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Molecular Pharmacology Dec 1992Junctional sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle were pretreated with 0.1-500 microM ryanodine under equilibrium conditions optimal...
Junctional sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle were pretreated with 0.1-500 microM ryanodine under equilibrium conditions optimal for receptor binding, followed by the removal of bound alkaloid by several washes in Ca(2+)- and ryanodine-free buffer. Pretreatment with > 100 nM ryanodine results in a concentration-dependent decrease in the Bmax of the high affinity sites and a complete loss of measurable low affinity binding sites that persist for > 48 hr. Quantitative analysis of residual ryanodine using three different methods demonstrates that the inhibition is not the result of residual ryanodine bound to its receptor. Ca2+ transport measurements made with antipyrylazo III show that actively loaded ryanodine-pretreated SR exhibits a persistent insensitivity to ryanodine- and daunomycin-induced Ca2+ release that is not seen with washed control vesicles. Lipid bilayer membranes fused with SR vesicles exhibit rapidly fluctuating single-channel events with a conductance of 468 pS in asymmetric CsCl solutions. Ryanodine (10 microM) produces a unidirectional transition to a slowly fluctuating half-conductance state that is not reversed by perfusion of the bilayer with Ca(2+)-free buffer and subsequent addition of dithiothreitol. However, dithiothreitol added in the ryanodine pretreatment medium offers marked protection against ryanodine-induced loss of binding sites and allows complete restoration of native gating behavior of single channels in bilayer lipid membrane. Using three different experimental approaches, the data demonstrate that the alkaloid at micromolar concentration persistently alters SR Ca2+ release channel function, perhaps by uncoupling four negatively cooperative binding sites. The oxidation of critical receptor thiols is implicated in the process.
Topics: Animals; Calcium Channel Blockers; Lipid Bilayers; Muscles; Rabbits; Receptors, Cholinergic; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sulfhydryl Compounds
PubMed: 1480132
DOI: No ID Found -
Biochemical and Biophysical Research... Feb 2015Ryanodine is a cell permeant plant alkaloid that binds selectively and with high affinity to ryanodine receptor (RyR) Ca(2+) release channels. Sub-micromolar ryanodine...
Ryanodine is a cell permeant plant alkaloid that binds selectively and with high affinity to ryanodine receptor (RyR) Ca(2+) release channels. Sub-micromolar ryanodine concentrations activate RyR channels while micromolar concentrations are inhibitory. Several reports indicate that neuronal synaptic plasticity, learning and memory require RyR-mediated Ca(2+)-release, which is essential for muscle contraction. The use of micromolar (inhibitory) ryanodine represents a common strategy to suppress RyR activity in neuronal cells: however, micromolar ryanodine promotes RyR-mediated Ca(2+) release and endoplasmic reticulum Ca(2+) depletion in muscle cells. Information is lacking in this regard in neuronal cells; hence, we examined here if addition of inhibitory ryanodine elicited Ca(2+) release in primary hippocampal neurons, and if prolonged incubation of primary hippocampal cultures with inhibitory ryanodine affected neuronal ER calcium content. Our results indicate that inhibitory ryanodine does not cause Ca(2+) release from the ER in primary hippocampal neurons, even though ryanodine diffusion should produce initially low intracellular concentrations, within the RyR activation range. Moreover, neurons treated for 1 h with inhibitory ryanodine had comparable Ca(2+) levels as control neurons. These combined findings imply that prolonged incubation with inhibitory ryanodine, which effectively abolishes RyR-mediated Ca(2+) release, preserves ER Ca(2+) levels and thus constitutes a sound strategy to suppress neuronal RyR function.
Topics: Animals; Calcium; Calcium Ionophores; Cells, Cultured; Cresols; Cytoplasm; Endoplasmic Reticulum; Hippocampus; Ionomycin; Neurons; Rats, Sprague-Dawley; Ryanodine; Ryanodine Receptor Calcium Release Channel; Thapsigargin
PubMed: 25623539
DOI: 10.1016/j.bbrc.2015.01.065 -
European Journal of Pharmacology Mar 1994The mechanisms of ryanodine-induced contractions were studied in strips of femoral arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats... (Comparative Study)
Comparative Study
The mechanisms of ryanodine-induced contractions were studied in strips of femoral arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Ryanodine (30 nM to 30 microM) alone contracted arterial strips in a dose-dependent manner. The maximum contraction in SHR was about 5 times greater than that in WKY. SHR strips had about a 30 times lower threshold than WKY strips. Pretreatment with ryanodine caused a more potent dose-dependent inhibition of 10 mM caffeine-induced contractions in SHR than in WKY. Ryanodine (10 microM) failed to contract SHR strips in a CaCl2-free solution, whereas caffeine induced a transient contraction in the solution. Under the conditions that the sarcoplasmic reticulum was depleted of Ca2+ by exposure of the strips to the CaCl2-free solution, ryanodine caused a contraction in the presence of extracellular Ca2+. Verapamil (1 nM) to 1 microM) relaxed the strips precontracted with ryanodine in a dose-dependent manner. In WKY strips exposed to a 15 mM K+ solution, ryanodine-induced contractions were potentiated and were not different from those of SHR strips in a normal K+ (5.9 mM) solution. These results suggest that ryanodine contracts rat femoral arteries by promoting Ca2+ influx through voltage-dependent Ca2+ channels, and that the ability of ryanodine to contract the arteries may depend on the state of the Ca2+ channels.
