We found that both KYNA and zaprinast, a phosphodiesterase (PDE) inhibitor and GPR35 agonist, did not modify either basal or peaks of [Ca2+]i induced by challenging the cells with ATP (30 µM). However, the [Ca2+]i plateau phase following peak was significantly attenuated by these compounds in a store-operated Ca2+ channel (SOC)-independent manner. The activation of GPR35 by KYNA and zaprinast was also studied at the CA3-CA1 synapse in the rat hippocampus. Evoked excitatory post synaptic currents (eEPSCs) were recorded from CA1 pyramidal neurons in acute brain slices. The action of KYNA on GPR35 was pharmacologically isolated by using NMDA and α7 nicotinic receptor blockers and resulted in a significant reduction of eEPSC amplitude. This effect was prevented in the presence of CID. Moreover, zaprinast reduced eEPSC amplitude in a PDE5- and cGMP-independent mechanism, thus suggesting that glutamatergic transmission in this area is modulated by GPR35. In conclusion, GPR35 is expressed in cultured astrocytes and its activation modulates cAMP production and [Ca2+]i. GPR35 activation may contribute to KYNA effects on the previously reported decrease of brain extracellular glutamate levels and reduction of excitatory transmission.Limited information is available on the brain expression and role of GPR35, a Gi/o coupled receptor activated by kynurenic acid (KYNA). In mouse cultured astrocytes, we detected GPR35 transcript using RT-PCR and we found that KYNA (0.1 to 100 µM) decreased forskolin (FRSK)-induced cAMP production (p<0.05). Both CID2745687 , a recently described GPR35 antagonist, and GPR35 gene silencing significantly prevented the action of KYNA on FRSK-induced cAMP production. In these cultures, we then evaluated whether GPR35 activation was able to modulate intracellular Ca The G protein coupled receptor 35 (GPR35) was identified approximately 15 years ago . HoweverKYNA has a number of other targets in the brain: it is a potent antagonist of the glycine allosteric site on the NMDA receptor complex and for several years it was assumed that the interaction between KYNA and the NMDA receptor could have a physiological role in brain function . It has In previous studies, we reported that GPR35 is abundantly expressed in the DRG and the spinal cord of the rodents and that experimental elevation of KYNA concentration in the blood or brain significantly decreased glutamate extracellular levels in the nervous tissue and reduced inflammatory pain . Since z2+ regulatory mechanisms in non-excitable cells -1-pyrazole-4-carboxylate (CID) was from Ryan Scientific . Tissue culture reagents were obtained from Gibco-BRL  and ICN Pharmaceuticals . Unless otherwise declared, all cell culture media were purchased from Sigma-Aldrich.2. These cultures contain a high percentage of viable astrocytes (>90%), as previously demonstrated with by means of glial fibrillary acidic protein (GFAP) immunostaining i and the modulation of this effect in the presence of the GPR35 agonist KYNA. GPR35 activation decreases the plateau phase of the increase of [Ca2+]i following an IP3 mediated stimulus (ATP 30 µM)  . Interess effect .2+ release from the stores. This influx has also been named capacitative Ca2+ entry and in cultured cerebellar astrocytes is modulated by cAMP intracellular concentrations [One of the mechanisms controlling the duration of the plateau phase of the waves is the opening of the so called “store operated calcium channels” after the IP3-induced Catrations . It is p2+ signaling in astrocytes [2+ free medium thapsigargin depletes Ca2+ stores passively by virtue of its ability to inhibit SERCA pumps on the endoplasmic reticulum and the ensuing entry of Ca2+ is therefore assumed to be the very definition of CCE [Since cAMP has been reported to increase capacitative calcium entry (CCE) and to modulate the length of the plateau phase of the Catrocytes  and the n of CCE ,26. The Previous studies demonstrated that KYNA reduces brain extracellular glutamate concentrations and that this action cannot be completely ascribed to NMDA or α7 nicotinic receptors . It has 2+ transients  FRSK-induced cAMP elevation , ii) ATPansients  and iii)ansients  and 5. Periments . TherefoAs previously mentioned, KYNA is one of the proposed endogenous ligand for GPR35 and zaprinast, a cGMP phosphodiesterase inhibitor, is another potent agonist of this Gi/o coupled receptor ,5. In thin vivo and in organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation in vitro [2+ transients  suggesting that KYNA may reduce the activity of brain excitatory transmission at concentrations unable to interact with NMDA receptors –42. It hin vitro . An incrin vitro ,45. Recein vitro . Similarin vitro . KYNA isin vitro  and a loin vitro ,49, althin vitro  and coulansients . While tur model ,51. In aur model . Therefoocol see  we notic release . In astrlarified . It is casticity . Our datasticity  and reduasticity ,7,11.


Recently, STIM1 was identified as a sarcoplasmic reticulum (SR) Ca2+ sensor that activates Orai1, the Ca2+ channel responsible for store-operated Ca2+ entry (SOCE). We investigated the role of STIM1 in [Ca2+]i and cell migration induced by platelet-derived growth factor (PDGF)-BB in human ASM cells. Cell migration was assessed by a chemotaxis chamber assay. Human ASM cells express STIM1, STIM2, and Orai1 mRNAs. SOCE activated by thapsigargin, an inhibitor of SR Ca2+-ATPase, was significantly blocked by STIM1 siRNA and Orai1 siRNA but not by STIM2 siRNA. PDGF-BB induced a transient increase in [Ca2+]i followed by sustained [Ca2+]i elevation. Sustained increases in [Ca2+]i due to PDGF-BB were significantly inhibited by a Ca2+ chelating agent EGTA or by siRNA for STIM1 or Orai1. The numbers of migrating cells were significantly increased by PDGF-BB treatment for 6 h. Knockdown of STIM1 and Orai1 by siRNA transfection inhibited PDGF-induced cell migration. Similarly, EGTA significantly inhibited PDGF-induced cell migration. In contrast, transfection with siRNA for STIM2 did not inhibit the sustained elevation of [Ca2+]i or cell migration induced by PDGF-BB. These results demonstrate that STIM1 and Orai1 are essential for PDGF-induced cell migration and Ca2+ influx in human ASM cells. STIM1 could be an important molecule responsible for airway remodeling.It is suggested that migration of airway smooth muscle (ASM) cells plays an important role in the pathogenesis of airway remodeling in asthma. Increases in intracellular Ca Airway remodeling due to repeated airway wall damage and repair plays an important role in the pathophysiology of severe asthma 2+ is a second messenger for ASM cell functions related to asthma, such as contraction, proliferation, and cytokine production 2+ entry (SOCE), originally introduced as capacitative Ca2+ entry by Putney 2+ influx pathway in various cell types including ASM cells 2+ concentration of the sarcoplasmic reticulum (SR) Ca2+ stores in muscle cells or endoplasmic reticulum (ER) in non-muscle cells through the binding of inositol–1,4,5-trisphosphate (IP3) to the IP3 receptor 2+ concentrations within the SR and reports this information to Orai1, a Ca2+-permeable channel responsible for SOCE Intracellular free Ca2+ concentrations ([Ca2+]i) mediated by a strong chemoattractant, PDGF, in human ASM cells. We demonstrated that both STIM1 and Orai1 are essential for cell migration and elevation of [Ca2+]i induced by PDGF in ASM cells.This study was designed to investigate the role of STIM1 in the cell migration and the regulation of intracellular Ca2 and 95% air at 37°C Primary cultures of normal human bronchial smooth muscle cells from multiple donors were obtained from Lonza . The cells were maintained in culture medium containing 5% FBS, human recombinant epidermal growth factor (1 ng/ml), insulin (10 mg/ml), human recombinant fibroblast growth factor (2 ng/ml), gentamycin (50 mg/ml), and amphotericin B (0.05 mg/ml)  in an atmosphere of 5% CO5′-CCAGAGCCTCAGCCATAGTC-3′ and 5′-CTTCAGCACAGTCCCTGTCA-3′, STIM2: 5′-GCTAAGGGAGGGAGCTGAAT-3′ and 5′-GCTAAGGGAGGGAGCTGAAT-3′, Orai1: 5′-TTCCTAGCTGAGGTGGTGCT-3′ and 5′-AATCCTCTTCCCTCCATGCT-3′ GAPDH: 5′-AACGGATTTGGTCGTATTGG-3′ and 5′-TGAGTCCTTCCACGATACCA-3′. Product sizes of the STIM1, STIM2, Orai1, and GAPDH were 481bp, 498bp, 483bp and 498bp, respectively.Total cellular RNA was extracted using RNeasy Mini Kit  −ddCt) method using Microsoft Excel 2010 Quantitative PCR was performed on a 7300 Real-Time PCR system  using the 3-stage program parameters provided by the manufacturer: 2 min at 50°C, 10 min at 95°C, and then 40 cycles of 15 s at 95°C and 1 min at 60°C. Relative changes in each mRNA expression compared to an unstimulated control and normalized to GAPDH were quantified by the comparative Ct  and the scrambled siRNA (negative control) were purchased from Invitrogen . Cells were transfected with 10 nM predesigned siRNA  targeting STIM1, STIM2, and Orai1 or with 10 nM scrambled siRNA (negative control). Lipofectamine RNAiMAX (Invitrogen) was used as a transfection vector. To minimize the possibility of off target effects, three different siRNAs targeting either gene were used. Cells were used for PCR, Western blotting, CaProtein concentrations of cellular lysates were measured by using a protein assay reagent kit . Equal amounts of lysates, adjusted for protein concentrations, were resolved by SDS-PAGE using a 5–20% linear gradient running gel . Proteins were transferred to nitrocellulose membranes, and the membranes were blocked in 5% skim milk for 2 h at room temperature, followed by overnight incubation at 4°C with primary antibodies. The membranes were incubated for 1 h at room temperature with a sheep anti-mouse or donkey anti-rabbit secondary antibody. The primary antibodies used were a mouse anti-GOK/Stim1 antibody , a rabbit anti-STIM2 antibody , and a mouse anti-Orai1 antibody . A polyclonal anti-actin antibody (Sigma-Aldrich) was used for the loading control. Detection was performed with an Enhanced Chemiluminescence (ECL) kit  2 2, MgCl2 1, glucose 10, and HEPES 10 (pH 7.40). After the cells were washed with normal physiological solution, the [Ca2+]i was assessed by the fluorescence of fura-2 using a fluorescence microscope  at room temperature. Data were analyzed using a digital fluorescence imaging system . The excitation wavelengths were set at 340 and 380 nm, and the emission was collected at 510 nm by a photomultiplier. The intensity of the fura-2 fluorescence due to excitation at 340 nm (F340) and 380 nm (F380) was measured after subtraction of the background fluorescence, and the ratio of F340 to F380 (F340/F380 ratio) was used as an indicator of the relative level of [Ca2+]iCells (approximately 50% confluence) grown on glass coverslips  were treated with 3 µM fura-2/AM  for 25 min at 37°C in normal physiological solution containing (in mM): NaCl 145, KCl 5, CaCl4) suspended in 400 μL of DMEM/F-12 containing 0.1% FBS were placed in the upper chamber. PDGF-BB (Sigma-Aldrich) dissolved in DMEM/F-12 containing 0.1% FBS was inserted in the wells of the lower chamber. The cells were transferred to the upper wells, and after incubation for 6 h at 37°C in a 5% CO2 incubator, the non-migrated cells on the upper surface of the filter were scraped off with a cotton-tipped applicator. The migrated cells were fixed and stained with Diff-Quik  and mounted onto glass slides. Cells in five fields per chamber were counted under a light microscope (x200). In the Results section, “n” refers to numbers of experiments tested. Each experimental condition was tested in duplicate. Solvents did not affect cell migration at the concentrations used (≤0.1%/vol.).Cell migration was measured using a modified Boyden chamber . Chambers with 8-μm pores were coated with type-1 collagen . Confluent ASM cells were brought to a quiescent state overnight by incubation in DMEM/F-12 cell culture medium (Invitrogen) containing 0.1% FBS before being used in a migration assay. Cells . P<0.05 was considered statistically significant.All data are expressed as means ± SD. Analysis of variance followed by the Bonferroni test for post hoc analysis or We initially measured the expression of STIM1, STIM2, another STIM protein similar in structure to STIM1 2+-ATPase (SERCA), induces SOCE by depleting SR Ca2+ stores in ASM cells 2+-free solution, a transient increase in the F340/F380 ratio, a measure of [Ca2+]i, due