PKA

Rp-8-Br-cAMPS (1 mM; 4-6 hours) can completely block the ability of 2-chloroadenosine to inhibit LAK cytotoxicity and significantly alleviate the inhibitory effect of 2-chloroadenosine on cytokine production [1]. Rp-8-Br-cAMPS (50-100 μM; 2 min) inhibits insulin secretion in 832/13 cells (derived from INS-1 cells) [2].

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In type 2 diabetes, beta-cells become glucose unresponsive, contributing to hyperglycemia. To address this problem, we recently created clonal insulin-producing cell lines from the INS-1 insulinoma line, which exhibit glucose responsiveness ranging from poor to robust. Here, mechanisms that determine secretory performance were identified by functionally comparing glucose-responsive 832/13 beta-cells with glucose-unresponsive 832/2 beta-cells. Thus, insulin secretion from 832/13 cells maximally rose 8-fold in response to glucose, whereas 832/2 cells responded only 1.5-fold. Insulin content in both lines was similar, indicating that differences in stimulus-secretion coupling account for the differential secretory performance. Forskolin or isobutylmethylxanthine markedly enhanced insulin secretion from 832/13 but not from 832/2 cells, suggesting that cAMP is essential for the enhanced secretory performance of 832/13 cells. Indeed, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, rp-isomer (Rp-8-Br-cAMPS) an inhibitor of protein kinase A (PKA), inhibited insulin secretion in response to glucose with or without forskolin. Interestingly, whereas forskolin markedly increased cAMP in 832/2 cells, 832/13 cells exhibited only a marginal rise in cAMP. This suggests that 832/13 cells are more sensitive to cAMP. Indeed, the cAMP-induced exocytotic response in patch-clamped 832/13 cells was 2-fold greater than in 832/2 cells. Furthermore, immunoblotting revealed that expression of the catalytic subunit of PKA was 2-fold higher in 832/13 cells. Moreover, when the regulatory subunit of PKA was overexpressed in 832/13 cells, to reduce the level of unbound and catalytically active kinase, insulin secretion and PKA activity were blunted. Our findings show that cAMP-PKA signaling correlates with secretory performance in beta-cells. [2]

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When 832/13 cells were stimulated by 15 mM glucose, Rp-8-Br-cAMPS inhibited secretion by 21% (P < 0.01; Fig. 3C); when cells were stimulated by forskolin in addition to glucose, the high concentration of Rp-8-Br-cAMPS completely blocked the potentiating effect of forskolin (P < 0.001; Fig. 3C). In 832/2 cells, neither concentration of Rp-8-Br-cAMPS exerted a significant effect on insulin secretion in the presence or absence of forskolin [2].

Rp-8-Br-cAMPS (1 mg; intraperitoneal injection; 10 days) can improve the immune function of mice in a mouse retroviral infection model[3].

