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  • 99
    Thermo Fisher ryanodine receptor 1
    TEMR–ADSC construct-repaired LD muscles display structural and functional hallmarks reminiscent of native muscles. Notes: LD muscles repaired with TEMR–ADSC constructs were retrieved 2 months postimplantation and analyzed for morphology and new tissue formation by IHC staining on paraffin-embedded histological sections. IHC staining demonstrated positivity for structural proteins, myosin heavy chain (MF20; A ) and titin (9D10; B ). Similarly IHC staining revealed the presence of functional proteins, Junctophilin (Jp1; C ) and <t>ryanodine</t> <t>receptor</t> 1 (RyR1; D ). Abbreviations: TEMR, tissue-engineered muscle repair; ADSC, adipose-derived stem cell; LD, latissimus dorsi; IHC, immunohistochemistry.
    Ryanodine Receptor 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ryanodine
    Effect of an mGluR antagonist and modulators of second messenger pathways on the mGluR-induced potentiation of NMDA receptors. Outlier box plots showing the amplitude of NMDA-induced calcium responses measured as Δ F / F. A, The mGluR antagonist CPCCOEt blocked the DHPG-induced potentiation of NMDA-induced calcium response. B, The potentiation persisted in the presence of the protein kinase inhibitor H-7. C, The potentiation persisted in the presence of the protein kinase inhibitor H-8. D, <t>Ryanodine</t> had no effect on the potentiation of NMDA response by DHPG.
    Ryanodine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 631 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Tocris ryanodine
    LTD-induced phosphorylation of CaMKII and Synapsin I. Induction of LTD for 60 min in control (C) slices caused a significant increase in the phosphorylation levels of Synapsin I (left panels), CaMKII-α (center panels) and CaMKII-β (right panels) relative to the levels displayed by unstimulated slices (naïve). Slices pre-incubated for 1 h with 20 μM <t>ryanodine</t> (Rya) before applying the LTD induction protocol displayed significantly lower increments in the phosphorylation levels of Synapsin I and CaMKII-α, whereas the phosphorylation levels of CaMKII-β were not significantly different from the levels displayed by unstimulated slices. Values represent Mean ± SE ( n = 3). Statistical analysis was performed with one-way ANOVA, followed by Tukey’s post hoc test. * p
    Ryanodine, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 288 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem ryanodine
    Depletion of the SR of Ca 2+ changes does not reduce the [Ca 2+ ] increases in subplasma membrane space or bulk cytoplasm in voltage-clamped single colonic myocytes. Depolarization (−70 to +10 mV; G) activated a voltage-dependent Ca 2+ current (I Ca ; F) to evoke a rise in [Ca 2+ ] (C and D). The rise in [Ca 2+ ], which occurred in some regions (B, left panels) of the subplasma membrane space (measured by TIRF; red line in inset in C), were more rapid in both onset and decline than that seen in average measured in the subplasma membrane space (black red line in inset in C; B and E). The same regions in the bulk cytoplasm (measured by wide-field epi-florescence; B, left panels) had approximately comparable rates of rise and decline (D, inset). 10 mM caffeine (E) applied by pressure ejection from a puffer pipette (A, left side; see also whole cell patch electrode [right side]) evoked increases in [Ca 2+ ] (C and D). 50 µM <t>ryanodine</t> (C, open bar above the trace) after repeated application of caffeine to open RYR abolished the [Ca 2+ ] increase to caffeine presumably because of SR store depletion. In contrast, the depolarization-evoked [Ca 2+ ] increase was not reduced in either subplasma membrane space or bulk cytoplasm. After inhibition of the caffeine-evoked [Ca 2+ ] increase, the depolarization-evoked [Ca 2+ ] PM or [Ca 2+ ] c increase were not reduced. Furthermore, localized increases in regions of the subplasma membrane space measured by TIRF (red line in inset in C) were more rapid in onset and decline than those seen in average measured in the subplasma membrane space (black red line in inset in C; B and E). This result suggests that SR Ca 2+ release does not contribute substantially to the depolarization-evoked [Ca 2+ ] PM or [Ca 2+ ] c increases. Changes in the fluorescence ratio with time (C and D) are derived from 1-pixel boxes (B) and from a larger region encompassing the entire TIRF region (B). The latter was used to obtain an average subplasma membrane and bulk average [Ca 2+ ] increases (C and D). (A; left) A bright field image of the cell; see also whole cell electrode (right side). B shows an expanded view of A (middle and right panels) to illustrate the regions of measurement. Note the pixel position is in a different position after SR depletion. The cell moved slightly during solution exchanges, and a new voltage-dependent Ca 2+ channel cluster may have emerged.
    Ryanodine, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ryr2  (Abcam)
    95
    Abcam ryr2
    Depolymerization of microtubules decreases the number of BKα and <t>RyR2</t> colocalization sites. ( A ) Wide-field image of a freshly isolated arterial myocyte immunolabeled for BKα ( n = 12 cells, n = 3 animals). Red box indicates the area where superresolution images were obtained. Scale bar, 10 μm. ( B ) Superresolution localization map obtained after immunolabeling with anti-BKμ (green) and anti-RyR2 (red) antibodies ( n = 12 cells, n = 3 animals). Scale bar, 3 μm. ROIs (yellow boxes) are shown in magnified view (I) and (II) below. Scale bars, 0.2 μm. ( C ) Cluster size distribution histograms of RyR2 and BKα ( n = 11,005 or n = 11,940 particles for RyR2 and BKα, respectively; 12 cells, n = 3 animals). ( D ) Summary of object-based analysis to determine the number of BKα protein cluster centroids per cell that overlay with RyR2 protein clusters in control cells, cells in which a random BKα distribution has been simulated, and cells treated with nocodazole (10 μM). * P ≤ 0.05 compared to control ( n =12 cells per group, 3 animals). Coloc., colocalization.
    Ryr2, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 311 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam ryanodine
    The ER Ca 2+ store regulates lysosome Ca 2+ stores. ( A ) In un-transfected HEK293T cells, ATP induced Ca 2+ release through IP3-receptors on the ER, and GPN induced lysosomal Ca 2+ release. ( B ) A 2-min application of TPEN, a membrane-permeable chelator of luminal ER Ca 2+ , attenuated Ca 2+ release from IP3-receptors stimulated by ATP in HEK293T cells. ( C ) A 2-min TPEN application did not significantly reduce GPN-induced lysosomal Ca 2+ release in HEK293T cells. ( D ) Long-term TPEN treatment (20 min) abolished ER Ca 2+ release upon ATP stimulation and GPN-induced lysosomal Ca 2+ release in HEK293T cells loaded with Fura-2. ( E ) In HEK293T cells transfected with the IP3R-ligand binding domain with ER targeting sequence (IP3R-LBD-ER), the responses to ATP and GPN were reduced compared to un-transfected cells on the same coverslip. ( F ) Caffeine stimulates Ca 2+ release from <t>ryanodine</t> receptors and ATP stimulates Ca 2+ release from IP3Rs in HEK-GCaMP3-ML1 cells loaded with Fura-2. Panels A – C and E show the average response of 30–40 cells from one representative experiment. DOI: http://dx.doi.org/10.7554/eLife.15887.011
    Ryanodine, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Alomone Labs ryanodine
    Action potential-induced CICR at somatic plasma membrane but not AIS or dendrites (A) Two-photon Ca 2+ imaging at somatic plasma membrane. (Ai) Maximum intensity projection of Alexa-594-filled cartwheel cell. The red boxed region is enlarged in Ai, inset. Regions of interest for segmented line scans are indicated by red lines. C: cytosolic side. M: membrane side. (Aii, top and middle panels) Spike trains evoked by current injection (top) elicited an increase of Fluo-5F fluorescence with no change in Alexa-594. (Aii, bottom) Ca 2+ transients induced by spike trains (6 simple spikes at 50 Hz). The transients are expressed as ΔG/R (change in Fluo-5F intensity divided by Alexa-594 intensity). Black, control; blue, in <t>ryanodine.</t> (Aiii) Averaged Ca 2+ transients from 10 regions of interest of 5 cells. Single spike or trains of simple spikes (6 spikes at 50 Hz) evoked by current injection. (Aiv) Summary of the changes in Ca 2+ transients. *** p
    Ryanodine, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam anti ryr2
    (A) Comparison of protein expression between the Casq2 R33Q/R33Q mice and the WT mice. (B) Quantification of different protein expression shows that CaMKII, p-CaMKII, <t>p-RyR2,</t> and NCX1.1 were promoted, but Casq2, JCT, and TRI were declined in the Casq2 R33Q/R33Q atria. CaMKII, calcium/calmodulin-dependent protein kinase II; RyR2, ryanodine receptor; NXC1.1, sodium–calcium exchanger 1.1; Casq2, calsequestrin 2; TRI, triadin; JCT, junctin; SERCA, sarcoplasmic reticulum calcium-ATPases; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ∗ P
    Anti Ryr2, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH ryanodine
    (A1–A2) Effects of 100 µM ACh on the relative fluorescence intensity of OHC [Ca 2+ ] i in Hank’s solution. (B1–B3) Effects of 100 µM ACh following 30 µM <t>ryanodine</t> on the relative fluorescence intensity of OHC [Ca 2+ ] i in Hank’s solution. (C1–C3) Effects of 100 µM ACh following 30 nM thapsigargin on the relative fluorescence intensity of OHC [Ca 2+ ] i in Hank’s solution. (D1–D2) Effects of 100 µM ACh on the relative fluorescence intensity of OHC [Ca 2+ ]i in D-Hank’s solution. (E1–E3) Effects of 100 µM ACh following 30 µM ryanodine on the relative fluorescence intensity of OHC [Ca 2+ ] i in D-Hank’s solution. (F1–F3) Effects of 100 µM ACh following 30 nM thapsigargin on the relative fluorescence intensity of OHC [Ca 2+ ] i in D-Hank’s solution.
    Ryanodine, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology ryanodine
    RyR function is required for Shh-dependent Gli-mediated gene expression. (A–H) Dorsal view images of live Tg(8xGli:mCherry-NLS-Odc1) embryos at 12hpf (A,C,E,G) or 24hpf (B,D,F,H) that had been treated with vehicle (0.5%DMSO), cyclopamine, azumolene, or <t>ryanodine.</t> At 12hpf, the position of the notochord (ntc) just ventral to the FP is outlined by dashed lines. (A) In control 12hpf embryos, mCherry is expressed in nuclei of cells responding to Shh, including adaxial cells (arrowhead) and FP cells (arrow). (B) At 24hpf, mCherry is expressed in nuclei of slow muscle cells (arrowhead) and cells in the ventral neural tube (arrow). (C–H) mCherry expression is reduced in embryos treated with each drug. (I) Tail-transected Tg(8xGli:mCherry-NLS-Odc1) embryos were soaked in vehicle or 4-CmC from 16 to 18hpf, fixed, and imaged. (J–L) Potentiation of RyR channel activity with 4-CmC treatment results in increased numbers of presumptive slow muscle nuclei that express the mCherry reporter (J and K are lateral views, arrowhead indicates nuclei of slow muscle cells and arrow indicates cells in the ventral neural tube). (L) Quantification of mCherry + nuclei per somite in 4-CmC-treated embryos. Each point represents a single somite and the horizontal line represents the mean. (M–Q) RyR activity affects endogenous ptch2 expression as detected by whole mount in situ hybridization in 24hpf embryos. (M–Q) Lateral views reveal expression in somites and (M′–Q′) transverse sections reveal expression in slow muscle cells surrounding the notochord and in the ventral neural tube. As compared with WT embryos, ptch2 expression is diminished azumolene-treated and MZryr1a (−/−) ;MZryr2a (−/−) ;MZryr3 (−/−) mutant embryos, and it is enhanced in 4-CmC-treated embryos. Scale bars indicate 25 μm.
    Ryanodine, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Nissen roux en y gastric bypass
    RyR function is required for Shh-dependent Gli-mediated gene expression. (A–H) Dorsal view images of live Tg(8xGli:mCherry-NLS-Odc1) embryos at 12hpf (A,C,E,G) or 24hpf (B,D,F,H) that had been treated with vehicle (0.5%DMSO), cyclopamine, azumolene, or <t>ryanodine.</t> At 12hpf, the position of the notochord (ntc) just ventral to the FP is outlined by dashed lines. (A) In control 12hpf embryos, mCherry is expressed in nuclei of cells responding to Shh, including adaxial cells (arrowhead) and FP cells (arrow). (B) At 24hpf, mCherry is expressed in nuclei of slow muscle cells (arrowhead) and cells in the ventral neural tube (arrow). (C–H) mCherry expression is reduced in embryos treated with each drug. (I) Tail-transected Tg(8xGli:mCherry-NLS-Odc1) embryos were soaked in vehicle or 4-CmC from 16 to 18hpf, fixed, and imaged. (J–L) Potentiation of RyR channel activity with 4-CmC treatment results in increased numbers of presumptive slow muscle nuclei that express the mCherry reporter (J and K are lateral views, arrowhead indicates nuclei of slow muscle cells and arrow indicates cells in the ventral neural tube). (L) Quantification of mCherry + nuclei per somite in 4-CmC-treated embryos. Each point represents a single somite and the horizontal line represents the mean. (M–Q) RyR activity affects endogenous ptch2 expression as detected by whole mount in situ hybridization in 24hpf embryos. (M–Q) Lateral views reveal expression in somites and (M′–Q′) transverse sections reveal expression in slow muscle cells surrounding the notochord and in the ventral neural tube. As compared with WT embryos, ptch2 expression is diminished azumolene-treated and MZryr1a (−/−) ;MZryr2a (−/−) ;MZryr3 (−/−) mutant embryos, and it is enhanced in 4-CmC-treated embryos. Scale bars indicate 25 μm.
    Roux En Y Gastric Bypass, supplied by Nissen, used in various techniques. Bioz Stars score: 88/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    FUJIFILM ryanodine
    Effects of nifedipine, BAY K 8644 and <t>ryanodine</t> on phenylephrine-induced mechanical responses in papillary muscles. (A) The representative response to phenylephrine (10 μM) in C57BL/6J. Actions of nifedipine (0.3 μM, B), BAY K 8644 (1 μM, C) and ryanodine (0.3 μM, D) on twitch tension in the absence and presence of phenylephrine in C57BL/6J. Phenylephrine exerted positive inotropic response in the presence of ryanodine (i.e., in situations where SR-function is greatly reduced). Summary of the effects of BAY K 8644 (1 μM, E) and ryanodine (0.3 μM, F) on phenylephrine-induced response (n = 4–6, * P
    Ryanodine, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam anti ryanodine receptor antibody c3 33
    Effects of nifedipine, BAY K 8644 and <t>ryanodine</t> on phenylephrine-induced mechanical responses in papillary muscles. (A) The representative response to phenylephrine (10 μM) in C57BL/6J. Actions of nifedipine (0.3 μM, B), BAY K 8644 (1 μM, C) and ryanodine (0.3 μM, D) on twitch tension in the absence and presence of phenylephrine in C57BL/6J. Phenylephrine exerted positive inotropic response in the presence of ryanodine (i.e., in situations where SR-function is greatly reduced). Summary of the effects of BAY K 8644 (1 μM, E) and ryanodine (0.3 μM, F) on phenylephrine-induced response (n = 4–6, * P
    Anti Ryanodine Receptor Antibody C3 33, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    DuPont de Nemours ryanodine
    Effect of 30 μ M thymol on the isolated RyRs. Incorporation was initiated in symmetric 250 mM KCl, holding potential was 61 mV; channel openings are upward deflections. Horizontal lines before each current record mark the closed state. ( A ) Single channel recordings in control conditions. ( B ) Representative segments of single channel behavior taken 5 min after the additions of thymol. ( C ) Single channel currents measured after the successive addition of 0.2 μ M <t>ryanodine</t> into the cis chamber. ( D ) Single channel currents plotted as a function of the holding potential. Open symbols represent measurements under control conditions, whereas solid symbols represent those in the presence of thymol. Straight lines correspond to a single channel conductance of 545 and 543 pS in control and in the presence of the drug, respectively. All recordings are from a single experiment.
    Ryanodine, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 92/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck & Co ryanodine
    (A) Representative recordings relative to fluo-8 fluorescence (expressed as DF/F0 (A. U.)) in resting WT (open circles) and mdx (filled circles) and in stretched WT (open squares) and mdx (filled squares) during a superfusion protocol starting initially with a calcium free Tyrode solution followed by the superfusion of 1.8 mM Ca 2+ Tyrode solution. (B) Maximal amplitude of fluo-8 fluorescence intensity in WT and mdx cardiomyocytes maintained in stretched condition with SACs inhibitors: cells were incubated with 300 µM streptomycin (Strp, gray bars) or 2.5 µM GsMTx-4 (Black bars) for SACs inhibition and with 10 µM nifedipine (vertical hatching) or 100 µM <t>ryanodine</t> (horizontal hatching) for EC coupling inhibition. Open bars represent the control. Declined hatching represents rest (non-stretched). Measurements are represented as mean normalized fluo 8 fluorescence intensity±SEM. Rest (WT: n =10 cells, N =4 hearts; mdx : n =12 cells, N =4 hearts). stretch control (WT: n =12 cells, N =4 hearts; mdx : n =12 cells, N =4 hearts). Strp (WT: n =7 cells, N =3 hearts; mdx : n =6 cells, N =3 hearts). Rest (WT: n =10 cells, N =4 hearts; mdx : n =12 cells, N =4 hearts). GsMTx-4 (WT: n =5 cells, N =3 hearts; mdx : n =5 cells, N =3 hearts). Nifedipine (WT: n =5 cells, N =4 hearts; mdx : n =5 cells, N =3 hearts). Ryanodine (WT: n =9 cells, N =4 hearts; mdx : n =7 cells, N =4 hearts). * Symbol represents the statistical difference with control, *** P
    Ryanodine, supplied by Merck & Co, used in various techniques. Bioz Stars score: 93/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher bodipy fl x ryanodine
    Confocal image of <t>ryanodine</t> receptor distribution in living endothelial cells Freshly isolated endothelial cells were loaded with 10 −7 mol l −1 <t>BODIPY</t> <t>FL-X</t> ryanodine for 10 min and images were collected using a confocal microscope. The figure shows a transmitted light image ( A ), the corresponding confocal image at approximately the middle depth of the cell ( B ) and a composite image of A and B ( C ). D shows a 3-dimensional reconstruction of 13 optical sections taken at 1 μm steps in the z -axis and the inset presents the single section in the x-y plane taken at the arrow. E , a 3-dimensional reconstruction above the x-y plane shown in B . The red circles and lines indicate corresponding areas in B and E . In F the plasmalemma is indicated by the outer red circle defined by the transmitted light image given in A and C . The inner red circle shows an erosion of 3 pixels from the plasmalemma towards the centre of the cell, which represents a distance of 0.6 μm from the plasmalemma.
    Bodipy Fl X Ryanodine, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    TEMR–ADSC construct-repaired LD muscles display structural and functional hallmarks reminiscent of native muscles. Notes: LD muscles repaired with TEMR–ADSC constructs were retrieved 2 months postimplantation and analyzed for morphology and new tissue formation by IHC staining on paraffin-embedded histological sections. IHC staining demonstrated positivity for structural proteins, myosin heavy chain (MF20; A ) and titin (9D10; B ). Similarly IHC staining revealed the presence of functional proteins, Junctophilin (Jp1; C ) and ryanodine receptor 1 (RyR1; D ). Abbreviations: TEMR, tissue-engineered muscle repair; ADSC, adipose-derived stem cell; LD, latissimus dorsi; IHC, immunohistochemistry.