to Ca2+ release from the SR Ca2+ stores was observed   , demonst340/F380 ratio in cells transfected with siSTIM1 and siOrai1 are shown in 340/F380 ratio in the nominally Ca2+-free solution due to Ca2+ release was still observed in the cells transfected with siSTIM1  (340/F380 due to SOCE (plateau phase) was not affected by siSTIM2 or scrambled siRNA (n = 6)  . SimilarP<0.001) . MoreoveP<0.001) . The inc (n = 6) .2+]i were investigated. Application of PDGF-BB (10 ng/mL) to the normal physiological solution containing 2 mM Ca2+ induced a transient increase in the F340/F380 ratio, followed by a sustained increase in the F340/F380 ratio  and nominally Ca2+-free solutions (n = 6)    . The susP<0.001) . These r2+]i by PDGF-BB. The transient increase in the F340/F380 ratio induced by PDGF-BB was still observed  (340/F380 ratio in the plateau phase was significantly lower in the siSTIM1- or siOrai1-transfected cells than in the cells transfected with scrambled siRNA (negative control)   (340/F380 ratio (340/F380 ratio was not inhibited by siSTIM2 (n = 6)  . The F34P<0.001) . Simulta P<0.05)  without 80 ratio , similar (n = 6) .The roles of STIM1 and Orai1 in PDGF-induced cell migration were investigated using a chemotaxis assay. Migrating cell numbers were significantly increased by treatment with PDGF-BB  compared with time-matched control cell cultures  . MoreoveNext, cells were transfected with siRNAs targeting STIM1, STIM2, Orai1, or the negative control (scrambled siRNA). Transfection with siSTIM1, siOrai1, or both siSTIM1 and siOrai1 significantly inhibited PDGF-BB-induced cell migration  . In cont2+]i induced by PDGF-BB was also inhibited by siSTIM1 and siOrai1, (3) STIM1 and Orai1 were essential for PDGF-induced ASM cell migration, and (4) STIM2 was not involved in these mechanisms. To our knowledge, this is the first report which demonstrates an essential role of STIM1 and Orai1 in migration and increases in [Ca2+]i induced by PDGF in human ASM cells.This study highlights the novel role of STIM1 in migration of ASM cells. The main findings are that (1) SOCE activation by thapsigargin was inhibited by siRNAs targeting STIM1 and Orai1, (2) a sustained increase in i due to PDGF-BB were significantly inhibited by knockdown of STIM1 and Orai1 with siRNA in human ASM cells  was regulated by SR Ca2+ store depletion and STIM1 in human ASM cells. In their report, the histamine-induced increase of [Ca2+]i was partially inhibited by the reverse mode NCX inhibitor KB-R7943 2+ influx is much less than that of Orai1 in ASM cells. In another report by Feng et al. 2+-ATPase 2 (SPCA2) independently of ER Ca2+ stores or STIM1 in breast cancer cells. By contrast to their results, we demonstrated that siOrai1 blocked the thapsigargin-induced SOCE and PDGF-induced [Ca2+]i elevation (2+]i elevation (2+ influx by PDGF-BB. Taken together, the PDGF-induced increase of [Ca2+]i is mostly via SOCE in human ASM cells. Nevertheless, possible involvement of store-independent mechanisms in PDGF-induced [Ca2+]i elevation cannot be ruled out.Recently, several reports have shown that STIM1 and Orai1 are also involved in mechanisms of Calevation  and 5. Mlevation . Therefo2+]i elevation evoked by PDGF-BB in human ASM cells.Activation of PDGF receptors strongly promotes migration of ASM cells 2+ signaling, are involved in processes of cell motility and migration 2+]i elevation in smooth muscle contraction Migration of ASM cells is not only essential for development of hollow airways and the respiratory system but also important for airway remodeling in asthma Because SOCE is a major influx pathway both in muscle and non-muscle cells, STIM1 and Orai1 proteins play critical roles in homeostasis of the immune system and normal development. Mutations in STIM1 and Orai1 genes are clinically characterized by severe immunodeficiency and congenital myopathy in human patients 2+ influx of human ASM cells. Our findings suggest that STIM1 and Orai1 may be important molecules responsible for airway remodeling in asthma.In summary, STIM1 and Orai1, key molecules for SOCE, regulate PDGF-induced migration and Ca


Contractions of isolated rat mesenteric lymphatic vessels were observed at a luminal pressure of 2 cm H2O in a 37°C bath. The expression of ROCK in isolated rat mesenteric lymphatic vessels was assessed by Western blotting and confocal microscopy. The role of ROCK in contractile function was tested using two specific yet structurally distinct inhibitors: H1152 (0.1–10 μM) and Y-27632 (0.5–50 μM). In addition, lymphatics were transfected with constitutively active (ca)-ROCK protein (2 μg/ml) to assess gain of contractile function. Vessel diameter and the concentration of intracellular free Ca2+ ([Ca2+]i) were simultaneously measured in a subset of isolated lymphatics loaded with the Ca2+-sensing dye fura-2. The results show expression of both the ROCK1 and ROCK2 isoforms in lymphatic vessels. Inhibition of ROCK increased lymphatic end diastolic diameter and end systolic diameter in a concentration-dependent manner. Significant reductions in lymphatic tone and contraction amplitude were observed after treatment 1–10 μM H1152 or 25–50 μM Y-27632. H1152 (10 μM) also significantly reduced contraction frequency. Transient increases in [Ca2+]i preceded each phasic contraction, however this pattern was disrupted by either 10 μM H1152 or 50 μM Y-27632 in the majority of lymphatics studied. The significant decrease in tone caused by H1152 or Y-27632 was not associated with a significant change in the basal [Ca2+]i between transients. Transfection with ca-ROCK protein enhanced lymphatic tone, but was not associated with a significant change in basal [Ca2+]i. Our data suggest that ROCK mediates normal tonic constriction and influences phasic contractions in lymphatics. We propose that ROCK modulates Ca2+ sensitivity of contractile proteins in lymphatics.The mechanisms that control phasic and tonic contractions of lymphatic vessels are poorly understood. We hypothesized that rho kinase ROCK, previously shown to increase calcium (Ca Lymphatics play a critical role in normal cardiovascular function, tissue fluid homeostasis, inflammation, adaptive immunity, digestive lipid uptake, metabolism, and the regulation of salt storage 2+ ([Ca2+]i) is considered the principal mechanism that initiates contraction and relaxation, respectively 2+]i, while a certain basal [Ca2+]i between contractions helps maintain tone The intrinsic pumping action of collecting lymphatics drives normal lymph flow, and is generated by their smooth muscle layer. Lymphatic pumping consists of a phasic, cardiac-like contractile cycle, superimposed over smooth muscle-like tone between the phasic contractions 2+ binds to calmodulin, and this complex activates the catalytic subunit of myosin light-chain kinase (MLCK). In turn, MLCK phosphorylates Ser19 and Thr18 on the regulatory myosin light chain (MLC) 2+]i inactivates MLCK and permits dephosphorylation of MLC by myosin light chain phosphatase (MLCP). A role for MLCK in establishing tone and phasic contractions in collecting lymphatics and the thoracic duct has previously been demonstrated For the maintenance of tone in smooth muscle, Ca2+ sensitivity in response to a variety of agonists, defined as the ability to change the level of tone generated at a given level of [Ca2+]i2+ sensitization in response to various agonists are thought to involve G-protein coupled inhibition of MLCP, shifting the kinase/phosphatase balance in favor of MLCK so that a higher level of MLC phosphorylation is achieved at a given [Ca2+]iIn addition, the contractile mechanisms in smooth muscle display a varying Ca2+ sensitivity. In addition, the expression of ROCK in lymphatic tissues has not previously been established. The goal of these studies was to test the hypothesis that ROCK increases Ca2+ sensitivity, leading to enhanced tone in isolated collecting lymphatic vessels. We performed studies to determine whether both ROCK isoforms are present in lymphatic smooth muscle cells. We also evaluated how pharmacologic inhibition of ROCK affects lymphatic [Ca2+]i and the lymphatic contractile cycle. Lastly, we investigated how experimentally enhancing ROCK activity affects [Ca2+]i and lymphatic pumping.What remains unclear is whether the reduction in tone in response to ROCK inhibitors represented a change in Cath Edition, 2011). The protocol was approved by the Louisiana State University Health Sciences Center – New Orleans Institutional Animal Care and Use Committee (permit number: 2875) and the University of South Florida Institutional Animal Care and Use Committee (permit number: RIS00000097). Rats were anesthetized with a mixture of ketamine (90 mg/kg) and xylazine (9 mg/kg) i.m. prior to surgery.All animal procedures were carried out in strict accordance as outlined in the National Institute of Health Guide for the Care and Use of Laboratory Animals  were housed in a controlled temperature (22°C) and controlled illumination (12∶12 h light dark cycle) environment. After arrival, the rats were submitted to a one-week acclimation period and were provided standard rat chow  and water 2·2H2O, 2 mM; MgSO4·7H2O, 1.2 mM; NaH2PO4, 1.2 mM; Na pyruvate, 2 mM; glucose, 5 mM; EDTA, 0.02 mM; MOPS, 3 mM and purified BSA 1 g/100 ml). Rats were euthanized with Euthasol/Somnasol  followed by opening of the chest to confirm death. In each experiment, a section of mesentery was pinned in a dissection chamber containing ice-cold APSS, and with the aid of a stereomicroscope, a collecting lymphatic vessel  was carefully dissected from surrounding adipose and connective tissue. The isolated lymphatic was transferred to an isolated vessel chamber  and was mounted onto two resistance-matched glass micropipettes containing APSS and secured with nylon thread. The vessels typically contained at least two valves, so that one complete lymphangion was available for study. The chamber was transferred to a Nikon Eclipse TS100 inverted microscope, equipped with a halogen lamp, 10× objective, CCD camera, DVD recorder, and a Living Systems Video Dimension Analyzer for video image acquisition, storage, and lymphatic diameter measurement. Adjustable reservoirs containing APSS, on a wall-mounted manometer, were connected to the pipettes to set the intraluminal pressure. All experiments were performed with the bath temperature set at 37°C and the reservoirs set at a height of +2 cm H2O relative to the isolated lymphatic. This luminal pressure is within the normal physiological range for lymphatics After anesthesia was induced in the rat, a midline laparotomy was performed, and the small intestine and mesentery were exteriorized and excised and placed in ice-cold albumin physiological salt solution . These concentrations ranges were chosen based on the reported IC50 values of 2.5 μM for H1152 and 12.8 μM for Y-27632 to inhibit ROCK-mediated phosphorylation in cultured cells 2+-free APSS), tone  =  100 * (MaxD – EDD)/MaxD, and Ejection Fraction (EF)  =  (EDD2-ESD2)/(EDD2). EDD, ESD, and AMP were normalized to MaxD to account for variability in the resting diameter of lymphatics. For baseline data, the means presented for each parameter represent the averages for the 5-min. period just prior to the addition of inhibitors. For each concentration of the ROCK inhibitors, the 5-min. increment after the first 2 min. post addition was used to calculate the mean data .The following parameters were determined from the lymphatic luminal diameter measurements 2+]i in isolated lymphatics has been previously described in detail 2+-sensing dye fura-2-acetoxymethyl ester (AM)  by exchanging the bath solution to APSS containing fura-2 AM (2 μM) and pluronic acid (0.2% wt/vol) for 30 minutes at 37°C. The fura-2 AM was only added to the abluminal side to restrict loading to the smooth muscle layer. The bath solution was then changed twice to normal APSS to wash out extracellular fura-2. The vessel was allowed to equilibrate for at least 20 min. to allow re-establishment of spontaneous contractions. A Lambda LS 300W Xenon lamp equipped with a LS-filter wheel  was used to alternately illuminate the vessel at 340 and 380 nm wavelengths for durations of 50 ms each. The emission light was passed through a band-pass filter  with a bandwidth of 80 nm and detected by a Photometrics HQ2 camera . The acquisition software used to capture images