The primary target of Ryanodine is the Ca²⁺ release channel (ryanodine receptor) of the sarcoplasmic reticulum in skeletal and cardiac muscle. High-affinity binding sites are present on heavy SR vesicle preparations, with a KD of 20-200 nM for skeletal muscle preparations, and cardiac preparations yield non-linear Scatchard plots indicating multiple receptor sites (0.5 pmol/mg protein, KD = 36 nM and 1.7 pmol/mg protein, KD = 340 nM). [1]

In heavy skeletal muscle SR vesicles passively loaded with ⁴⁵Ca²⁺, Ryanodine (0.01 μM) rendered vesicles permeable to ⁴⁵Ca²⁺ when diluted into a medium containing the channel inhibitors Mg²⁺ and ruthenium red. At concentrations greater than 10 μM, ⁴⁵Ca²⁺ efflux was inhibited. An optimal stimulatory effect was observed when vesicles were incubated with ryanodine at 37°C in media that caused partial opening of the channel. Similar results were obtained using cardiac SR vesicles. [1]
Using the slowly permeating molecule L-[³H]glucose, ryanodine did not appreciably change the regulation of efflux rates by external Ca²⁺, Mg²⁺, and adenine nucleotide. L-glucose-permeable vesicles released L-[³H]glucose with a first-order rate constant of about 25 min⁻¹ in the presence of 5 μM free Ca²⁺ and 2.5 mM AMP-PCP. Ryanodine increased the L-glucose efflux rate by a factor of about 2 in media containing low Ca²⁺ concentrations and decreased the rate constant from about 25 to 10 min⁻¹ when the channel was nearly fully activated. [1]
In isolated SR Ca²⁺ channels incorporated into lipid bilayers, ryanodine decreases their conductance twofold and locks them in an open subconductance state. [2]

In heavy skeletal muscle SR vesicles passively loaded with ⁴⁵Ca²⁺, Ryanodine (0.01 μM) rendered vesicles permeable to ⁴⁵Ca²⁺ when diluted into a medium containing the channel inhibitors Mg²⁺ and ruthenium red. At concentrations greater than 10 μM, ⁴⁵Ca²⁺ efflux was inhibited. An optimal stimulatory effect was observed when vesicles were incubated with ryanodine at 37°C in media that caused partial opening of the channel. Similar results were obtained using cardiac SR vesicles. [1]
Using the slowly permeating molecule L-[³H]glucose, ryanodine did not appreciably change the regulation of efflux rates by external Ca²⁺, Mg²⁺, and adenine nucleotide. L-glucose-permeable vesicles released L-[³H]glucose with a first-order rate constant of about 25 min⁻¹ in the presence of 5 μM free Ca²⁺ and 2.5 mM AMP-PCP. Ryanodine increased the L-glucose efflux rate by a factor of about 2 in media containing low Ca²⁺ concentrations and decreased the rate constant from about 25 to 10 min⁻¹ when the channel was nearly fully activated. [1]
In isolated SR Ca²⁺ channels incorporated into lipid bilayers, ryanodine decreases their conductance twofold and locks them in an open subconductance state. [2]

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In a dose-related manner, ryanodine (100-5000 nM, 30-120 minutes) irreversibly suppresses both slow- and fast-twitch twitching as well as tonic tone [1]. Ryanodine generates frightening, slowly developing respiratory contractions at a dose of 250 nM, which cannot be stopped by 5 mM Co2+[1].

