Large Ca Research Articles

A human pluripotent stem cell model of catecholaminergic polymorphic ventricular tachycardia recapitulates patient-specific drug responses

ABSTRACTAlthough β-blockers can be used to eliminate stress-induced ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), this treatment is unsuccessful in ∼25% of cases. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from these patients have potential for use in investigating the phenomenon, but it remains unknown whether they can recapitulate patient-specific drug responses to β-blockers. This study assessed whether the inadequacy of β-blocker therapy in an individual can be observed in vitro using patient-derived CPVT iPSC-CMs. An individual with CPVT harboring a novel mutation in the type 2 cardiac ryanodine receptor (RyR2) was identified whose persistent ventricular arrhythmias during β-blockade with nadolol were abolished during flecainide treatment. iPSC-CMs generated from this patient and two control individuals expressed comparable levels of excitation-contraction genes, but assessment of the sarcoplasmic reticulum Ca2+ leak and load relationship revealed intracellular Ca2+ homeostasis was altered in the CPVT iPSC-CMs. β-adrenergic stimulation potentiated spontaneous Ca2+ waves and unduly frequent, large and prolonged Ca2+ sparks in CPVT compared with control iPSC-CMs, validating the disease phenotype. Pursuant to the patient's in vivo responses, nadolol treatment during β-adrenergic stimulation achieved negligible reduction of Ca2+ wave frequency and failed to rescue Ca2+ spark defects in CPVT iPSC-CMs. In contrast, flecainide reduced both frequency and amplitude of Ca2+ waves and restored the frequency, width and duration of Ca2+ sparks to baseline levels. By recapitulating the improved response of an individual with CPVT to flecainide compared with β-blocker therapy in vitro, these data provide new evidence that iPSC-CMs can capture basic components of patient-specific drug responses.

R- and S-terbutaline activate large conductance and Ca2 + dependent K+ (BKCa) channel through interacting with β2 and M receptor respectively

This study investigated the effect of the β2 receptor agonist terbutaline on the single channel activity of BKCa channel. The effects of racemate and two isomers of terbutaline were all assessed. β2 adrenoceptors were stably overexpressed on HEK293 cells by lentiviral transduction method and chicken BKCa channels were transiently expressed on normal HEK293 cell line or HEK293 cells overexpressing β2 receptors. Data showed that terbutaline significantly increased the single channel open probability of BKCa channel within 10min. The channel activating effects of terbutaline are stereoselective and mainly stay with the R-enantiomers. The opening probability of BKCa channel at 10min after drug application normalized to that just before drug application (Po10/Po0s) for R- and S-terbutaline were 7.85±3.20 and 1.06±0.45 respectively at 1μM concentration, corresponding to 28.37±9.96 and 2.68±1.09 at the higher concentration of 10μM. ICI 118551 blocked the effect of R- but not S-terbutaline (10μM), whereas atropine blocked the channel activating effects of S-terbutaline of higher concentration. In addition, the muscarinic receptor agonist carbachol increased the BKCa channel activity in an atropine-sensitive manner as an positive control experiment, which indicate the involvement of M receptor in the channel activating effect of S-terbutaline.

Reactive transport modelling of groundwater-bentonite interaction: Effects on exchangeable cations in an alternative buffer material in-situ test

