 20× or 40× (oil immersion) Nikon objective, equipped with fluorescent filter sets. A Till polychrome II monochromator was used to excite the fluorescence of Fura-2 at 340 nm and 380 nm, and data were acquired using OptoFluor software . Fura-2AM emission was collected at 505 nm.Plated DRG cells were washed with Ca2.52, 1 CaCl2, 4 MgCl2, 10 HEPES–Na, 10 EGTA, 4.5 MgATP and 0.4 GTP–Na, adjusted to pH 7.3 and osmolarity 300 mOsm/l using CsOH and sucrose, respectively. Recordings were performed in voltage-clamp mode and currents were low-pass filtered at 5 kHz and sampled at 10 kHz. Values for series resistance (Rs) and capacitance (Cm) were read directly from the amplifier after subtraction of capacitative transients. Series resistances were compensated to the maximum extent possible  and voltages were not corrected for liquid junction potentials. External solution consisting of (in mM) 162.5 tetraethylammonium (TEA)–Cl, 5 CaCl2, 1 MgCl2, 10 glucose and 10 HEPES with pH adjusted to 7.4 using TEA–OH was used to isolate calcium currents. Lyso-Gb3 was applied to cells via a perfusing system and the speed of perfusing solution was set at 1 ml/min. Only small-diameter DRG neurons (with capacitance less than 25 pF) were chosen for recording. Off-line data analysis was performed using ClampFit v9.0 software .Whole-cell patch-clamp recordings were performed at room temperature using an Axopatch 200B amplifier and Digidata board 1320A controlled by pClamp v9.0 software . Recording pipettes were pulled from borosilicate glass microcapillaries  with a range of pipette resistances between 2 and 4 MΩ after filling with intracellular solution consisting of (in mM) 146 CsClDrugs stocks of Fura-2AM  and lyso-Gb3  were dissolved in DMSO and HBSS containing 2 mM Ca2+ and 1 mM Mg2+, respectively. Stocks of capsaicin  were prepared in ethanol. For intraplantar administration, Gb3 or lyso-Gb3  were prepared in saline. 50 mM KCl was prepared in Ca2+ recording buffer on the same day for use.2.6post-hoc test when statistical significance was observed. Comparison between two groups was performed with independent samples T-test, and a Student’s paired T-test was used to compare two different conditions in the same cell unless otherwise stated. Data were presented as the mean ± SEM. All statistical analyses were performed using Prism 5 for Windows.One way ANOVA was followed by a Bonferroni 33.1P < 0.05). The increased sensitivity lasted up to 6 h. All the mice completely recovered from the acute mechanical allodynia 24 h post injection.We administered 20 μl of 30 μM Gb3 or lyso-Gb3 to the hind paws of wild type mice and tested sensitivity at different time points. As seen in 3.22+ of DRG neurons. Increased Ca2+ levels were observed following lyso-Gb3 application at concentrations higher than 0.1 μM  evoked a depolarization-mediated rise in intracellular Ca2+ level in every tested neuron  and during application of lyso-Gb3 by eliciting currents with a set of 250-ms voltage steps from −60 to +50 mV (Vt) in 5 mV increments (holding potential: –70 mV, see 2+ currents were significantly augmented in small-diameter DRG neurons at most of the tested membrane potentials (2+ currents was significantly increased from Vt −15 mV to +35 mV (n = 8/membrane potential; P < 0.05; Next we examined whether the lyso-Gb3-evoked increases in cytoplasmic Catentials A and B. 42+ levels associated with the functional upregulation of voltage-activated Ca2+ channels.This study investigated a potential link between high levels of the Fabry lipid lyso-Gb3 and pain. In healthy mice administration of lyso-Gb3 induced mechanical allodynia. In peptidergic DRG neurons, lyso-Gb3 evoked an increase in intracellular Ca2+ levels in a subpopulation of DRG neurons that express markers associated with damage sensing. The origin of excessive lysoGb3 is not clearly identified, although drugs that stabilise the GLA enzyme also reduce the levels of lyso-Gb3 in plasma Plasma circulation of lyso-Gb3 and the concomitant lysosomal accumulation of Gb3 have been proposed to lead to pain in Fabry disease 2+ influx may cause Ca2+-dependent excitotoxicity 2+ rises in small-diameter DRG neurons of the IB4 nonbinding, peptidergic sub-class.This study is the first to show a direct link between lyso-Gb3, intracellular changes in peripheral sensory neurons and pain. The pain syndrome in Fabry disease was thought to arise from small fibre dysfunction, such as length dependent neuropathy which is typically linked to a loss or decrease of myelinated Aδ fibres and unmyelinated C fibres 2+ signalling regulates many cellular functions. In the present study, 1 μM lyso-Gb3, a possible concentration reported in patients with Fabry disease 2+ influx. Further, the lipid enhanced voltage-dependent Ca2+ current densities, indicating functional upregulation of voltage-dependent Ca2+ channels as a plausible mechanism of functional changes in peripheral nociceptors. Voltage-dependent Ca2+ channels in DRG neurons play a role in transmission of nociceptive signals from the periphery, and neuropathic manifestations including increased pain sensitivity have also been shown to be associated with increased Ca2+ entry in disease states, such as diabetes Intracellular Ca2+ influx mediated by upregulated voltage-dependent Ca2+ channels in nociceptive DRG neurons, following lyso-Gb3 administration. This study shows that Fabry lipids may cause pain through direct actions on sensory neurons, and that promoted Ca2+-dependent excitability of nociceptors is a possible mechanism. These observations support further investigation of the effects of Fabry-disease associated lipids on ion fluxes in sensory neurons and their potential significance in the pain associated with this condition.This is the first report showing the onset of mechanical allodynia, and augmented Ca


