Journal: The Journal of Neuroscience

Article Title: Unique Postsynaptic Signaling at the Hair Cell Efferent Synapse Permits Calcium to Evoke Changes on Two Time Scales

doi: 10.1523/JNEUROSCI.17-01-00428.1997

Figure Lengend Snippet: Effect of ryanodine on CM and CAP. A and B are records of normalized CAP and CM amplitude in a case that produced marked decrease in the CAP. Baseline data were taken while the scala tympani was perfused with artificial perilymph. The boxes along the time axis indicate the period during which the cochlea was perfused with the drug. The acoustic stimulus was 30 dB above threshold to produce a measurable CM. Obviously, accurate measurement of the slow effect is difficult in the face of such large changes in the CAP.

Article Snippet: Pharmacological agents thapsigargin, cyclopiazonic acid (CPA), and ryanodine were obtained from Research Biochemicals (Natick, MA).

Techniques: Produced

Journal: American Journal of Physiology - Cell Physiology

Article Title: Malignant hyperthermia-associated mutations in the S2-S3 cytoplasmic loop of type 1 ryanodine receptor calcium channel impair calcium-dependent inactivation

doi: 10.1152/ajpcell.00134.2016

Figure Lengend Snippet: Ca 2+ -dependent activities of MH/CNM-associated RyR1 mutants. Ca 2+ -dependent changes in the activities of wild-type (WT) and mutant RyR1 were determined by the [ 3 H]ryanodine binding method. Activities of mutant RyR1s carrying disease-associated mutations in EF-hand domain ( A ), S2–S3 loop ( B ), and S4–S5 loop ( C ) are shown together with that of WT-RyR1 (dotted line, mean values). A dashed line in B represents a mean value of WT-RyR2. D : IC 50 values of wild-type and mutant RyRs. E : activities at 40 nM free Ca 2+ were normalized to the peak activities for wild-type and each mutant RyR1. * P

Article Snippet: Nonspecific binding of [3 H]ryanodine was determined in the presence of 2.5 mM EGTA and 2.5 μM unlabeled ryanodine.

Techniques: Mutagenesis, Binding Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Malignant hyperthermia-associated mutations in the S2-S3 cytoplasmic loop of type 1 ryanodine receptor calcium channel impair calcium-dependent inactivation

doi: 10.1152/ajpcell.00134.2016

Figure Lengend Snippet: Effect of Mg 2+ on MH/CNM-associated RyR1 mutants. Mg 2+ -dependent changes in the activities of wild-type and mutant RyR were determined at 100 μM Ca 2+ by the [ 3 H]ryanodine binding method. Activities of wild-type RyRs ( A ), S2–S3 loop mutants ( B ), S4–S5 loop mutants ( C ), and EF-hand mutants ( D ) are shown. Dotted and dashed lines in B , C , and D are mean values of WT-RyR1 and WT-RyR2, respectively. The smaller dotted line indicates the level at 100%. * P

Article Snippet: Nonspecific binding of [3 H]ryanodine was determined in the presence of 2.5 mM EGTA and 2.5 μM unlabeled ryanodine.

Techniques: Mutagenesis, Binding Assay

Journal: Scientific Reports

Article Title: Promiscuous attraction of ligands within the ATP binding site of RyR2 promotes diverse gating behaviour

doi: 10.1038/s41598-018-33328-8

Figure Lengend Snippet: Investigating the ability of ATP fragments to stimulate [ 3 H]ryanodine binding as effectively as ATP alone. For clarity, in each graph, the ATP data from Fig. 3B is shown as a solid blue line. ( A ) Comparison of the effects of PPPi alone (pink), adenosine alone (green) and adenosine in the presence of 10 mM PPPi (black dashed line). ( B ) Comparison of the effects of PPi alone (pink), AMP alone (green) and AMP in the presence of 10 mM PPi (black dashed line). ( C ) Comparison of the effects of Pi alone (pink), ADP alone (green) and ADP in the presence of 10 mM Pi (black dashed line). Where combinations of ligands were used, they were added simultaneously at the start of the incubation period. The addition of Pi to ADP and PPi to AMP did not significantly increase binding above that observed with ADP (p = 0.2101) or AMP (p = 0.1917) alone whereas the addition of PPPi significantly increased binding above that observed with adenosine alone (**p

Article Snippet: Materials [3 H]ryanodine was purchased from New England Nuclear Ltd. Unlabelled ryanodine was purchased from Calbiochem (Nottingham, UK).