Bentonite clays are regarded a promising material for engineered barrier systems for the encapsulation of hazardous wastes because of their low hydraulic permeability, swelling potential, ability to self-seal cracks in contact with water and their high sorption potential. SKB (Svensk Kärnbränslehantering) has been conducting long term field scale experiments on potential buffer materials at the Äspö Hard Rock Laboratory for radioactive waste disposal in Sweden.The Alternative Buffer Material (ABM) test examined buffer properties of eleven different clay materials under the influence of groundwater and at temperatures reaching up to 135 °C, replicating the heat pulse after waste emplacement. Clay materials were emplaced into holes drilled in fractured granite as compacted rings around a central heater element and subsequently brought into contact with groundwater for 880 days. After test termination, and against expectations, all clay materials were found to have undergone large scale alterations in the cation exchange population. A reactive-diffusive transport model was developed to aid the interpretation of the observed large-scale porewater chemistry changes. It was found, that the interaction between Äspö groundwater and the clay blocks, together with the geochemical nature of the clays (Na vs Ca-dominated clays) exerted the strongest control on the porewater chemistry. A pronounced exchange of Na by Ca was observed and simulated, driven by large Ca concentrations in the contacting groundwater. The model was able to link the porewater alterations to the fracture network in the deposition hole. The speed of alterations was in turn linked to high diffusion coefficients under the applied temperatures, which facilitated the propagation of hydrochemical changes into the clays. With diffusion coefficients increased by up to one order of magnitude at the maximum temperatures, the study was able to demonstrate the importance of considering temperature-dependant diffusion in understanding and predicting geochemical alterations of engineered barriers systems after relatively short exposure times following waste emplacement.

Differential Somatic Ca2+ Channel Profile in Midbrain Dopaminergic Neurons.

Dopaminergic (DA) neurons located in the ventral midbrain continuously generate a slow endogenous pacemaker activity, the mechanism of which is still debated. It has been suggested that, in the substantia nigra pars compacta (SNc), the pacemaking relies more on Ca(2+) channels and that the density of L-type Ca(2+) channels is higher in these DA neurons than in those located in the ventral tegmental area (VTA). This might lead to a higher Ca(2+) load in SNc DA neurons and explain their higher susceptibility to degeneration. However, direct evidence for this hypothesis is lacking. We found that the L-type current and channel density are indeed higher in the somata of rat SNc DA neurons and that this current undergoes less inactivation in this region. Nonstationary fluctuation analysis measurements showed a much higher number of L-type channels in the soma of SNc DA neurons, as well as a smaller single-channel conductance, pointing to a possible different molecular identity of L-type channels in DA neurons from the two areas. A major consequence of this is that pacemaking and, even more so, bursting are associated with a larger Ca(2+) entry through L-type channels in SNc DA neurons than in their VTA counterparts. Our results establish a molecular and functional difference between two populations of midbrain DA neurons that may contribute to their differential sensitivity to neurodegeneration. Dopamine neurons from the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) are involved in various brain functions, such as movement initiation and goal directed behavior, respectively. This work shows that, although both neurons fire in a similar regular and slow pacemaker mode, this firing activity is supported by different calcium channel landscapes. Indeed, the L-type calcium current is larger in the soma of dopamine neurons of the SNc, leading to a higher charge transfer through L-type channels during pacemaking and bursting. Therefore, these neurons may be physiologically exposed to a larger stress than their neighbors from the VTA.

Nucleotide homeostasis and purinergic nociceptive signaling in rat meninges in migraine-like conditions

Extracellular ATP is suspected to contribute to migraine pain but regulatory mechanisms controlling pro-nociceptive purinergic mechanisms in the meninges remain unknown. We studied the peculiarities of metabolic and signaling pathways of ATP and its downstream metabolites in rat meninges and in cultured trigeminal cells exposed to the migraine mediator calcitonin gene-related peptide (CGRP). Under resting conditions, meningeal ATP and ADP remained at low nanomolar levels, whereas extracellular AMP and adenosine concentrations were one-two orders higher. CGRP increased ATP and ADP levels in meninges and trigeminal cultures and reduced adenosine concentration in trigeminal cells. Degradation rates for exogenous nucleotides remained similar in control and CGRP-treated meninges, indicating that CGRP triggers nucleotide release without affecting nucleotide-inactivating pathways. Lead nitrate-based enzyme histochemistry of whole mount meninges revealed the presence of high ATPase, ADPase, and AMPase activities, primarily localized in the medial meningeal artery. ATP and ADP induced large intracellular Ca(2+) transients both in neurons and in glial cells whereas AMP and adenosine were ineffective. In trigeminal glia, ATP partially operated via P2X7 receptors. ATP, but not other nucleotides, activated nociceptive spikes in meningeal trigeminal nerve fibers providing a rationale for high degradation rate of pro-nociceptive ATP. Pro-nociceptive effect of ATP in meningeal nerves was reproduced by α,β-meATP operating via P2X3 receptors. Collectively, extracellular ATP, which level is controlled by CGRP, can persistently activate trigeminal nerves in meninges which considered as the origin site of migraine headache. These data are consistent with the purinergic hypothesis of migraine pain and suggest new targets against trigeminal pain.