Roseobacter denitrificans is a member of the widespread marine Roseobacter genus. We report the first characterization of a truncated hemoglobin from R. denitrificans (Rd. trHb) that was purified in the heme-bound form from heterologous expression of the protein in Escherichia coli. Rd. trHb exhibits predominantly alpha-helical secondary structure and absorbs light at 412, 538 and 572 nm. The phylogenetic classification suggests that Rd. trHb falls into group II trHbs, whereas sequence alignments indicate that it shares certain important heme pocket residues with group I trHbs in addition to those of group II trHbs. The resonance Raman spectra indicate that the isolated Rd. trHb contains a ferric heme that is mostly 6-coordinate low-spin and that the heme of the ferrous form displays a mixture of 5- and 6-coordinate states. Two Fe-His stretching modes were detected, notably one at 248 cm-1, which has been reported in peroxidases and some flavohemoglobins that contain an Fe-His-Asp (or Glu) catalytic triad, but was never reported before in a trHb. We show that Rd. trHb exhibits a significant peroxidase activity with a (kcat/Km) value three orders of magnitude higher than that of bovine Hb and only one order lower than that of horseradish peroxidase. This enzymatic activity is pH-dependent with a pKa value ~6.8. Homology modeling suggests that residues known to be important for interactions with heme-bound ligands in group II trHbs from Mycobacterium tuberculosis and Bacillus subtilis are pointing toward to heme in Rd. trHb. Genomic organization and gene expression profiles imply possible functions for detoxification of reactive oxygen and nitrogen species in vivo. Altogether, Rd. trHb exhibits some distinctive features and appears equipped to help the bacterium to cope with reactive oxygen/nitrogen species and/or to operate redox biochemistry. Roseobacters . Th. ThFe-Hirequency ,67,68. Ioxidases , which ilignment , we noteRd. trHb  cannot bobacters , suggestal model . The lowrin ring , a line (CβCaCb)  Table 2Fe-His inRd. trHb is 6-coordinate and low-spin with ν3 at 1496 cm-1 and ν2 at 1582 cm-1. The relatively high-frequency of the oxidation state marker ν4 at 1373 cm-1 is typical of Fe(II)CO complexes of heme proteins and is indicative of π-back-bonding from the reduced iron to CO. Even at relatively low power (less than 2 mW), a small amount of photo-dissociation of CO occurred as revealed by the detection of the ν4 (~1356 cm-1) and ν3 (1471 cm-1) lines of a 5-coordinate reduced state (FeII). These band increased in intensity at higher laser power (13 mW) and decreased at lower intensity (0.5 mW) indicating that this process was reversible (data not shown). The frequency of the ν4 line of the photo-dissociated reduced state (1356 cm-1) differed somewhat from that of the equilibrium reduced state (1362 cm-1) suggesting that only the 5-coordinate state may be present if the sixth ligand to the 6-coordinate state observed in the equilibrium sample had not formed yet.Rd. trHb (Fe-CO) was identified at 509 cm-1 (496 cm-1 with 13C18O). The frequency of the νFe-CO mode remained the same (509 cm-1) for spectra recorded at 0.5 mW (not shown) and 2 mW laser power (Rd. trHb is lower than that of wild-type Mt. trHbO (525 cm-1)  and compared the results with those of the well characterized HRP  and approximately one order of magnitude lower kcat,app (apparent kcat) and kcat,app/Km,app values compared to HRP (Rd. trHb (50 nM) was also much higher than that of bovine Hb (77.5 nM)  (Inset in Mt. trHbO (1.4 x 103 M-1s-1) and Tf. trHbO (1.9 x 103 M-1s-1). This slope is also a measure of the apparent kcat/Km when the concentration of the variable substrate is small relative to Km which reduces the Michaelis-Menten equation to V = Vmax/Km or k = kcat/Km when the initial rates are divided by the concentration of enzyme. Such an analysis gives a kcat/Km ratio of 3.0 x 104 M-1s-1 which is consistent with the kcat/Km ratio calculated from the apparent kcat and Km values  , which iubstrate ,79. To cf. trHbO ,79. ThisInset in  comparedm values . Thus, oRd. trHb, we performed steady-state kinetic measurements with H2O2/ABTS in buffers of different pHs (pH 6 to 10.5) . Data fitted with equations Ka value: 6.5 ± 0.2 , 7.0 ± 0.3  and 6.8 ± 0.2  , is observed in kcat,app, Km,app and kcat,app/Km,app. If the pKa value of ~6.8 represents the pKa of the proximal histidine of Rd. trHb, it could explain that a significant fraction of the protein displayed a proximal histidine with imidazolate character as detected from the resonance Raman spectrum collected in a buffer at pH 8.0  . The perld range . The kcigher pH . Neverth smaller . The smaKm,app) . Thus, at pH 8.0 .Rd. trHb displays clear differences of its active site compared to Mt. and Tf. trHbO, it shares with the latter a significant peroxidase activity (see above), and thus