    Journal: International Journal of Nanomedicine

    Article Title: Evaluation of adipose-derived stem cells for tissue-engineered muscle repair construct-mediated repair of a murine model of volumetric muscle loss injury

    doi: 10.2147/IJN.S101955

    Figure Lengend Snippet: TEMR–ADSC construct-repaired LD muscles display structural and functional hallmarks reminiscent of native muscles. Notes: LD muscles repaired with TEMR–ADSC constructs were retrieved 2 months postimplantation and analyzed for morphology and new tissue formation by IHC staining on paraffin-embedded histological sections. IHC staining demonstrated positivity for structural proteins, myosin heavy chain (MF20; A ) and titin (9D10; B ). Similarly IHC staining revealed the presence of functional proteins, Junctophilin (Jp1; C ) and ryanodine receptor 1 (RyR1; D ). Abbreviations: TEMR, tissue-engineered muscle repair; ADSC, adipose-derived stem cell; LD, latissimus dorsi; IHC, immunohistochemistry.

    Article Snippet: IHC stainings were performed using antibodies to detect myosin (MF-20, 1:10), titin (9D10, 1:10), ryanodine receptor 1 (RyR1; 34C, 1:10), and Junctophilin 1 (Jp1; Thermo Fisher Scientific 40-5100, 1:120).

    Techniques: Construct, Functional Assay, Immunohistochemistry, Staining, Derivative Assay

    Effect of an mGluR antagonist and modulators of second messenger pathways on the mGluR-induced potentiation of NMDA receptors. Outlier box plots showing the amplitude of NMDA-induced calcium responses measured as Δ F / F. A, The mGluR antagonist CPCCOEt blocked the DHPG-induced potentiation of NMDA-induced calcium response. B, The potentiation persisted in the presence of the protein kinase inhibitor H-7. C, The potentiation persisted in the presence of the protein kinase inhibitor H-8. D, Ryanodine had no effect on the potentiation of NMDA response by DHPG.

    Journal: The Journal of Neuroscience

    Article Title: Interaction between Metabotropic and Ionotropic Glutamate Receptors Regulates Neuronal Network Activity

    doi: 10.1523/JNEUROSCI.20-14-05382.2000

    Figure Lengend Snippet: Effect of an mGluR antagonist and modulators of second messenger pathways on the mGluR-induced potentiation of NMDA receptors. Outlier box plots showing the amplitude of NMDA-induced calcium responses measured as Δ F / F. A, The mGluR antagonist CPCCOEt blocked the DHPG-induced potentiation of NMDA-induced calcium response. B, The potentiation persisted in the presence of the protein kinase inhibitor H-7. C, The potentiation persisted in the presence of the protein kinase inhibitor H-8. D, Ryanodine had no effect on the potentiation of NMDA response by DHPG.

    Article Snippet: H-7, H-8, and ryanodine were purchased from Research Biochemicals (Natick, MA).

    Techniques:

    Reactive oxygen species (ROS) activates oxidized Ca 2+ /calmodulin-dependent protein kinase II (ox-CaMKII) increasing serine 2814 on RyR2, and the inhibitory effect of apocynin. (A–F) Representative western blots and quantification of anti-calmodulin-dependent protein kinases II (CaMKII), anti-CaMKⅡ (phospho T286, p -CaMKII), oxidized CaMKII, Ryanodine Receptor 2 (RyR2), RyR2-Ser2814 expression in the atrial tissues of mice in the control group, ibrutinib group, and apocynin group with GAPDH as a loading control (n = 3 mice per group, one way ANOVA). Values are presented as mean ± SD. * P

    Journal: Redox Biology

    Article Title: Enhanced cardiomyocyte reactive oxygen species signaling promotes ibrutinib-induced atrial fibrillation

    doi: 10.1016/j.redox.2020.101432

    Figure Lengend Snippet: Reactive oxygen species (ROS) activates oxidized Ca 2+ /calmodulin-dependent protein kinase II (ox-CaMKII) increasing serine 2814 on RyR2, and the inhibitory effect of apocynin. (A–F) Representative western blots and quantification of anti-calmodulin-dependent protein kinases II (CaMKII), anti-CaMKⅡ (phospho T286, p -CaMKII), oxidized CaMKII, Ryanodine Receptor 2 (RyR2), RyR2-Ser2814 expression in the atrial tissues of mice in the control group, ibrutinib group, and apocynin group with GAPDH as a loading control (n = 3 mice per group, one way ANOVA). Values are presented as mean ± SD. * P

    Article Snippet: After incubation in closed buffer (0.5% Tween-20 in TBS, 5% bovine serum albumin (BSA)), the membrane was incubated with the following antibodies for 1 h at room temperature: anti-calmodulin-dependent protein kinases II (CaMKII, ab181052), anti-CaMKⅡ (phospho T286, ab32678), oxidized CaMKII (methionine 281/282 oxidation, GTX36254), Ryanodine Receptor 2 (RyR2, Millipore, AB9080), RyR2-Ser2814 (badrilla, A010-31), anti-xanthine oxidase (XO, ab109235), anti-NOX4 (ab133303), anti-transforming growth factor-β1 (TGF-β1, ab190503), anti-NOXA2/p67-phox (NOX2, ab109366), and anti-Cytochrome b245 Light Chain/p22-phox (ab75941, Abcam, Cambridge, UK).

    Techniques: Western Blot, Expressing, Mouse Assay

    Identification and quantitative analysis of differentially expressed atrial tissue proteins in the control group vs. the ibrutinib group. (A) Differential protein expression was verified using Student's t-test and a volcano plot was constructed. (B) Gene Ontology (GO) annotation and enrichment of differentially expressed proteins. (C) KEGG Pathway analysis. (D) The clustering heat map analysis. (E) Protein-protein interaction (PPI) network showing twenty-three major nodes. n = 3 mice per group. NCX, calcium and sodium exchangers; RyR2, Ryanodine Receptor 2; CaMKII, Ca 2+ /calmodulin-dependent protein kinase II; TGF-β, transforming growth factor-β; NOS, nitric oxide synthase; NADPH oxidase, nicotinamide adenine dinucleotide phosphate oxidase; MPO, myeloperoxidase; HOCL, hypochlorous; MHCI, MHC class I molecule.

    Journal: Redox Biology

    Article Title: Enhanced cardiomyocyte reactive oxygen species signaling promotes ibrutinib-induced atrial fibrillation

    doi: 10.1016/j.redox.2020.101432

    Figure Lengend Snippet: Identification and quantitative analysis of differentially expressed atrial tissue proteins in the control group vs. the ibrutinib group. (A) Differential protein expression was verified using Student's t-test and a volcano plot was constructed. (B) Gene Ontology (GO) annotation and enrichment of differentially expressed proteins. (C) KEGG Pathway analysis. (D) The clustering heat map analysis. (E) Protein-protein interaction (PPI) network showing twenty-three major nodes. n = 3 mice per group. NCX, calcium and sodium exchangers; RyR2, Ryanodine Receptor 2; CaMKII, Ca 2+ /calmodulin-dependent protein kinase II; TGF-β, transforming growth factor-β; NOS, nitric oxide synthase; NADPH oxidase, nicotinamide adenine dinucleotide phosphate oxidase; MPO, myeloperoxidase; HOCL, hypochlorous; MHCI, MHC class I molecule.

    Article Snippet: After incubation in closed buffer (0.5% Tween-20 in TBS, 5% bovine serum albumin (BSA)), the membrane was incubated with the following antibodies for 1 h at room temperature: anti-calmodulin-dependent protein kinases II (CaMKII, ab181052), anti-CaMKⅡ (phospho T286, ab32678), oxidized CaMKII (methionine 281/282 oxidation, GTX36254), Ryanodine Receptor 2 (RyR2, Millipore, AB9080), RyR2-Ser2814 (badrilla, A010-31), anti-xanthine oxidase (XO, ab109235), anti-NOX4 (ab133303), anti-transforming growth factor-β1 (TGF-β1, ab190503), anti-NOXA2/p67-phox (NOX2, ab109366), and anti-Cytochrome b245 Light Chain/p22-phox (ab75941, Abcam, Cambridge, UK).

    Techniques: Expressing, Construct, Mouse Assay

    Effects of CLIC2 on [ 3 H]-ryanodine binding to skeletal heavy SR and purified RyR1 (A) CLIC2 increases [ 3 H]-ryanodine binding to skeletal heavy SR. Equilibrium binding experiments were performed in the absence or presence of CLIC2. The binding buffer contained 2 nM [ 3 H]-ryanodine and 10 μM Ca 2+ . CLIC2 increased [ 3 H]-ryanodine binding to skeletal heavy SR from 1.31±0.1 pmol/ml (buffer, n=6) to 1.57±0.03 pmol/ml (15 μM CLIC2, n=6), and to 1.7±0.01 pmol/ml (30 μM CLIC2, n=6). (B) CLIC2 increases [ 3 H]-ryanodine binding to purified RyR1. Equilibrium [ 3 H]-ryanodine binding experiments were carried out in binding buffer containing 2 nM [ 3 H]-ryanodine and various concentrations of Ca 2+ , in the absence or presence of 15 μM CLIC2. [Ca 2+ ] was maintained, in a range between 0.1 μM and 10 mM, by a combination of EGTA and CaCl 2 . Free Ca 2+ Data points shown are the mean ± S.E.M. from three separate experiments. (C) and (D) Equilibrium saturation assay of [ 3 H]-ryanodine binding to purified RyR1. Experiments were carried out in binding buffer containing 10 μM Ca 2+ , and various concentrations of [ 3 H]-ryanodine (from 1 nM to 24 nM), in the absence or presence of 15 μM CLIC2, as described in the “Materials and methods”. Panel C shows the saturation curves for [ 3 H]-ryanodine binding to purified RyR1. Inset are the best-fit values of B max and K d . Panel D shows the Scatchard analysis of panel C. Data points shown are the mean ± S.E.M., from three separate experiments.

    Journal: Journal of molecular biology

    Article Title: CLIC2-RyR1 interaction and structural characterization by cryo-electron microscopy

    doi: 10.1016/j.jmb.2009.01.059

    Figure Lengend Snippet: Effects of CLIC2 on [ 3 H]-ryanodine binding to skeletal heavy SR and purified RyR1 (A) CLIC2 increases [ 3 H]-ryanodine binding to skeletal heavy SR. Equilibrium binding experiments were performed in the absence or presence of CLIC2. The binding buffer contained 2 nM [ 3 H]-ryanodine and 10 μM Ca 2+ . CLIC2 increased [ 3 H]-ryanodine binding to skeletal heavy SR from 1.31±0.1 pmol/ml (buffer, n=6) to 1.57±0.03 pmol/ml (15 μM CLIC2, n=6), and to 1.7±0.01 pmol/ml (30 μM CLIC2, n=6). (B) CLIC2 increases [ 3 H]-ryanodine binding to purified RyR1. Equilibrium [ 3 H]-ryanodine binding experiments were carried out in binding buffer containing 2 nM [ 3 H]-ryanodine and various concentrations of Ca 2+ , in the absence or presence of 15 μM CLIC2. [Ca 2+ ] was maintained, in a range between 0.1 μM and 10 mM, by a combination of EGTA and CaCl 2 . Free Ca 2+ Data points shown are the mean ± S.E.M. from three separate experiments. (C) and (D) Equilibrium saturation assay of [ 3 H]-ryanodine binding to purified RyR1. Experiments were carried out in binding buffer containing 10 μM Ca 2+ , and various concentrations of [ 3 H]-ryanodine (from 1 nM to 24 nM), in the absence or presence of 15 μM CLIC2, as described in the “Materials and methods”. Panel C shows the saturation curves for [ 3 H]-ryanodine binding to purified RyR1. Inset are the best-fit values of B max and K d . Panel D shows the Scatchard analysis of panel C. Data points shown are the mean ± S.E.M., from three separate experiments.

    Article Snippet: Non-specific binding was measured in the presence of a 1000-fold excess of unlabeled ryanodine (Calbiochem, La Jolla, California, USA).

