L-type calcium channel
L-type calcium channels (Ca channels) in guinea-pig ventricular heart cells [2].
EC₅₀: 0.8 μM (for blocking L-type calcium current) [2].
Maximum inhibition: 85% at 5 μM [2].
Hill coefficient: 2.37 [2].
Sodium channels (Na channels) in guinea-pig ventricular heart cells [2].
At 10 μM, blocked 26.7 ± 4.3% of sodium current [2].
At 1 μM, blocked 7.6 ± 2.7% of sodium current [2].
Ruthenium red targets sarcoplasmic reticulum Ca2+ release channels, specifically ryanodine receptors. It inhibits Ca2+ release from the sarcoplasmic reticulum, blocks Ca2+ uptake and release from mitochondria, and prevents Ca2+ release from ryanodine-sensitive stores. The compound modifies both cardiac and skeletal muscle Ca2+ release channels by multiple mechanisms. It also binds to various cellular structures due to its polycationic nature.

The staining of intracellular organelles and structures with ruthenium red appears to be dependent on the dose and duration of exposure to the reagent [2]. With an EC50 of 0.8 μM, ruthenium red efficiently and dose-dependently inhibits L-type calcium currents in isolated guinea pig ventricular heart cells [2]. In isolated guinea pig ventricular heart cells, ruthenium red (10 μM) inhibits 26.7% of sodium current, slows down its inactivation time course, and blocks sarcoplasmic Ca2+ release channels or mitochondrial Ca2+ absorption [2].

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Block of L-type calcium current (I_Ca): Ruthenium red blocked the L-type calcium current in a dose-dependent manner. Significant block was observed at concentrations as low as 0.3 μM. The EC₅₀ was 0.8 μM, maximum inhibition of 85% was reached at 5 μM, and the Hill coefficient was 2.37 [2].
Voltage-dependence of I_Ca: Ruthenium red did not shift the voltage-dependence of calcium current activation or steady-state inactivation (determined with a 1-second prepulse). However, removal of calcium current inactivation at positive voltages was considerably reduced at concentrations above 1 μM. A slowing of the inactivation time course of the calcium current was also observed [2].
Block of sodium current (I_Na): At 10 μM (a concentration commonly used to block sarcoplasmic Ca release channels or mitochondrial Ca uptake), ruthenium red blocked 26.7 ± 4.3% (n=8) of the sodium current and slowed its inactivation time course. No effect was observed on the voltage-dependence of current activation or inactivation. At 1 μM, the peak sodium current was decreased by 7.6 ± 2.7% (n=3) [2].
Kinetics of I_Na: Ruthenium red (10 μM) significantly increased the half-width (width at 50% of peak amplitude) of the transient sodium current for potentials between -30 and +30 mV, and almost abolished the voltage-dependence of the maximum inactivation rate of the sodium current. The time to peak current was not significantly affected [2].

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In vitro, Ruthenium red is used as a pharmacological tool to study calcium signaling pathways. It inhibits sarcoplasmic reticulum Ca2+ release, blocks Ca2+ uptake and release from mitochondria, and prevents Ca2+ release from ryanodine receptors. The compound is also used as a histochemical stain for electron microscopy, where it binds to negatively charged structures. Its polycationic nature allows it to interact with various cellular components, making it useful for studying calcium homeostasis and cellular structure.

In vivo, Ruthenium red is used as a pharmacological tool to study calcium signaling in animal models. By inhibiting Ca2+ release from intracellular stores, the compound can modulate muscle contraction, neurotransmitter release, and other calcium-dependent processes. However, its use in vivo is limited due to its polycationic nature and potential toxicity. The compound is primarily used in research settings rather than for therapeutic applications. Comprehensive in vivo pharmacological studies are limited.

Whole-cell patch-clamp technique: Freshly isolated guinea-pig ventricular heart cells were used. The whole-cell patch-clamp technique was employed to study L-type calcium and sodium currents at room temperature (22-25°C). To record specific ionic currents, cells were locally superfused with solutions that blocked all other currents. For calcium current experiments, the extracellular solution contained (in mM): TEACl 140, CaCl₂ 1.8, MgCl₂ 1, HEPES 10, glucose 11 (pH 7.3 with TEAOH). The intracellular pipette solution contained (in mM): CsCl 110, TEACl 30, MgCl₂ 1, MgATP 5, EGTA 10, HEPES 10 (pH 7.3 with TEAOH). For sodium current recordings, the extracellular solution contained (in mM): NaCl 30, TEACl 110, CaCl₂ 1, MgCl₂ 1, CoCl₂ 2, GdCl₃ 0.1, HEPES 10, glucose 11 (pH 7.3 with TEAOH). Data were acquired at 6.7 kHz (Ca current) or 25 kHz (Na current) and filtered with an 8-pole lowpass Bessel filter at 5 kHz. A P/5 subtraction protocol was used to remove linear capacitive currents during Na current experiments [2].

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For non-cell-based receptor binding assays, Ruthenium red can be evaluated using purified ryanodine receptors or sarcoplasmic reticulum membrane preparations. Radioligand binding displacement experiments are performed using [3H]-ryanodine as the radiolabeled ligand. Membrane preparations are incubated with increasing concentrations of the test compound and a fixed concentration of the radioligand at appropriate conditions. Bound radioligand is separated from free by rapid filtration. Nonspecific binding is determined in the presence of excess unlabeled ryanodine. IC50 values are calculated from displacement curves.