the ability to oxidize an organic substrate using hydrogen peroxide [katG gene), which has been detected in several Roseobacters [gpo gene), which is known to detoxify ROS in aerobic organisms [Rd. trHb in vivo using gene expression experiments  and the transcript levels of glbO (RD1_4240), katG (RD1_2195) and gpo (RD1_0599) were examined during the exponential growth phase. Significant up-regulation of the gpo and katG genes, along with moderate increment of glbO, were observed in the cultures exposed to H2O2, compared with cultures without H2O2, implying potential detoxification of reactive oxygen via Rd. trHb in vivo.Although peroxide . Meanwhiobacters ,82,83, mobacters ,86,87,88rganisms . Herein,eriments . R. deniM. tuberculosis was proposed to use Mt. trHbN to detoxify NO produced by macrophages [Mt. trHbO was shown to have a 1,200-fold reduced NO dioxygenase activity compared with Mt. trHbN making it unlikely that it is involved in such a reaction [Rd. trHb distinguishes this protein. However, the imidazolate character of the Fe-His stretching mode of flavohemoglobins and the single chain hemoglobin Cgb of C. jejuni, which is believed to favor their NO dioxygenase activity, is a feature shared by Rd. TrHb [Mycobacterium lepreae is involved in both H2O2 and NO scavenging [2O2 would lead to the formation of a ferryl species that would then react with NO to produce nitrite. Such a reaction bypasses the need of a reductase to reduce the heme and to allow oxygen binding as occurs in flavohemoglobins as they carry out the NO dioxygenase reaction. The relatively efficient reaction with peroxide catalyzed by Rd. trHb discussed above may be relevant to such a NO detoxification reaction.Different globins are known to possess a NO dioxygenase activity that converts NO to harmless nitrate from the reaction of the oxygenated state of these protein with NO ,89,90,91rophages , whereasreaction . It was reaction . The sinRd. TrHb . Interesavenging . For thiR. denitrificans. While aerobic denitrification is no longer recognized as an uncommon process in nature [R. denitrificans has not yet been shown to grow under strictly denitrification condition [R. denitrificans cannot grow aerobically without the supply of nitrate (data not shown), the biological and physiological roles of its aerobic denitrification activity, in addition to consuming excess reducing equivalents to minimize the production of ROS [glbO and genes involved in denitrification, biomass production and carbon assimilation are stimulated by light, whereas genes involved in carbohydrate metabolism and the TCA cycle are repressed. These observations are in agreement with the fact that Roseobacters perform photosynthesis during phototrophic growth and obtain energy from the oxidation of organic carbons via aerobic respiration during chemotrophic growth [cd1 complex) [An enzyme with a NO detoxification activity, either through a NO dioxygenase reaction or through a reaction involving a ferryl heme and NO, would be helpful during aerobic denitrification in n nature , R. deniondition  and thusn of ROS , remainsc growth . Moreovecomplex)  and acticomplex) , suggestc oxidase activity and thus could down-regulate aerobic respiration, as reported in mitochondria [R. denitrificans breaks down sugars to produce pyruvate molecules and synthesizes ATP, NADH and NADPH to preserve energy [Roseobacters have been suggested recently to be the major source of ROS in ocean [R. denitrificans produces more trHb to convert excess NO and O2 to nitrate/nitrite during phototrophic growth. A similar mechanism was also suggested recently to link the activity of THB1, which is a trHb from Chlamydomonas reinhardtii that exhibits NO dioxygenase activity, to nitrogen assimilation in this organism [NO produced by aerobic denitrification may function as a signal transducer to inhibit the cytochrome chondria . NO is achondria . The late energy . Alternae energy , which ce energy  and Rosein ocean . ROS areorganism . This hyRoseobacters. In this paper, we characterized the recombinant trHb from R. denitrificans using functional, spectral and modeling studies. The sequence alignment suggests that Rd. trHb belongs to group II trHbs and contains distal amino acids known to participate in ligand stabilization in this group of heme proteins. Resonance Raman spectra reveal a heme pocket structure consistent with the very significant peroxidase activity measured by steady-state kinetics with Rd. trHb. Together, these properties suggest a role for Rd. trHb in mechanisms of defense against reactive oxygen and/or nitrogen species or in redox biochemical reactions that remain to be explored further. Gene expression analyses also suggest that Rd. trHb contributes the detoxification of reactive oxygen and nitrogen species in vivo. Current genomic sequence data indicate that trHbs genes are widely distributed in the ecologically important marine Roseobacters. Some of the distinctive properties reported in this paper set the stage for further in-depth analyses of this interesting enzyme.Hemoglobins are known to bind various gas molecules and some of them also possess enzymatic activities. Analysis using bioinformatic tools suggest that putative globin-like proteins belonging to the truncated globin family are present in S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.