    Techniques: Binding Assay, Purification, Saturation Assay

    Characterization of muscle–nerve co-cultures. Notes: ( A and B ) Characterization of motor neuron formation. ( A ) A representative image of the co-culture cells stained for βIII-tubulin (green) and DAPI (blue); scale bars: 25 µm. ( B ) A representative image of the co-culture cells stained for ChAT (green), α-BTX (red), and DAPI (blue); scale bars: 75 µm. ( C and D ) Characterization of advanced differentiated myotubes. ( C ) A representative image of the co-culture cells stained for DHPR (red), RyR (green), and DAPI (blue); scale bars: 2.5 µm. ( D ) A representative image of the co-culture cells stained for DHPR (red), RyR (green), and DAPI (blue); scale bars: 7.5 µm. ( E ) Characterization of functional NMJs formation. A representative image of the co-culture cells stained for βIII-tubulin (TuJ1; green), α-BTX (red), and DAPI (blue); scale bars: 25 µm. Abbreviations: α-BTX, α-bungarotoxin; ChAT, choline acetyltransferase; DHPR, dihydropyridine receptor; NMJ, neuromuscular junction; RyR, ryanodine receptor.

    Journal: Stem Cells and Cloning : Advances and Applications

    Article Title: A functional human motor unit platform engineered from human embryonic stem cells and immortalized skeletal myoblasts

    doi: 10.2147/SCCAA.S178562

    Figure Lengend Snippet: Characterization of muscle–nerve co-cultures. Notes: ( A and B ) Characterization of motor neuron formation. ( A ) A representative image of the co-culture cells stained for βIII-tubulin (green) and DAPI (blue); scale bars: 25 µm. ( B ) A representative image of the co-culture cells stained for ChAT (green), α-BTX (red), and DAPI (blue); scale bars: 75 µm. ( C and D ) Characterization of advanced differentiated myotubes. ( C ) A representative image of the co-culture cells stained for DHPR (red), RyR (green), and DAPI (blue); scale bars: 2.5 µm. ( D ) A representative image of the co-culture cells stained for DHPR (red), RyR (green), and DAPI (blue); scale bars: 7.5 µm. ( E ) Characterization of functional NMJs formation. A representative image of the co-culture cells stained for βIII-tubulin (TuJ1; green), α-BTX (red), and DAPI (blue); scale bars: 25 µm. Abbreviations: α-BTX, α-bungarotoxin; ChAT, choline acetyltransferase; DHPR, dihydropyridine receptor; NMJ, neuromuscular junction; RyR, ryanodine receptor.

    Article Snippet: The following antibodies were used: mouse anti-βIII-tubulin (MAB1195, clone #TuJ1, 1/400; R & D Systems), rabbit anti-ryanodine receptor (anti-RyR; AB9078, 1/200; Millipore), goat anti-choline acetyltransferase (anti-ChAT; ABN100, 1/200; Millipore), goat anti-ChAT (AB144, 1/200; Millipore), and mouse anti-dihydropyridine receptor (anti-DHPR; Ab2864, 1/400; Abcam).

    Techniques: Co-Culture Assay, Staining, Functional Assay

    LTD-induced phosphorylation of CaMKII and Synapsin I. Induction of LTD for 60 min in control (C) slices caused a significant increase in the phosphorylation levels of Synapsin I (left panels), CaMKII-α (center panels) and CaMKII-β (right panels) relative to the levels displayed by unstimulated slices (naïve). Slices pre-incubated for 1 h with 20 μM ryanodine (Rya) before applying the LTD induction protocol displayed significantly lower increments in the phosphorylation levels of Synapsin I and CaMKII-α, whereas the phosphorylation levels of CaMKII-β were not significantly different from the levels displayed by unstimulated slices. Values represent Mean ± SE ( n = 3). Statistical analysis was performed with one-way ANOVA, followed by Tukey’s post hoc test. * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Ryanodine Receptor-Mediated Calcium Release Has a Key Role in Hippocampal LTD Induction

    doi: 10.3389/fncel.2018.00403

    Figure Lengend Snippet: LTD-induced phosphorylation of CaMKII and Synapsin I. Induction of LTD for 60 min in control (C) slices caused a significant increase in the phosphorylation levels of Synapsin I (left panels), CaMKII-α (center panels) and CaMKII-β (right panels) relative to the levels displayed by unstimulated slices (naïve). Slices pre-incubated for 1 h with 20 μM ryanodine (Rya) before applying the LTD induction protocol displayed significantly lower increments in the phosphorylation levels of Synapsin I and CaMKII-α, whereas the phosphorylation levels of CaMKII-β were not significantly different from the levels displayed by unstimulated slices. Values represent Mean ± SE ( n = 3). Statistical analysis was performed with one-way ANOVA, followed by Tukey’s post hoc test. * p

    Article Snippet: Stock solutions of ryanodine (Tocris, Bristol, UK) and caffeine (Sigma, St. Louis, MO, USA) were dissolved in water and stored in aliquots at −20°C before thawing and dilution to their final concentrations in artificial cerebrospinal fluid (ACSF) solution (in mM: 124 NaCl, 5 KCl, 1 MgCl2 , 2 CaCl2 , 1.25 NaH2 PO4 , 26 NaHCO3 , pH 7.4, 10 glucose).

    Techniques: Incubation

    Effects of stimulatory and inhibitory ryanodine concentrations and of caffeine on fEPSP rise times, half-widths and decay constants tau measured in CA3–CA1 hippocampal synapses. Left panels: slices were treated with 1 μM ryanodine. (A) fEPSP rise times vs. stimulus intensity. (B) fEPSP half-widths vs. stimulus intensity; (C) fEPSP decay constants (tau) vs. stimulus intensity. Central panels: slices were treated with 20 μM ryanodine for 60 min. (D) fEPSP rise times vs. stimulus intensity; (E) fEPSP half-widths vs. stimulus intensity; (F) fEPSP decay constants (tau) vs. stimulus intensity. Right panels: slices were treated for 15 min with 1 mM caffeine. (G) fEPSP rise times vs. stimulus intensity; (H) fEPSP half-widths vs. stimulus intensity; (I) fEPSP decay constants (tau) vs. stimulus intensity. Values represent Mean ± SE; (12, 3). The first number in parentheses indicates the number of hippocampal slices and the second the number of animals used. Statistical significance of values was assessed Mann-Whitney test (* p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Ryanodine Receptor-Mediated Calcium Release Has a Key Role in Hippocampal LTD Induction

    doi: 10.3389/fncel.2018.00403

    Figure Lengend Snippet: Effects of stimulatory and inhibitory ryanodine concentrations and of caffeine on fEPSP rise times, half-widths and decay constants tau measured in CA3–CA1 hippocampal synapses. Left panels: slices were treated with 1 μM ryanodine. (A) fEPSP rise times vs. stimulus intensity. (B) fEPSP half-widths vs. stimulus intensity; (C) fEPSP decay constants (tau) vs. stimulus intensity. Central panels: slices were treated with 20 μM ryanodine for 60 min. (D) fEPSP rise times vs. stimulus intensity; (E) fEPSP half-widths vs. stimulus intensity; (F) fEPSP decay constants (tau) vs. stimulus intensity. Right panels: slices were treated for 15 min with 1 mM caffeine. (G) fEPSP rise times vs. stimulus intensity; (H) fEPSP half-widths vs. stimulus intensity; (I) fEPSP decay constants (tau) vs. stimulus intensity. Values represent Mean ± SE; (12, 3). The first number in parentheses indicates the number of hippocampal slices and the second the number of animals used. Statistical significance of values was assessed Mann-Whitney test (* p

    Article Snippet: Stock solutions of ryanodine (Tocris, Bristol, UK) and caffeine (Sigma, St. Louis, MO, USA) were dissolved in water and stored in aliquots at −20°C before thawing and dilution to their final concentrations in artificial cerebrospinal fluid (ACSF) solution (in mM: 124 NaCl, 5 KCl, 1 MgCl2 , 2 CaCl2 , 1.25 NaH2 PO4 , 26 NaHCO3 , pH 7.4, 10 glucose).

    Techniques: MANN-WHITNEY

    Effects of stimulatory and inhibitory ryanodine concentrations and of caffeine on paired-pulse (PP) responses measured in CA3–CA1 hippocampal synapses. (A) Representative fEPSP traces showing PP responses before and after addition of 1 μM ryanodine (left panels), 20 μM ryanodine (center panels) and 1 mM caffeine (right panels). (B) Effects of 1 μM ryanodine applied for 15 min on PP facilitation. The graph illustrates the facilitation ratio vs. inter-stimulus intervals. (D) Effects of 1 mM caffeine applied for 15 min on PP facilitation. The graph illustrates the facilitation ratio vs. inter-stimulus intervals. (C) Effects of 20 μM ryanodine applied for 60 min on PP facilitation. The graph illustrates the facilitation ratio vs. inter-stimulus intervals. Values represent Mean ± SE; (13, 4) for ryanodine-treated slices; (12, 3) for caffeine-treated slices and (16, 4) for the control. The first number in parentheses indicates the number of hippocampal slices and the second the number of animals used.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Ryanodine Receptor-Mediated Calcium Release Has a Key Role in Hippocampal LTD Induction

    doi: 10.3389/fncel.2018.00403

    Figure Lengend Snippet: Effects of stimulatory and inhibitory ryanodine concentrations and of caffeine on paired-pulse (PP) responses measured in CA3–CA1 hippocampal synapses. (A) Representative fEPSP traces showing PP responses before and after addition of 1 μM ryanodine (left panels), 20 μM ryanodine (center panels) and 1 mM caffeine (right panels). (B) Effects of 1 μM ryanodine applied for 15 min on PP facilitation. The graph illustrates the facilitation ratio vs. inter-stimulus intervals. (D) Effects of 1 mM caffeine applied for 15 min on PP facilitation. The graph illustrates the facilitation ratio vs. inter-stimulus intervals. (C) Effects of 20 μM ryanodine applied for 60 min on PP facilitation. The graph illustrates the facilitation ratio vs. inter-stimulus intervals. Values represent Mean ± SE; (13, 4) for ryanodine-treated slices; (12, 3) for caffeine-treated slices and (16, 4) for the control. The first number in parentheses indicates the number of hippocampal slices and the second the number of animals used.

    Article Snippet: Stock solutions of ryanodine (Tocris, Bristol, UK) and caffeine (Sigma, St. Louis, MO, USA) were dissolved in water and stored in aliquots at −20°C before thawing and dilution to their final concentrations in artificial cerebrospinal fluid (ACSF) solution (in mM: 124 NaCl, 5 KCl, 1 MgCl2 , 2 CaCl2 , 1.25 NaH2 PO4 , 26 NaHCO3 , pH 7.4, 10 glucose).

    Techniques:

    Stimulatory and inhibitory ryanodine concentrations and caffeine modify the long-term depression (LTD) response. (A) Time course of fEPSP slopes recorded (CA3–CA1) before and after application of the low frequency stimulation (LFS) protocol to control hippocampal slices (14, 4) or to slices treated with 1 μM ryanodine (14, 3) or 20 μM ryanodine (13, 4). Representative fEPSP traces recorded 1–5 min before (trace 1) and 60 min after applying the LFS protocol (trace 2) to control slices, or recorded in slices treated with 1 μM or 20 μM ryanodine are shown on top of the graph. Open symbols: control slices; gray symbols: slices treated with 1 μM ryanodine; black symbols: slices treated with 20 μM ryanodine. (B) Average magnitudes of fEPSP slopes recorded during the last 10 min after stimulation. (C) Time course of fEPSP slopes recorded (CA3–CA1) before and after application of the LFS protocol to control hippocampal slices (15, 3) or to slices treated with 1 mM caffeine (12, 4). The first number in parentheses indicates the number of hippocampal slices and the second the number of animals used. Representative fEPSP traces recorded 1–5 min before (trace 1) and 60 min (trace 2) after applying the LFS protocol to control slices and to slices treated with 1 mM caffeine are shown on top of the graph. Open symbols: control slices; black symbols: slices treated with 1 mM caffeine. (D) Average magnitudes of fEPSP slopes recorded during the last 10 min after stimulation of control or caffeine-treated slices. Values represent Mean ± SE. Statistical significance of values was assessed by Mann-Whitney test (* p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Ryanodine Receptor-Mediated Calcium Release Has a Key Role in Hippocampal LTD Induction

    doi: 10.3389/fncel.2018.00403

    Figure Lengend Snippet: Stimulatory and inhibitory ryanodine concentrations and caffeine modify the long-term depression (LTD) response. (A) Time course of fEPSP slopes recorded (CA3–CA1) before and after application of the low frequency stimulation (LFS) protocol to control hippocampal slices (14, 4) or to slices treated with 1 μM ryanodine (14, 3) or 20 μM ryanodine (13, 4). Representative fEPSP traces recorded 1–5 min before (trace 1) and 60 min after applying the LFS protocol (trace 2) to control slices, or recorded in slices treated with 1 μM or 20 μM ryanodine are shown on top of the graph. Open symbols: control slices; gray symbols: slices treated with 1 μM ryanodine; black symbols: slices treated with 20 μM ryanodine. (B) Average magnitudes of fEPSP slopes recorded during the last 10 min after stimulation. (C) Time course of fEPSP slopes recorded (CA3–CA1) before and after application of the LFS protocol to control hippocampal slices (15, 3) or to slices treated with 1 mM caffeine (12, 4). The first number in parentheses indicates the number of hippocampal slices and the second the number of animals used. Representative fEPSP traces recorded 1–5 min before (trace 1) and 60 min (trace 2) after applying the LFS protocol to control slices and to slices treated with 1 mM caffeine are shown on top of the graph. Open symbols: control slices; black symbols: slices treated with 1 mM caffeine. (D) Average magnitudes of fEPSP slopes recorded during the last 10 min after stimulation of control or caffeine-treated slices. Values represent Mean ± SE. Statistical significance of values was assessed by Mann-Whitney test (* p

    Article Snippet: Stock solutions of ryanodine (Tocris, Bristol, UK) and caffeine (Sigma, St. Louis, MO, USA) were dissolved in water and stored in aliquots at −20°C before thawing and dilution to their final concentrations in artificial cerebrospinal fluid (ACSF) solution (in mM: 124 NaCl, 5 KCl, 1 MgCl2 , 2 CaCl2 , 1.25 NaH2 PO4 , 26 NaHCO3 , pH 7.4, 10 glucose).

    Techniques: MANN-WHITNEY

    Effects of stimulatory and inhibitory ryanodine concentrations and of caffeine on basal synaptic transmission in the CA3–CA1 hippocampal synapse. Left panels: slices were treated with 1 μM ryanodine; representative field excitatory postsynaptic potential (fEPSP) traces registered before and 15 min after ryanodine addition are illustrated on top of the panels. (A) Fiber volley (FV) amplitude vs. stimulus intensity; (B) amplitude vs. stimulus intensity; (C) fEPSP slopes vs. stimulus intensity. Central panels: slices were treated with 20 μM ryanodine; representative fEPSP traces registered before and 60 min after ryanodine addition are illustrated on top. (D) FV amplitude vs. stimulus intensity; (E) fEPSP amplitude vs. stimulus intensity; (F) fEPSP slopes vs. stimulus intensity. Right Panels: slices were treated with 1 mM caffeine; representative fEPSP traces registered before and 15 min after caffeine addition are illustrated on top. (G) FV amplitude vs. stimulus intensity; (H) fEPSP amplitude vs. stimulus intensity; (I) fEPSP slopes vs. stimulus intensity. Values represent Mean ± SEM; (12, 3). The first number in parentheses indicates the number of hippocampal slices and the second the number of animals used. Statistical significance of values was assessed by Mann-Whitney test ( p > 0.05 in all cases).

