However, it remains unclear whether SACs express any specific Syt isoform mediating retinal waves. Moreover, it is unknown how Ca2+ binding to C2A and C2B of Syt affects wave dynamics. Here, we investigated the expression of Syt I in the neonatal rat retina and examined the roles of C2A and C2B in regulating wave dynamics.In neonatal binocular animals, the developing retina displays patterned spontaneous activity termed retinal waves, which are initiated by a single class of interneurons  that release neurotransmitters. Although SACs are shown to regulate wave dynamics, little is known regarding how altering the proteins involved in neurotransmitter release may affect wave dynamics. Synaptotagmin (Syt) family harbors two Ca2+ sensor mediating retinal waves. By combining a horizontal electroporation strategy with the SAC-specific promoter, we specifically expressed Syt I mutants with weakened Ca2+-binding ability in C2A or C2B in SACs. Subsequent live Ca2+ imaging was used to monitor the effects of these molecular perturbations on wave-associated spontaneous Ca2+ transients. We found that targeted expression of Syt I C2A or C2B mutants in SACs significantly reduced the frequency, duration, and amplitude of wave-associated Ca2+ transients, suggesting that both C2 domains regulate wave temporal properties. In contrast, these C2 mutants had relatively minor effects on pairwise correlations over distance for wave-associated Ca2+ transients.Immunostaining and confocal microscopy showed that Syt I was expressed in neonatal rat SACs and cholinergic synapses, consistent with its potential role as a Ca2+ binding to C2A or C2B, the Ca2+ sensor Syt I in SACs may regulate patterned spontaneous activity to shape network activity during development. Hence, modulating the releasing machinery in presynaptic neurons (SACs) alters wave dynamics.Through Ca Accordingly, the intracellular Ca2+ concentrations exhibit spontaneous and correlated increases, producing wave-like Ca2+ oscillations across the RGC layer 2+ transients in RGCs has been widely used to elucidate the synaptic mechanisms underlying retinal waves Binocular visual circuit refinement requires a robust patterned spontaneous activity in the developing retina . Retinal waves are spontaneous, correlated action potentials propagating through the retinal ganglion cell (RGC) layer during a developmental critical period prior to visual experience. In particular, retinal waves are mediated via synaptic transmission with a periodicity on the order of minutes. In the developing mammalian retina, three different stages of retinal waves have been classified, of which the stage-II waves are the best studied 2+-regulated exocytosis. Members of the Ca2+ sensor protein family synaptotagmin (Syt) transduce the Ca2+ signal to trigger fusion between vesicle and plasma membranes 2+ through their two Ca2+-binding C2 domains (C2A and C2B), with three binding to C2A and two binding to C2B 2+ binding, Syts interact with distinct effectors, such as lipids, SNARE (soluble NSF attachment protein receptor) proteins, assembled SNARE complex, and other Syt molecules to trigger exocytosis Synaptic transmission is initiated by neurotransmitter release via Ca2+ binding to distinct C2 domains in Syt controls the choice of exocytotic modes 2+ binding to C2A reduces full fusion by limiting fusion pore dilation and preventing release of some vesicle content. By contrast, weakened Ca2+ binding to C2B abolishes both full fusion and kiss-and-run 2+ binding to C2A or C2B in Syt I 2+ binding to various C2 domains of Syt confers a repertoire of functions in controlling neurotransmitter release and synaptic transmission Previous studies revealed that preferential Ca2+ spikes and Ca2+-dependent afterhypolarizations 2+-dependent. Borges et al. reported that a small number of primed vesicles, very likely one per release site, can be quickly exocytosed upon Ca2+ influx into SACs 2+ influx into SACs may cause Ca2+ binding to its sensor Syt to drive exocytosis during stage-II waves.Many studies have characterized the role of SACs in regulating wave dynamics during the stage-II period. However, the release mechanism in SACs has not been well understood. Immunolabeling and confocal microscopy suggested that several SNARE proteins are expressed in SACs, such as syntaxin 1 and 2 2+ imaging to investigate the expression of Syt I in SACs and determine the roles of C2AB domains in regulating stage-II waves during the development of neural circuits.Although the human genome encodes more than seventeen Syt isoforms 2+ sensor in triggering neurotransmitter release during stage-II waves.Syt I is the major Syt isoform found in many parts of the nervous system 2+ imaging to measure wave-associated Ca2+ transients in both acutely isolated retinas and retinal explants after 3–4 day in vitro culture. The characteristics of wave-associated Ca2+ transients were not significantly changed after in vitro culture   . These d2+ transients  promoter has been shown to target SACs specifically To manipulate neurotransmitter release from SACs during stage-II waves, we aimed to transfect SACs with Syt I mutants with the weakened CaThe immunoreactivities from retinal cross-sections were used to evaluate our transfection in SACs. The HA/EGFP immunoreactivity represented all transfected cells , whereas ChAT immunoreactivity (in red) indicated SACs. Therefore the transfected SACs would display yellow color . We first determined our transfection efficiency in SACs, which was calculated as the number of cells with yellow signal divided by the number of cells with red signal . The transfection efficiency with the mGluR2 promoter was 57.14±4.56% , compared to 30.25±3.45%  with the CMV promoter.right insets). With the mGluR2 promoter, the promoter specificity for SACs was 83.87±1.61% , compared to 8.79±1.22% (mean ± S.E.M.) with the CMV promoter (N = 6 sections). Thus, the mGluR2 promoter achieved a significantly higher SAC specificity compared to the CMV promoter . This indicates that the mGluR2 promoter can be used to target SACs for molecular perturbation in the developing rat retina.We then calculated the promoter specificity, which was defined as the number of cells with yellow signal divided by the number of cells with green signal  , compared to relatively large RGCs (∼20 µm) stained by the Ca2+ indicator fura-2 , whereas D363N confers a Ca2+-binding site mutation in the C2B domain of Syt I (designated Syt I-C2B*). Each mutated residue abolishes the first ligand of the third loop in the Ca2+-binding pocket of the respective Syt C2 domain 2+ transient frequency in the developing rat retina, live Ca2+ imaging was conducted in the retinal explants after transfection. Spontaneous, correlated Ca2+ transients in individual cells revealed retinal waves in the RGC layer of explants transfected with control vector, wild-type Syt I, Syt I-C2A* or Syt I-C2B*  and the fraction of cells displaying Ca2+ transients . In contrast, both Syt I mutants reduced the mean frequency of Ca2+ transients by 2-fold compared to wild-type Syt I . Thus, weakening Ca2+ binding to either the C2A or C2B domain of Syt I reduced Ca2+ transient frequency. In addition, the cumulative probability for Ca2+ transient frequency was constructed from all recorded cells for control, Syt I, Syt I-C2A* and Syt I-C2B*  was 0.58 for control, 0.55 for Syt I, 0.35 for Syt I-C2A*, and 0.35 for Syt I-C2B* . The mean fraction of cells with Ca2+ transients in control was indistinguishable from intact, untransfected culture retinas . Wild-type Syt I left the fraction of cells with Ca2+ transients identical to that seen in control cells . The mean fraction of cells with Ca2+ transients was significantly reduced in both Syt I-C2A* and Syt I-C2B* compared to control and wild-type Syt I . Hence, weakened Ca2+ binding to either C2 domain in Syt I reduced the fraction of cells with Ca2+ transients. With fewer cells displaying Ca2+ transients and less frequent Ca2+ transients in most cells, these results suggest that weakened Ca2+ binding to the C2AB domains of Syt I may reduce the frequency of stage-II waves.Second, to determine whether Syt I mutants change the fraction of cells participating in waves, we calculated the fraction of cells with Ca2+ transients associated with waves can last for tens of seconds following relatively brief spontaneous depolarizations 2+ transients in an unbiased way . Moreover, cumulative probability distributions were constructed to get the median Ca2+ transient durations   , and the retinas . The curpe Syt I . Thus, w2+ transient amplitude, we compared the mean amplitude from different transfection groups. Similar to the effects on Ca2+ transient duration, wild-type Syt I did not change the Ca2+ transient amplitude compared to control (p>0.05). Both Syt I-C2A* and Syt I-C2B* significantly reduced Ca2+ transient amplitude compared to wild-type Syt I and control  were also reduced in Syt I-C2A* and Syt I-C2B* . To assess wave correlation structure across cells, we computed cross-correlograms over cell pairs Both Syt I-C2A* and Syt I-C2B* dampened wave-associated Ca2+ binding to the C2AB domain of Syt I may not alter the spatial correlation structure of stage-II waves.To further quantify the strength of the correlations, the correlation index (C.I.) 2+ binding in the C2AB domains of Syt I, we tested the effects of presynaptic perturbation on wave-associated Ca2+ transients. Our Ca2+ imaging results provide the first demonstration that Ca2+ binding to C2A or C2B of Syt I regulates the temporal properties of stage-II waves. In contrast, Ca2+ binding to C2AB in Syt I has relatively minor effects on the spatial properties of stage-II retinal waves. These results suggest that the C2AB domains of Syt I regulate the dynamics of patterned spontaneous activity during the development of neural circuits.In this study, we showed that Syt I is strongly expressed in cholinergic neurons (SACs) and synapses (the IPL) in retinas exhibiting stage-II waves. We further established a molecular perturbation method targeted to presynaptic SACs. By weakening Ca2+ transients via Ca2+ binding to its C2AB domains. Together with the previous findings 2+ oscillations in SACs 2+ binding to its sensor Syt I to trigger exocytosis While the importance of Syt I in synaptic transmission has been recognized for a couple of years 2+-dependent manner 2+ binding to Syt I warrants further investigation, especially in the form of kiss-and-run or full fusion.Developing SACs corelease ACh and GABA in a CaManipulations that alter the spatiotemporal properties of stage-II waves result in defective retinogeniculate and retinocollicular projections The roles of the C2AB domains in Syt I have been intensively investigated at the molecular and functional levels. Biochemical analysis showed that both Syt I-C2A* and Syt I-C2B* greatly reduce their binding to t-SNARE heterodimers (corresponding to target proteins on plasma membrane) and PS-containing liposomes (corresponding to vesicle membrane constituent) 2+ dynamics in SACs and thus reduce exocytosis. However, several in vitro and in vivo studies have demonstrated that overexpression of Syt I with Ca2+-binding site mutations can not alter whole-cell Ca2+ current or Ca2+ dynamics 2+ channels or dynamics in SACs.Alternative interpretation for the effects of Syt I mutations on wave dynamics is that overexpression of Syt I-C2A* and Syt I-C2B* might alter cytoplasmic Ca2+ transients in nearby RGCs. We observed that Syt I-C2A* and Syt I-C2B* dampen the temporal properties of stage-II waves  promoter was used for SAC-specific expression  imaging . For Wes imaging , the pCM imaging , the pCMad libitum access to food and water. Rat pups (P0–P2) were deeply anesthetized with ice before decapitation with all efforts to minimize suffering.