Techniques: Binding Assay, Incubation

Journal: Scientific Reports

Article Title: Promiscuous attraction of ligands within the ATP binding site of RyR2 promotes diverse gating behaviour

doi: 10.1038/s41598-018-33328-8

Figure Lengend Snippet: Irreversible RyR2 inactivation by PPPi. ( A ) The top trace shows a typical single RyR2 channel activated by 10 μM cytosolic Ca 2+ alone. The subsequent trace shows an example of the inactivating effects of 10 mM PPPi. Occasional, very brief openings were observed initially as shown in the middle trace but after 140 s no further openings were detected. The bottom trace shows that after perfusing away the PPPi from the cytosolic chamber, the channel remains shut (no openings during the 3 min recording period). The holding potential was 0 mV. O and C represent the open and closed channel levels respectively. ( B ) Comparison of the stimulation of [ 3 H]ryanodine binding to vesicles of sheep cardiac heavy SR by ATP (blue) and PPPi (pink). The results are expressed as a percentage of the control binding at 10 μM cytosolic Ca 2+ which was 0.18 ± 0.02 pmol [ 3 H]/mg protein (SEM; n = 10). Where not shown, error bars are within the symbol. The optimum stimulation of [ 3 H]ryanodine binding was significantly greater for ATP than for PPPi (**p

Article Snippet: Materials [3 H]ryanodine was purchased from New England Nuclear Ltd. Unlabelled ryanodine was purchased from Calbiochem (Nottingham, UK).

Techniques: Binding Assay

Journal: The journal of pain : official journal of the American Pain Society

Article Title: Regulation of expression of hyperalgesic priming by estrogen receptor alpha in the rat

doi: 10.1016/j.jpain.2016.12.017

Figure Lengend Snippet: AMP-, but not CPA-, induced mechanical hyperalgesia in female primed rats is dependent on estrogen receptor alpha (EsRα) A : Male (left panel) and female (right panel) rats received an intradermal injection of ryanodine (100 ng in males and 1 pg in females) on the dorsum of both hind paws. One week later, intrathecal treatment with ODN antisense or mismatch against EsRα mRNA was performed for 3 consecutive days. On the 4 th day, AMP (1 μg) was injected at the same site as ryanodine, and mechanical nociceptive threshold evaluated 10, 20 and 30 min later. While AMP induced significant hyperalgesia in both male and female rats treated with mismatch and in the male antisense group, in the group of females that had been treated with EsRα antisense, AMP-induced hyperalgesia was prevented [males: F 1 ,10 = 1.179, p = 0.3030 (non-significant); females: F 1 ,10 = 52.35, **** p

Article Snippet: The drugs used in this study were: adenosine 5′-monophosphate disodium salt (AMP); the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX); the A1 adenosine receptor agonist N6 -cyclopentyladenosine (CPA) and the ryanodine receptor modulator ryanodine, all from Sigma-Aldrich (St. Louis, MO); and, the ecto-5′-nucleotidase inhibitor α,β-methyleneadenosine 5′-diphosphate sodium salt (AMPCP) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Injection

Journal: The journal of pain : official journal of the American Pain Society

Article Title: Regulation of expression of hyperalgesic priming by estrogen receptor alpha in the rat

doi: 10.1016/j.jpain.2016.12.017

Figure Lengend Snippet: AMP induces mechanical hyperalgesia in primed male and female rats Male and female rats were divided in two groups, primed (filled symbols) and non-primed (open symbols). The priming inducer, ryanodine (100 ng in males and 1 pg in females), or its vehicle (saline) were injected on the dorsum of the right or left hind paw, respectively. One week later, AMP (1 μg) was injected at the same site. Mechanical nociceptive thresholds were then evaluated 10, 20 and 30 min after AMP injection. We observed that, in non-primed paws, AMP did not induce significant change in the mechanical nociceptive threshold, whereas in primed paws, hyperalgesia was observed at all time points in both male and female rats. Two-way repeated measures ANOVA followed by the Bonferroni post-hoc test showed significant difference between the effect of AMP in primed and non-primed paws in both male and female groups (males: F 3,30 = 18.18, **** p

