Ca2+ shifts the absorption spectrum of heme a, which allowed previously to determine the kinetics and equilibrium characteristics of the binding. However, no effect of Ca2+ on the functional characteristics of cytochrome oxidase was revealed earlier. Here we report that Ca2+ inhibits cytochrome oxidase activity of isolated bovine heart enzyme by 50–60% with Ki of ∼1 µM, close to Kd of calcium binding with the oxidase determined spectrophotometrically. The inhibition is observed only at low, but physiologically relevant, turnover rates of the enzyme (∼10 s−1 or less). No inhibitory effect of Ca2+ is observed under conventional conditions of cytochrome c oxidase activity assays (turnover number >100 s−1 at pH 8), which may explain why the effect was not noticed earlier. The inhibition is specific for Ca2+ and is reversed by EGTA. Na+ ions that compete with Ca2+ for binding with the Cation Binding Site, do not affect significantly activity of the enzyme but counteract the inhibitory effect of Ca2+. The Ca2+-induced inhibition of cytochrome c oxidase is observed also with the uncoupled mitochondria from several rat tissues. At the same time, calcium ions do not inhibit activity of the homologous bacterial cytochrome oxidases. Possible mechanisms of the inhibition are discussed as well as potential physiological role of Ca2+ binding with cytochrome oxidase. Ca2+- binding at the Cation Binding Site is proposed to inhibit proton-transfer through the exit part of the proton conducting pathway H in the mammalian oxidases.Cytochrome  The enzyme catalyses reduction of molecular oxygen to water coupled to translocation of protons across the coupling membrane Cytochrome c2+ + O2 + 8H+inside = 4 cyt c3+ + 2H2O + 4H+outside.4 cyt a and high-spin a3, and two copper centers, a binuclear CuA and a mononuclear CuB. The high-spin heme a3 iron and CuB are located within ∼5 A from each other and form a di-nuclear site of oxygen reduction to water. The sequence of electron transfer through the enzyme is described by a scheme.The electron transfer in the oxidase is mediated by four metal redox centers: two A-type hemes, low-spin c → CuA → heme a → heme a3/CuB → O2.cyt 2+ (or Mn2+) ion which holds together subunits I and II and may be part of the exit pathway for the pumped protons and for water formed in the active site 2+ or Na+ ion.In addition to the redox centers, cytochrome oxidases from mitochondria and many bacteria contain non-redox metal ions, revealed by chemical analysis 2+ with the mitochondrial COX was described originally by Wikstrom and Saari a α-absorption band induced by EDTA and showed that the effect was due to reversal of a red shift induced by adventitious calcium ions acting from the outer side of the mitochondrial membrane. The specific CBS was identified later on in the crystal structures of the A1-class P. denitrificansR.sphaeroides (PDB entry 1M56)) and bovine heart Reversible binding of Caa. In the bacterial oxidases, the X-ray structure and chemical analysis reveal tightly bound Ca2+ at the site 2+ does not induce spectral shift of heme a in COX from R. sphaeroidesP. denitrificans2+ in COX from P.denitrificansR. sphaeroides2+ with the bacterial enzyme, making the bacterial oxidases a useful model for the studies of CBS in the mammalian oxidase.As shown in 2+ and proton + as revealed by competition of the latter with Ca2++ rather than Ca2+ bound at the site , carbonyl cyanide m-chlorophenyl hydrazone (CCCP), cyclosporine A, nagarse, cytochrome c type III, N,N,N’,N’ – tetrametyl-p-phenylenediamine (TMPD), potassium salts of ferrocyanide and ferricyanide, and calcium buffers: ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), nitrilotriacetic acid (NTA), 1,2-bis(2-aminophenoxy)-ethane- N,N,N’,N’- tetraacetic acid (BAPTA) and N-(2-hydroxyethyl)ethylenediamine- N,N’,N’-triacetic acid (HEDTA) were from Sigma-Aldrich. pH buffers, sodium chloride and magnesium sulfate were purchased from Amresco. Dodecylmaltoside (DM) “SOL-GRADE” was from Anatrace.Sodium dithionite, CaClR. sphaeroides was purified from bacterial membranes  on a column with Ni2+-NTA Sepharose (Qiagen) P.denitrificans was kindly provided by Dr. Anne Puustinen . Concentration of COX was determined from the “dithionite-reduced minus oxidized” difference absorption spectra using Δε605–630 of 27 mM−1cm−1. Mitochondria from rat tissues were isolated from outbred white male rats by conventional methods as used in this laboratory c and calcium ions. After the treatment, mitochondria were washed thoroughly to remove Nagarse. Animal protocols were approved by the Institutional Review Board. Handling of the animals and experimental procedures were conducted in accordance with the international guidelines for animal care and use and were approved by the Institutional Ethics Committee of A.N. Belozersky Institute of Physico-Chemical Biology at Moscow State University.“Fast” COX was purified from bovine heart mitochondria using a modified protocol by Fowler et al. c with COX, in some experiments we had to increase the ionic strength of the reaction mixtures. To this end, choline chloride was chosen as the main salt component of the reaction buffers because, in our experience, this salt as available from Sigma-Aldrich (C-1879) proved to have very low contamination with Na+. Concentration of sodium ions in the media has to be minimized because Na+ competes with Ca2+ for binding with COX 2, Ca2+-buffering chelators  and other specific conditions was calculated as earlier Experiments with the isolated COX were performed in a basic medium containing 50 mM Tris/MES pH 8.0–8.2, 0.05% dodecyl maltoside, choline chloride (50 mM or higher as indicated), and also 100 µM EGTA to bind adventitious calcium and strip off the bound calcium from the mitochondrial oxidase. In order to slow down reaction of cytochrome c was measured in a dual-wavelength mode at 550 nm vs a reference wavelength at 535 or 540 nm. Turnover numbers (TN) for COX  are expressed in electrons per second per cytochrome oxidase monomer, if not stated otherwise. The Ca2+-induced red shift of heme a absorption spectrum was measured in the SLM-Aminco 2000C spectrophotometer operated in a split beam mode. COX was pre-reduced by 5 mM ascorbate and 100 µM TMPD in the presence of 5 mM KCN that resulted in complete reduction of heme a. The difference spectra induced in the α-band of heme a by increasing concentrations of CaCl2 in the presence of Ca2+-buffers were recorded. The amplitude of the derivative-shaped difference spectra at 613 nm minus 599–600 nm 2+. The data were processed with OriginLab 7e software package . The figures with the crystal structure of COX domains were prepared with the aid of PyMOL software.Spectrophotometric measurements were made in a Varian Cary300Bio or an SLM-Aminco 2000C instrument in 10 mm optical pathway cells at 25°C. Oxidation of ferrocytochrome 2+ on COX activity, if in existence, would be more pronounced under the conditions of the enzyme turnover close to those in the respiring mitochondria. COX turnover in the respiring mitochondria , whereas in the mitochondria respiring on succinate or NADH-dependent substrates, COX turns over much slower, ca. 10 s−1 or less, even in the fully uncoupled state. Second, in the standard assays, cytochrome c is kept almost fully reduced  and its redox potential, Eh, is much lower than Em, whereas in mitochondria respiring with succinate or NADH-dependent susbstrates, cytochrome c is typically less than half-reduced in the steady-state 2+ on COX activity at slow turnover rates of the enzyme and at high redox potential of the electron donors.Our conjecture was that the putative regulatory effect of Cadria cf.  differs ch, of the donor. As shown in c-catalyzed oxidation of ferrocyanide by COX is significantly inhibited by Ca2+ (trace 2) but not by Mg2+ (trace 3), and the inhibition is reversed by EGTA  and is most likely due to stimulation of direct interaction of ferrocyanide anion with the negatively charged electron entry site of the enzyme by divalent cations 2+ in 2+ according to a hyperbolic curve with Ki of 0.9 µM and maximal inhibition of ∼60% .In the first series of experiments, slow aerobic turnover of purified bovine COX with ferrocyanide as the high-potential electron donor was studied in the presence of catalytic amount (40 nM) of cytochrome 2+-induced inhibition of COX is also observed with artificial electron donors, such as TMPD and ferrocyanide, in the absence of cytochrome c . The inhibition observed with TMPD  seems to be somewhat less than measured with the other electron donors tested (50–60%). However, it will increase to ∼50% if corrected for ca. 25% of KCN-insensitive oxidation of TMPD in the presence of COX.The Ca2+ on oxidation of excess ferrocytochrome c by COX was studied in the absence of artificial redox compounds. A representative experiment is shown in c in the medium. In a standard assay (“high turnover conditions”), no effect of Ca2+ is observed . However, if the conditions are set so as to decrease the rate of COX activity to ∼5–10 s−1 . The inhibitory effect of Ca2+ at turnover rates below ∼10 s−1 has been documented for several large scale preparations of bovine CcO with but minor differences in the extent of inhibition among the preparations. In replicate experiments with the same sample of the enzyme at initial turnover rate of 6.2±0.05 s−1, the inhibition induced by 100 µM free calcium gives a value of 58±2% (15 experiments). No such effect is induced by Mg2+ (2). Similarly to the results shown in 2+ on ferrocytochrome c oxidation was reversed and prevented by excess EGTA and was not observed with COX from R. sphaeroides (data not included). The inhibitory effect of Ca2+ titrates according to a hyperbolic curve with the maximal inhibition of 50–60% and Ki of ∼1.4 µM  in good agreement with the data on ferrocyanide oxidation . At the same time, 50 mM Na+ largely prevents inhibition of the activity by Ca2+  or D477A mutant COX from P.denitrificans . Stronger inhibition is observed with mitochondria from rat liver . The inhibition is reversed by addition of excess EGTA (3). No inhibition was induced by Mg2+ (not shown). The inhibition of COX in liver mitochondria titrates with [I]50 of ∼0.5 µM . Thus, like in the case of the purified bovine heart oxidase, the inhibition of COX activity in rat liver mitochondria correlates with calcium binding at the site responsible for the spectral shift of heme a. As with the soluble cytochrome oxidase, no significant effect of Na+ on the activity could be observed, but sodium ions prevented the Ca2+-induced inhibition (data not shown).As shown in Table summarizes the inhibitory effects of Ca2+ on the COX activity of mitochondria from several rat tissues. The inhibition in mitochondria from the non-excitable tissues (liver and kidney) appears to be stronger than in the heart or skeletal muscle mitochondria. It is noted that the incomplete inhibition of COX by Ca2+ observed in the experiments may be explained, as usual, either by partial inhibition of the entire population of the enzyme or by complete inhibition of variable fraction of the enzyme (or both). More experiments are required to distinguish between these possibilities.The 2+ ions at concentrations of few µM inhibit mitochondrial cytochrome c oxidase, both in the soluble state and in the mitochondrial membrane. The inhibition is associated with Ca2+ binding at the special cation binding site of COX The principal finding of this work is that Cac and turnover rate of ∼102 s−1 or higher at pH 8, no inhibitory effect of Ca2+ is observed . The reaction media used in experiments may easily contain some 5–10 µM of adventitious Ca2+ unless special precautions are taken. Therefore, the CBS of COX may be saturated with Ca2+ already before addition of exogenous calcium. In order to control free Ca2+ concentration in the µM range, Ca2+ buffers have to be used which was not often the case in the past works on Ca2+ interaction with COX .As a matter of fact, scattered evidence for inhibition of the mitochondrial respiration