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol for all animal experiments was approved by the Institutional Animal Care and Use Committee of National Taiwan University (Permit Number: 97-27 and 99-82). Neonatal (P0–P2) Sprague-Dawley rats  were used in the study. All rat pups were housed with their own mothers in the individually ventilated cages with well-controlled conditions  and 3, pH 7.35]. The retinal pieces were attached onto nitrocellulose membranes (Millipore) with the RGC layer facing up. Retinal explants were cultured at 35°C in a 5% CO2 humidified incubator, and daily supplied with fresh Retinal Serum-Free Culture Medium (SFCM-A), containing Neurobasal-A (GIBCO #10888), 0.6% Glucose, 2 mM L-Glutamine (Sigma #G6392), 1X B-27 (GIBCO #17504-044), 10 mM HEPES, 1 mM Sodium Pyruvate (GIBCO #11360-070), 2.5 µg/mL Insulin (Sigma #I1882), 100 µg/mL Penicillin/100 µnits/mL Streptomycin (GIBCO #15140-122), and 6 µM Forskolin The retina was isolated and cut into thirds in dissection buffer  for 1 hr. After fixation, the explants were blocked at RT for 1 hr in 3% donkey-serum blocking solution (DBS), containing 3% donkey serum (Jackson Lab #017000121), 0.5% Triton X-100, and 0.1% sodium azide in PBS. The explants were first incubated with primary antibodies in 1% DBS [goat polyclonal anti-ChAT  and mouse anti-HA ] at RT for 2 days, and then washed with PBS. Secondary antibodies in 1% DBS [donkey-anti-goat IgG conjugated with Alexa Fluor 568  and donkey-anti-mouse IgG conjugated with Alexa Fluor 488 ] were further added at RT for 1 day, and then washed out with PBS. The anti-fade reagent Fluoromount G (Electron Microscopy Sciences #17984-25) was added to retinal explants on the slides. The slides were sealed by coverslips. Fluorescent images were acquired by confocal microscopy .For whole-mount retina staining, the transfected retinal explants were placed on poly-lysine-coated slides, fixed with 4% paraformaldehyde (PFA) at RT for 30 min, and washed with phosphate buffered saline  at 4°C overnight. Membranes were washed three times with TBST and then incubated with secondary antibody in 5% milk in TBST at RT for 1 hr. Membranes were washed three times with TBST, and the signals were visualized by enhanced chemiluminescence followed by autoradiography.The P0–P2 rat retinal explants transfected with pCMV-HA or pCMV-HA-Syt I were spun down in dissection buffer at ∼2,000 2+ imaging, retinal explants were placed in the forskolin-free SFCM-A containing 10 µM fura-2-AM (Molecular Probes #F1221), 0.02% pluronic acid (Molecular Probes #P3000MP), and 1% DMSO for 30–60 min. During Ca2+ imaging experiments, retinal explants were perfused continuously with artificial cerebrospinal fluid (ACSF) containing (in mM): 119 NaCl, 26.2 NaHCO3, 2.5 KCl, 1.0 K2HPO4, 1.3 MgCl2, 2.5 CaCl2, and 11 D-glucose bubbled with 95% O2/5% CO2. The temperature of ACSF was maintained at 30°C with a TC-344B controller (Warner Instruments). Live Ca2+ imaging was performed on an upright Olympus BX51WI, using a 20X water immersion objective (Olympus). The transfected regions in the retinal explants were identified by EGFP fluorescence . The fura-2 fluorescence was excited at 380 nm (Chroma #D380xv2) via a xenon arc lamp  and a 455DCLP dichotic mirror (Chroma). The fura-2 emission fluorescence was collected at 510 nm (Chroma #D510/40 m) and captured by a CCD camera . To measure spontaneous Ca2+ transients associated with retinal waves, the fura-2 fluorescence images were continuously acquired at 1 sec-intervals for 10 min , with 100–150 msec exposure times and two as binning number. One or two transfected regions from each retinal explant were chosen for 10-min recordings. Digitized imaging data were subsequently processed by MetaMorph software. Further data analysis was conducted by Igor Pro (WaveMetrics), Excel and Orgin 5 or 8 (OriginLab).Retinal explants were transferred to forskolin-free SFCM-A overnight before imaging. Prior to Ca2+ imaging data for each transfection group were acquired by the following procedures. First, 49 RGCs were randomly selected from the 7×7 divisions in one imaged region (340×460 µm) using MetaMorph. The wave data from single RGCs were acquired from the fluorescence changes across all the time frames, previously background subtracted for each frame. The fluorescence signals were further corrected for photobleaching by using Igor Pro to set the baseline. Second, to analyze wave characteristics (frequency/duration/amplitude) in an unbiased way, an Igor procedure was written in this laboratory to automatically pick the wave peaks, with their fluorescence intensity 2-fold greater than the RMS noise (∼0.25% ΔF/F). The definitions of the duration and amplitude for individual waves are presented in The Ca2+ waves between nearby cells was calculated from pairs of RGCs in the same imaged region separated by various distances (10–500 µm). The positions of individual cells were calculated using MetaMorph. The intercellular distance was taken as the shortest distance between selected cells. The lag times of the waves in these two nearby cells were acquired by individually subtracting from the presenting time of every wave in the paired cells. All the subtracted times were collected for each cell pair to construct cross-correlograms from one imaged region (t  = 3 sec), as given by the following equation:NAB is the wave number for which cell B fires with a time ± Δt from cell A, NA and NB are the total numbers of waves fired by cells A and B, respectively, during the total recording time (both NA and NB ≥ 3 in 10 min), T is total recording time (600 sec), and Δt is the correlation time interval (3 sec). The cell pairs from one imaged region were grouped according to their intercellular distances with a bin width of 50 µm. The median C.I. values were computed from each distance group out of the same imaged region. The averaged C.I. values were computed from the same distance group out of many imaged regions for each transfection group, and then plotted against the upper limit of intercellular distance for each distance group .All data were presented as mean ± S.E.M. Differences between means of different groups (≥3 groups) were evaluated for statistical significance with One-way ANOVA followed by Figure S12+ transients in acutely isolated retinas (Acute) and retinal explants after 3–4 day Comparison of wave-associated Cain vitro culture (Culture). (A) Ca2+ transient frequency. (B) Fraction of cells with Ca2+ transients. (C) Ca2+ transient duration. (D) Ca2+ transient amplitude. (E) Pairwise C.I. values as a function of intercellular distance. The methods to measure the properties of Ca2+ transients were described in p<0.05, unpaired t-test for comparison at a given distance; n.s., not significantly different from acutely isolated retinas)(TIF)Click here for additional data file.


Hepatic angiomyolipoma (AML) is notoriously difficult to diagnose without an invasive surgery even with the recent development of the various imaging modalities. Additionally, recent reports showed its malignant behavior after the surgery; it is important to diagnose the character of each tumor including the possible malignant potential and determine the postoperative management for each case. For this purpose, we have reviewed reports and focused on the immunohistochemical staining with p53 and ki67 of the tumors showing the representative case of 60-year-old female. The imaging study of her tumor showed the character similar to the hepatocellular carcinoma, and she underwent the hepatectomy. The resected tumor stained positive for HMB-45 that is a marker of the AML, and 30–50% of the tumor cells were positively stained with Ki67 that is a mitotic marker. Also, the atypical epithelioid cells displayed p53 immunoreactivity. These results suggest the malignant potential of our tumor based on the previous reports; therefore the careful followup for this case is necessary for a long period whether it shows metastasis, sizing up, and so forth. Angiomyolipoma of the liver is rare and has been considered a benign tumor since Ishak  first deAmong the various benign tumors, angiomyolipoma (AML) is a well-known renal tumor associated with the tuberous sclerosis complex. However, such tumors have been reported to occur in various extrarenal sites, including the liver, uterus, and retroperitoneum. Hepatic AML is a rare benign tumor of mixed mesenchymal origin, which Ishak  firstly The biological behavior of hepatic AML requires further investigation. Because hepatic AML has been considered as benign tumor, most clinicians advocated conservative treatment. In 2000, Dalle et al. reported the first case of malignant hepatic AML , in a 70α-SMA and HMB-45. In the nine cases that have been published, all of which clearly involved metastasis and/or recurrence, the malignant hepatic AML was >8 cm in diameter. Some cases involved portal vein thrombus and necrosis. A recent study by Maklouf et al. showed that CD117 staining was positive in all cases of benign renal and hepatic AML [We reviewed the literature , 21 and atic AML . Nguyen atic AML . Deng etatic AML . AlthougOur representative case was a 60-year-old woman. She had no history of malignant disease, and the blood exams were negative for tumor markers and viral markers.The abdominal plain CT revealed a low-density area  in segmeα-SMA (Figures A partial hepatectomy of segment 6 was performed. Upon macroscopic examination, the surgical specimen appeared as a solid whitish mass consisting of a single nodule free of necrosis. There were no signs of chronic inflammation or fibrosis in the surrounding liver tissue. The three defining characteristics of AML were identified, including mature fat, blood vessels, and epithelioid-spindle cells Figures . The rou Figures . Fat cel Figures , and 30% Figures . Based oHepatic AML usually follows a benign clinical course. However, the tumor malignancy cannot be ignored. Therefore, we recommend that excision or fine-needle biopsy be accompanied by subsequent histological staining for markers such as Ki67 and p53. This information will be helpful for the management of the tumor.