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Initial experiments were performed to evaluate the biodistribution of Rp-8-Br-cAMPS. The concentration of the compound was measured by HPLC on liver, spleen and plasma of the animals implanted with the osmotic pumps set to deliver 0.7 mg Rp-8-Br-cAMPS/24h. As shown in Table 1, Rp-8-Br-cAMPS was found at significant concentration in all the organs analysed demonstrating that the compound was delivered to relevant tissues such as spleen. The toxicity of the Rp-8-Br-cAMPS was also evaluated and as shown in Table 2, there were no unscheduled deaths during the study. Furthermore, there were no clinical signs observed in the mice throughout the time of administration in the different experiments (Table 2). All animals were considered to have achieved satisfactory bodyweight gains through the study. Macroscopic examination at time of sacrifice of the animals did not reveal any abnormalities. Thus, we conclude that Rp8-Br-cAMPS was well tolerated in control non-infected animals as well as in infected mice. In a next set of experiments performed with iterative injections of Rp-8-Br-cAMPS during 10 days, we evaluated the effects of the compound in mice with established RadLV-Rs infection. Typically, each experiment encompassed four groups of seven to ten mice (2 groups with mice inoculated with RadLV-Rs eight weeks earlier and 2 groups with age-matched sham-injected controls). Mice were treated either with Rp-8-Br-cAMPS for 10 days (daily intraperitoneal injections of 1mg/mouse) or equivalent sham-injections with 300 μl PBS. At the end of the 10-days injection period, mice were sacrificed. Treatment with Rp-8-Br-cAMPS had no significant effect on the extent of lymphadenopathy and splenomegaly which is typical of RadLV-Rs retroviral infection. Indeed, weights of the lymphoid organs were always similar in infected mice treated with Rp-8-Br-cAMPS and in infected mice receiving PBS (Fig. 1). Size, and cellularity were also similar in both groups (data not shown). After preparation of cell suspensions from the peripheral lymph nodes of each mouse from the different experimental groups, we measured the proliferative responses to soluble anti-CD3 mAb. The cells were cultured for 72 hours in the presence of anti-CD3 mAb (2C11: 4μg/ml). During this 72-hour culture period, Rp-8-Br-cAMPS (1 mM) was added to the cells isolated from mice treated with this compound. Administration of Rp-8-Br-cAMPS had no effect on the response to antiCD3 mAb in non-infected mice (not shown). As expected, in RadLV-Rs retrovirus-infected mice, proliferative responses to anti-CD3 mAb were nearly abolished, with stimulatory indexes (defined as stimulated/non stimulated CPMs) typically around 10% of values reached with the cells of normal mice (Fig. 2). Treatment of non-infected mice with Rp-8-BrcAMPS did not significantly modify their proliferative responses to the anti-CD3 mAb (not shown). In contrast, i.p. administration of the PKA type I inhibitor Rp-8-Br-cAMPS to RadLV-Rs infected mice strongly increased their responses to the anti-CD3 mAb (31.97 ± 4.21 n=10 vs 5.979 ± 0.882 n=8, p<0.0001). In fact, stimulatory indexes values reached more than 50% of control values in most experiments (Fig. 2). When the cells of infected and treated mice were activated in vitro in the absence Rp-8-Br-cAMPS, the effect of the treatment was partially lost and became non significant in certain experiments (not shown). When cells from untreated, retrovirus-infected mice were incubated in the presence of Rp-8-Br-cAMPS in vitro, a significant improvement of T cell responses also occurred (12.45 ± 2.02 n=8 vs 5.98 ± 0.88 n=8) (p=0.013) as demonstrated in our previous studies but stimulation index remained much lower than those of infected mice treated with IP injections of the compound (p=0.0014) demonstrating the in vivo impact of PKA type I inhibition on the restoration of proliferative responses in infected mice. [3]

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Despite its specific virological features and the preferential tropism of RadLV-Rs for B cells rather than CD4 T cells, MAIDS presents with striking similarities with HIV infection such as polyclonal and sustained immune activation involving CD4 as well as CD8 T cells and leading to the impairment of T cell responses. Indeed, recent evidence suggests that abnormal immune activation rather than direct cytopathic effects of HIV plays a major role in the pathogenesis of HIV infection, at least during its early stages. The PGE2-cAMP-PKA type I pathway is most probably involved in this abnormal activation, as shown by the reduction of activated CD8 CD38 T cells in HIV-infected patients treated with celecoxib and by in vitro data demonstrating partial restoration of T cell responses after incubation with Rp-8-BrcAMPS. Although COX-2 is indeed overexpressed in HIV infection, it is likely that other soluble factors might play a role in the activation of adenylate cyclase. CCR5 plays a paramount importance in the pathogenesis of HIV infection and this could be partly independent of its function as an entry coreceptor for HIV. Interestingly, HIV coreceptors such as CCR5 and CXCR4 are coupled to protein G and ligand binding activates adenylate cyclase. Gp120 binding to its coreceptor could therefore directly increase cAMP concentration by a PGE2-independent mechanism. It is therefore important to design pharmacological approaches acting downstream of adenylate cyclase activation [3].