by micromolar CaSeveral possible explanations may be considered.c with COX is essentially electrostatic, so one could propose that Ca2+ competes with cytochrome c for the anionic binding site at CuA. However, the observed inhibitory effect of Ca2+ requires the cation binding at the specific Cation Binding Site separate from the cytochrome c docking site; the effect is not mimicked by Mg2+ and is not observed with the bacterial oxidases. Furthermore, the inhibition of bovine oxidase by Ca2+ can be observed with the artificial electron donors - anionic ferrocyanide ((i) is not likely.Binding of cytochrome 2+ could be related to a positive shift of Em of heme a induced by the cation 2+ does not inhibit activity of mutant COX from R. sphaeroides (D485A) or P.denitrificans (D477A)  cf. Figure 6a.As discussed in 2+-bound CBS in bovine COX has not been published yet, but it was modeled 2+ and Na+ in bovine COX and taking into account the resolved structure of the Ca2+-loaded site in the bacterial oxidases , but it does not exclude the role of redox potential of cytochrome c. Much more experiments are required to clarify the quantitative aspects of the Ca2+-induced inhibition. In any case, it is worth noting that, firstly, the low turnover rates at which the inhibition is observed most clearly (∼10 s−1 or less) are close to typical turnover rates of COX in the respiratory chain of mitochondria (ca. 100 ng-at O2 • min−1 • (mg protein) −1, i.e. ∼10 s−1, assuming 0.3 nmol of aa3 per mg of protein of heart mitochondria c is only partly reduced in the actively respiring mitochondria  indicate that the turnover rate may be itself important  Stimulation of Reactive Oxygen Species (ROS) production. Activation of the dehydrogenases and concomitant inhibition of COX should act synergistically in increasing the reduction of the respiratory chain carriers at given NAD(P)H/NAD(P)+ and GSH/GSSG ratios in the mitochondrial matrix. In this way Ca2+ is expected to stimulate ROS production by the respiratory chain (cf. Cadase cf. .(ii) Sti(iii) Modulation of calcium uptake by mitochondria. Ca2+ is taken up actively by respiring mitochondria via the so-called calcium uniporter, a highly specific Ca2+ channel, down the Δψ formed by the respiratory chain . A likely role of the inhibition might consist in damping the effects of the periodical [Ca2+free] spikes in the cytoplasm 2+ during the spikes. In the non-excitable tissues, mitochondria that are able to suck in rapidly calcium released to the cytoplasm from the endoplasmic reticulum cisterns or delivered from the extracellular stores, are active players in the Ca2+ -mediated signal transmission and participate in “shaping in time and space” the cytoplasmic Ca2+ signals 2+ could then be involved in modulation of the multiple regulatory pathways in which mitochondria interfere with the cellular response to Ca2+.In the excitable tissues like heart or skeletal muscle, the Ca2+ on mammalian cytochrome oxidase is an important novel factor potentially involved in regulation of oxidative phosphorylation, mitochondrial ROS production and intracellular transmission of the calcium signals.1. The direct inhibitory effect of Ca2+ on the mammalian, but not on the bacterial, cytochrome oxidases, favour functional significance of the “H-channel” in cytochrome oxidase from mammalian mitochondria. Ca2+ may be proposed to be a physiological specific inhibitor of the H-channel, whatever the function of the channel is.2. Location of the calcium binding site near the exit of the so-called proton conducting pathway H along with the specific inhibitory effect of CaFigure S1c. The reaction mixture contained 50 mM tris-MES buffer, pH 8, with 0.4 M choline chloride, 0.05% DM, 100 µM EGTA and 1∶1 mixture of the oxidized and reduced forms of cytochrome c at concentrations indicated (1–55 µM of each). The cytochrome oxidase reaction was initiated by addition of 6 nM purified bovine COX, and oxidation of cytochrome c2+ was followed spectrophotometrically in a dual-wavelength mode at 550 nm vs the 535 nm reference. Where indicated, 200 µM Ca2+ (100 µM excess over EGTA) was also present. The curves are drawn through the points to guide the eye. 2+?Is turnover rate an essential factor that determines cytochrome c oxidase sensitivity to inhibition by Ca In a pilot experiment shown in Figure S1, concentration of cytochrome c was varied to change the cytochrome c oxidase reaction rate, while the redox potential was kept constant by using equimolar mixture of the reduced and oxidized cytochrome c for each point. It can be seen that the curves in the absence and in the presence of calcium tend to converge as the reaction rate increases. For instance, the inhibition induced by Ca2+ is 57% at the lowest rate of electron transfer (1.2 s−1), 44% at 6.1 s−1 and 21% at 12 s−1. In agreement with the data in −1; the latter point  was measured with this sample of COX in a separate experiment with 15 µM ferrocytochrome as the electron donor but without ferric cytochrome c and at choline concentration of 50 mM. Results of the experiment confirm that the turnover rate is indeed an essential factor affecting the sensitivity of COX to inhibition by calcium. Conceivably, the data do not exclude contribution of other factors, such as redox potential. It is noted that the dependencies in are to be considered as preliminary data and apply just to one of the several COX samples studied in this work. The exact profile for the Ca2+-induced inhibition as a function of turnover rate may vary for different preparations. Click here for additional data file.


Factors affecting [Ca2+]i include Ca2+-release from intracellular stores (CRIS), store-operated Ca2+-entry (SOCE) through STIM1/STIM2-regulated Orai1, K+-dependent Na+/Ca2+-exchangers (NCKX), K+-independent Na+/Ca2+-exchangers (NCX) and calbindin-D28k. The present study explored whether PKB/SGK-dependent GSK3α, β-activity impacts on CRIS, SOCE, NCKX, NCX or calbindin. DCs were isolated from KIgsk3 mice and respective wild-type mice (WTgsk3), [Ca2+]i estimated from Fura2 fluorescence, Orai1, STIM1, STIM2 as well as calbindin-D28k protein abundance determined by Western blotting and mRNA levels quantified by real time PCR. As a result, thapsigargin-induced CRIS and SOCE were significantly blunted by GSK3-inhibitors SB216763  or GSK-XIII  but were significantly lower in WTgsk3 than in KIgsk3DCs. Orai1, STIM1 and STIM2 protein abundance was significantly lower and calbindin-D28k abundance significantly higher in KIgsk3 than in WTgsk3DCs. Activity of NCKX and NCX was significantly higher in KIgsk3 than in WTgsk3DCs and was significantly increased by SB216763  or GSK-XIII . Treatment of WTgsk3 DCs with SB216763  or GSK-XIII  did not significantly modify the protein abundance of Orai1, STIM1 and STIM2. The present observations point to a dual role of GSK3 in the regulation of Ca2+ in DCs. Acute inhibition of GSK3 blunted the increase of [Ca2+]i following CRIS and SOCE and stimulated NCKX/NCX activity. However, expression of PKB/SGK-resistant GSK3α, β downregulated the increase of [Ca2+]i following CRIS and SOCE, an effect at least partially due to downregulation of Orai1, STIM1 and STIM2 expression as well as upregulation of Na+/Ca2+-exchanger activity and calbindin D28k expression.Dendritic cells (DCs), key players of immunity, are regulated by glycogen synthase kinase GSK3. GSK3 activity is suppressed by PKB/Akt and SGK isoforms, which are in turn stimulated by the PI3K pathway. Exposure to bacterial lipopolysaccharides increases cytosolic Ca Cultures were supplemented with GM-CSF  and fed with fresh medium containing GM-CSF on days 3 and 6. Experiments were performed on DCs at days 7–9.Dendritic cells (DCs) were cultured from bone marrow of 7–12 week old mice. Bone marrow derived cells were flushed out of the cavities from the femur and tibia with PBS. Cells were then washed twice with RPMI and seeded out at a density of 2×10KIgsk3 or WTgsk3 mice were washed with ice cold phosphate-buffered saline (PBS) and cells were lysed with cell lysis buffer . The extracts were centrifuged at 13,000 rpm for 20 min at 4°C and the protein concentration of the supernatant was determined. Total protein (30 µg) was subjected to 10% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane (VWR) and the membranes were then blocked for 1 h at room temperature with 10% non- fat dried milk in tris-buffered saline (TBS) containing 0.1% Tween-20. For immunoblotting the membranes were incubated overnight at 4 °C with antibodies directed against GSK3α/β , phospho-GSK3α/β , Orai1 , STIM1 , STIM2  or calbindin-D28k . A GAPDH antibody  was used for a loading control. Specific protein bands were visualized after subsequent incubation with a 1∶3000 dilution of anti-rabbit IgG conjugated to horseradish peroxidase and a Super Signal Chemiluminescence detection procedure . Specific bands were quantified by Quantity one software . Levels of each protein were expressed as the ratio of signal intensity for the target protein relative to that of GAPDH.The protein expression levels were analyzed by Western blotting. In brief, DCs from Total RNA was extracted from mouse dendritic cells in TriFast  according to the manufacturer's instructions. After DNAse digestion reverse transcription of total RNA was performed using Transcriptor High Fidelity cDNA Synthesis kit (Roche). Polymerase chain reaction (PCR) amplification of the respective genes were set up in a total volume of 20 µl using 40 ng of cDNA, 500 nM forward and reverse primer and 2x GoTaq® qPCR Master Mix SYBR Green  according to the manufacturer's protocol. Cycling conditions were as follows: initial denaturation at 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec, 55°C for 15 sec and 72°C for 20 sec. For the amplification the following primers were used (5′->3′orientation):CATGGTAGCGATGGTGGAAGTC rev TGCTfCATCGTCTTTAGTGCCT;Orai1, fw ATGGTGGCCATGGTGGAGGT rev ATTGCCTTCAGCGCCTGCA;Orai2, fw CTTGGCCTGGGATCTCAGAG rev TCAGCCATTGCCTTCTTGCC;STIM1, fw GCAGGATCTTTAGCCAGAAG rev ACATCTGCTGTCACGGGTGA;STIM2, fw TCCCTCACCTAGAGATAGAAGCAGCGCAGCalbindin, fw AGACAGCAGAATCGAGGAGTCTGCTGCTC;rev CAAGCTGGAGGTGATCATCG rev TCCACAGTGCTCTTGAATTCG.Tbp, fw Specificity of PCR products was confirmed by analysis of a melting curve. Real-time PCR amplifications were performed on a CFX96 Real-Time System (Bio-Rad). All experiments were done in duplicate. Amplification of the house-keeping gene Tbp (TATA binding protein) was performed to standardize the amount of sample RNA. Relative quantification of gene expression was achieved using the Δct method as described earlier 2+ concentration, the cells were loaded with Fura-2/AM  for 15 min at 37°C. Fluorescence measurements were carried out with an inverted phase-contrast microscope . Cells were excited alternatively at 340 or 380 nm and the light was deflected by a dichroic mirror into either the objective  or a camera . Emitted fluorescence intensity was recorded at 505 nm and data acquisition was accomplished by using specialized computer software . The corresponding ratios (F340/F380) were used to obtain intracellular Ca2+ concentrations. The following equation was used: [Ca2+]free  =  Kd x ((R-Rmin)/(Rmax-R)) x Sf . As a measure for the increase of cytosolic Ca2+ concentration, the slope and peak of the changes in intracellular Ca2+ concentration were determined in each experiment.To determine cytosolic Ca2+ were monitored upon depletion of the intracellular Ca2+ stores. Experiments were carried out prior to and during exposure of the cells to the Ca2+-free solution (see below). In the absence of Ca2+, the intracellular Ca2+ stores were depleted by inhibition of the vesicular Ca2+ pump by thapsigargin . Readdition of Ca2+ allowed assessing the SOCE. To monitor the activity of K+ dependent (NCKX) or K+ independent (NCX) Na+/Ca2+ exchangers, changes in cytosolic Ca2+ were determined upon replacement of extracellular Na+ by N-methyl-D-glucamine (NMDG) in Ringer solution for NCX or NCKX. In some experiments DCs were treated with GSK3 inhibitors SB2167633  or GSK3 