A thermal shift assay was not described. However, the effect of Ryanodine on SR Ca²⁺ release channels was studied using ⁴⁵Ca²⁺ efflux from passively loaded vesicles. Heavy SR vesicles (5-10 mg protein/ml) were incubated at 22°C in a loading medium containing 20 mM K Pipes, pH 7, 0.1 M KCl, 0.1 mM EGTA, and 0.2-5.1 mM Ca²⁺. After 2 h, samples received 0.1 volume of loading medium containing dissolved ryanodine, followed by incubation at 22°C and 37°C. Ca²⁺ efflux was assessed by diluting vesicles into iso-osmolal unlabeled Ca²⁺ release channel-inhibiting (10 mM Mg²⁺ plus 10 μM ruthenium red) or channel-activating (1 mM EGTA plus 0.93 mM Ca²⁺; 5 μM free Ca²⁺) media. Untrapped and released Ca²⁺ were separated by filtration, and retained radioactivity was determined. [1]
A rapid quench apparatus was used to determine initial ⁴⁵Ca²⁺ efflux rates. Vesicles (2 mg protein/ml) incubated with 1 mM ⁴⁵Ca²⁺ for 2 h at 22°C were diluted with 4 volumes of release medium containing 6.25 mM EGTA, 5.55 mM Ca²⁺, and 0, 1.25, or 187.5 μM ryanodine. Efflux was inhibited at indicated times by adding 4 volumes of quench solution containing 22.5 mM Mg²⁺ and 22.5 μM ruthenium red. Vesicles were then filtered and rinsed. [1]
The effect of Ryanodine on SR Ca²⁺ permeability was also assessed by ⁴⁵Ca²⁺ influx. Vesicles (5 mg protein/ml) were incubated at 37°C with 5 μM ryanodine for indicated times in media containing various free Ca²⁺ and AMP-PCP concentrations. Vesicles were then diluted with 9 volumes of ⁴⁵Ca²⁺ influx medium yielding 1 mM ⁴⁵Ca²⁺ and 0.25 mM AMP-PCP. Influx was stopped at 15 s by adding 25 volumes of a medium containing 10 mM Mg²⁺ and 10 μM ruthenium red, followed by filtration and rinsing. [1]

Rat extensor digitorum longus (EDL) or soleus muscles were used. A small bundle of 10-40 fibres was dissected and transferred to a temperature-controlled bath (25±0.2°C) perfused with normal Krebs solution. Muscles were stimulated directly (0.5 ms duration, supramaximal voltage) by platinum electrodes set 5 mm apart. Force was measured isometrically. Ryanodine was dissolved in double-distilled water as a 1 mM stock and diluted to the required concentration in buffer. Only a single concentration was used in each dose-response experiment because ryanodine's action is irreversible with normal washing. [2]
For contracture studies, muscles were perfused with Ca²⁺-free EGTA solution (normal Krebs without CaCl₂ plus 1 mM EGTA) to relax contractures. For Co²⁺ experiments, HEPES-buffered solutions containing 5 mM CoCl₂ were used. All solutions were equilibrated with 95% O₂-5% CO₂ and contained (+)-tubocurarine (15×10⁻³ mM) at pH 7.4 and 25°C. [2]
For charge movement experiments, the triple Vaseline-gap technique was used to voltage clamp a segment of a single EDL fibre. From a holding potential of -90 mV, the fibre was subjected to a 20 mV hyperpolarizing control step (35 ms) followed 50 ms later by a 35 ms depolarizing test step. Charge movement was recorded in fibres with or without internal EGTA (10 mM). Ryanodine (5 or 10 μM) was added to the external solution. [2]

Rat extensor digitorum longus (EDL) or soleus muscles were used. A small bundle of 10-40 fibres was dissected and transferred to a temperature-controlled bath (25±0.2°C) perfused with normal Krebs solution. Muscles were stimulated directly (0.5 ms duration, supramaximal voltage) by platinum electrodes set 5 mm apart. Force was measured isometrically. Ryanodine was dissolved in double-distilled water as a 1 mM stock and diluted to the required concentration in buffer. Only a single concentration was used in each dose-response experiment because ryanodine's action is irreversible with normal washing. [2]
For contracture studies, muscles were perfused with Ca²⁺-free EGTA solution (normal Krebs without CaCl₂ plus 1 mM EGTA) to relax contractures. For Co²⁺ experiments, HEPES-buffered solutions containing 5 mM CoCl₂ were used. All solutions were equilibrated with 95% O₂-5% CO₂ and contained (+)-tubocurarine (15×10⁻³ mM) at pH 7.4 and 25°C. [2]
For charge movement experiments, the triple Vaseline-gap technique was used to voltage clamp a segment of a single EDL fibre. From a holding potential of -90 mV, the fibre was subjected to a 20 mV hyperpolarizing control step (35 ms) followed 50 ms later by a 35 ms depolarizing test step. Charge movement was recorded in fibres with or without internal EGTA (10 mM). Ryanodine (5 or 10 μM) was added to the external solution. [2]
Toxicity Summary: Ryanodine has extremely high affinity to the open-form ryanodine receptor, a group of calcium channels found in skeletal muscle, smooth muscle, and heart muscle cells. It binds with such high affinity to the receptor that it was used as a label for the first purification of that class of ion channels and gave its name to it. At nanomolar concentrations, ryanodine locks the receptor in a half-open state, whereas it fully closes them at micromolarconcentration. The effect of the nanomolar-level binding is that ryanodine causes release of calcium from calcium stores as thesarcoplasmic reticulum in the cytoplasm, leading to massive muscular contractions.

