SQ22536 (NSC-53339)

adenylate cyclase (AC)

体外活性：SQ22536 (250 µmol/L) 将腺苷对 ADP 诱导的血小板聚集的抑制作用分别从 8±5% 减弱至 57±5% (p<0.001)。 SQ22536 还可将腺苷引起的血小板内 cAMP 水平从 29±2 pmol/108 血小板减弱至 9±1 pmol/108 个血小板 (p<0.05)。对肌苷（1～4mmol/L）的血小板抗聚集活性和ADP诱导的血小板聚集无影响。激酶测定：SQ22536 比 8-Br-cAMP 诱导的 Elk 激活 (IC50=170 μM) 更有效地抑制毛喉素诱导的 Elk 激活 (IC50=10 μM)。细胞测定：将 HMC-1 细胞和 hCBMC 铺板于 48 孔板中，并进行血清饥饿过夜。第二天，将细胞与指定浓度的 SQ22536 预孵育 30 分钟，然后在存在或不存在 SQ22536 的无血清培养基中用 CRH（HMC-1 为 100 nM，hCBMC 为 1 μM）刺激 3 分钟。然后制备细胞裂解物并使用 ELISA 测定蛋白激酶 A 活性。

SQ22536 消除了利拉鲁肽对 KK/Ta-Akita 小鼠的肾脏保护作用。在用利拉鲁肽联合SQ22536治疗的KK/Ta-Akita小鼠中，利拉鲁肽对肾小球组织病理学损伤的改善被消除。用 SQ22536 治疗后肾 cAMP 不增加。总之，利拉鲁肽治疗肾病的有益作用被腺苷酸环化酶抑制剂SQ22536抑制。

SQ22536 抑制毛喉素诱导的 Elk 活化比 8-Br-cAMP 诱导的 Elk 活化更有效（IC50 = 170 μM；IC50 = 10 μM）。

大鼠 PAC1hop 受体由转导 HEK293 CRE-luc2P GloResponse 荧光素酶报告细胞的逆转录病毒载体表达。通过使用有限稀释克隆，获得单个细胞系。然后繁殖表达 PAC₁ 的克隆系并用于 CRE 荧光素酶测定。总之，使用检测培养基（补充有 1% 胎牛血清的 DMEM）将 HEK293 CRE-luc2P 细胞铺在 96 孔板中（每孔 80 μL 培养基中 10,000 个细胞）。铺板一天后，用 AC 抑制剂（测定介质/孔中 10 μL）处理细胞 30 分钟，然后用激动剂（测定介质/孔中 10 μL）处理细胞，并孵育 4 小时。添加 100 μL/孔的 Bright-Glo 荧光素酶检测试剂后，即可测量荧光素酶活性。在室温下搅拌 2 分钟后，在 Victor3 微量滴定板读数器中测量发光 (RLU)。利用 NS-1 细胞对环 AMP 进行定量。 NS-1 细胞实质上是在 96 孔板中接种并生长一整夜。第二天，将细胞在含有 3-异丁基-1-甲基黄嘌呤 (0.5 mM) 磷酸二酯酶抑制剂（含或不含 SQ22536）的培养基中预处理 20 分钟。以 10× 溶液的形式添加激动剂，并在用抑制剂预处理细胞后刺激细胞 20 分钟。然后使用 cAMP Biotrak 酶免疫分析技术测量细胞内 cAMP，从而实现非乙酰化 cAMP 的定量。

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

78.85

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (12.18 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (12.18 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (12.18 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 25 mg/mL (121.82 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。