53,639 research outputs found

    Undirectional calcium and nucleotide fluxes in cardiac sarcoplasmic reticulum. II. Experimental results

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    Unidirectional calcium influx and efflux were evaluated in cardiac sarcoplasmic reticulum (SR) by 45Ca- 0Ca exchange at steady state calcium uptake in the absence of calcium precipitating anions. Calcium efflux was partitioned into a pump-mediated efflux and a parallel passive efflux by separately measuring passive efflux referable to the steady state. Unidirectional and net ATP-ADP fluxes were measured using [3H]-ATP -- ADP and [3H]- ADP -- ATP exchanges. Methods are presented that take into account changing specific activities and sizes of the nucleotide pools during the measurement of nucleotide fluxes. The contribution of competent and incompetent vesicles to the unidirectional and net nucleotide fluxes was evaluated from the specific activity of these fluxes in incompetent vesicles and from the fraction of vesicles that were incompetent. The results indicate that, in cardiac SR, unidirectional calcium fluxes are larger than the unidirectional nucleotide fluxes contributed by competent vesicles. Because the net ATPase rate of competent vesicles is similar to the parallel passive efflux, it appears that cardiac SR Ca-ATPase tightly couples ATP hydrolysis to calcium transport even at static head, with a coupling ratio near 1.0

    IP3 receptor isoforms differently regulate ER-mitochondrial contacts and local calcium transfer

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    Contact sites of endoplasmic reticulum (ER) and mitochondria locally convey calcium signals between the IP3 receptors (IP3R) and the mitochondrial calcium uniporter, and are central to cell survival. It remains unclear whether IP3Rs also have a structural role in contact formation and whether the different IP3R isoforms have redundant functions. Using an IP3R-deficient cell model rescued with each of the three IP3R isoforms and an array of super-resolution and ultrastructural approaches we demonstrate that IP3Rs are required for maintaining ER-mitochondrial contacts. This role is independent of calcium fluxes. We also show that, while each isoform can support contacts, type 2 IP3R is the most effective in delivering calcium to the mitochondria. Thus, these studies reveal a non-canonical, structural role for the IP3Rs and direct attention towards the type 2 IP3R that was previously neglected in the context of ER-mitochondrial calcium signaling

    Phytochrome modulation of calcium fluxes in wheat (Triticum aestivum L.) protoplasts

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    Employing the metallochromic dye murexide and by monitoring the uptake of radiolabelled calcium, photoreversible calcium fluxes were measured in wheat leaf protoplast suspensions. Results obtained by both methods were identical - red light promoted and subsequent far-red irradiation reversed an influx of Ca++ ions into the protoplasts. These findings imply phytochrome regulation of Ca++ fluxes across the plasma membrane. The influx of Ca++ stimulated by 2 min red irradiation could be maintained in total darkness for the initial 16-18 min after illumination, after which a 6-8 min efflux process was triggered and the basal Ca++ level restored. Verapamil, a calcium channel blocker, inhibited the red-promoted influx, whereas the far-red mediated efflux could be checked by the use of the ATPase inhibitor vanadate, and also by the calmodulin antagonist chlorpromazine, thus suggesting a role of ion channels and pumps in phytochrome-controlled Ca++ fluxes. The possible involvement of phosphoinositides in phytochrome-modulated calcium fluxes was also investigated

    Reverse Mode of the Sarcoplasmic Reticulum Calcium Pump and Load-Dependent Cytosolic Calcium Decline in Voltage-Clamped Cardiac Ventricular Myocytes

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    AbstractWe have characterized [Ca]i decline in voltage-clamped rabbit ventricular myocytes with progressive increases in sarcoplasmic reticulum (SR) calcium load. “Backflux” through the SR calcium pump is a critical feature which allows realistically small values for SR calcium leak fluxes to be used. Total cytosolic calcium was calculated from the latter part of [Ca]i decline using rate constants for cellular calcium buffers. Intra-SR calcium buffering characteristics were also deduced. We found that the net SR calcium pump flux and rate of [Ca]i decline decreased as the SR free [Ca] rose, with pump parameters held constant. We have therefore characterized for the first time in intact myocytes both forward and reverse SR calcium pump kinetics as well as intra-SR calcium buffering and SR calcium leak. We conclude that the reverse flux through the SR calcium pump is an important factor in comprehensive understanding of dynamic SR calcium fluxes

    Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

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    Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest

    Modeling effects of L-type ca(2+) current and na(+)-ca(2+) exchanger on ca(2+) trigger flux in rabbit myocytes with realistic T-tubule geometries.

