On the one hand, elevated myocyte Ca2+ can cause delayed afterdepolarizations, triggered activity, and consequently life-threatening ventricular tachyarrhythmias in various pathological conditions such as digitalis toxicity, myocardial ischemia, or heart failure. On the other hand, VF itself directly and rapidly causes progressive myocyte Ca2+ overload that maintains VF and renders termination of VF increasingly difficult. Accordingly, energy levels for successful electrical defibrillation (defibrillation thresholds) increase as both VF and Ca2+ overload progress. Furthermore, VF-induced myocyte Ca2+ overload can promote re-induction of VF after defibrillation and/or postfibrillatory myocardial dysfunction (postresuscitation stunning) due to reduced myofilament Ca2+ responsiveness. The probability of these adverse events is best reduced by early detection and rapid termination of VF to prevent or limit Ca2+ overload. Early additional therapy targeting transsarcolemmal Ca2+ entry, particularly during the first 2 min of VF, may partially prevent myocyte Ca2+ overload and thus, increase the likelihood of successful defibrillation as well as prevent postfibrillatory myocardial dysfunction.Based on recent experimental studies, this review article introduces the novel concept that  cardiomyocyte Ca In. In2+ anchannels . This is2+ source, VF-induced myocyte Ca2+ overload contributes to maintain VF, leading to a self-maintaining vicious circle in which termination of VF becomes increasingly difficult  we could demonstrate a causal relationship between myocyte Ca2+ concentrations and defibrillation success. Thus, the longer VF lasts, the higher both myocyte Ca2+ concentration and defibrillation threshold rise. This relationship was not due to prolonged myocardial ischemia because the hearts were continuously perfused during VF  , a , a 2+ ant hearts ,15. Accoalpains) . The subalpains) .2+ responsiveness following VF-induced myocyte Ca2+ overload. Therefore, postfibrillatory myocardial dysfunction presumably is a consequence of reduced myofilament Ca2+ responsiveness following myocyte Ca2+ overload caused by both VF and consequent ischemia. Further contributions to postfibrillatory myocardial dysfunction may arise from ischemia-induced oxygen free radicals and/or from the impact of electrical defibrillation shocks. However, it seems that only high energy shocks of monophasic waveform may precipitate myocardial injury and dysfunction. Biphasic shocks of normal energy  [Postresuscitation stunning may be clinically relevant because defibrillation frequently results in a depressed myocardial function , particual mass) .2+ and VF may also apply to atrial fibrillation (AF). It has been proposed that with increasing duration of paroxysmal AF, the likelihood that it will become chronic increases in parallel[electrical remodeling of the atria involving myocyte Ca2+ overload[2+ channel blocker [2+ release blocker) [2+ agonist BAY K8644, [2+ influx contributed to this dysfunction. All these observations fit in our vicious circle concept, whereby AF-induced Ca2+ overload may be responsible for the progressive nature of AF and for the re-initiation of AF as well as atrial contractile dysfunction after cardioversion.The same vicious circle described above for myocyte Ca parallel. This so overload. Atrial  blocker -28 or ryblocker)  attenuatblocker) -28 and rblocker)  or even blocker)   re-initblocker)  even thoY K8644,  suggesti2+ and VF forming a vicious circle, arise from studies in cardiomyocytes or in isolated hearts of rats or ferrets. Importantly, the central role of Ca2+ in excitation-contraction coupling involving Ca2+-induced Ca2+ release in cardiac muscle physiology [2+ overload in most species including adult human beings. However, important inter-species and developmental differences exist regarding Ca2+-induced Ca2+ release from the sarcoplasmic reticulum and regarding Ca2+ removal processes [2+- induced Ca2+ release was absent in frog or prenatal rat ventricle, intermediate in human ventricle, and most prominent in adult rat ventricular myocytes [2+ in cardiac muscle of various species depends on different contributions from the sarcoplasmic reticulum, from L-type Ca2+ channels, and from forward Na+/Ca2+ exchange [2+ overload may therefore vary among species and be part of the reason why some species are better protected against sustained VF than others . Based on the foregoing, VF in adult human beings most likely induces cardiomyocyte Ca2+ overload and the Ca2+ sources of this overload may be slightly but not fundamentally different from adult rat ventricles. Finally, similarities of myofilament Ca2+ sensitivity [2+ overload is an important and species-independent part of postfibrillatory myocardial stunning. This is why the proposed vicious circle of Ca2+ overload is likely to be of importance in human VF (and may be in AF too).Extrapolation of the experimental findings in this article to the clinical setting should consider inter-species differences of excitation-contraction coupling. Most of the findings that underlie the proposed concept of Caysiology  suggestsrocesses . For examyocytes . Consequexchange . The kinsitivity  and molesitivity  suggest 2+ and VF are mutually related forming a self-maintaining vicious circle in the initiation, maintenance, and termination of VF. On the one hand, various experimental studies have shown that elevated myocyte Ca2+ can cause delayed afterdepolarizations, triggered activity, and consequently life-threatening ventricular tachyarrhythmias. On the other hand, VF itself directly and rapidly causes progressive myocyte Ca2+ overload that maintains VF and renders termination of VF increasingly difficult. Accordingly, VF-induced myocyte Ca2+ overload can cause electrical defibrillation to fail. Furthermore, VF-induced myocyte Ca2+ overload can promote re-induction of VF after defibrillation and/or postfibrillatory myocardial dysfunction (postresuscitation stunning) due to reduced myofilament Ca2+ responsiveness. The same vicious circle may apply to AF during electrical remodeling to cause re-initiation of AF and atrial contractile dysfunction after cardioversion. The experimental studies reviewed here suggest that the probability of these adverse events is best reduced by early detection and rapid termination of VF to prevent or limit Ca2+ overload. In other words, rapid termination of VF not only reduces the risk of cerebral injury but also the likelihood of failed defibrillation and postfibrillatory myocardial dysfunction. Early additional therapy targeting transsarcolemmal Ca2+ entry, particularly during first 2 min of VF, may partially prevent myocyte Ca2+ overload and thus, increase the likelihood of successful defibrillation as well as prevent postfibrillatory myocardial dysfunction. Thereafter, the vicious circle of myocyte Ca2+ overload is established and therapeutic attempts have an inherently lower likelihood of success.Cardiomyocyte Ca


Freely diffusible hydrogen peroxide (H2O2) is implied to alter Ca2+ homeostasis, which further enhances abnormal cellular activity, causing changes in signal transduction, and cellular dysfunction. Whether H2O2 could affect [Ca2+]i in HSV-1-infected cells had not been investigated.It was reported that elevation of the intracellular concentration of free Ca2O2 treatment increased the amount of cell-free virus and decreased the proportion of viable cells. After the treatment, an elevation in [Ca2+]i was observed and the increase in [Ca2+]i was suppressed when intracellular and cytosolic Ca2+ were buffered by Ca2+ chelators. In the presence of Ca2+ chelators, H2O2-mediated increases of cell-free virus and cell death were also diminished. Electron microscopic analysis revealed enlarged cell junctions and a focal disintegration of the plasma membrane in H2O2-treated cells.H2O2 can elevate [Ca2+]i and induces non-apoptotic cell death with membrane lesions, which is responsible for the increased release of HSV-1 from epithelial cells.These results indicate that H Polymorphonuclear leukocytes (PMNs) have been detected in the early cellular infiltrate at sites of herpes simplex virus (HSV) infection . It was 2O2) as an oxygen radical can damage DNA directly by penetrating the cell nucleus or indirectly by increasing the intracellular concentration of free Ca2+ ([Ca2+]i). The peroxidation of membrane phospholipids leads to alterations in Ca2+ homeostasis, which further enhances abnormal cellular activity, causing changes in signal transduction, and cellular dysfunction [2O2 was cytotoxic to renal tubular epithelial cells and caused a sustained and uncontrolled rise in [Ca2+]i that preceded substantial cell injury or irreversible cell death i that preceded substantial cell injury or irreversible cell death [2O2 could affect the [Ca2+]i was examined at concentrations to enhance the virus release. FI cells were infected with HSV-1 at an MOI of 2 PFU/cell and cultured for 18 h. The mean level of [Ca2+]i in HSV-1-infected cells was approximately 200 nM. When the infected cells were treated with 1 mM H2O2, a significant rise in [Ca2+]i beginning approximately 30 sec after the exposure to H2O2 was observed. Subsequently, there was a secondary rise in [Ca2+]i, that appeared within 40 sec; a maximal level (460 nM) was attained in 6 min ethane- N',N',N',N'-tetraacetic acid (BAPTA) or quin-2, for 20 min prior to the H2O2 treatment, both the initial and secondary rises in [Ca2+]i were suppressed. Although the secondary rise was suppressed by this treatment, the level of [Ca2+]i gradually increased to 300–350 nM in 8 min -N',N',N',N'-tetraacetic acid (EGTA), for 20 min until 18 h p.i., and then Hvel Fig. . When HSmin Fig.  and 2D.2+ depletion on the release of HSV-1 was examined. Eighteen hours after infection, cells were pretreated with 10 mM EGTA for 20 min to deplete extracellular Ca2+. Thereafter, treatment with 1 mM H2O2 for 2 h was initiated. In this condition, the amount of cell-free virus was 150% of that in the untreated control, whereas it was increased to 450% of the control value by the treatment with H2O2 When HSV-1-infected cells were treated with 1 mM H2O2, electron microscopy was used. The cultures were fixed in situ and sections parallel to the dish surface were prepared. HSV-1-infected cells had large vesicular nuclei with dispersed chromatin. In the portion where cell-to-cell interaction was tight, a large number of viral particles were pooled in a narrow intercellular space . Cells were grown on glass-based plastic dishes and incubated with 4 μM fura-2/AM in DMEM for 30 min at 37°C. Cells were then washed in modified Hank's solution (Sigma) containing 137 mM NaCl, 3.5 mM KCl, 0.44 mM KH2PO4, 25 mM NaHCO3, 0.33 mM Na2HPO4 and 0.5 mM CaCl2 for a further 20 min at room temperature. To deplete extracellular Ca2+, cells were treated with 10 mM EGTA  for 10 min prior to the H2O2 treatment. For buffering [Ca2+]i, cells were pretreated with 50 μM of the acetoxymethyl ester of BAPTA  or 50 μM of the acetoxymethyl ester of quin-2  for 10 min. After the addition of H2O2, [Ca2+]i was measured in individually identified fura-2-loaded cells using alternating excitation wavelengths (340 and 380 nm) with an AQUACOSMOS ratio imaging application software  and an inverted epifluorescence microscope . In order to evaluate its ability to quantify [Ca2+]i, the instrument was tested on Ca2+ buffer solutions (Molecular Probes) with known values of [Ca2+]i, using fura-2/AM i; TS performed flow cytometric analysis; YO carried out electron microscopic study. All authors read and approved the final manuscript.EA and YY conceived of the study, analyzed the results and wrote the manuscript. SI measured [Ca