and for data analysis was Nikon Elements 4.0 .The protocol for measuring [Ca2+]i, were measured. To determine the 340/380 ratio, a rectangular region of interest that included the entire lymphatic vessel and surrounding area was selected, so that the vessel was fully tracked during relaxation and contraction. We were careful to exclude regions on lymphatic vessels where there appeared to be a capillary attached to the exterior, as our target was to estimate smooth muscle [Ca2+]i. An increase in 340/380 indicates an increase in [Ca2+]i. Using the 380 nm channel of the same image set, we used a contrast threshold protocol in Nikon Elements to track the vessel within a region of interest around a short segment of the vessel. This allowed us to automatically measure the distance between outer edges of the vessel. The resulting external diameter data was then exported to Microsoft Excel for further analysis. All [Ca2+]i data are expressed using the raw 340/380 ratio values Changes in both vessel diameter and the ratio of fluorescence at 340 nm and 380 nm (340/380), indicative of  using GraphPad Prism 6 software. When comparing two time-matched groups, two-way repeated-measures ANOVA followed by Bonferroni t-tests to compare groups at individual time points was the analysis of choice. For a single comparison between baseline and treatment, a paired t-test was used. Significance was accepted at P<0.05.Representative tracings are shown for the ROCK inhibitor protocols. Summarized data are presented as mean ± SE. The responses of lymphatic vessels over time to various treatments were evaluated with repeated measures ANOVA, followed by Dunnett's test to compare individual time points to baseline when appropriate. In addition, concentration-response relationships were investigated using nonlinear regression analysis. Data were normalized to the absolute percentage values based on the maximum (Max) and minimum (Min) responses observed, and curves were fit to the equation Y  =  Max + i or ROCK; which of these myosin isoforms are involved in tonic versus phasic contractions; and what degree of cooperation may there be between myosin isoforms in both tonic and phasic contraction generation.In summary, our data suggests a role for ROCK in the CaFigure S1Representative tracing of an untreated isolated rat mesenteric lymphatic. The vessel was kept at 2 cm H2O during the entire protocol, and is representative of three separate experiments.(TIF)Click here for additional data file.Figure S2Concentration-response of lymphatic contractile parameters in response to H1152. All data are normalized to the maximum responses observed.(TIFF)Click here for additional data file.Figure S3Concentration-response of lymphatic contractile parameters in response to Y-27632. All data are normalized to the maximum responses observed.(TIFF)Click here for additional data file.Movie S1Confocal image stack of an isolated rat mesenteric collecting lymphatic labeled for ROCK1. Confocal slices were obtained in single photon mode at 2 μm intervals, which are indicated at the top left of each frame. Images of individual channels for ROCK1 (green) and nuclei, (white), and an overlay of these two channels are shown in the top row. Images of the glycocalyx (red) and smooth muscle (SM) actin (blue) channels, and an overlay are shown in the left column. All other combinations of overlays fill out the remaining rows and columns. In the bottom red/white overlay (bottom left), magenta areas indicate overlap. Just to the right, in the red/blue/white overlay, the endothelium and smooth muscle layers can be distinguished further due to the orientation of the nuclei. Endothelial nuclei are located in the inner layer and are oriented longitudinally. Smooth muscle nuclei are elongated and oriented perpendicular to the vessel, in the same fashion as the smooth muscle cells. In the green/red images, yellow pixels indicate overlap. In the green/blue images, cyan pixels indicate overlap. In the green/red/blue image, grey/white pixels indicate overlap. ROCK1 labeling overlaps with some areas of smooth muscle, to a lesser extent in the endothelial layer, and is also present within vasa vasorum on the outer surface of the lymphatic vessel. The labeling becomes weaker at the far end of the z-stack because no correction for z-distance was used in the image capturing. This vessel is representative of three separate experiments.(AVI)Click here for additional data file.Movie S2Confocal image stack of an isolated rat mesenteric collecting lymphatic labeled for ROCK2. Confocal slices were obtained in single photon mode at 2 μm intervals, which are indicated at the top left of each frame. Images of individual channels for ROCK2 (green) and nuclei, (white), and an overlay of these two channels are shown in the top row. Images of the glycocalyx (red) and smooth muscle (SM) actin (blue) channels, and an overlay of these two channels are shown in the left column. All other combinations of overlays fill out the remaining rows and columns. In the red/blue overlay, magenta pixels indicate overlap. In the green/red overlay, yellow pixels indicate overlap. In the green/blue overlay, cyan pixels indicate overlap. In the green/red/blue image, grey/white pixels indicate overlap. Overlays including nuclei are also included to help distinguish the endothelial and smooth muscle layers. Strong ROCK2 labeling is evident in the smooth muscle layer and endothelium, and is also present within the vasa vasorum on the outer surface of the lymphatic vessel. The labeling becomes weaker at the far end of the z-stack because no correction for z-distance was used in the image capturing. This vessel is representative of three separate experiments.(AVI)Click here for additional data file.Movie S3Time-lapse 340/380 ratio images of a pumping mesenteric collecting lymphatic during baseline.(AVI)Click here for additional data file.Movie S4Time-lapse 340/380 ratio images of a pumping mesenteric collecting lymphatic immediately after adding 10 μM H1152.(AVI)Click here for additional data file.


The risk of overwhelming postsplenectomy infection (OPSI) prompted the evolution toward preservation of the injured spleen.Nonoperative management (NOM) of blunt injury to the spleen in adults has become the standard of care in hemodynamically stable patients. This modality of treatment began in the 1970’s in paediatric patients. It is highly successful with overall failures rates from 2% to 31% (average 10.8%) - with the majority of failures occurring in the first 24 hours. Current, NOM of splenic trauma includes splenic artery embolization.However, the criteria for NOM are controversial. In this study we present the current criteria, the evolution and failure rates of this type of management viewed through the general knowledge and, particularly, our experience. He submitted this theory following the post-mortem findings in a 43 year-old woman, who fell from height during work and died 5 days later from brain and abdominal injuries. The autopsy revealed a splenic injury without an obvious sign of recent bleeding. Therefore, Billroth concluded that ”the lesion healed completely judging by the macroscopic appearance of the lesion and the reduced amount of intraperitoneal blood”.Billroth suggested over 100 years ago that the injured spleen has the ability of self-healing [2]. The treatment for blunt abdominal trauma has significantly changed thanks to new diagnostic methods and the accurate assessment of organ damage. When it comes to visceral injuries following abdominal trauma, there is nothing as radical as the nonoperative management (NOM) of hepatic and splenic injuries . OPSI is most frequent during the first 2 years of asplenia but there is a permanent risk of infection with a mortality of over 80%.This major therapeutic change was the consequence of many clinical studies indicating that splenectomy increases the risk of infection susceptibility with its most deadly manifestation-OPSI (overwhelming postsplenectomy sepsis), which appears in 0.5% of all splenectomies in trauma patients and in over 20% of elective splenectomies for hematologic disorders .In 1882, Gross indicated NOM for spleen injuries and recommends bed rest and mild diet for minor injuries and lead acetate, ergot and opium for severe lesions; surgery will be performed only if necessary. “A System of Surgery” [1] the pioneer for non-operative management (NOM) in splenic trauma was Wanborough, in 1940 .According to Lucas [4]. This fact is explained by varied mechanisms: hypotension, clot formation, the gluing effect of the great omentum, perisplenic hematoma containing the actual bleeding, intact splenic capsule.In 1968, Upadhyaya observed that “often, children with splenic injuries do not exhibit the signs of an important blood loss. It is intriguing that in most children with splenic lesions the bleeding had stopped by the time of laparotomy “ .However, employing non-operative treatment for splenic injuries in adults was initially a challenge for surgeons for several reasons: the post-splenectomy sepsis is less frequent and less severe compared to children; structural and vascular splenic changes according to age and possibly the type of force inducing the lesion make a spontaneous hemostatis unlikely; the risk of overlooked associated injuries; the ensuing possibility of delayed rupture of the spleen (DRS), splenosis or post-traumatic cyst  first uses the term of “alternative surgery” to define non-operative management or the selective approach of trauma patients.Patients with traumatic splenic injuries can be treated surgically or conservatively according to the surgeon’s or hospital’s characteristics. Britt . 54.8% of the patients included in this study have been successfully treated conservatively; there were 10.8% failed approaches with 60.9% of them materializing in the first 24 hrs.Eastern Association for Surgery of Trauma (EAST) infers that “non-operative management of splenic and/or hepatic trauma in hemodinamically stable patients is feasible” :The standard criteria for NOM are .The only absolute indication for emergency laparotomy is hemodynamic instability ; Garber [18] is the author of a multicentric retrospective study, made in Ontario (Canada) which validates that NOM is the preferred therapeutic method (in 69% of patients), followed by splenectomy (28%) and splenorrhaphy (4%) in non-trauma centers and 65%, 33% and 2% respectively in trauma centers. The incidence of NOM has increased from 59% (1991) to 75% (1994) and that of splenectomy has decreased from 35% (1991) to 24% (19914). The incidence of splenorrhaphy has significantly dropped from 6% to 1%.“NOM for blunt splenic injuries replaces splenorrhaphy which was the usual method for preserving the spleen” [3].Even 2 units of transfused blood during the first 48 hrs (in order to maintain a HGB level above 8 g/dl) is compatible with a successful NOM .