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    The transverse tubular system of rabbit ventricular myocytes consists of cell membrane invaginations (t-tubules) that are essential for efficient cardiac excitation-contraction coupling. In this study, we investigate how t-tubule micro-anatomy, L-type Ca(2+) channel (LCC) clustering, and allosteric activation of Na(+)/Ca(2+) exchanger by L-type Ca(2+) current affects intracellular Ca(2+) dynamics. Our model includes a realistic 3D geometry of a single t-tubule and its surrounding half-sarcomeres for rabbit ventricular myocytes. The effects of spatially distributed membrane ion-transporters (LCC, Na(+)/Ca(2+) exchanger, sarcolemmal Ca(2+) pump, and sarcolemmal Ca(2+) leak), and stationary and mobile Ca(2+) buffers (troponin C, ATP, calmodulin, and Fluo-3) are also considered. We used a coupled reaction-diffusion system to describe the spatio-temporal concentration profiles of free and buffered intracellular Ca(2+). We obtained parameters from voltage-clamp protocols of L-type Ca(2+) current and line-scan recordings of Ca(2+) concentration profiles in rabbit cells, in which the sarcoplasmic reticulum is disabled. Our model results agree with experimental measurements of global Ca(2+) transient in myocytes loaded with 50 μM Fluo-3. We found that local Ca(2+) concentrations within the cytosol and sub-sarcolemma, as well as the local trigger fluxes of Ca(2+) crossing the cell membrane, are sensitive to details of t-tubule micro-structure and membrane Ca(2+) flux distribution. The model additionally predicts that local Ca(2+) trigger fluxes are at least threefold to eightfold higher than the whole-cell Ca(2+) trigger flux. We found also that the activation of allosteric Ca(2+)-binding sites on the Na(+)/Ca(2+) exchanger could provide a mechanism for regulating global and local Ca(2+) trigger fluxes in vivo. Our studies indicate that improved structural and functional models could improve our understanding of the contributions of L-type and Na(+)/Ca(2+) exchanger fluxes to intracellular Ca(2+) dynamics

    The spatial variation of Asian dust and marine aerosol contributions to glaciochemical signals in central Asia

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    Short-term (6 months to 17 years) glaciochemical records have been collected from several glacier basins in the mountains of central Asia. The spatial distribution of snow chemistry in central Asia is controlled by the influx of dust from the large expanse of arid and semiarid regions in central Asia. Glaciers in the Northern and Western Tibetan Plateau show elevated concentrations and elevated annual fluxes of calcium, sodium, chloride, sulphate and nitrate due to the influx of desert dust from nearby arid and semi-arid regions. Glaciers in the Southeastern Tibetan Plateau show lower concentrations and lower annual fluxes of major ions due to longer transport distances of dust from the arid and semi-arid regions of Western China. Snow from the Karakoram and Western Himalaya show ion concentrations similar to those in Southeastern Tibetan Plateau, but much higher annual fluxes suggesting that much of the aerosol and moisture transported with the westerly jet stream is removed as it ascends the Southwest margin of the Tibetan Plateau. Snow from the Southern slopes of the Eastern Himalayas shows very low concentrations and very low annual fluxes of major ions, indicating that this region is relatively free from the chemical influence of Asian dust. The glaciochemical data suggest that glaciers which are removed from large source areas of mineral aerosol, such as those in the Himalaya, the Karakoram, and the Southeastern Tibetan Plateau, are the ones most likely to contain longer-term glaciochemical records which detail annual to decadal variation in the strength of the Asian monsoon and long-range transport of Asian dust

    The distribution of charged amino acid residues and the Ca(2+) permeability of nicotinic acetylcholine receptors: a predictive model

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    Nicotinic acetylcholine receptors (nAChRs) are cation-selective ligand-gated ion channels exhibiting variable Ca(2+) permeability depending on their subunit composition. The Ca(2+) permeability is a crucial functional parameter to understand the physiological role of nAChRs, in particular considering their ability to modulate Ca(2+)-dependent processes such as neurotransmitter release. The rings of extracellular and intracellular charged amino acid residues adjacent to the pore-lining TM2 transmembrane segment have been shown to play a key role in the cation selectivity of these receptor channels, but to date a quantitative relationship between these structural determinants and the Ca(2+) permeability of nAChRs is lacking. In the last years the Ca(2+) permeability of several nAChR subtypes has been experimentally evaluated, in terms of fractional Ca(2+) current (Pf, i.e., the percentage of the total current carried by Ca(2+) ions). In the present study, the available Pf-values of nAChRs are used to build a simplified modular model describing the contribution of the charged residues in defined regions flanking TM2 to the selectivity filter controlling Ca(2+) influx. This model allows to predict the currently unknown Pf-values of existing nAChRs, as well as the hypothetical Ca(2+) permeability of subunit combinations not able to assemble into functional receptors. In particular, basing on the amino acid sequences, a Pf > 50% would be associated with homomeric nAChRs composed by different α subunits, excluding α7, α9, and α10. Furthermore, according to the model, human α7β2 receptors should have Pf-values ranging from 3.6% (4:1 ratio) to 0.1% (1:4 ratio), much lower than the 11.4% of homomeric α7 nAChR. These results help to understand the evolution and the function of the large diversity of the nicotinic receptor family
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