How this influx relates to steady-state contractions elicited by α1-adrenoreceptor stimulation and how it is modulated by small variations in resting membrane potential (Vm) of VSMC is not clear yet. Here, we show that α1-adrenoreceptor stimulation of aortic segments of C57Bl6 mice with phenylephrine (PE) causes phasic and tonic contractions. By studying the relationship between Ca2+ mobilisation and isometric tension, it was found that the phasic contraction was due to intracellular Ca2+ release and the tonic contraction determined by Ca2+ influx. The latter component involves both Ca2+ influx via VGCC and via non-selective cation channels (NSCC). Influx via VGCC occurs only within the window voltage range of the channel. Modulation of this window Ca2+ influx by small variations of the VSMC Vm causes substantial effects on the contractile performance of aortic segments. The relative contribution of VGCC and NSCC to the contraction by α1-adrenoceptor stimulation could be manipulated by increasing intracellular Ca2+ release from non-contractile sarcoplasmic reticulum Ca2+ stores. Results of this study point to a complex interactions between α1-adrenoceptor-mediated VSMC contractile performance and Ca2+ release form contractile or non-contractile Ca2+ stores with concomitant Ca2+ influx. Given the importance of VGCC and their blockers in arterial stiffening and hypertension, they further point toward an additional role of NSCC (and NSCC blockers) herein.L-type Ca Tension was measured simultaneously, acquired at 1 Hz and reported in mN mm-1 i from 0.91±0.03 to 0.81±0.02 RU  and of basal tension from 0.52±0.02 to 0.40±0.05 mN/mm . Application of 1 μM PE in 0Ca induced transient parallel [Ca2+]i and isometric tension increase . Peak PE-elicited tension of 0.72±0.09 mN/mm was significantly decreased compared with the peak tension of 1.13±0.15 mN/mm (p<0.05) in the presence of Ca2+.Removal of external Caincrease . The pea2+ to 0Ca containing PE -induced contraction , verapamil (30–3000 nM) or diltiazem (0.1–10 μM), as representatives of the different classes of L-type Catraction , but eff2+ influx via L-type and non-L-type Ca2+ channels to the contraction by PE was further investigated by incubation of the segments with the L-type Ca2+ channel blocker verapamil and/or the putative non-selective cation channel blocker 2-aminoethoxydiphenyl borate (2-APB) [2+, 3 μM verapamil did not affect the PE-induced phasic contraction, but significantly reduced the +Ca contraction  . In 0Ca2traction . At 600 traction . Becausereceptor   and decreases window Ca2+ influx [2+ (2+ influx and contraction could be completely reversed by adding 50 μM glibenclamide (inhibitor of K+(ATP) channels, data not shown), by increasing extracellular K+ from 5.9 to 12 and 18 mM . PE-induced isometric contraction at the different K+ concentrations displayed a bi-exponential time course  caused a large influx of Ca2+ from the extracellular medium for CPA alone when compared with PE alone but this large Ca2+ influx was accompanied by a significantly smaller increase in force than by PE. Finally, even this large increase of intracellular Ca2+, could in the presence of 1 μM PE, not substantially increase the force induced by PE alone. As summarized in the calcium-force graph of 2+ release and influx without majorly affecting force, whereas Ca2+ release and influx by PE causes a significant temporally-related increase in force. When CPA and PE were combined, both a large Ca2+ influx and a large force increase were simultaneously present, but not significantly larger than these of CPA (Ca2+) or PE (tension) alone.Caa2+ pump  and leadthe VSMC . When VSof force . In cont2+ from non-contractile Ca2+ stores and induces non-contractile Ca2+ influx. CPA did not significantly increase the tension induced by PE alone, but it empties Ca2+ stores, and thereby may affect the contraction induced by PE. 2+ (control) when segments were first incubated with 0Ca + 10 μM CPA + 1μM PE to empty CPA-sensitive Ca2+ stores completely. In this condition, contraction increased to 124±2%  of the contraction in the presence of PE alone. Diltiazem (35 μM) inhibited this contraction by 65±1% in the absence and by 36±2%  in the presence of 10 μM CPA. Although CPA caused the mean contraction by PE to increase with about 24%, the contraction changed from mainly VGCC-mediated to mainly NSCC-mediated. Combining diltiazem with 50 μM 2-APB inhibited the contraction completely, suggesting that in the presence of CPA the relative contribution of NSCC was significantly increased from 35 to 64%.According to these data, CPA mobilizes Ca2+ influx was nearly voltage-independent for K+ concentrations between 2 and 15 mM. Because NSCC have their reversal potential around 0 mV, contractions by 1 μM PE were measured at more depolarized membrane potentials. These were calculated to be −66 mV at 5.9 mM K+, −34 mV at 35 mM K+, −19 mV at 65 mM K+ and −3 mV at 124 mM K+ [+ concentrations. Thereby, 35 μM diltiazem prevented the activation of L-type Ca2+ influx upon re-addition of extracellular Ca2+ to the depolarized segments. Moreover, the relative contribution of NSCC was increased by emptying CPA-sensitive Ca2+ stores  to 60%  inhibited by VGCC blockers, which indicates the occurrence of a non-L-type Ca2+ influx. The tonic component of PE-contracted segments was temporally related to a Ca2+ signal resulting from the influx of Ca2+ via different Ca2+ permeable ion channels  and 2-APB (50%)-sensitive receptor-operated channels, as well as Ca2+ sensitization mechanisms [2+ influx via VGCC; 2) Vm-independent Ca2+ influx probably via direct activation of VGCC; 3) increased Ca2+ sensitivity of the contractile elements and 4) release of Ca2+ from intracellular stores [2+ [2+ influx with diltiazem and that 2) the contraction remaining in the presence of diltiazem was voltage-independent within a small voltage range or did not change with addition of levcromakalim, suggest that PE elicits Ca2+ influx via NSCC.Force by high Ksegments  or by α1l artery  was compchannels , event 2chanisms . Of the r stores , the secores [2+ . One shoores [2+ ,30. Nevem or physiological K+ concentrations below 20 mM [+ or decreased with hyperpolarization induced by the ATP-dependent K+ channel opener levcromakalim , the baseline and PE-induced force are voltage-dependent mainly because Vm of VSMC resides in the window voltage range of the VGCC. Hyperpolarization of Vm with levcromakalim or low K+ avoids or diminishes activation of window Ca2+ influx and causes contraction by Ca2+ influx mainly via NSCC. By causing intracellular Ca2+ release from non-contractile Ca2+ stores with CPA, PE-induced contractions can be turned from mixed VGCC/NSCC-mediated to majorly NSCC-mediated. Results of this study emphasize the important role of VGCC and NSCC in mouse aortic performance and further indicate that although the contractile performance of aortic segments in different conditions may be the same, the relative contributions of VGCC or NSCC Ca2+ influx may significantly differ. The extreme voltage-dependent VGCC window contractions and the complex interactions with NSCC and non-contractile SR Ca2+ stores may have consequences for the development of arterial de-stiffening and antihypertensive therapies.In conclusion, αS1 Text(DOCX)Click here for additional data file.S1 Fig50 of 34±4 μM 2-APB. B. Tonic contractions by 1 μM PE upon re-addition of 3.5 μm Ca2+ to the 0Ca solution containing 1 μM PE. The concentration-response (isometric force) curve in D revealed an IC50 of 38±5 μM and was not significantly different from the IC50 for inhibition of the tonic contraction. (n = 5)A. Phasic contractions by 1 μM PE were measured 3 minutes after applying 0Ca. The concentration-response  curve in C revealed an IC(TIF)Click here for additional data file.


This study aims to investigate the role of Sirt3 in H2O2 induced oxidative neuronal injury in primary cultured rat cortical neurons. We found that H2O2 treatment significantly increased the expression of Sirt3 in a time-dependent manner at both mRNA and protein levels. Knockdown of Sirt3 with a specific small interfering RNA (siRNA) exacerbated H2O2-induced neuronal injury, whereas overexpression of Sirt3 by lentivirus transfection inhibited H2O2-induced neuronal damage reduced the generation of reactive oxygen species (ROS), and increased the activities of endogenous antioxidant enzymes. In addition, the intra-mitochondrial Ca2+ overload, but not cytosolic Ca2+ increase after H2O2 treatment, was strongly attenuated after Sirt3 overexpression. Overexpression of Sirt3 also increased the content of mitochondrial DNA (mtDNA) and the expression of mitochondrial biogenesis related transcription factors. All these results suggest that Sirt3 acts as a prosurvival factor playing an essential role to protect cortical neurons under H2O2 induced oxidative stress, possibly through regulating mitochondrial Ca2+ homeostasis and mitochondrial biogenesis.Oxidative stress is a well-established event in the pathology of several neurobiological diseases. Sirt3 is a nicotinamide adenine nucleotide (NAD The endutophagy ,4. H2O2  disease ,6,7,8.c from mitochondria into the cytoplasm [c binds to the apoptosis protease activation factor (APAf-1) and forms a complex to induce the activation of pro-caspase 9 and initiate an enzymatic reaction cascade leading to the execution of apoptosis [It has become increasingly clear that there are many complex signaling pathways involved in apoptosis after oxidative neuronal injury, including the intrinsic and extrinsic pathways. The intrinsic pathway is mediated by various stimuli that cause the release of cytochrome ytoplasm ,10. In tpoptosis ,12,13. Spoptosis ,15.2O2-induced neuronal injury in primary cultured cortical neurons, as well as the potential mechanisms with focus on mitochondrial calcium metabolism and mitochondrial biogenesis.The sirtuins (or Sir2-like proteins) are a conserved family of class III histone deacetylases (HDACs), and have been reported to be involved in transcriptional silencing, genetic control of aging and longevity of organisms ranging from yeasts to humans . Among t2O2 (0.1 mM) for 24 h, and the expression of sirt3 mRNA and protein was detected by RT-PCR or Western blot at different time points . The levels of sirt3 mRNA and protein were both significantly increased within 24 h of the start of H2O2 treatment, and peaked at 6 or 12 h, respectively  or control siRNA (Si-control). Western blot analysis indicated that Sirt3 expression was significantly reduced in neurons after their transfection with Si-Sirt3  or control lentivirus (LV-control). The results of Western blot analysis indicated that the expression of Sirt3 was significantly increased by LV-Sirt3 transfection as compared to LV-control  or control lentivirus (LV-control) for 72 h, and the ROS production was measured by Hstaining A. The reed group B. We alsreatment C. As sho2+ concentrations after H2O2 treatment. 