• no orifice/multiple orifice bleeding [19]. Kluger [20] performed an experimental study on young rats and adult rats in order to clear up the cellular mechanism of splenic scarring after trauma and the influence of patient’s age on the success of NOM.It was initially considered that patients undergoing NOM or splenorrhaphy require bed rest for 1 week and avoidance of physical activity for 6 months; the experimental studies performed on dogs and pigs by Dulchavsky and co. showed that splenic scarring consists of an extensive capsular fibrosis and fibrous reaction at splenorrhaphy site and paralleling intrasplenic septs  conducted a study that included children with grade I-II splenic injuries with complete resolution on CT scans after 4 months from the initial injury; for severe lesions the healing time is extended to over 6 months for grade III and over 11 months for grade IV injuries.Accelerated splenic healing that grants a successful NOM in children and young adults is explained by this early accumulation of myofibroblasts at the lesion site. Benya [The author considers a complete resolution on CT scan when there are no abnormal areas in or around the spleen or when there is a mild residual deformation of the splenic outline (without the obvious presence of a hematoma/ perisplenic fluid collection).22].Patients of at least 50 years of age do not represent a contraindication for NOM, although they are at risk for an unsuccessfull conservative approach [23] predictive parameters for a successful NOM include:According to Longo, Uranüs and Sartorelli  states that 33% of the patients with blunt aortic injury have associated simultaneous hepatic/ splenic lesions. Recent NOM protocols for splenic injuries debunk the “removal of spleen from the equation” myth. Santaniello’s study shows that minor splenic injuries (grade I-II) associated with aortic lesions pose a minimum/no risk for anticoagulation therapy. In this article’s editorial Kenneth Mattox disagrees upon unrecognized these findings when dealing with aortic injury associated with major splenic lesions.Blunt aortic injuries accompany hepatic and splenic lesions in 15-20% of cases ; Santaniello’s study [23] considers that the outcome of NOM in multiple parenchymal trauma patients is not different from that of NOM in unique organ involvement. Furthermore, NOM in patients with associated brain injuries to hepatic/ splenic lesions is safe . Garber [18] observed that chest injuries account for most of the associated lesions (77%), followed by head injuries (59%).Sartorelli [17] quotes 2 articles written by Morgenstern and published between 1983 and 1979. Morgenstern and Uyeda (1983) assert that “splenic hemostatis is tempered by age, children and young adults having functional smooth muscle tunic and elastic tunic” whilst elderly patients exhibit structural changes that “restrict the contraction and retraction of damaged blood vessels within the splenic parenchyma”.An age over 55 years was considered a criterion for an unsuccessful NOM . Why? Elderly patients have diminishing biological reserves; structural alterations concordant with age make a spontaneous hemostasis unlikely, increased splenic frailty. In an attempt to decipher these statements, Barone [23] reported favorable results for NOM in 83.3% of all patients >55 years old, similar to those conveyed by Barone (83%- 17), Myers [26], Brasel (71% -15) and Cocanour [12]. Furthermore, Clancy [27] declared that the percentage of conserved spleens in patients over 65 years of age is similar to that of younger patients (40 patients over 65 years of age have been treated successfully by NOM). It is not the age but the grade of splenic injury that increases the risk of failure for NOM [28]. The use of BOAST (Bedside Organ Assessment with Sonography for Trauma) as well as permanent and careful monitoring of these patients ensures the success of favorable outcome with NOM [29].In 1979, Morgenstern and Shapiro suggested that splenorrhaphy should be contraindicated in elderly patients. In 1964, Gross observed the structural distinction between the splenic capsule in young adults and elderly patients, stating that “after the age of 60 years the splenic capsule is thickening”. Perhaps Gross’s studies should be reviewed and set as a standard protocol for NOM in elderly patients. (Barone-17). Sartorelli [30] old age has no influence on the final outcome of elderly multiple trauma patients; trauma is not only a disease of the young and “age is the problem, not the injury”. According to Frizis,  and Keller’s [25] juvenile studies did not warrant the existence of undiagnosed complications in children. Rozycki’s study [29] corroborates Archer’s findings, including for patients with a GCS ≤ 8, stating that ”NOM is not only perfectly feasible in patients with severe brain damage, but efficient and safe”. According to Pal [32] the CT scans represent a very effective diagnostic method for hemodinamically stable patients with altered mental status and equivocal abdominal exam, having a sensitivity of 97.7%, a specificity of 98.5% and an overall accuracy of 99.4%. Authors consider that DPL is not necessary in this group of patients. 12] considers that brain injuries are not a contraindication for NOM.Archer’s results  are confirmed by the rate of success of NOM in patients with GCS<13 (93%). Likewise, Cocanour [23] about multiple intraabdominal parenchymal injuries established a rate of success for NOM of 94.1%, therefore confirming its safety; similarly, Goan [14] considers that NOM in patients with hepatic lesions is secure when careful clinical and imagistic monitoring is provided. Sartorelli’s study [The severity of splenic injury- it appears that NOM is effective in splenic injuries with an average lesional AAST score of 3 [33]. There are a few studies [34]  signaling the fact that splenic injuries have an unpredictable progress and proving there is no obvious correlation between the anatomical lesion severity and clinical outcome. Velmahos debated these results based on his conclusions: AIS is a flawed system of staging intra-abdominal visceral injuries; a useful prediction model should be simple.The severity of hemoperitoneum- it is considered to be correlated with the injury score; Hiatt and Federico (cit. 14) considered the exact opposite to be true.Blood transfusions > 4 units; all patients included in Sartorelli’s study with early failure of NOM required more than 4 units of transfused blood. A hemoglobin level of < 9 g/dl and a heart rate of > 100 beats per minute is an indicator for blood transfusion.Recent protocols for NOM are applicable in all multiple trauma patients with splenic injuries (but without hemorrhage), requiring more than 4 units of transfused units  only in trauma centers. It is important to remember that prolonged bleeding may cause clotting disturbances, affecting the overall outcome of NOM, thus emphasizing the importance of an accurate clinical assessment.Multiple transfusions are actually the hallmark of failed NOM.36] consider that pre-existent splenic diseases do not represent an absolute contraindication for NOM . The splenomegaly induced by tropical diseases  require a conservative approach in the event of a trauma (NOM or splenorrhaphy). In Papua, New Guinea malaria is endemic with a high prevalence of ruptured pathological spleen but with a high preservation rate of over 70% (Waters). Guth and Patcher [37]. NOM failure is explained by altered spontaneous hemostasis associating with pre-existent portal hypertension syndrome (which leads to increased hydrostatic pressure within the parenchyma); there is also a clotting factor deficit in decompensated hepatic cirrhosis with a subsequent coagulopathy. Therefore, the mortality rate is directly correlated with increased PT values (prothrombin time), high lesion score and low serum albumin levels. Coagulopathy is a risk factor for a trauma patient with cirrhosis . It is imperative to operate to stop the bleeding if the patient has a pre-existent coagulopathy worsened by the ongoing hemorrhage. When preexistent coagulopathy is the one responsible for the bleeding following trauma, then the bleeding disorder should be tackled first and then decide whether or not surgical intervention is still required. Fang considers that cirrhosis is a contraindication for NOM.92% of all the patients with cirrhosis had an unsuccessfull NOM with 55% of fatal cases after surgery (splenectomy as a consequence of failed NOM) [38].Patients with a prolonged PT should not be approached by NOM in case of splenic trauma even if cirrhosis is not present [Religionrepresents an important factor when treating splenic injury. Zieg and co. [39] presented the case of a type A hemophiliac patient, a Jehovah witness, with splenic trauma and favorable NOM outcome that was treated with recombinant factor VIII. There are 10 cases in English literature of hemophiliac patients and splenic trauma out of whom, 3 had an excellent outcome for NOM.40] which are basically criteria for a more cautious attitude when assessing and establishing the adequate treatment :We now present the relative contraindications for NOM .• another associated lesion interfering with the splenic lesion and possibly requiring surgical intervention ; in 1.7% splenic injury is associated with diaphragmatic lesion (Miller-41) and less than 1% of patients with blunt abdominal trauma exhibit hollow viscus injury [43];• age>55 years [• diseased spleen.The only absolute contraindication is represented by hemodynamic instability.45] are:The benefits of NOM .Allen and co (cit. 46) observed that 2.3% of NOM patients have had other associated injuries that were initially overlooked and required surgery later on , but with many intra-abdominal complications. In Sartorelli’s study overlooked hollow viscus injuries totalized 0.8% of all cases  identified 4 independent risk factors for an unsuccessfull NOM: splenic injury severity score, hemoperitoneum of over 300 ml, positive FAST, necessary blood transfusions. Statistically speaking, when all 4 factors are present, NOM will fail in 96% of cases.Delayed surgical exploration could be increase the risk of hemorrhagic shock, major blood disorders, excessive blood transfusions and potential death. In 90% of cases the failure of NOM is evident in the first 50 hrs from the initial insult. Velmahos [26], Uranus [48] and Wisner [49] pinpointed the following criteria for mandatory emergency surgery:Meyers  complications following NOM occurred in 40% of cases and consist of:In Velmahos’s study  the minimum time period necessary for a patient to be included in NOM protocol is 3 hrs.According to Velmahos [22] with subsequent prolonged hospital stay (an average of 11.2 days). 67% of patients with unsuccessful NOM had contrast blush  [40]. Therefore, he concluded that the risk for failing NOT when contrast blush is present is 24-fold increased.The time interval between onset and reported NOM failure ranged between 6 and 94 hrs [11].NOM failure can be explained by complications and by the constant pressure physicians find themselves to discharge patients as soon as possible; some failures are evident after discharge which means it is very important to identify any problem before that. Velmahos identified 2 independent risk factors for failing NOM: splenic injury ≥ 3 and more than 1 unit of transfused blood. When both factors are present NOM failing rate is as high as 97%; when none of these factors is present then NOM failing rate is 3% [54]. In Fang’s study [37] this rate was of 21.9% because 92% of his patients had liver cirrhosis.Unsuccessful NOM rate ranges between 2% and 31%.[Gavant’s and Federle’s retrospective studies (cit. 44) showed that contrast extravasation/ post-traumatic vascular injuries (contrast blush) visible on CT scans/ spiral CT scans with IV contrast are usually associated with an increased rate for unsuccessful NOM .40].Failing NOM in adults is equivalent with increased blood transfusions (with its risk) and impending surgery [Successful NOM55]In adults it ranges between 61.5% and 97%. [36] reports the following results: 53% in grade II injuries; 29% in grade III; 4% in grade IV; 1% in grade V lesions. The high percentage (97%) reported by Sclafani [55] is subsequent to the use of angiography and proximal angioembolization. NOM is successful in 97% of cases in children no matter the injury score (Velanovich cit. 8).Pachter [Hospital stay22].It varies between 3 to 7 days when no other injuries are present to elicit a prolonged stay [Discharge recommendations [56]:Grade I-II lesions:• o Avoidance of strenuous activities and sport ,o Avoidance of construction work for 6-8 weeks;o Light activities  2 weeks after the initial injury.o CT scan/US will be performed only if the clinical exam requires it.Grade ≥III lesions :• o Minimal activity for 1 week;o Light activity 4-8 weeks;o Avoidance of strenuous activities and sport for 10-12 weeks.Grade IV, V lesions:• o Avoidance of strenuous activities and sport for > 3 months.o Mandatory CT scans or US.Splenic angiography (diagnostic and therapeutic)57]. Angiography can have a diagnostic purpose as well as therapeutic (vascular embolization and hemostasis).Recent NOM protocols for splenic trauma include angiography (diagnostic and therapeutic) as an efficient alternative [58].The first angiographic embolization used Gelfoam  and temporary balloon occlusion  and were performed for hemostatic purposes prior splenectomy [Vascular lesions visible on angiography are [60]:• contrast extravasation inside or outside of spleen;• vascular damage of terminal arteries (complete vascular transection);• intraparenchymal arterio-venous fistula;• intrasplenic pseudoaneurysm;• vascular compression by subcapsular hematoma;• variable degree of devascularization and irregularities in contrast filling .Indications for splenic angiography [62]:• grade 3, 4, 5 splenic injuries;• vascular lesions visible on initial CT scan;• active bleeding upon CT scan or contrast blush in a hemodinamically stable patient (upon repeated CT scans);• unexplained decrease of hemoglobin level when no other lesions are present.Splenic angioembolization (SAE) can be:distal (supraselective)55] considers that the preservation of immunological functions is compatible with this procedure and even splenorrhaphy is facilited in case of surgical intervention.