We found that mean Ca2+ spark amplitude, duration, size and rate-of-rise were significantly smaller in Fluo-3 loaded db/db compared to control CASMCs, with a subsequent decrease in the total amount of Ca2+ released through Ca2+ sparks in db/db CASMCs, though Ca2+ spark frequency remained. Interestingly, the frequency of large-amplitude Ca2+ sparks was also significantly reduced in db/db cells. In addition, the frequency and amplitude of STOCs were markedly reduced at all voltages tested (from −50 to 0 mV) in db/db CASMCs. The latter correlates with decreased BK channel β1/α subunit ratio found in db/db vascular tissues. Taken together, Ca2+ spark alterations lead to inappropriate BK channels activation in CASMCs of db/db mice and this condition is aggravated by the decrease in the BK β1 subunit/α subunit ratio which underlies the significant reduction of Ca2+ spark/STOC coupling in CASMCs of diabetic animals.Diabetes is a major risk factor for stroke. However, the molecular mechanisms involved in cerebral artery dysfunction found in the diabetic patients are not completely elucidated. In cerebral artery smooth muscle cells (CASMCs), spontaneous and local increases of intracellular Ca2+ due to the opening of ryanodine receptors (Ca2+ sparks) activate large conductance Ca2+-activated K+ (BK) channels that generate spontaneous transient outward currents (STOCs). STOCs have a key participation in the control of vascular myogenic tone and blood pressure. Our goal was to investigate whether alterations in Ca More than 65% of patients with diabetes die from cardiovascular disease or stroke db/db mouse, a genetic model of non-insulin dependent type-2 diabetes exhibits cerebral vascular dysfunction gracilis muscle arterioles db/db mice. Cerebral arterioles of db/db mice show impaired response to vasodilators and reduced baseline arteriolar diameter gracilis muscle arterioles of db/db mice show impaired response to vasodilators, enhanced response to vasoconstrictors and enhanced basal myogenic tone db/db mice are smaller than the diameters of corresponding control arteries db/db mice in the presence of sodium nitroprusside The 2+ due to the opening of Ryanodine Receptors (RyRs), visualized as Ca2+ sparks, activate large conductance Ca2+ sensitive K+ channels (BK channels) that generate spontaneous transient outward currents (STOCs) 2+ influx through L-type Ca2+ channels, diminishes global intracellular Ca2+ concentration ([Ca2+]i), and opposes vasoconstriction 2+ sparks and BK channels, by producing STOCs regulates arterial tone favoring vasorelaxation 2+ spark generation is also regulated by Ca2+ influx due to an indirect coupling between L-type Ca2+ channels and RyRs 2+ ATPase (SERCA) participates in this indirect coupling by redirecting the Ca2+ coming from the extracellular medium towards its luminal stores, located mainly inside the sarcoplasmic reticulum (SR) of CASMCs. Navedo and collaborators 2+ currents and Ca2+sparklet activity –brief plasma membrane Ca2+ fluxes due to the activation of clusters of L-type Ca2+ channels– are enhanced in CASMCs of db/db mice; suggesting that the mechanisms that participate in the closure and tight regulation of L-type Ca2+ channels could be impaired in diabetic CASMCs. Therefore, we sought to investigate whether BK channels and their functional pair, the RyRs, might contribute to worsening the condition of diabetic CASMCs.In cerebral artery smooth muscle cells (CASMCs), spontaneous and local increases of intracellular Ca2+ spark and STOC in CASMCs from db/db mice due to Ca2+ spark alterations which lead to inappropriate BK channels activation, and the latter is aggravated by the decrease in the BK β1 subunit/α subunit ratio also found in CASMCs of db/db mice.Our data represent the first demonstration of altered Ca2+ sparks of CASMCs represent the spontaneous and coordinated opening of an undefined number of RyRs within a cluster i.e. amplitude, duration, size and mean rise rate– are used as markers of in situ RyR activity db/db mice in situ activity of vascular RyR in single, freshly isolated CASMCs of diabetic mice.Ca2+sparks (right images) recorded in intact control and diabetic (db/db) CASMCs that conserve their typical relaxed spindle shape (left images). We found that the db/db CASMCs produced similar Ca2+ spark frequency to control cells /dt: 0.181±0.006 vs. 0.163±0.005 in 460 Ca2+ sparks from 43 control CASMCs vs. 423 Ca2+ sparks from 41 db/db CASMCs, P<0.05).ectively B, with ectively . The maimplitude , full dumplitude , full wimplitude  and mean2+ spark parameters –plotted as a function of probability density (PDf)– were asymmetrical for both control and diabetic events  released through Ca2+ sparks was decreased in db/db cells . This could be due to a decreased number of RyR opening simultaneously in a cluster. We estimated RyR expression by western blots in aorta homogenates and found a decrease in db/db mice . Global [Ca2+]i responses were measured in CASMCs from control and db/db mice using fura-2 based Ca2+ microfluorometry. Examples of such traces are shown in left traces. No significant differences in the peak amplitude of the caffeine-evoked [Ca2+]i transients were observed  (left bars) thus ruling out a difference on the SR Ca2+ load at rest between db/db and control CASMCs underlying the weaker Ca2+ sparks. To observe the time dependence of luminal Ca2+ replenishment 2+]i transient was significantly reduced in the CASMCs of diabetic animals   Ca P<0.05) B. The n2+sparks activate large conductance K+ (BK) channels, which play a critical role in controlling vascular tone 2+ sparks in diabetic CASMCs had an impact on BK channel activity by analyzing spontaneous transient outward currents (STOCs). Intact CASMCs were patch-clamped and progressively depolarized from a holding potential of −50 mV to 0 mV in 10 mV voltage increments , suggesting that voltage sensitivity of BK channel activation is unaltered in type 2 diabetes. Amplitude was also significantly reduced in db/db CASMCs as compared to control cells, with statistical difference from −30 mV to 0 mV. However, linear fitting of these data demonstrated that the voltage dependence of STOC amplitude was diminished 0.55 fold in db/db CASMCs  nor to a change in zero current potential . Further analysis of STOC kinetics revealed that both STOC time-to-rise ; suggesting that composition stoichiometry of BK channels is modified by the diabetic condition; with decreased proportion of BK channel β1-subunits to α-subunits. Separate analysis of BK α and β1 subunit expression levels were slightly but not significantly modified .We next sought to determine whether the diminished frequency and amplitude of STOCs in the diabetic CASMCs might be related to a change in BK channel composition. Vascular BK channels are composed of four pore-forming α-subunits and four accessory β-subunits, β1 being the predominant isoform expressed in vascular smooth muscle unoblots A and baunoblots B show t2+ sparks have a vasorelaxing effect by activating STOCs, which hyperpolarizes the cell, leading to inactivation of VDCCs. The STOC/Ca2+spark coupling ratio was indirectly estimated by dividing the number of STOCs at near resting membrane potential (−40 mV) by the number of spontaneous Ca2+ sparks in single quiescent cells. db/db CASMCs, suggesting that many of the Ca2+ sparks produced in the diabetic CASMCs lack STOCs, contrary to what is observed in normal vascular myocytes It has been shown that Cadb/db mice. We have demonstrated: 1) Ca2+ spark properties are significantly decreased in the CASMCs of diabetic animals; 2) STOC frequency, amplitude, kinetics and area were further reduced; and 3) diminution of the BK channel β1/α subunit ratio, resulting in an abnormal Ca2+ spark-STOC coupling in db/db CASMCs.In this work we present the first compelling evidence of the RyR and BK channel functional loss in native CASMCs of 2+ sparks in db/db CASMCs, probably due to 1) smaller and shorter Ca2+ sparks with reduced amplitude than then could be STOCless; 2) a compromised SR Ca2+ load recovery; and 3) decreased STOC frequency, amplitude, area, time-to-rise and time-to-decay. The latter is probably due to reduced Ca2+ sparks because of decreased RyRs expression and further enhanced by the altered subunit composition of BK channels. Taken together, the reduced efficacy of Ca2+ sparks to trigger STOCs might contribute to an increase in vascular tone of db/db arteries.Taken together, our data suggests the scenario schematized in db/db mice is characterized by reduced baseline arteriolar diameter, increased basal tone and reduced response to acetylcholine, in part due to enhanced Rho-kinase activity, superoxide production 2+ handling and STOCs which might contribute to these alterations, since it has been previously shown that spontaneous Ca2+ sparks contribute to vascular relaxation by activating STOCs.Previous studies have shown that the function of cerebral arteries is impaired in experimental and genetic models of type-2 diabetes 2+ spark frequency was not significantly altered, but the Ca2+ spark amplitude, duration, width, and as a consequence Ca2+ spark mass were decreased in db/db CASMCs. To our knowledge, this is the first study of Ca2+ sparks in CASMCs of type 2 diabetes. In type 1 diabetes, Ca2+ sparks frequency has been found to be unaltered 2+ spark amplitude.We found that the overall Ca2+ load in vascular myocytes reduces Ca2+ spark properties 2+ load in the conditions of Ca2+ sparks recording , which are increased in the db/db mice, depress SERCA function 2+ spark amplitude, duration, spatial spread, and mass of the Ca2+ spark. This could be supported by the reduced RyR expression in db/db arteries .All experiments were carried out according to European Union Council Directives (86/609/EEC) for the care of laboratory animals. Animal Protocol was approved by the db/db) mice used in the current study were on a C57BL/KsJ genetic background. We used male animals at the age of 14 to 16 weeks. db/db mice were significantly heavier than WT controls .The diabetic (db/db) mice and their controls (+/+), using a previously standardized method 2, 10 glucose and 10 HEPES, pH 7.4). The cerebral basilar arteries were dissected, transferred to a 1.5 ml tube and incubated in the shaker for 16 min at 37 °C in HBDS containing in mg/ml: 1.0 papain , 1.0 bovine serum albumin (BSA), and 1.0 dithiothreitol. Then, the tissue was transferred to a collagenase solution  plus 100 µmol/L CaCl2 and incubated for 8 min at 37 °C with shaking. Enzyme digestion was stopped by washing the tissue 5 times with HBDS. Digested tissue was triturated with a fire-polished glass Pasteur pipette to yield single smooth muscle cells. Myocytes used for confocal experiments were loaded with 10 µmol/L Fluo-3 AM for 30 min at room temperature, washed and kept at 4 °C in physiological saline solution  to be used within the same day.Cerebral artery smooth muscle cells (CASMCs) were enzymatically isolated from 14 to 16 week-old male C57BL/KsJ-db  equipped with an x63 water-immersion objective (N.A. 1.2) in the line scan mode (5 images of 1000 lines at speed of 1.92 ms/line). Fluo-3 was excited at 488 nm with an Argon laser (3% intensity), and emission measured at above 510 nm. Ca2+ sparks were reconstructed by stacking consecutive line scans and performing a time-intensity plot. Ca2+ sparks were automatically detected and properties of amplitude (F/F0), duration  and width  measured with a custom-made program running in IDL 5.5 software (Research Systems Inc.), with a detection threshold of 4.3×S.D 2+ sparks were normalized by dividing the fluorescence intensity of each pixel (F) by the average resting fluorescence intensity (F0) to generate an F/F0 image. Mean rate of rise was determined as reported by Shen et alSpontaneous and local Ca2+]i transients were elicited consecutively with a 5-minute interval in between to allow refilling of intracellular Ca2+ stores. Images of Fura-2 fluorescence were captured by a cooled CCD camera  with an oil-immersion x40 objective mounted on an inverted microscope , and acquired at 510 nm emission after excitations at 340 nm and 380 nm using a lambda-DG4 excitation system . Ca2+ signals were analyzed using Metafluor software . [Ca2+]i (in nmol/L) was calculated from the ratio of 340/380 nm after correcting for background. Fluorescence emission ratios were converted to [Ca2+]i according to the Grynkiewicz equation in situ calibration were used for Rmin, Rmax, and β, respectively.Myocytes were loaded with 2.5 µmol/L fura 2-AM. Cells were transferred to a bath perfusion chamber and continuously superfused with PSS. Two caffeine-induced [Ca2, 3 MgATP, 0.1 NaGTP, 0.05 KEGTA, 20 HEPES, pH 7.2 with KOH. CASMCs were superfused with PSS and clamped at −50 mV or stepped from −50 to −10 mV in 10 mV increments. Membrane capacitance (Cm) was determined from the current amplitude elicited in response to a hyperpolarizing voltage pulse from a holding potential of −80 mV . Currents were filtered at 500 Hz and digitalized at 2 kHz (500 µs/point). STOC analysis was performed off line, using the event detection tool of Clampfit 9.2 ; where both STOC time-to-rise and time-to-decay were measured at half peak of STOC in ms.STOCs were recorded in the whole cell configuration of the patch clamp technique using solutions and protocol previously described g for 15 min at 4°C. Supernatants were fractionated on 15% SDS-PAGE gels, transferred onto nitrocellulose membranes  and probed with anti-BK α antibody , anti BK β1 antibody , anti RyR  and anti-actin antibody  in phosphate-buffered saline solution containing Tween-20 . Membranes were incubated 1 h with secondary peroxidase-conjugated goat anti-rabbit (1∶15000). Signal was detected by chemiluminiscence. Densitometry was done with Kodak ID Software . Actin signals were detected in the same blots. Protein concentration was assessed by the Bradford method.Aorta homogenates were used because they provide more protein. They were prepared with homogenization buffer  plus protease inhibitors . Aortas were homogenized on ice with a glass homogenizer and spun at 2,000 P<0.05 were considered statistically significant. The probability density function analysis2+ spark parameter a, totalN is the total number of Ca2+ sparks, and Δ is the bin width. Bimodal Gaussian distributions were done as previously reported Data are expressed as the mean ± SE. Statistical significance was assessed using SigmaStat 3.0 software by Student’s t-tests, one way ANOVA test or by nonparametric Mann-Whitney Rank-sum test for cases in which data failed the Shapiro-Wilk normality test. Values of Figure S12+ sparks were recorded within the same cell during the recording period (9.6 seconds). Firing sites were counted as the sites where we recorded at least one Ca2+ spark. B, Ca2+ spark frequency within each firing site reported as number of events recorded within each site/s. C, Probability in each cell to present sites that fire repetitively. D, Maximum number of Ca2+sparks recorded within the same site. N = 43 for control CASMCs (white bars) and n = 41 for db/db cells (gray bars).A, Average of sites where Ca(DOCX)Click here for additional data file.