2+ as monitored by Rhod-2 AM probe and expressed as fold of the baseline for up to 120 min following H2O2 treatment. Overexpression of Sirt3 attenuated the increase of mitochondrial Ca2+ induced by H2O2 treatment from 30 to 120 min after neurotoxicity. In addition, we also measured the changes of cytosolic Ca2+ after H2O2 treatment with or without Sirt3 overexpression  was measured  or control lentivirus (LV-control) for 72 h before Hmeasured A, and thr injury B. In addr injury C. The reN-methyl-d-aspartic acid (NMDA) treatment and protects cortical neurons against excitotoxic injury through inhibiting NAD depletion and subsequent oxidative stress [2O2 treatment in a time-dependent manner in primary cultured cortical neurons. Knockdown of Sirt3 by specific siRNA transfection aggravated H2O2 induced neuronal injury, and overexpression of Sirt3 exerted neuroprotective effects. These data indicated that Sirt3 may be an endogenous protective mechanism under oxidative stress, and overexpression of Sirt3 would provide an incremental protective effect against H2O2 injury.Mammalian sirtuin members were originally identified as histone deacetylases that are associated with numerous physiological roles, such as stress response, regulation of metabolism, gene silencing, and aging ,23. Recee stress . In the c release from mitochondria into cytoplasma after H2O2 treatment in cortical neurons, indicating the anti-apoptotic activity of Sirt3 in oxidative neuronal injury. In consistent with our findings, a previous study showed that Sirt3 is required for the regulation of cytochrome c superoxide-scavenging capacity via deacetylation and activation of complex IV [The mitochondrion plays a prominent role in the induction of apoptotic cell death after oxidative stress . It is wmplex IV . In addimplex IV ,40. All 2+, induced by receptor-mediated Ca2+ influx or Ca2+ release from intracellular stores, and followed mitochondrial Ca2+ overload [2+ uptake seems to play a two-faced role in the regulation of mitochondrial function: be beneficial for mitochondrial function in the processes of oxidation phosphorylation and ATP synthesis, or be detrimental in instigating subsequent pathological cascades [2+ overload as a key effector in the apoptotic response induced by stimuli like oxygen and glucose deprivation (OGD), H2O2, or arachidonic acid [2O2 treatment significantly increased the intracellular and intra-mitochondrial Ca2+ concentrations, which confirmed the involvement of intracellular Ca2+ overload and mitochondrial Ca2+ uptake in our in vitro neuronal injury model. The mechanism underlying mitochondrial Ca2+ loading in apoptotic cell death under oxidative stress is associated with nitric oxide production, cytochrome c dissociation, mitochondrial permeability transition pore (mPTP) opening with release of cytochrome c, and Ca2+-calmodulin dependent protein kinases activation [2+ uptake by decreasing the releasable endocytoplasmic reticulum (ER) Ca2+ pool, whereas the ER resident fraction of Bax amplified Ca2+-mediated cell death [2+ transport into mitochondria through targeting the mitochondrial calcium uniporters (mCU), such as uncoupling proteins (UCPs), has been demonstrated to protect neurons from cell death mediated by glutamate [2+ uptake after H2O2 treatment. Intriguingly, the increased intracellular Ca2+ concentration after oxidative injury was not altered by Sirt3 overexpression compared with that in LV-control transfected neurons. Intracellular Ca2+ concentration is differently regulated by Ca2+ influx from extracellular spaces, Ca2+ release from the ER stores and mitochondrial Ca2+ uptake, as well as the interaction between these factors [2+ transfer from cytoplasm to mitochondria through the regulation of mCU without effects on the Ca2+ influx from extracellular spaces, or the intra-mitochondrial Ca2+ overload was due to the Ca2+ influx from the ER stores into the mitochondria, which has been demonstrated in ischemic neuronal injury models [One of the key events that cause mitochondria related apoptosis is an abnormal increase in intracellular Caoverload ,42. Intrcascades . Recent nic acid ,45. In ctivation . The antll death ,48,49. Ilutamate ,51,52. H factors ,53. Thusy models .2O2 treatment, indicating damaged mitochondrial biogenesis function in oxidative stress conditions. The capacity for mitochondrial biogenesis decreases with increasing age [2O2 insult, which was also demonstrated in recent studies [In our study, a decline in intracellular ROS generation after Sirt3 overexpression was accompanied by increased mRNA levels and enzymatic activities of MnSOD and IDH2. Increased activity of endogenous antioxidant enzymes, an important marker to assess mitochondrial function, can be the result of increased mitochondrial biogenesis, which is defined as the growth and division of mitochondria . Mitochosing age , and