inhibitor XIII  To measure SOCE, changes in cytosolic Ca4, 32.2 Hepes, 2 Na2HPO4, 2 CaCl2, and 5 glucose at pH 7.4 (NaOH). Ca2+-free solution contained (in mM): 125 NaCl, 5 KCl, 1.2 MgSO4, 2 Na2HPO4, 32.2 Hepes, 0.5 EGTA, and 5 glucose at pH 7.4 (NaOH). Ringer solution for NCX contained (in mM): 130 NaCl, 2 CaCl2, 2 MgCl2, 10 HEPES, and 10 glucose at pH 7.4 (NaOH). Ringer solution for NCKX contained (in mM): 90 NaCl, 40 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, and 10 glucose at pH 7.4 (NaOH). The Na+-free solutions were identical, except that NaCl was replaced by 130 or 90 mM NMDG to measure NCX or NCKX, respectively.The Ringer solution contained (in mM): 125 NaCl, 5 KCl, 1.2 MgSOn represents the number of independent experiments. All data were tested for significance using unpaired Student t-test, Mann–Whitney U test or ANOVA . Only results with p<0.05 were considered statistically significant.Data are provided as means ± SEM, 2+ concentration ([Ca2+]i) in dendritic cells (DCs) following stimulation of intracellular Ca2+ release by inhibition of the sarco/endoplasmic Ca2+ ATPase (SERCA) with thapsigargin in the absence of extracellular Ca2+ and/or the increase of [Ca2+]i during store operated Ca2+ entry (SOCE) following readdition of extracellular Ca2+. The first series of experiments was performed in the presence and absence of the GSK3 inhibitor SB216763  added at different concentrations (100 nM – 10 µM) 30 min before the experiment. As illustrated in 2+ resulted in a transient increase of [Ca2+]i reflecting intracellular Ca2+ release. The subsequent readdition of extracellular Ca2+ was followed by a rapid increase of [Ca2+]i reflecting SOCE. Following addition of SB216763 the increase of [Ca2+]i upon thapsigargin treatment (Ca2+ release) and following readdition of extracellular Ca2+ (SOCE) were blunted by 1 µM and 10 µM of SB216763 (2+ release and SOCE (The present study explored whether glycogen synthase kinase 3 (GSK3) modifies the increase of cytosolic CaSB216763 . Those rand SOCE .2+]i following intracellular Ca2+ release and subsequent SOCE was monitored in DCs isolated from mice expressing PKB/Akt and SGK insensitive GSK3α,β (KIgsk3) and DCs isolated from wild type mice (WTgsk3). As illustrated in 2+]i due to Ca2+ release and readdition of Ca2+ triggered SOCE, effects both significantly blunted in KIgsk3 DCs as compared to WTgsk3 DCs.In a second series of experiments the increase of [Ca2+]i following intracellular Ca2+ release and/or SOCE is expected to be blunted by stimulation of Ca2+ extrusion, such as activation of Na+/Ca2+ exchangers. The increase of [Ca2+]i following removal of extracellular Na+ was taken as evidence for Na+/Ca2+ exchanger activity. In order to discriminate between K+-independent (NCX) and K+-dependent (NCKX) Na+/Ca2+ exchangers, experiments were performed in the absence (0 mM) or in the presence of high (40 mM) extracellular K+ concentrations. In the absence and in the presence of K+, the increase of [Ca2+]i following removal of extracellular Na+ was significantly higher in KIgsk3 DCs than in WTgsk3 DCs  moiety Orai1 and its regulators STIM1 and STIM2 in WTgsk3 and KIgsk3 DCs. As illustrated in KIgsk3 DCs than in WTgsk3 DCs. The transcript abundance of STIM1, and STIM2 were not significantly different between genotypes. To confirm that the mutation in KIgsk3 DCs disrupted GSK3α/β phosphorylation, phosphorylated GSK3 and total GSK3 were determined in WTgsk3 and KIgsk3 DCs. Indeed, phosphorylated GSK3 was observed in DCs from WTgsk3 mice but not in DCs from KIgsk3 mice  following inhibition of the sarco/endoplasmic Ca2+ ATPase (SERCA) with thapsigargin in the absence of extracellular Ca2+ as well as the store operated Ca2+ entry (SOCE) following readdition of extracellular Ca2+. On the other hand, disruption of PKB/Akt and SGK dependent GSK3α,β phosphorylation downregulates both, intracellular Ca2+ release and SOCE. Phosphorylation of GSK3 by PKB/Akt The present observations reveal a dual role of glycogen synthase kinase 3 (GSK3) in the regulation of dendritic cell (DC) CaGSK3 is apparently active in immature DCs 2+]i following treatment of DCs with bacterial LPS was virtually abolished in the presence of GSK3 inhibitor SB216763. Those observations are consistent with inhibition of SOCE by the GSK3 inhibitor. Notably, the inhibitor interferes with both, intracellular Ca2+ release and SOCE. Thus, GSK3 may be involved in the signaling leading to emptying of intracellular stores. Since the GSK3 inhibitor SB216763 was effective within a few minutes it apparently disrupted activation or inactivation of existing proteins. Moreover, as shown in the present study, both K+ dependent (NCKX) and K+ independent (NCX) Na+/Ca2+ exchangers are stimulated by short-term inhibition of GSK3 by either SB216763 or GSK-XIII. Enhanced extrusion of Ca2+ via these transporters could also underlie the reduced SOCE.As shown earlier KIgsk3 mice is at least partially due to decreased expression of Orai1, STIM1 and STIM2. Moreover, the blunted increase of [Ca2+]i in DCs from KIgsk3 mice during intracellular Ca2+ release and SOCE are in part due to enhanced Ca2+ buffering due to increased expression of calbindin-D28k. The differences between DCs from KIgsk3 mice and DCs from WTgsk3 mice is thus partially due to altered gene expression. The blunted increase of [Ca2+]i during intracellular Ca2+ release and SOCE is further in part due to enhanced activity of both K+ dependent (NCKX) and K+ independent (NCX) Na+/Ca2+ exchangers. In contrast, inhibition of GSK3 by either SB216763 or GSK-XIII did not modify expression of Orai1, STIM1 and STIM2.In contrast, the decreased SOCE in DCs isolated from KIgsk3 mice and DCs from WTgsk3 mice may be an indirect result of the altered regulation of GSK3 activity in other cell types, which in turn influence gene expression in DCs. Along those lines PKB/Akt and SGK1 resistance of GSK3 influences steroid hormone release in vivo prior to isolation, the DCs could remain altered following isolation and subsequent culture.At least in theory, the differences between DCs from 2+ concentration in dendritic cells. Acutely, GSK3 activity is required for the full effect of LPS stimulation and SERCA inhibition on cytosolic Ca2+ activity. On the other hand, disruption of PKB/Akt and SGK dependent phoshorylation of GSK3 downregulates Orai1, STIM1 and STIM2 expression, upregulates calbindin-D28k expression and enhances the activity of K+ dependent (NCKX) and K+ independent (NCX) Na+/Ca2+ exchangers in dendritic cells.In conclusion, GSK3 plays a dual role in the regulation of cytosolic Ca


In addition, recent studies have shown that combinations of such agents with cytarabine result in enhanced antileukemic responses in vitro, raising the prospect and potential of use of these agents in combination regimens for the treatment of AML.Despite recent advances in the field, the treatment of patients with acute myeloid leukemia (AML) remains challenging and difficult. Although chemotherapeutic agents induce remissions in a large number of patients, many of them eventually relapse and die. A major goal for the development of new approaches for the treatment of AML is to enhance the antileukemic effects of standard chemotherapeutics and to design effective combinations targeting non-overlapping cellular pathways. The PI3K/Akt/mTOR signaling pathway plays a critical role in survival and growth of malignant cells and its targeting has been the focus of extensive work and research efforts over the last two decades. It now appears possible that a major limitation of the first generation of mTOR inhibitors can be overcome by a new class of catalytic inhibitors of mTOR. There is emerging evidence that such compounds target both TORC1 and TORC2 and elicit much more potent responses against early leukemic precursors  Untreated, acute myeloid leukemia (AML) is a fatal hematological malignancy. Although remissions can be achieved with intensive chemotherapy, the disease relapses in a large number of cases and progression and death frequently occurs -5. The cA major problem in efforts to treat and cure AML is the inability to efficiently target and eliminate leukemia initiating cells (LICs), which are the cells that initiate and maintain the leukemic phenotype , 11. TheThe PI3’ kinase/AKT/mTOR pathway is a key regulatory network of signaling cascades in mammalian cells, whose coordinated operations regulate and promote important cellular activities and functions , 16. ViaAs aberrant activation of the PI3’K/AKT/mTOR pathway promotes malignant cell proliferation and survival , 26, 27,The mammalian target of rapamycin (mTOR) is a central element of the pathway and a key kinase activated downstream of PI3K/AKT. This kinase was originally identified in yeast , and subIn contrast to TORC1, whose major function is control of signals for the initiation of mRNA translation, the TORC2 complex plays a different role in normal and malignant cells. The TORC2 complex includes mTOR, Rictor (rapamycin-insensitive companion of mTOR), SIN1, and mLST8 , 46-48. Because of the high relevance of the mTOR pathway in malignancies, first generation mTOR inhibitors, including rapamycin and related drugs  have been tried extensively in various clinical contexts for the treatment of tumors of diverse cellular origin. Two rapalogs, temsirolimus (CCI-779) and everolimus (RAD001) have shown major activity and have been approved by the FDA for the treatment of renal cell carcinoma , 56. SinThe simultaneous deregulation of pathways that control both transcription and mRNA translation of genes encoding for oncogenic proteins appears to play key roles in the pathogenesis and pathophysiology of AML. There has been extensive evidence that the PI3’K/AKT/mTOR pathway is aberrantly activated and deregulated in AML . There iin vitro and in AML mouse models in vivo [Recognition of aberrations in the AKT/mTOR pathway has led to clinical trials with rapalogs in AML. Recher et al showed that rapamycin resulted in blast clearance in some patients with AML. However, the length of response was limited and not all patients responded . There h in vivo . However in vivo .in vitro [in vivo [Although approaches to optimize the administration of rapalogs with chemotherapy , in variin vitro , 81 and The potent suppressive effects of dual TORC1/TORC2 inhibitors on BCR-ABL-transformed cells, have raised the possibility that such agents may have activity in other leukemias and prompted us to perform additional studies to examine the spectrum of the antileukemic properties of OSI-027 in AML. In recently published work , we examAltogether, the results of such work raise the prospect of future clinical trials using dual TORC1/TORC2 inhibitors for the treatment of AML. Beyond OSI-027 there are additional TORC1/2 inhibitors in clinical or pre-clinical development -77, 85 tWhile inhibiting mTOR is a promising strategy for the treatment of malignancies, agents that selectively target TORC1  have limited clinical activity and are unlikely to have major impact in the treatment of AML. The development of selective ATP-catalytic inhibitors, which have the capacity to block the functions of both TORC1 and TORC2 has resulted in new momentum in the research field of mTOR targeting in AML and is igniting important work with major therapeutic implications. Approaches to overcome the limitations of rapalogs for the treatment of leukemias are now possible, using either dual TORC1/2 inhibitors or pan–PI3K-TORC1/2 inhibitors. Our recent studies have established that beyond exhibiting potent antileukemic effects, dual TORC1/2 catalytic inhibition enhances the effects of cytarabine on primitive leukemic precursors from AML patients. These studies are very encouraging and suggest a potential role for these agents in the treatment of AML patients. They also raise the possibility that combinations of dual TORC1/2 inhibitors with chemotherapeutic agents may provide a novel approach to target leukemic initiating stem cells and increase the probability of cure for AML patients.