Our long time experience and recent research give us grounds to believe that increasing the doses of nonsedating H1 antihistamines up to fourfold improves significantly the chances of successful treatment. Our data suggest that the urticaria-associated discomfort is relieved by higher than conventional doses of levoce-tirizine and desloratadine in about 75% of the patients and that sedation/somnolence does not seem to be a major deterrent. The dose increase also improves the urticaria-specific quality of life. Contrary to the belief that individual patients may benefit from one antihistamine or another, we demonstrate that the drug with better ability to suppress the histamine skin effects in experiments in healthy volunteers (levocetirizine) is also superior in improving the different aspects of control of chronic urticaria  and that increasing its dose of up to fourfold may even paradoxically reduce the sense of sedation/somnolence in parallel with the relief of urticaria discomfort.The term "chronic idiopathic urticaria" denotes a spectrum of conditions with different poorly understood pathogenetic mechanisms in which the release of histamine plays a role. Nonsedating second-generation H Awide spectrum of different conditions share a seemingly common clinical feature: appearance of urticarial lesions. The latter are described as red, raised, itchy wheals  resulting from vasodilatation, increased blood flow, and increased vascular permeability. They haεRI) on basophils and mast cells[1 antihistamines (nsAH) (table Chronic urticaria (CU) is a relatively common condition, which has a profound effect on the quality of life of those suffering. The neeast cells-6. Anothast cells. It appeast cells. The staWhen analyzing the state-of-the-art of urticaria management, the guidelines have recognized deficits in the supporting evidence. In a recent overview of his extensive experience in urticaria treatment, Allen Kaplan, a leading expert in the field of urticaria, lists a number of misconceptions among the medical community and the lay public related to urticaria management and pinpoints controversies relating to the use of AH, which need to be addressed by specifically designed clinical studies. The preL-histidine by the enzyme histidine decarboxylase and plays a vital role in the regulation of many important functions of the organism related to circadian influences, adaptation to the environment, and stress. Conversely, histamine is intimately implicated in the pathogenesis of allergic and other diseases, which develop as a defective systemic trait and may have different organ expressions. Histamine exerts its multiple effects by coupling to receptors expressed on the membrane of cells in different tissues. Four different receptors have been identified and sequenced so far denoted sequentially as H1 to H4[1 receptors located on nerves and endothelial cells[4 receptors in the generation of pruritus and eosinophil chemotaxis[Histamine is a low molecular weight amine synthesized from  H1 to H4. The hisial cells. A role hemotaxis. The hishemotaxis. However1 AH to be proposed as first-line and mainstay treatment  adverse effects, and became subsequently known as first-generation H1 AH[1 receptors, and had longer half-life, allowing a once daily dosing regimen[1-receptor antagonists, and whereas 2 of the earlier compounds (terfenedine and astemisol) were abandoned because of serious cardiac side effects, the "newest arrivals"  took up the lead in prescription practices as "modern" second-generation drugs.Within a period of 40 years since 1937, when Bovet and Staub described the first pharmaceutical formulation to counter the effects of histamine in a guinea pig, about 40 AH had been introduced for the treatment of nasal allergic and skin symptoms. They all had a short plasma half-life, were not very Hion H1 AH. Some ofion H1 AH. A signig regimen. They we1 receptors to ensure the best effect. Determinants for this to happen are the applied dose and the pharmoacokinetic properties of the separate formulations: bioavailability, plasma half-life, volume of distribution, and receptor affinity. Following this rationale, the second-generation nsAH should have a clear cut advantage over the first-generation ones. This is not, however, the case in everyday practice where nonexperts treat urticaria and prescribe first-generation drugs contrary to guidelines[Ideally, an AH drug should bind to a maximal number of Huidelines. At leas1 AH is still alive because of some other arguments prompted by empirical experience. Thus, Allen Kaplan is favoring high doses of hydroxyzine and diphenhydramine if doubling the doses of second-generation nsAH does not achieve satisfactory control of symptoms[The debate about the use of first-generation H symptoms,15,17. I1 receptors may prove an advantage as they may exert some additional beneficial effects through other mechanisms: antimuscarinic, anti-α-adrenergic, antiserotonin effects, and activity against H4 receptors.• The relatively lower specificity of the first-generation AH for the H1 AH are considered a limitation to their use. However, unlike other conditions where histamine plays a role, these features in urticaia patients are intimately associated with the nature of the disease, as sleep deprivation because of nighttime itch can be the real cause underlying them. Thus, sedating AH may turn out to have a paradoxal positive effect on the state of alertness.• Sedation, somnolence, and performance impairment with H• The sedative effects on initial intake are short-lived and gradually wear off by day 4 of treatment with first- generation AH.• The alternative to sedation would be the prescription of corticosteroids and other more hazardous and expensive drugs like cyclosporine, methotrexate, sulfasalazine, intravenous gamma globulin, dapsone, hydroxychloroquine, colchicine, and cytoxan, with the ensuing deleterious risks in the long run.1 receptors. Thus, Allen Kaplan advocates that up to 4 × 50 mg of hydroxyzine and diphenhydramine should be applied to increase the chances of success if doubling the standard dose of nsAH fails. This leads us to a total daily dose of 200 mg of these first-generations drugs, which is way too high compared with the 20-mg daily dose of cetirizine, if a speculative approximation of 10 mg of cetirizine corresponding to 30 mg of hydroxyzine (used in comparative studies) is assumed. The question emerges whether increasing the dose of nsAH high enough would prove helpful in controlling the urticaria symptoms. Studies with fexofenadine failed to show that increasing the dose from 60 to 240 mg makes a meaningful difference[This sound logic is backed up by decades of first-generation AH prescriptions, indicating that really high doses of some of the older drugs can be used to saturate all skin Hifference. Howeverifference a threefifference and a fo1 receptor antagonists desloratadine and levocetirizine of up to fourfold could increase the success rate of treatment of chronic urticaria and improve the health-related quality of life without compromising safety[Led by the plausible statement of Allen Kaplan that defining the level of refractoriness to antihistamines is the key to effective therapy, we carried out a study to check if a dose increase of the modern second-generation Hng safety. We recrng safety. The resA common belief is that patients might respond to one or another AH depending on their specific phenotype, rather than on the pharmacological characteristics of the different formulations. This type of reasoning prompts switching from one drug to another and also simultaneous intake of different AH. In a preceding study we compared the ability of increasing doses of levocetirizine and desloratadine to suppress the effects of intradermal histamine in healthy men. The resThe relationship between subjective perceptions like discomfort because of urticaria, sedation/somnolence, and disease-specific quality of life is rather complex. On the basis of the relief of the discomfort because of urticaria, we could differentiate "low-dose responders," approximately 10% of the patients who responded to the standard dose of 5 mg, "nonresponders," about 15% who did not experience any improvement even at the highest 20-mg dose, and "high-dose responders," ranging in the gray zone between those 2 extremes making up 75% of the patients. In the 1 AH in chronic urticaria could be given an ultimate answer only if specifically designed comparative studies are carried out.The quoted study and the experience we have acquired during the past years in the treatment of chronic urticaria give us ground to believe that high enough doses of nsAH give the best chance of achieving control of the urticaria symptoms before resorting to other potentially more hazardous approaches. The higher doses of these drugs may also be associated with additional anti-inflammatory effects that may


Putative early targets of these ions, both of which are linked to increased reactive oxygen species (ROS) generation, are mitochondria and the cytosolic enzyme, NADPH oxidase (NOX). The present study uses primary cortical neuronal cultures to examine respective contributions of mitochondria and NOX to ROS generation in response to Ca2+ or Zn2+ loading. Induction of rapid cytosolic accumulation of either Ca2+ (via NMDA exposure) or Zn2+ (via Zn2+/Pyrithione exposure in 0 Ca2+) caused sharp cytosolic rises in these ions, as well as a strong and rapid increase in ROS generation. Inhibition of NOX activation significantly reduced the Ca2+-induced ROS production with little effect on the Zn2+- triggered ROS generation. Conversely, dissipation of the mitochondrial electrochemical gradient increased the cytosolic Ca2+ or Zn2+ rises caused by these exposures, consistent with inhibition of mitochondrial uptake of these ions. However, such disruption of mitochondrial function markedly suppressed the Zn2+-triggered ROS, while partially attenuating the Ca2+-triggered ROS. Furthermore, block of the mitochondrial Ca2+ uniporter (MCU), through which Zn2+ as well as Ca2+ can enter the mitochondrial matrix, substantially diminished Zn2+ triggered ROS production, suggesting that the ROS generation occurs specifically in response to Zn2+ entry into mitochondria. Finally, in the presence of the sulfhydryl-oxidizing agent 2,2'-dithiodipyridine, which impairs Zn2+ binding to cytosolic metalloproteins, far lower Zn2+ exposures were able to induce mitochondrial Zn2+ uptake and consequent ROS generation. Thus, whereas rapid acute accumulation of Zn2+ and Ca2+ each can trigger injurious ROS generation, Zn2+ entry into mitochondria via the MCU may do so with particular potency. This may be of particular relevance to conditions like ischemia in which cytosolic Zn2+ buffering is impaired due to acidosis and oxidative stress.Excessive “excitotoxic” accumulation of Ca Mitochondria, which can take up and buffer large cytosolic Ca2+ loads, have long been considered to be critical targets of the Ca2+ loads, with a number of studies finding NMDA receptor mediated Ca2+ rises to result in release of ROS from the mitochondria into the cytosol i and [Ca2+]i imaging, cultures were loaded in the dark with 5 μM of either the relatively low-affinity Zn2+ indicator, Newport Green diacetate , the high affinity Zn2+ indicator, FluoZin-3 , or the low-affinity Ca2+ indicator, Fura-2FF  in HSS containing 0.2 % Pluronic F-127 and 1.5 % dimethlyl sulfoxide (DMSO) for 30 min at 25°C, then washed into HSS for Ca2+ imaging or Ca2+-free HSS for Zn2+ imaging, and kept in the dark for an additional 30 min. For Newport Green and FluoZin-3, the green fluorescence cube was used, while for Fura-2FF, excitation was via the filter wheel (340 and 380 nm filters), and emission via the Fura-2 filter cube. Experiments were carried out in a static bath of HSS or Ca2+-free HSS and drugs were added to the buffer for pre-treatments as indicated. For intracellular Zn2+, fluorescence measurements for each cell (Fx) were normalized to the average fluorescence intensity for that cell during the first 10 min of the experiment (F0). Changes in intracellular Ca2+ were assessed as the ratio of the emission intensity (at 510 nm) when excited at 340 nm to that upon excitation at 380 nm (“340/380 emission ratio”).  