Impact of current speed on mass flux to a model flexible seagrass blade

AbstractSeagrass and other freshwater macrophytes can acquire nutrients from surrounding water through their blades. This flux may depend on the current speed (U), which can influence both the posture of flexible blades (reconfiguration) and the thickness of the flux‐limiting diffusive layer. The impact of current speed (U) on mass flux to flexible blades of model seagrass was studied through a combination of laboratory flume experiments, numerical modeling and theory. Model seagrass blades were constructed from low‐density polyethylene (LDPE), and 1, 2‐dichlorobenzene was used as a tracer chemical. The tracer mass accumulation in the blades was measured at different unidirectional current speeds. A numerical model was used to estimate the transfer velocity (K) by fitting the measured mass uptake to a one‐dimensional diffusion model. The measured transfer velocity was compared to predictions based on laminar and turbulent boundary layers developing over a flat plate parallel to flow, for which and , respectively. The degree of blade reconfiguration depended on the dimensionless Cauchy number, Ca, which is a function of both the blade stiffness and flow velocity. For large Ca, the majority of the blade was parallel to the flow, and the measured transfer velocity agreed with laminar boundary layer theory, . For small Ca, the model blades remained upright, and the flux to the blade was diminished relative to the flat‐plate model. A meadow‐scale analysis suggests that the mass exchange at the blade scale may control the uptake at the meadow scale.

Luminal Ca2+ dynamics during IP3R mediated signals

The role of cytosolic Ca2+ on the kinetics of Inositol 1,4,5-triphosphate receptors (IP3Rs) and on the dynamics of IP3R-mediated Ca2+ signals has been studied at large both experimentally and by modeling. The role of luminal Ca2+ has not been investigated with that much detail although it has been found that it is relevant for signal termination in the case of Ca2+ release through ryanodine receptors. In this work we present the results of observing the dynamics of luminal and cytosolic Ca2+ simultaneously in Xenopus laevis oocytes. Combining observations and modeling we conclude that there is a rapid mechanism that guarantees the availability of free Ca2+ in the lumen even when a relatively large Ca2+ release is evoked. Comparing the dynamics of cytosolic and luminal Ca2+ during a release, we estimate that they are consistent with a 80% of luminal Ca2+ being buffered. The rapid availability of free luminal Ca2+ correlates with the observation that the lumen occupies a considerable volume in several regions across the images.

2세 남아의 항문과 항문주위 첨형 콘딜로마 치험 1례

Abstract Anal and perianal condyloma acuminatum(CA), which is a large ca uliflower-like tumor, is a manifestation of human papillomavirus (HPV) infection. HPV may be acquired via sexual transmission, vertical transmission or nonsexual contact. It is an uncommon condition in children but the incidence in ch ildren has been increasing recently. There are many therapies for CA including chemical or physica l destruction, immunological therapy, or a surgical excision. All these procedures have some degree of limitations such as limited clearance rate, high recurrence rate, pain, bleeding, release of potentially infectious aerosols and scar. It may be traumatic mentally to the children as well as physically. Korean Medicine included herbal treatment and herba l ointment for CA is no pain, and it doesn't need an anesthesia. We present a case of anal and perianal CA in a 2 years old bo y that was treated successfully with Korean Medicine, with no recurrence during the follow up 3 months. Key words : anal and perianal; condyloma acuminatum; children; korean medic ine