In polymorphonuclear leukocytes (PMNL), mobilization of calcium ions is one of the early events triggered by binding of chemoattractant to its receptors. Besides chemotaxis, a variety of other functional responses are dependent on calcium ion mobilization. PMNL from chronic myeloid leukaemia (CML) patients that were morphologically indistinguishable from normal PMNL were found to be defective in various functions stimulated by a chemoattractant – fMLP. To study the mechanism underlying defective functions in CML PMNL, we studied calcium mobilization in CML PMNL in response to two different classical chemoattractants, fMLP and C5a.2+]i levels were lower in CML PMNL as compared to those in normal PMNL. But, both normal and CML PMNL showed maximum [Ca2+]i in response to fMLP and C5a at 10 sec and 30 sec, respectively. Spectrofluorimetric analysis of the total calcium release in chemoattractant treated PMNL indicated more and faster efflux of [Ca2+]i in CML PMNL as compared to normal PMNL.Release of calcium estimated by flow cytometry and spectrofluorimetry using fluo-3 as an indicator showed that the [Ca2+ homeostasis was altered in CML PMNL. The altered Ca2+ homeostasis may contribute to the defective functions of CML PMNL.Fine-tuning of Ca Migratiemotaxis . PMNL miemotaxis  and anapemotaxis . Specifiemotaxis . Mobiliz1) and presence of a large number of mature and immature myeloid cells in the peripheral blood [2+]i levels lead to fragmentation of actin network by disrupting the cross bridges of actin network. Increased [Ca2+]i levels cause fragmentation of F-actin by activation of actin severing and capping proteins such as gelsolin and macrophage capping protein i over the basal levels. Further treatment with ionophore increased the mean ratio to 2.5 ± 0.36. Subsequent addition of EGTA and MnCl2 decreased [Ca2+]i levels to 21% and 64% of the maximum levels, respectively; ultimately quenching down the fluorescence to about 30% lower than the basal levels , ligand-gated non-specific cation channels (LGCCs) and receptor-activated Ca2+ channels (RACCs). Calcium can also be released from internal Ca2+ stores through inositol 1,4,5-triphosphate (IP3) or ryanodine receptors and is replenished by store-operated channels (SOCs)  Fig.  16]. Pr. Pr2+ ch2+]i occur. Two distinct [Ca2+]i storage sites have been identified in PMNL. One site was located peripherally under the plasma membrane and the other in the juxtanuclear space. The central [Ca2+]i storage site released [Ca2+]i in response to fMLP, whereas engagement and clustering of CD11b/CD18 integrins caused [Ca2+]i release from the peripheral stores [2+]i are known to be regulated by the cytoskeleton interactions [2+]i concentration is caused by the mobilization of Ca2+ from intracellular stores to the cytosol followed by an immediate increase in plasma membrane permeability to extracellular calcium. Efflux of [Ca2+]i is mediated by calmodulin dependent calcium adenosine triphosphatase (Ca-ATPase). Ca-ATPase probably serves as the regulatory and homeostatic mechanism required to maintain low [Ca2+]i concentrations. When the cytosolic [Ca2+]i concentration is increased either by fMLP or ionophore, efflux of [Ca2+]i from the cell occurs as a result of calmodulin mediated activation of Ca-ATPase i levels and [Ca2+]i levels reached after stimulation. On further stimulation of the chemoattractant stimulated PMNL with calcium ionophore, the heterogeneity in [Ca2+]i levels decreased. Vast heterogeneity was seen in PMNL with respect to the sensitivity of quenching of [Ca2+]i by EGTA and MnCl2. Similar heterogeneity was reported earlier. Elsner et al reported heterogeneity in [Ca2+]i levels in human PMNL [2+]i [2+]i levels and subsequent stimulation with fMLP resulted in further elevation of [Ca2+]i. LPS augmented the overall responsiveness of a population of PMNL by causing a subpopulation of cells to become highly responsive and able to generate changes in [Ca2+]i upon low level stimulation [2+]i mediated signal transduction may be important in the fine control of the non-specific immune system in response to weak environmental signals.Our studies showed altered calcium homeostasis in CML PMNL. The analysis of fluo-3 loaded normal and CML PMNL showed heterogeneity with respect to the basal [Caman PMNL . AdditioNL [2+]i . Yee et mulation . This he2+]i release on fMLP stimulation differed from that of C5a stimulation. The response to C5a was delayed as compared to that seen for fMLP. This was true for both CML and normal PMNL. This could be because of different time kinetics for internalization and recycling of FPR and C5aR in human PMNL i from the intracellular stores and efflux of [Ca2+]i from the cell cytoplasm. Earlier we have observed that in CML PMNL actin polymerization in response to chemoattractant stimulation was delayed and significantly lower. Moreover, distribution of F-actin in CML PMNL was altered as compared to that in normal PMNL [2+]i, following stimulation with fMLP or zymosan activated serum [2+]i levels cause fragmentation of F-actin by activation of actin severing and capping proteins such as gelsolin and macrophage capping protein [2+]i and the increased extent of [Ca2+]i release seen in CML PMNL in response to chemoattractants could be because of altered regulation of [Ca2+]i stores by altered actin network.Ras related GTPases – rho, rac and cdc42 regulate polymerization of actin to produce stress fibres and lamellipodia . Expressmal PMNL ,12. Sached serum . In PMNLed serum . In turn2+ transporters lead to plethora of diseases. In skeletal muscle pathology, mutations in ryanodine receptors cause malignant hyperthermia and porcine stress syndrome. Mutations in calcium pump cause Brody disease. Various abnormalities in calcium handling proteins have been reported in the heart during aging, hypertrophy, diabetes, etc. [2+]i levels of PMNL are associated with various disorders. Lower [Ca2+]i levels were observed in PMNL from asthmatic patients, in response to PAF stimulation [2+]i were due to low influx of Ca2+. In localized juvenile periodontitis (JP), the basal levels of [Ca2+]i in PMNL were comparable to that of normal. But, fMLP and C5a induced [Ca2+]i levels were reduced in JP. This was due to decreased Ca2+ influx. These reduced [Ca2+]i levels were associated with reduced PKC levels and defective chemotaxis [2+]i levels that were associated with defective chemotaxis and oxidative burst [2+]i levels were often associated with altered chemotaxis. In contrast to this, calcium transients were not required for other PMNL functions such as an oxidative burst. For example, PMNL from Myelodisplastic syndrome (MDS) patients are defective in respiratory burst. Studies by Nakaseko et al showed that basal and fMLP stimulated [Ca2+]i levels in MDS PMNL were comparable to those in normal PMNL [2+]i transients potentiated PMNL degranulation and respiratory burst [2+]i is also known to regulate chemoattractant receptor availability in PMNL. In PMNL from asthmatic patients, lower [Ca2+]i levels were associated with lower PAF receptor [2+]i depleted PMNL showed decrease in receptors for fMLP, LTB4 and PAF [2+]i were lower in unstimulated CML PMNL as compared to normal PMNL. These [Ca2+]i levels in CML PMNL were lower up to 1 min after stimulation with fMLP and C5a. However, the total calcium release was more in CML PMNL than that in normal PMNL. This suggested that efflux of [Ca2+]i was more and faster in CML PMNL than that in normal PMNL. Earlier we have shown that CML PMNL showed a lower number of FPR, defects in actin and tubulin polymerization, pinocytosis and degranulation. But, stimulation of oxidative burst in CML PMNL was comparable to that in normal PMNL. Thus, our observations are in agreement with earlier reports that [Ca2+]i levels regulate availability of chemoattractant receptors and low [Ca2+]i levels are associated with defects in actin polymerization, chemotaxis and degranulation. However, [Ca2+]i transients are not essential for stimulation of an oxidative burst. Additionally our results suggest that the levels of [Ca2+]i maintained in the cell immediately after stimulation and not the amount of total calcium released are important in stimulation of various functional events in PMNL. PKC is one of the important messengers leading to various functional events in PMNL. In signal transduction pathway, PKC is downstream to Ca2+ mobilization. In CML PMNL, expression of calcium dependent PKC isoform α was lowered ireceptor . O'Flahe and PAF . Our stu lowered . Various2+ homeostasis in CML PMNL is altered. This could be one of the contributing factors for the reduced responses seen in CML PMNL. Further studies of the calcium oscillations in CML PMNL and the Ca-ATPase would help in pin pointing the defects in calcium homeostasis in CML PMNL.In summary, our studies show that Ca2), n-formyl-methionyl-leucyl-phenylalanine (fMLP), complement factor 5a (C5a) and Calcium ionophore A23187 were from Sigma. Dimethyl sulfoxide (DMSO) was from Seezle, Germany. Fluo-3-pentapotassium salt and fluo-3AM – the pentaacetoxymethyl ester of fluo-3 and calcium calibration kit were from Molecular Probes .Ficoll-Hypaque, 4-(2-hydroxyethyl)-1-piperazine ethane sulphonic acid (HEPES), ethyleneglycol-bis-(3-aminoethyl)-tetra acetate (EGTA), manganese chloride . Viability of cells was more than 99% as checked by the erythrocin B dye exclusion test. The PMNL fraction was 95% enriched as checked by Giemsa's staining.PMNL from peripheral blood were isolated on a ficoll-hypaque gradient . To lyse7 cells per ml in fluo-3 loading buffer  containing 5 μM fluo-3AM. PMNL were incubated for 30 min at 37°C with gentle agitation. Cells were then washed twice with Ca2+ estimation buffer (i.e. fluo-3 loading buffer with 1 mM CaCl2) to remove extra cellular fluo-3AM. Finally the cell density was adjusted to 1 × 107 cells per ml in the calcium estimation buffer. The cells were kept in dark at 4°C till use.One millimolar stock solution of fluo-3AM was prepared in dry DMSO. Fluo-3 loading of PMNL was carried out as described by Vandenberghe et al with some modifications . Briefly6 PMNL were suspended in one ml Ca2+ estimation buffer and incubated in a 37°C water bath, for 5 min before each assay. The samples were run on FACScalibur  using Cell-Quest software. The samples were excited by an argon ion laser at 488 nm and emission was measured at 525 nm on a logarithmic scale. Before addition of the stimuli, fluorescence of the fluo-3 loaded PMNL was measured. Optimum stimulation of PMNL in suspension with fMLP was seen at a concentration of 10-8 M i/[(F-Fmin)/(Fmax-F)]     equation I [Kd = [Cauation I The experiment was done four times independently.6 fluo-3 loaded PMNL were suspended in two ml Ca2+ estimation buffer and incubated in a water bath at 37°C for 5 min, before each assay. PMNL were then stimulated with 10-8 M fMLP or 10-9 M C5a. The fluorimetric reading was taken before stimulation and at 10 sec, 30 sec and 60 sec after stimulation.10max, the maximum fluorescence was obtained by treating the cells with 10 μM of calcium ionophore A23187. F represents the fluorescence of the test sample. Quenching of external and internal fluo-3 fluorescence was estimated using 0.4 M EGTA and 2 mM MnCl2, respectively. Fluo-3-Mn2+ complex is eight times less fluorescent as compared to fluo-3-Ca2+ complex. Under experimental conditions, the quenching of fluo-3 by MnCl2 was low as compared to autofluorescence of PMNL, hence autofluorescence was considered as Fmin [2+ by using equation I.Autofluorescence was calculated using unloaded PMNL. F as Fmin . The speNon-parametric tests were applied for statistical analysis of the data. Wilcoxon signed rank test was used to compare the median fluorescence channel and absolute calcium concentrations within normal and CML samples. The Mann-Whitney Wilcoxon test was used to compare the median fluorescence channel and absolute calcium concentrations of normal and CML samples. The Mann-Whitney Wilcoxon test was also used to compare the median fluorescence channel and absolute calcium concentration values of fMLP stimulated PMNL with that of C5a stimulated PMNL.The author(s) declare that they have no competing interests.CMR participated in the standardization of the techniques, acquisition of the data, analysis and drafting the manuscript. SHA was a clinical collaborator and was involved in diagnosis of the patients and providing clinical samples for the studies. NRN conceived the study, participated in the design of the study, standardization of the techniques, acquisition of the flow cytometry data, coordination and helped to draft the manuscript. All the authors have read and approved the final manuscript.