Topics: Animals; Caffeine; Calcium Chloride; Femoral Artery; Male; Muscle Contraction; Muscle, Smooth, Vascular; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Ryanodine; Verapamil
PubMed: 8206110
DOI: 10.1016/0014-2999(94)90383-2 -
Pflugers Archiv : European Journal of... Apr 1987The effects of ryanodine on isometric tension development, net cellular Ca content and 45Ca efflux were measured in the isolated rabbit aorta. Pretreatment of the tissue...
The effects of ryanodine on isometric tension development, net cellular Ca content and 45Ca efflux were measured in the isolated rabbit aorta. Pretreatment of the tissue with 10 microM ryanodine for 30 min reduced the norepinephrine (NE)-induced tension of the aortic rings bathed in the absence of extracellular Ca2+ in parallel with a reduction in the NE-stimulated 45Ca efflux. Ryanodine alone caused a delayed moderate increase in 45Ca efflux, slowly increasing the fractional loss from 0.02/min to 0.03/min, without any increase in tension. The increased 45Ca efflux was accompanied by a decrease in the net cellular Ca content. When ryanodine and NE were administered in sequence during 45Ca efflux, we found a quantitative correlation between the ryanodine induced loss of 45Ca and the inhibition of the NE-induced 45Ca release. We conclude that the inhibition of the NE-induced contraction by 10 microM ryanodine is the result of depletion of Ca from intracellular stores rather than inhibition of Ca2+ release.
Topics: Alkaloids; Animals; Aorta, Thoracic; Calcium; Calcium Radioisotopes; In Vitro Techniques; Muscle Contraction; Muscle, Smooth, Vascular; Rabbits; Ryanodine
PubMed: 3588253
DOI: 10.1007/BF00581127 -
The Journal of Biological Chemistry Nov 2020Structural analyses identified the central domain of ryanodine receptor (RyR) as a transducer converting conformational changes in the cytoplasmic platform to the RyR...
Structural analyses identified the central domain of ryanodine receptor (RyR) as a transducer converting conformational changes in the cytoplasmic platform to the RyR gate. The central domain is also a regulatory hub encompassing the Ca-, ATP-, and caffeine-binding sites. However, the role of the central domain in RyR activation and regulation has yet to be defined. Here, we mutated five residues that form the Ca activation site and 10 residues with negatively charged or oxygen-containing side chains near the Ca activation site. We also generated eight disease-associated mutations within the central domain of RyR2. We determined the effect of these mutations on Ca, ATP, and caffeine activation and Mg inhibition of RyR2. Mutating the Ca activation site markedly reduced the sensitivity of RyR2 to Ca and caffeine activation. Unexpectedly, Ca activation site mutation E3848A substantially enhanced the Ca-independent basal activity of RyR2, suggesting that E3848A may also affect the stability of the closed state of RyR2. Mutations in the Ca activation site also abolished the effect of ATP/caffeine on the Ca-independent basal activity, suggesting that the Ca activation site is also a critical determinant of ATP/caffeine action. Mutating residues with negatively charged or oxygen-containing side chains near the Ca activation site significantly altered Ca and caffeine activation and reduced Mg inhibition. Furthermore, disease-associated RyR2 mutations within the central domain significantly enhanced Ca and caffeine activation and reduced Mg inhibition. Our data demonstrate that the central domain plays an important role in channel activation, channel regulation, and closed state stability.
Topics: Adenosine Triphosphate; Binding Sites; Caffeine; Calcium; Calcium Signaling; HEK293 Cells; Humans; Magnesium; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Myocardium; Protein Binding; Protein Stability; Protein Structure, Tertiary; Ryanodine; Ryanodine Receptor Calcium Release Channel
PubMed: 32878990
DOI: 10.1074/jbc.RA120.013512 -
Life Sciences 1993[3H]Ryanodine is shown to specifically bind to cultured myotubes from 10 day quail embryo pectoralis. The binding of [3H]ryanodine increases in a time-dependent manner...