• proximal (splenic artery)- achieved by using metal spirals (coils). It produces hemostasis by decreasing the blood flow and intrasplenic pressure; the viability of the remaining spleen is ensured by collateral blood flow . Sclafani [• Combined.61].Diagnostic and therapeutic (embolization) angiography is performed after CT scans showed intrasplenic vascular damage. Embolization is carried out only if there is angiographic confirmation of the lesion [63] also believes that “delayed vascular emergencies” (term first introduced by the Memphis group) are basically delayed diagnoses that become evident when performing angiography for severe splenic injuries . The Memphis group  proved that initial CT and angiographic scans can skip vascular injury due to arterial spasm at the moment of the examination but can later become clinically detectable; spiral CT scans identified 80% of all vascular lesions that were initially unnoticeable . The only statistically significant failure risk for NOM is the arterio-venous fistula which is treated not only by proximal SAE but by a more direct approach-distal SAE [64]. Second-look angiography is useful in recurrent bleeding and after an initially negative angiography (10%) (63 Haan). Haan employed preferentially distal SAE for small grade lesions and combined SAE for severe injuries . Haan [65]:The conclusions inferred by Haan’s study are .SAE is a useful and efficient method for NOM but it is necessary in only 7% of cases [SAE indications [66]:Proximal SAE: it is indicated in hilar lesions;• o >3 distinct peripheral vascular lesions;o the injury affect more than 50% of the splenic parenchyma.Selective SAE: limited vascular injuries. It is proficient because it allows proper hemostasis and adequate perfusion to remaining organ.• Combined SAE: for multiple vascular injuries (high injury scores).• It is recommended to perform multiple CT scans after SAE in order to monitor the vascular damage, pseudoaneurysm formation, size of infarcted area and existence of localized infection (splenic abscess).SAE represents an elegant alternative and is now part of all NOM protocols in trauma centers.SAE induced complications [71]o Major complications (19%-28.5 %)o Bleeding- it is the most common complication caused by delayed diagnosis of pseudoaneurysms and late pseudoaneurysm formation;o Overlooked injuries: usually diaphragmatic, pancreatic;o Infection- splenic abcess, sepsis;o Splenic atrophy;o Iatrogenic arterial damage;o Acute renal failure after contrast administrationo Deep venous thrombosis.o Minor complications (23%-61.9 %)o Splenic infarction: in 27% of cases after distal SAE and in 20% of cases after proximal SAE. Most of them are asymptomatic but it is believed that a splenic infarction is significant when a devascularization of >25% of splenic parenchyma occurs (upon repeated CT scans);o Migration of embolic material: spiral that migrates in proximal SAE needs extraction.o Angiographic vascular dissection: it is usually asymptomatic and non-occlusive .o Vascular damage when inserting the catheter( arterio-venous fistula)o Persistent pain at the catheter insertion siteo Hematoma on the puncture site. o Post-emobolization syndrome- includes symptoms such as general discomfort, fever, local pain and/or leucocytosis which generally persist for 3-5 days; if blood cultures are negative and no signs of infection are present then it is considered to be a rather benign complication. It is self-limiting and it is caused by extensive tissue necrosis or intravascular thrombosis subsequent to a successful embolization.o Pleural and pulmonary complications;o Thrombocytosis;o Allergic reactions to contrast71] 28.5 % of patients had major complications including 4 cases of postprocedural bleeding that might be attributed to the use of Gelfoam as an embolizing agent. In Shih series [CT findings after SAE [72]Areas of spleen infarction appear after SAE that have certain characteristics:• Infarction appeared in 63% of cases after proximal SAE, but only in 20% of cases the area extended over more than 50% of splenic parenchyma.These areas are usually small in size, multiple, situated at the splenic border and heal completely.• Infarction areas after distal SAE occur in 100% of cases with only 9% of cases affecting over 50% of the splenic parenchyma. They are usually a unique, large area immediately beneath the embolized blood vessel and heal completely in most cases.Statistically speaking distal SAE triggered more splenic infarctions than proximal SAE.• Combined SAE trigger splenic infarction in 71% of cases; in 20% of them more than 50% of splenic parenchyma was affected.When air bubbles are visible within the splenic parenchyma it is necessary to rule out a splenic abscess. Likewise, the presence of air-fluid level in a subcapsular collection suggests the development of a splenic abscess (which can be drained percutaneously).71] showed that SAE dysregulates the nuclear factor (NF)-kB translocations and aggravates the cytokine response in patients with spleen injury. Nakae [73], in a recent study finds that splenic preservation  not have advantages over splenectomy in immunologic indices including levels of IgM and 14 serotypes of anti-Streptococcus Pneumoniae antibodies. Tominaga’s results [74] suggest that the immunologic profile of embolized patients is similar to controls. He tested IgM, IgG, C 3 complement, complement factor B, CD3, CD4, CD8 (helper and suppressor T-cells), complete blood counts and HIV status and found that splenic immunocompetence is preserved at a minimum 3 months after embolization. Consequently the immunization may not be necessary. However, larger studies are useful to make definitive vaccination recommendations.The immune alteration after SAE remains unclear. In a recent study Shih et al. [76]. When dealing with splenic trauma NOM is the rule and not the exception [9] with its success relying upon adequate clinical assessment.NOM represents an effective and safe alternative for selected patients with splenic trauma [The utilization of mobile digital subtraction angiography directly into the trauma resuscitation area shortened the time required to restore normal physiology  (Morozumi-77).79] identified 2 independent predictors of NOM failure: grade V blunt splenic injuries and the presence of a brain injury. Jeremitsky [80] has evaluated the role of splenic embolization as an adjunct for NOM and found that it increased splenic preservation success. In his opinion the markers of greater injury severity are associated with an increased risk for NOM failure and substance abuse represents an independent predictor of NOM failure. Splenic angioembolization represents a valid and effective option in patients with severe splenic injuries and/or active bleeding .Recent studies suggests that early surgical intervention should be considered in blunt splenic injured patients with contrast extravasation and ISS ≥ 25 (Fu-78); Velmahos [The use of splenic angioembolization for traumatic injuries was initiated at our institution in 2009. The first successful splenic angioembolization in trauma in Romania was performed at Emergency Hospital Bucharest and published in “Chirurgia” in 2010 (Venter-83).As a conclusion: “ in a hemodinamically stable patient, with a major splenic injury, proximal SAE has the same effectiveness as splenectomy but with a low number of units of transfused blood and a low mortality rate” -Salvatore Sclafani.Acknowledgement. Our entire gratitude to Dr. Ioana-Iftimie Nastase for helping us translate this manuscript.


A process implicated in cancer metastasis that describes the conversion to a more invasive phenotype is epithelial-mesenchymal transition (EMT). In this study we show that EGF-induced EMT in MDA-MB-468 breast cancer cells is associated with a reduction in agonist-stimulated and store-operated Ca2+ influx, and that MDA-MB-468 cells prior to EMT induction have a high level of non-stimulated Ca2+ influx. The potential roles for specific Ca2+ channels in these pathways were assessed by siRNA-mediated silencing of ORAI1 and transient receptor potential canonical type 1 (TRPC1) channels in MDA-MB-468 breast cancer cells. Non-stimulated, agonist-stimulated and store-operated Ca2+ influx were significantly inhibited with ORAI1 silencing. TRPC1 knockdown attenuated non-stimulated Ca2+ influx in a manner dependent on Ca2+ influx via ORAI1. TRPC1 silencing was also associated with reduced ERK1/2 phosphorylation and changes in the rate of Ca2+ release from the ER associated with the inhibition of the sarco/endoplasmic reticulum Ca2+-ATPase (time to peak [Ca2+]CYT = 188.7±34.6 s (TRPC1 siRNA) versus 124.0±9.5 s (non-targeting siRNA); P<0.05). These studies indicate that EMT in MDA-MB-468 breast cancer cells is associated with a pronounced remodeling of Ca2+ influx, which may be due to altered ORAI1 and/or TRPC1 channel function. Our findings also suggest that TRPC1 channels in MDA-MB-468 cells contribute to ORAI1-mediated Ca2+ influx in non-stimulated cells.In addition to their well-defined roles in replenishing depleted endoplasmic reticulum (ER) Ca For example rapid Ca2+ influx through voltage-gated Ca2+ channels at the neurological synapse regulates the exocytosis of neurotransmitters from presynaptic neurons 2+ entry 2+ reserves following Ca2+ store depletion, include the store-operated Ca2+ channel pore-forming subunits ORAI1, ORAI2 and ORAI3 2+ sensors STIM1 and STIM2 2+ influx by other proteins has also been identified; this includes regulation by pre-STIM2 2+ store-independent mechanism important in some breast cancers. Although ORAI proteins are now recognized as the central components of store-operated Ca2+ entry, the canonical-type TRP channels (TRPCs), in particular TRPC1, are also reported to interact with STIM proteins and in some cell types appear to regulate agonist-stimulated and store-operated Ca2+ entry 2+ reserves, such as the ion channels involved and their activation, may therefore be context-dependent and vary between cell types.Plasma membrane Cain vitro and metastasis formation in vivoIn breast cancer cells, ORAI1 regulates processes important for carcinogenesis and is enriched in some breast cancer cell lines relative to non-tumorigenic breast epithelial cells A process implicated in breast cancer metastasis is the transition from an epithelial state to a more invasive mesenchymal phenotype, termed epithelial-mesenchymal transition (EMT) 2+ influx pathways, including elevated store-operated Ca2+ entry 2+ signaling requirements are also likely to differ as cells undergo EMT and transition from an epithelial phenotype to a mesenchymal state. Indeed, EGF-induced EMT in MDA-MB-468 breast cancer cells is associated with a remodeling of purinergic receptor-mediated Ca2+ signaling 2+ influx 2+ entry in a well characterized model of EMT mediated by EGF in MDA-MB-468 breast cancer cells. A greater understanding of the changes in Ca2+ influx associated with EMT in breast cancer cells may help identify novel therapeutic targets for the control of breast cancer metastasis.A cellular conversion comparable to EMT that is particularly important in atherosclerotic vascular disease, involves the transformation of vascular smooth muscle cells from a contractile (quiescent) to a synthetic (proliferative) phenotype 2 and were routinely screened for mycoplasma contamination . Cyclopiazonic acid (CPA), trypsin and ATP were purchased from Sigma Aldrich. Fluo-4 AM and BAPTA were purchased from Invitrogen. TaqMan Gene Expression Assays (Applied Biosystems) included: human Twist1 (Hs00361186_m1), Snail (Hs00195591 _m1), fibronectin (Hs01549976_m1), vimentin (Hs00185584_m1), ORAI1 (Hs00385627_m1) and TRPC1 (Hs01553152_m1). Dharmacon On-TARGET plus SMARTpool™ siRNAs were used at a final concentration of 100 nM: non-targeting (D-001810-10-05), TRPC1 (L-004191-00-0005) and ORAI1 (L-014998-00-0005). The following Cell Signaling antibodies were used: mouse monoclonal anti-phospho-ERK1/2  at 1∶2000 and rabbit polyclonal anti-ERK1/2 (9102) at 1∶1000, incubated overnight; and from BioRad, anti-mouse horseradish peroxidase-conjugated and anti-rabbit horseradish peroxidase-conjugated secondary antibodies .MDA-MB-468 2+ responses in MDA-MB-468 and MDA-MB-231 cells were assessed using a fluorometric imaging plate reader . MDA-MB-468 breast cancer cells were seeded at 3×104 cells/well in 96-well microplates (Corning Costar). Cells were serum-deprived  (2+ assays in siRNA-transfected MDA-MB-468 cells a seeding density of 1.5×104 cells/well was used and 4×103 cells/well for MDA-MB-231 cells. Cells were loaded for 1 h at 37°C with 2 µM Fluo-4 AM Ca2+ indicator in a solution containing 500 µM probenecid and 5% (v/v) PBX Signal Enhancer (BD Biosciences) in physiological salt solution  2+-free PSS. Intracellular Ca2+ measurements were performed with 470/95 and 515/75 nm excitation and emission filters. Data analysis was performed using ScreenWorks Software . The ratio of influx to store release was calculated as follows: [peak 2 amplitude/peak 1 area under the curve (AUC)]*1000. The % reduction in Ca2+ influx  was calculated by first subtracting the baseline, as follows: [(siNT peak 2 amplitude −1)/(siORAI1 peak 2 amplitude −1)]*100%.Global cytosolic CaS, 24 h) , 2 & 3, S, 24 h)  & 3 [19]2+ oscillations, MDA-MB-468 cells were grown to confluency and cells were loaded with Fluo-4 AM as described above. Images were acquired using a Nikon Eclipse TE 300 inverted epifluorescence microscope with a 40× oil objective and 488/550 nm excitation/emission wavelengths. A 33 ms exposure time and 2×2 binning was used; images were acquired every 5 s for approximately 120 cycles prior and subsequent to BAPTA (1.7 mM) addition.For live cell imaging of Ca4 cells/well. Cells were serum deprived and treated with EGF (50 ng/mL) for 12 h. Total RNA was isolated using the Qiagen RNeasy Mini Kit. Omniscript RT (Qiagen) was used for reverse transcription and resulting cDNA was amplified using TaqMan Fast Universal PCR Master Mix with TaqMan Gene Expression Assays.For quantitation of changes in gene expression as a consequence of EGF-induced EMT, MDA-MB-468 cells were plated in 6-well plates at seeding density of 8.5×10plus SMARTpool™ siRNA was used as per the manufacturer's instructions. This product consists of four rationally designed siRNAs with both sense and anti-sense strand modification to reduce off-target effects 4 cells/well in antibiotic-free media. DharmaFECT4 transfection reagent was used at a concentration of 0.2 µL/well. Knockdown for Ca2+ assays and protein isolations was confirmed 48 h post-transfection as per manufacturer's instructions, given that changes in mRNA levels are expected to precede changes in protein expression and functional responses (assessed 96 h post-transfection).Dharmacon ON-TARGETCell extracts were prepared using protein lysis buffer supplemented with protease and phosphatase inhibitors (Roche Applied Science). Gel electrophoresis was performed under reduced denatured conditions using 4–12% gradient Bis-Tris gels with MOPS running buffer (Invitrogen), and transferred to PVDF membranes. Membranes were blocked for 1 h at room temperature using 5% (w/v) skim milk powder in phosphate buffered saline (PBS) containing 0.1% (v/v) Tween-20. Bands were visualized with chemiluminescence using Super-Signal West Dura substrate (Thermo Fisher Scientific). Image acquisition was performed on a VersaDoc Imaging System (BioRad) and quantitation using ImageJ , as per the gel analysis method outlined in the ImageJ documentation. Brightness and contrast adjustment were applied uniformly to all gels.3 cells/well and treated with siRNA for 96 h. Cells were incubated with EdU (10 mM) for 1 h, fixed with 3.7% (v/v) formaldehyde in PBS and permeabilized with 0.5% (v/v) Triton X-100. Cells were then incubated with the Click-iT reaction cocktail  for 30 min and DAPI (400 nM) for 90 min. Cells were imaged with a 10× objective using the ImageXpress Micro automated epifluorescence microscope (Molecular Devices) based on the following excitation and emission wavelengths: 377/50 and 447/60 nm for DAPI and 531/40 and 593/40 for EdU (Cy3). Cell number and percentage of EdU positive cells was measured using the multi-wavelength cell scoring application module .MDA-MB-468 cells were seeded in 96-well imaging plates (BD Biosciences) at 5×10Statistical analysis was performed using GraphPad Prism (v5.03 for Windows). Statistical tests used in this study are outlined in each figure legend.2+ entry in MDA-MB-468 breast cancer cells compared to the previously characterized MDA-MB-231 breast cancer cell line 2+ ATPase (SERCA) inhibitor) in nominally Ca2+-free conditions, to assess ATP-stimulated Ca2+ entry and store-operated Ca2+ entry, respectively. ATP and CPA both produced an initial (peak 1) transient increase in cytosolic free Ca2+ levels ([Ca2+]CYT) and Ca2+ influx (peak 2) following the re-addition of extracellular Ca2+ in MDA-MB-468  was observed in MDA-MB-468-EMT cells  (2+ entry with EGF-induced EMT. Similar reductions in agonist-stimulated Ca2+ entry were observed with the protease-activated receptor-2 (PAR-2) activator trypsin (2+ entry as assessed with the SERCA inhibitor CPA (A reduction in ATP-mediated CaMT cells . We quank 1 AUC) . These r trypsin  as well itor CPA .2+]CYT responses we quantified the time to reach peak [Ca2+]CYT levels after ATP, trypsin and CPA addition (P<0.05); this may be explained by altered purinergic-mediated signaling with EGF-induced EMT, as previously reported 2+]CYT was observed with trypsin-activation. However, a pronounced delay was seen in the time taken to reach peak [Ca2+]CYT with CPA treatment in MDA-MB-468 cells after EMT induction . These results are reflective of a substantial change in the nature of the response to CPA, and altered ER Ca2+ homeostasis associated with EGF-induced EMT.To examine the nature of the [Caaddition . A modes2+ influx with EGF-induced EMT were associated with reduced mRNA levels of TRPC1 and ORAI1 channels. No change in TRPC1 mRNA levels was observed with EGF-induced EMT  in MDA-MB-468 cells (2+ influx in MDA-MB-468 cells.Silencing of both ORAI1 (siORAI1) and TRPC1 (siTRPC1)  inhibite68 cells . These r2+ entry in MDA-MB-468 breast cancer cells (2+ influx (peak 2 relative [Ca2+]CYT response) (2+ entry with siORAI1. Silencing of TRPC1 produced a modest but significant increase in Ca2+ influx (peak 2 relative [Ca2+]CYT response), however, this effect was associated with increased ATP-mediated Ca2+ store release (peak 1) and so was not associated with alterations in agonist-stimulated Ca2+ entry as assessed by the peak ratio (2+ entry mediated by trypsin was greatly attenuated by ORAI1 siRNA (2+]CYT response (2+ entry as assessed by the peak ratio (2+ entry mediated by Ca2+ store-depletion using CPA (Silencing of ORAI1 inhibited ATP-stimulated Caer cells . Quantitesponse)  and the esponse)  showed sak ratio . SimilarI1 siRNA . TRPC1 sresponse ; this waak ratio . Only ORsing CPA . These r2+]CYT with CPA stimulation  or via inhibition of SERCA activity (with CPA) was assessed in two phenotypically distinct breast cancer cell lines—MDA-MB-468 and MDA-MB-231. Both cell lines demonstrated Ca2+ influx via ATP-stimulated and store-operated Ca2+ entry. Surprisingly MDA-MB-468 breast cancer cells also showed pronounced Ca2+ influx in the absence of agents to deplete ER Ca2+ reserves. This non-stimulated Ca2+ influx is not a universal characteristic of breast cancer cell lines 2+ influx. These results suggest that in addition to a remodeling of purinergic receptor-mediated Ca2+ signaling 2+ influx. EGF-induced EMT in MDA-MB-468 breast cancer cells was also associated with reduced agonist-stimulated and store-operated Ca2+ entry, in contrast with a recent study by Hu et al. 2+ entry with TGFβ-induced EMT in MCF7 breast cancer cells. Further studies are therefore required to determine how store-operated Ca2+ entry changes in other cancer cell lines and using other methods to induce EMT, for example hypoxia or via the expression of EMT-inducing transcription factors Ca2+-permeable ion channels are implicated in constitutive Ca2+ influx, including TRP channels with reported basal activity  2+ influx; suggesting that altered activity of these channels may be a feature of EGF-induced EMT. Mechanistically, the many activators of ORAI1, including STIM1 and −2 2+ influx. Reduced non-stimulated influx via TRPC1 may also arise through multiple mechanisms such as altered gene expression, channel gating 2+ influx pathways during this phenotypic switch.An array of Ca2+ influx may depend on a complex interplay between TRPC1 and ORAI1 channels. The 93% inhibition of non-stimulated Ca2+ influx by ORAI1 knockdown would suggest that other plasma membrane Ca2+ channels make up only a small (∼7%) component of non-stimulated Ca2+ influx. However, TRPC1 knockdown inhibited non-stimulated Ca2+ influx by more than 50%. These results suggest that TRPC1 and ORAI1 cooperate to regulate non-stimulated Ca2+ influx, and that TRPC1-mediated regulation of non-stimulated Ca2+ influx is dependent on Ca2+ influx through ORAI1 channels.In addition to these channel-specific mechanisms of regulation, control of non-stimulated Ca2+]CYT after SERCA inhibition, providing some evidence for a role for TRPC1 in the regulation of resting ER Ca2+. Such a result can be explained by the cellular expression of TRPC1 at sites other than the plasma membrane. A non-plasmalemmal distribution for TRPC1 has been shown in human salivary gland cells 2+ leak 2+ channels may promote ER Ca2+ leak, similar to the increased Ca2+ leak reported with ER-localized TRPP2 Ca2+ channels in kidney cells 2+ leak regulated by Bcl-2 2+ leak would trigger Ca2+ influx via ORAI1 to replenish lower ER Ca2+ reserves. Therefore, the non-stimulated Ca2+ influx observed in MDA-MB-468 breast cancer cells, which is sensitive to both TRPC1 and ORAI1 knockdown, may be due to enhanced ER Ca2+ leak through TRPC1, the consequence of which is increased store-regulated Ca2+ influx through ORAI1.Unlike ORAI1 knockdown, silencing of TRPC1 was associated with significant delays in the time to reach peak CYT, as both agents reduced non-stimulated Ca2+ influx. Given that both ERK and upstream Ras-GTPases may localize to the ER membrane 2+ signal regulated by TRPC1.Differences in the functional consequences of ORAI1 and TRPC1 knockdown in MDA-MB-468 cells were also reflected in effects on constitutive ERK1/2 phosphorylation. Knockdown of ORAI1 in MDA-MB-468 breast cancer cells had no effect on the level of constitutively active ERK1/2. This is in contrast to MCF7 breast cancer cells, where ORAI1 silencing almost completely abolishes constitutive ERK1/2 activity 2+ signaling via TRPC1 regulates a constitutive ERK1/2 phosphorylation and cell proliferation in MDA-MB-468 cells breast cancer cells.To explore the possible consequences of TRPC1-mediated reductions in ERK1/2 signaling, we assessed cell proliferation in MDA-MB-468 breast cancer cells with TRPC1 silencing. Recent studies show that sustained (but not transient) ERK1/2 activation is a regulator of S-phase entry in NIH3T3 mouse embryonic fibroblasts 2+ influx in MDA-MB-468 human breast cancer cells and assessed alterations in Ca2+ signaling as a consequence of EGF-induced EMT. MDA-MB-468 breast cancer cells in the epithelial state have a high degree of non-stimulated Ca2+ influx through ORAI1, potentially due to alterations in ER Ca2+ reserves. Our studies suggest that some of the disparities in the reported roles for TRPC1 in store-operated Ca2+ entry may be explained by the context-specific signaling of these channels ; factors which are likely to vary between cell types and experimental conditions. Our studies also define a specific role for TRPC1 in the regulation of the constitutively active pool of ERK1/2 in MDA-MB-468 cells. Given the potential for Ca2+ to regulate processes important in carcinogenesis 2+ channels in cancer cells, and the molecular mechanisms governing their cellular distribution.The current study has characterized mechanisms regulating CaMovie S12+ oscillations in MDA-MB-468 breast cancer cells.Ca Relates to (AVI)Click here for additional data file.