T-bet knock-out (KO) mice show key features of allergic asthma such as a shift towards TH2-lymphocytes and display a broad spectrum of asthma-like histological and functional characteristics. In this study, we aimed at investigating whether Ca2+-homeostasis of ASMC is altered in T-bet KO-mice as an experimental model of asthma.Airway smooth muscle cells (ASMC) play a key role in bronchial hyperresponsiveness (BHR). A major component of the signaling cascade leading to ASMC contraction is calcium. So far, agonist-induced Ca2+-signaling in single ASMC of lung slices was assessed using two-photon-microscopy.Lung slices of 100 to 200 μm thickness were obtained from T-bet KO- and wild-type mice. Airway contraction in response to acetylcholine (ACH) was measured by video-microscopy and Ca2+-concentrations, whereas BHR correlated with higher ACH-induced Ca2+-transients and an increased proportion of ASMC showing Ca2+-oscillations. Emptying intracellular Ca2+-stores using caffeine or cyclopiazonic acid induced higher Ca2+-elevations in ASMC from T-bet KO- compared to wild-type mice.Airways from T-bet KO-mice showed increased baseline airway tone (BAT) and BHR compared to wild-type mice. This could be mimicked by incubation of lung slices from wild-type mice with IL-13. The increased BAT was correlated with an increased incidence of spontaneous changes in intracellular Ca2+-homeostasis of ASMC contributes to increased BAT and BHR in lung slices from T-bet KO-mice as a murine asthma model. We propose that a higher Ca2+-content of the intracellular Ca2+-stores is involved in the pathophysiology of these changes.Altered Ca Calcium binds to calmodulin, thereby activating myosin light chain (MLC) kinase, which phosphorylates MLC and initiates actomyosin cross-bridge cycling. Consequently, agonist-induced Ca2+-signaling has been regarded as a possible target in the pathophysiology of BHR c, whereas increased BHR correlated with elevated acetylcholine (ACH)-induced Ca2+-transients and a higher proportion of ASMC showing Ca2+-oscillations. Emptying intracellular Ca2+-stores induced higher Ca2+-elevations in ASMC from T-bet KO- compared to wild-type mice. We therefore propose that a higher Ca2+-content of the intracellular Ca2+-stores is involved in the pathophysiology of these changes.T-bet is a TH1-cells . Naïve mH1-cells . Recentlituation ,28. In tCell culture reagents were obtained from Life Technologies . Other reagents were bought from Sigma-Aldrich  unless stated otherwise. Balb/C mice were purchased from Harlan-Winkelmann  and T-bet KO-mice on a Balb/C background from Charles River . All procedures had been approved by the Ethics Committee of the Ludwig-Maximilians-University Munich.2+-indicator dyes, slices were cut ~100 μm thick for the use with two-photon microscopy. The slices were maintained by floating them in DMEM supplemented with Penstrep®  and Fungizone®  at 37°C in 5 % CO2. Experiments were performed on day 2 to 4 of culture and each slice was used for one experiment only. For each group of experiments, slices from 5 to 6 different mice were used.Lung slices were prepared as described previously . Briefly2 without significant differences between T-bet KO- and wild-type mice. This size reflects an airway level below the segmental bronchi and above the respiratory bronchioles. Contraction velocity was defined as maximal change in cross-sectional area per second.To measure airway cross-sectional area, lung slices were placed in culture dishes immersed in sHBSS and held in position by a piece of a nylon mesh. Phase-contrast images were recorded using a digital CCD camera . Frames were captured in time-lapse (0.5 frame/s) and the cross-sectional area of the airway was measured by pixel summing using the image analysis software "Scion" . The mean cross-sectional area of all airways used was 39326 ± 9390 μm2+]c in ASMC, slices were loaded for 1 h at 37°C with 10-5 M Fluo-4-AM  in sHBSS containing 0.2 % Pluronic  and 10-4 M sulfobromophthalein. Sulfobromophthalein was used to block unspecific ion pumps and cation channels thereby reducing the loss of de-esterified Fluo-4. After loading, slices were incubated for at least 30 min in sHBSS containing 10-4 M sulfobromophthalein to allow for complete dye de-esterification. The bath solution for all experiments was sHBSS without sulfobromophthalein. The slices were placed in a custom made Plexiglas chamber and held in position by a platinum mesh. Detection of fluorescent signals of single ASMC within the slices was performed with a custom-built two-photon microscope based on an Olympus microscope  to ensure the detection of potentially high frequency Ca2+-oscillations. Regions of interest of 10 × 10 pixels were defined in single ASMC excluding the nucleus and fluorescence intensities were analyzed frame by frame using custom written macros in the image analysis software "Scion". Final fluorescence values were expressed as fluorescence ratio (F/Fo) normalized to the fluorescence immediately prior to the addition of an agonist (Fo).To determine c occurred , the initial Ca2+-transient was followed by Ca2+-oscillations above the baseline level . When analyzing the frequency of the Ca2+-oscillations, there was a concentration-dependent increase with increasing concentration of ACH in the absence of significant differences between T-bet KO- and wild-type mice . Increain vivo with the ability to observe single cell properties, which is usually only rewarded by single cell preparations.The major advantage of lung slices is that the in situ organization of the lung tissue and the contractility of the ASMC are maintained for several days. Lung slices in combination with video-microscopy have been used before to study airway responses ,33. Usinin vivo situation.Similar to previous observations , we foun2-cytokines especially IL-13 in the lavage of T-bet KO-mice c. Depending on the frequency, these changes could be regarded as spontaneous Ca2+-oscillations or as spontaneous Ca2+-transients, although sometimes the differentiation between slow frequency Ca2+-oscillations and Ca2+-transients was arbitrary. The higher incidence of changes in [Ca2+]c in T-bet KO-mice may have led to an increased BAT by either a frequency modulated process integrating Ca2+-oscillations or simply by increasing overall [Ca2+]c.ASMC from T-bet KO- and wild-type mice showed spontaneous changes in c has to be high enough to prevent dephosphorylation of MLC and thereby maintaining contraction. The latch state describes a situation, where MLC is dephosphorylated but nevertheless remains attached to actin, which results in a much slower relaxation rate c as observed in ASMC from T-bet-KO mice.An increased Ca2+-puffs . The ele3R play a critical role in the induction of Ca2+-oscillations because these Ca2+-channels repetitively allow or inhibit Ca2+-release from the Ca2+-pools. Shifting the Ca2+-sensitivity of RyR and IP3R by increasing the Ca2+-content of the Ca2+-pools could sensitize the Ca2+-signaling apparatus to allow a higher proportion of cells to display Ca2+-oscillations in response to ACH as seen in ASMC from T-bet KO-mice.RyR and IP2+-transient has been shown to be caused by Ca2+-release from Ca2+-pools with Ca2+-influx across the cell membrane playing a minor role c, whereas increased BHR correlates with elevated ACH-induced Ca2+-transients and a higher proportion of ASMC showing Ca2+-oscillations. We propose that a higher Ca2+-content of the intracellular Ca2+-stores is involved in the pathophysiology of these changes. Pharmacological interventions targeting altered Ca2+-homeostasis may therefore constitute a new therapeutic tool in asthma.Lung slices from T-bet KO-mice as an asthma model preserve the key characteristics of the living animal in terms of increased BAT and BHR. Increased BAT is correlated with an increased incidence of spontaneous changes in [CaThe author(s) declare that they have no competing interests.2+-signaling in lung slices and drafted the manuscript. JK performed studies on airway contraction in lung slices and contributed substantially to the analysis and interpretation of the data. AK also performed in vitro studies and contributed substantially to the analysis and interpretation of the data. FG revised the manuscript and gave valuable intellectual input in the course of the study. RMH participated in the study design and made substantial contributions to the interpretation of the data. All authors read and approved the final manuscript.AB laid out the principal conception and design of the study, carried out experiments on Ca2+-signaling in ASMC in lung slices have been added to the manuscript as online supplement.Two videos showing airway contraction and CaClick here for file2+-signaling in ASMC in lung slices have been added to the manuscript as online supplement.Two videos showing airway contraction and CaClick here for file