Mapping the Ca2 + induced structural change in calreticulin

Calreticulin is a highly conserved multifunctional protein implicated in many different biological systems and has therefore been the subject of intensive research. It is primarily present in the endoplasmatic reticulum where its main functions are to regulate Ca2+ homeostasis, act as a chaperone and stabilize the MHC class I peptide-loading complex. Although several high-resolution structures of calreticulin exist, these only cover three-quarters of the entire protein leaving the extended structures unsolved. Additionally, the structure of calreticulin is influenced by the presence of Ca2+. The conformational changes induced by Ca2+ have not been determined yet as they are hard to study with traditional approaches. Here, we investigated the Ca2+-induced conformational changes with a combination of chemical cross-linking, mass spectrometry, bioinformatics analysis and modelling in Rosetta. Using a bifunctional linker, we found a large Ca2+-induced change to the cross-linking pattern in calreticulin. Our results are consistent with a high flexibility in the P-loop, a stabilization of the acidic C-terminal and a relatively close interaction of the P-loop and the acidic C-terminal. Biological significanceThe function of calreticulin, an endoplasmatic reticulin chaperone, is affected by fluctuations in Ca2+concentration, but the structural mechanism is unknown. The present work suggests that Ca2+-dependent regulation is caused by different conformations of a long proline-rich loop that changes the accessibility to the peptide/lectin-binding site. Our results indicate that the binding of Ca2+ to calreticulin may thus not only just be a question of Ca2+ storage but is likely to have an impact on the chaperone activity.

Membrane channel gene expression in human costal and articular chondrocytes

ABSTRACTChondrocytes are the uniquely resident cells found in all types of cartilage and key to their function is the ability to respond to mechanical loads with changes of metabolic activity. This mechanotransduction property is, in part, mediated through the activity of a range of expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to musculoskeletal diseases such as osteoarthritis and Albers-Schönberg. In this study we analyzed the consistency of gene expression between channelomes of chondrocytes from human articular and costal (teenage and fetal origin) cartilages. Notably, we found 14 ion channel genes commonly expressed between articular and both types of costal cartilage chondrocytes. There were several other ion channel genes expressed only in articular (6 genes) or costal chondrocytes (5 genes). Significant differences in expression of BEST1 and KCNJ2 (Kir2.1) were observed between fetal and teenage costal cartilage. Interestingly, the large Ca2+ activated potassium channel (BKα, or KCNMA1) was very highly expressed in all chondrocytes examined. Expression of the gap junction genes for Panx1, GJA1 (Cx43) and GJC1 (Cx45) was also observed in chondrocytes from all cartilage samples. Together, this data highlights similarities between chondrocyte membrane channel gene expressions in cells derived from different anatomical sites, and may imply that common electrophysiological signaling pathways underlie cellular control. The high expression of a range of mechanically and metabolically sensitive membrane channels suggest that chondrocyte mechanotransduction may be more complex than previously thought.

Chromium, vanadium, and titanium valence systematics in Solar System pyroxene as a recorder of oxygen fugacity, planetary provenance, and processes

Pyroxene is arguably the most powerful, single-phase geochemical and petrologic recorder of Solar System processes, from nebular condensation through planetary evolution, over a wide range of temperatures, pressures, and f o 2 . It is an important mineral phase in the crusts and mantles of evolved planets, in undifferentiated and differentiated asteroids, and in refractory inclusions—the earliest Solar System materials. Here, we review the valence state partitioning behavior of Cr (Cr 2+ , Cr 3+ ), Ti (Ti 3+ , Ti 4+ ), and V (V 2+ , V 3+ , V 4+ , V 5+ ) among crystallographic sites in pyroxene over a range of f o 2 from approximately fayalite-magnetite-quartz (FMQ) to ~7 log units below iron-wustite (IW-7), and decipher how pyroxene can be used as a recorder of conditions of planetary and nebular environments and planetary parentage. The most important crystallographic site in pyroxene with respect to its influence on mineral/melt partitioning is M2; its Ca content has a huge effect on partitioning behavior, because the large Ca cation expands the structure. As a result, distribution coefficients ( D s) for Cr and V increase with increasing Ca content from orthopyroxene to pigeonite to augite. In addition, it is noted that V 3+ is favored over V 4+ in olivine and pyroxene. In pyroxene in refractory inclusions, Ti 3+ is favored over Ti 4+ and incorporation of Ti is facilitated by the high availability of Al for coupled substitution. The most important results from analysis of pyroxene in martian meteorites (e.g., QUE 94201) are the oxygen fugacity estimates of IW+0.2 and IW+0.9 derived from partitioning and valence data for Cr and V, respectively, obtained from experiments using appropriate temperatures and melt compositions. In angrites, changes in V valence state may translate to changes in f o 2 , from IW-0.7 during early pyroxene crystallization, to IW+0.5 during later episodes of pyroxene crystallization. In addition to f o 2 , the partitioning behavior of Cr, V, and Ti between pyroxene and melt is also dependent upon availability of other cations, especially Al, for charge-balancing coupled substitutions.