For [Zn2XRos (MTR-CMH2)  for 30 min at 37°C in the dark, and then switched to 0 Ca2+ HCSS and 10 µM pyrithione for the imaging experiment using the confocal. An image was acquired prior to Zn2+ treatment, immediately after a 5 min exposure to 300 µM Zn2+/pyrithione, and again 5 min after washout. Images were scanned sequentially with a 488 nm line Argon laser for MitoTracker Green followed by a 543 nm line Green HeNe laser for MTR-CMH2.  Confocal microscopy of live dissociated cell culture was performed using an Olympus IX70 microscope equipped with a Bio-Rad Radiance 2000 laser scanning system. Images were collected with a 100 X oil-immersion objective using Zeiss LaserSharp 2000 software. Briefly, cells were loaded with 200 nM MitoTracker green  and 500 nM MitoTracker Red CM-H Traces (with the exception of


Moreover, influx of Ca2+ through L-type Ca2+ channels (LTCCs) activates Ca2+ signaling pathways, which in turn contribute to numerous cellular processes. Although LTCCs are expressed in undifferentiated cells, very little is known about its contributions to the transcriptional regulation of mesodermal and cardiac genes. This study aimed to examine the contribution of LTCCs and the effect of nifedipine on the commitment of pluripotent stem cells toward the cardiac lineage in vitro. The murine embryonic stem  and induced pluripotent stem  cells were differentiated into enhanced green fluorescence protein (EGFP) expressing spontaneously beating cardiomyocytes (CMs). Early treatment of differentiating cells with 10 µM nifedipine led to a significant inhibition of the cardiac mesoderm formation and cardiac lineage commitment as revealed by gene regulation analysis. This was accompanied by the inhibition of spontaneously occurring Ca2+ transient and reduction of LTCCs current density (ICaL) of differentiated CMs. In addition, nifedipine treatment instigated a pronounced delay of the spontaneous beating embryoid body (EB) and led to a poor surface localization of L-type Ca2+ channel α1C (CaV1.2) subunits. Contrary late incubation of pluripotent stem cells with nifedipine was without any impact on the differentiation process and did not affect the derived CMs function. Our data indicate that nifedipine blocks the determined path of pluripotent stem cells to cardiomyogenesis by inhibition of mesodermal commitment at early stages of differentiation, thus the proper upkeep Ca2+ concentration and pathways are essentially required for cardiac gene expression, differentiation and function.Dihydropyridines (DHP), which nifedipine is a member of, preferentially block Ca The LTCCs, like other membrane channels and receptors, is a heteromultimetric complex, which is also connected with signaling molecules, enzymes, or proteins 1 subunit of the L-type Ca2+ channel and prevent Ca2+ influx, therefore reduce the cytosolic Ca2+ concentration and in turn reduce excitation/contraction coupling.Although the mechanisms controlling the temporal aspects of mesoderm induction are poorly understood, both the sarcolemmal LTCCs and Ca2+ through LTCCs is the major boost mechanism for rise of intracellular Ca2+ levels. This mechanism is accomplished via activation of the ryanodine receptors, localized in the sarcoplasmic reticulum (SR), which in turn induces Ca2+ release from SR 2+ binding to the myofilament regulatory protein, known as troponin C, which then switches on the contractile elements for CMs contraction. In embryonic CMs, mainly transarcolemmal Ca2+ influx via LTCCs modulates the intracellular Ca2+ concentration [Ca2+]i during systole and diastole 2+ handling facilitators are structurally and functionally underdeveloped in embryonic CMs In adult CMs, the influx of Ca2+ channel subtypes have been shown to allow modulation of intracellular Ca2+ signals capable to influence cellular physiology in terms of proliferation, apoptosis, signal transduction and membrane potential 2+ signaling resulted in inhibition of mRNA expression and activity of cardiac-specific genes 2+ influx through LTCCs, especially at a very early stage of cell fate specification and differentiation may be of importance for activation and regulation of the Ca2+-dependent cardiac differentiation program.In early developmental stages of embryonic CMs, voltage-gated Ca2+ influx as a regulatory key event in cardiac fate determination, providing a significant improvement in our mechanistic understanding of the critical role of Ca2+ signals/pathways plays in cardiomyogenesis.Using the well established in vitro model system of ES and iPS cells, here we provide by molecular, histochemical and electrophysiological approaches, some preliminary evidence of the mechanism by which LTCCs blockage represses differentiation of mES cell-derived CMs. This study was also conducted to unravel the effects of CaWe have used the conventional murine ES cell line D3 (clone α-pig44) For detailed design protocol, please see Supplementary materials. Briefly, saturating concentration of Nifedipine (10 µM), Verapamil (10 µM) or (−) BayK8644  was added to undifferentiated cells (day 0) and after EB formation at different days of culture. Nifedipine was thoroughly washed out 24 hours before analysis.Three independent total RNA samples were extracted from EBs at day 4 and beating clusters of EBs at day 12 of differentiation using the TRIzol reagent. The characteristics of these samples are summarized in Biotin-labeled cRNAs were prepared using the Ambion® Illumina RNA amplification kit (Ambion Europe), 1.5 µg was hybridized to Sentrix® whole genome bead chips  carrying 45281 probe sets and scanned on the Illumina® BeadStation 500x. Data analysis procedure and statistical methods were performed as previously described For reverse-transcription (RT) polymerase chain reaction (PCR) and real-time PCR analysis, total RNA was isolated from undifferentiated cells (day 0) or EBs at day 2–5, 8 and day 12 contracting areas (CAs) using the RNeasy mini kit  or TRIzol (Invitrogen) and analyzed as previously described 5 cells/ml in PBS and then analyzed with the FACScan flow cytometer . The emitted fluorescence of GFP was measured at 530 nm (FITC band pass filter) and analyses were performed using the Win MDI 2.8 software.For flow cytometric (FC) analysis, EBs were washed with PBS and then dissociated to a single cell suspension by trypsin treatment (120 µl of trypsin/EDTA solution) for 2–3 min. Then, 1 ml of DMEM +20% FCS was added to the single-cell suspension. After centrifugation (1000 rpm) for 5 min, cells were resuspended to a concentration of 5 × 10Immunofluorescence was carried out using standard techniques. Detailed methodology is described in Supplementary materials. For analysis the Zeiss AxioVision 4.5 software package  was used.To characterize the electrophysiological properties of ES cell-derived CMs cultured under nifedipine-treated conditions, we used the whole cell configuration of the patch-clamp technique 2+ imaging was performed, using the cell membrane-permeant Ca2+ indicator fura-2AM  according to standard protocols . Intracellular Ca2+ were measured using an imaging system from TILL-Photonics  and the emission data as well as the method used to access sarcoplasmic reticulum (SR) Ca2+ content, were analyzed as described in Supplementary materials.Single-cell Cat test for grouped data (in case of two groups) or one-way analysis of variance (in case of multiple groups). A P-value of <0.05 was considered statistically significant.Data are presented as mean±SEM. Statistical analysis was performed using Student’s V1.2 family)). After 7 days in culture, enhanced Green Fluorescent Protein (EGFP)-positive clusters of differentiated CMs could be detected in EBs generated under control conditions , nifedipine, verapamil (LTCCs blockers) or BayK8644  in nifedipine-treated cultures when added at day 0 and to 40.4±11% (P<0.01) at day 2 of differentiation, whereas BayK8644 treatment was without significant effect as compared to control condition  and EGTA , the number of spontaneously beating EBs was significantly decreasing (data not shown). Similar results were obtained in Ca2+-free condition, providing strong evidence of the role of both extracellular and intracellular Ca2+ sources for cardiac differentiation in mammalian cells.Moreover, in the presence of BAPTA-AM  revealed that 47 probe sets (1.03%) at day 4 and 192 probe sets (4.2%) at day 12 of differentiation significantly differ in their expression levels between control and nifedipine-treated cells. Log ratio analysis revealed significant differences  of genes involved in regulation of cardiomyocytes progenitor and development (data not shown), confirming the effect of nifedipine on ES cells during differentiation towards CMs.To compare the morphological characteristics of the differentiated CMs generated under nifedipine-treated conditions, we performed immunostaining for different cardiac markers. The analysis of EGFP-positive cells confirmed the above mentioned reduction of CMs by nifedipine . Moreove1c), CACNAB2 (β2) and CACNA2D2 (α2δ2)CACNA1C  with higher expression at day 12. We also found that the cardiac markers α-MHC or Myh6, ANF or Nppa, MLC2v or Myl2, GATA4 and Nkx2.5 were lower expressed in cells generated under nifedipine-treated cultures at day 12 of ES and iPS differentiation . In control CMs (n = 32) and in CMs derived from nifedipine-treated group (n = 21) we could measured APs of all 3 major cardiomyocytes subtypes. These could be classified as nodal-like (9.3% vs. 9.5%), embryonic atrial-like (12.50% vs. 14.2%) and ventricular-like (78.1% vs. 76.2%) APs , left, rAs critical determinant of normal CM function is the intact response to hormones and transmitters of the central nervous system, we also assessed the functional integrity of the cells. In ES cell-derived CMs generated under control  and nifeFor a detailed