[3H]Ryanodine is shown to specifically bind to cultured myotubes from 10 day quail embryo pectoralis. The binding of [3H]ryanodine increases in a time-dependent manner reaching 38 +/- 3 fmol/mg protein at 4 h. A level of theophylline (THEO; 5mM) that induces propagated wave-like contractures, doubles the capacity of the myotubes to bind [3H]ryanodine (78 +/- 7 fmol/mg protein at 4 h). Polycationic ruthenium red (100 microM) only partially inhibits (56%) [3H]ryanodine-binding, whereas the membrane permeable channel antagonist [2,6-dichloro-4-dimethyl-amino-phenyl]-isopropylamine (20 microM) inhibits occupancy > 80%. Ryanodine (10 microM) interferes with THEO-induced contractures. Pretreatment with micromolar ryanodine for 48 h, followed by washout for 48 h, causes a persistent decrease in [3H]ryanodine-binding sites. Persistent [3H]ryanodine receptor blockade coincides with a dramatic shift in AChE forms found in the myotubes. A transition from the embryonic 4S and 7S globular forms to the 20S collagen-tailed (adult) form is evident within 12 hr exposure to ryanodine and progresses after removal of the alkaloid from the culture medium, mimicking the transition that normally occurs during myocyte maturation in vivo. These results suggest that SR Ca++ movements and excitation-contraction coupling may, at least in part, contribute to AChE maturation.
Topics: Acetylcholinesterase; Animals; Calcium Channels; Cells, Cultured; Coturnix; Muscle Proteins; Pectoralis Muscles; Phenethylamines; Radioligand Assay; Ruthenium Red; Ryanodine; Ryanodine Receptor Calcium Release Channel; Theophylline
PubMed: 7681926
DOI: No ID Found -
The Journal of Pharmacology and... Mar 1991The distribution of [3H]ryanodine binding in subcellular fractions isolated from rat vas deferens (RVD) paralleled that of NADPH cytochrome C reductase activity...
The distribution of [3H]ryanodine binding in subcellular fractions isolated from rat vas deferens (RVD) paralleled that of NADPH cytochrome C reductase activity indicating an endoplasmic reticulum origin of the binding sites. Scatchard analysis of [3H] ryanodine binding indicated an homogenous site with a Kd value of 6.4 nM. The maximum number of ryanodine binding sites was 488 fmol of [3H]ryanodine per milligram of protein. Norepinephrine (NE) or ATP endogenously released after electrical field stimulation (tetrodoxin-sensitive responses), both produced a biphasic contraction of the RVD when the action of the other was blocked. When NE was the agonist (prazosin-sensitive response), the initial transient contraction was suppressed by 30 microM ryanodine whereas the secondary component was abolished by 2 microM nifedipine. When ATP was the agonist (P2 tachyphylaxis-sensitive response), both phases of the contraction were suppressed by 2 microM nifedipine. However, the initial phasic component of the contraction induced by endogenously released ATP was also inhibited by 30 microM ryanodine except at high stimulation frequency (10 Hz). Exogenously added NE and alpha, beta methylene ATP produced concentration-dependent contractions of the RVD. The effect of both agonists was inhibited by 2 microM nifedipine whereas 30 microM ryanodine had little effect except at high concentrations of NE. We conclude that ryanodine binding sites reside in RVD endoplasmic reticulum. There was a lack of uniformity in the effect of ryanodine and nifedipine against alpha adrenoceptor stimulation and purinoceptor stimulation indicating a difference in the stimulation-contraction coupling process between these two modes of stimulation.
Topics: Animals; Binding Sites; Calcium; Electric Stimulation; Kinetics; Male; Muscle Contraction; Muscle, Smooth; Nifedipine; Radioligand Assay; Rats; Rats, Inbred Strains; Receptors, Purinergic; Ryanodine; Vas Deferens
PubMed: 2005572
DOI: No ID Found -
The International Journal of... Aug 2005The ryanodine receptor (RyR)/Ca2+ release channel mobilizes Ca2+ from internal calcium stores to support a variety of neuronal functions. To investigate the presence of...