Synthesis and characterization of the epoxy-functionalized quaternary ammonium chloride

Abstract A kind of surface active epoxy-functionalized quaternary ammonium salt, N,N-dimethyl-N-dodecyl-N-(1,2-epoxy propyl) ammonium chloride (DDEAC), was synthesized. The surface activity and aggregation behavior were investigated by surface tension, dynamic light scattering, conductivity, steady state fluorescence measurements and by molecular dynamics simulations. As a control, the surface properties of dodecyl trimethyl ammonium chloride (DTAC) were also studied. Compared to DTAC, DDEAC showed higher surface activity and more ready aggregation formation as well as larger Ca2+ and Na+ tolerance. The data from molecular dynamics simulations showed that the DDEAC hydrated more easily than DTAC. The conductivity results indicated that the micellization of DDEAC is enthalpy-driven, however that of DTAC is enthalpy-driven only at temperatures higher than 25 °C and entropy-driven at temperatures lower than 25 °C. The reaction between DDEAC and chitosan implied that DDEAC could endow chitosan both hydrophilicity and hydrophobicity by one step reaction method and expand the application of chitosan.

Role of BKCa Potassium Channels in the Mechanisms of Modulatory Effects of IL-10 on Hypoxia-Induced Changes in Activity of Hippocampal Neurons

We studied the contribution of large conductance Ca(2+)-activated potassium channels (BKCa) in the mechanisms of neuromodulatory effects of anti-inflammatory cytokine IL-10 on hypoxiainduced changes in activity of CA1 pyramidal neurons in rat hippocampus. We used the method of registration of population spikes from CA1 pyramidal neurons in hippocampal slices before, during, and after exposure to short-term episodes of hypoxia. Selective blocker (iberiotoxin) and selective activator of BKCa (BMS-191011) were used to evaluate the contribution of these channels in the mechanisms of suppressive effects of IL-10 on changes in neuronal activity during hypoxia and development of post-hypoxic hyperexcitability. It was shown that BKCa are involved in the modulatory effects of IL-10 on hypoxia-induced suppression of activity of CA1 pyramidal neurons in the hippocampus and development of post-hypoxic hyperexcitability in these neurons.

A quantitative description of tubular system Ca2+ handling in fast‐ and slow‐twitch muscle fibres