Urticaria and angioedema are common clinical conditions representing a major concern for physicians and patients alike. The World Allergy Organization (WAO), recognizing the importance of these diseases, has contributed to previous guidelines for the diagnosis and management of urticaria. The Scientific and Clinical Issues Council of WAO proposed the development of this global Position Paper to further enhance the clinical management of these disorders through the participation of renowned experts from all WAO regions of the world. Sections on definition and classification, prevalence, etiology and pathogenesis, diagnosis, treatment, and prognosis are based on the best scientific evidence presently available. Additional sections devoted to urticaria and angioedema in children and pregnant women, quality of life and patient-reported outcomes, and physical urticarias have been incorporated into this document. It is expected that this article will supplement recent international guidelines with the contribution of an expert panel designated by the WAO, increasing awareness of the importance of urticaria and angioedema in medical practice and will become a useful source of information for optimum patient management worldwide. Albanian Society of Allergology and Clinical ImmunologyAmerican Academy of Allergy, Asthma and ImmunologyAmerican College of Allergy, Asthma and ImmunologyArgentine Association of Allergy and Clinical ImmunologyArgentine Society of Allergy and ImmunopathologyAustralasian Society of Clinical Immunology and AllergyAustrian Society of Allergology and ImmunologyAzerbaijan Society for Asthma, Allergy and Clinical ImmunologyBrazilian Society of Allergy and ImmunopathologyBritish Society for Allergy and Clinical ImmunologyBulgarian National Society of AllergologyCanadian Society of Allergy and Clinical ImmunologyColombian Allergy, Asthma, and Immunology AssociationCroatian Society of Allergology and Clinical ImmunologyCuban Society of AllergologyCzech Society of Allergology and Clinical ImmunologyDanish Society for AllergologyDutch Society of AllergologyEgyptian Society of Allergy and Clinical ImmunologyEgyptian Society of Pediatric Allergy and ImmunologyFinnish Society of Allergology and Clinical ImmunologyGerman Society for Allergology and Clinical ImmunologyHonduran Society of Allergy and Clinical ImmunologyHong Kong Institute of AllergyHungarian Society of Allergology and Clinical ImmunologyIcelandic Society of Allergy and ImmunologyIndian College of Allergy, Asthma and Applied ImmunologyIndonesian Society for Allergy and ImmunologyIsrael Association of Allergy and Clinical ImmunologyItalian Society for Allergology and Clinical ImmunologyJapanese Society of AllergologyJordanian Society for Allergy and Clinical ImmunologyKorean Academy of Allergy, Asthma and Clinical ImmunologyKuwait Society of Allergy and Clinical ImmunologyLatvian Association of AllergistsLebanese Society of Allergy and ImmunologyMalaysian Society of Allergy and ImmunologyMexican College of Pediatricians Specialized in Allergy and Clinical Immunology Mongolian Society of AllergologyNorwegian Society of Allergology and ImmunopathologyPanamanian Association of Allergology and Clinical Immunology.Philippine Society of Allergy, Asthma and ImmunologyPolish Society of AllergologyRomanian Society of Allergology and Clinical ImmunologyRussian Association of Allergology and Clinical Immunology(Singapore) Allergy and Clinical Immunology Society of SingaporeSlovenian Association for Allergology and Clinical Immunology(South Africa) Allergy Society of South AfricaSpanish Society of Allergology and Clinical Immunology(Sri Lanka) Allergy and Immunology Society of Sri LankaSwiss Society of Allergology and Immunology(Thailand) Allergy, Asthma and Immunology Society of ThailandTurkish National Society of Allergy and Clinical ImmunologyUruguayan Society of AllergologyVenezuelan Society of Allergy and ImmunologyAmerican Academy of Allergy, Asthma and ImmunologyAmerican College of Allergy, Asthma and ImmunologyAsia Pacific Association of Allergy, Asthma and Clinical ImmunologyEuropean Academy of Allergy and Clinical ImmunologyLatin American Society of Allergy and ImmunologyUrticaria is a highly prevalent condition resulting in large numbers of medical consultations worldwide. Its prevalence ranges between 0.3 and 11.3% depending on the study population , and in recent years, an increase in the rate of hospitalizations due to urticaria and angioedema has been observed in some countries [Although urticaria has a tremendous impact on patient's quality of life, it is often disregarded as a trivial disease by many physicians . TherefoThis position paper provides updates on recent advances in the understanding of etiologic factors, pathogenic mechanisms, diagnostic methods, and medical management of acute and chronic urticaria (CU) and angioedema.The World Allergy Organization (WAO) is an international federation of 89 regional and national allergy and clinical immunology societies dedicated to raising awareness and advancing excellence in clinical care, research, education, and training in allergy and clinical immunology. This WAO position paper on the diagnosis and treatment of urticaria and angioedema was developed as a document presenting a worldwide perspective encompassing the participation and input of leaders from all WAO regional member societies.This position paper includes sections on the definition, prevalence, classification, mechanisms, diagnosis, treatment, and prognosis of urticaria and angioedema. In addition, special chapters dealing with particularly important issues have been included to review physical urticarias, urticaria in childhood, urticaria and pregnancy, and quality of life and patient-reported outcomes (PROs). The concept of disease control for CU, similar to other chronic allergic diseases such as asthma and rhinitis, is highlighted and the importance of patient education on the possible mechanisms, causes, prognosis, and treatment of acute and CU is emphasized.National and regional guidelines for the diagnosis and treatment of urticaria and angioedema have been previously published -5. BecauThe objectives of this WAO position paper on urticaria and angioedema are to provide updated information on the assessment and treatment that should be applied in health care settings worldwide to obtain a better symptom control, improve patients' quality of life, contribute to patient education, and enhance accessibility to more effective therapies. This information is designed for use by both allergy and immunology specialists as well as physicians in general practices who daily observe patients with urticaria and angioedema.This position paper was developed by a special steering committee of internationally recognized experts appointed by the WAO Scientific and Clinical Issues Council.Recommendations are based on the best evidence presently available. Urticaria and angioedema guidelines previously published in indexed peer-reviewed journals were reviewed. Drafts were developed through e-mail correspondence among authors, distributed to all members of WAO Board of Directors for comment, and then circulated to WAO Member Societies for review, comments, and approval. In all, more than 50 allergy and immunology specialists on 5 continents contributed to the development of this position paper.Urticaria is characterized by the sudden appearance of wheals and/or angioedema, defining wheals as a cutaneous swelling of variable size, almost invariably surrounded by a reflex erythema, with associated itching or, sometimes, a burning sensation, and of transient nature, with the skin returning to its normal appearance in usually 1 to 24 hours.Angioedema can be defined as a sudden and pronounced swelling of the deep dermis and subcutaneous tissue or mucous membranes, with a painful rather than an itching sensation and a slower resolution than for wheals that can take up to 72 hours ,6.Urticaria can be classified on the basis of its duration and in the presence or absence of inducing factors (induced vs spontaneous).AU is characterized by the occurrence of hives and/or angioedema for < 6 weeks, whereas episodes lasting longer than 6 weeks are regarded as CU . This soUrticaria pigmentosa (cutaneous mastocytosis), urticarial vasculitis, familial cold urticaria, and nonhistaminergic angioedema  are no longer considered as subtypes of urticaria, due to their distinctly different pathomechanisms .Finally, there are syndromes that can be associated with wheals:• Muckle-Wells syndrome: a combination of wheals, deafness, and amyloidosis, characterized by sensorineural deafness, recurrent urticaria, fever, and arthritis .• Schnitzler syndrome: chronic wheals and monoclonal gammopathy  associated with at least 2 of the following components: fever, arthralgia, or arthritis, bone pain, hepatomegaly or splenomegaly or hepatospleenomegaly, lymphadenopathy, elevated erythrocyte sedimentation rate, leukocytosis, and/or abnormal findings on bone morphological investigations .• Gleich syndrome: episodic angioedema with eosinophilia .• Well syndrome or eosinophilic cellulitis: granulomatous dermatitis with eosinophilia .The prevalence of urticaria and angioedema varies according to the population under investigation. Lifetime prevalence rates of 8.8% have been reported, with a 1.8% rate for CU . ApproxiIn a study carried out in Spain, the prevalence of urticaria in the past 12 months was 0.8%, and the prevalence of CU was 0.6%. Urticaria was present more often in female patients among the 35 to 60 years age-group . Duration of the disease was 1 to 5 years in 8.7% of the patients and more than 5 years in 11.3% .Autoimmune disturbances are present in 40 to 45% of patients with chronic spontaneous urticaria . AngioedSymptoms of chronic spontaneous urticaria appear seemingly spontaneously, that is, in most patients, there is no identifiable exogenous stimulus for hive production. In some patients, however, nonspecific exogenous triggers for the development of wheals and/or angioedema, such as exercise, environmental changes, and stress are present. We now consider this group to be chronic "spontaneous" urticaria ,17; thusFor decades, theories regarding etiology would appear and disappear, but none proved to be credible. In the 1950s and 1960s, many considered chronic spontaneous urticaria to be an anxiety disorder, and even now, there is some limited data to suggest worsening of symptoms with anxiety. It is now generally accepted that mental illnesses, such as depression and anxiety, influence the quality of life of chronic spontaneous urticaria patients and those are important comorbidities in such patients. However, it cannot be considered to be a cause, and a clear distinction between less tolerance of symptoms and actual worsening of skin inflammation has not been made.The relation between food allergy/pseudoallergy and CU is controversial because some experts do not recommend elimination diets for such condition, whereas others have observed the improvement of symptoms by means of pseudoallergen-free diets in about one third of patients with chronic spontaneous urticaria .