PKA[1]

In the absence of IL-1β, vascular smooth muscle cells release three times as much nitrite when exposed to Sp-8-CPT-cAMPS (100 μM; 24 hours) as when they are stimulated by IL-1β[2]. In rat aortic smooth muscle cells, Sp-8-CPT-cAMPS (100 μM; 24 h) boosts IL-1β-induced production of iNOS protein[2]. The guinea pig trachealis shows anti-spasmogenic efficacy against ACh-induced tension development when Sp-8-CPT-cAMPS (10 μM; 30 min) is applied[3].

IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors Clostridium difficile toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used[2].

[1]. Probing the cyclic nucleotide binding sites of cAMP-dependent protein kinases I and II with analogs of adenosine 3',5'-cyclic phosphorothioates. J Biol Chem. 1990 Jun 25;265(18):10484-91.

[2]. Effect of cyclic GMP-dependent vasodilators on the expression of inducible nitric oxide synthase in vascular smooth muscle cells: role of cyclic AMP. Br J Pharmacol. 1996 Oct;119(4):707-15.

[3]. Evidence that the anti-spasmogenic effect of the beta-adrenoceptor agonist, isoprenaline, on guinea-pig trachealis is not mediated by cyclic AMP-dependent protein kinase. Br J Pharmacol. 2001 Aug;133(8):1201-12.

series of cAMP analogs were synthesized by introducing exocyclic sulfur substitutions at the equatorial (Rp) or axial (Sp) positions of the cyclic phosphate ring and modifying the adenine bases of cAMP. The ability of these compounds to inhibit the binding of [3H]cAMP to the A and B sites of type I (rabbit skeletal muscle) and type II (bovine cardiac muscle) cAMP-dependent protein kinases was quantitatively determined. On average, the Sp isomer showed a 5-fold lower affinity for the A site of the type I isoenzyme and a 30-fold lower affinity for the B site compared to its cyclic phosphate homologue. Affinities for the corresponding sites of the type II isoenzymes were reduced by an average of 20-fold and 4-fold, respectively. Compared to the A and B sites of isoenzyme II, the Rp isomer showed approximately 400-fold and 200-fold decreased affinity for the A and B sites of isoenzyme I, respectively, while its affinity for the A and B sites of isoenzyme II decreased by approximately 200-fold and 45-fold, respectively. Therefore, Sp substitution increased the relative preference for the A site of isoenzyme I and the B site of isoenzyme II. On the other hand, Rp substitution increased the relative preference for the B sites of both isoenzymes. These data suggest that the two intrachain sites of isoenzymes I and II exhibit different tolerances to Rp and Sp substitutions. They also support the hypothesis that, in all four binding sites, the negative charge interacting with conserved arginine is provided by the axial oxygen atom (rather than the previously proposed equatorial oxygen atom). Furthermore, they demonstrated that the combined modification of the cAMP adenine ring and the cyclic phosphate ring enhances the ability to distinguish between the A and B sites of isoenzymes and between isoenzymes I and II. Since it is known that Rp analogs of cAMP can inhibit the activation of cAMP-dependent protein kinases, the findings of this study are of great significance for the synthesis of analogs with high selectivity for isoenzyme I or II. [1]

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Background: Airway smooth muscle participates in the pathogenesis of lung diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). The production of IL-8 is partially regulated by the activation of Gq and Gs coupled receptors. This study investigated the role of cyclic adenosine monophosphate (cAMP) effector protein kinase A (PKA) and cAMP-directly activated exchange proteins (Epac1 and Epac2) in bradykinin-induced IL-8 release in human airway smooth muscle cell lines and their potential molecular mechanisms. [2]Results: The β2 receptor agonist fenoterol enhanced bradykinin-induced IL-8 release. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-O-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-O-Me-cAMPS mimics the action of 8-pCPT-2'-O-Me-cAMP, while the negative control 8-pCPT-2'-O-Me-cGMP does not. Fenoterol, foskeline, and 6-Bnz-cAMP induce VASP phosphorylation, while the PKA inhibitor Rp-8-CPT-cAMPS attenuates this phosphorylation. 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP induce GTP binding of Rap1, but have no effect on Rap2. Treatment of cells with toxins B-1470 and U0126 significantly reduces bradykinin-induced IL-8 release, whether used alone or in combination with PKA and Epac activators. Interestingly, the inhibition of PKA by Rp-8-CPT-cAMPS and the silencing of Epac1 and Epac2 expression by specific siRNAs significantly reduced Rap1 activation and the enhancing effect of PKA and Epac on bradykinin-induced IL-8 release. [2] Conclusion: In summary, our data suggest that PKA, Epac1, and Epac2 work synergistically to regulate the inflammatory properties of airway smooth muscle by signaling to Ras-like GTPases Rap1 and ERK1/2. [2]

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Sudden death following venom poisoning in some Australian cobra species is a cause of death that is not fully understood. We have previously demonstrated that venom from Oxyuranus scutellatus causes cardiovascular failure in anesthetized rats. Pre-administration of a sublethal dose of venom attenuates the response to subsequent administration of a higher (lethal) dose of venom. This study explores the possible mechanisms mediating this "protective effect". Babbita tamarin venom (5 μg/kg, intravenously) induced mild, transient hypotension in anesthetized rats, while a dose of 10 μg/kg resulted in a 73 ± 12% decrease in arterial blood pressure. The venom (20 μg/kg or 50 μg/kg) induced cardiovascular failure in all tested animals (n=12). Pre-administration of “starting” doses of venom (5, 10, and 20 μg/kg) prevented cardiovascular failure induced by 50 μg/kg venom. Furthermore, pre-administration of indomethacin (30 mg/kg) or heparin (300 IU/kg) also prevented sudden death induced by venom (20 μg/kg). The venom had no effect on isolated hearts, suggesting that direct cardiac effects are unlikely to be the cause of “sudden death.” The venom induced endothelium-dependent and endothelium-independent relaxation in pre-constricted rat mesenteric arterial rings, which was inhibited by indomethacin, IbTx, and Rp-8-CPT-cAMPs. This vasodilatory effect was significantly reduced after the second exposure to the venom. Our results suggest that cardiovascular failure induced by O. scutellatus venom may be the result of the combined effects of the release of multiple substances. [3]

508.976

129693-13-6

Rp-8-CPT-cAMPS sodium;221905-35-7;Rp-8-CPT-cAMPS;129735-01-9

23679060

Typically exists as solid at room temperature

3.773

2

11

3

31

701

4

IVNQJYQKSYRLTE-IYKFWPKASA-N

InChI=1S/C16H15ClN5O5PS2/c17-7-1-3-8(4-2-7)30-16-21-10-13(18)19-6-20-14(10)22(16)15-11(23)12-9(26-15)5-25-28(24,29)27-12/h1-4,6,9,11-12,15,23H,5H2,(H,24,29)(H2,18,19,20)/t9-,11-,12-,15-,28?/m1/s1

(4aR,6R,7R,7aS)-6-[6-amino-8-(4-chlorophenyl)sulfanylpurin-9-yl]-2-hydroxy-2-sulfanylidene-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-7-ol

Sp-8-CPT-cAMPS; Rp-8-CPT-cAMPS; 129693-13-6; (4AR,6R,7R,7aS)-6-(6-amino-8-((4-chlorophenyl)thio)-9H-purin-9-yl)-7-hydroxy-2-mercaptotetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinine 2-oxide; Rp-8-CPT-Cyclic AMP (sodium salt); Sp-8-CPT-Cyclic AMPS (sodium salt); CHEMBL1412152;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)