Ca2+-ATPase
The primary target of Rp-8-Br-cGMPS is cGMP-dependent protein kinase (PKG), specifically the Ialpha and Ibeta isoforms. It acts as a competitive inhibitor by binding to the regulatory domain of PKG without activating the enzyme, thus blocking cGMP-induced activation. It inhibits cGKI and cGKII with Ki values of 35 nM and 30 nM, respectively, and shows weaker inhibition of PKA (Ki=11 uM).

The effects of 8-bromo-cGMP on intracellular calcium concentrations in cultured rat aortic smooth muscle cells were studied. Both angiotensin II and depolarizing concentrations of K+ stimulated Ca2+ accumulation in the cytoplasm. The increase in Ca2+ due to angiotensin II was associated with an increase in inositol phosphates, while that due to K+ was not. Preincubation of cells with 8-bromo-cGMP (100 microM) caused an inhibition of peak Ca2+ accumulation to either angiotensin II or K+. To probe the mechanism of action of cGMP in vascular smooth muscle, the effects of cGMP-dependent protein kinase on Ca2+-ATPase from the cultured cell particulate material were investigated. Ca2+-activated ATPase was stimulated approximately equal to 2-fold by exogenous calmodulin and up to 4-fold by low concentrations of purified cGMP-dependent protein kinase. The inclusion of both calmodulin and cGMP-dependent protein kinase resulted in an additive stimulation of Ca2+-ATPase. Stimulation of Ca2+-ATPase activity was observed at all Ca2+ concentrations tested (0.01-1.0 microM). cAMP-dependent protein kinase catalytic subunit and protein kinase C were either ineffective or less effective than cGMP-dependent protein kinase in stimulating the Ca2+-ATPase from rat aortic smooth muscle cells. These results suggest a possible mechanism of action for cGMP in mediating decreases in cytosolic Ca2+ through activation of a Ca2+-ATPase and the subsequent removal of Ca2+ from the cell.[1]

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In non-cellular assays, Rp-8-Br-cGMPS potently inhibits the activation of cGKI and cGKII by cGMP with Ki values of 35 nM and 30 nM, respectively. It shows significantly weaker inhibition of PKA (Ki=11 uM) and does not activate cyclic nucleotide-gated channels. Its resistance to mammalian phosphodiesterases ensures no metabolic side effects in experimental settings.

In vivo, Rp-8-Br-cGMPS blocks the relaxation of rat tail arteries induced by the nitric oxide donor SIN-1 at 10 uM, confirming its functional antagonism of the NO/cGMP/PKG pathway. It has also been shown to influence oxytocin (OT) output in porcine ovarian granulosa cells, with effects varying by concentration (increased at 1-10 nM, decreased at 100 nM).

Cyclic nucleotide-gated cation channels have been studied intensively in the primary sensory neurons of the visual and olfactory systems. Using both anatomical and physiological methods we have shown that they have a much more widespread distribution in the nervous system. In many retinal ganglion cells cGMP, but not cAMP, activates a non-selective conductance that has many of the properties of CNG channels. As many neurons also contain cGMP-dependent protein kinases (PKGs), we have used a variety of cGMP analogues to distinguish the actions of cGMP. Sp-8-Br-PET-cGMPS is a potent non-hydrolyzable cGMP analogue that is an agonist of PKG. We found that Sp-8-Br-PET-cGMPS acts as a competitive inhibitor of at least the rod CNG channel. Rp-8-Br-cGMPS has shown the opposite effects, namely as an agonist of the rod CNG channel and an inhibitor of PKG. In dissociated cell cultures and slices of rodent visual cortex cGMP had multiple rapid and reversible effects on transmission at glutamatergic synapses. Extracellular application of 8-Br-cGMP or Sp-8-Br-PET-cGMPS reduced stimulus evoked EPSPs in cortical slices. In cortical cultures both analogs reduced the frequency of spontaneous EPSCs, but not their amplitude. The effects on both EPSPs and EPSCs were presynaptic. The effects on evoked EPSPs may be due, in part, to reduced calcium influx through voltage-gated calcium channels. The effects on spontaneous EPSCs may be due, in part, to modulation of calcium fluxes through internal stores. Similar modulations of synaptic transmission have been found at gabaergic synapses. On postsynaptic cells, PKG activation produced a dramatic enhancement of the responses to applied NMDA. No effects were detected on applied AMPA/kainate or GABA. Together the results suggest that cGMP may use multiple mechanisms to modulate synaptic efficacy and that its actions may include regulating synaptic plasticity and the relative strength of excitatory and inhibitory drive through neural pathways.[2]

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The standard non-cellular protocol uses a radiometric assay. Recombinant human cGKI or cGKII is incubated with [gamma-32P]-labeled substrate peptide (e.g., BPDEtide) and varying concentrations of Rp-8-Br-cGMPS (0.1 nM to 10 uM) in a buffer containing MgCl2 and ATP. The reaction is terminated after 10 minutes at 30degC, and phosphorylated substrate is separated using phosphocellulose paper for scintillation counting.