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Ryanodine Receptor-Mediated Calcium Release Has a Key Role in Hippocampal LTD Induction

    doi: 10.3389/fncel.2018.00403

    Figure Lengend Snippet: Effects of stimulatory and inhibitory ryanodine concentrations and of caffeine on basal synaptic transmission in the CA3–CA1 hippocampal synapse. Left panels: slices were treated with 1 μM ryanodine; representative field excitatory postsynaptic potential (fEPSP) traces registered before and 15 min after ryanodine addition are illustrated on top of the panels. (A) Fiber volley (FV) amplitude vs. stimulus intensity; (B) amplitude vs. stimulus intensity; (C) fEPSP slopes vs. stimulus intensity. Central panels: slices were treated with 20 μM ryanodine; representative fEPSP traces registered before and 60 min after ryanodine addition are illustrated on top. (D) FV amplitude vs. stimulus intensity; (E) fEPSP amplitude vs. stimulus intensity; (F) fEPSP slopes vs. stimulus intensity. Right Panels: slices were treated with 1 mM caffeine; representative fEPSP traces registered before and 15 min after caffeine addition are illustrated on top. (G) FV amplitude vs. stimulus intensity; (H) fEPSP amplitude vs. stimulus intensity; (I) fEPSP slopes vs. stimulus intensity. Values represent Mean ± SEM; (12, 3). The first number in parentheses indicates the number of hippocampal slices and the second the number of animals used. Statistical significance of values was assessed by Mann-Whitney test ( p > 0.05 in all cases).

    Article Snippet: Stock solutions of ryanodine (Tocris, Bristol, UK) and caffeine (Sigma, St. Louis, MO, USA) were dissolved in water and stored in aliquots at −20°C before thawing and dilution to their final concentrations in artificial cerebrospinal fluid (ACSF) solution (in mM: 124 NaCl, 5 KCl, 1 MgCl2 , 2 CaCl2 , 1.25 NaH2 PO4 , 26 NaHCO3 , pH 7.4, 10 glucose).

    Techniques: Transmission Assay, MANN-WHITNEY

    Depletion of the SR of Ca 2+ changes does not reduce the [Ca 2+ ] increases in subplasma membrane space or bulk cytoplasm in voltage-clamped single colonic myocytes. Depolarization (−70 to +10 mV; G) activated a voltage-dependent Ca 2+ current (I Ca ; F) to evoke a rise in [Ca 2+ ] (C and D). The rise in [Ca 2+ ], which occurred in some regions (B, left panels) of the subplasma membrane space (measured by TIRF; red line in inset in C), were more rapid in both onset and decline than that seen in average measured in the subplasma membrane space (black red line in inset in C; B and E). The same regions in the bulk cytoplasm (measured by wide-field epi-florescence; B, left panels) had approximately comparable rates of rise and decline (D, inset). 10 mM caffeine (E) applied by pressure ejection from a puffer pipette (A, left side; see also whole cell patch electrode [right side]) evoked increases in [Ca 2+ ] (C and D). 50 µM ryanodine (C, open bar above the trace) after repeated application of caffeine to open RYR abolished the [Ca 2+ ] increase to caffeine presumably because of SR store depletion. In contrast, the depolarization-evoked [Ca 2+ ] increase was not reduced in either subplasma membrane space or bulk cytoplasm. After inhibition of the caffeine-evoked [Ca 2+ ] increase, the depolarization-evoked [Ca 2+ ] PM or [Ca 2+ ] c increase were not reduced. Furthermore, localized increases in regions of the subplasma membrane space measured by TIRF (red line in inset in C) were more rapid in onset and decline than those seen in average measured in the subplasma membrane space (black red line in inset in C; B and E). This result suggests that SR Ca 2+ release does not contribute substantially to the depolarization-evoked [Ca 2+ ] PM or [Ca 2+ ] c increases. Changes in the fluorescence ratio with time (C and D) are derived from 1-pixel boxes (B) and from a larger region encompassing the entire TIRF region (B). The latter was used to obtain an average subplasma membrane and bulk average [Ca 2+ ] increases (C and D). (A; left) A bright field image of the cell; see also whole cell electrode (right side). B shows an expanded view of A (middle and right panels) to illustrate the regions of measurement. Note the pixel position is in a different position after SR depletion. The cell moved slightly during solution exchanges, and a new voltage-dependent Ca 2+ channel cluster may have emerged.

    Journal: The Journal of General Physiology

    Article Title: Elevations of intracellular calcium reflect normal voltage-dependent behavior, and not constitutive activity, of voltage-dependent calcium channels in gastrointestinal and vascular smooth muscle

    doi: 10.1085/jgp.200810189

    Figure Lengend Snippet: Depletion of the SR of Ca 2+ changes does not reduce the [Ca 2+ ] increases in subplasma membrane space or bulk cytoplasm in voltage-clamped single colonic myocytes. Depolarization (−70 to +10 mV; G) activated a voltage-dependent Ca 2+ current (I Ca ; F) to evoke a rise in [Ca 2+ ] (C and D). The rise in [Ca 2+ ], which occurred in some regions (B, left panels) of the subplasma membrane space (measured by TIRF; red line in inset in C), were more rapid in both onset and decline than that seen in average measured in the subplasma membrane space (black red line in inset in C; B and E). The same regions in the bulk cytoplasm (measured by wide-field epi-florescence; B, left panels) had approximately comparable rates of rise and decline (D, inset). 10 mM caffeine (E) applied by pressure ejection from a puffer pipette (A, left side; see also whole cell patch electrode [right side]) evoked increases in [Ca 2+ ] (C and D). 50 µM ryanodine (C, open bar above the trace) after repeated application of caffeine to open RYR abolished the [Ca 2+ ] increase to caffeine presumably because of SR store depletion. In contrast, the depolarization-evoked [Ca 2+ ] increase was not reduced in either subplasma membrane space or bulk cytoplasm. After inhibition of the caffeine-evoked [Ca 2+ ] increase, the depolarization-evoked [Ca 2+ ] PM or [Ca 2+ ] c increase were not reduced. Furthermore, localized increases in regions of the subplasma membrane space measured by TIRF (red line in inset in C) were more rapid in onset and decline than those seen in average measured in the subplasma membrane space (black red line in inset in C; B and E). This result suggests that SR Ca 2+ release does not contribute substantially to the depolarization-evoked [Ca 2+ ] PM or [Ca 2+ ] c increases. Changes in the fluorescence ratio with time (C and D) are derived from 1-pixel boxes (B) and from a larger region encompassing the entire TIRF region (B). The latter was used to obtain an average subplasma membrane and bulk average [Ca 2+ ] increases (C and D). (A; left) A bright field image of the cell; see also whole cell electrode (right side). B shows an expanded view of A (middle and right panels) to illustrate the regions of measurement. Note the pixel position is in a different position after SR depletion. The cell moved slightly during solution exchanges, and a new voltage-dependent Ca 2+ channel cluster may have emerged.

    Article Snippet: Ryanodine was purchased from Enzo Biochem, Inc. Fluo-5F acetoxymethylester was purchased from Invitrogen.

    Techniques: Transferring, Inhibition, Fluorescence, Derivative Assay

    Depolymerization of microtubules decreases the number of BKα and RyR2 colocalization sites. ( A ) Wide-field image of a freshly isolated arterial myocyte immunolabeled for BKα ( n = 12 cells, n = 3 animals). Red box indicates the area where superresolution images were obtained. Scale bar, 10 μm. ( B ) Superresolution localization map obtained after immunolabeling with anti-BKμ (green) and anti-RyR2 (red) antibodies ( n = 12 cells, n = 3 animals). Scale bar, 3 μm. ROIs (yellow boxes) are shown in magnified view (I) and (II) below. Scale bars, 0.2 μm. ( C ) Cluster size distribution histograms of RyR2 and BKα ( n = 11,005 or n = 11,940 particles for RyR2 and BKα, respectively; 12 cells, n = 3 animals). ( D ) Summary of object-based analysis to determine the number of BKα protein cluster centroids per cell that overlay with RyR2 protein clusters in control cells, cells in which a random BKα distribution has been simulated, and cells treated with nocodazole (10 μM). * P ≤ 0.05 compared to control ( n =12 cells per group, 3 animals). Coloc., colocalization.

    Journal: Science signaling

    Article Title: Microtubule structures underlying the sarcoplasmic reticulum support peripheral coupling sites to regulate smooth muscle contractility

    doi: 10.1126/scisignal.aan2694

    Figure Lengend Snippet: Depolymerization of microtubules decreases the number of BKα and RyR2 colocalization sites. ( A ) Wide-field image of a freshly isolated arterial myocyte immunolabeled for BKα ( n = 12 cells, n = 3 animals). Red box indicates the area where superresolution images were obtained. Scale bar, 10 μm. ( B ) Superresolution localization map obtained after immunolabeling with anti-BKμ (green) and anti-RyR2 (red) antibodies ( n = 12 cells, n = 3 animals). Scale bar, 3 μm. ROIs (yellow boxes) are shown in magnified view (I) and (II) below. Scale bars, 0.2 μm. ( C ) Cluster size distribution histograms of RyR2 and BKα ( n = 11,005 or n = 11,940 particles for RyR2 and BKα, respectively; 12 cells, n = 3 animals). ( D ) Summary of object-based analysis to determine the number of BKα protein cluster centroids per cell that overlay with RyR2 protein clusters in control cells, cells in which a random BKα distribution has been simulated, and cells treated with nocodazole (10 μM). * P ≤ 0.05 compared to control ( n =12 cells per group, 3 animals). Coloc., colocalization.

    Article Snippet: Cells were fixed with 3.2% formaldehyde/0.1% glutaraldehyde–phosphate-buffered saline (PBS), permeabilized and blocked with 0.2% saponin/5% horse serum–PBS, and incubated with primary antibodies against α-tubulin (1:100; MA1-80017, Life Technologies), BKα (1:100; APC-021, Alomone Labs), and RyR2 (1:100; ab2868, Abcam).

    Techniques: Isolation, Immunolabeling

    RyR2 protein clusters selectively align with microtubules. ( A ) Representative images (offive cells from n = 3 animals) of an isolated native cerebral arterial myocyte immunolabeled with anti-α-tubulin (red). The image on the left is a wide-field image. The ROI in the yellow box was imaged using GSDIM. Scale bar, 10 μm. Center: Superresolution image of the ROI. Scale bar, 3 μm. Magnified views of the indicated ROIs depicting arching microtubule structures are shown on the right. Scale bar, 0.2 μm. ( B ) Representative superresolution images (of five cells from n = 3 animals) of an isolated native cerebral arterial myocyte immunolabeled with anti-α-tubulin antibody (red), anti-RyR2 antibody (green), and the overlay. Scale bar, 3 μm. ROIs (yellow boxes) are shown at the right. Scale bar, 0.2 μm.

    Journal: Science signaling

    Article Title: Microtubule structures underlying the sarcoplasmic reticulum support peripheral coupling sites to regulate smooth muscle contractility

    doi: 10.1126/scisignal.aan2694

    Figure Lengend Snippet: RyR2 protein clusters selectively align with microtubules. ( A ) Representative images (offive cells from n = 3 animals) of an isolated native cerebral arterial myocyte immunolabeled with anti-α-tubulin (red). The image on the left is a wide-field image. The ROI in the yellow box was imaged using GSDIM. Scale bar, 10 μm. Center: Superresolution image of the ROI. Scale bar, 3 μm. Magnified views of the indicated ROIs depicting arching microtubule structures are shown on the right. Scale bar, 0.2 μm. ( B ) Representative superresolution images (of five cells from n = 3 animals) of an isolated native cerebral arterial myocyte immunolabeled with anti-α-tubulin antibody (red), anti-RyR2 antibody (green), and the overlay. Scale bar, 3 μm. ROIs (yellow boxes) are shown at the right. Scale bar, 0.2 μm.

    Article Snippet: Cells were fixed with 3.2% formaldehyde/0.1% glutaraldehyde–phosphate-buffered saline (PBS), permeabilized and blocked with 0.2% saponin/5% horse serum–PBS, and incubated with primary antibodies against α-tubulin (1:100; MA1-80017, Life Technologies), BKα (1:100; APC-021, Alomone Labs), and RyR2 (1:100; ab2868, Abcam).

    Techniques: Isolation, Immunolabeling

    Ryanodine receptor immunoreaction in the SCN and the SON of WT and BMAL1 −/− mice. a , Representative microphotographs and quantification of the RyR-IR in the SCN of BMAL1 +/+ (black circles) and BMAL1 −/− (white circles) mice determined every 6 h during a 12 h L/D schedule. Scale bar, 100 μm. oc, Optic chiasm; 3V, third ventricle. ** p = 0.01; *** p = 0.001. b , Representative microphotographs and quantification of the RyR-IR in the SON of BMAL1 +/+ (black bars) and BMAL1 −/− (white bars) mice determined every 6 h during a 12 h L/D schedule. Scale bar, 50 μm. oc, Optic chiasm.

    Journal: The Journal of Neuroscience

    Article Title: The Mammalian Molecular Clockwork Controls Rhythmic Expression of Its Own Input Pathway Components

    doi: 10.1523/JNEUROSCI.0275-09.2009

    Figure Lengend Snippet: Ryanodine receptor immunoreaction in the SCN and the SON of WT and BMAL1 −/− mice. a , Representative microphotographs and quantification of the RyR-IR in the SCN of BMAL1 +/+ (black circles) and BMAL1 −/− (white circles) mice determined every 6 h during a 12 h L/D schedule. Scale bar, 100 μm. oc, Optic chiasm; 3V, third ventricle. ** p = 0.01; *** p = 0.001. b , Representative microphotographs and quantification of the RyR-IR in the SON of BMAL1 +/+ (black bars) and BMAL1 −/− (white bars) mice determined every 6 h during a 12 h L/D schedule. Scale bar, 50 μm. oc, Optic chiasm.

    Article Snippet: Immunohistochemistry was performed with a primary anti-ryanodine receptor antibody (ab2868; Abcam) in a dilution of 1:100.

    Techniques: Mouse Assay

    The ER Ca 2+ store regulates lysosome Ca 2+ stores. ( A ) In un-transfected HEK293T cells, ATP induced Ca 2+ release through IP3-receptors on the ER, and GPN induced lysosomal Ca 2+ release. ( B ) A 2-min application of TPEN, a membrane-permeable chelator of luminal ER Ca 2+ , attenuated Ca 2+ release from IP3-receptors stimulated by ATP in HEK293T cells. ( C ) A 2-min TPEN application did not significantly reduce GPN-induced lysosomal Ca 2+ release in HEK293T cells. ( D ) Long-term TPEN treatment (20 min) abolished ER Ca 2+ release upon ATP stimulation and GPN-induced lysosomal Ca 2+ release in HEK293T cells loaded with Fura-2. ( E ) In HEK293T cells transfected with the IP3R-ligand binding domain with ER targeting sequence (IP3R-LBD-ER), the responses to ATP and GPN were reduced compared to un-transfected cells on the same coverslip. ( F ) Caffeine stimulates Ca 2+ release from ryanodine receptors and ATP stimulates Ca 2+ release from IP3Rs in HEK-GCaMP3-ML1 cells loaded with Fura-2. Panels A – C and E show the average response of 30–40 cells from one representative experiment. DOI: http://dx.doi.org/10.7554/eLife.15887.011

    Journal: eLife

    Article Title: The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes

    doi: 10.7554/eLife.15887

    Figure Lengend Snippet: The ER Ca 2+ store regulates lysosome Ca 2+ stores. ( A ) In un-transfected HEK293T cells, ATP induced Ca 2+ release through IP3-receptors on the ER, and GPN induced lysosomal Ca 2+ release. ( B ) A 2-min application of TPEN, a membrane-permeable chelator of luminal ER Ca 2+ , attenuated Ca 2+ release from IP3-receptors stimulated by ATP in HEK293T cells. ( C ) A 2-min TPEN application did not significantly reduce GPN-induced lysosomal Ca 2+ release in HEK293T cells. ( D ) Long-term TPEN treatment (20 min) abolished ER Ca 2+ release upon ATP stimulation and GPN-induced lysosomal Ca 2+ release in HEK293T cells loaded with Fura-2. ( E ) In HEK293T cells transfected with the IP3R-ligand binding domain with ER targeting sequence (IP3R-LBD-ER), the responses to ATP and GPN were reduced compared to un-transfected cells on the same coverslip. ( F ) Caffeine stimulates Ca 2+ release from ryanodine receptors and ATP stimulates Ca 2+ release from IP3Rs in HEK-GCaMP3-ML1 cells loaded with Fura-2. Panels A – C and E show the average response of 30–40 cells from one representative experiment. DOI: http://dx.doi.org/10.7554/eLife.15887.011

    Article Snippet: 2-APB, ATP, Con-A, CPA, Doxycycline, DHBP, TG, TPEN were from Sigma; GPN and U73122 were from Santa Cruz; Ryanodine was from Abcam; LysoTracker, Fura-2, Mitotracker, Plurionic F-127, and Fura-Dextran were from Invitrogen; Baf-A was from LC Laboratories; ML-SA1 was from Chembridge; and Xestospongin-C was from Cayman Chemical, AG Scientific, and Enzo; Oregon Green 488 BAPTA-1 dextran was from life technologies.