PKA Activity [2]
Confluent 832/13 cells in 12-well dishes kept in RPMI-1640 medium were infected with an equal titer of AdCMV-PKAreg or AdCMV-βGal as described above. The medium was removed, and the cells were washed in HBSS-3 mM glucose and subsequently incubated for 10 min in HBSS containing 15 mM glucose and 2.5 μM forskolin. Then, the cells were washed once in ice-cold PBS and placed on ice, and 150 μl homogenization buffer [50 mM N-[Tris(hydroxymethyl)methyl]-2-aminoethane-sulfonic acid (TES), 1 mM EDTA, 0.1 mM EGTA, 250 μM sucrose; pH 7.4] containing protease inhibitors (1 μg/ml pepstatin, 10 μg/ml leupeptin, 1 μg/ml antipain), phosphatase inhibitor (0.1 μM okadaic acid), and phosphodiesterase inhibitor (1.67 mM IBMX) were added. Homogenates were prepared by aspirating cells through a syringe five times followed by centrifugation at 5000 rpm for 10 min at 4 C. PKA in 10-μl aliquots of the homogenates was allowed to phosphorylate a synthetic peptide substrate (13 μg Kemptide) in a mixture of 39 mM TES, 0.5 M sucrose, 0.1 M MgSO4, 10 mM dithioerytritol, 0.4 mM ATP (pH 7.4), and 15 μCi [γ32P]ATP; the reactions were carried out in duplicate at 30 C in the presence or absence of protein kinase inhibitor (17 μM). The reaction was terminated after 30 min by addition of 10 μl 1% BSA-1 mM ATP and 10 μl 12.5% trichloroacetic acid and subsequently put on ice for 10 min. The reaction mixture was centrifuged at 3000 rpm for 3 min, after which 10 μl homogenate were transferred to a Whatman P81 membrane. The membrane was washed three times in H3PO4 and once in acetone. Activity was determined by scintillation counting. PKA activity was calculated by subtracting the activity in reactions to which protein kinase inhibitor had been added. In each experiment, PKA activity was expressed as percent of that in controls.

Insulin Secretion Studies [2]
For assay of insulin secretion, the cells were grown to confluence in 24-well dishes, and the glucose concentration in the culture medium was switched to 5 mM 18 h before assay. When assayed, the cells were washed in HEPES balanced salt solution (HBSS; 114 mM NaCl; 4.7 mM KCl; 1.2 mM KH2PO4; 1.16 mM MgSO4; 20 mM HEPES; 2.5 mM CaCl2; 25.5 mM NaHCO3; 0.2% BSA, pH 7.2) supplemented with 3 mM glucose for 2 h at 37 C. Insulin secretion was then measured by static incubation of the cells for 1 h in 0.8 ml HBSS containing the glucose concentration indicated in the figure legends. When KATP-independent glucose sensing was examined, the K+ concentration in the HBSS during the static incubation was increased to 35 mM, whereas the Na+ concentration was reduced to 89.8 mM, and 250 μM diazoxide was added. Insulin was measured by the Coat-a-Count kit (DPC), which recognizes human insulin and cross reacts approximately 20% with rat insulin. Insulin content of cells was determined after acid ethanol extraction of the hormone.

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Western Blot Analysis [2]
Cells were homogenized in 0.25 M sucrose; 1 mM EDTA, pH 7.0; 1 mM dithioerytritol (DTE); 20 μg/ml leupeptin; 20 μg/ml antipain; and 1 μg/ml pepstatin A. A cytosolic fraction was prepared by centrifugation at 110,000 × g for 45 min at 4 C. Proteins were resolved by SDS-PAGE and electroblotted to nitrocellulose membranes. Western blot analysis was performed by the enhanced chemiluminescence system, using polyclonal rabbit antihuman PKAcat or PKAreg antibodies (sc-903 and sc 907, respectively). In the experiments for quantitation, 5 μg protein (determined by the BCA Protein Assay Kit; Pierce) from each cell line was loaded onto the gel. Expression was determined by densitometric analysis of the Western blots (n = 5 for each line).