The ryanodine receptor (RyR)/Ca2+ release channel mobilizes Ca2+ from internal calcium stores to support a variety of neuronal functions. To investigate the presence of such a protein in mammalian retina, we applied ryanodine binding, PCR and antibodies against known RyRs. Surprisingly, ryanodine-binding properties of retinal endoplasmic reticulum-enriched membrane fraction were vastly different from those of skeletal and cardiac muscles ryanodine-binding proteins. In common with the skeletal and cardiac muscle, ryanodine bound with high-affinity to two or more types of binding site (Kd1 = 20.6 and Kd2 = 114 nM); binding was strongly stimulated by high concentrations of NaCl; it was inhibited by tetracaine and the protein appeared to possess an ATP-binding site. Unlike cardiac and skeletal muscle, RyRs in retina binding was Ca2+-independent; inhibited by caffeine and dantrolene; less sensitive to ruthenium red; and unaffected by La3+. Also, in retina, ryanodine rapidly associated to and dissociated from its binding sites. Furthermore, although the protein bound the ATP analog BzATP, retinal ryanodine binding was not stimulated by nucleotides. Immunostaining of bovine retinal sections with anti-RyR2 showed a strong staining of amacrine, horizontal and ganglion cells. Finally, using RT-PCR, the three known RyR isoforms were identified in retina. However, consistent with the novel binding properties, the peptide maps yielded by trypsin treatment and Western blotting demonstrate different patterns. Together, the results suggest that retina expresses a novel ryanodine-binding protein, likely to be a ryanodine receptor. Its presence in retina suggests that this protein might play a role in controlling intracellular Ca2+ concentration.
Topics: Animals; Blotting, Western; Calcium; Cattle; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Kinetics; Polymerase Chain Reaction; Protein Binding; RNA, Messenger; Receptors, Retinoic Acid; Retina; Ryanodine; Temperature
PubMed: 15896674
DOI: 10.1016/j.biocel.2005.03.001 -
Bulletin of Experimental Biology and... Apr 2023The effect of the compound N-(2,3,4-trimethoxy)-N-{2-[(2,3,4-trimethoxybenzyl)amino]ethyl}-1,2-ethane-diamine (code ALM-802) on the amplitude of the Ca response in the...
The effect of the compound N-(2,3,4-trimethoxy)-N-{2-[(2,3,4-trimethoxybenzyl)amino]ethyl}-1,2-ethane-diamine (code ALM-802) on the amplitude of the Ca response in the cell was studied in in vitro experiments. The concentration of intracellular calcium was assessed using a Fura-2 two-wave probe. The experiments were performed on a culture of isolated rat hippocampal neurons. The effect of compound ALM-802 on the activity of ryanodine receptors (RyR2) was studied on an isolated strip of rat myocardium. The compound ALM-802 (69.8 μM) in hippocampal neurons causes a significant decrease in the amplitude of the Ca response induced by addition of KCl to the medium. Experiments performed on an isolated myocardial strip showed that compound ALM-802 (10 M) almost completely blocked the positive inotropic reaction of the strip to the RyR2 agonist caffeine (5×10 M). The data obtained indicate that the decrease in the concentration of Ca ions in the cell caused by ALM-802 is due to its ability to block RyR2 located on the membrane of the sarcoplasmic reticulum, which can be associated with the antiarrhythmic activity of the compound.
Topics: Rats; Animals; Ryanodine Receptor Calcium Release Channel; Myocardium; Anti-Arrhythmia Agents; Caffeine; Sarcoplasmic Reticulum; Calcium; Ryanodine
PubMed: 37170020
DOI: 10.1007/s10517-023-05781-7 -
Pflugers Archiv : European Journal of... Sep 1988The caffeine-sensitive intracellular Ca store was characterized and the mechanism of action of ryanodine in the store was studied using K-depolarized guinea-pig taenia...
The caffeine-sensitive intracellular Ca store was characterized and the mechanism of action of ryanodine in the store was studied using K-depolarized guinea-pig taenia caecum. (1) After incubation of the preparation with CaCl2 (Ca loading), caffeine was applied in Ca-deprived medium, to produce a transient contraction and to monitor the amount of the stored Ca. As duration of Ca deprivation was prolonged, the amplitude of the caffeine-induced contraction was decreased. When ryanodine was applied during Ca deprivation, the rate of the decrease was remarkably accelerated. (2) The rate of rise of the contraction induced by external Ca [Ca)o) was slowed by preceding depletion of the stored Ca by caffeine, compared with that observed in the Ca loaded preparation. However, in the presence of ryanodine, even if stored Ca was depleted by caffeine, the rate of rise of the (Ca)o-induced contraction remained at a higher level. (3) These results suggest that ryanodine stimulates a leak of the stored Ca, and that the contraction induced by the transmembrane influxed Ca could be modulated by the amount of Ca in, or leakiness of, the caffeine-sensitive Ca store.
Topics: Alkaloids; Animals; Caffeine; Calcium; Cecum; Female; Guinea Pigs; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; Ryanodine
PubMed: 3174395
DOI: 10.1007/BF01907555