analysis of the functional expression of the main voltage-gated ion channels, which control the electrical impulses in the heart and to evaluate the side effect of nifedipine, whole-cell voltage-clamp experiments were performed in cells generated under nifedipine at day 4 and 12 of ES cells differentiation.INa was not different between both untreated and nifedipine-treated groups  between both groups at day 4  and 12  of differentiation. density  was simi+ currents. Peak currents (Ipeak) and late currents (Isus) were measured as described in methods S1 + current (I/V curves) of Ipeak (Isus (data not shown) was not significantly different. At both day 4 and 12 of differentiation, Ipeak and Isus densities of cells generated under nifedipine were similar to control groups. Control experiments to tests for intact pharmacology of INa and Ik of CMs generated in the presence of nifedipine revealed no acute effect of nifedipine on INa density an at maximum outward Ik. However TTX (1 µM), a powerful and specific voltage-gated Na+ channel blocker and TEA (10 mM), a non-selective K+ channel blocker significantly reduced current density of INa  in control CMs to 5.2±0.69 pA/pF  in CMs generated under nifedipine treatment at day 12 of differentiation. As also shown in ICaL density during differentiation with significance difference between untreated and nifedipine-treated CMs at day 8  and 12  of differentiation, suggesting the maturation of ICaL density during differentiation. Additionally, the mean I/V relationship at the maximum of ICaL was decreased by approximately 75% and shifted the maximum peak of I/V curve from 0 mV to +10 mV  was more positive in CMs from nifedipine group   as well as the channel availability  and slow (τs) phase of ICaL inactivation revealed that τf (P<0.05). However, nifedipine treatment did not significantly affect τs  (1C subunit (CaV1.2) in CMs derived from untreated  . In addintreated  and nifentreated  groups a2+ transients are important events in the regulation of contraction and relaxation of CMs, we, therefore, determine the consequences on overall Ca2+ homeostasis by investigating the Ca2+ transients of single CMs at different time points of differentiation. Spontaneously evoked Ca2+ transients of shorter amplitude were detected in CMs generated under nifedipine-treatment at day 12 of differentiation  on to flura-2 AM loaded CMs. As can be observed in 2+ from the intracellular stores, resulting in a high amplitude caffeine-induced Ca2+ transient in control (n = 11) as well as in nifedipine-treated (n = 7) CMs (P<0.05). The maximum amplitude of caffeine-induced Ca2+ transient  an heterogeneous sarcolemmal surface expression of the α1C subunit, (ii) a significant reduction of ICaL density at late developmental stages, and (iii) a prolongation of fast inactivation time constant of ICaL. The inhibition of ICaL density is possibly mediated by downregulation of surface expression of the channels by nifedipine.In the remaining CMs derived from nifedipine-treated group, the cell functions were altered, i.e. reduction of AP and Ca2+ influx in different cell types, but the exact molecular details that underlie this regulation are still unclear. Previous studies have shown that Ca2+ entering through LTCCs can diffuse to the cell nucleus and activate nuclear Ca2+-dependent enzymes 2+-calmodulin-dependent protein kinase II (CaMKII) 2+]i into corresponding levels of kinase activity and regulate the activity of transcription factors and co-regulators In fact, activation of LTCCs can lead to the activation of the transcription factors such as CRE-binding protein CREB, GATA-4, Nkx2.5 MEF and NFAT 2+ signalling is caused by altered expression of Ca2+-depending proteins, among these is the reduced LTCCs expression, activity and density Cacna1c) expression was apparently normal in the remaining CMs generated under nifedipine-treatment, the alteration of function is presumably explained by deficits in [Ca2+]i and/or Ca2+ sparklets activity due to the inhibition of Ca2+ influx via LTCCs 2+-free medium, in the presence of Ca2+ chelators BAPTA and EGTA, confirming that Ca2+ ions are at least partially involved in maintenance of normal differentiation process of ES cells toward CMs in vitro. This observation is in accordance to previous studies demonstrating that intracellular Ca2+ level play an important role ahead in embryogenesis in the specification and commitment steps of the cardiac precursor cell population In addition, impaired CaAs additional prove for an alteration of the transcription process by LTCCs blockage, we found cell proliferation and apoptosis unaltered. Likewise, previous studies demonstrated that nifedipine inhibits rather than induces cell apoptosis of CMs 2+]i2+-activated K channels which has been shown to drives the fate of pluripotent cells toward cardiomyocytes specification The finding of remaining but functionally altered CMs obtained despite LTCCs blockage could be attributed either to the combined input of low transcription factors, which might provide sufficient information to activate the cardiac program, or to a combined unknown compensatory mechanism + and several types of K+ channels that had previously been reported + and K+ currents at early and late stages of differentiation. The significant increase of both Na+ and K+ current densities from day 4 to 12 in CMs generated under both culture conditions reflects an ongoing maturation process.To exclude unspecific side effects of nifedipine on other channels such as NaICaL in whole cell patch clamp experiments. We assume that the minor expression level of LTCCs in early stage of differentiated cells and the presumably small current flow at that level of differentiation may account for these findings. To further address the functional roles of ion channels in ES or iPS cells, specific knockdown of Ca2+ channels with interfering RNA during differentiation into CMs could be used in future studies.Despite the clear expression of LTCCs protein in undifferentiated ES cells, we were not able to measure stable 2+ influx pathways such as the Na+/Ca2+ exchanger shown to be highly expressed, functionally active, and working in the forward and the reverse mode in early stage CMs, to the uphold of the cardiac differentiation program, cannot be excluded Considering the complexity of the mechanisms (e.g. genes interactions) involved in stem cell differentiation, to attribute these impacts  solely to nifedipine or LTCCs activities would be an exaggeration. A potential contribution of some CaICaL allows sufficient Ca2+ entry to induce a cardiac directed differentiation program. This study led us to identify a not previously described critical importance function of LTCCs and nifedipine effect at the level of mesoderm specification for the establishment of the cardiac lineage using ES or iPS cells. These informations could provide insights into the signaling pathways involving in the establishment of the cardiac lineage from human ES/iPS cells.These results raise the possibility that in the earliest stages of cardiac differentiation Figure S1(A) Representative eGFP+ CMs obtained from dissociated EBs cultured under control conditions or in the presence of nifedipine or BayK8644. (B) eGFP-positive cells (CMs) at day 12 of differentiation generated under nifedipine- and BayK8644-treatment. CMs were manually counted and are expressed as a percentage of total CMs generated under control condition (C) The yield of CMs generated under control and nifedipine-treated conditions. Beating EBs were dissected, enzymatically dissociated and cultured on fibronectin- or gelatine-coated plates. Thereafter, cells were staining with nuclei dye Hoechst 33342 and CMs were investigated for α-actinin by means of immunocytochemistry. Alpha-actinin positive cells are represented as a percentage of total Hoechst 33432-labelled (positive nuclei) cells. (D)Time course of the incidence of ES and iPS cell-derived contracting EBs generated in the absence (control) or presence of nifedipine, verapamil or BayK8644. The mean±SEM of the percentage of EBs with contracting areas during differentiation is depicted. (E) Percentage of CMs  obtained by FACS analysis at day 12 of differentiation. Results are reported as the means±SEM (n = 3). * denote significant differences to control. Scale bars: 20 µm.(TIF)Click here for additional data file.Figure S2(A) Hierarchical clustering and (B) Principal component analysis of variable genes expression during induction of CMs under control and nifedipine-treated conditions at day 4 and 12 of differentiation. (C–D) Proliferation and viability assay of untreated and nifedipine-treated ES cells. Nifedipine did not alter cell proliferation (C) or viability (D) measured after 4 and 12 days of differentiation. A total of 3 independent experiments were studied for each time point .(TIF)Click here for additional data file.Figure S32+ transients obtained from control and nifedipine-treated CMs.Representative Ca (A) Developmental changes of spontaneous Ca2+ transients of cells generated from EBs at day 2, 4, 6, 8, and 10 of differentiation. Note the time-dependent increase of the Ca2+ transients’ amplitude of cell generated under control and nifedipine-treated conditions. (B) Effect of nifedipine on spontaneaous Ca2+ transients during differentiation. 10 µM Nifedipine was applied at days 0, 2, 4, 6, 8, and 10 during the differentiation process. Thereafter cells were isolated on day 11 or 12 and measured 24 to 48 h later by Ca2+ imaging.  Acute effect of nifedipine on spontaneous Ca2+ transients. Representative tracings of spontaneous Ca2+ transients in untreated (C) and nifedipine-treated (D) cells before (control) and after application of nifedipine (+nifedipine).(TIF)Click here for additional data file.Table S1List of samples used for global gene expression profiling.(TIF)Click here for additional data file.Table S2Primer sequences used for normal and quantitative RT-PCR analysis of total RNA isolated from ES cell-derived CMs cultured under control and nifedipine-treated conditions. Abbreviations:ANF/Nppa, natriuretic peptide precursor A; ES cell, embryonic stem cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; α-MHC/Myh6, myosin heavy chain 6, MLC2v/Myl2, myosin light chain 2 ventricular transcripts; Myocd, myocardin; Mest, mesoderm specific transcript; Mesp1, mesoderm posterior 1; CACNA1C, calcium channel, voltage-dependent, L type, alpha 1C subunit; KCNH2, potassium voltage-gated channel subfamily H member 2; SCN5A, sodium channel, voltage-gated, type V, alpha subunit; PECAM-1, platelet/endothelial cell adhesion molecule 1; AFP, alpha fetoprotein; NF-H, neurofilament H; aSMA, alpha-smooth muscle actin.(TIF)Click here for additional data file.Methods S1(DOC)Click here for additional data file.Video S1Representative EB showing EGFP-positive CMs differentiated under control conditions.(MPG)Click here for additional data file.Video S2After nifedipine treatment. The videos show the 3D structure of the entire EB.(MPG)Click here for additional data file.