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Determination of cAMP [2]
For determination of cAMP accumulation, cells were grown to confluence in 12-well dishes in complete RPMI-1640 medium, as described above. The cells were then kept in RPMI-1640 medium containing 5 mM glucose for 12 h, followed by a 2-h incubation in HBSS supplemented with 3 mM glucose. Two minutes after a switch to HBSS containing either 3 or 15 mM glucose with or without 2.5 μM forskolin, cAMP was extracted from cells by adding 0.5 ml 80% ethanol to the cells. cAMP levels were determined after centrifugation of the cellular extracts by RIA.

Animal/Disease Models: Murine leukemia retrovirus RadLV-Rs treated male C57BL/6 mice[3]
Doses: 1 mg
Route of Administration: Intraperitoneal injection (i.p.); 10 days
Experimental Results: Had no significant effect on the extent of lymphadenopathy and splenomegaly which is typical of RadLV-Rs retroviral infection. Strongly increased responses to the anti-CD3 mAb. Improved T cell responses.

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Male C57BL/6 mice were bred in our facility. Mice were injected twice intraperitoneally (i.p.) at the age of 4 and 5 weeks with 0.25 ml of the cell free viral extract. Agematched control mice were injected twice i.p. with 0.25 ml phosphate buffered saline (PBS). At different times post infection, mice were killed by CO2 asphyxiation. Peripheral lymph nodes (inguinal, axillary and cervical) were dissociated with syringes to obtain single cell suspensions and passed through a nylon cell strainer, washed three times with complete RPMI 1640 medium and counted on Thoma cytometer after trypan blue exclusion prior to further analysis or cell culture. For in vivo experiments, Rp-8-Br-cAMPS 1 mg qd was injected i.p. to the mice during 10 days. In initial experiments aimed at evaluating Rp-8-Br-cAMPS biodistribution, groups of infected and healthy mice were treated by subcutaneous implantation of Alzet osmotic pumps filled with 10 mg Rp-8-Br-cAMPS dissolved in PBS and set to deliver 0.7 mg/24h. The pumps were implanted 14 days before sacrifice. [3]

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Quantitative Determination of Rp-8-Br-cAMPS Concentration Suitable sample preparation and HPLC methods were developed for quantitative determination of Rp-8-Br-cAMPS in mice tissues and serum samples that allows evaluation of drug concentrations of in vivo experiments. Calibrations were done with Rp-8-Br-cAMPS and 8-Br-cAMP. Both compounds gave sufficient linearity in a range between 0 ng/ml and 1000 ng/ml. Each mice sample was transferred into a borosilicate micro mortar (1000
L) followed by addition of 250
l water. After manual homogenization and addition of 750
l water the resulting suspension was transferred into 1,5 mL sarstedt-tubes with screw cap. After a minimum period of 4 hours at –70 °C all samples were freeze-dried in a Speed-Vac under oil-pump vacuum overnight. The freezedried material was suspended in 1000
L MeOH/H20 (1:1;v.v) and placed for 15 min in an ultrasonic bath, followed by centrifugation for 15 min (13000 rpm). 0.85 mL of the supernatant was loaded on an anion exchanger SPE cartridge (Chromafix 400mg SB/ArtNr.: 731835/Machery-Nagel), washed twice with 2 ml of water and then eluted with 1 ml 0,6 M NaCl. The resulting solution was directly used for HPLC analytics. For complete loading 300
l of the solute were applied on the 200
l sample loop. This volume of solute produced reproducible data during calibration of the HPLC method [3].