Our objective was to mathematically model this phase-space relationship between [Ca2+] and LVP with a focus on the changes in cross-bridge kinetics and myofilament Ca2+ sensitivity responsible for alterations in Ca2+-contraction coupling due to inotropic drugs in the presence and absence of ischemia reperfusion (IR) injury.The phase-space relationship between simultaneously measured myoplasmic [Ca2+] transients from unpaced, isolated guinea pig hearts as the model input. Values of model parameters were estimated by minimizing the error between experimentally measured LVP and model-predicted LVP.We used a four state computational model to predict LVP using experimentally measured, averaged myoplasmic [Ca2+ sensitivity, and decreased cross-bridge association and dissociation rates. Dopamine (8 μM) reduced myofilament Ca2+ sensitivity before, but enhanced it after ischemia while improving cross-bridge kinetics before and after IR injury. Dobutamine (4 μM) reduced myofilament Ca2+ sensitivity while improving cross-bridge kinetics before and after ischemia. Digoxin (1 μM) increased myofilament Ca2+ sensitivity and cross-bridge kinetics after but not before ischemia. Levosimendan (1 μM) enhanced myofilament Ca2+ affinity and cross-bridge kinetics only after ischemia.We found that IR injury resulted in reduced myofilament Ca2+-contraction coupling due to IR injury, specifically the inefficient utilization of Ca2+ for contractile function with diastolic contracture (increase in resting diastolic LVP). The model parameters also reveal drug-induced improvements in Ca2+-contraction coupling before and after IR injury.Estimated model parameters reveal mechanistic changes in Ca The model parameters were estimated using data obtained from guinea pig spontaneously beating, isolated hearts at physiologic temperature) ("upstream" mechanism), their effects on Ca2+ binding to troponin C  and actinomyosin cross-bridge cycling ("downstream" mechanism) remain unexplored or controversial.Our objective was to determine how positive inotropic drugs with differing pharmacological mechanisms of action affect estimated rates of cross-bridge cycling and myofilament Caperature). Three p2+ affinity for troponin C is unclear. However, another Na+/K+ ATPase inhibitor, ouabain, was shown to increase cross-bridge kinetics and myofilament Ca2+ cycling in ventricular myocardium from patients with heart failure [2+ sensitizers are believed to improve myofilament Ca2+ sensitivity by enhancing troponin C sensitivity for Ca2+ and may also alter cross-bridge attachment and detachment rate constants [2+ and to stabilize the Ca2+- troponin C complex without increasing Ca2+ binding affinity with troponin C [2+] into myocardial contraction and relaxation before and after IR injury in the presence and absence of these inotropic drugs.Several investigators have reported an increase in cross-bridge formation by post-synaptic adrenoceptor agonists ,10, but  failure . Ca2+ seonstants . One of oponin C . In guinoponin C , acceleroponin C . It is uGuide for the Care and Use of Laboratory Animals from the US National Institutes of Health . Prior approval was obtained from the Medical College of Wisconsin and Marquette University Animal Studies Committees. Guinea pig heart isolation and our fluorescence technique to measure myoplasmic free [Ca2+] has been detailed in previously published work  (1.25 mM) allowed a wider range of inotropic responses at a lower control LVP.The investigation conformed to the hed work -24, and LVP was measured isovolumetrically with a transducer connected to a thin, saline-filled latex balloon inserted into the LV through the mitral valve from an incision in the left atrium. Balloon volume was adjusted initially to a diastolic LVP of zero mmHg so that any subsequent increase in diastolic LVP reflected an increase in LV wall stiffness i.e., diastolic contracture. Pairs of bipolar electrodes were placed in the right atrial appendage, right ventricular apex and LV base to monitor spontaneous heart rate and atrial-ventricular conduction time. Coronary flow  was measured at constant temperature and perfusion pressure (55 mmHg) by an ultrasonic flowmeter  placed directly into the aortic inflow line.2+ indicator indo-1 AM  was dissolved in vehicle solution consisting of 1 mL of dimethyl sulfoxide (DMSO) containing 16 % (w/v) Pluronic I-127  and diluted to 165 mL with modified KR solution. Each heart was then loaded with indo-1 AM for 30 min with the re-circulated KR solution, which had a final indo-1 AM concentration of 6 μM. Loading was stopped when the fluorescence (F) intensity at 385 nm increased by about 10 fold. Residual interstitial indo-1 AM was washed out by perfusing the heart with KR for another 20 min. Probenecid (100 μM) was present in the perfusate to retard cell leakage of indo-1. Fluorescence emissions at 385 and 456 nm (F385 and F456) were recorded using a modified luminescence spectrophotometer . The LV region of the heart was excited with light from a xenon arc lamp and the light was filtered through a 360 nm monochromator with a bandwidth of 16 nm. Although both F385 and F456 decline over time, time control studies showed that the F385/F456 ratio remained stable indicating that the effective measured [Ca2+] was unchanged [® for off-line signal processing of the recorded data. The F385/F456 ratio was converted to [Ca2+] after correcting for background autofluorescence and non-cytosolic fluorescence as described in our previously published work.  and LVP were obtained at selected intervals before and during administration of inotropic drugs and IR  myofilament (TnCA) and myosin (M) for cross-bridge formation and force development in the presence of Ca2+ ,6-8,25. Mod), is proportional to the number of cross-bridges formed: [Ca•TnCA•M] + [TnCA•M] (t = 0) = 20 μM, [Ca•TnCA](t = 0) = 0 μM, [Ca•TnCA•M](t = 0) = 0 μM, and [TnCA•M](t = 0) = 0 μM.The experimentally measured and averaged Canditions ,8,25,26:Mod and LVP at the sampled time points.Model rate constants were optimized using commercially available algorithms based on constrained quasi-Newton methods that guarantee linear convergence, and were estimated to minimize the root mean square (RMS) error between LVPst order rate constants were set at 0 and 2000 /sec (0.5 ms step size) respectively. Initial values for the parameters were obtained from Baran et al.  have been previously reported [The effects of drugs and ischemia on heart rate and phasic LVP and [Careported  and are 2+], is represented by symbols for all interventions before (A) and after (B) ischemia in figure Mod, plotted as a function of [Ca2+], is represented by the dashed line for all interventions. Typical plots of the estimated cooperative parameters K1 (A) and Ka (B) as a function of the total number of cross-bridges formed ([Ca•TnCA•M]+[TnCA•M]) for all groups before and after ischemia are shown in figure Experimentally measured LVP, plotted as a function of  and LVP during dobutamine administration with low errors of 5.0 ± 0.5% before and 3.0 ± 0.4% after ischemia. The model predicted:The model successfully described the relationship between  and LVP; 2) IR injury in the absence of inotropic agents resulted in reduced Ca2+ affinity for TnCA, and decreased cross-bridge kinetics; 3) dopamine enhanced cross-bridge kinetics before and after ischemia; 4) dopamine decreased myofilament Ca2+ affinity before ischemia but enhanced this affinity after ischemia; 5) in contrast, dobutamine reduced myofilament Ca2+ sensitivity, and increased cross-bridge kinetics before and after ischemia; and 6) digoxin and levosimendan improved Ca2+ affinity for TnCA and cross-bridge association after ischemia but neither drug substantially affected these parameters before ischemia.The results indicate: 1) the four-state model with cooperativity is capable of interpreting changes in central and downstream regulation of contractility due to inotropic drugs in the presence and absence of IR injury in guinea pig isolated hearts from the phase-space relationship between  that have been analyzed qualitatively [2+ binding associated with IR injury. The model's results suggest that IR injury causes inefficient utilization of available [Ca2+] for contractile function. The model-estimated parameters revealed that IR injury results in reduced myofilament sensitivity for Ca2+ binding; however, the basal rate constant of Ca2+ binding to TnCA was increased. This may reflect excess myoplasmic Ca2+ loading despite concomitant reduction of myofilament Ca2+ sensitivity. IR injury also resulted in attenuation of model-predicted cross-bridge kinetics and formation of fewer cross-bridges. Decreased ATP production and hydrolysis may account for the reduction in cross-bridge dissociation rate constants, Kd and Kd'. These alterations in intracellular Ca2+ homeostasis may have precipitated the rise in diastolic LVP and the reduction in LV compliance as noted from the phase-space diagram after ischemia in isolated hearts.The present investigation used dynamic changes in Catatively , to exam1-adrenergic receptors by dopamine and dobutamine has been shown to increase myoplasmic cyclic adenosine 3', 5'-monophosphate (cAMP) levels and to activate cAMP-dependent protein kinase A. This protein kinase phosphorylates sarcolemmal Ca2+ channels, SR Ca-ATPase, and troponin I on actin myofilaments  (upstream mechanism) in normal myocardium. In contrast, after ischemia digoxin was predicted to increase myofilament Ca2+ sensitivity and cross-bridge kinetics, and to increase the estimated number of cross-bridges formed; however, no significant changes were seen in phase-space diagram size and shape from control after ischemia. This indicates that though the phase-space relationship between LVP and [Ca2+] appears unchanged by digoxin administration from control after IR injury  when administered before ischemia  and LVP during the cardiac cycle in the guinea pig isolated heart when exposed to positive inotropic drugs before and after global ischemia. The changes in estimated values of rate constants before and after ischemia appeared to coincide with known changes in cross-bridge formation and myofilament Ca2+ sensitivity and helped to resolve controversial findings regarding drug effects on central and downstream mechanisms after ischemia. The model also predicted differential changes in contractile protein interactions based on post-synaptic receptor adrenergic or dopaminergic activation. Moreover, the model parameters predicted that digoxin and levosimendan have little effect on central and downstream mechanisms prior to ischemia but that both significantly improve myofilament Ca2+ sensitivity and cross-bridge cycling after ischemia. These mathematical characterizations should facilitate future studies that aim to analyze the effects of preconditioning or heart failure on contractile kinetics. The phase-space relationship between experimentally measured [Ca2+] and LVP is useful as a framework to model the theoretical effects of these interventions on Ca2+-contraction coupling.In summary, we used a 4-state computational model to predict the relationship between [Ca


