adenylate cyclase (AC)

SQ22536 eliminates the renal protective effects of liraglutide in KK/Ta-Akita mice. In KK/Ta-Akita mice treated with liraglutide plus SQ22536, the amelioration of glomerular histopathological damage by liraglutide is eliminated. Renal cAMP does not rise following SQ22536 treatment. In summary, the adenylate cyclase inhibitor SQ22536 inhibits the beneficial effects of liraglutide for the treatment of nephropathy[5].

SQ22536 inhibits Elk activation induced by forskolin more potently than Elk activation induced by 8-Br-cAMP (IC50 = 170 μM; IC50 = 10 μM).

Rat PAC1hop receptors are expressed by retroviral vectors that transduce HEK293 CRE-luc2P GloResponse luciferase reporter cells. By using limiting dilution cloning, individual cell lines are obtained. A clonal PAC₁-expressing line is then propagated and employed in CRE luciferase assays. To summarize, assay media (DMEM supplemented with 1% fetal bovine serum) is used to plate HEK293 CRE-luc2P cells in 96-well plates (10,000 cells in 80 μL media per well). One day after plating, cells are treated with AC inhibitors (10 μL in assay media/well) for 30 minutes, followed by agonists (10 μL in assay media/well), and are incubated for 4 hours. Once 100 μL/well of Bright-Glo Luciferase Assay Reagent has been added, luciferase activity is measured. Luminescence (RLU) is measured in a Victor3 microtiter plate reader after 2 minutes of agitation at room temperature. Utilizing NS-1 cells, cyclic AMP is quantified. NS-1 cells are, in essence, seeded and grown in 96-well plates for an entire night. Cells are pretreated in media containing 3-isobutyl-1-methylxanthine (0.5 mM) phosphodiesterase inhibitor with or without SQ22536 for 20 minutes the following day. Agonists are added as 10× solutions and let to stimulate cells for 20 minutes after the cells have been pretreated with inhibitors. The quantification of nonacetylated cAMP is then achieved by measuring intracellular cAMP using the cAMP Biotrak enzyme immunoassay technique.

Male guinea-pigs (Duncan Harver, 250–300 g) were killed by cervical dislocation and the upper thoracic aorta removed and placed in physiological saline solution (PSS) containing in mm; NaCl 112, KCl 5, CaCl2 1.8, MgCl2 1, NaHCO3 25, KH2PO4 0.5, NaH2PO4 0.5, glucose 10 and 0.03 phenol red (pH 7.4 with 95%O2/5% CO2). The tissue was cut into rings (∼2 mm wide, and cut longitudinally) or helical strips (∼2 mm wide, 10 mm long). The endothelium was denuded by gently rubbing the lumen of the muscle with filter paper. Tissues were mounted in an organ bath (0.3 ml) and tension measured isometrically. Muscle strips were subjected to a basal tension of 1.25 g and continuously perfused with PSS (1.0 ml min−1 at 37°C) for 30 min before being precontracted with phenylephrine (1 or 6 μm). Removal of the endothelium was confirmed by lack of relaxation to acethylcholine (10 μm for 2 min). Concentration-response curves were obtained by cumulatively applying iloprost (1–1000 nm) in the absence or presence of the adenylyl cyclase inhibitor, SQ22536 (100 μm). SQ22536 was given 30 min before the addition of iloprost and remained throughout. A 1 h wash-out period was allowed between concentration-response curves.[6]

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The stable prostacyclin analogue, iloprost relaxes a variety of blood vessels and increases cyclic AMP, although the relationship between adenosine 3': 5'-cyclic monophosphate (cyclic AMP) and vasorelaxation remains unclear. We therefore investigated the effect of the adenylyl cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) on iloprost-mediated relaxation and cyclic AMP elevation in endothelium-denuded aortic strips. Iloprost (1-1000 nM) caused a concentration-dependent inhibition of phenylephrine (1-6 microM) contractions, the responses being unaffected by pre-incubation with SQ22536 (100 microM) for 30 min. In other experiments 60 nM iloprost caused a 64% inhibition of phenylephrine contractions concomitant with a 3 fold rise in cyclic AMP. SQ22536 completely abolished the iloprost-induced elevation in cyclic AMP while having no significant effect on relaxation. Our results therefore strongly suggest that cyclic AMP-independent pathways are responsible for the vasorelaxant effects of iloprost in guinea-pig aorta.[6]

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Dissolved in saline; 10 mg/kg; s.c.

Male C57BL/6J mice

[1]. Eur J Pharmacol . 2002 Feb 2;436(3):227-33.

[2]. Mol Pharmacol . 2006 Mar;69(3):998-1006.

[3]. J Endocrinol . 2015 Mar;224(3):225-34.

[4]. PLoS One . 2014 Nov 13;9(11):e112741.

[5]. Kidney Int . 2014 Mar;85(3):579-89.