[1]. Adenosine-mediated inhibition of cytotoxic activity and cytokine production by IL-2/NKp46-activated NK cells: involvement of protein kinase A isozyme I (PKA I). Immunol Res. 2006;36(1-3):91-9.

[2]. Enhanced cAMP protein kinase A signaling determines improved insulin secretion in a clonal insulin-producing beta-cell line (INS-1 832/13). Mol Endocrinol. 2004 Sep;18(9):2312-20.

[3]. In vivo administration of a PKA type I inhibitor (Rp-8-Br-cAMPS) restores T-cell responses in retrovirus-infected miceJ. The Open Immunology Journal, 2008, 1(1).

Adenosine inhibits the production of a variety of cytokines/chemokines and suppresses the cytotoxic activity of mouse and human NK cells activated by IL-2 or Ly49D, NKp46 receptor crosslinking. These effects are mediated by A2A type adenosine receptors, with mechanisms including stimulation of adenylate cyclase, increased cAMP production, and activation of PKA. PKA I, but not PKA II, is involved in the inhibitory effect of adenosine. Blocking the regulatory subunit (but not the catalytic subunit) of PKA I eliminates the inhibitory effect of adenosine. These findings suggest that tumor-produced adenosine inhibits the activity of NK cells and other effector cells, thereby protecting tumors from immune-mediated destruction. [1]

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Mice AIDS (MAIDS) is caused by infection with the murine leukemia retrovirus RadLV-Rs, characterized by T cell unresponsiveness and severe immunodeficiency, accompanied by increased susceptibility to a variety of experimental opportunistic infections, similar to that of HIV infection. T cell unresponsiveness is associated with elevated intracellular cAMP levels, which trigger a multi-step pathway involving the activation of type I protein kinase A (PKA I), ultimately leading to inhibition of proximal T cell receptor (TCR) signaling. We have previously demonstrated that blocking PKA I with the selective inhibitor Rp-8-Br-cAMPS can restore in vitro T cell function in MAIDS mice and HIV-infected mice. In this report, we investigated the effects of parenteral administration of Rp-8-Br-cAMPS on MAIDS mice. We found that the compound was non-toxic and partially restored the proliferative response of anti-CD3 monoclonal antibodies in vitro, but had no effect on the characteristic lymphadenopathy and splenomegaly of MAIDS syndrome. [3] This is the first report of improved immune function after short-term parenteral administration of a type I PKA inhibitor in a retroviral infection model. Our observations provide a validation of the principle for reversing retrovirus-induced immunodeficiency by novel drugs that act directly on the proximal steps of T cell signaling. Further research is needed to determine whether PKA type I blockade can also improve antigen-specific immune responses and other immune parameters in infected mice, such as CD4 cytokine secretion and CD8 function. [3]

C10H10BRN5NAO5PS

446.15

925456-59-3

Sp-8-Br-cAMPS sodium;1573115-90-8;Rp-8-Br-cAMPS;129735-00-8

Typically exists as solids at room temperature

0

BrC1=NC2=C(N=CN=C2N1[C@@H]1O[C@]2([H])CO[P@](S)(=O)O[C@@]2([H])[C@H]1O)N.[NaH]

925456-59-3; 1573115-90-8; Rp-8-bromo-Cyclic AMPS (sodium salt); Rp-8-bromo-Cyclic AMPS sodium salt; 8-Bromoadenosine 3',5'-cyclic monophosphothiaoate, Sp-isomer sodium salt; sodium;(4aR,6R,7R,7aS)-6-(6-amino-8-bromopurin-9-yl)-2-oxido-2-sulfanylidene-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-7-ol; 8-Bromoadenosine3',5'-CyclicMonophosphothioateSp-IsomerSodiumSalt; 8-bromo-adenosinecyclic3',5'-[hydrogen[P(R)]-phosphorothioate],monosodiumsalt;

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.)