[1]. Ryanodine activation and inhibition of the Ca2+ release channel of sarcoplasmic reticulum. J Biol Chem. 1986 May 15;261(14):6300-6.

[2]. The action of ryanodine on rat fast and slow intact skeletal muscles. J Physiol. 1989 Jul;414:399-413.

Ryanodine binds to the Ca²⁺ release channel of sarcoplasmic reticulum. Stimulation or inhibition of Ca²⁺ efflux is influenced by divalent cation concentration, temperature, time of incubation, and concentration of ryanodine. At 37°C and 0.1 mM Ca²⁺, essentially all skeletal muscle SR release vesicles were rendered permeable to ⁴⁵Ca²⁺ at a ryanodine concentration of 0.01 μM when diluted into a medium containing Mg²⁺ and ruthenium red. Inhibition of ⁴⁵Ca²⁺ efflux was observed only at ryanodine concentrations in excess of 10 μM. The effectiveness of ryanodine appeared dependent on whether the Ca²⁺ release channel was present in an open or closed configuration. [1]
In intact rat skeletal muscle, ryanodine (100 nM) potentiates caffeine contractures, and at higher concentrations (>250 nM) induces a slow contracture that is dependent on the presence of extracellular calcium but is not blocked by 5 mM Co²⁺. A second type of contracture, induced by readdition of Ca²⁺ after relaxing the first contracture in Ca²⁺-free EGTA solution, is dependent on millimolar extracellular Ca²⁺ and is prevented by 5 mM Co²⁺. [2]
Ryanodine (10 μM) had virtually no effect on asymmetric charge movement or calcium current in cut EDL fibres with 10 mM EGTA internally over 30 minutes. In fibres without internal EGTA, ryanodine caused the fibre to contract and be lost in 21±4 minutes (compared to 46±5 minutes in controls), suggesting that the eventual loss is related to a rise in myoplasmic calcium exacerbated by ryanodine. [2]

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Rhinodine is an insecticidal alkaloid isolated from the South American plant Ryania speciosa. It is a Rhinodine receptor modulator and a plant-derived insecticide. It is an alkaloid and also a cyclic hemiacetal. Rhinodine has been reported to exist in Brassica napus and Ryania speciosa, and relevant data are available. Rhinodine is a methylpyrroleic acid ester derived from plants in the Rhinodine genus. It disrupts the Rhinodine receptor calcium release channel, thereby altering calcium release from the sarcoplasmic reticulum and ultimately leading to changes in muscle contraction. It has been used in insecticides. Currently, it is often used in combination with thiopyruvate and other calcium ATPase inhibitors to inhibit calcium entry into the sarcoplasmic reticulum.

C25H35NO9

493.54700

493.231

15662-33-6

11317883

White to off-white solid powder

1.6±0.1 g/cm3

714.2±60.0 °C at 760 mmHg

385.7±32.9 °C

0.0±2.4 mmHg at 25°C

1.685

3.86

7

9

4

35

1010

11

O[C@@]1([C@@]([C@@]2(O)O3)4C)[C@@]35[C@](O)(CC[C@H](C)[C@H]5O)[C@@](C2)(C)[C@@]1([C@H](OC(C6=CC=CN6)=O)[C@]4(O)C(C)C)O

JJSYXNQGLHBRRK-SFEDZAPPSA-N

InChI=1S/C25H35NO9/c1-12(2)22(31)17(34-16(28)14-7-6-10-26-14)23(32)18(4)11-21(30)19(22,5)25(23,33)24(35-21)15(27)13(3)8-9-20(18,24)29/h6-7,10,12-13,15,17,26-27,29-33H,8-9,11H2,1-5H3/t13-,15+,17+,18-,19+,20-,21-,22+,23+,24+,25+/m0/s1

[(1R,2R,3S,6S,7S,9S,10R,11S,12R,13S,14R)-2,6,9,11,13,14-hexahydroxy-3,7,10-trimethyl-11-propan-2-yl-15-oxapentacyclo[7.5.1.01,6.07,13.010,14]pentadecan-12-yl] 1H-pyrrole-2-carboxylate

ryanodine; 15662-33-6; Ryanodin; Ryanodol, 3-(1H-pyrrole-2-carboxylate); Ryania;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~101.31 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.53 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.25 mg/mL (2.53 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1.25 mg/mL (2.53 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)