[6]. Br J Pharmacol . 1999 Feb;126(4):845-7.

9-(tetrahydrofuryl)adenine is a nucleoside analogue that is adenine in which the nitrogen at position 9 has been substituted by a tetrahydrofuran-2-yl group. It is an adenylate cyclase inhibitor. It has a role as an EC 4.6.1.1 (adenylate cyclase) inhibitor. It is a nucleoside analogue and a member of oxolanes. It is functionally related to an adenine.

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The effects of inhibition of adenylyl cyclase on isoproterenol-induced relaxation were determined in isolated pulmonary veins of newborn lambs (7-12 days old). In veins constricted with endothelin-1, isoproterenol at concentrations < or = 3 x 10(-9) M had no effect on the cyclic AMP (cAMP) content but caused up to 56% relaxation. At higher concentrations (> or = 10(-8) M), isoproterenol elevated cAMP content and caused further relaxation. In veins constricted with endothelin-1 or U46619 (9,11-dideoxy-11, 9-epoxymethanoprostaglandin prostaglandin F2alpha), the cAMP elevation but not relaxation caused by isoproterenol was abolished by SQ 22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine; an adenylyl cyclase inhibitor]. The effects of isoproterenol on vessel tension and cAMP content were inhibited by propranolol. Rp-8-CPT-cAMPS [8-(4-Chlorophenylthio)-adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer] and Rp-8-Br-PET-cGMPS [beta-phenyl-1, N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer], inhibitors of cAMP- and guanosine-3',5'-cyclic monophosphate (cGMP)-dependent protein kinases, respectively, attenuated relaxation caused by a cAMP analog but not that by isoproterenol. In the crude membrane preparations of pulmonary veins, an increase in the activity of adenylyl cyclase caused by isoproterenol was abolished by propranolol and SQ 22536. These results suggest that cAMP may not play a critical role in isoproterenol-induced relaxation of pulmonary veins of newborn lambs. [1]

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Mast cells are involved in allergic reactions but also in innate immunity and inflammation. Corticotropin-releasing hormone (CRH), the key regulator of the hypothalamic-pituitary-adrenal axis, also has proinflammatory effects, apparently through mast cells. We showed recently that CRH selectively stimulates human leukemic mast cells and human umbilical cord blood-derived mast cells to release newly synthesized vascular endothelial growth factor (VEGF) without release of either preformed mediators or cytokines. This effect was mediated through the activation of CRH receptor-1 and adenylate cyclase with increased intracellular cAMP. However, the precise mechanism by which CRH induces VEGF secretion has not yet been defined. Here, we show that CRH-induced VEGF release was dose-dependently inhibited by the specific protein kinase A inhibitor N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89) or the p38 mitogen-activated protein kinase (MAPK) inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) but not by the specific inhibitor 2'-amino-3'-methoxyflavone (PD98059) of mitogen-activated protein kinase kinase, the upstream kinase of the extracellular signal-regulated protein kinase (ERK) or the c-Jun N-terminal kinase (JNK) inhibitor 1,9-pyrazoloanthrone anthra-(1,9-cd)pyrazol-6(2H)-one (SP600125). Furthermore, CRH significantly increased protein kinase A activity, which could be mimicked by the cell-permeable cAMP analog 8-bromo-cAMP, and was blocked by H89 or the adenylate cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536). CRH also induced rapid phosphorylation of p38 MAPK, which was mimicked by 8-bromo-cAMP and was inhibited by H89 or SB203580. CRH did not stimulate ERK or JNK phosphorylation and did not increase intracellular calcium levels. These results indicate that CRH induces VEGF release in human mast cells via selective activation of the cAMP/protein kinase A/p38 MAPK signaling pathway, thereby providing further insight into the molecular mechanism of how CRH affects the release of a key proinflammatory mediator.[2]

C9H11N5O

205.22

205.096

C, 52.67; H, 5.40; N, 34.13; O, 7.80

17318-31-9

5270

White to off-white solid powder

1.7±0.1 g/cm3

474.8±55.0 °C at 760 mmHg

160-161ºC

241.0±31.5 °C

0.0±1.2 mmHg at 25°C

1.831

-0.17

1

5

1

15

239

0

O1C([H])([H])C([H])([H])C([H])([H])C1([H])N1C([H])=NC2=C(N([H])[H])N=C([H])N=C12

UKHMZCMKHPHFOT-UHFFFAOYSA-N

InChI=1S/C9H11N5O/c10-8-7-9(12-4-11-8)14(5-13-7)6-2-1-3-15-6/h4-6H,1-3H2,(H2,10,11,12)

9-(oxolan-2-yl)purin-6-amine

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (12.18 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 25.0 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: ≥ 2.5 mg/mL (12.18 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 25.0 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: ≥ 2.5 mg/mL (12.18 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 25 mg/mL (121.82 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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