    Techniques: Transfection, Ligand Binding Assay, Sequencing

    Lysosomal Ca2+ refilling is compromised in IP3R TKO DT40 cells. ( A ) Ryanodine receptor blocker DHBP (50 μM) did not block Ca 2+ refilling of lysosomes. ( B ) Quantification of the 1st, 2nd and 3rd ML-SA1 responses in GCaMP3-ML1-transfected WT and IP3R-TKO DT40 cells ( Figure 3 — source data 1 ). ( C ) Time-dependence of lysosomal Ca 2+ store refilling in WT and IP3R TKO DT40 cells. ( D ) GCaMP3-ML1-transfected IP3R-TKO DT40 cells still showed refilling after 5 min of DHBP application to block RYRs. ( E ) RYR inhibitors Diltiazem (50 µM) and Dantrolene (50 µM) did not block lysosomal Ca 2+ refilling in GCaMP3-ML1-transfected IP3R-TKO DT40 cells. Panels A , D and E show the average response of 30–40 cells from one representative experiment. DOI: http://dx.doi.org/10.7554/eLife.15887.015

    Journal: eLife

    Article Title: The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes

    doi: 10.7554/eLife.15887

    Figure Lengend Snippet: Lysosomal Ca2+ refilling is compromised in IP3R TKO DT40 cells. ( A ) Ryanodine receptor blocker DHBP (50 μM) did not block Ca 2+ refilling of lysosomes. ( B ) Quantification of the 1st, 2nd and 3rd ML-SA1 responses in GCaMP3-ML1-transfected WT and IP3R-TKO DT40 cells ( Figure 3 — source data 1 ). ( C ) Time-dependence of lysosomal Ca 2+ store refilling in WT and IP3R TKO DT40 cells. ( D ) GCaMP3-ML1-transfected IP3R-TKO DT40 cells still showed refilling after 5 min of DHBP application to block RYRs. ( E ) RYR inhibitors Diltiazem (50 µM) and Dantrolene (50 µM) did not block lysosomal Ca 2+ refilling in GCaMP3-ML1-transfected IP3R-TKO DT40 cells. Panels A , D and E show the average response of 30–40 cells from one representative experiment. DOI: http://dx.doi.org/10.7554/eLife.15887.015

    Article Snippet: 2-APB, ATP, Con-A, CPA, Doxycycline, DHBP, TG, TPEN were from Sigma; GPN and U73122 were from Santa Cruz; Ryanodine was from Abcam; LysoTracker, Fura-2, Mitotracker, Plurionic F-127, and Fura-Dextran were from Invitrogen; Baf-A was from LC Laboratories; ML-SA1 was from Chembridge; and Xestospongin-C was from Cayman Chemical, AG Scientific, and Enzo; Oregon Green 488 BAPTA-1 dextran was from life technologies.

    Techniques: Blocking Assay, Transfection

    Blocking ER IP3-receptors Ca 2+ channels refill lysosome Ca 2+ stores to prevent lysosomal dysfunction. ( A ) Upper panels: Western blotting analyses of Lamp1 in HEK293T cells treated with 2-APB (50 μM), TPEN (0.1 μM), Xesto (10 μM), and DHBP (5 μM) compared to DMSO for 24 hr (n=4 separate experiments for each condition). Lower panel: treating HEK293T cells with 2-APB (p=0.05) and Xesto (p=0.013), as well as TPEN (p=0.02), significantly increased Lamp1 expression. DHBP did not significantly change Lamp1 expression (p=0.23) ( Figure 4 — source data 1 ). ( B ) The effects of Xesto (10 μM, 18 hr; p=0.0001) and DHBP (50 μM, 18 hr; p=0.063) treatment compared to DMSO on the lysosomal compartments detected by LysoTracker staining in HEK293T cells (average of 20–30 cells in each of 3 experiments; Figure 4 — source data 1 ). Scale bar = 15 μm. ( C ) The effect of Xesto (10 μM, 18 h) treatment on accumulation of the autofluorescent lipofuscin materials in non-transfected HEK293T cells. Autofluorescence was observed in a wide spectrum but shown at two excitation wavelengths (488 and 561 nm). ML1 KO MEFs are shown for comparison. Scale bar = 15 μm. ( D ) A proposed model of Ca 2+ transfer from the ER to lysosomes. The ER is a Ca 2+ store with [Ca 2+ ] ER ~ 0.3–0.7 mM; lysosomes are acidic (pH Ly ~ 4.6) Ca 2+ stores ([Ca 2+ ] Ly ~ 0.5 mM). IP3Rs on the ER release Ca 2+ to produce a local high Ca 2+ concentration, from which an unknown low-affinity Ca 2+ transport mechanism refills Ca 2+ to a lysosome. Unidentified tether proteins may link the ER membrane proteins directly with the lysosomal membrane proteins to maintain contact sites of 20–30 nm for purposes of Ca 2+ exchange. Ca 2+ released from lysosomes or a reduction/depletion in [Ca 2+ ] Ly may, through unidentified mechanisms, 'promote' or 'stabilize' ER-lysosome interaction ( Phillips and Voeltz, 2016 ; Eden, 2016 ). At the functional ER-lysosome contact sites, Ca 2+ can be transferred from the ER to lysosomes through a passive Ca 2+ transporter or channel based on the large chemical gradient of Ca 2+ that is created when lysosome stores are depleted. Baf-A and Con-A are specific V-ATPase inhibitors; Xesto and 2APB are IP3R blockers; U73122 is a PLC inhibitor that blocks the constitutive production of IP3; DHBP and Ryanodine ( > 10 μM) are specific RyR blockers; TG and CPA are SERCA pump inhibitors; and TPEN is a luminal Ca 2+ chelator. DOI: http://dx.doi.org/10.7554/eLife.15887.017 10.7554/eLife.15887.018 Source data of Figure 4A,B : Quantifications of Lamp-1 protein levels ( A ) or LysoTracker staining ( B ) under different experimental conditions and manipulations. DOI: http://dx.doi.org/10.7554/eLife.15887.018

    Journal: eLife

    Article Title: The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes

    doi: 10.7554/eLife.15887

    Figure Lengend Snippet: Blocking ER IP3-receptors Ca 2+ channels refill lysosome Ca 2+ stores to prevent lysosomal dysfunction. ( A ) Upper panels: Western blotting analyses of Lamp1 in HEK293T cells treated with 2-APB (50 μM), TPEN (0.1 μM), Xesto (10 μM), and DHBP (5 μM) compared to DMSO for 24 hr (n=4 separate experiments for each condition). Lower panel: treating HEK293T cells with 2-APB (p=0.05) and Xesto (p=0.013), as well as TPEN (p=0.02), significantly increased Lamp1 expression. DHBP did not significantly change Lamp1 expression (p=0.23) ( Figure 4 — source data 1 ). ( B ) The effects of Xesto (10 μM, 18 hr; p=0.0001) and DHBP (50 μM, 18 hr; p=0.063) treatment compared to DMSO on the lysosomal compartments detected by LysoTracker staining in HEK293T cells (average of 20–30 cells in each of 3 experiments; Figure 4 — source data 1 ). Scale bar = 15 μm. ( C ) The effect of Xesto (10 μM, 18 h) treatment on accumulation of the autofluorescent lipofuscin materials in non-transfected HEK293T cells. Autofluorescence was observed in a wide spectrum but shown at two excitation wavelengths (488 and 561 nm). ML1 KO MEFs are shown for comparison. Scale bar = 15 μm. ( D ) A proposed model of Ca 2+ transfer from the ER to lysosomes. The ER is a Ca 2+ store with [Ca 2+ ] ER ~ 0.3–0.7 mM; lysosomes are acidic (pH Ly ~ 4.6) Ca 2+ stores ([Ca 2+ ] Ly ~ 0.5 mM). IP3Rs on the ER release Ca 2+ to produce a local high Ca 2+ concentration, from which an unknown low-affinity Ca 2+ transport mechanism refills Ca 2+ to a lysosome. Unidentified tether proteins may link the ER membrane proteins directly with the lysosomal membrane proteins to maintain contact sites of 20–30 nm for purposes of Ca 2+ exchange. Ca 2+ released from lysosomes or a reduction/depletion in [Ca 2+ ] Ly may, through unidentified mechanisms, 'promote' or 'stabilize' ER-lysosome interaction ( Phillips and Voeltz, 2016 ; Eden, 2016 ). At the functional ER-lysosome contact sites, Ca 2+ can be transferred from the ER to lysosomes through a passive Ca 2+ transporter or channel based on the large chemical gradient of Ca 2+ that is created when lysosome stores are depleted. Baf-A and Con-A are specific V-ATPase inhibitors; Xesto and 2APB are IP3R blockers; U73122 is a PLC inhibitor that blocks the constitutive production of IP3; DHBP and Ryanodine ( > 10 μM) are specific RyR blockers; TG and CPA are SERCA pump inhibitors; and TPEN is a luminal Ca 2+ chelator. DOI: http://dx.doi.org/10.7554/eLife.15887.017 10.7554/eLife.15887.018 Source data of Figure 4A,B : Quantifications of Lamp-1 protein levels ( A ) or LysoTracker staining ( B ) under different experimental conditions and manipulations. DOI: http://dx.doi.org/10.7554/eLife.15887.018

    Article Snippet: 2-APB, ATP, Con-A, CPA, Doxycycline, DHBP, TG, TPEN were from Sigma; GPN and U73122 were from Santa Cruz; Ryanodine was from Abcam; LysoTracker, Fura-2, Mitotracker, Plurionic F-127, and Fura-Dextran were from Invitrogen; Baf-A was from LC Laboratories; ML-SA1 was from Chembridge; and Xestospongin-C was from Cayman Chemical, AG Scientific, and Enzo; Oregon Green 488 BAPTA-1 dextran was from life technologies.

    Techniques: Blocking Assay, Western Blot, Expressing, Staining, Transfection, Concentration Assay, Functional Assay, Planar Chromatography

    Changes in anti-glial fibrillary acidic protein (GFAP)-positive astrocytes (a), GFAP-positive area fraction (b), and ionized calcium binding adaptor molecule 1 (Iba-1)-positive microglia (c) in the intragyral white matter of the first and second parasagittal gyri (IGWM 1 and IGWM 2, respectively) and the periventricular white matter (PVWM). Sham-control group, white bars ( n = 7), asphyxia-vehicle group, black bars ( n = 6) and asphyxia-hAEC group, striped bars ( n = 6). Photomicrographs from the IGWM of GFAP (d) and Iba-1-positive microglia (e). Asphyxia was associated with a significant increase in GFAP-positive astrocyte area and numbers of Iba-1-positive microglia. hAEC treatment significantly attenuated this increase in GFAP-positive astrocytes and Iba-1-positive microglia. Arrows show GFAP-positive or Iba-1-positive cells, respectively. Data are mean ± SEM. * P

    Journal: Journal of Cerebral Blood Flow & Metabolism

    Article Title: Delayed intranasal infusion of human amnion epithelial cells improves white matter maturation after asphyxia in preterm fetal sheep

    doi: 10.1177/0271678X17729954

    Figure Lengend Snippet: Changes in anti-glial fibrillary acidic protein (GFAP)-positive astrocytes (a), GFAP-positive area fraction (b), and ionized calcium binding adaptor molecule 1 (Iba-1)-positive microglia (c) in the intragyral white matter of the first and second parasagittal gyri (IGWM 1 and IGWM 2, respectively) and the periventricular white matter (PVWM). Sham-control group, white bars ( n = 7), asphyxia-vehicle group, black bars ( n = 6) and asphyxia-hAEC group, striped bars ( n = 6). Photomicrographs from the IGWM of GFAP (d) and Iba-1-positive microglia (e). Asphyxia was associated with a significant increase in GFAP-positive astrocyte area and numbers of Iba-1-positive microglia. hAEC treatment significantly attenuated this increase in GFAP-positive astrocytes and Iba-1-positive microglia. Arrows show GFAP-positive or Iba-1-positive cells, respectively. Data are mean ± SEM. * P

    Article Snippet: Sections were labeled with monoclonal antibodies; 1:200 mouse anti- neuronal nuclear antigen (NeuN, Chemicon International, Temecula, CA, USA), 1:200 mouse anti-synaptophysin (Merck-Millipore, Billerica, MA, USA), 1:200 rabbit anti-cleaved caspase-3 (Chemicon International), 1:400 rabbit anti-oligodendrocyte transcription factor 2 (Olig-2, Merck-Millipore), 1:200 mouse anti-CNPase (Merck-Millipore), 1:200 rabbit anti-myelin basic protein (MBP, Merck-Millipore), 1:200 rabbit anti-glial fibrillary acidic protein (GFAP, Abcam, Cambridge, United Kingdom), 1:200 goat anti-Iba-1 (Abcam), 1:200 mouse anti-tumor necrosis factor alpha (TNFα, AbD Serotec, Pucheim, Germany) or 1:200 mouse anti-human cytoplasmic marker STEM121 (Stem Cells Inc. Newark, CA, USA) overnight at 4℃.

    Techniques: Binding Assay

    Representative immunohistochemistry for phosphorylated neurofilament (SMI31) ( A, C ), myelin basic protein (MBP) ( B, D ), glial fibrillary acidic protein (GFAP) ( E, H ), ionized calcium-binding adaptor molecule 1 (IBA1) ( F, I ) and rat endothelial cell antigen (RECA1) ( G, J ) in the corpus callosum of normal ( A, B, E-G ) and ventriculomegaly ( C, D, H-J ) rats. The increased water content was associated with the loss of SMI31 staining in the corpus callosum of ventriculomegaly rats ( A vs. C ). No significant difference is found in the MBP, GFAP, Iba1, and RECA1 immunostaining between the normal and hydrocephalic rats. Ventriculomegaly rats have significantly thinner corpus callosum (0.47 ± 0.03 mm) vs. the normal rats (0.61 ± 0.04 mm) ( B, D ). The high-magnification images in A-J were taken from the region indicated in A . Scale bars: A-J , 250 μm (30 μm in magnified insets in A-J ).