+ with a mixture of TH1 and TH2 subtypes [α subunit of the IgE receptor [Autoreactivity (see below) represents one major approach to elucidating the initiating stimulus for persisting hive formation. It is clear that cutaneous mast cell degranulation induces hive formation and on biopsy, a nonnecrotizing perivascular infiltration of cells is seen, which resembles a cutaneous late phase reaction -21. Thersubtypes . This issubtypes  and subsreceptor . The theThe remaining 55 to 60% of patients lacking such autoimmunity are considered to have chronic nonautoimmune or idiopathic (but nevertheless spontaneous) urticaria. In vitro studies support antireceptor antibody binding to the a subunit of the IgE receptor to activate the classical complement pathway with release of C5a, which further activates basophils and mast cells and contributes to recruitment of granulocytes and monocytes by its chemotactic activity . Marked α subunit can be added to serum to bind the antireceptor antibody so that HR is prevented [The presence of these antibodies does not prove causality, although their role as a pathogenic mechanism is debated with evidence pro and con ,27. Clearevented ,30. In mThere are also publications suggesting the presence of vasoactive factors in IgG-depleted serum of patients with CU, but no fα subunit cannot cross-link receptors and activation of basophil and mast cell would not occur. In practice, the IgE receptor reduction via omalizumab occurs rapidly for blood basophils and much more slowly on skin mast cells, yet omalizumab does not eliminate either cell's capacity to respond to a cross-linking stimulus [Finally, it was theorized that anti-IgE therapy with omalizumab might be effective in patients with hives. The thesis was that as IgE levels decrease toward zero, IgE receptors are downregulated, and if the spacing and surface density is sufficiently low, the IgG anti-stimulus -35. Thusstimulus -38 and pstimulus ,40. Thusstimulus  that is,stimulus -44; furtstimulus .D-dimer levels but without any abnormal coagulopathy [There are additional observations regarding chronic spontaneous urticaria possibly related to pathogenesis. Early on, an association with Hashimoto thyroiditis, and more specifically, with the presence of autoantibodies was reported,,47 incluulopathy ,51. Tissulopathy . It has ulopathy ,54. Leukulopathy -57. It iεRI pathway [εRI-induced HR and are called responders [Another approach to understanding chronic spontaneous urticaria, whether associated with autoantibodies, is to focus on possible abnormalities within the cell, and the basophil is a prime candidate. A hallmark of CU is the unique relationship of disease activity to altered blood basophil phenotypes . Since t pathway -62. CSU  pathway ,63. Fiftsponders . These bsponders ,66. Furtsponders . Systemisponders ,69. In Csponders ,63.The goal of diagnostic measures is to (1) identify urticaria type and subtype and (2) identify underlying causes (in long-standing or severe chronic sponganeous urticaria only). Urticaria of either acute or chronic type is a common disease that manifests with heterogeneous phenotypes. It poses a high socioeconomic burden for patients . In geneAU is more common than the chronic form and is associated with a rapid recovery, but the identification of its etiology can be helpful to prevent recurrence especially when allergy is suspected to be the cause. Although chronic spontaneous urticaria has various etiologies and subtypes, routine patient evaluation comprising the careful acquisition of patient history, physical examination, and ruling out of systemic diseases should be considered. Specific provocation and laboratory tests are needed to confirm the underlying causes whenever the clinical history is supportive. These extensive diagnostic procedures should be considered on an individual basis in patients with long-standing, severe, or persistent urticaria.Although both a detailed history and physical examination remain essential, the etiology of acute spontaneous urticaria can be suggested by a patient's history. Upper respiratory tract and viral infections are the most common etiology in children. Foods and drugs such as antibiotics and nonsteroidal anti-inflammatory drugs (NSAIDs) can be cIn patients with CSU, it is necessary to obtain a thorough history, including all possible eliciting factors, and to identify the significant aspects of the disease. This initial assessment is helpful in the identification of the subtype of urticaria . The overall duration of CSU is likely to be longer in patients with high disease severity, angioedema, positive autologous serum skin test (ASST) results, or comorbidity with physical urticaria. Next, the impact of the disease on the patient and the disease activity should be evaluated using the urticaria activity score and the CU quality of life questionnaire . The patient should be asked about the time of onset; frequency and duration of wheals; presence of diurnal variation; shape, size, and distribution of wheals; associated angioedema; family and personal history of urticaria; atopy; medications ; and observed correlation with food and stress. The first step is to exclude major comorbid disorders and physical urticaria, and the second step is to identify the underlying cause. Patient questioning together with physical examination and laboratory and provocation tests may be useful to identify associated diseases and comorbidities, and in some cases, the underlying cause. Routine laboratory testing in the absence of a clinical history is rarely helpful in determining an etiology for patients with CSU ,73. NeveHelicobacter pylori in the causation of chronic urticarial is controversial, and the evidence is weak and conflicting [Routine hematological tests, including complete blood count and liver function tests, the determination of erythrocyte sedimentation rate, and C-reactive protein levels may be considered. The role for infectious agents such as flicting . Screeniflicting . Some CSflicting . The ASSSecond-generation antihistamines  at licensed doses represent the mainstay of treatment for urticaria. A number of high-quality, randomized, controlled trials have been carried out with these drugs in patients with mild/moderate urticaria ,5,75. EvThe higher efficacy of cetirizine (10 mg) over fexofenadine (180 mg) has been shown in a randomized, double-blind study . In anotFinally, in a series of in vivo comparative studies assessing suppression of histamine-induced wheal and flare responses of different second-generation antihistamines, cetirizine and its derivative levocetirizine were always superior to other nonsedating antihistamines in terms of efficacy -81. HoweDouble-blind placebo-controlled studies have demonstrated efficacy for several first-generation antihistamines in CU with overall similar efficacy to second-generation antihistamines -87. FirsMany patients with CU may not respond adequately to the recommended doses of second-generation antihistamines. Limited data are available on dosing second-generation antihistamines at higher than the recommended amounts -96. An oMost studies demonstrating efficacy of H2-antagonists added to H1-antagonists in CU have been performed with cimetidine -102. StuThe effectiveness of these drugs has been reported in several relatively small, randomized, double-blind studies,-112 but Although it is clinically recognized that oral corticosteroids are effective for H1-antihistamine-resistant CU, controlled studies are lacking . In viewOne published protocol suggests using prednisone 15 mg daily (preferably 10 mg) and decrease by 1 mg (using 1-mg tablets) each week. Considerable efficacy can be achieved, and subsequent responsiveness to other modalities can be enhanced. If higher doses are needed to significantly lessen symptoms, the drug should not be used . In concAlthough the evidence for efficacy in the treatment of CU for many of the following anti-inflammatory agents is limited, the favorable cost and relatively safe side effect profiles warrant their consideration before using more expensive or more toxic agents.Case reports and case series have found dapsone to be effective in the treatment of CU, idiopathic angioedema, delayed pressure urticaria, and urticarial vasculitis -124. A rCase reports and case series have suggested that sulfasalazine is efficacious in patients with CU and delayed pressure urticaria -130. A rThe most common side effects include nausea, vomiting, dyspepsia, anorexia, and headache . These sLimited data are available on the use of hydroxychloroquine in CU. A case report suggested efficacy in a patient with hypocomplementemic urticarial vasculitis . A randoCU patients with neutrophilic inflammation responded to colchicine, and caseCase reports and case series have described benefit of cyclosporine to patients with CU unresponsive to antihistamines -144. TheTacrolimus, another calcineurin inhibitor, has been reported in an observational study to be effective in CU patients unresponsive to antihistamines, one of which was also unresponsive to cyclosporine .Several other immunosuppressive drugs, including methotrexate, cyclophosphamide, azathioprine, sirolimus, and mycophenolate mofetil, have been used to treat H1-antihistamine-resistant CU. However, most experience relies on case reports or single-center uncontrolled studies. Two recent retrospective studies have been published showing that methotrexate at a weekly mean dosage of 15 mg is effective and safe in the majority of CU patients who are not responsive to conventional therapy ,152. AccRecently, a growing number of studies evaluating the effectiveness of omalizumab  in different subsets of antihistamine unresponsive CU/angioedema patients have been reported ,159-166.Success in CU was first reported in an open-label trial of 10 CU patients with positive ASST and basophil HR tests who failed other therapies at a dose of 0.4 g/kg per day for 5 consecutive days ; 9 of 10Anticoagulants have recently been found to be effective in patients with refractory CU ,177-180.α inhibitors, calcium channel blockers, gold, plasmapheresis,[Other therapies have been reported as cases or case series for the treatment of CU, and very little information is known about their effectiveness and therefore is not recommended for routine use. These treatments include theophylline, androgens, β-agonists, nonsteroidal antinflammatory drugs, tumor necrosis factor-pheresis, phototheAll existing systems to grade the quality of evidence and the strength of recommendations have their own weaknesses. In the present article, the GRADE system  has beenThe prognosis of AU is excellent, with most cases resolving within days; however, the prognosis of CU is variable. If angioedema is present, the prognosis is worsened (see below). CU is more common in adults and unusual in children.Few studies are available on the prognosis of AU ,184. TwoHospital admissions for urticaria were approximately 3 times higher in children aged 0 to 4 years than for other ages. Between 1993-1994 and 2004-2005, there were significant increases in the rate of hospital admissions for urticaria in all ages [In adults, longer disease duration is an important risk for poorer prognosis . AU causStudies in multiple countries report complete resolution in approximately one third of patients with idiopathic CU for more than 1 to 5 years and partial improvement in another third .Spontaneous remission occurs in 30 to 50% of patients within 1 year, and another 20% within 5 years. Nearly 20% of patients still have symptoms after 5 years. Almost half of patients with CU lasting 6 months are likely to have wheals 10 years later . Those wPatients younger than 30 years with more severe symptoms, or symptoms with physical causes, fared less well . For thoIn an Amsterdam prospective study of 220 patients with CU and angioedema, 35% of pA prospective study published in 2004 found that duration of urticaria was longer in patients who had associated angioedema or positive anti-IgE receptor antibody . DiseaseMalignancy has been linked with urticaria and may suggest a relapse of the malignancy. There is no strong evidence to confirm an association between malignancy and uncomplicated CU, except occasionally in urticarial vasculitis and, more frequently, in acquired C1 esterase inhibitor deficiency ,205. AltIn cases involving recurrent angioedema without urticaria, hereditary and acquired angioedema must be differentiated. Acquired angioedema includes, among other etiologies, ACE inhibitor-induced angioedema and angioedema due to acquired C1-inhibitor deficiency. Much like CU, the majority of cases involving acquired angioedema, with some exceptions such as ACE-inhibitor angioedema, can be adequately controlled with daily doses of nonsedating antihistamines . AngioedIn Australia, over an 8-year period, there were 106 deaths associated with anaphylaxis or angioedema. According to this study, there was a continuous increase in the rate of hospital admissions for angioedema (3.0% per year) and urticaria (5.7% per year). The rate of hospitalization for angioedema was highest in persons aged 65 years and older and lowest in children between 5 and 14 years. Although the rate of hospital admissions for angioedema remained relatively constant for most age groups between 1993-1994 and 2004-2005, the rate in persons aged 65 years and older doubled from 10 to 20 per 100,000 population. This represented an average annual increase of 5.6% in the rate of admissions for angioedema in this older age group. For those aged 15 to 34 years, the average annual increase was 4.3%. There was no significant change in the rate of hospital admissions for angioedema in those younger than 15 years or from 35 to 64 years. Among older persons, angioedema is becoming an increasing problem [The prognosis for patients with acquired angioedema associated with C1 inhibitor deficiency is variable and depends on control of the underlying disorder. Even with appropriate treatment of the underlying disease, patients may only temporarily be free of symptoms. In