However, genetic mutants in Ca2+ sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca2+-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca2+ activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca2+-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca2+-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca2+ oscillation experiments revealed that Ca2+-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca2+-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca2+-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca CDPK genes were found to be expressed inArabidopsis guard cells, where they are involved in ion channel regulation in response to abscisic acid signaling.Two At1g18890) cause constitutive stress signaling cyt-activated S-type anion channel currents were observed in wild-type guard cells in the presence of elevated (2 μM) free Ca2+ in the patch pipette (cytosolic) solution  [2+]cyt were significantly reduced in the cpk3–1 mutant (n = 11 guard cells) compared to wild-type . cpk3–2 showed similar results to cpk3–1 . S-type anion channel currents were further reduced in cpk6–1 (n = 11) and cpk6–2 (n = 4) single mutant guard cells . In cpk3-1cpk6–1 double mutant guard cells (n = 17), S-type anion channel currents were substantially reduced compared to wild-type controls (p < 0.0005). Essentially similar results were obtained in cpk3-2cpk6–2 . Nevertheless, background anion currents did remain in the double mutants (CPK6 disruptions compared to CPK3 disruptions. Thus CPK3 and CPK6 are important for Ca2+ activation of S-type anion channels.As illustrated in solution A and 2C t shown) . S-type  mutants B and 2C.2+-dependent and -independent signaling branches . In cpk3-1cpk6–1 (n = 7) and cpk3-2cpk6–2 (n = 7) double mutant guard cells, ABA regulation of S-type anion currents was impaired (p < 0.01). These data show that, despite the complex network of ion channel regulation mechanisms in guard cells cyclo-octa-trinden-1-one], 20 min prior to, and continuously during, patch clamping. Moreover, the cytoplasm of whole cells was dialyzed in the absence of ATP in whole cell recordings for longer than 15 min prior to extracellular ABA exposure. Ionic currents were recorded in the same guard cells prior to and after exposure to ABA. In negative controls, treatment with K252a alone did not cause constitutive activation of ICa channel currents without ABA treatment (n = 3). Interestingly, pretreatment with K252a (2 μM) did not disrupt ABA activation of ICa channels . These findings indicate that the effect of the cpk3 and cpk6 mutations on ABA activation of ICa channel currents are most likely not caused by direct ABA-induced upstream CDPK activation in the NADPH-dependent activation branch of ICa channels cyt oscillations on stomatal closure in cpk3cpk6 double mutant plants cyt ratio increase per period was also determined and found to be similar for wild-type and cpk3-1cpk6–2 guard cells . These data are consistent with findings that external Ca2+-induced [Ca2+]cyt elevations in guard cells include intracellular Ca2+ release from guard cell organelles ), and the leaf segment was incubated for 2.5 h in light (125 μmol m−2 s−1) to open stomata. [Ca2+]cyt transients were imposed by rapid exchange of the chamber buffer between depolarizing buffer and hyperpolarizing buffer . Depolarizing buffer decreases [Ca2+]cyt levels, whereas hyperpolarizing buffer increases [Ca2+]cyt levels [To measure imposed Cat levels . For eac2+ concentration changes during imposed [Ca2+]cyt transient experiments were confirmed by using the improved Yellow Cameleon 3.6 (YC3.6) fluorescence reporter cyt changes [−, as imposing the same chloride changes while removing extracellular Ca2+ does not produce any YC2.1 ratio changes . In these experiments, extracellular Ca2+ was chelated with 10 mM EGTA, which lowers external Ca2+ to nanomolar levels, to physically inhibit Ca2+ influx that would otherwise occur at strongly hyperpolarized potentials. In further controls, replacing CaCl2 with MgCl2 in the bath did not produce cameleon ratio changes [2+ changes were imposed but not Cl− changes, by using the impermeant counteranion imminodiacetate (+ imminodiacetate (no added Ca2+) (pH 6.15) or 1 mM K+ imminodiacetate and 1 mM Ca2+ imminodiacetate (pH 6.15). These controls showed YC3.6 ratio changes in the absence of imposed Cl− changes, demonstrating that Ca2+ changes are being reported (Cytosolic Careporter . Col-0 aomophore  and ther H+ ions ,105. In  changes . Previou data to ; http:// changes . Furtheriacetate . Stomatareported , as prevGermination assays were performed as described previously . Mutant


In a similar and consistent manner with the preservation of motor function, UCN 'protected' the nigra from both DA depletion and loss of TH activity, indicating preservation of DA cells. The effects of UCN were antagonised by the non-selective CRF receptor antagonist α-helical CRF and were not replicated by the selective CRF2 ligand UCN III. This suggests that UCN is acting via CRF1 receptors, which have been shown to be anti-inflammatory in the periphery. Our data therefore indicate that UCN is capable of maintaining adequate nigrostriatal function in vivo, via CRF1 receptors following a neuro-inflammatory challenge. This has potential therapeutic implications in PD.We have recently observed that the corticotrophin releasing hormone (CRF) related peptide urocortin (UCN) reverses key features of nigrostriatal damage in the hemiparkinsonian 6-hydroxydopamine lesioned rat. Here we have studied whether similar effects are also evident in the lipopolysaccaride (LPS) neuroinflammatory paradigm of Parkinson's disease (PD). To do this we have measured restoration of normal motor behaviour, retention of nigral dopamine (DA) and also tyrosine hydroxylase (TH) activity. Fourteen days following intranigral injections of LPS and UCN, rats showed only modest circling after DA receptor stimulation with apomorphine, in contrast to those given LPS and vehicle where circling was pronounced. In separate experiments, rats received UCN seven days  Before the disease presents clinically, death of dopamine (DA) neurons occurs in the substantia nigra pars compacta (SNc) asymptomatically. This has traditionally been ascribed to concurrent apoptotic, excitotoxic and free-radical mediated events ,2 Recenteriphery . We haveeriphery . UCN suberiphery . Althougeriphery ,4. There agonist , on indiad libitum. Animals were anaesthetised, secured in a stereotaxic frame and given injections of LPS (2 μg/2 μl) into SNc . Animals co-treated with UCN, UCN III or α-helical CRF received injections (20 fmols/2 μl) directly into the ipsilateral SNc whereas those not receiving UCN were given vehicle. UCN itself was administered either concomitantly with LPS or 7 days later  seen after apomorphine treatment. However, a determination of actual DA receptor population would be required to prove this.The current findings demonstrate that UCN greatly attenuates the development of PD-like pathology in a recently proposed ,4 paradi lesions ,14. Futh1 subtype [2 sites has been cited as important [Evidence has shown that UCN protects some neurons via activation of the CRF subtype ,6, whilsmportant ,15. The mportant  and the mportant ,16. We amportant . Additioafter the lesion has become established by a molecule known to have antiinflammatory properties [1 sites can be activated is clearly essential as may be refinement to ensure an appropriate pharmacological response. While UCN is a relatively large molecule with poor blood brain barrier penetration, recently small, lippophillic, CRF1 selective antagonists have become available and CRF receptor pharmacology is a rapidly expanding field. As such we consider it highly likely that CRF1 agonists will become available offering new possibilities in the study of UCN mediated neuroprotection as well as being of potential therapeutic value in PD.In summary, our data constitutes the first report of a restoration of key indicators of nigrostriatal damage in a neuroinflammatory model of PD operties . Althougoperties . In ordehypothalamopituitary-adrenal (HPA), lipopolysaccaride (LPS), Parkinson's disease (PD), tyrosine hydroxylase (TH), urocortin (UCN).Dopamine (DA), corticotrophin releasing factor (CRF), The author(s) declare that they have no competing interests.AA, AH, CSB and PSW were responsible for the planning and actual experimentation involved in this study. RL contributed to the interpretation of the data and writing of the manuscript.