Article Snippet: The drugs used in this study were: adenosine 5′-monophosphate disodium salt (AMP); the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX); the A1 adenosine receptor agonist N6 -cyclopentyladenosine (CPA) and the ryanodine receptor modulator ryanodine, all from Sigma-Aldrich (St. Louis, MO); and, the ecto-5′-nucleotidase inhibitor α,β-methyleneadenosine 5′-diphosphate sodium salt (AMPCP) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Injection

Journal: Biophysical Journal

Article Title: Lanthanides Report Calcium Sensor in the Vestibule of Ryanodine Receptor

doi: 10.1016/j.bpj.2017.03.023

Figure Lengend Snippet: The effect of Ln 3+ on the [3H] ryanodine binding properties of HSR vesicles. ( A ) Relative ryanodine binding of HSR vesicles as a function of [Eu 3+ ] ( open squares ) is shown. ( Inset ) Shown here is relative ryanodine binding in control ( solid squares ), and then at 7.5 μ M ( shaded diamonds ), at 18 μ M ( open triangles ), and at 20 μ M ( open spheres ) Eu 3+ . ( B ) Relative ryanodine binding of HSR vesicles as a function of [Sm 3+ ] ( open squares ) is given. Inset shows the relative ryanodine binding in control ( solid squares ), and then at 4 μ M ( shaded diamonds ), at 11 μ M ( open triangles ), and at 45 μ M ( open spheres ) Sm 3+ . ( C and D ) Shown here is the relative ryanodine binding as a function of [Ca 2+ ] in the absence ( solid squares ) and in the presence of different Eu 3+ ( C ) and Sm 3+ ( D ) concentrations. Eu 3+ was applied at 7.5 μ M ( shaded triangles ) and 20 μ M ( open spheres ). Sm 3+ was applied at 4 μ M ( shaded triangles ) and 45 μ M ( open spheres ).

Article Snippet: Ryanodine-binding assay was carried out using [3H] ryanodine in a medium composed of 1 M NaCl, 25 mM Na/PIPES (pH 7.1), 1 mM Pefabloc (Sigma-Aldrich), and Chelex-treated (Bio-Rad) water.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: A Metabotropic-Like Flux-Independent NMDA Receptor Regulates Ca2+ Exit from Endoplasmic Reticulum and Mitochondrial Membrane Potential in Cultured Astrocytes

doi: 10.1371/journal.pone.0126314

Figure Lengend Snippet: Analysis of i Ca 2+ source. ( A ) ΔF/F 0 averaged response in Fluo-4-AM-labeled rCCA perfused with 1 mM NMDA in the presence of MK-801. ( B ) ΔF/F 0 averaged response in Fluo-4-AM-labeled rCCA perfused with 1 mM NMDA under extracellular Ca 2+ -free conditions. ( C ) ΔF/F 0 averaged response in Fluo-4-AM-labeled rCCA perfused with 1 mM NMDA in the presence of XestosponginC. ( D ) ΔF/F 0 averaged response in Fluo-4-AM-labeled rCCA perfused with 1 mM NMDA in the presence of Ryanodine. Line above traces indicates perfusion with 1 mM NMDA after 120 sec basal recording. Line below (if applicable) indicates the inhibitor used throughout the recording time. ( E ) ∫(ΔF/F 0 ) distribution histogram for cell population responses in MK-801 and extracellular Ca 2+ -free conditions, NMDA distribution is included for comparative purposes. ( F ) ∫(ΔF/F 0 ) distribution histogram for cell population responses in XestosponginC and Ryanodine conditions; the NMDA treated population distribution is included for comparison. The RTV value is indicated by the black arrowhead (see text). Statistical analysis for these distributions with the number of cells and the number of experiments are shown in Table 1 . Representative images from i Ca 2+ responses are shown in S8 Fig .