This link was confirmed in platelets stimulated with the physiological agonist, thrombin, and experiments were performed to examine how Ca2+ removal by the NCX modulates platelet dense granule secretion. In cells loaded with the near-membrane indicator FFP-18, thrombin stimulation was observed to elicit an NCX-dependent accumulation of Ca2+ in a pericellular region around the platelets. To test whether this pericellular Ca2+ accumulation might be responsible for the influence of NCXs over platelet function, platelets were exposed to fast Ca2+ chelators or had their glycocalyx removed. Both manipulations of the pericellular Ca2+ rise reduced thrombin-evoked Ca2+ signals and dense granule secretion. Blocking Ca2+-permeable ion channels had a similar effect, suggesting that Ca2+ exported into the pericellular region is able to recycle back into the platelet cytosol. Single cell imaging with extracellular Fluo-4 indicated that thrombin-evoked rises in extracellular [Ca2+] occurred within the boundary described by the cell surface, suggesting their presence within the open canalicular system (OCS). FFP-18 fluorescence was similarly distributed. These data suggest that upon thrombin stimulation, NCX activity creates a rise in [Ca2+] within the pericellular region of the platelet from where it recycles back into the platelet cytosol, acting to both accelerate dense granule secretion and maintain the initial rise in cytosolic [Ca2+].We have previously demonstrated that Na Using this information we have been able to refine our understanding of how platelet Ca2+ signaling is affected by a variety of experimental interventions.The importance of rises in cytosolic Ca+/Ca2+ exchanger (NCX) influences platelet Ca2+ signaling and thus platelet function. NCX3 has been shown to be the predominant isoform of this exchanger expressed in human platelets in both a proteomic screen of the platelet plasma membrane , we and others have demonstrated that the NCX plays a key role in regulating rises in [Ca2+]cyt evoked by a variety of agonists , but instead influenced the agonist-evoked rise in [Ca2+]cyt at a point downstream of the activation of these ion channels. By analysis of a number of the factors that influence platelet Ca2+ signal generation, it was concluded that the NCX exerted its influence by regulating the rate of autocoid secretion from dense granules  or by addition of releasate from uninhibited, activated platelets  creates a pericellular Ca2+ source from which Ca2+ is able to recycle back into the platelet cytosol down its concentration gradient through Ca2+-permeable ion channels. Furthermore, we demonstrate that experimental interference with the thrombin-evoked pericellular Ca2+ signal inhibits both platelet dense granule secretion and thrombin-evoked rises in [Ca2+]cyt. These observations provide new insights into how the NCX influences platelet Ca2+ signaling and dense granule secretion in human platelets.Although these results provided an interesting insight into the role that NCX3 plays in regulating human platelet function, our earlier experiments principally used nonphysiological agonists to isolate specific CaThis study was approved by the University of Cambridge Human Biology Research Ethics Committee. Blood was donated by healthy, drug-free volunteers who gave written informed consent. The experiments conformed to the guidelines stated in the Declaration of Helsinki.+ salt was from Cambridge Biosciences . FFP-18 K+ salt , Fura-2/AM and SBFI/AM were from TEFlabs Inc. . Thrombin was from Merck Chemicals . Fluo-5N/AM and K+ salts of BAPTA and Fluo-4 were from Invitrogen . KB-R7943, MRS-1845, and SN-6 were from Tocris Bioscience . Apyrase, luciferin–luciferase, NMDG, and RGDS peptide were from Sigma Aldrich . 5′-Iodo-resiniferatoxin was from LC laboratories . All other reagents were of analytical grade.5-5′-Dimethyl-BAPTA K−1 sodium citrate, 78 mmol L−1 citric acid, and 111 mmol L−1 D-glucose) and platelet-rich plasma (PRP) prepared by centrifugation for 5 min at 700 g, before aspirin (100 μmol L−1) and apyrase (40 μg mL−1) were added.Blood was collected by venepuncture and mixed with one-sixth volume of acid citrate dextrose anticoagulant , 10 mmol L−1d-glucose, 5 mmol L−1 KCl, 1 mmol L−1 MgSO4, pH 7.45) supplemented with 0.1% w/v bovine serum albumin, 200 μmol L−1 CaCl2 and 40 μg mL−1 apyrase (supplemented HBS). Fluorescence was recorded from 1 mL stirred aliquots of platelet suspension at 37°C using a Cairn Research Spectrophotometer  with excitation at 340 and 380 nm and emission at 515 nm. Changes in [Ca2+]cyt were monitored using the 340/380 nm fluorescence ratio and calibrated according to the method of Grynkiewicz et al.  was mixed with 10% pluronic F-127 dissolved in dimethylsulfoxide (DMSO) to give a stock solution of 5 mmol L−1. This was added to washed platelet suspensions to give a final concentration of 10 μmol L−1 SBFI/AM. After incubation at 37°C for 40 min, 10% v/v ACD was added and the cells were recollected by centrifugation in a microcentrifuge at 8000 g for 30 sec. The platelets were then resuspended in supplemented HBS and SBFI fluorescence measurements were made as for Fura-2 above. Changes in [Na+]cyt were monitored using the SBFI 340/380 nm fluorescence ratio. We have previously described a small quenching effect of KB-R7943 on SBFI fluorescence . This was then subject to nonlinear regression to fit to a one-phase association equation (R2 between 0.96 and 0.99). The derived equation was then used to calibrate the Fluo-4 data.In some experiments, Fluo-4 data were calibrated by measuring the fluorescence of a cell-free aliquot of supplemented HBS to which 2.5 μmol L2+ to the extracellular medium in the presence of 300 μmol L−1 extracellular Ca2+ was monitored by addition of 5 μmol L−1 Rhod-5N to washed platelet suspensions immediately prior to the start of experiments. Fluorescence was monitored as for Fura-2 but with excitation at 550 nm and collecting emitted light of wavelengths between 570 and 640 nm.The release of Ca2+]peri was monitored by treating washed platelets with 5 μmol L−1 FFP-18 salt for 5 min at room temperature. Cells were then separated into 1-mL aliquots to which 250 μL of ACD was added. Excess dye was removed by centrifugation for 30 sec at 8000 g. The supernatant was removed and the pelleted cells were resuspended in supplemented HBS. Fluorescence was recorded as for Fura-2 above. Changes in [Ca2+]peri were monitored using the 340/380 nm fluorescence ratio. FFP-18 loading was designed to specifically label the extracellular membrane with the indicator (see Results).cyt, [Ca2+]ext, [Ca2+]peri, [Ca2+]st, and [Na+]cyt were quantified by integration of the change in fluorescence records from basal with respect to time for 3 min after thrombin addition unless stated.[Ca2+ removal across the plasma membrane of single platelets was recorded using an FV300 laser-scanning confocal microscope  with a PLAPON 60× oil immersion objective and a 300-μm confocal aperture. Chambered coverslips (Nunc) were coated with collagen-related peptide  or fibrinogen (10 μg mL−1) overnight at 4°C. Slides were washed with Ca2+-free Tyrodes solution and mounted on the microscope stage. Platelets at a density of 1 × 108 mL−1 in a Ca2+-free Tyrodes solution containing 2.5 μmol L−1 Fluo-4 were pipetted into the chambered coverslip and allowed to adhere to the substrate for 5 min. Images were recorded at a frequency of 0.9 Hz for 5 min with excitation at 488 nm and emission at 510–530 nm.Net Ca−1) and SN-6 (50 μmol L−1) had no detectible effect on the assay.Luciferin–luciferase  was added to 1.5 mL stirred aliquots of platelets prior to the start of experiments. ATP secretion was measured by monitoring the light emitted from the sample in a light-protected cuvette holder connected to a high gain photomultiplier tube. Measurements of ATP secretion were taken as the integral over basal light levels (average light emitted 30 sec before stimulation) for 20 sec after stimulation. KB-R7943 (50 μmol Ln) indicated. Analysis of statistical significance was performed using Student's t-test. P < 0.05 was considered significant.Values stated are mean ± SEM of the number of observations  in human platelets without directly affecting the Ca2+ signaling apparatus of these cells (Harper and Sage 2+]cyt in the presence of extracellular Ca2+ were reduced to 64.7 ± 12.6% or 53.2 ± 10.7% of control after treatment with SN-6 (50 μmol L−1) or KB-R7943 (50 μmol L−1), respectively  on the thrombin-evoked change in [Ca2+]cyt in the absence of extracellular Ca2+ were investigated in the presence and absence of valinomycin (3 μmol L−1), which clamps the platelet membrane potential near the reversal potential of K+  and the Na+/H+ exchanger (NHE), both of which have previously been suggested to play a role in platelet Ca2+ signaling (Siffert and Akkerman −1) evoked the expected rise in SBFI fluorescence  inhibited agonist-evoked Ca2+ signaling as well as dense granule secretion  or KB-R7943 (50 μmol L−1) reduced the extent of dense granule secretion during the first 20 sec after thrombin addition . The cause of this difference is currently unknown; it could be due to either the loss of Na+ or the presence of NMDG in the extracellular medium exerting an effect on either the activity of the luciferin–luciferase reporter or the ATP-scavenging apyrase. Experiments to distinguish these possibilities are needed, but are beyond the scope of this current work. Previous work by other investigators has shown that removal of extracellular Na+ inhibits platelet dense granule secretion , the P2X1 antagonist Ro-0437626 (25 μmol L−1), the P2Y1 antagonist 2-methylthioadenosine 5′-monophosphate (100 μmol L−1), and the P2Y12 antagonist MRS-2179 (75 μmol L−1) inhibited thrombin-evoked rises in [Ca2+]cyt  or removal of extracellular Na+  consistently abolished this response  receptors, sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) or other Ca2+ removal pathways that complicate the interpretation of the effects of these agents. Therefore, we examined the effects on thrombin-evoked Ca2+ signaling of MRS-1845 or 5′-Iodo-resiniferatoxin . MRS-1845 (30 μmol L−1), 5′-Iodo-RTX (20 μmol L−1) or both in combination reduced thrombin-evoked rises in [Ca2+]cyt in the absence of extracellular Ca2+ to 76.2 ± 6.0%, 70.1 ± 5.0% or 54.4 ± 3.0% of control, respectively . As the K+ salts of the chelators are cell impermeant, the effects on pericellular Ca2+ must cause the effects on [Ca2+]cyt. BAPTA chelators have been shown to have nonspecific effects on K+ channels when loaded intracellularly . These data therefore suggest that intracellular signal transduction leading to Ca2+ permeable ion channel activation by thrombin is normal in the presence of BAPTA, but extracellular interference with the Ca2+ flux leads to a reduction in the rise in [Ca2+]cyt.To further test whether a pericellular Cah et al.  utilizinh et al. ). When c2+ signal affects the thrombin-evoked fall in [Ca2+]st in an analogous manner to NCX inhibition or blockage of Ca2+-permeable ion channels in the plasma membrane  These results indicate that although Ca2+ release from the intracellular stores was not inhibited by chondroitinase treatment, Ca2+ that was removed from the cells, instead of staying close to the plasma membrane, rapidly diffused away into the bulk extracellular medium, as indicated by the greater rise in [Ca2+]ext . Addition of thrombin evoked pericellular Ca2+ signals at some point during the 5 min of observation .To further elucidate the presence and location of the pericellular Caion Fig. . Pericel2+ signals in fibrinogen-bound platelets are reported to rise almost instantaneously throughout the entire cell  and the background fluorescence . In contrast, the mean background fluorescence was not significantly different from that observed in the pericellular region around the stimulated cells (P < 0.05). Therefore, the pericellular Ca2+ signals appeared to be generated in a restricted volume within the platelet which was contiguous with the extracellular medium. Platelets contain a tubular invagination of their plasma membrane, the open canalicular system (OCS), which resembles the transverse-tubule system of striated muscle types  and measured 758 ± 39 nm in diameter (n = 17). Interestingly, the microdomains of high [Ca2+]peri in stimulated cells were found to be of an equivalent size . The agonist-evoked pericellular Ca2+ signals observed in the extracellular Fluo-4 experiments and the hotspots observed in the FFP-18 images were both observed in eccentric microdomains of the cells. One might conjecture that the cellular structure associated with the FFP-18 accumulation is likely to be responsible for the generation of the pericellular Ca2+ microdomain. As the DTS is the site of IP3-mediated Ca2+ release in platelets, the membrane complexes formed by the association of the OCS and DTS would provide a suitable cellular structure for