    Journal: Journal of neuropathology and experimental neurology

    Article Title: Imaging of Spontaneous Ventriculomegaly and Vascular Malformations in Wistar rats: implications for Preclinical Research

    doi: 10.1097/NEN.0000000000000140

    Figure Lengend Snippet: Representative immunohistochemistry for phosphorylated neurofilament (SMI31) ( A, C ), myelin basic protein (MBP) ( B, D ), glial fibrillary acidic protein (GFAP) ( E, H ), ionized calcium-binding adaptor molecule 1 (IBA1) ( F, I ) and rat endothelial cell antigen (RECA1) ( G, J ) in the corpus callosum of normal ( A, B, E-G ) and ventriculomegaly ( C, D, H-J ) rats. The increased water content was associated with the loss of SMI31 staining in the corpus callosum of ventriculomegaly rats ( A vs. C ). No significant difference is found in the MBP, GFAP, Iba1, and RECA1 immunostaining between the normal and hydrocephalic rats. Ventriculomegaly rats have significantly thinner corpus callosum (0.47 ± 0.03 mm) vs. the normal rats (0.61 ± 0.04 mm) ( B, D ). The high-magnification images in A-J were taken from the region indicated in A . Scale bars: A-J , 250 μm (30 μm in magnified insets in A-J ).

    Article Snippet: The antibodies used were to: ionized calcium-binding adaptor molecule 1 (Iba1) (Wako, Richmond, VA) at 1/200 (for activated microglia), glial fibrillary acidic protein (GFAP) (Abcam, Cambridge, MA) at 1/1500, (for astrocytes), (SMI31) (Covance, Princeton, NJ) at 1/1500, (for phosphorylated neurofilament H), hexaribonucleotide binding protein-3 (NeuN) (Abcam) at 1/1000, (for neurons), rat endothelial cell antigen (RECA1) (Abcam) at 1/750 (for vasculature), or myelin basic protein (MBP) (Abcam) at 1/500.

    Techniques: Immunohistochemistry, Binding Assay, Staining, Immunostaining

    Action potential-induced CICR at somatic plasma membrane but not AIS or dendrites (A) Two-photon Ca 2+ imaging at somatic plasma membrane. (Ai) Maximum intensity projection of Alexa-594-filled cartwheel cell. The red boxed region is enlarged in Ai, inset. Regions of interest for segmented line scans are indicated by red lines. C: cytosolic side. M: membrane side. (Aii, top and middle panels) Spike trains evoked by current injection (top) elicited an increase of Fluo-5F fluorescence with no change in Alexa-594. (Aii, bottom) Ca 2+ transients induced by spike trains (6 simple spikes at 50 Hz). The transients are expressed as ΔG/R (change in Fluo-5F intensity divided by Alexa-594 intensity). Black, control; blue, in ryanodine. (Aiii) Averaged Ca 2+ transients from 10 regions of interest of 5 cells. Single spike or trains of simple spikes (6 spikes at 50 Hz) evoked by current injection. (Aiv) Summary of the changes in Ca 2+ transients. *** p

    Journal: Neuron

    Article Title: Double nanodomain coupling of calcium channels, ryanodine receptors and BK channels controls generation of burst firing

    doi: 10.1016/j.neuron.2017.10.014

    Figure Lengend Snippet: Action potential-induced CICR at somatic plasma membrane but not AIS or dendrites (A) Two-photon Ca 2+ imaging at somatic plasma membrane. (Ai) Maximum intensity projection of Alexa-594-filled cartwheel cell. The red boxed region is enlarged in Ai, inset. Regions of interest for segmented line scans are indicated by red lines. C: cytosolic side. M: membrane side. (Aii, top and middle panels) Spike trains evoked by current injection (top) elicited an increase of Fluo-5F fluorescence with no change in Alexa-594. (Aii, bottom) Ca 2+ transients induced by spike trains (6 simple spikes at 50 Hz). The transients are expressed as ΔG/R (change in Fluo-5F intensity divided by Alexa-594 intensity). Black, control; blue, in ryanodine. (Aiii) Averaged Ca 2+ transients from 10 regions of interest of 5 cells. Single spike or trains of simple spikes (6 spikes at 50 Hz) evoked by current injection. (Aiv) Summary of the changes in Ca 2+ transients. *** p

    Article Snippet: The amplitudes of current pulses were carefully adjusted so as not to induce burst firings even in the presence of ryanodine (Alomone labs) or CPA, and therefore each pulse evoked single action potential.

    Techniques: Imaging, Injection, Fluorescence

    The effects of ryanodine and IbTX on action potential properties (A and B) Evoked action potentials recorded in perforated patch mode. Resting potential was slightly hyperpolarized by injecting negative current to suppress spontaneous firing (A). Traces recorded in control (black) and ryanodine (gray) are superimposed. Injected currents in (Ai) and (Aii) were 500 and 900 pA, respectively. Duration: 5 ms. Asterisk indicates fAHP between 1st and 2nd spikelets. The region surrounded with box in (Ai) was expanded in the inset. The inset in (Aii) includes a longer segment of the recording to illustrate the slow afterpotential. (Aiii) Summary of the change of burst firing probability by ryanodine. Three to four successive trials were used to obtain averaged probability in each experiment. (B) The effect of ryanodine was occluded by IbTX. Bath application of IbTX (100 nM) alone broadened 1st action potentials (Bi and Bii, gray traces) and made the fAHP less negative (Bii,, asterisk). Subsequent application of ryanodine in the presence of IbTX did not affect the waveform (Bi’ and Bii’, black traces). Inset in (Bii) is the same sweep but displayed with longer time base, with spikes truncated. (Biii) Summary of the change of burst firing probabilities by IbTX. Statistical significance was tested between control and IbTX.

    Journal: Neuron

    Article Title: Double nanodomain coupling of calcium channels, ryanodine receptors and BK channels controls generation of burst firing

    doi: 10.1016/j.neuron.2017.10.014

    Figure Lengend Snippet: The effects of ryanodine and IbTX on action potential properties (A and B) Evoked action potentials recorded in perforated patch mode. Resting potential was slightly hyperpolarized by injecting negative current to suppress spontaneous firing (A). Traces recorded in control (black) and ryanodine (gray) are superimposed. Injected currents in (Ai) and (Aii) were 500 and 900 pA, respectively. Duration: 5 ms. Asterisk indicates fAHP between 1st and 2nd spikelets. The region surrounded with box in (Ai) was expanded in the inset. The inset in (Aii) includes a longer segment of the recording to illustrate the slow afterpotential. (Aiii) Summary of the change of burst firing probability by ryanodine. Three to four successive trials were used to obtain averaged probability in each experiment. (B) The effect of ryanodine was occluded by IbTX. Bath application of IbTX (100 nM) alone broadened 1st action potentials (Bi and Bii, gray traces) and made the fAHP less negative (Bii,, asterisk). Subsequent application of ryanodine in the presence of IbTX did not affect the waveform (Bi’ and Bii’, black traces). Inset in (Bii) is the same sweep but displayed with longer time base, with spikes truncated. (Biii) Summary of the change of burst firing probabilities by IbTX. Statistical significance was tested between control and IbTX.

    Article Snippet: The amplitudes of current pulses were carefully adjusted so as not to induce burst firings even in the presence of ryanodine (Alomone labs) or CPA, and therefore each pulse evoked single action potential.

    Techniques: Injection, Mass Spectrometry

    CICR triggers BK channel-mediated transient outward currents (A) IbTX-sensitive transient outward currents. (Ai) In control, transient currents followed by sustained currents were evoked by depolarizing voltage steps (−30 to −10 mV from −70 mV holding potential, 10-mV increment). All transient current was inhibited by 100 nM IbTX (traces in IbTX and subtraction). IbTX-sensitive currents were obtained by subtracting traces in IbTX from control traces. (Aii) Summary of the peak current densities (left panel), and rise time and decay time constant of IbTX-sensitive currents (right panel). In (A) and (B), capacitive artifacts were blanked for clarity. Here and in following figures, dashed lines in current traces indicate zero current levels. (B) RyRs are involved in the transient outward currents. Same voltage protocol as in (A). (Bi) Note that some transient outward currents remain in ryanodine (Bi, ryanodine). (Bii) Summary of peak current densities (left panel) and rise time and decay time constant of ryanodine-sensitive currents (right panel). (Ci) Most of the transient current is suppressed by ω-Agatoxin-IVA (Aga-IVA, a P/Q-type blocker, 200 nM; trace in Aga-IVA and Aga-IVA-sensitive). Subsequent application of nonspecific Ca v channel blockers (200 µM CdCl 2 and 500 µM NiCl 2 ) blocked transient currents almost completely (traces in lower panel in Ci). Data in (C) were recorded in the presence of TTX, synaptic blockers, and 1 mM 4-AP. (Cii) Summary of peak current densities (left panel), and the rise time and decay time constant of Aga-VIA-sensitive currents (right panel). (Ciii) Summary of effects of subtype-specific Ca v blockers on transient currents. Aga-VIA inhibited transient currents more potently than nimodipine or TTA-P2. Effects are expressed as 100 × (selective blocker-sensitive current)/(nonspecific Ca v blockers-sensitive current). ***p

    Journal: Neuron

    Article Title: Double nanodomain coupling of calcium channels, ryanodine receptors and BK channels controls generation of burst firing

    doi: 10.1016/j.neuron.2017.10.014

    Figure Lengend Snippet: CICR triggers BK channel-mediated transient outward currents (A) IbTX-sensitive transient outward currents. (Ai) In control, transient currents followed by sustained currents were evoked by depolarizing voltage steps (−30 to −10 mV from −70 mV holding potential, 10-mV increment). All transient current was inhibited by 100 nM IbTX (traces in IbTX and subtraction). IbTX-sensitive currents were obtained by subtracting traces in IbTX from control traces. (Aii) Summary of the peak current densities (left panel), and rise time and decay time constant of IbTX-sensitive currents (right panel). In (A) and (B), capacitive artifacts were blanked for clarity. Here and in following figures, dashed lines in current traces indicate zero current levels. (B) RyRs are involved in the transient outward currents. Same voltage protocol as in (A). (Bi) Note that some transient outward currents remain in ryanodine (Bi, ryanodine). (Bii) Summary of peak current densities (left panel) and rise time and decay time constant of ryanodine-sensitive currents (right panel). (Ci) Most of the transient current is suppressed by ω-Agatoxin-IVA (Aga-IVA, a P/Q-type blocker, 200 nM; trace in Aga-IVA and Aga-IVA-sensitive). Subsequent application of nonspecific Ca v channel blockers (200 µM CdCl 2 and 500 µM NiCl 2 ) blocked transient currents almost completely (traces in lower panel in Ci). Data in (C) were recorded in the presence of TTX, synaptic blockers, and 1 mM 4-AP. (Cii) Summary of peak current densities (left panel), and the rise time and decay time constant of Aga-VIA-sensitive currents (right panel). (Ciii) Summary of effects of subtype-specific Ca v blockers on transient currents. Aga-VIA inhibited transient currents more potently than nimodipine or TTA-P2. Effects are expressed as 100 × (selective blocker-sensitive current)/(nonspecific Ca v blockers-sensitive current). ***p

    Article Snippet: The amplitudes of current pulses were carefully adjusted so as not to induce burst firings even in the presence of ryanodine (Alomone labs) or CPA, and therefore each pulse evoked single action potential.

    Techniques:

    SMOCs are induced by CICR triggered by P/Q-type Ca 2+ channels (A–C) Representative current traces containing SMOCs evoked by 10-mV depolarization from −70 mV in the presence of TTX and synaptic blockers. SMOCs were blocked completely by IbTX (A), ryanodine (B), and Aga-IVA (P/Q-type Ca 2+ blocker, 200 nM). (D) Summary of change of SMOC frequency (Di) and amplitude (Dii). Frequencies and amplitudes were normalized by using control data obtained before drug application. ** p

    Journal: Neuron

    Article Title: Double nanodomain coupling of calcium channels, ryanodine receptors and BK channels controls generation of burst firing

    doi: 10.1016/j.neuron.2017.10.014

    Figure Lengend Snippet: SMOCs are induced by CICR triggered by P/Q-type Ca 2+ channels (A–C) Representative current traces containing SMOCs evoked by 10-mV depolarization from −70 mV in the presence of TTX and synaptic blockers. SMOCs were blocked completely by IbTX (A), ryanodine (B), and Aga-IVA (P/Q-type Ca 2+ blocker, 200 nM). (D) Summary of change of SMOC frequency (Di) and amplitude (Dii). Frequencies and amplitudes were normalized by using control data obtained before drug application. ** p

    Article Snippet: The amplitudes of current pulses were carefully adjusted so as not to induce burst firings even in the presence of ryanodine (Alomone labs) or CPA, and therefore each pulse evoked single action potential.

    Techniques:

    Blockade of CICR induces spontaneous spike bursts (A) Loose cell-attached recordings of a spontaneously firing cell in the presence of synaptic blockers. (Ai, left) Control, all action potentials are simple spikes. (Ai, right) The boxed region in (Ai, left) with expanded time base. (Aii, left) Bursting (*) observed in the presence of 20 µM ryanodine. (Aii, right) The boxed region (Aii, left), showing one burst of 5 spikelets. (B) Instantaneous firing frequency over time. The data were obtained from the same cell in (A). Ryanodine was bath-applied during time marked by gray box. (C) Summarized data of instantaneous frequencies in ryanodine or CPA (10 µM). Simple: data from spontaneous simple spike-firing cells; Burst: data from spontaneous burst-firing cells; Simple+Burst: pooled data from both firing types of cells. Here and elsewhere, error bars indicate SEM, and statistical significance was tested using paired t -test unless otherwise stated (significance, p

    Journal: Neuron

    Article Title: Double nanodomain coupling of calcium channels, ryanodine receptors and BK channels controls generation of burst firing

    doi: 10.1016/j.neuron.2017.10.014

    Figure Lengend Snippet: Blockade of CICR induces spontaneous spike bursts (A) Loose cell-attached recordings of a spontaneously firing cell in the presence of synaptic blockers. (Ai, left) Control, all action potentials are simple spikes. (Ai, right) The boxed region in (Ai, left) with expanded time base. (Aii, left) Bursting (*) observed in the presence of 20 µM ryanodine. (Aii, right) The boxed region (Aii, left), showing one burst of 5 spikelets. (B) Instantaneous firing frequency over time. The data were obtained from the same cell in (A). Ryanodine was bath-applied during time marked by gray box. (C) Summarized data of instantaneous frequencies in ryanodine or CPA (10 µM). Simple: data from spontaneous simple spike-firing cells; Burst: data from spontaneous burst-firing cells; Simple+Burst: pooled data from both firing types of cells. Here and elsewhere, error bars indicate SEM, and statistical significance was tested using paired t -test unless otherwise stated (significance, p

    Article Snippet: The amplitudes of current pulses were carefully adjusted so as not to induce burst firings even in the presence of ryanodine (Alomone labs) or CPA, and therefore each pulse evoked single action potential.

    Techniques:

    (A) Comparison of protein expression between the Casq2 R33Q/R33Q mice and the WT mice. (B) Quantification of different protein expression shows that CaMKII, p-CaMKII, p-RyR2, and NCX1.1 were promoted, but Casq2, JCT, and TRI were declined in the Casq2 R33Q/R33Q atria. CaMKII, calcium/calmodulin-dependent protein kinase II; RyR2, ryanodine receptor; NXC1.1, sodium–calcium exchanger 1.1; Casq2, calsequestrin 2; TRI, triadin; JCT, junctin; SERCA, sarcoplasmic reticulum calcium-ATPases; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ∗ P

    Journal: Frontiers in Physiology

    Article Title: Calcium-Mediated Oscillation in Membrane Potentials and Atrial-Triggered Activity in Atrial Cells of Casq2R33Q/R33Q Mutation Mice

    doi: 10.3389/fphys.2018.01447

    Figure Lengend Snippet: (A) Comparison of protein expression between the Casq2 R33Q/R33Q mice and the WT mice. (B) Quantification of different protein expression shows that CaMKII, p-CaMKII, p-RyR2, and NCX1.1 were promoted, but Casq2, JCT, and TRI were declined in the Casq2 R33Q/R33Q atria. CaMKII, calcium/calmodulin-dependent protein kinase II; RyR2, ryanodine receptor; NXC1.1, sodium–calcium exchanger 1.1; Casq2, calsequestrin 2; TRI, triadin; JCT, junctin; SERCA, sarcoplasmic reticulum calcium-ATPases; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ∗ P

    Article Snippet: Western blot was performed under one of the unique antibodies including anti-RyR2 (abcam), anti-phospho-RyR2 (Badrilla), anti-CaMKII (abcam), anti-phospho-CaMKII (abcam), anti-CASQ (ABR), anti-triadin (ABR), anti-junctin (ABR), anti-NCX1.1 (abcam), and anti-SERCA (abcam), and then incubated overnight at 4°C for specialty binding.