Sirsing age . In the  studies . All the2+ uptake is considered as an important regulator of intracellular Ca2+ homeostasis, it is well-known that the ER Ca2+ stores may also be pivotal for Ca2+ overload associated neuronal cell death [2+ homeostasis and its interaction with mitochondrial proteins need more extensive study after Sirt3 knockdown or overexpression.There are some limitations to our study. First, Sirt3 is shown to be expressed in both neurons and glial cells , which pll death ,63. Sevell death ,65. Thuset al. [l-Glutamine and plated at a density of 3 × 105 cells/cm2. Before seeding, culture vessels, consisting of 96-well plates, 1.5 cm glass slides or 6 cm dishes were coated with poly-l-lysine  at room temperature overnight. Neurons were maintained at 37 °C in a humidified 5% CO2 incubator and half of the culture medium was changed every other day.Cortical neurons were cultured from Sprague–Dawley rats using a modified method reported by Redmond et al. . BrieflyImmunocytochemistry was used to detect the expression of Sirt3 in cortical neurons. Briefly, after being fixed with 4% paraformaldehyde for 15 min at room temperature, neurons were permeabilized with 0.2% Triton X-100, and incubated with primary antibody  overnight at 4 °C. The neurons were then incubated with the Alexa-488-conjugated mouse-anti-rabbit secondary antibody  for 2 h at 37 °C. 4,6-diamidino-2-phenylindole  was used to stain nucleus. Mito-tracker  was used to stain the mitochondria. Images were captured with an Olympus FV10i Confocal Microscope .2O2 (0.1 mM) for 24 h and subjected to various measurements.The specific siRNA targeted Sirt3  and control siRNA , which should not knock down any known proteins, were purchased from Santa Cruz Biotechnology, Inc. . The above siRNA molecules were transfected with Lipofectamine 2000  in 6-well plates for 72 h. After transfection, the cortical neurons were treated with HThe coding sequence of Sirt3 was amplified by RT-PCR. The primer sequences were: forward, 5'-TACTTCCTTCGGCTGCTTCA-3'; reverse, 5'-AAGGCGAAATCAGCCACA-3'. The PCR fragments and the pGC-FU plasmid  were digested with Age I and then ligated with T4 DNA ligase to produce pGC-FU-Sirt3. To generate the recombinant Lentivirus LV-Sirt3, 293T cells were co-transfected with the pGC-FU plasmid (20 μg) with a cDNA encoding Sirt3, pHelper1.0 plasmid (15 μg) and pHelper 2.0 plasmid (10 μg) by using Lipofectamine 2000 (100 μL). The supernatant was harvested and the viral titer was calculated by transducing 293T cells. As a control, we also generated a control lentiviral vector that expresses GFP alone (LV-control). Cortical neurons were transfected with lentivirus vectors for 72 h and subjected to various treatments.4 in microplates in a final volume of 100 μL/well culture medium. After various treatments, 10 μL cell proliferation reagent, WST-1, was added into each well and incubated for 4 h at 37 °C. Then, 100 μL/well culture medium and 10 μL WST-1 was added into one well in the absence of neurons, and its absorbance was used as a blank position for the ELISA reader. Cells were shaken thoroughly for 1 min on a shaker and the absorbance of the samples was measured using a microplate reader.Neuronal viability assay was performed by using the Cell Proliferation Reagent WST-1 following the manufacture’s protocol . Briefly, cortical neurons were cultured at a concentration of 5 × 10Cytotoxicity was determined by the release of LDH, a cytoplasmic enzyme released from cells. LDH release into the culture medium was detected using a diagnostic kit according to the manufacturer’s instructions . Briefly, 50 µL of supernatant from each well was collected to assay LDH release. The samples were incubated with reduced form of nicotinamide-adenine dinucleotid (NADH) and pyruvate for 15 min at 37 °C and the reaction was stopped by adding 0.4 M NaOH. The activity of LDH was calculated from the absorbance at 440 nm and background absorbance from culture medium that was not used for any cell cultures was subtracted from all absorbance measurements. The results were presented as the fold of control.c release into the cytoplasm was assessed after subcellular fraction preparation. Cortical neurons were washed with ice-cold phosphate buffered sodium (PBS) for three times and lysed with a lysis buffer containing protease inhibitors. The cell lysate was centrifuged for 10 min at 750× g at 4 °C, and the pellets containing the nuclei and unbroken cells were discarded. The supernatant was then centrifuged at 15,000× g for 15 min. The resulting supernatant was removed and used as the cytosolic fraction. The pellet fraction containing mitochondria was further incubated with PBS containing 0.5% Trition X-100 for 10 min at 4 °C. After centrifugation at 16,000× g for 10 min, the supernatant was collected as mitochondrial fraction. The levels of cytochrome c in cytosolic and mitochondrial fractions were measured using the Quantikine M Cytochrome c Immunoassay kit obtained from R&D Systems . Data were expressed as ng/mg protein.Cytochrome 6 cells were mixed with 32 μL of assay buffer and 2 μL of 10 mM Ac-DEVD-pNA substrate. Absorbance at 405 nm was measured after incubation at 37 °C for 4 h. Absorbance of each sample was determined by subtraction of the mean absorbance of the