Within a given tumour, only a subset of cells have the capacity to self-renew, give rise to more differentiated progeny and maintain the tumour long-term  using xenotransplantation experiments in immunodeficient mice. Subsequent work has demonstrated that CSCs in most cases of AML comprise a subpopulation of cells that express several cell surface markers in common with normal hematopoietic stem and progenitor cells, as well as other leukaemia stem cell (LSC)-specific surface markers Majeti, . AML LSCLike AML, precursor B-acute lymphoblastic leukaemia (B-ALL) is thought to develop from malignant transformation of immature hematopoietic progenitor cells. By analogy to AML, it has been postulated that a rare LSC population must exist in B-ALL. However, despite considerable investigative effort, prospective isolation and characterization of B-ALL LSC has remained elusive , CD34+/CD19+ and CD34−/CD19+ populations  mice, and vice versa. This is true of the markers CD10 and CD20 as well.In this issue of EMBO Molecular Medicine, Rehe et al  build on»…the findings of Rehe et al suggest that B-ALL cells are not arranged in a strict hierarchy…«ex vivo and then must overcome a xenotransplantation barrier, it is likely that the true LSC frequency is higher. Indeed, in some B-ALL cases, most or even all of the leukemic blasts may be functional LSC. Taken together, the findings of Rehe et al , a component of the telomerase holoenzyme that is required to maintain telomeres during cell division. TERT is expressed in normal hematopoietic stem cells; thus, these findings are consistent with the notion that both CD34+ and CD34− B-ALL blasts retain stem cell function.In the case of AML, LSC were initially isolated based on cell surface marker expression. More recently, AML LSC were shown to have a gene expression profile distinct from the bulk of the leukemic blasts (Eppert et al, The findings of Rehe et al  contrastTaken together, the authors' findings suggest that the hierarchical LSC model of AML does not apply to B-ALL . The autThese findings provide significant insights into the pathogenesis of B-ALL, yet also raise many new questions. For example, if B-ALL subpopulations are not distinct with regard to their leukemogenicity, why do they differ immunophenotypically? Do immunophenotypically defined subpopulations differ in other properties, for example chemoresistance? What do these findings tell us, if anything, about the cell of origin in B-ALL? Does the plasticity in immunophenotype reflect an underlying plasticity of B-ALL blasts? Most fundamentally, why does AML maintain a hierarchy similar to normal hematopoiesis while B-ALL does not appear to follow such a paradigm?


However, the mechanism by which L-type VGCCs couple to the ERK signalling pathway in pancreatic β-cells is poorly understood. In this report, we characterise the relationship between L-type VGCC mediated changes in intracellular Ca2+ concentration ([Ca2+]i) and the activation of ERK, and demonstrate that the sustained activation of ERK (up to 30 min) in response to GLP-1 requires the continual activation of the L-type VGCC yet does not require a sustained increase in global [Ca2+]i or Ca2+ efflux from the endoplasmic reticulum. Moreover, sustained elevation of [Ca2+]i induced by ionomycin is insufficient to stimulate the prolonged activation of ERK. Using the cell permeant Ca2+ chelators, EGTA-AM and BAPTA-AM, to determine the spatial dynamics of L-type VGCC-dependent Ca2+ signalling to ERK, we provide evidence that a sustained increase in Ca2+ within the microdomain of the L-type VGCC is sufficient for signalling to ERK and that this plays an important role in GLP-1- stimulated ERK activation.Glucagon like peptide-1 (GLP-1) is released from intestinal L-cells in response to nutrient ingestion and acts upon pancreatic β-cells potentiating glucose-stimulated insulin secretion and stimulating β-cell proliferation, differentiation, survival and gene transcription. These effects are mediated through the activation of multiple signal transduction pathways including the extracellular regulated kinase (ERK) pathway. We have previously reported that GLP-1 activates ERK through a mechanism dependent upon the influx of extracellular Ca GLP-1R agonists stimulate an increase in the production of cAMP, which potentiates glucose-stimulated rises in intracellular free [Ca2+] ([Ca2+]i), and thus insulin secretion through a number of mechanisms including the inactivation of K+ATP channels. GLP-1 also potentiates glucose-stimulated ERK activation GLP-1 is a hormone secreted from intestinal L-cells in response to nutrients  2+]i, changes in [Ca2+]i were assessed by single-cell epifluorescence microscopy in response to glucose, GLP-1 plus glucose or GLP-1 plus glucose in the presence of nifedipine. Application of 16.7 mM glucose alone caused a rise in [Ca2+]i initiating approximately 5 min after application which, like ERK activation, was potentiated by 10 nM GLP-1  from intracellular stores triggered by Ca2+ entry through L-type VGCCs. Indeed, it has been reported that glucose plus- GLP-1-stimulated ERK activation in INS-1 cells is dependent upon CICR 2+ stores in GLP-1 plus glucose-stimulated ERK activation, MIN6 cells were pre-incubated with ryanodine, to inhibit ryanodine receptors and thus CICR, or thapsigargin to inhibit ER Ca2+-ATPase and to cause ER store depletion , which caused a much lower increase in [Ca2+]i through membrane depolarisation and L-type VGCC channel activation  the open probability of the channel is low and therefore Bay-K 8644 only increases the open probability of a small proportion of L-type VGCCs. These results suggests that a local increase in [Ca2+]i within the vicinity of the L-type VGCC is sufficient to couple to the ERK signalling pathway. To explore this possibility, MIN6 cells were loaded with BAPTA-AM or EGTA-AM that differ in their rate of Ca2+ binding and can reveal information regarding the spatial dynamics of Ca2+ signalling  2+, predicted to be within 20 nm of the site of Ca2+ entry (i.e. the L-type VGCC) To further investigate how L-type VGCCs signal to ERK, MIN6 cells were incubated with Bay-K 8644, an activator of the L-type channel, and changes in the phosphorylation of ERK and [Catermined . Bay-K 8termined  yet caused  whereas the acute activation of ERK is mediated by both local and global rises in [Ca2+].To investigate whether the GLP-1 plus glucose-stimulated ERK activation was also dependent upon local increases in [Careatment . GLP-1 p EGTA-AM . In addit 30 min . This in2+ through L-type VGCC  whereas local increases in [Ca2+] are responsible for the prolonged activation of ERK (30 min post-GLP-1 plus glucose stimulation). This indicates that there are two mechanistically distinct phases of ERK activation. Interestingly, two phases of ERK activation in response to GLP-1 plus glucose have been reported previously: a PKA-dependent pathway mediating rapid and transient ERK1/2 phosphorylation and a second β-arrestin 1-dependent phase The present study shows that GLP-1 plus glucose-stimulated ERK activation in pancreatic β-cells is dependent upon the influx of Caype VGCC  and thatype VGCC , demonstCC  pore forming subunit of the channel v1.2, the LA motif, within the pore of the α1 subunit in a Ca2+-independent fashion 2+] activates the bound CaM, which is then displaced from the LA motif and re-binds to a second Ca2+-CaM binding site (the IQ motif) within the intracellular C-terminal tail of the channel In cultured cortical neurones, the L-type VGCC has been implicated in the activation of ERK via a mechanism that requires calmodulin (CaM) binding to the αThere are several downstream effectors of CaM, which may in turn activate ERK indirectly, including the CaM kinases, CaMKII and IV 2+] close to the VGCCs 2+], thus ensuring that exocytosis is triggered by the localized increases in [Ca2+]i evoked by an action potential. Functional studies using dihydropyridine resistant α1D and α1C channels expressed in pancreatic β-cells indicate that GLP-1 potentiation of glucose-induced insulin secretion is preferentially coupled to the α1D channel in INS-1 cells via a mechanism that requires the activation of ERK1/2 Insulin secretion also depends upon a localized increase in [Ca2+]i or Ca2+ efflux from the ER. Interestingly, based on the studies with BAPTA-AM and EGTA-AM, we provide evidence that elevation of [Ca2+]i within 20 nm of the site of entry through L-type VGCCs is sufficient for ERK activation and plays an important role in ERK activation in response to GLP-1.In conclusion, we show that that the sustained activation of ERK in response to GLP-1 in the presence of an elevated glucose concentration requires the continual activation of the L-type VGCC yet does not require a sustained increase in global [CaNifedipine, dantrolene, ryanodine and thapsigargin were purchased from Calbiochem . Ionomycin was purchased from Tocris Bioscience . EGTA-AM and BAPTA-AM were purchased from Invitrogen . All other chemicals (unless stated) were obtained from Sigma-Aldrich .Mouse Insulinoma-6 cells (MIN6 cells) 3, 2.5 mM MgCl2, 2.5 mM CaCl2, 20 mM HEPES, pH 7.4). The cells were then incubated for 1 h at 37°C in KRB containing 1 mM glucose prior to treatments for the times indicated. All treatments were stopped by the addition of ice cold lysis buffer . The lysates were then centrifuged for 10 min at 16,000×g, supernatants kept, and total protein concentrations determined by the Bradford assay ). The protein lysates were stored at Prior to treatment, the medium was removed and cells washed twice with HEPES-buffered Krebs-Ringer bicarbonate (KRB) , prepared in dye-loading buffer  and 0.1% Pluronic F-127) for 30 min at 20°C. The cells were then washed and further incubated for 10 min in KRB to allow de-esterification of the indicator. Changes in fluorescence were monitored using a Nikon Diaphot 200 inverted epifluorescence microscope with an oil immersion objective (40×) and a SpectraMASTER II module . Cells were excited alternatively with light at 340 nm and 380 nm with emission detected at 510 nm. Raw fluorescence data were exported to Microsoft Excel and expressed as the 340/380 ratio for each cell and further analysed in GraphPad Prism. A ratio value was collected each 2 s. Data are reported as either the 340/380 ratio (R) or R/R0 where R0 is the basal (unstimulated) ratio.For Ca2+ fluorescence measurements were performed using a NOVOstar microplate reader  equipped with a pipettor and 2 injectors. MIN6 cells in a 96-well plate format were washed twice with KRB buffer then loaded with 2 µM fluo-4-AM as described above. Cells were washed with KRB after fluo-4-AM loading. Details of treatments are provided in the figure legends. Fluo-4 was excited using a high energy xenon flash lamp through a 485 nm filter and emission detected by a photomultiplier after passing through a 520 nm filter. Measurements were collected at 2 s intervals. Raw fluorescence data were exported to Microsoft Excel and expressed as F/Fo  and further analysed in GraphPad Prism. A single well of cells without fluo-4-AM loading was used as an auto-fluorescence control and subtracted from the fluorescence values obtained. Data are reported as the average of at least three independent experiments ± S.E.M..Population-based Ca2SO4, 10 mM NaCl, 10 mM KCl, 1 mM MgCl2, and 5 mM HEPES (pH 7.35 with KOH) and then back-filled with the same solution including amphotericin B (240 µg/ml). The bath solution contained: 140 mM NaCl, 3.6 mM KCl, 2 mM NaHCO3, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 2.6 mM CaCl2, 5 mM HEPES and 2.5 mM D-glucose (pH 7.4 with NaOH).MIN6 cells were preincubated for 1 h in KRB buffer prior to electrophysiological measurements. Membrane potential was recorded using an Axopatch 200 amplifier ). Signals were digitized using a DIGIDATA 1322A interface, and records acquired and analysed using pClamp9.2 software ). All electrophysiological recordings were made at 28°C using the perforated-patch, whole-cell configuration. Patch pipettes (3–5 MΩ) were made from thick-walled borosilicate glass. Pipettes were initially dipped in a solution containing: 76 mM KStatistical differences between multiple groups were analysed by either one-way or two-way analysis of variance (ANOVA), followed by post-hoc analysis. Comparison between two sets of data have been analysed by Student's t-test. Statistical analyses were performed in GraphPad Prism and significance was only confirmed when p<0.05.