Current methods do not allow a quantitative description of Ca(2+) movements across the tubular (t-) system membrane without isolating the membranes from their native skeletal muscle fibre. Here we present a fluorescence-based method that allows determination of the t-system [Ca(2+) ] transients and derivation of t-system Ca(2+) fluxes in mechanically skinned skeletal muscle fibres. Differences in t-system Ca(2+) -handling properties between fast- and slow-twitch fibres from rat muscle are resolved for the first time using this new technique. The method can be used to study Ca(2+) handling of the t-system and allows direct comparisons of t-system Ca(2+) transients and Ca(2+) fluxes between groups of fibres and fibres from different strains of animals. The tubular (t-) system of skeletal muscle is an internalization of the plasma membrane that maintains a large Ca(2+) gradient and exchanges Ca(2+) between the extracellular and intracellular environments. Little is known of the Ca(2+) -handling properties of the t-system as the small Ca(2+) fluxes conducted are difficult to resolve with conventional methods. To advance knowledge in this area we calibrated t-system-trapped rhod-5N inside skinned fibres from rat and [Ca(2+) ]t-sys , allowing confocal measurements of Ca(2+) -dependent changes in rhod-5N fluorescence during rapid changes in the intracellular ionic environment to be converted to [Ca(2+) ] transients in the t-system ([Ca(2+) ]t-sys (t)). Furthermore, t-system Ca(2+) -buffering power was determined so that t-system Ca(2+) fluxes could be derived from [Ca(2+) ]t-sys (t). With this new approach, we show that rapid depletion of sarcoplasmic reticulum (SR) Ca(2+) induced a robust store-operated Ca(2+) entry (SOCE) in fast- and slow-twitch fibres, reducing [Ca(2+) ]t-sys to <0.1mm. The rapid activation of SOCE upon Ca(2+) release was consistent with the presence of STIM1L in both fibre types. Abruptly introducing internal solutions with 1mm Mg(2+) and [Ca(2+) ]cyto (28nm-1.3μm) to Ca(2+) -depleted fibres generated t-system Ca(2+) uptake rates dependent on [Ca(2+) ]cyto with [Ca(2+) ]t-sys reaching final plateaus in the millimolar range. For the same [Ca(2+) ]cyto , t-system Ca(2+) fluxes of fast-twitch fibres were greater than that in slow-twitch fibres. In addition, simultaneous imaging of t-system and SR Ca(2+) signals indicated that both membrane compartments accumulated Ca(2+) at similar rates and that SOCE was activated early during SR Ca(2+) depletion.

Neuronal processing of noxious thermal stimuli mediated by dendritic Ca(2+) influx in Drosophila somatosensory neurons.

Adequate responses to noxious stimuli causing tissue damages are essential for organismal survival. Class IV neurons in Drosophila larvae are polymodal nociceptors responsible for thermal, mechanical, and light sensation. Importantly, activation of Class IV provoked distinct avoidance behaviors, depending on the inputs. We found that noxious thermal stimuli, but not blue light stimulation, caused a unique pattern of Class IV, which were composed of pauses after high-frequency spike trains and a large Ca(2+) rise in the dendrite (the Ca(2+) transient). Both these responses depended on two TRPA channels and the L-type voltage-gated calcium channel (L-VGCC), showing that the thermosensation provokes Ca(2+) influx. The precipitous fluctuation of firing rate in Class IV neurons enhanced the robust heat avoidance. We hypothesize that the Ca(2+) influx can be a key signal encoding a specific modality.

SERCA2a upregulation ameliorates cellular alternans induced by metabolic inhibition.

Cardiac alternans has been associated with the incidence of ventricular tachyarrhythmias and sudden cardiac death. The aim of this study was to investigate the effect of impaired mitochondrial function in the genesis of cellular alternans and to examine whether modulating the sarcoplasmic reticulum (SR) Ca(2+)ameliorates the level of alternans. Cardiomyocytes isolated from control and doxycyline-induced sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a)-upregulated mice were loaded with two different Ca(2+)indicators to selectively measure mitochondrial and cytosolic Ca(2+)using a custom-made fluorescence photometry system. The degree of alternans was defined as the alternans ratio (AR) [1 - (small Ca(2+)intensity)/(large Ca(2+)intensity)]. Blocking of complex I and II, cytochrome-coxidase, F0F1synthase, α-ketoglutarate dehydrogenase of the electron transport chain, increased alternans in both control and SERCA2a mice (P< 0.01). Changes in AR in SERCA2a-upregulated mice were significantly less pronounced than those observed in control in seven of nine tested conditions (P< 0.04).N-acetyl-l-cysteine (NAC), rescued alternans in myocytes that were previously exposed to an oxidizing agent (P< 0.001). CGP, an antagonist of the mitochondrial Na(+)-Ca(2+)exchanger, had the most severe effect on AR. Exposure to cyclosporin A, a blocker of the mitochondrial permeability transition pore reduced CGP-induced alternans (P< 0.0001). The major findings of this study are that impairment of mitochondrial Ca(2+)cycling and energy production leads to a higher amplitude of alternans in both control and SERCA2a-upregulated mice, but changes in SERCA2a-upregulated mice are less severe, indicating that SERCA2a mice are more capable of sustaining electrical stability during stress. This suggests a relationship between sarcoplasmic Ca(2+)content and mitochondrial dysfunction during alternans, which may potentially help to understand changes in Ca(2+)signaling in myocytes from diseased hearts, leading to new therapeutic targets.