The acidic stores may comprise the contractile vacuole network (CV), the endosomal compartment and acidocalcisomes. Here the role of CV in respect to function as a potential Ca2+-store was investigated.cAMP-induced Ca2+. Release of Ca2+ was elicited by arachidonic acid or the calmodulin antagonist W7, the latter due to inhibition of the pump. The characteristics of Ca2+-transport and Ca2+-release of CV were compared to similarly purified vesicles of the ER labeled by calnexin-GFP. Since the CV proved to be a highly efficient Ca2+-compartment we wanted to know whether or not it takes part in cAMP-induced Ca2+-influx. We made use of the LvsA--mutant expected to display reduced Ca2+-transport due to loss of calmodulin. We found a severe reduction of cAMP-induced Ca2+-influx into whole cells.Dajumin-GFP labeled contractile vacuoles were purified 7-fold by anti-GFP-antibodies in a magnetic field. The purified CV were shown for the first time to accumulate and release CaDictyostelium represent a highly efficient acidic Ca2+-store that is required for cAMP-induced Ca2+-influx.The contractile vacuoles in  Dictyostelium consists of tubes and bladders. It transiently fuses with the plasma membrane to expel water and ions and thereby serves as an efficient osmoregulatory organelle 2+ev) of 0.53 ± 0.02 μM uptake ceased to 0.10 ± 0.05 μM [Ca]2+ev in the presence of 32 ± 12 μg protein. In agreement with previous results obtained for acidic Ca2+-stores the calmodulin antagonist W7 as well as arachidonic acid (AA) caused Ca2+-release in P0E. The response to W7 (30–100 μM) amounted to 21.4 ± 10.2 nmol Ca2+/mg protein (n = 4) and to AA (10–20 μM) 23.9 ± 7.2 nmol Ca2+/mg protein (n = 4).Since both purifications had given a strong enrichment of the GFP-label as well as in Ca2+-uptake by P1E, the eluate of calnexin-GFP-labeled cells containing ER, is shown. In contrast to P0E P1E was less responsive to AA. In three out of four experiments no Ca2+-release was measured. The one that responded to AA had a lower purification factor with respect to GFP fluorescence compared with the other three, 3.8 versus 7.8 ± 0.96 (n = 3). IP3, most of the times, was inefficient to elicit Ca2+-release. In general, Ca2+-uptake was slower in P1E as compared to P0E starting at 0.56 ± 0.15 μM [Ca]2+ev and ceased to extravesicular Ca2+-concentrations of about 0.36 ± 0.08 μM in the presence of 27 ± 12 μg protein (n = 4), indicating that the CV is more relevant in maintaining a basal cytosolic Ca2+-concentration of 50 to 60 nM i and stimulus-induced [Ca2+]i-transients ev at the beginning of the experiment is not known and Ca2+-ATPase activity is increasing with increasing Ca2+-concentrations the results shown in Table 2+]ev in order to relate the outcome of different experiments to each other. This was done by using a calibration curve at different [Ca2+]ev obtained with either P0 or P1, respectively, and adjusting the measured value to a [Ca2+]ev of 1 μM. The Ca2+-ATPase activities of both, P0 and P1, increased by a factor of about 3.3 from 0.5 μM to 1.5 μM [Ca2+]ev. Statistical data are expressed throughout as the mean ± S.D.Caescribed . In brie+-ATPase antibody (kindly provided by Margaret Clarke) preferentially reacting with the 37/42 kD subunits of the proton pump.Seven microgram of protein per lane were resolved by SDS-PAGE in 12.5% gels, blotted and immunoblotted with a V-type H2+-influx was measured as described [- and DH1) during every washing step was shortened without any loss of cells. Protein concentrations were determined with the Coomassie protein assay reagent (Pierce) using bovine serum albumin as standard. Endosomal content was determined by fluid phase uptake of RITC-dextran as described, except that labeling was performed in phosphate buffer pH 6.0 [cAMP-induced Caescribed . The cenr pH 6.0 .AA: arachidonic acid; BHQ: 2,5-di(tert-butyl)-1,4-hydroquinone; CV: contractile vacuole; CMA: concanamycin A; ER: endoplasmic reticulum; Tg: thapsigargin2+-influx and responses of living cells to AA; CS cultured the strains and prepared the figures. ADL contributed the strains LvsA- and DH1 and participated in the preparation of the manuscript as did AMT, who also contributed the GFP-strains and Figure DM performed the organelle experiments and in collaboration with CS and DFL designed the experiments and wrote the manuscript. DFL measured receptor-mediated Ca


Sir,This is with reference to the article entitled “Use of patch testing for identifying allergen causing chronic urticaria” which appeared in the March–April issue of IJDVL. The artiet al. and Bajaj, et al. mentions about positive autologous serum skin test in 34 and 49.5% of patients, respectively.[35in vivo in patients with chronic urticaria. ASST is the only practicable test available to clinicians to detect autoimmune urticaria in India.[et al., in the Blue journal.[None of the patients underwent autologous serum skin test, which can be done with the help of pathology laboratory. Mamatha, tively.35 Autologoin India. The real journal.Observations of Sharma are of interest but need to be confirmed by other controlled studies before routine patch testing and avoidance measures are recommended in the diagnosis and treatment of chronic urticaria.


Less is known about the dynamics of other intracellular ions. The present study investigated the intracellular source of zinc (Zn2+) that has been reported to play a role in cell signaling.Changes in ionic concentration have a fundamental effect on numerous physiological processes. For example, IP2+ indicators, we showed that intracellular regions of Zn2+ staining co-localized with the endoplasmic reticulum (ER). The latter was identified with ER-tracker Red, a marker for ER. The colocalization was abolished upon exposure to the Zn2+ chelator TPEN, indicating that the local Zn2+ fluorescence represented free Zn2+ localized to the ER in the basal condition. Blockade of the ER Ca2+ pump by thapsigargin produced a steady increase of intracellular Zn2+. Furthermore, we determined that the thapsigargin-induced Zn2+ increase was not dependent on extracellular Ca2+ or extracellular Zn2+, suggesting that it was of intracellular origin. The applications of caged IP3 or IP3-3Kinase inhibitor (to increase available IP3) produced a significant increase in intracellular Zn2+.In primary cultured cortical cells (neurons) labeled with intracellular fluorescent Zn2+ is sequestered into thapsigargin/IP3-sensitive stores and is released upon agonist stimulation.Taken together, these results suggest that Zn As such, Zn2+ levels are normally tightly regulated, limiting the extent of cytosolic labile (or free) Zn2+ concentrations [2+ are several orders of magnitude less than that of Ca2+ [2+ may act as a cellular messenger in physiological and cytotoxic signaling, and the changes in Zn2+ homeostasis have a fundamental effect in cell function [2+ to precede cell death or neurodegeneration in response to cytotoxic stress [2+-mediated signaling pathways or Zn2+-induced cytotoxicity, it is important to determine the source(s) of intracellular free Zn2+ in response to specific stimuli or injury.Zntrations ,2. For e of Ca2+ . Zn2+ mafunction ,5. Many c stress ,7. To ch2+ from the cytosol by means of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) or so-called endoplasmic Ca2+ pump [2+ can be released into the cytosol upon a variety of stimuli including inositol 1,4,5-trisphosphate (IP3). It is IP3 that mobilized Ca2+ from the ER Ca2+ store following interaction with specific IP3 receptors (IP3R). A commonly used tool in studying Ca2+ homeostasis is thapsigargin, a plant derived compound that specifically inhibits SERCA activity [2+ into the ER, thapsigargin causes these stores to become depleted and thereby raise the cytosolic Ca2+ concentration.The endoplasmic reticulum (ER) is an intracellular organelle that has been shown to sequester Caa2+ pump . This seactivity . By bloc2+ homeostasis are not well established, available data support that, like Ca2+, intracellular Zn2+ levels are determined by the interaction of membrane Zn2+ transporters and cytoplasmic Zn2+ buffers [2+, particularly, if thapsigargin can trigger the release of Zn2+. This possibility is supported by recent evidence that Zn2+ can be released from intracellular sources upon stimulation [2+ is released from thapsigargin-sensitive and IP3R-mediated stores.While the mechanisms responsible for regulating Zn buffers ,10. The mulation -13. Our 2 and the fetuses were removed and placed in ice-cold Hank's Balanced Salt Solution without Ca2+ or Mg2+ (HBSS). The brains of fetuses were removed and placed into cold HBSS for further dissection. Using a dissecting microscope and blunt dissection, the meninges were gently separated away. The cerebral cortex was then removed and each cortical hemisphere was cut into four pieces and trypsinized in HBSS at 37°C. Following trypsinization, cells were separated by trituration through the opening of a fire polished Pasteur pipette. The suspensions were then passed through a 70 μm cell strainer. The dissociated cells were added to the bottom of 35mm glass-bottomed petri dishes previously coated with polyethyleneimine  diluted 1:1000 in borate buffer. The cortical neurons were then allowed to attach to the surface at 37°C, 5% CO2 in 2 ml of MEM solution  supplemented with 10% (v/v) heat-inactivated fetal bovine serum. After 3-6 hrs, solutions were replaced with fresh supplemented MEM which was later replaced (24 hrs) with Neurobasal medium  supplemented with 2% B-27.Pregnant Sprague-Dawley rats (E17-E18) were anaesthetized with CO4, 1 Na2HPO4, 25 Glucose, 20 HEPES, 1 Na-Pyruvate; pH adjusted to 7.4. Cortical cells grown in glass-bottomed petri dishes were washed with fresh HEPES medium. The cells were then incubated at 37°C for 30 min with ER-Tracker Red  and Newport Green  or ZinPyr-1 . Cells were incubated with 10 μM of the specified fluorescent Zn2+ indicator either alone or in conjunction with ER-Tracker red for 30 min then were washed 3x with fresh HEPES medium and placed into a custom 35mm stage adapter and continuously perfused with fresh HEPES medium on the stage of a Zeiss LSM 510  microscope. Cultures were examined using a Plan-Neofluar 100x/1.3 NA oil immersion objective. For ER-Tracker Red excitation was done with a HeNe Laser line of 543nm and an LP560nm emission filter. Newport Green and ZinPyr-1 were imaged using an Argon Laser line of 488nm for excitation and a BP505-550nm emission filter. Separate fluorescent channels were employed for each indicator, and channels were scanned sequentially to minimize crosstalk. Cells were imaged by serial z-scans progressively from bottom to top, in increments of 500 nm. Colocalization of ER-Tracker Red and Newport Green or ZinPyr-1 was measured using Zeiss software [All imaging experiments were performed in HEPES medium containing the following (in mM): 130 NaCl, 5 KCl, 8 MgSOsoftware . Colocal3Rs we used the membrane-permeable UV light-sensitive caged IP3 analogue, ci-IP3/PM  benzyl-myo-inositol 1,4,5-trisphosphate-hexakis(propionoxymethyl)ester  [3 and Newport Green for 30 min at 37°C, then washed and incubated for an additional 30 min to allow for complete de-esterification.To directly activate IPGermany) ,16. Cell3 was photoreleased by flashes of 364 nm light focused uniformly throughout the field of view.IP3K inhibitor