several small studies, patients with acquired angioedema associated with C1 inhibitor deficiency had approximately 20% incidence of non-Hodgkin lymphoma .In summary, the prognosis of urticaria and angioedema is improved with prompt and proper treatment. Using available medications, the condition is usually manageable.There is little published information on the prevalence, diagnosis, or management of urticaria in children. Even in the current EAACI/GA2LEN/EDF/WAO and BSACI guidelines on the diagnosis and management of urticaria,,4 the seA very recent review on urticaria in children was published by Church et al, which coFor AU, infections appear to play a more significant role in infants  and chilIn a recent study in 80 children with CU, dietician supervised elimination diets of all candidate food allergies suspected by the history and specific IgE levels did not result in any participants reducing or eliminating their requirement for ongoing antihistamine medication, suggesting that food allergy is not an important etiological factor in CU in childhood .Three independent studies have shown that CSU in children can be caused by autoreactivity as assessed by use of the ASST -218.In 1 pediatric study of children between 3 and 17 years, 12 of 16 (75%) were diagnosed with additive-induced urticaria, occurring mainly in response to coloring agents, preservatives, monosodium glutamate, and sweeteners, in the absence of atopy .Chlamydia pneumonia and H. pylori [Although some authors suggest that urinary tract infections followed by . pylori  were ass. pylori ,217. Wed. pylori  has suggAlthough thyroid autoimmunity occurs as a comorbidity in between 14 and 33% of adults with chronic spontaneous urticaria, it has bε receptor.In the cohort of pediatric patients with chronic spontaneous urticaria followed for 3 years by Du Toit et al, no clearBy contrast, 58% of children became free of urticarial symptoms in a study of 94 children, of whom 29 were considered "idiopathic" after 16 months, whereas the remaining 42% continued to have recurrent symptoms . A very In view of the marked adverse effects on the quality of life, ability to play, and school attendance, treatment is necessary in nearly all children with chronic spontaneous urticaria. CU negatively affects school performance. First-generation antihistamines, although effective, are no longer recommended for the management of children with chronic spontaneous urticaria .Second-generation antihistamines are the treatment of choice. In a study of antihistamine treatment given to infants with atopic dermatitis, continuoPediatric suspensions of desloratadine, fexofenadine, rupatadine, and loratadine are available, but pediatric studies on these second-generation H1 antihistamines, which are effective in adult urticaria particularly at standard and higher than standard doses, are still to be performed.In the follow-up study by Du Toit et al, all chilThere are no pediatric studies on the use of leukotriene receptor antagonists, H2 antihistamines, cyclosporine, or omalizumab for the treatment of urticaria. Experience with cyclosporine in children with severe resistant chronic spontaneous urticaria is similar to that reported in adults. It has been found to be safe and highly effective when indicated .There is no evidence in the literature that children with persistent spontaneous urticaria, who do not go into spontaneous remission within a few years, go on to develop other autoimmune diseases. Long-term follow-up studies (more than 10 years) of urticaria in children are awaited.Urticaria may occur in pregnancy as a result of any of the causes seen in nonpregnant women. In women with preexisting CU, the condition may worsen in some patients and appears to improve in others .Urticaria occurring only in pregnancy is rare, but when it occurs, it suggests that sensitivity to hormones may be the basis of the condition. It may recur with each pregnancy in a predisposed woman. Gestational urticaria must be distinguished from other pruritic dermatoses of pregnancy, such as prurigo of pregnancy, PUPPP, PEP, or autoimmune progesterone dermatitis of pregnancy.This condition is relatively common occurring in approximately 1 in 300 pregnancies. Characteristically, it begins in the second or third trimester. Patients usually present with marked excoriations with erythematous nodules or papules on the extensor surfaces of the limbs and the trunk. Usually total remission occurs immediately postpartum. Management is usually with topical corticosteroids .PUPPP occurs in 1 in 160 to 1 in 300 pregnancies and usually presents in the third trimester ,232. It This condition is similar to the rare autoimmune progesterone urticaria that occurs in a cyclical pattern in nonpregnant women. In pregnancy, it is characterized by a papulopustular eruption, transient arthritis, eosinophilia, and delayed hypersensitivity to intradermal progesterone. It may be associated with spontaneous abortion -236.Pregnant women with urticaria should be treated with the least amount of medication possible. Most patients can be treated with H1 antihistamines alone, with occasional short courses of oral glucocorticosteroids (GCS) for severe flares.Antihistamine treatment is the mainstay of management in urticaria. The intense itch experienced by patients demands relief, whereas soothing baths and emollients offer minor comfort; most patients require symptomatic relief with an antihistamine.There are no oral antihistamines with a category A listing for pregnancy. Categories are based on the results of animal studies, human data, and whether the use of the drug has a positive risk-benefit ratio in pregnancy. Category "B" drugs possess reassuring animal data, but there are no controlled clinical human trials.A number of studies -240 haveChlorpheniramine, loratadine, cetirizine, and levocetirizine have all been assigned category B by the US Food and Drugs Administration. As with any medication use, antihistamines should only be used if clearly needed and when the benefits outweigh the potential risk to the fetus. Use of the lowest dose that gives relief is advisable. There are several thousand reports of chlorpheniramine use in pregnancy with no evidence of increased incidence of congenital abnormality. No rate of increased congenital defects was reported in prospectively collected data from 1769 women exposed to loratadine. Small sample size studies are available for cetirizine, and there is a meta-analysis available for loratadine ,238. HydThe second-generation antihistamines of choice in pregnancy are loratadine 10 mg daily or cetirizine 10 mg daily because there is a body of evidence of their use in pregnancy with reassuring safety profiles .In special cases where a sedative effect is required along with an antihistaminic effect, chlorpheniramine is the first-generation antihistamine of choice. Recommended dosing is 4 mg 3 to 4 times a day. Diphenhydramine shows higher efficacy and can be used as an alternative to chlorpheniramine if the use of a first-generation antihistamine is being considered.Significant amounts of some antihistamines are detected in breast milk. Again, antihistamines should only be used during lactation when the benefit outweighs the potential harm to the infant, and in this circumstance, use the lowest dose possible for the shortest duration to give relief from symptoms. Loratadine and cetirizine appear safer than others with very low levels recorded in breast milk ,242.Systemic corticosteroids may be required periodically to gain temporary control of symptoms during severe exacerbations of urticaria that significantly impair the quality of life. These rescue courses are generally added to the medications the patient is already taking.The optimal dose and duration of GCS used for urticarial exacerbations has not been systematically studied, and recommendations vary among specialists. In addition, patients differ in their responsiveness to GCS in both the dose and duration of treatment required to control symptoms. Because of their importance in the treatment of a variety of inflammatory conditions, systemic GCS have been used fairly extensively during pregnancy.Three potential areas of concern have been raised: congenital malformations , neonatal adrenal insufficiency, and low birth weight . The comNeonatal adrenal insufficiency following maternal administration of steroids is unusual. The rapid maternal metabolism of prednisolone binding to serum proteins and conversion to inactive metabolites by placental 11 beta-hydroxysteroid dehydrogenase results in relatively low fetal compared with maternal concentrations . As a reMultiple studies have observed low birth weight in offspring of animals given GCS during pregnancy. However, this association has been rarely reported in humans . It is dLow levels of prednisone and prednisolone can be measured in breast milk. A nursing infant of a mother consuming a daily dose of 80 mg of prednisolone would ingest < 0.1%, which is equivalent to < 10% of endogenous cortisol production . As a reDuring the past 20 years, relevant progresses have been made in defining and evaluating PROs, with growing recognition of their importance in health outcomes research. The expression PROs refers to all health-related reports coming from the patient, without involvement or interpretation by a physician or others  [ie, heaPROs have recently gained great attention in clinical research and by regulatory bodies due to their relevance in the overall treatment efficacy assessment -255. A cHRQoL in CU has been assessed by generic, dermatologic-specific, and disease-specific tools. Several generic tools have been used to compare HRQoL of CU patients and healthy subjects--Medical Outcomes Study, SF-36,,258 WorlThe available questionnaires aimed at assessing HRQoL in skin diseases, the Dermatology Quality of Life Index, and the The Chronic Urticaria Quality of Life Questionnaire is the only validated specific instrument for CSU and was originally developed in Italian, German, Spanish,Available data show that from a subjective viewpoint, CU is more than an annoying disease. CU subjects report lower HRQoL when compared with healthy subjects or with patients suffering from other medical conditions. A pioneer study by O'Donnell et al  comparedFurthermore, HRQoL levels in patients with chronic spontaneous urticaria are lower than in healthy subjects and in patients with respiratory allergy . A studyMore recently, an article by Staubach et al  showed tAs yet, the effect of treatment on HRQoL of CU patients has been explored only in 11 trials ,270-276.CU symptoms can be specifically evaluated with the Urticaria Activity Score . This isAlthough PROs evaluation is relevant for a more global comprehension of a disease and its treatment, the available literature on CU is still poor. The following unexplored areas should be further investigated:• Other PROs besides HRQoL and symptoms• CU impact on caregivers and partners• Impact of treatment on HRQoL by a specific questionnaire• Relationships among different PROs and between PROs and psychological variables• Relation of PROs with other clinical measures of health impact.According to the current international EACCI/GA2LEN/EDF/WAO guidelines on urticaria,,5 physicPhysical urticaria needs to be distinguished from both spontaneous urticaria and other inducible urticaria types, such as aquagenic urticaria or cholinergic urticaria, where wheal formation is not induced by a physical stimulus. Physical urticarias usually have a chronic course, but patients can be free of symptoms for weeks or months when the physical stimulus is avoidable and avoided. This is a clear-cut difference to chronic spontaneous urticaria. One point of confusion in the past has been between physical urticaria and cholinergic urticaria. Cholinergic urticaria symptoms can be elicited through a hot shower or bath. The underlying mechanism in cholinergic urticaria, however, is not the external stimulus but the increase in body core temperature; cholinergic urticaria can also be elicited by exercise or emotional distress and is, therefore, included in the urticaria subgroup "other inducible urticarias."Table 2LEN/EDF/UNEV consensus panel recommendations."