    Techniques: Expressing, Mouse Assay

    (A1–A2) Effects of 100 µM ACh on the relative fluorescence intensity of OHC [Ca 2+ ] i in Hank’s solution. (B1–B3) Effects of 100 µM ACh following 30 µM ryanodine on the relative fluorescence intensity of OHC [Ca 2+ ] i in Hank’s solution. (C1–C3) Effects of 100 µM ACh following 30 nM thapsigargin on the relative fluorescence intensity of OHC [Ca 2+ ] i in Hank’s solution. (D1–D2) Effects of 100 µM ACh on the relative fluorescence intensity of OHC [Ca 2+ ]i in D-Hank’s solution. (E1–E3) Effects of 100 µM ACh following 30 µM ryanodine on the relative fluorescence intensity of OHC [Ca 2+ ] i in D-Hank’s solution. (F1–F3) Effects of 100 µM ACh following 30 nM thapsigargin on the relative fluorescence intensity of OHC [Ca 2+ ] i in D-Hank’s solution.

    Journal: European Journal of Histochemistry : EJH

    Article Title: Differential expression of ryanodine receptor in the developing rat cochlea

    doi: 10.4081/ejh.2009.e30

    Figure Lengend Snippet: (A1–A2) Effects of 100 µM ACh on the relative fluorescence intensity of OHC [Ca 2+ ] i in Hank’s solution. (B1–B3) Effects of 100 µM ACh following 30 µM ryanodine on the relative fluorescence intensity of OHC [Ca 2+ ] i in Hank’s solution. (C1–C3) Effects of 100 µM ACh following 30 nM thapsigargin on the relative fluorescence intensity of OHC [Ca 2+ ] i in Hank’s solution. (D1–D2) Effects of 100 µM ACh on the relative fluorescence intensity of OHC [Ca 2+ ]i in D-Hank’s solution. (E1–E3) Effects of 100 µM ACh following 30 µM ryanodine on the relative fluorescence intensity of OHC [Ca 2+ ] i in D-Hank’s solution. (F1–F3) Effects of 100 µM ACh following 30 nM thapsigargin on the relative fluorescence intensity of OHC [Ca 2+ ] i in D-Hank’s solution.

    Article Snippet: The final concentration of thapsigargin (Biomol, USA) and ryanodine (Biomol, USA) in Petri dishes were 30 nM and 30 µM, respectively.

    Techniques: Fluorescence

    RyR function is required for Shh-dependent Gli-mediated gene expression. (A–H) Dorsal view images of live Tg(8xGli:mCherry-NLS-Odc1) embryos at 12hpf (A,C,E,G) or 24hpf (B,D,F,H) that had been treated with vehicle (0.5%DMSO), cyclopamine, azumolene, or ryanodine. At 12hpf, the position of the notochord (ntc) just ventral to the FP is outlined by dashed lines. (A) In control 12hpf embryos, mCherry is expressed in nuclei of cells responding to Shh, including adaxial cells (arrowhead) and FP cells (arrow). (B) At 24hpf, mCherry is expressed in nuclei of slow muscle cells (arrowhead) and cells in the ventral neural tube (arrow). (C–H) mCherry expression is reduced in embryos treated with each drug. (I) Tail-transected Tg(8xGli:mCherry-NLS-Odc1) embryos were soaked in vehicle or 4-CmC from 16 to 18hpf, fixed, and imaged. (J–L) Potentiation of RyR channel activity with 4-CmC treatment results in increased numbers of presumptive slow muscle nuclei that express the mCherry reporter (J and K are lateral views, arrowhead indicates nuclei of slow muscle cells and arrow indicates cells in the ventral neural tube). (L) Quantification of mCherry + nuclei per somite in 4-CmC-treated embryos. Each point represents a single somite and the horizontal line represents the mean. (M–Q) RyR activity affects endogenous ptch2 expression as detected by whole mount in situ hybridization in 24hpf embryos. (M–Q) Lateral views reveal expression in somites and (M′–Q′) transverse sections reveal expression in slow muscle cells surrounding the notochord and in the ventral neural tube. As compared with WT embryos, ptch2 expression is diminished azumolene-treated and MZryr1a (−/−) ;MZryr2a (−/−) ;MZryr3 (−/−) mutant embryos, and it is enhanced in 4-CmC-treated embryos. Scale bars indicate 25 μm.

    Journal: Developmental cell

    Article Title: Intracellular calcium mobilization is required for Sonic hedgehog signaling

    doi: 10.1016/j.devcel.2018.04.013

    Figure Lengend Snippet: RyR function is required for Shh-dependent Gli-mediated gene expression. (A–H) Dorsal view images of live Tg(8xGli:mCherry-NLS-Odc1) embryos at 12hpf (A,C,E,G) or 24hpf (B,D,F,H) that had been treated with vehicle (0.5%DMSO), cyclopamine, azumolene, or ryanodine. At 12hpf, the position of the notochord (ntc) just ventral to the FP is outlined by dashed lines. (A) In control 12hpf embryos, mCherry is expressed in nuclei of cells responding to Shh, including adaxial cells (arrowhead) and FP cells (arrow). (B) At 24hpf, mCherry is expressed in nuclei of slow muscle cells (arrowhead) and cells in the ventral neural tube (arrow). (C–H) mCherry expression is reduced in embryos treated with each drug. (I) Tail-transected Tg(8xGli:mCherry-NLS-Odc1) embryos were soaked in vehicle or 4-CmC from 16 to 18hpf, fixed, and imaged. (J–L) Potentiation of RyR channel activity with 4-CmC treatment results in increased numbers of presumptive slow muscle nuclei that express the mCherry reporter (J and K are lateral views, arrowhead indicates nuclei of slow muscle cells and arrow indicates cells in the ventral neural tube). (L) Quantification of mCherry + nuclei per somite in 4-CmC-treated embryos. Each point represents a single somite and the horizontal line represents the mean. (M–Q) RyR activity affects endogenous ptch2 expression as detected by whole mount in situ hybridization in 24hpf embryos. (M–Q) Lateral views reveal expression in somites and (M′–Q′) transverse sections reveal expression in slow muscle cells surrounding the notochord and in the ventral neural tube. As compared with WT embryos, ptch2 expression is diminished azumolene-treated and MZryr1a (−/−) ;MZryr2a (−/−) ;MZryr3 (−/−) mutant embryos, and it is enhanced in 4-CmC-treated embryos. Scale bars indicate 25 μm.

    Article Snippet: Ryanodine (Santa Cruz Biotechnology), azumolene (Santa Cruz Biotechnology), NAC (Sigma-Aldrich), and thapsigargin (Sigma-Aldrich) were prepared in DMSO; aldrithiol (DTDP, Sigma-Aldrich) and cyclopamine (LC Labs) were prepared in ethanol; and tricaine (MS-222, Sigma) and 4-CmC were prepared in water.

    Techniques: Expressing, Activity Assay, In Situ Hybridization, Mutagenesis

    RyR function is required for development of Shh-dependent neural crest-derived neurons of the dorsal root ganglia (DRGs) and the enteric nervous system (ENS). (A–L) Lateral views of live 72hpf Tg(isl2b:GFP) embryos with GFP-labeled Rohon Beard neurons (arrow) and DRGs (arrowhead). Embryos were treated between 24 and 48hpf with cyclopamine (cyc), azumolene (azum), ryanodine (ryan), N-acetyl cysteine (NAC), aldrithiol, thapsigargin (thaps), or tricaine at indicated concentrations or injected at the one-cell stage with ryr1a and ryr3 MOs. Arrowheads in C and G indicate the presence of small, faint DRGs. (M) Quantification of embryos treated as in A–L. DRGs present in somites 11–15 were counted. For each condition, the number of embryos analyzed is indicated. Comparisons are to control vehicle-treated embryos unless otherwise indicated. Data are represented as mean ±SEM. (N–Q) Lateral views of live 78hpf Tg(phox2bb:GFP) embryos with the ENS (arrow) labeled by GFP in control, cyclopamine-treated, ryr1a,3 .

    Journal: Developmental cell

    Article Title: Intracellular calcium mobilization is required for Sonic hedgehog signaling

    doi: 10.1016/j.devcel.2018.04.013

    Figure Lengend Snippet: RyR function is required for development of Shh-dependent neural crest-derived neurons of the dorsal root ganglia (DRGs) and the enteric nervous system (ENS). (A–L) Lateral views of live 72hpf Tg(isl2b:GFP) embryos with GFP-labeled Rohon Beard neurons (arrow) and DRGs (arrowhead). Embryos were treated between 24 and 48hpf with cyclopamine (cyc), azumolene (azum), ryanodine (ryan), N-acetyl cysteine (NAC), aldrithiol, thapsigargin (thaps), or tricaine at indicated concentrations or injected at the one-cell stage with ryr1a and ryr3 MOs. Arrowheads in C and G indicate the presence of small, faint DRGs. (M) Quantification of embryos treated as in A–L. DRGs present in somites 11–15 were counted. For each condition, the number of embryos analyzed is indicated. Comparisons are to control vehicle-treated embryos unless otherwise indicated. Data are represented as mean ±SEM. (N–Q) Lateral views of live 78hpf Tg(phox2bb:GFP) embryos with the ENS (arrow) labeled by GFP in control, cyclopamine-treated, ryr1a,3 .

    Article Snippet: Ryanodine (Santa Cruz Biotechnology), azumolene (Santa Cruz Biotechnology), NAC (Sigma-Aldrich), and thapsigargin (Sigma-Aldrich) were prepared in DMSO; aldrithiol (DTDP, Sigma-Aldrich) and cyclopamine (LC Labs) were prepared in ethanol; and tricaine (MS-222, Sigma) and 4-CmC were prepared in water.

    Techniques: Derivative Assay, Labeling, Injection

    Effects of nifedipine, BAY K 8644 and ryanodine on phenylephrine-induced mechanical responses in papillary muscles. (A) The representative response to phenylephrine (10 μM) in C57BL/6J. Actions of nifedipine (0.3 μM, B), BAY K 8644 (1 μM, C) and ryanodine (0.3 μM, D) on twitch tension in the absence and presence of phenylephrine in C57BL/6J. Phenylephrine exerted positive inotropic response in the presence of ryanodine (i.e., in situations where SR-function is greatly reduced). Summary of the effects of BAY K 8644 (1 μM, E) and ryanodine (0.3 μM, F) on phenylephrine-induced response (n = 4–6, * P

    Journal: PLoS ONE

    Article Title: New Isoform of Cardiac Myosin Light Chain Kinase and the Role of Cardiac Myosin Phosphorylation in α1-Adrenoceptor Mediated Inotropic Response

    doi: 10.1371/journal.pone.0141130

    Figure Lengend Snippet: Effects of nifedipine, BAY K 8644 and ryanodine on phenylephrine-induced mechanical responses in papillary muscles. (A) The representative response to phenylephrine (10 μM) in C57BL/6J. Actions of nifedipine (0.3 μM, B), BAY K 8644 (1 μM, C) and ryanodine (0.3 μM, D) on twitch tension in the absence and presence of phenylephrine in C57BL/6J. Phenylephrine exerted positive inotropic response in the presence of ryanodine (i.e., in situations where SR-function is greatly reduced). Summary of the effects of BAY K 8644 (1 μM, E) and ryanodine (0.3 μM, F) on phenylephrine-induced response (n = 4–6, * P

    Article Snippet: Materials The following chemicals were used: phenylephrine hydrochloride (Sigma Chemical Co.), guanethidine (Tokyo Kasei, Tokyo, Japan), atenolol (Sigma Chemical Co.), nifedipine (Sigma Chemical Co.), BAY K 8644 (Tocris Bioscience, Bristol, UK), ryanodine (Wako Pure Chemical, Osaka, Japan), calyculin A (Wako Pure Chemical, Osaka, Japan).

    Techniques:

    Effect of 30 μ M thymol on the isolated RyRs. Incorporation was initiated in symmetric 250 mM KCl, holding potential was 61 mV; channel openings are upward deflections. Horizontal lines before each current record mark the closed state. ( A ) Single channel recordings in control conditions. ( B ) Representative segments of single channel behavior taken 5 min after the additions of thymol. ( C ) Single channel currents measured after the successive addition of 0.2 μ M ryanodine into the cis chamber. ( D ) Single channel currents plotted as a function of the holding potential. Open symbols represent measurements under control conditions, whereas solid symbols represent those in the presence of thymol. Straight lines correspond to a single channel conductance of 545 and 543 pS in control and in the presence of the drug, respectively. All recordings are from a single experiment.

    Journal: Biophysical Journal

    Article Title: Altered Elementary Calcium Release Events and Enhanced Calcium Release by Thymol in Rat Skeletal Muscle

    doi:

    Figure Lengend Snippet: Effect of 30 μ M thymol on the isolated RyRs. Incorporation was initiated in symmetric 250 mM KCl, holding potential was 61 mV; channel openings are upward deflections. Horizontal lines before each current record mark the closed state. ( A ) Single channel recordings in control conditions. ( B ) Representative segments of single channel behavior taken 5 min after the additions of thymol. ( C ) Single channel currents measured after the successive addition of 0.2 μ M ryanodine into the cis chamber. ( D ) Single channel currents plotted as a function of the holding potential. Open symbols represent measurements under control conditions, whereas solid symbols represent those in the presence of thymol. Straight lines correspond to a single channel conductance of 545 and 543 pS in control and in the presence of the drug, respectively. All recordings are from a single experiment.

    Article Snippet: Lipids were obtained from Avanti Polar Lipids (Alabaster, AL), [3 H]ryanodine was from Dupont (Boston, MA), and all other chemicals were from Sigma.

    Techniques: Isolation

    Effects of thymol on ryanodine binding to heavy SR vesicles. ( A ) Ryanodine binding determined in the absence (○) and presence (▴) of 300 μ M thymol at different [ 3 H]ryanodine concentrations. Assuming a single binding site the Hill fit resulted in k 50 = 25.3 ± 1.4 nM and B max = 8.67 ± 0.26 pmol/mg protein in control, whereas k 50 = 15.5 ± 1.0 nM and B max = 14.65 ± 0.45 pmol/mg protein in the presence of the drug. ( B ) The data presented in A were used for constructing the Scatchard plot. Straight lines were drawn using the parameters given above. ( C ) Concentration-dependent effect of thymol on ryanodine binding. Ryanodine binding was determined in the absence and presence of various concentrations of thymol at 6 nM [ 3 H]ryanodine concentrations. Fitting the Hill equation resulted in a half-activating concentration of 144 ± 11 μ M, a Hill coefficient of 1.89 ± 0.27, and approximately three times higher ryanodine binding at saturating thymol concentration than in control.