blank and corrected by the protein concentration of the cell lysate. The results were described as relative activity to that of control group.The activity of caspase-3 was measured using the colorimetric assay kit according to the manufacturer’s instructions . Briefly, after being harvested and lysed 102DCF-DA) in a microscope and the fluorescence was read using an excitation wavelength of 480 nm and an emission wavelength of 530 nm.Briefly, cortical neurons were incubated with 2,7-dichlorofluorescein diacetate (DCF-DA)  (10 μM) for 1 h at 37 °C in the dark, and then resuspended in PBS. Intracellular ROS production was detected using the fluorescence intensity of the oxidant-sensitive probe 2,7-dichlorodihydro-fluorescein diacetate (HThe enzymatic activity of manganese superoxide dismutase (MnSOD) and isocitrate dehydrogenase 2 (IDH2) was measured by use of a commercially available assay kits following the manufacturer’s instruction . Protein concentration was determined by using BCA protein kit . The enzyme activities were then normalized to the corresponding protein concentration for each sample.2O2 treatment at room temperature. Cells were then placed in an open-bath imaging chamber containing Dulbecco’s NaCl/Pi  supplemented with 20 mM glucose. With a Nikon inverted fluorescence microscope , cells were excited at 345 and 385 nm, and the emission fluorescence at 510 nm was recorded. Images were collected and analyzed with METAFLUOR image-processing software. The Ca2+ concentration values were then calculated, and Ca2+-insensitive fluorescence was subtracted from each wavelength before calculations were performed. Cell permeable Rhod-2 AM was used as a mitochondria selective Ca2+ indicator. The cells were loaded with 2 mM Rhod-2 AM for 30 min before various treatments, and then washed three times. The fluorescence intensities were immediately analyzed with a Nikon inverted fluorescence microscope. Results are presented as the fold of the control.Intracellular calcium concentrations were measured by Fura-2-AM . CorticaLong fragment PCR was used to quantify the relative abundance of intact mtDNA as previously described . As an i2, pH 7.4) with 0.1% BSA, 1 mM malate, 1 mM glutamate, and buffer B (containing 0.8 mM luciferin and 20 mg/mL luciferase in 0.5 M Tris-acetate pH 7.75). The reaction was initiated by addition of 0.1 mM ADP and monitored for 5 min using a microplate reader.Isolated mitochondria were utilized to measure ATP synthesis with a luciferase/luciferin-based system as described elsewhere . Thirty Total RNA was isolated from cortical neurons using Trizol reagent . After the equalization of the RNA quantity in each group, the mRNA levels were quantitated using a Bio-Rad iQ5 Gradient Real-Time PCR system , and GAPDH was used as an endogenous control. Primers for all Real-Time PCR experiments were listed as follow: Sirt3: forward: 5'-TACTTCCTTCGGCTGCTTCA-3', reverse: 5'-AAGGCGAAATCAGCCACA-3'; MnSOD: forward: 5'-TTAACGCGCAGATCATGCA-3', reverse: 5'-GGTGGCGTTGAGATTGTTCA-3'; IDH2: forward: 5'-AATTTTAGGACCCCCGTCTG-3', reverse: 5'-GGGGTGAAGACCATTTTGAA-3'; NRF-1: forward: 5'-GAGTGACCCAAACCGAACA-3', reverse: 5'-GGAGTTGAGTATGTCCGAGT-3'; PGC-1α: forward: 5'-GTGCAGCCAAGACTCTGTATGG-3', reverse: 5'-GTCCAGGTCATTCACATCAAGTTC-3'; TFAM: forward: 5'-GGTGTATGAAGCGGATTT-3', reverse: 5'-CTTTCTTCTTTAGGCGTTT-3'; GAPDH: forward: 5'-AAGGTGAAGGTCGGAGTCAA-3', reverse: 5'-AATGAAGGGGTCATTGATGG-3'. Samples were tested in triplicates and data from five independent experiments were used for analysis.Equivalent amounts of protein (40 μg per lane) were loaded and separated by 10% sodium dodecyl sulfate (SDS)-PAGE gels, and transferred to polyvinylidene difluoride (PVDF) membranes . The membranes were blocked with 5% nonfat milk solution in tris-buffered saline with 0.1% Triton X-100  for 1 h, and then incubated overnight at 4 °C with the primary Sirt3 antibody (1:1000) or β-actin (1:600) antibody dilutions in TBST. After that the membranes were washed and incubated with secondary antibody for 1 h at room temperature. Immunoreactivity was detected with Super Signal West Pico Chemiluminescent Substrate . An analysis software named Image J  was used to quantify the optical density of each band.p < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS 16.0 , a statistical software package. Statistical evaluation of the data was performed by one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons. A value of 2O2 treatment in primary cultured cortical neurons. Down-regulation of Sirt3 using specific targeted siRNA exacerbated the H2O2-induced neuronal injury, whereas overexpression of Sirt3 exerted protective effects through attenuating ROS generation and activation of endogenous antioxidant enzymes. The increased expression of Sirt3 induced by oxidative stress might be an endogenous protective mechanism, which is partly dependent on the preservation of mitochondrial calcium homeostasis and enhancement of mitochondrial biogenesis. Thus, metabolic rescue observed upon overexpression of Sirt3 may represent an appropriate strategy to avoid neuronal death in a broad range of oxidative stress related brain disorders.In conclusion, our results showed that the expression of Sirt3 was significantly increased at both mRNA and protein levels after H