Article Snippet: Reagents and antibodies Salts, reagents, NMDA, adenosine tri-phosphate (ATP), D(-)-2-amino-5-phosphonopentanoic acid (APV), Kynurenic acid (KYNA), Dizocilpine (MK-801), XestosponginC (XesC), Ryanodine (Ry), Genistein (GX) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) were from Sigma Chemical Co. (St. Louis, MO, USA).

Techniques: Labeling, Size-exclusion Chromatography

Journal: Biophysical Journal

Article Title: Slow Calcium Signals after Tetanic Electrical Stimulation in Skeletal Myotubes

doi:

Figure Lengend Snippet: The slow calcium signal is blocked by TTX and is refractory to ryanodine. The myotubes were exposed to 400 pulses of 1 ms at 45 Hz. ( A ) The experiments were performed in normal saline with calcium ( solid circles ) and 10 μ M TTX ( solid squares ). The mean of at least seven independent experiments ± SE is shown. ( B ) The fluorescence signals after the exposure to 10 μ M ( open squares ) and 30 μ M ( open circles ) of ryanodine, and the control condition without extracellular calcium (0.5 mM EGTA, open triangles ), are shown. The mean of at least 10 independent experiments ± SE is shown. The lower trace shows the timing of the stimulation protocol.

Article Snippet: Before a specific experiment, the cells were incubated from 30 to 60 min with 10 μ M tetrodotoxin (TTX) (Sigma-Aldrich), 10–30 μ M ryanodine (Sigma-Aldrich), 1 μ M nifedipine (Sigma-Aldrich), 2 μ M (−)S-Bay K 8644 (RBI, Natick, MA), 5 μ M xestospongin C (Calbiochem, La Jolla, CA), or 30 μ M (Sigma-Aldrich), in Krebs buffer (145 mM NaCl, 5 mM KCl, 1 mM CaCl2 , 1 mM MgCl2 , 10 mM HEPES-Na, 5.6 mM glucose, pH 7.4).

Techniques: Mass Spectrometry, Fluorescence

Journal: Frontiers in Molecular Neuroscience

Article Title: Cerebellar Kainate Receptor-Mediated Facilitation of Glutamate Release Requires Ca2+-Calmodulin and PKA

doi: 10.3389/fnmol.2018.00195

Figure Lengend Snippet: Facilitation of glutamate release mediated by presynaptic kainate receptor (KAR) activation requires an increase of Ca 2+ in the cytosol at PF-PuC synapses. (A) Time-course of KA (3 μM) effect on eEPSCs amplitude in control condition (circles) and in slices treated with philanthotoxin (squares). (B) Quantification of modulation observed in (A) . (C) Time-course of the effect of KA on eEPSCs amplitude in control slices (circles) and in thapsigargin-treated slices (squares). (D) In slices treated with thapsigargin or ryanodine, the increase of eEPSCs amplitude induced by KA is prevented. The number of slices (from two to three mice) is indicated in parenthesis at the top of each bar. Results are expressed as means ± SEM (** P

Article Snippet: Salts and general reagents were purchased from Sigma (St. Louis, MO, USA); GYKI 53655, D-AP5, NBQX, bicuculline, Rp-Br-cAMP, H-89, forskolin, philanthotoxin, ryanodine, thapsigargin, kainate, Pertussis toxin CMZ and W-7 were obtained from Tocris (Bristol, UK).

Techniques: Activation Assay, Mouse Assay

Journal: Biophysical Journal

Article Title: Biochemical and Electrophysiological Modification of Amyloid Transthyretin on Cardiomyocytes