[Although the current international guidelines on the classification, definition, and diagnosis of urticaria give general recommendations, more detailed recommendations for diagnostic testing in physical urticaria are published in the "European guideline definition and diagnostic testing of physical and cholinergic urticariasd--EACCI/GAdations."A general principle in the diagnosis of physical urticaria is to mimic the physical stimulus, which leads to the formation of wheals and angioedema and at the same time if possible determine the threshold. Threshold measurements are important because they can help to give the patient practical advice on how to avoid or reduce above threshold stimulus exposure. Threshold testing also allows for the objective evaluation and monitoring of patients who receive treatment. Figure When performing provocation tests in patients with physical urticaria, it is recommended to have the same standard of emergency treatment available as for other kinds of allergy skin testing because rare cases of systemic anaphylactic reactions, especially in cold urticaria, have been described.Physical urticaria can vary considerably in severity between individuals. In a number of patients, signs of physical urticaria only occur with unusually strong external stimulation of the skin, for example, very cold, windy, winter days in cold urticaria, and depending on the usual geographic location and everyday living habits, the required strength of the stimulus to elicit symptoms is not usually reached. However, in other cold urticaria patients, the eliciting temperature of the skin can be as high as 28°C, a temperature which is easily reached in usual daily activities in moderate climates, and even in warm climates, if there is a mild wind because wind chill temperature increases the cooling effect on the skin. In cold urticaria, systemic reactions have been described in the case of a rapid change of skin temperature, for example, when patients jumped into cold water. Another risk factor in cold urticaria is the rapid ingestion of cold food such as ice cream or cold beverages, which may lead to swellings of the upper airways and in the esophagus and to systemic histamine liberation and subsequent anaphylactic reactions.Physical urticaria can also have an impact on occupation. It has been recognized as an occupational disease .A general principle of the international urticaria guidelines on the management of urticaria is the identification and elimination of the underlying cause and/or trigger . AlthougWith the exception of cold contact urticaria where in rare cases infectious diseases, such as hepatitis, have been described as an underlying cause, it is not recommended to invest too many resources into the investigation of causes. In physical urticaria, the routine diagnostic program should be limited at the most to differential blood count and the determination of erythrocyte sedimentation rate. However, with the identification of the eliciting trigger, it is in many cases easy to help the patient by in detail explanation of the possibilities for avoidance. For example, pressure is defined as force per area and simply increasing the size of a handle of a bag may help in patients with pressure urticaria to avoid symptoms.The treatment in physical urticaria is aimed at the prevention and reduction of symptoms. This follows in general the algorithm, which has been published for urticaria in the international consensus guidelines Figure .The level of evidence for first-line treatment with nonsedating antihistamines is very good both in chronic spontaneous urticaria and physical urticaria. The updosing of nonsedating antihistamines has been widely studied in physical urticaria.Thus, the level of evidence to use higher than standard doses as the preferred second-line treatment is very high in this group of urticarias. Siebenhaar et al  studied In general, however, the level of antihistamine treatment required may be different from day to day depending on the strength of the external stimuli and patients' needs to be very thoroughly counseled on the daily use of the drug treatment.Alternative treatments in physical urticaria have only been scarcely studied and knowledge needs to be extrapolated from what we know from chronic spontaneous urticaria. However, physical urticarias are distinct from other urticaria subtypes in that it is possible to achieve a reduction of repetitive mast cell responses to the specific physical stimulus by long-term controlled exposure to the stimulus. For example, in cold contact urticaria, the occurrence of symptoms can be prevented by administering daily cold baths, and for solar urticaria, UV light treatment can raise UV tolerance. However, this kind of treatment is time consuming for the patient and in the case of cold bath is not always very well liked. Furthermore, it is recommended to start at the threshold level and increase slowly the strength of the physical stimulus because generalized reactions may occur.World Allergy Organization Journal  at http://www.WAOJournal.org to facilitate rapid access by all 3000 WAO members. The WAO Member Societies are encouraged to contribute with the dissemination of this position paper through discussion at national and international meetings, and translation and publication in national allergy society journals.The WAO urticaria and angioedema position paper is being published in the This Position Paper presents recommendations for the proper diagnosis and treatment of urticaria and angioedema, highly prevalent diseases in all areas of the world. Although there have recently been important advances in the elucidation of the pathogenesis, allowing the implementation of innovative diagnostic and therapeutic procedures for patients suffering urticaria, the basic mechanisms remain elusive.Second-generation nonsedating antihistamines at usual or increased doses are presently recommended as first-line therapy for patients with acute and chronic spontaneous urticaria and angioedema. Alternative treatments include H2-antagonists, corticosteroids, leukotriene receptor antagonists, other antiinflammatory drugs, immunosuppressants, omalizumab, and intravenous immunoglobulins.About one third of patients with CU will continue to experience symptoms after 5 years of follow-up. Consequently, it is important to provide early treatment to improve patient's quality of life. Reduction of the exposure to precipitating and aggravating factors is also important, especially in patients with physical urticarias.Abstract, Introduction, WAO Global Position Papers, Methods, Prevalence, Dissemination and implementation of the Position Paper, Summary: M. Sánchez-Borges. Definition and Classification: P. Tassinari. Etiology and Pathogenesis: A. P. Kaplan and S. Saini. Diagnostic Approach: H. S. Park, Y.M. Ye, and I. Treatment: A. R. Asero, J. A. Bernstein, D. A. Khan, and A. Tedeschi. Prognosis: R. Gower. Urticaria and Angioedema in Children: P. Potter. Urticaria and Pregnancy: C. Katelaris. Quality of Life and Patient's Reported Outcomes: I. Baiardini and G. W. Canonica. Special Considerations of Physical Urticarias: M. Maurer and T. Zuberbier.All authors reviewed and approved this manuscript.


Ca2+-handling abnormalities may promote ectopic activity, conduction abnormalities facilitating reentry, and AF-related remodeling. In this review article, we summarize the Ca2+-handling derangements occurring in AF and discuss their impact on fundamental arrhythmogenic mechanisms. We focus in particular on the role of the multifunctional Ca2+/calmodulin-dependent protein kinase type-II (CaMKII), which acts as a major link between Ca2+-dysregulation and arrhythmogenesis. CaMKII expression and activity are increased in AF and promote arrhythmogenesis through phosphorylation of various targets involved in cardiac electrophysiology and excitation-contraction coupling. We discuss the implications for potential novel therapeutic strategies for AF based on CaMKII and Ca2+-handling abnormalities.Atrial fibrillation (AF) is the most frequently encountered clinical arrhythmia and is associated with increased morbidity and mortality. Ectopic activity and reentry are considered major arrhythmogenic mechanisms contributing to the initiation and maintenance of AF. In addition, AF is self-reinforcing through progressive electrical and structural remodeling which stabilize the arrhythmia and make it more difficult to treat. Recent research has suggested an important role for Ca Repolarizing currents include the transient-outward K+-current (Ito), delayed-rectifier K+-currents with slow, rapid or ultra-rapid kinetics , as well as the Na+/K+-ATPase current (INaK). In addition, AP duration (APD) and resting membrane potential are influenced by basal and acetylcholine-activated inward-rectifier K+-currents . The IKur and IK,ACh currents are predominantly expressed in the atria, thereby providing potential atrial-specific therapeutic targets.The atrial action potential (AP) is determined by depolarizing and repolarizing ionic currents  through type-2 ryanodine receptor channels (RyR2), producing the systolic Ca2+-transient responsible for initiating contraction of atrial cardiomyocytes -receptor-mediated Ca2+-release may contribute to Ca2+-induced Ca2+-release by activating neighboring RyR2, although direct IP3-receptor-mediated activation of NCX1 has also been described recently , or is taken back up into the SR by the type-2a SR Ca2+-ATPase (SERCA2a). The affinity of SERCA2a for intracellular Ca2+ is largely determined by the inhibitory proteins phospholamban (PLB) and sarcolipin. The expression of sarcolipin is atrial-specific, whereas PLB is more strongly expressed in the ventricles than in the atria . EADs are caused primarily by recovery from inactivation of ICa,L during excessive APD-prolongation, for example due to loss of repolarizing K+-currents. DADs are likely the most common mechanism underlying ectopic (triggered) activity and result from intracellular Ca2+-handling abnormalities. Spontaneous diastolic SR Ca2+-release events resulting from SR Ca2+-overload or intrinsic RyR2-dysfunction can activate NCX1, resulting in a transient-inward current that depolarizes the membrane potential as Ca2+ is extruded from the atrial cardiomyocyte  and one without such NLS sequence (CaMKIIδC). The latter was traditionally considered cytosolic  , which have been implicated in the initiation of AF in some animal models  activity, promoting AF initiation. Genetic mouse models have revealed that intrinsic RyR2-dysfunction is sufficient to increase the susceptibility to pacing-induced AF, as reviewed in . These mice also have an increased susceptibility to pacing-induced AF .Atrial cardiomyocytes from cAF patients have unaltered RyR2 protein expression levels and SR CaF Figure . Whereas2+-handling abnormalities also play a role in AF-promoting reentry. APD-shortening is a hallmark feature of AF-related remodeling that facilitates the maintenance of reentrant circuits. It is largely mediated by a reduction in depolarizing ICa,L and an increase in several repolarizing K+-currents. Various mechanisms contribute to reduced ICa,L in AF   K+-current (ISK) is upregulated in atria of cAF patients, which might contribute to APD shortening  is a critical regulator of myofilament function  develop age-dependent progression from spontaneous atrial ectopy to paroxysmal and long-lasting AF episodes  in CREM mice prevents Ca2+-handling abnormalities and spontaneous AF, as well as atrial dilatation and conduction abnormalities  activity and reentry that contribute to AF initiation and maintenance. The expected increase in the incidence of AF and the limited efficacy and safety of currently available antiarrhythmic drugs, make a better understanding of these AF-modulating processes critical for the development of improved therapeutic strategies. Ca2+-handling abnormalities provide a novel set of potential antiarrhythmic targets for the treatment of AF. However, due to the multitude of etiologies and complexity of mechanisms underlying clinical AF, it is likely that tailored therapeutic strategies for specific groups of patients that target multiple pathophysiological processes will be necessary. Cardiac-specific inhibition of CaMKII could be a promising therapeutic strategy for certain groups of AF patients.CaThe authors' work is supported by the European–North American Atrial Fibrillation Research Alliance (07CVD03 to Dobromir Dobrev) and the Alliance for Calmodulin Kinase Signaling in Heart Disease  grants of Fondation Leducq, the European Network for Translational Research in Atrial Fibrillation , the German Federal Ministry of Education and Research through the DZHK , the American Heart Association (13EIA14560061 to Xander H. T. Wehrens), Muscular Dystrophy Association (186530), and National Institutes of Health grants HL089598, HL091947, and HL117641 (to Xander H. T. Wehrens).The 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.