N2-(m-trifluorobenzyl),N6-(p-nitrobenzyl)purine , N,N,N',N'-tetrakis (2 pyridylmethyl) ethylenediamine (TPEN) , the InsP3 receptor blocker 2-aminoethoxydiphenyl-borate (2-APB), were applied by bath application in HEPES medium.Thapsigargin , the IP2+ indicators and examined under basal conditions showed consistent regions of elevated fluorescent intensity in the soma and processes, and particularly in a region that was identified as the endoplasmic reticulum by a fluorescent marker for the organelle. The elevated levels of Zn2+ were seen to represent labile or free Zn2+ because they were sensitive to both low and high affinity fluorescent Zn2+ indicators Newport Green (KDZn2+ ≈ 10-6M)    Figure  and ZinP) Figure . Before  of Ca2+ ,18. Fluo1 Figure . Both fl2+ release from thapsigargin-sensitive stores, cells incubated with Newport Green were exposed to thapsigargin, which inhibits SERCA pump. The application of thapsigargin depletes the ER Ca2+ stores in cells and raises cytosolic Ca2+ concentration. The experiments above suggested that Zn2+ may be also transported into and sequestered in ER. If this was true, the accumulation of Zn2+ by blocking SERCA with thapsigargin should also produce Zn2+ signals. Indeed, exposure to thapsigargin resulted in a gradual increase of cytosolic or intracellular Zn2+ . Next, we examined the contribution of extracellular Ca2+ on thapsigargin-induced elevation of intracellular Zn2+. We found that the removal of extracellular Ca2+ had no effect on the thapsigargin-induced Zn2+ rises  located on regions of the ER. When IP3 binds to and activates IP3Rs, the channel portion of the receptor opens and Ca2+ is released from the ER to the cytosol. The experiment was therefore performed utilizing ci-IP3/PM, a cell-permeable form of caged IP3 to directly induce Zn2+ release [2+ fluorophore Newport Green and caged IP3, IP3 uncaging resulted in a rapid increase in intracellular Zn2+, which persisted for 30 s and followed by a roughly exponential decay -tetrakisphosphate P4) by the enzyme InsP3 3-kinase (IP3-3Kinase or IP3-3K) [3-3K has been shown to elevate intracellular levels of IP3 by halting its conversion into InsP4 [3 signaling on intracellular Zn2+ dynamics, N2-(m-trifluorobenzyl),N6-(p-nitrobenzyl)purine, a membrane-permeable inhibitor of IP3-3K was employed to confirm the results observed using the caged IP3. Upon bath application of the inhibitor, Newport Green fluorescence showed a gradual, significant increase  . Inhibit3,4,5)P4 . Here, te Figure  &5D, sup2+ in ER-like storage, and Zn2+ is released into the cytosol in a thapsigargin- and IP3-sensitive manner. These findings suggest a new model of intracellular Zn2+ homeostasis where cellular organelles like the ER act as sites of intracellular Zn2+ storage.The major findings of the present study are the following: Neuronal cells maintain a substantial concentration of Zn2+ levels can be determined by the interaction of membrane Zn2+ transporters and cytoplasmic Zn2+ buffers [2+ has been found to be in the range of a few hundred micromolars [2+ is, however, protein bound or sequestered into organelles, which results in free cytosolic Zn2+ concentrations being in the picomolar to nanomolar range [2+ fluorescence between ER-like lumen and cytosolic space . Cols Figure  that may2+ is buffered by the abundant luminal resident chaperone protein calreticulin which binds Ca2+. Although calreticulin was first identified as a Ca2+ binding protein [2+ with multiple binding sites [2+ also binds with several other luminal proteins [2+, recent work indicates that mitochondria take up cytosolic Zn2+ and that Zn2+ accumulation leads to a loss of mitochondrial membrane potential . There function . It rema2+ trafficking remain elusive, there is little doubt that the intracellular free Zn2+ level must be maintained within a physiological limit. Zn2+ has been shown to activate a number of protein kinases such as protein kinase C, CaMKII, TrkB, Ras and MAP kinase [2+ may lead to either Zn2+-induced toxicity or Zn2+ deficiency-induced apoptosis [2+ in response to stimuli, and is likely to play an important role in the regulation of intracellular levels of Zn2+.While the mechanism(s) that govern ZnP kinase -43. On tpoptosis . TherefoThe authors declare that they have no competing interests.CS carried out the fluorescence imaging experiments, analysed data, and participated in the experimental design and the preparation of manuscript. YL conceived of the study, and participated in its design, and drafted and prepared manuscript, and coordination. All authors read and approved the final manuscript.


A patient who had received temozolomide (TMZ) as a single agent in treatment of malignant glioma developed therapy-induced myelodysplasia (T-MDS). TMZ is an orally active imidazotetrazine which methylates guanine residues in DNA, ultimately causing single and double-strand DNA breaks leading to apoptotic cell death. TMZ does not chemically cross-link DNA and is considered a nonclassical alkylating agent, similar in structure and activity to dacarbazine. Observations on this patient, and on similarly treated others, suggest that the cumulative dose threshold (CDT) for TMZ that predisposes to T-MDS and which may potentially lead to acute myeloid leukemia (T-AML) is around 18000 to 20000 mg/sq m. Although the incidence of T-MDS and the predisposing CDT of TMZ may differ from that of other potentially leukemogenic compounds currently and formerly used as chemotherapeutic agents, all alkylating agents, including TMZ, should be considered potentially leukemogenic when administered long term. Most instances of myelodysplasia (MDS) and acute myeloid leukemia (AML) are de novo or primary illnesses, and occur without known exogenous or environmental cause and at an increasing incidence with advancing age –3. FailuSecondary forms of MDS/AML are less common and may occur in the setting of pre-existing hematological or systemic disorders, after exposure to environmental toxins, or consequent to treatment of malignancy or circumstances requiring prolonged immunosuppression [secondary MDS/AML may have particular predisposing genetic loci [secondary forms of MDS/AML are more refractory to therapy than are de novo illnesses, and as recently reported, even with equivalent low-risk chromosome aberrations [secondary MDS consequent to excessive occupational exposures to benzene, an obsolete chemotherapy drug [pression –7. More tic loci , 9. It irrations –12. The rrations . This seapy drug –17.secondary AML syndromes include a MDS phase in as many as 70% of cases [Alkylating agent-induced MDS/AML was first described after treatment of multiple myeloma with melphalan . Many suof cases , 4, 5. Bof cases –5, 10. Mof cases , 20. Expof cases , 22. Unfof cases , 24. Thefluorescent in situ hybridization) probes confirmed isolated monosomy 7 in 80% of the marrow metaphases and no alterations in chromosome 5. A diagnosis of high-risk MDS was evident. Allogeneic bone marrow transplantation was contemplated, but 5 months after his diagnosis of MDS, recurrence of the brain tumor was identified in the left frontal lobe behind the optic nerve. Re-biopsy confirmed astrocytoma, and he received additional radiation therapy along with a course of azacitidine for his MDS. This latter treatment resulted in a severe pancytopenia, but eight months later, his blood counts were considerably improved from those obtained at the time of his bone marrow examination, and he was essentially asymptomatic. He died from progression of the cerebral neoplasm 43 months following its initial diagnosis without conversion from MDS to AML.A 64-year-old previously healthy right-handed man experienced the sudden onset of grand mal seizures. Cranial CT scans demonstrated a small left temporal lobe lesion. Microscopic examination after its excision showed histology consistent with astrocytoma, and clear tumor margins. He received postoperative radiation therapy, along with small oral doses of TMZ as a radiosensitizer. This drug was then continued at 200 mg/sq m/day, 5 days each month, for 18 courses . Pretreatment blood counts gave entirely normal values. Transient leukopenia and thrombocytopenia occurred during chemotherapy, but the patient did not require use of hematological growth promoting factors or blood component transfusion. His blood counts returned to normal, but several months after completion of all therapy, a mild pancytopenia became evident, and gradually worsened. Three years after treatment, the peripheral blood film demonstrated macrocytosis and typical bilobed Pelger-Huet granulocytes . The bonsecondary AML was soon appreciated with analysis of studies reporting the incidence of AML following increasing cycles of MOPP  chemotherapy for Hodgkin's lymphoma [secondary AML [secondary AML in patients treated for breast cancer and receiving cyclophosphamide in doses above 8,000 mg/sq m along with other agents [secondary AML/MDS, such as topoisomerase II inhibitors, the mean latency is shorter, and different chromosome aberrations such as balanced translocations are common [Kyle, in 1970, first called attention to the leukemogenic potential of the alkylating agent, melphalan, when used in the treatment of multiple myeloma . The impdary AML . The potr agents . This ine common . The exie common , 7, 11. TMZ is a novel chemotherapy drug that is structurally related to dacarbazine. It has the advantage of a rapid absorption following oral administration with almost 100% bioavailability. Moreover, drug levels in the central nervous system are 30–40% of TMZ plasma concentrations , 28. As 6-methylguanine generates single and/or double strand DNA breaks when the affected cell line attempts to repair this alteration via mismatch repair mechanisms and apoptosis results. O6–methylguanine-DNA methyltransferase (MGMT) plays a critical role in protecting cells from alkylating agents that target O6-guanine, including TMZ [TMZ is considered a non-classical alkylating agent in that it does not cross-link DNA strands. Its metabolites methylate DNA, particularly on guanine sites. The formation of Oding TMZ , 30. Thiding TMZ . Thus, Tding TMZ , 31. Fording TMZ , 33.secondary AML, in comparison with de novo AML and may have something to do with the lack of response to standard chemotherapy in these syndromes and could also favor the induction of T-MDS/AML [Lacking ability to cross-link DNA, TMZ has less direct toxicity to hematopoietic progenitor cells than classical alkylating agents. As a result, myelosuppression is relatively mild and not often dose-limiting. However, scattered reports describe prolonged myelosuppression and even aplastic anemia, in addition to the delayed syndromes of T-MDS/AML following therapy with TMZ –36. Hype-MDS/AML . Currently, TMZ is used in treatment of various types of gliomas, typically in concert with radiation therapy and as a radiosensitizing agent . In more The patient reported here received TMZ at a total dose of 20,000 mg/sq m prior to the diagnosis of T-MDS. In another instance where TMZ was used primarily as a single agent, the total dose administered before onset of T-MDS was quite similar, at 18,750 mg/sq m . In a sesecondary MDS/AML via occupational exposures at high cumulative dose levels [secondary MDS/AML [Benzene is an obsolete chemotherapeutic agent briefly used in the early approach to treatment of chronic myeloid leukemia . It conte levels , 44–47. e levels . Regulat MDS/AML , 47, 48. MDS/AML , 50.3 = 1 ppm) inhalation over an 8 hour daily work schedule during a 250 day work year and continuing over a 40 year period employing standard measurements for respiratory rate, tidal volume and absorbed dose, would yield a value around 92,000 mg. Assuming the worker measured 2 sq m in body surface area, this would suggest that the minimum CDT necessary to predispose to secondary MDS/AML would be about 46,000 mg/sq m. With controls on the amount of benzene allowable as a trace contaminant in modern solvents, it has become more difficult to achieve the cumulative dose levels required to initiate leukemogenesis in workers in developed countries [secondary MDS/AML for selected and well-studied modern chemotherapeutic agents including TMZ and in comparison with benzene is shown in In order to compare potentially leukemogenic doses of benzene with those of other chemicals, the typical exposure metric of ppm-years must be converted to the more clinically relevant mg/sq m. An estimate of the mg absorbed dose of benzene given a constant 1 ppm (32 mg/mountries . The widMost individuals receiving exceptionally large doses of alkylating agents over an extended period do not develop T-MDS/AML. This also is true in patients receiving TMZ . However