B subtype or the slightly less abundant δC subtype. The CaMKIIδB mRNA sequence contains a 33bp insert not present in δC that codes for an 11-amino acid nuclear localization sequence. This review focuses on the localization and function of the CaMKIIδ subtypes δB and δC and the role of these subtypes in arrhythmias, contractile dysfunction, gene transcription, and the regulation of Ca2+ handling.In this review we discuss the localization and function of the known subtypes of calcium/calmodulin dependent protein kinase IIδ (CaMKIIδ) and their role in cardiac physiology and pathophysiology. The CaMKII holoenzyme is comprised of multiple subunits that are encoded by four different genes called CaMKIIα, β, γ, and δ. While these four genes have a high degree of sequence homology, they are expressed in different tissues. CaMKIIα and β are expressed in neuronal tissue while γ and δ are present throughout the body, including in the heart. Both CaMKIIγ and δ are alternatively spliced in the heart to generate multiple subtypes. CaMKIIδ is the predominant cardiac isoform and is alternatively spliced in the heart to generate the CaMKIIδ Calcium/calmodulin dependent protein kinase II (CaMKII) is a multimeric enzyme consisting of distinct subunits encoded by four different genes known as CaMKIIα, β, γ, and δ. These genes have a high degree of sequence homology but show differential tissue expression. CaMKIIα and β are predominantly expressed in neuronal tissue while γ and δ are present throughout the body, including the heart . CaMKIIδ1-4. CaMKIIδ2, and CaMKIIδ3 were shown to be identical except for the insertion of an 11-amino acid sequence in the variable domain of CaMKIIδ3, the more abundant of the two subtypes in the heart (2+/CaM). They refer to the predominant cardiac subtypes as CaMKIIδB and CaMKIIδC (the convention that will be used in this review), which correspond to the δ3 and δ2 subtypes, respectively. The structure of these proteins is shown in Figure 1. CaMKIIδB and δC possess similar catalytic activity and sensitivity to Ca2+/CaM. Furthermore, both subtypes can undergo autophosphorylation and acquire a similar degree of Ca2+-independent or autonomous activity  is also present in some splice variants of CaMKIIα and γ; this conservation suggests an important function .The 11-amino acid insert in CaMKIIδfunction . Srinivalization . A simillization . FurtherB to δC in a multimer could regulate the localization of the holoenzyme. This has been demonstrated experimentally. When CaMKIIδB and δC are cotransfected into fibroblasts or NRVMs, the localization of the expressed protein can be shifted in accordance with the ratio of the expressed CaMKIIδ subtypes, i.e., highly expressed δC sequesters δB in the cytosol and blocks its nuclear localization  immediately adjacent to the NLS of CaMKIIδB was shown to be a site of phosphorylation by CaMKI and CaMKIV in vitro. Phosphorylation prevents association of δB with the NLS receptor m-pendulin and thus limits localization of CaMKIIδB to the nucleus -mediated ASF/SF2 phosphorylation has been correlated with alternative splicing of CaMKIIδ in heart and brain  injury . In huma failure . The alt failure . Specifind brain . Additiond brain . CaMKIIδmulation . The RNAmulation  and factB in NRVMs induced atrial natriuretic factor (ANF) expression and led to increased myofilament organization, both hallmarks of cardiac hypertrophy, while expression of CaMKIIδC did not , GATA4, and heat shock factor 1  mice (B under the control of the cardiac-specific α-myosin heavy chain (α-MHC) promoter, demonstrate the enhanced expression of hypertrophic markers observed in NRVMs expressing CaMKIIδB. CaMKIIδBTG animals develop hypertrophy and moderate cardiac dysfunction by 4 months of age. Thus, CaMKIIδB expression appears to be sufficient to induce cardiac hypertrophy. Surprisingly, despite the increased CaMKII activity in the CaMKIIδBTG mouse heart, phosphorylation of the canonical cardiac CaMKII substrate phospholamban (PLN) at its CaMKII site (Thr17) was not increased but rather was decreased relative to WT mice. PLN phosphorylation at the PKA site (Ser16) was similarly reduced. These data were related to increases in phosphatase activity , was not increased by cardiac CaMKIIδB expression  mice . These aactivity , but alspression . This fiB has also been suggested to regulate expression of the Na+/Ca2+ exchanger (NCX1) during the development of cardiac dysfunction following trans-aortic constriction  simulates autophosphorylation. Interestingly, the authors found that this construct was excluded from the nucleus . The observation that mutation of Ser332 to Ala restores nuclear localization of constitutively active CaMKIIδB  is diminished in δCTG mice as occurs in other models of heart failure. Since SERCA regulates Ca2+ reuptake into the sarcoplasmic reticulum (SR), this decrease would diminish SR Ca2+ loading. On the other hand, the CaMKIIδCTG mice show hyperphosphorylation of PLN at Thr17, which should improve SERCA function. In addition δCTG animals display marked increases in phosphorylation of the RyR2, the channel through which Ca2+ exits the SR. Taken together, these changes would predict dysregulation of SR Ca2+ cycling and excitation–contraction coupling. This was substantiated in an accompanying paper that systematically analyzed and demonstrated dysregulation of cardiac Ca2+ handling in mice expressing δC .Expression of the cardiac sarco/endoplasmic reticulum Cassing δC . Specifihandling . In othe loading . These fCTG mice  contributes to arrhythmias and SR Ca2+ leak is supported by the finding that mutation of Ser2814 to Ala (S2814A) blocks the ability of CaMKII to induce Ca2+ sparks (C expression and activity are implicated in the etiology of premature death in CPVT as expression of CaMKIIδC exacerbates the effects of the R4496C mutation (2814 phosphorylation is increased by expression of CaMKIIδC (but not by δB) in vivo , which can prolong the duration of the action potential and contribute to arrhythmias. Additionally, increased INa can lead to Na+-overloading of the cardiomyocyte, which contributes to diminished diastolic contractile performance  and repolarizing potassium currents  is an initiating event in δC-induced heart failure. Specifically, as a consequence of SR Ca2+ leak and SERCA downregulation, the SR Ca2+ load is diminished which would compromise contractile function. To determine whether diminished SR Ca2+ load is the primary causal event leading to contractile dysfunction and premature death in response to δC overexpression, we crossed the δCTG mice with mice in which the SERCA regulatory protein PLN was deleted (PLN-KO). Deletion of PLN in the context of δC overexpression normalized SR Ca2+ levels and the contractile function of isolated myocytes was restored  and cell death  as a potential target of CaMKII  promoter and subsequent gene expression , resulting in activation of the transcription factor MEF2  in NRVM. The ability of CaMKIIδC to decrease nuclear NFAT was blocked by coexpression of a dominant-negative construct of CaMKIIδC and was shown to be elicited by phosphorylation and inhibition of the Ca2+/CaM dependent phosphatase calcineurin  expression (C activation is implicated in cell death elicited by a variety of stimuli (C (but not δB) upregulates the proapoptotic transcription factor p53  and since IKK activation can also upregulate p53 not present in δB or δC and thus may possess unique properties that merit further investigation, as the function and localization of δ9in vivo has not been explored. Along similar lines, CaMKIIδA expression is increased in a model of cardiac hypertrophy (Of additional interest is the neglected CaMKIIδ work of  identifiertrophy , but theThe 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.


Purpose: Higher order chromatin structure progressively changes with cell differentiation and seems to play an important role in DNA double-strand break (DSB) induction and repair (reviewed in [1]). We compared DNA damage in heterochromatin (Hc) upon the action of qualitatively different radiations. We also studied, how is the sensitivity to DSB induction, assembly of repair foci and processing of DSBs influenced by the differentiation-induced changes in chromatin structure and composition.Materials and methods: Formation, localization (relative to higher-order chromatin domains) and mutual colocalization of γH2AX and p53BP1 repair foci have been studied together with DSB repair kinetics in spatially fixed human skin fibroblast and differently differentiated white blood cells (WBC) irradiated with gamma rays, protons of different energies , and 20Ne ions (submitted). Immunostaining and ImmunoFISH were used in combination with high-resolution confocal microscopy  and living cell imaging [4].Results: We found that less DSBs appear in Hc after irradiating cells with gamma rays and protons but not 20Ne ions (preliminary results). In addition, contrary to γ-irradiated human skin fibroblasts and lymphocytes, mature granulocytes neither express DSB repair proteins nor form functional repair foci [5]. At least some DSB repair proteins (e.g. 53BP1) are expressed and γH2AX foci still occur in immature granulocytes and monocytes ; however, the colocalization of γH2AX with 53BP1 is low and the majority of DSBs are not repaired. Despite this fact, γH2AX foci protrude from Hc into nuclear subcompartments with low chromatin density. Our living cell observations suggest that 53BP1 can penetrate into the interior of dense Hc domains only after their decondensation [2].Conclusions: We show that Hc is less sensitive to DSB induction by gamma rays but not heavy ions; lower Hc hydratation and higher protein density (when compared with euchromatin) probably reduce formation of free radicals and increase their sequestration, respectively. This mechanism can protect cells against the indirect effect of ionizing radiation (marked for gamma rays and protons but not heavy ions). Hc features, however, preclude DSB repair, which is best illustrated by its absence in differentiated WBC but not their immature precursors. The protrusion of Hc-DSBs into low-density chromatin nuclear subdomains, however, appears also in differentiated WBC, so the process might simply follow physical forces (e.g. as suggested by M Durante's group).There is no Clinical Trial Registration number.