Enhanced Cardiac Contractility and Ca2+ Signalling Following Dietary Nitrate Supplementation in Mice

Inorganic nitrate (NO3-) that is naturally enriched in green diet has emerged as an important dietary component, which can regulate blood pressure, mitochondrial efficiency and skeletal muscle function. It is not known if dietary nitrate also affects cardiac function. To test this, mice were put on a low nitrate diet for 1 week. Then, some mice were supplemented for 7-14 days with 1 mM sodium nitrate in the drinking water. Compared to control, hearts from the nitrate treated mice displayed increased peak pressure development rate and left ventricular developed pressure as measured with the Langendorff heart technique. Cardiomyocytes were isolated from hearts of nitrate treated and control animals, then incubated with the fluorescent indicator fluo-3 to measure cytoplasmic free [Ca2+] and fractional shortening. In the nitrate group, cardiomyocytes displayed increased fractional shortening and larger Ca2+ transients. Furthermore, the nitrate group displayed increased protein expression of the L-type Ca2+ channel/dihydropyridine receptor in the heart. This was paralleled with an increase in the peak L-type Ca2+ channel current, measured with patch clamp in cardiomyocytes from nitrate treated mice. Moreover, the expression of the Na+/Ca2+ exchanger was found reduced in nitrate compared to control hearts. Our findings indicate that dietary nitrate influences the expression of important Ca2+ handling proteins in the heart, which results in increased cardiomyocyte Ca2+ transients and enhanced left ventricular contractile function. This adds understanding to potential health effects of a nitrate enriched diet.

The WNT Proteins Induce Ca2+ Signaling through the Activation of the Polycystin Complex

WNT ligands can induce Ca2+ signaling on target cells. PKD1 (Polycystin 1) is an atypical G protein coupled receptor complexed with TRPP2 (Polycystin 2 or PKD2), a Ca2+-permeable ion channel. Inactivating mutations in the PKD1 or PKD2 genes cause autosomal dominant polycystic kidney disease (ADPKD), one of the most common genetic diseases of unknown mechanism. Here, we show that secreted WNTs specifically bind to the extracellular domain of PKD1 and induce large whole cell currents and Ca2+ influx. Pathogenic PKD1 or PKD2 mutations that abrogate complex formation, compromise cell surface expression of PKD1, or diminish TRPP2 channel activity suppress activation by a WNT protein. Cells derived from Pkd2-/- mice show complete loss of WNT-induced Ca2+ currents and are unable to polarize in response to WNT-induced cell migration. These data define PKD1 as a new class of WNT (co)receptors coupled to Ca2+ signaling and directly implicate defective WNT/Ca2+ pathway as the primary cause of ADPKD.

Functional Coupling between TRPV1 and ANO1 in Sensory Neurons Requires Ca2+-Release from the Endoplasmic Reticulum