Here, we describe the pharmacological characteristics and clinical applications of K201.K201 is a 1,4-benzothiazepine derivative that is a promising new drug with a strong cardioprotective effect. We initially discovered K201 as an effective suppressant of sudden cardiac cell death due to calcium overload. K201 is a nonspecific blocker of sodium, potassium and calcium channels, and its cardioprotective effect is more marked than those of nicorandil, prazosine, propranolol, verapamil and diltiazem. Recently, K201 has also been shown to have activities indicated for treatment of atrial fibrillation, ventricular fibrillation, heart failure and ischemic heart disease, including action as a multiple-channel blocker, inhibition of diastolic Ca Ischemic heart disease, heart failure and severe arrhythmia still have a poor outcome despite recent advances in drug therapy. These diseases interact with one another and consequently treatment options are often limited. In two large-scale clinical trials, the CAST  and SWOR25H32N2O2S•HCl; MW: 461.07 ] is a promising new drug that has cardioprotective effects. K201 was initially discovered by Kaneko et al. as an effective suppressant of sudden cardiac cell death  for prevention of onset of myocardial infarction -7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine monohydrochloride; C1 receptors and intracellular Ca2+ pathways [K201 is a 1,4-benzothiazepine derivative with a different structural scaffold from the 1,5-benzothiazepine, diltiazem, and is not classified as a calcium channel blocker. Rather than acting as a β receptor blocker, K201 blocks αpathways  and has 2). We note that the drug is currently referred to as K201 or JTV519. K201 was the original name of the compound during development, whereas the drug was referred to as JTV519 in later clinical trials. Here, we describe the pharmacological characteristics and clinical applications of K201. The effects of K201 on the cardiovascular system have been studied extensively and the sites of action of K201 in membranes and intracellular organs in cardiomyocytes are shown in Fig. (1.+ current (INa) in a voltage- and frequency-dependent manner with IC50 values of approximately 1.2 and 2 μM at holding potentials of -60 and -90 mV, respectively. The time-course of INa blocking by K201 is slower than that of lidocaine and similar to that of quinidine; therefore, K201 is categorized as having “intermediate” but not “fast” kinetics.K201 is a non-specific blocker of sodium, potassium and calcium channels -6. The e2+ current (ICa) and the inwardly rectifying K+ current (IK1) with IC50 values of 3 and 5 μM, respectively. K201 blocks the rapidly activating component of the delayed rectified K+ current (IKr) with an IC50 of 1.2 μM [Ks) [KAch), and K201 inhibits the carbachol-, adenosine-, and GTP gamma S-induced IKAch with IC50 values of 0.12, 2.29 and 2.42 μM, respectively [K201 also slightly blocks the Caf 1.2 μM , but not μM [Ks) -6. Recen2.2+ required for excitation-contraction (EC) coupling. Membrane depolarization activates the L-type Ca2+ influx channel, which results in Ca2+ influx that then triggers a large Ca2+ release from the SR by opening the cardiac ryanodine receptor (RyR2). This mechanism is known as Ca2+-induced Ca2+ release [Calcium release from the sarcoplasmic reticulum (SR) is the major source of Ca release . 2+ leak that triggers arrhythmias [2+-induced activation [2+ release from the sarcoplasmic reticulum, since the loss of FKBP12.6 has no significant effect on the conduction and activation of RyR2 or the propensity for spontaneous Ca2+ release and stress-induced ventricular arrhythmias [2+ release in heart failure [2+ overload rat model, treatment of ventricular myocytes with FK506 to dissociate FKBP12.6 from RyR2 did not affect the suppression of spontaneous Ca2+ release by K201 [ K201 is believed to stabilize the closed state of RyR2 by increasing its affinity for the 12.6 kDa FK506-binding protein (FKBP12.6), which prevents the Cahythmias . Proteintivation -10. Hypetivation . However failure . However failure . In a Ca by K201 .2+ spark frequency and size is entirely attributable to phospholamban phosphorylation and consequent enhanced SR Ca2+ loading, and PKA does not seem to have any appreciable effects on resting RyR2 function in ventricular myocytes [2+ leak. K201 reverses this domain unzipping and stops Ca2+ leak completely, even after FKBP12.6 has mainly dissociated from RyR2 due to hyperphosphorylation of RyR2 [The phosphorylation status of RyR2 in heart failure and its effect on the RyR2-FKBP12.6 interaction are controversial. The PKA-dependent increase in myocyte Camyocytes . In cardmyocytes . FKBP12. of RyR2 . 3.2+ sparks) are elementary SR Ca2+-release events. A Ca2+ spark is a local Ca2+ release from the SR that also occurs in normal ventricular myocytes. Ca2+ spark accumulation propagates to an adjacent SR as a Ca2+ wave, with consequent induction of Ca2+-induced Ca2+ release [2+ wave activity is the underlying cause of afterdepolarization-induced electrical activity in surviving subendocardial Purkinje cells. Drugs that block inward sodium or calcium currents  have no effect on Ca2+ activity in Purkinje cells, but K201 strongly suppresses spontaneous Ca2+ release in Purkinje cells dispersed from the subendcardium of the infarct zone (IZPCs) [2+ channel activity and reduce Ca2+ waves can have antiarrhythmic effects in arrhythmogenic Purkinje cells in the infarcted heart [2+ release and the frequency and velocity of SR Ca2+ waves, despite the absence of a change in SR Ca2+ content, and also reduces Ca2+ spark amplitude and frequency and inhibits SR Ca2+ uptake and release. K201 inhibits spontaneous diastolic Ca2+ release during Ca2+ overload by a dual inhibitory action on sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) and RyR2, without significantly affecting the transient Ca2+ amplitude [In heart muscle, local elevations in intracellular calcium  . Agents ed heart . K201 at4.2+-binding proteins that have activity that depends on the Ca2+ concentration. In vitro, annexin V binding to F-actin depends on the calcium concentration and the presence of phosphatidylserine, a membrane phospholipid [2+ “hole” of annexin V  [4(a),(b)). K201 may restrain the hinge movement of annexin V in an allosteric manner, resulting in inhibition of calcium movement across the annexin V molecule [2+ channels and K201 inhibits this Ca2+ influx in an artificial lipid membrane. Annexin V also induces Ca2+ channel activity in large unilamellar vesicles (LUVs) and K201 shows dose-dependent inhibition of annexin V-induced inward Ca2+ movement into LUVs [Annexin V is a member of the annexin family of Capholipid -23. K201 . K201 bimolecule . We havento LUVs .2+ leakage does not depend on the facilitated binding of FKBP12.6 to RyR2 [A recent study has shown a similarity of the amino-acid sequences of RyR2 and annexin V: the K201 binding site in annexin is present as domain 2114-2149 of RyR2, and domain 2059-2156 of RyR2 showed a striking similarity to the corresponding sequence of annexin V . Interru to RyR2 . Another to RyR2 .5.2+ loading induces severe diastolic dysfunction [5) [2+ increase in the diastolic phase is an important factor in development of diastolic dysfunction, but it has also been reported that an intracellular Ca2+ increase in the diastolic phase alone does not increase the left ventricular end-diastolic pressure [We have shown that norepinephrine with Cafunction . Under ttion [5) . K201 sition [5) . Norepinpressure .1.KAch and IKr in atrial muscle cells is a potential mechanism for atrial fibrillation, and IKAch is often generated in the sinus node and atrium, and particularly in the atrial appendage of the atrium. K201 directly inhibits IKAch [Kr with an IC50 of 0.41 μM. Therefore, K201 is thought to improve atrial fibrillation by inhibiting IKAch and IKr [K201 inhibits carbachol-induced atrial fibrillation in guinea pig atrial muscle . An incrts IKAch  and also and IKr , and has and IKr .Abnormal calcium homeostasis with ryanodine receptor dysfunction may underlie arrhythmogenic activity in pulmonary veins. Both pretreatment and rapid administration of K201 (0.3 μM) decrease isoproterenol-induced arrhythmogenesis in pulmonary veins through reduction of the arrhythmogenic activity of pulmonary vein cardiomyocytes. These findings indicate the anti-arrhythmic potential of K201 .2.2+. Classic antiarrhythmic drugs tend to inhibit cardiac function and induce arrhythmia. The class III drug clofilium prolongs QT and induces torsades de pointes (Tdp) and ventricular tachycardia when combined with an α1-adrenoceptor stimulator, due to EAD generated from the third phase of the action potential [1-adrenoceptor stimulation. The inhibition of clofilium-induced Tdp by K201, despite QT prolongation, suggests that QT prolongation alone is not a proarrhythmic signal. K201 suppresses Tdp dose-dependently by blocking the α1-adrenoceptor and inhibiting abnormal Ca2+ release from the SR [Triggered activity is a major cause of arrhythmias, in addition to abnormal automaticity and reentry. Triggered activity is classified into early after depolarization (EAD), a transient depolarization that is generated immediately after or from the second or third phase of the preceding action potential; and delayed after depolarization (DAD) generated from the fourth phase of the preceding action potential. EAD develops due to a prolonged duration of the action potential and DAD is induced by an abnormal increase in intracellular Caotential -35. We hm the SR .3.2+ loading or old myocardial infarction [Diastolic dysfunction refers to an abnormality in diastolic distensibility, filling, and relaxation of the left ventricle , 38. Thefarction . K201 sifarction , 29, 39.4.K201 was initially developed as a drug that prevents onset of myocardial infarction and sudden cardiac death. K201 provides myocardial protection more effectively than existing calcium channel blockers and β blockers and is less negatively inotropic and chronotropic . K201 in