Cell isolation: Guinea-pig heart cells were enzymatically isolated from the left ventricle. Dissociated cells were placed in a small chamber on an inverted microscope and continuously superfused with normal Tyrode solution [2].
Calcium current recordings: Currents were elicited by 150 ms depolarizing pulses from a holding potential of -80 mV to 0 mV at 0.1 Hz. Current-voltage relationships were obtained by applying depolarizing pulses from -80 mV to various test potentials. Steady-state inactivation was determined using a double-pulse protocol: 1-second inactivating prepulses from -100 mV to various potentials, followed by a 400 ms test pulse to 0 mV after an 8 ms repolarization interval to the holding potential [2].
Sodium current recordings: Currents were elicited by 20 ms depolarizing pulses from a holding potential of -110 mV to test potentials. Current-voltage relationships were obtained by applying pulses from -50 to +60 mV. Steady-state inactivation was determined using a double-pulse protocol: 500 ms inactivating prepulses from -100 mV to -20 mV, followed by a 30 ms test pulse to -10 mV after a 1 ms repolarization interval to the holding potential [2].

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For in vitro cellular assays, cells or isolated organelles (e.g., sarcoplasmic reticulum vesicles, mitochondria) are used. Calcium release assays are performed by loading cells or organelles with calcium-sensitive fluorescent dyes such as Fluo-4 or Fura-2. After dye loading, cells are treated with Ruthenium red and calcium release is stimulated by agonists or depolarizing agents. Fluorescence intensity is measured using a fluorescence plate reader or spectrophotometer. The reduction in calcium signal compared to control indicates inhibition of calcium release.

For in vivo animal studies, Ruthenium red can be administered to rodents via intraperitoneal or intravenous injection. In muscle physiology studies, the compound's effects on muscle contraction and calcium release are assessed. In cardiovascular studies, its effects on cardiac function are evaluated. However, the compound's polycationic nature and potential toxicity limit its use in vivo. Comprehensive in vivo studies are limited. The compound is primarily used in research settings rather than for therapeutic applications.

Ruthenium red has a molecular weight of 786.35 and a molecular formula of Cl6H42N14O7Ru3. It is a polycationic dye that is soluble in water. The compound should be stored at -20°C for long-term stability. It is supplied as a crystalline solid. The compound is not for human or veterinary use. It is used as a histochemical stain and a pharmacological tool for studying calcium signaling.

The toxicity profile of Ruthenium red has not been extensively reported. As a polycationic compound, it may have toxic effects at high concentrations due to its interactions with cellular structures. The compound is not for human or veterinary use. Standard safety precautions should be observed when handling the compound, including appropriate personal protective equipment. Potential adverse effects may include disruption of calcium homeostasis and cellular toxicity. The compound is for research use only.

[1]. Ruthenium red staining for ultrastructural visualization of a glycoprotein layer surrounding the spore of Bacillus anthracis and Bacillus subtilis. J Microbiol Methods. 2004 Jul;58(1):23-30.

[2]. Ruthenium red as an effective blocker of calcium and sodium currents in guinea-pig isolated ventricular heart cells. Br J Pharmacol. 1998 Jun; 124(3): 465-472.

Ruthenium red (structural formula: (NH₃)₅Ru-O-Ru(NH₃)₄-O-Ru(NH₃)₅Cl₆) is a synthetic crystalline inorganic polycationic dye [2].
The ruthenium red used in this study was obtained from a commercial source and was approximately 50% pure. The actual concentration was estimated to be about 50% of the indicated concentration [2].
At concentrations used to assess intracellular Ca movements (e.g., to block sarcoplasmic Ca release channels or mitochondrial Ca uptake), ruthenium red induced significant block of both Ca and Na channels in heart cells [2].
The rapid onset of calcium current block suggests an extracellular site of action, possibly involving binding to negatively charged amino acid residues at the outer mouth of Ca channels. The slower phase of block may indicate an intracellular effect or a progressive dephosphorylation process [2].

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Ruthenium red is a polycationic dye that inhibits sarcoplasmic reticulum Ca2+ release, blocks Ca2+ uptake and release from mitochondria, and prevents Ca2+ release from ryanodine receptors. It modifies the cardiac and skeletal muscle Ca2+ release channels (ryanodine receptors) by multiple mechanisms. The compound is used as a histochemical stain and a pharmacological tool for studying calcium signaling. It has a molecular weight of 786.35. It is for research use only and not for human consumption.

CL6H42N14O2RU3

786.35

785.887

11103-72-3

117587625

Brown to black solid powder

3.11

>500ºC

14

22

0

25

0

0

N.N.N.N.N.N.N.N.N.N.N.N.N.N.[O-2].[O-2].[Cl-].[Cl-].[Cl-].[Cl-].[Cl-].[Cl-].[Ru+3].[Ru+3].[Ru+4]

GOOXRYWLNNXLFL-UHFFFAOYSA-H

InChI=1S/6ClH.14H3N.2O.3Ru/h6*1H;14*1H3;;;;;/q;;;;;;;;;;;;;;;;;;;;2*-2;2*+3;+4/p-6

azane;bis(oxygen(2-));bis(ruthenium(3+));ruthenium(4+);hexachloride

KU6M163B0R; DTXSID80108179; C.I. 77800; Red, Ruthenium; ...; 11103-72-3;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

H2O: 10 mg/mL (12.72 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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