Results showed that intracellular Ca2+ concentration increased rapidly when extracellular salinity suddenly decreased or increased, but the increase could be inhibited by pretreatment of Ca2+ channel blockers LaCl3, verapamil or ruthenium red. The changes of glycerol content and G3pdh activity in D. salina to respect to hypoosmotic or hyperosmotic stress were also inhibited in different degrees by pretreatment of Ca2+ channel blockers, indicating that the influx of Ca2+ via Ca2+ channels are required for the transduction of osmotic signal to regulate osmotic responses of D. salina to the changes of salinity. Differences of the three blockers in block effect suggested that they may act on different channels or had different action sites, including influx of Ca2+ from the extracellular space via Ca2+ channels localized in the plasma membrane or from intracellular calcium store via the mitochondrial. Other Ca2+-mediated or non-Ca2+-mediated osmotic signal pathway may exist in Dunaliella in response to hypoosmotic and hyperosmotic stresses.The effect of Ca The aim of this paper is to study the role of Ca2+ by using Ca2+ channel blockers LaCl3, verapamil (VP) and ruthenium red (RR) to elucidate the osmotic stress signal transduction pathway in D. salina.In order to obtain direct evidence of the involvement of CaD. salina strain (FACHB-435) was obtained from Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academic of Sciences. Cells of D. salina were cultivated in the culture medium containing 2.0 M NaCl at 26°C and 108 µmol m−2 s−1 provided by cool-white fluorescent lamps, under a 14/10 h light/dark cycle with shaking at 96 rpm according to Chen et al. D. salina cells were loaded with the fluorescent dyes 5 µmol/L final concentration of acetoxymethyl ester form (AM) of fluo-3 and 50 mmol/L final concentration of sorbitol at 25°C in the dark for 1 h. Loaded cells were subsequently rinsed 3 times in fresh iso-osmotic medium, and then harvested by centrifugation. Harvested loaded cells were resuspended in fresh iso-osmotic and iso-volumetric medium and cultivated for 2 h again.The algal cells at log phase were harvested by centrifugation at 5,000 g for 15 min at room temperature. The algal pellet was resuspended in fresh iso-osmotic and iso-volumetric medium and cultivated for 1 h. Then 2+ channel blockers respectively and preincubated for 10 min. These channel blockers were a non-specific Ca2+ channel blocker LaCl3, a voltage-dependent Ca2+ channel blocker verapamil and a putative mitochondrial and endoplasmic reticulum Ca2+ channnel inhibitor ruthenium red, whose final concentration were 0.2 mmol/L, 10 µmol/L and 10 µmol/L.The loaded cells with fluo-3 AM were added three CaD. salina cells preincubated with Ca2+ channel blockers were harvested by centrifugation at 5,000 g for 15 min at room temperature and then treated with hypoosmotic or hyperosmotic stress in isovolumetric fresh medium, which contained 0.5 or 4.5 M NaCl.D. salina cells loaded with fluo-3 AM was detected immediately under CLSM . D. salina culture was dropped on the slide with a groove that was filled with culture. The intact D. salina cells in the groove of the slide covered with coverslip were chosen by microscope for detection. Imaging of the cells were obtained with excitation by argon laser and monitored with an intensified CCD camera. The excitation wavelength and emission wavelength were 488 nm and 525 nm. Data of image and fluorescence were detected every 10 s under CLSM with soft LAS AF 2.1.1 build 4443 and the total time was 10 min. An intact D. salina cell was chosen to calculate single cell cytosolic free calcium concentration. Concentration of cytosolic free calcium was calculated from the following equation: Kd is the dissociation constant of the fluo-3 AM, which is 450 nM according to the manufacturer's instructions; F is the fluorescence of sample; Fmin is the fluorescence in the absence of calcium and Fmax is the fluorescence of the sample at saturated calcium concentration. To obtain Fmax, the cells were exposed to a solution contained 10 µmol/L A-23187, an ionophore that is commonly used for intracellular calibration of calcium indicators. The cells were then exposed to the Ca-free solution with 1 mmol/L EGTA to obtain Fmin.After treatment with hypoosmotic or hyperosmotic stress, fluorescence from 2+ channel blockers respectively and preincubated for 10 min. Afterwards cells were harvested by centrifugation and treated with hypoosmotic or hyperosmotic shock in isovolumetric fresh medium, which contained 0.5 or 4.5 M NaCl. After 2 h, resuspended algae cells were harvested again by centrifugation for the measurement of glycerol content. According to the method of Chen et al. The algal cells in the culture containing 2.0 M NaCl at log phase were harvested by centrifugation at 5,000 g for 15 min at room temperature. The algal pellet was resuspended in fresh iso-osmotic and iso-volumetric medium, then added the three Ca2+ channel blockers respectively for 10 min and then treated with hypoosmotic or hyperosmotic stress for 2 h according to the method above for the extraction of enzyme and the measurement of (NAD+)-dependent G3pdh activity. The crude enzyme extract from cells of each sample were obtained according to the method of Chen et al. The algal cells in the culture containing 2.0 M NaCl were also preincubated with the three CaThe activity of G3pdh, catalyzing a reversible reaction, was analyzed according to the method of Wei et al. y) against OD595 value (x) was plotted and the protein concentration was calculated according to the regression equation y = 1.5746x−0.0170, R2 = 0.9969. From the relationship curve between OD340 and NADH concentration regression equation, the NADH concentration was obtained by determining OD340: Y = 235.84X+0.0118, 2R = 0.99002, where Y represents NADH concentration (nmol/mL) and X represents OD340 value.A relationship curve of protein concentration (mg/mL)  in cells of the nitrogen-fixing filamentous cyano-bacterium Anabaena sp. PCC7120. Ca2+ transients induced by NaCl and sucrose were completely blocked by the calcium chelator ethylene glycol-bis(β-aminoethylether)N,N,N′,N′-tetraacetic acid (EGTA) and were partially inhibited by the calcium channel blocker verapamil. However, Karimova et al. 2+ and Na+ cross the membranes of two Dunaliella species , the Ca2+ uptake depended on the intracellular Na+ release, and the agents blocking Ca2+ channels did not affect the transport of Ca2+ and Na+.The increase in cytosolic free Ca2+ concentration in the samples treated by verapamil were small and the trends were stable at the end, suggesting that verapamil may also take block effect in the process of the extrusion of Ca2+ from cytoplasm at later stage, which perhaps was resulted from non-specific deleterious effects of this pharmacological inhibitor.From 2+ measurements using fluo-3, fluo-4, and related indicators are hampered if there are significant Ca2+-independent fluorescence intensity fluctuations from cell to cell due to variations in the intracellular indicator concentration 2+ fluorescence was detected on single cell by using fluo-3, and all fluorescence pictures in 2+-independent fluorescence fluctuations from cell to cell were minor and the conclusions were reasonable.Quantitative CaD. salina cells treated by Ca2+ channel blockers under hypoosmotic or hyperosmotic stress. It was found that the shapes of algal cells in all samples were normal and intact, which illuminated that all blockers had no toxic or less toxic on the algal cells. It was also found that there was no marked difference on the shape of cells treated by the same salinity between the cells with blocks and the cells without blockers, indicating that these Ca2+ channel blockers might have no effect on the variation of cell shape.3 or ruthenium red, the decrease of glycerol content was less than the sample without Ca2+ channel blocker  but increased by high concentrations of Ca2+ (10 mM) 2+ could regulate intracellular glycerol content under osmotic stress.As a result, in D. salinacell reduced, but the activity in algae cell treated with LaCl3 or verapamil raised, which suggested that LaCl3 and verapamil could block the regulation of Ca2+-mediated osmotic signal on G3pdh forward reaction activity under hypoosmotic stress -dependent G3pdh activity in D. salina under salt stress. Similarly, in a study by Kadota et al. 2O2-induced [Ca2+]cyt rose the expression of antioxidant enzymes. Glutathione peroxidase (GPX) and ascorbate peroxidase (APX) in tobacco BY-2 cells were inhibited by the cosuppression of NtTPC1A/B as well as Al ion, a specific blocker for NtTPC1s, the oxidative stress-responsive putative voltage-dependent Ca2+ permeable channels, suggesting that NtTPC1s are the major Ca2+-permeable channels activated by H2O2 and that Ca2+ influx via NtTPC1s is involved in induction of H2O2-triggered gene expression.Ca2+ channel blockers under salt stress were not in line with the corresponding block effect treated by other blockers (2+ concentration was ruthenium red >LaCl3> verapamil. Ruthenium red blocked the variations of both glycerol content and G3pdh activity except G3pdh activity under hypoosmotic stress, LaCl3 blocked the variations of both glycerol content and G3pdh activity except G3pdh backward reaction activity under hyperosmotic stress. Verapamil had no block effect on glycerol content and G3pdh backward reaction activity under hypoosmotic stress and G3pdh forward reaction activity under hyperosmotic stress, which suggested that the three blockers had different block effect on Ca2+ concentration, glycerol content and G3pdh activity. The signal transduction was mediated by Ca2+ influx via different Ca2+ channels, which were blocked by different blockers, and may have different regulatory effects on osmotic responses. Single signal transduction could not regulate whole osmotic response of glycerol and G3pdh, and there might be a synergistic effect of various osmotic signal transductions by Ca2+ influx via all Ca2+ channels or other Ca2+ influx mechanism. Future work should focus on the combined effects of the three blockers to determine whether they act on a single or multiple Ca2+ channels. By observing the combined effects, we can determine whether there is other Ca2+-mediated or non-Ca2+-mediated osmotic signal pathway in Dunaliella under hypoosmotic or hyperosmotic stress.However, it was also found that the variations of glycerol content and G3pdh activity in samples treated by some Cablockers . From Fi