the efficient creation of the pericellular Ca2+ signals observed in our experiments.The hotspots in the FFP-18 images were found to contain 23.2% ± 1.6% of the total cellular FFP-18 fluorescence  as Ca2+ leaves the cells. Any change in free [Ca2+] in any compartment inside the cell represents a small proportion of the Ca2+ moving into or out of that compartment, due to the ability of proteins and membranes to bind Ca2+. To estimate the total Ca2+ passing into or out of a given cellular compartment requires an estimate of the Ca2+-binding capacity of that compartment. We therefore used a minor modification in the methodology of Mogami et al. . In addition, [Ca2+]ext was measured in some experiments by addition of 2.5 μmol·mL−1 Fluo-4 K+ salt to the extracellular medium. In these experiments, there was no interference in the Fluo-4 signal from the Fluo-5N due to the extracellular dye being monitored at a significantly lower photomultiplier tube voltage, which means that changes in Fluo-5N signal were not detectable under these conditions.Platelets coloaded with Fluo-5N and either Fura-2, Fura-4F or Fura-FF were used to make measurements of ext as well as the rate of decline in both [Ca2+]cyt and [Ca2+]st over the final 3 min of the recordings. This timeframe was chosen as the store was largely depleted at this point, such that Ca2+ influx into the cytosol was not the dominant Ca2+ flux at this time, and the rates of change in [Ca2+]cyt and [Ca2+]ext are both well approximated by linear functions, as shown in Figure The Ca2+ flux out of the cytosol  was compared against the net rate of free Ca2+ decline in the cytosol using the following equation:The net CaKst was taken as 1039 and Vcyt was calculated as the difference between total platelet volume and Vst such that:Where Kcyt for the cells of each of the five donors loaded with the different cytosolic dyes. These data showed that Kcyt in the presence of Fura-2 was 18,311 ± 4064. This is significantly greater than the value obtained in cells loaded with Fura-4F , suggesting that this widely used indicator significantly adds to the Ca2+ buffering of platelets. Curiously, Kcyt in Fura-FF-loaded cells was not significantly different from that of cells loaded either of the other indicators . As the mean Kcyt obtained from Fura-FF-loaded cells is not lower than that observed with Fura-4F, the measurement of Kcyt from the medium-affinity dye is likely to be a reasonable initial estimate of the endogenous Ca2+-binding capacity of the platelet cytosol.Using this value we calculated 2+ dynamics observed in all areas of the cell compared to those observed in cells loaded with Fura-4F, with the rises in [Ca2+]cyt and [Ca2+]ext after addition of nigericin and SERCA inhibitors being reduced to 63.3 ± 6.9% or 19.9 ± 11.2% of control, respectively, while the corresponding fall in [Ca2+]st was reduced to 69.5 ± 6.8% of control .As can be observed in Figure 2+-binding capacities of the cytosol and stores obtained above are both noticeably high compared to previously described values in other cells  in the range 0.6–6 μmol L−1 . Although we have heavily buffered the extracellular medium with 1 mmol L−1 EGTA, the addition of further Ca2+ upon agonist stimulation will cause a small increase in the free Ca2+ concentration of the extracellular medium. We therefore used the MaxChelator program (http://maxchelator.stanford.edu) to calculate the increase in free [Ca2+]ext that would be expected in response to these agonist-evoked changes in total Ca2+.Our measurements of thrombin-evoked increase in extracellular Ca−1 CaCl2 to prevent platelet store depletion on resuspension, to this we added 1 mmol L−1 EGTA in all experiments which would chelate the vast majority of the free Ca2+ ions. When calibrating the Fluo-4 salt measurements of total [Ca2+]ext, the fluorescence taken from a platelet-free sample of supplemented HBS was taken as the baseline fluorescence. We then created a calibration curve through the addition of Ca2+ to the medium to give known total [Ca2+]ext. This calibration curve was then used in a linear regression fit to calibrate the additional Ca2+ added to the medium following agonist stimulation. As shown in Figure −1 . After thrombin stimulation for 10 sec, the measured [Ca2+]ext increased to 32.1 μmol L−1 . Utilizing the two chelators, two metal version of MaxChelator with the standard conditions of T = 37°C, pH = 7.4, Ionic concentration = 0.154N, EGTA = 0.001 mol L−1, Mg = 0.001 mol L−1, and ATP = 0 mol L−1, the calculated increase in total extracellular Ca2+ concentration would lead to an increase in free [Ca2+]ext of 2.9 nmol L−1. This is much smaller than the 31.1 nmol L−1 increase in the pericellular free Ca2+ concentration observed over the same time period, suggesting that our measurement is due to a genuine accumulation of Ca2+ specifically in the pericellular region, rather than being an artifact of a generalized increase in free Ca2+ concentration through the bulk extracellular medium.The HBS used in our experiments contained 200 μmol L2+ recycling back into the platelet through Ca2+-permeable ion channels there must be an electrochemical gradient created between the pericellular region and the cytosol. Given the platelet membrane potential does not significantly depolarise during thrombin stimulation  and KB-R7943-treated cells (3.365 μmol L−1 sec−1) were significantly different (P < 0.0001). Using the calculated volume of the intracellular stores (4.32 × 10−7 L) as well as Kst (1039), it was possible to use these data to estimate the total amount of Ca2+ released per second from the stores using the following equation:The above analysis fails to explain how KB-R7943 pretreatment causes a reduction in the initial spike in cyt of 693 nmol L−1 for the control cells and a rise of only 392 nmol L−1 for the KB-R7943-pretreated cells. These values are in reasonable agreement with the rises in [Ca2+]cyt measured in Fura-2-loaded cells  mechanism, allowing maximal Ca2+ release from the intracellular stores. Consistent with this possibility, research in murine cardiac myocytes has demonstrated that the NCX can play a role in ensuring synchronous Ca2+ release from dyadic release sites . Both of these compounds also markedly reduced the thrombin-evoked decrease in [Ca2+]st . Both chelators caused a marked reduction in the net Ca2+ release over the first 15 sec of the response to thrombin. The magnitude of inhibition of net thrombin-evoked Ca2+ release by BAPTA or DM-BAPTA loading appeared to be inversely correlated with the Kd of the buffer concerned, with DM-BAPTA having a lower effective Kd than BAPTA  As pointed out above, the cytosolic Na+ accumulation and Ca2+ efflux observed are only possible if the NCX is working in forward mode at near full capacity, (ii) Unlike cardiac myocytes, platelets are nonexcitable cells and so there is only a minimal membrane depolarization observable upon agonist stimulation  and OCS are intimately associated . This is consistent with previous work by Brass and Shattil  at the end of the recording period, suggesting that this might represent the total capacity of the additional extracellular Ca2+ buffer. The final calculated values are 137.5% and 129.5% of the measured values in KB-R7943- and DMSO-treated cells, respectively, which is similar to the 128.7% increase in the Fluo-4 signal observed in chondroitinase-treated cells. Further experiments will be needed to confirm these initial analyses, but the results are consistent with a role for the glycocalyx in buffering Ca2+ in the pericellular region.Interestingly, both KB-R7943- and DMSO-treated cells show a similar difference between the measured and calculated Ca2+ removal data may more accurately resemble the amount of Ca2+ translocating across the platelet plasma membrane than the removal measured using extracellular Fluo-4. As KB-R7943-treated cells show no observable pericellular signal, the difference in the calculated Ca2+ removed from DMSO- and KB-R7943-treated cells should represent a good estimate of the amount of Ca2+ responsible for generating the pericellular signal. As shown in Figure −1 during this time. As the OCS is continuous with the bulk extracellular medium, we assumed that the pericellular Ca2+ concentration would reflect the rate of Ca2+ removal as the bulk extracellular medium can rapidly exchange with the OCS. This assumption is supported by our experiments showing that EGTA addition to FFP-18-loaded cells suspended in high external Ca2+ results in a rapid fall in the FFP-18 fluorescence ratio to a new steady value (data not shown) suggesting that there is good exchange between these two compartments.The above results suggest that the calculated Ca2+ was introduced into the volume of the total OCS found in our samples  this would be expected to cause the total [Ca2+] of the OCS ([Ca2+]OCS) to increase by 446 μmol L−1. If we assume that the OCS contents are identical to the rest of the extracellular medium, we can again use the Maxchelator program to calculate the expected increase in free [Ca2+]OCS that increasing the total Ca2+ concentration from 200 μmol L−1 to 646 μmol L−1 would elicit . This indicates that the calculated rate of Ca2+ entry into the OCS would be expected to increase the free [Ca2+]OCS from 29 nmol L−1 to 197 nmol L−1. This fits reasonably well with our FFP-18 measurements of [Ca2+]peri which increase from a basal level of 91 nmol L−1 to 141 nmol L−1 after 30 sec of thrombin stimulation  all caused a similar inhibition in thrombin-evoked Ca2+ store depletion. A later effect appeared to be the direct maintenance of the [Ca2+]cyt at raised levels by the recycled Ca2+, creating the plateau phase observed after the initial spike. This interpretation is supported by quantitative analysis of calibrated data sets obtained from a single donor.Ca2+ recycling system in human platelets, there remain a number of questions about the cellular mechanisms underlying this system as well as its physiological relevance. One challenge to the model described above is the possibility that the Ca2+ accumulated in the pericellular space is not removed from the cytosol by the NCX but is the result of the exocytosis of Ca2+ stored in dense granules. Although at present we cannot definitively exclude this possibility, we favor the hypothesis that the NCX is the source of the Ca2+ accumulated pericellularly for the following reasons. First, the NCX inhibitor KB-R7943 abolished the creation of the pericellular Ca2+ signal, but only slowed and did not abolish dense granule secretion suggesting that dense granule secretion and the pericellular signal are not causally related. Second, previous work on platelets from Hermansky–Pudlak syndrome patients, whose platelets lack dense granules, has shown that the deficit in the thrombin-evoked Ca2+ signal can be reversed by the addition of exogenous adenine nucleotides  is less able to influence thrombin-evoked dense granule secretion and rises in [Ca2+]cyt than Ca2+ removed from the cytosol and then recycled back into the cell. In other cells in which a Ca2+ recycling system has been demonstrated, such as arterial smooth muscle cells  than a Ca2+ puff elicited by the opening of a small group of IP3 receptors  or strong  agonists, suggesting that some such mechanism must exist. Previous studies have demonstrated the presence of a localized Ca2+ gradient in single platelets that would be consistent with some structure controlling the spread of platelet Ca2+ signals  being intimately intertwined to form membrane complexes White . The OCSecretion . This mo2+-free media, so there remains a significant question as the physiological relevance of a pericellular recycling system. There is a need to overcome the difficulty of visualizing pericellular Ca2+ signals when platelets are suspended in extracellular Ca2+ concentrations that will saturate pericellular and extracellular Ca2+ indicators. Preliminary studies using the low-affinity Ca2+ indicator Rhod-5N have shown that it is possible to monitor changes in [Ca2+]ext in the presence of near-physiological extracellular Ca2+ concentrations and that events such as reduced thrombin-evoked Ca2+ signaling and dense granule secretion following NCX inhibition are observed under these conditions  reduces the both the initial rate and maximal extent of platelet aggregation , our data suggest that Ca2+ recycling plays a physiological role in platelet function.Finally, our data largely concern investigations conducted in Canditions . As NCX 2+ in the pericellular region of the platelet might provide a novel method for creating antithrombotic drugs. If this could be achieved specifically it might be possible to reduce platelet dense granule secretion and thus indirectly interfere with autocrine ADP signaling, a factor known to be important for thrombus growth (Nesbitt et al. 2+.Our data suggest that creating compounds that could disrupt the accumulation of Ca