Cell-based assays are not the primary focus for this PKG inhibitor. However, functional antagonism can be studied in primary vascular smooth muscle cells pre-contracted with phenylephrine. The nitric oxide donor SIN-1 is added to induce cGMP production and relaxation. Co-incubation with Rp-8-Br-cGMPS (10 uM) blocks this relaxation, and the reduction in vessel relaxation is quantified by measuring isometric tension.

In vivo pharmacological validation typically uses the rat tail artery model. Rat tail arterial rings are mounted in an organ bath, pre-contracted with phenylephrine, and then relaxed with SIN-1 (a NO donor) to induce cGMP-dependent relaxation. Rp-8-Br-cGMPS (10 uM) is added to the bath prior to SIN-1, and the inhibition of relaxation is recorded via a force transducer.

Rp-8-Br-cGMPS is significantly more lipophilic (LogP=1.29) and membrane-permeant than cyclic GMP or Rp-cGMPS, allowing for better cell penetration. It is also highly resistant to hydrolysis by mammalian cyclic nucleotide-dependent phosphodiesterases, ensuring a long intracellular half-life without metabolic side effects in typical assay durations.

No detailed toxicological data is provided, as this is a research tool, not a therapeutic agent. It is classified as a PDE-resistant compound with no reported metabolic side effects in standard in vitro assays. Standard laboratory safety precautions should be followed, and it is not for use in humans.

[1]. Effects of 8-bromo-cGMP on Ca2+ levels in vascular smooth muscle cells: possible regulation of Ca2+-ATPase by cGMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5685-9.

[2]. Modulation of synaptic function by cGMP and cGMP-gated cation channels. Neurochem Int. 2004 Nov;45(6):875-84.

(Sp)-8-Br-cGMPS is a nucleoside 3',5'-cyclic thiophosphate with the nucleobase 8-bromoguanine (Sp- stereoisomer). It is a protein kinase agonist. It is a nucleoside 3',5'-cyclic thiophosphate, belonging to the organobromine class of compounds and also to the purine family. Its function is related to 3',5'-cyclic GMP.

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Rp-8-Br-cGMPS is distinguished from other PKG inhibitors by its combination of high potency, competitive mechanism, and exceptional resistance to phosphodiesterases. The Rp configuration of the exocyclic sulfur atom is critical for its inhibitory activity. It is a standard tool in cardiovascular research for studying the role of PKG in vasodilation, platelet aggregation, and cardiac contractility.

C10H10BRN5NAO6PS

462.15

460.917

208445-06-1

135511711

Typically exists as solid at room temperature

-0.6

4

9

1

24

655

4

[C@@H]1(N2C3N=C(N)NC(=O)C=3N=C2Br)O[C@@H]2CO[P@@](=O)([S-])O[C@H]2[C@H]1O.[Na+]

KRYIOQOBMVFLBO-CIZWMVDRSA-N

InChI=1S/C10H11BrN5O6PS/c11-9-13-3-6(14-10(12)15-7(3)18)16(9)8-4(17)5-2(21-8)1-20-23(19,24)22-5/h2,4-5,8,17H,1H2,(H,19,24)(H3,12,14,15,18)/t2-,4-,5-,8-,23?/m1/s1

9-[(4aR,6R,7R,7aS)-2,7-dihydroxy-2-sulfanylidene-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-6-yl]-2-amino-8-bromo-1H-purin-6-one

Rp-8-Br-Cgmps; 208445-06-1; Rp-8-bromo-Cyclic GMPS (sodium salt); Rp-8-Br-cGMPS sodium salt, >=98% (HPLC); J-008703; Guanosine,8-bromo-,cyclic 3',5'-[hydrogen(R)-phosphorothioate](9ci); Guanosine,8-bromo-,cyclic 3',5'-[hydrogen(S)-phosphorothioate](9ci); Guanosine, 8-bromo-, cyclic 3',5'-[(R)-(hydrogen phosphorothioate)], monosodium salt

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)

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.)