    Journal: Biophysical Journal

    Article Title: Altered Elementary Calcium Release Events and Enhanced Calcium Release by Thymol in Rat Skeletal Muscle

    doi:

    Figure Lengend Snippet: Effects of thymol on ryanodine binding to heavy SR vesicles. ( A ) Ryanodine binding determined in the absence (○) and presence (▴) of 300 μ M thymol at different [ 3 H]ryanodine concentrations. Assuming a single binding site the Hill fit resulted in k 50 = 25.3 ± 1.4 nM and B max = 8.67 ± 0.26 pmol/mg protein in control, whereas k 50 = 15.5 ± 1.0 nM and B max = 14.65 ± 0.45 pmol/mg protein in the presence of the drug. ( B ) The data presented in A were used for constructing the Scatchard plot. Straight lines were drawn using the parameters given above. ( C ) Concentration-dependent effect of thymol on ryanodine binding. Ryanodine binding was determined in the absence and presence of various concentrations of thymol at 6 nM [ 3 H]ryanodine concentrations. Fitting the Hill equation resulted in a half-activating concentration of 144 ± 11 μ M, a Hill coefficient of 1.89 ± 0.27, and approximately three times higher ryanodine binding at saturating thymol concentration than in control.

    Article Snippet: Lipids were obtained from Avanti Polar Lipids (Alabaster, AL), [3 H]ryanodine was from Dupont (Boston, MA), and all other chemicals were from Sigma.

    Techniques: Binding Assay, Concentration Assay

    Immunoblot analysis and [ 3 H]ryanodine binding to cortical microsomes of sea urchin eggs A , Western blot analysis with RyR antibodies. Cortical microsomal proteins and cardiac and skeletal SR microsomes were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with a rabbit monoclonal skeletal RyR antibody and cardiac RyR antibody, as indicated. Lane 1, 30 μg rabbit skeletal microsomes; Lane 2, 50 μg sea urchin egg microsomes; Lane 3, 30 μg pig cardiac microsomes. B , [ 3 H]ryanodine saturation binding curve. Between 1 and 10 μg of L. pictus cortical microsomes were incubated with the indicated concentration of [ 3 H]ryanodine as indicated in Methods. Non-specific binding has been subtracted from each data point. Data were fitted with the equation: B = B max [ [ 3 H]ryanodine]/( K d + [ [ 3 H]ryanodine]), where B corresponds to specific binding of [ 3 H]ryanodine, B max is the maximal density of receptor sites and K d is the apparent dissociation constant of the [ 3 H]ryanodine-RyR complex. C , Ca 2+ dependence of [ 3 H]ryanodine binding to sea urchin egg microsomes. Binding conditions were as in B except that 1 mM EGTA and varying concentrations of CaCl 2 were added to the medium to bring [free Ca 2+ ] to the specified level. [ 3 H]Ryanodine concentration was 7 nM.

    Journal: The Journal of Physiology

    Article Title: Detection and functional characterization of ryanodine receptors from sea urchin eggs

    doi: 10.1111/j.1469-7793.1998.155bz.x

    Figure Lengend Snippet: Immunoblot analysis and [ 3 H]ryanodine binding to cortical microsomes of sea urchin eggs A , Western blot analysis with RyR antibodies. Cortical microsomal proteins and cardiac and skeletal SR microsomes were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with a rabbit monoclonal skeletal RyR antibody and cardiac RyR antibody, as indicated. Lane 1, 30 μg rabbit skeletal microsomes; Lane 2, 50 μg sea urchin egg microsomes; Lane 3, 30 μg pig cardiac microsomes. B , [ 3 H]ryanodine saturation binding curve. Between 1 and 10 μg of L. pictus cortical microsomes were incubated with the indicated concentration of [ 3 H]ryanodine as indicated in Methods. Non-specific binding has been subtracted from each data point. Data were fitted with the equation: B = B max [ [ 3 H]ryanodine]/( K d + [ [ 3 H]ryanodine]), where B corresponds to specific binding of [ 3 H]ryanodine, B max is the maximal density of receptor sites and K d is the apparent dissociation constant of the [ 3 H]ryanodine-RyR complex. C , Ca 2+ dependence of [ 3 H]ryanodine binding to sea urchin egg microsomes. Binding conditions were as in B except that 1 mM EGTA and varying concentrations of CaCl 2 were added to the medium to bring [free Ca 2+ ] to the specified level. [ 3 H]Ryanodine concentration was 7 nM.

    Article Snippet: [3 H]Ryanodine (60 Ci mmol−1 , Dupont NEN) was incubated with cortical microsomes (0.01-1.0 mg ml−1 ) in medium containing 0.2 M KCl, 20 mM Mops (pH 7.2), 1 mM EGTA, and different amounts of CaCl2 to set [free Ca2+ ] in the range of 0.08-100 μM.

    Techniques: Binding Assay, Western Blot, SDS Page, Incubation, Concentration Assay

    Reconstitution of sea urchin egg RyR channels in planar lipid bilayers All traces were recorded at a holding potential of −25 mV. At this voltage, Cs + flows from the trans (lumenal) to the cis (cytosolic) chamber and channel openings correspond to downward deflections of the baseline current. A , traces labelled Control: only brief and sparse openings were observed under our standard recording conditions (symmetrical 300 mM caesium methanesulphonate, 10 mM Mops, pH 7.2). C indicates the closed state. + ATP and total homogenate ( cis side): the same channel after addition of 10 μl of sea urchin egg total homogenate (0.1 mg protein ml −1 ) supplemented with 5 mM ATP. For this particular channel, P o increased from ≤ 0.01 to ≈0.6. This is one of the most dramatic responses. Typical and consistent responses to homogenate addition were an increase in P o from ≤ 0.01 to 0.2-0.5 ( n = 9). + 20 mM Ca 2+ ( trans side): addition of 20 mM CaCl 2 to the trans (lumenal) side decreased single channel conductance; + 5 μM ryanodine: ryanodine induced the appearance of a long-lived subconductance state. The transition to this modified state was not reversible within the duration of the experiment (≈20 min). B , current-voltage relation for the RyR channel before and after addition of ryanodine.

    Journal: The Journal of Physiology

    Article Title: Detection and functional characterization of ryanodine receptors from sea urchin eggs

    doi: 10.1111/j.1469-7793.1998.155bz.x

    Figure Lengend Snippet: Reconstitution of sea urchin egg RyR channels in planar lipid bilayers All traces were recorded at a holding potential of −25 mV. At this voltage, Cs + flows from the trans (lumenal) to the cis (cytosolic) chamber and channel openings correspond to downward deflections of the baseline current. A , traces labelled Control: only brief and sparse openings were observed under our standard recording conditions (symmetrical 300 mM caesium methanesulphonate, 10 mM Mops, pH 7.2). C indicates the closed state. + ATP and total homogenate ( cis side): the same channel after addition of 10 μl of sea urchin egg total homogenate (0.1 mg protein ml −1 ) supplemented with 5 mM ATP. For this particular channel, P o increased from ≤ 0.01 to ≈0.6. This is one of the most dramatic responses. Typical and consistent responses to homogenate addition were an increase in P o from ≤ 0.01 to 0.2-0.5 ( n = 9). + 20 mM Ca 2+ ( trans side): addition of 20 mM CaCl 2 to the trans (lumenal) side decreased single channel conductance; + 5 μM ryanodine: ryanodine induced the appearance of a long-lived subconductance state. The transition to this modified state was not reversible within the duration of the experiment (≈20 min). B , current-voltage relation for the RyR channel before and after addition of ryanodine.

    Article Snippet: [3 H]Ryanodine (60 Ci mmol−1 , Dupont NEN) was incubated with cortical microsomes (0.01-1.0 mg ml−1 ) in medium containing 0.2 M KCl, 20 mM Mops (pH 7.2), 1 mM EGTA, and different amounts of CaCl2 to set [free Ca2+ ] in the range of 0.08-100 μM.

    Techniques: Modification

    (A) Representative recordings relative to fluo-8 fluorescence (expressed as DF/F0 (A. U.)) in resting WT (open circles) and mdx (filled circles) and in stretched WT (open squares) and mdx (filled squares) during a superfusion protocol starting initially with a calcium free Tyrode solution followed by the superfusion of 1.8 mM Ca 2+ Tyrode solution. (B) Maximal amplitude of fluo-8 fluorescence intensity in WT and mdx cardiomyocytes maintained in stretched condition with SACs inhibitors: cells were incubated with 300 µM streptomycin (Strp, gray bars) or 2.5 µM GsMTx-4 (Black bars) for SACs inhibition and with 10 µM nifedipine (vertical hatching) or 100 µM ryanodine (horizontal hatching) for EC coupling inhibition. Open bars represent the control. Declined hatching represents rest (non-stretched). Measurements are represented as mean normalized fluo 8 fluorescence intensity±SEM. Rest (WT: n =10 cells, N =4 hearts; mdx : n =12 cells, N =4 hearts). stretch control (WT: n =12 cells, N =4 hearts; mdx : n =12 cells, N =4 hearts). Strp (WT: n =7 cells, N =3 hearts; mdx : n =6 cells, N =3 hearts). Rest (WT: n =10 cells, N =4 hearts; mdx : n =12 cells, N =4 hearts). GsMTx-4 (WT: n =5 cells, N =3 hearts; mdx : n =5 cells, N =3 hearts). Nifedipine (WT: n =5 cells, N =4 hearts; mdx : n =5 cells, N =3 hearts). Ryanodine (WT: n =9 cells, N =4 hearts; mdx : n =7 cells, N =4 hearts). * Symbol represents the statistical difference with control, *** P

    Journal: Data in Brief

    Article Title: Data on calcium increases depending on stretch in dystrophic cardiomyocytes

    doi: 10.1016/j.dib.2016.08.011

    Figure Lengend Snippet: (A) Representative recordings relative to fluo-8 fluorescence (expressed as DF/F0 (A. U.)) in resting WT (open circles) and mdx (filled circles) and in stretched WT (open squares) and mdx (filled squares) during a superfusion protocol starting initially with a calcium free Tyrode solution followed by the superfusion of 1.8 mM Ca 2+ Tyrode solution. (B) Maximal amplitude of fluo-8 fluorescence intensity in WT and mdx cardiomyocytes maintained in stretched condition with SACs inhibitors: cells were incubated with 300 µM streptomycin (Strp, gray bars) or 2.5 µM GsMTx-4 (Black bars) for SACs inhibition and with 10 µM nifedipine (vertical hatching) or 100 µM ryanodine (horizontal hatching) for EC coupling inhibition. Open bars represent the control. Declined hatching represents rest (non-stretched). Measurements are represented as mean normalized fluo 8 fluorescence intensity±SEM. Rest (WT: n =10 cells, N =4 hearts; mdx : n =12 cells, N =4 hearts). stretch control (WT: n =12 cells, N =4 hearts; mdx : n =12 cells, N =4 hearts). Strp (WT: n =7 cells, N =3 hearts; mdx : n =6 cells, N =3 hearts). Rest (WT: n =10 cells, N =4 hearts; mdx : n =12 cells, N =4 hearts). GsMTx-4 (WT: n =5 cells, N =3 hearts; mdx : n =5 cells, N =3 hearts). Nifedipine (WT: n =5 cells, N =4 hearts; mdx : n =5 cells, N =3 hearts). Ryanodine (WT: n =9 cells, N =4 hearts; mdx : n =7 cells, N =4 hearts). * Symbol represents the statistical difference with control, *** P

    Article Snippet: Tranilast (Trn) was purchased from Calbiochem (53902-12-8), GsMTx-4 from Abcam (ab141871), probenecid (Prb, P8761), Streptomycin sulfate (Strp, s9137) and 4-methyl-4′-[3.5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-1.2.3-thiadiazole-5-carboxanilide (YM-58483, y4895) from Sigma, ryanodine from Merck (15662-33-6), fura-2-AM (108964-32-5) and fluo-8-AM (sc-362561) from Santa-Cruz.

    Techniques: Fluorescence, Incubation, Inhibition

    Confocal image of ryanodine receptor distribution in living endothelial cells Freshly isolated endothelial cells were loaded with 10 −7 mol l −1 BODIPY FL-X ryanodine for 10 min and images were collected using a confocal microscope. The figure shows a transmitted light image ( A ), the corresponding confocal image at approximately the middle depth of the cell ( B ) and a composite image of A and B ( C ). D shows a 3-dimensional reconstruction of 13 optical sections taken at 1 μm steps in the z -axis and the inset presents the single section in the x-y plane taken at the arrow. E , a 3-dimensional reconstruction above the x-y plane shown in B . The red circles and lines indicate corresponding areas in B and E . In F the plasmalemma is indicated by the outer red circle defined by the transmitted light image given in A and C . The inner red circle shows an erosion of 3 pixels from the plasmalemma towards the centre of the cell, which represents a distance of 0.6 μm from the plasmalemma.

    Journal: The Journal of Physiology

    Article Title: Submaximal stimulation of porcine endothelial cells causes focal Ca2+ elevation beneath the cell membrane

    doi: 10.1111/j.1469-7793.1998.109bx.x

    Figure Lengend Snippet: Confocal image of ryanodine receptor distribution in living endothelial cells Freshly isolated endothelial cells were loaded with 10 −7 mol l −1 BODIPY FL-X ryanodine for 10 min and images were collected using a confocal microscope. The figure shows a transmitted light image ( A ), the corresponding confocal image at approximately the middle depth of the cell ( B ) and a composite image of A and B ( C ). D shows a 3-dimensional reconstruction of 13 optical sections taken at 1 μm steps in the z -axis and the inset presents the single section in the x-y plane taken at the arrow. E , a 3-dimensional reconstruction above the x-y plane shown in B . The red circles and lines indicate corresponding areas in B and E . In F the plasmalemma is indicated by the outer red circle defined by the transmitted light image given in A and C . The inner red circle shows an erosion of 3 pixels from the plasmalemma towards the centre of the cell, which represents a distance of 0.6 μm from the plasmalemma.

    Article Snippet: BODIPY FL-X ryanodine, Calcium Green-1 acetoxymethyl ester form (AM), Calcium Green-1 hexapotassium salt, Calcium Crimson, 3,3′-dihexyloxacarbocyanine iodine (DiOC6), 5-(and 6)-carboxy SNARF-1® AM, (4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p -toluenesulphonate (TMA-DPH), and sodium-binding benzofura isophthalate acetoxymethyl ester (SBFI AM) were obtained from Molecular Probes.

    Techniques: Isolation, Microscopy

    Effect of a nocodazole treatment on 3-dimensional organization of the endoplasmic reticulum in porcine endothelial cells using BODIPY FL-X ryanodine Using deconvolution microscopy images were collected from cultured endothelial cells (passage 1) treated for 16 h in the absence ( A ) or presence ( C ) of 10 μmol l −1 nocodazole. The corresponding 1 μm thick x - y slice in middle depth after deconvolution is shown in B for image A and in D for image C .

    Journal: The Journal of Physiology

    Article Title: Submaximal stimulation of porcine endothelial cells causes focal Ca2+ elevation beneath the cell membrane

    doi: 10.1111/j.1469-7793.1998.109bx.x

    Figure Lengend Snippet: Effect of a nocodazole treatment on 3-dimensional organization of the endoplasmic reticulum in porcine endothelial cells using BODIPY FL-X ryanodine Using deconvolution microscopy images were collected from cultured endothelial cells (passage 1) treated for 16 h in the absence ( A ) or presence ( C ) of 10 μmol l −1 nocodazole. The corresponding 1 μm thick x - y slice in middle depth after deconvolution is shown in B for image A and in D for image C .

    Article Snippet: BODIPY FL-X ryanodine, Calcium Green-1 acetoxymethyl ester form (AM), Calcium Green-1 hexapotassium salt, Calcium Crimson, 3,3′-dihexyloxacarbocyanine iodine (DiOC6), 5-(and 6)-carboxy SNARF-1® AM, (4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p -toluenesulphonate (TMA-DPH), and sodium-binding benzofura isophthalate acetoxymethyl ester (SBFI AM) were obtained from Molecular Probes.

    Techniques: Microscopy, Cell Culture