Ca2+ activated Cl- channel ANO1 (TMEM16A) is implicated in important physiological processes such as secretion, olfaction and inflammatory pain. Due to the low Ca2+ sensitivity, ANO1 channels require close association with the Ca2+ source to allow activation and preferential coupling of ANO1 to the endoplasmic reticulum (ER) in sensory neurons has been demonstrated. TRPV1 is another sensory ion channel implicated in inflammatory pain, which also activates ANO1. To investigate how TRPV1 Ca2+ signals may activate ANO1, we simultaneously monitored activity of ANO1 in dorsal root ganglion neurons using a halide-sensitive EYFP mutant (H148Q/I152L) and fura-2 Ca2+ imaging. Capsaicin application produced large Ca2+ transients with concurrent significant EYFP quenching (13.6±0.03%) compared to that induced by bradykinin (8.8±0.01%) and voltage-gated Ca2+ channel (VGCC) activation (6.02±0.006%). However, depleting intracellular Ca2+ stores with thapsigargin significantly reduced the Ca2+ rises and EYFP quenching. Interestingly, application of capsaicin in Ca2+-free extracellular solution did not abolish Ca2+ transients, which were significantly smaller (R/R0=1.6±0.17) compared to those produced by capsaicin in Ca2+-containing extracellular solutions (R/R0=2.6±0.29) or by bradykinin in Ca2+-free solutions (R/R0=2.0±0.31). Capsaicin applied in the Ca2+-free solution was unable to produce significant YFP quenching. Ca2+ imaging with the low affinity Ca2+ indicator fura-FF showed that capsaicin induced Ca2+ rises were significantly greater compared to bradykinin- or VGCC-induced rises. Finally, we demonstrated that the ER moved towards the plasma membrane after capsaicin application to TRPV1-overexpressing HEK cells, as revealed by TIRF microscopy. Taken together, these results indicate that for effective coupling of TRPV1 and ANO1 channels both Ca2+ influx and Ca2+ release from the ER are necessary. We further suggest that TRPV1 activation results in mobilisation of internal Ca2+ in which the ER may move closer to the membrane to facilitate ANO1 activation.

Functional coupling of diverse voltage-gated Ca(2+) channels underlies high fidelity of fast dendritic Ca(2+) signals during burst firing.

In neurons, the Ca(2+) signal associated with the dendritic back-propagating action potential codes a chemical message to the different dendritic sites, playing a crucial role in electrical signalling, synaptic transmission and synaptic plasticity. The study of the underlying Ca(2+) current, mediated by different types of voltage-gated Ca(2+) channels, cannot be achieved by using the patch clamp technique. In this article, we used a recently developed cutting-edge optical technique to investigate the physiological behaviour of local Ca(2+) currents along the apical dendrite of CA1 hippocampal pyramidal neurons. We directly measure, for the first time, the synergistic activation and deactivation of the diverse dendritic voltage-gated Ca(2+) channels operating during bursts of back-propagating action potentials to precisely control the Ca(2+) signal. We demonstrate that the Ca(2+) loss via high-voltage-activated channels is compensated by the Ca(2+) entry via the other channels translating in high fidelity of Ca(2+) signalling. In CA1 hippocampal pyramidal neurons, the dendritic Ca(2+) signal associated with somatic firing represents a fundamental activation code for several proteins. This signal, mediated by voltage-gated Ca(2+) channels (VGCCs), varies along the dendrites. In this study, using a recent optical technique based on the low-affinity indicator Oregon Green 488 BAPTA-5N, we analysed how activation and deactivation of VGCCs produced by back-propagating action potentials (bAPs) along the apical dendrite shape the Ca(2+) signal at different locations in CA1 hippocampal pyramidal neurons of the mouse. We measured, at multiple dendritic sites, the Ca(2+) transients and the changes in membrane potential associated with bAPs at 50μs temporal resolution and we estimated the kinetics of the Ca(2+) current. We found that during somatic bursts, the bAPs decrease in amplitude along the apical dendrite but the amplitude of the associated Ca(2+) signal in the initial 200μm dendritic segment does not change. Using a detailed pharmacological analysis, we demonstrate that this effect is due to the perfect compensation of the loss of Ca(2+) via high-voltage-activated (HVA) VGCCs by a larger Ca(2+) component via low-voltage-activated (LVA) VGCCs, revealing a mechanism coupling the two VGCC families of K(+) channels. More distally, where the bAP does not activate HVA-VGCCs, the Ca(2+) signal is variable during the burst. Thus, we demonstrate that HVA- and LVA-VGCCs operate synergistically to stabilise Ca(2+) signals associated with bAPs in the most proximal 200μm dendritic segment.