doi: 10.1016/j.bpj.2016.09.010

Figure Lengend Snippet: Effects of different TTR samples on cytoplasmic calcium levels in automatic HL-1 CMs. ( A ) Time course of cytosolic Ca 2+ signals detected in automatic HL-1 CMs exposed to vehicle ( A ), 10 μ M TTR ( B ), TTR-ol ( C ), TTR-ol in Ca 2+ -free medium ( D ), TTR-ol plus ryanodine ( E ), TTR-ol plus 2-APB ( F ), TTR-fib ( G ), TTR-fib in Ca 2+ -free medium ( H ), TTR-fib plus ryanodine ( I ), TTR-fib plus 2-APB ( J ), TTR-T4 ( K ), TTR-T4 in Ca 2+ -free medium ( L ), TTR-T4 plus ryanodine ( M ), or TTR-T4 plus 2-APB ( N ) for 20 min before SR depletion with 10 mM caffeine. Abbreviations: Veh, vehicle; TTR-ol, oligomeric transthyretin; TTR-fib, fibrillar transthyretin; TTR-T4, native TTR complex with thyroid hormone; TTR-ol 0 Ca, oligomeric TTR in Ca 2+ -free medium; TTR-fib 0 Ca, fibrillar TTR in Ca 2+ -free medium; TTR-T4 0 Ca, TTR-T4 in Ca 2+ -free medium; TTR-ol Rya, oligomeric TTR plus ryanodine; TTR-ol 2-APB, oligomeric TTR plus 2-APB; TTR-fib Rya, fibrillar TTR plus ryanodine; TTR-fib 2-APB, fibrillar TTR plus 2-APB; TTR-T4 Rya, TTR-T4 plus ryanodine; TTR-T4 2-APB, TTR-T4 plus 2-APB.

Article Snippet: Ca2+ release from sarcoplasmic reticulum (SR) stores was studied using 5.0 μ M ryanodine (Rya; Tocris Bioscience, Ellisville, MO) or 5.0 μ M 2-aminoethyl diphenylborinate (2-APB; Sigma-Aldrich) to block the Rya and IP3 receptors, respectively.

Techniques:

Journal: American Journal of Physiology - Cell Physiology

Article Title: Hypoxia selectively upregulates cation channels and increases cytosolic [Ca2+] in pulmonary, but not coronary, arterial smooth muscle cells

doi: 10.1152/ajpcell.00272.2017

Figure Lengend Snippet: Comparison of increases in [Ca 2+ ] cyt due to inward Ca 2+ transportation through Na + /Ca 2+ exchanger (NCX) in CASMC and PASMC. A : representative records showing different patterns of [Ca 2+ ] cyt changes ( patterns 1 and 3 ) in CASMC and PASMC by removal of extracellular Na + (0Na). B : summarized data (means ± SE) showing the amplitude ( left ) and AUC ( middle ) of increases in [Ca 2+ ] cyt and frequency ( right ) of Na + -free medium-induced [Ca 2+ ] cyt oscillations in CASMC and PASMC. C : summarized data showing the percentage of cells exhibiting different patterns of [Ca 2+ ] cyt changes induced by removal of extracellular Na + in CASMC and PASMC. D : summarized data (means ± SE) showing the percentage of responsive cells in CASMC ( n = 212 cells from 8 coverslips) and PASMC ( n = 179 cells from 8 coverslips). E : representative records showing different patterns of [Ca 2+ ] cyt changes induced by removal of extracellular Na + in the presence of 50 μM ryanodine (an RyR inhibitor) in CASMC and PASMC. F : summarized data (means ± SE) showing the amplitude ( left ) and AUC ( middle ) of increases in [Ca 2+ ] cyt and the frequency ( right ) of Na + -free medium-induced [Ca 2+ ] cyt oscillations in CASMC and PASMC. G : summarized data showing the percentage of cells exhibiting a different pattern of [Ca 2+ ] cyt changes induced by removal of extracellular Na + in the presence of 50 μM ryanodine in CASMC and PASMC. H : summarized data (means ± SE) showing the percentage of responsive cells in CASMC ( n = 385 cells from 6 coverslips) and PASMC ( n = 379 cells from 6 coverslips). *** P

Article Snippet: Ryanodine was purchased from Abcam and soluble in ethanol.

Techniques:

Journal: Molecular Pharmacology

Article Title:

doi: 10.1124/mol.112.079707

Figure Lengend Snippet: EGCG sensitizes RyR2 channels to activation by cytosolic Ca 2+ . A, concentration-effect curve of EGCG on specific binding of [ 3 H]ryanodine to cardiac SR membranes. B, EGCG (10 μM) significantly increases the sensitivity of [ 3 H]ryanodine binding

Article Snippet: [3 H]Ryanodine was purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA); nonradioactive ryanodine was from Abcam (Cambridge, MA).

Techniques: Activation Assay, Concentration Assay, Binding Assay