U73122

phospholipase C (PLC); 5-LO (5-lipoxygenase)

在与 PMN 分离的膜中，U-73122 有效抑制 PLC 的受体偶联激活 [1]。 U-73122 抑制 N-甲酰基-甲硫氨酰-亮氨酰-苯丙氨酸诱导的人多形核中性粒细胞 (PMN) 聚集以及相应的二酰基甘油和 IP3 的合成 [2]。在洋地黄皂苷通透的细胞中，U-73122 显着抑制由氧化震颤素-M 或鸟苷-5'-O-（3-硫代三磷酸）引起的磷酸肌醇释放，但不通过添加 Ca2+ 来抑制 [3]。

在内毒素血症小鼠中，U73122 显着增强心脏做功、收缩和舒张率，而不改变心率，而对假手术动物则没有影响，并且大大降低了 TNF-α mRNA 的表达 [4]。与注射到 VTA 中的载体相比，U73122 (400 nM/μL) 显着缩短了雌激素和黄体酮诱导的仓鼠脊柱前凸的总长度。活动监测器中仓鼠的运动行为不受 U73122 VTA 输注的影响；然而，与给予 SKF38393 的仓鼠相比，蝇蕈醇显着减少了光束中断的总数 [5]。

1-[6-[[17β-3-甲氧基雌二醇-1,3,5（10）-三烯17-基]氨基]己基]-1H-吡咯-2,5-二酮（U-73122）（IC50值1-5微M）抑制了由多种激动剂诱导的人血小板的聚集，但不受其中吡咯二酮被吡咯二酮取代的紧密类似物1-[6-][17β-3-乙氧基雌醇-1,3,5。U-73122的抑制不是由细胞内环状AMP的增加介导的。相反，由凝血酶或血栓素模拟物（5Z，9α，11α，13E，15S）15-羟基-11,9-（环氧甲氧基）前列腺素-5，13，-二烯-1-酸（U-46619）诱导的1，4，5-三磷酸肌醇（IP3）的产生和随后胞浆Ca++的快速增加被U-73122抑制，但不被U-73343抑制。IP3水平的降低似乎反映了对IP3产生的抑制，因为U-73122（Ki分别为9和40微M）抑制了由血小板的可溶性部分催化的磷脂酰[3H]肌醇和磷脂酰[3H]肌醇4,5-二磷酸的水解。此外，U-73122抑制胶原诱导的血栓素B2的产生，但不抑制外源性添加的花生四烯酸支持的血栓素，这表明U-73122也抑制花生四烯酸的受体偶联动员。在血小板与[3H]花生四烯酸预孵育后，U-73122减弱了凝血酶诱导的[3H]磷脂酰肌醇的损失和[3H]磷酸的积累。U-73122不抑制从猪胰腺或从金刚蛙和Naja Naja的静脉中纯化的磷脂酶A2的活性。尽管U-73122既不抑制外源性花生四烯酸转化为血栓素B2，也不抑制血栓素受体拮抗剂[1S-[1α，2β（5Z），3β，4α]]-7-[3-[[2-[2-[（苯基氨基）-羰基]-肼基]甲基]-7-氧杂双环[2.2.1]-庚-2-基-5-庚烯酸与血小板膜的结合，但它是花生四烯酸诱导的血小板聚集的有效抑制剂。这些数据与观察到的U-73122通过抑制信号转导的磷脂酶C依赖性成分的机制抑制血栓素受体激动剂U-46619的血小板活化的作用一致。U-73122，而不是U-73343，也抑制N-甲酰基-甲酰基-亮氨酸诱导的人类多形核中性粒细胞（PMN）的聚集以及相关的IP3和二甘醇的产生。在用N-甲酰基-甲酰基-亮氨酸苯丙氨酸刺激的PMN中产生的二烷基甘油可皂化74+/-7%，并被U-73122（Ki=2 microM）抑制[2]。

激动剂诱导的PMN中IP3的产生是通过使用竞争性放射结合测定法来测量的。PMN（2 x 106-107）溶于0.2 mL磷酸盐缓冲盐水中，pH 7.4[NaC1（138 mM），Na2HPO4（8.1 mM）、KH2PO4（1.5 mM）和KCI（2.7 mM）。CaCl2（1.0 mM）；MgC12（1.0 mM M）和葡萄糖（0.1%，w/v）]在锥形聚丙烯管中于37°C的摇动水浴中孵育。在加入激动剂FMLP（0.1μM）加细胞松弛素B（5μg/mL）前3分钟加入U-73122或U-73343（在1μL DMSO中）。FMLP和细胞松弛素B分别加入DMSO和乙醇各1μL中。每个实验中都包括适当的车辆控制。PMN孵育混合物通过加入0.07 mL冰冷的TCA（20%，w/v）来骤冷，并且一部分（0.2 mL）TCA提取物通过如上所述的血小板的竞争性放射结合进行处理以测量IP3[2]。

Hamsters are hormone-primed with 17β-oestradiol at h 0 and progesterone at h 45. At h 48, hamsters are pretested for motor behaviour, followed by sexual behaviour testing, and bilateral infusions of U73122 (400 nM/μL) or saline vehicle. Thirty minutes after infusions, hamsters are re-tested for sexual behaviour (post inhibitor infusion test) and, immediately after testing, infused bilaterally with SKF38393 (100 ng/μL), muscimol (100 ng/μL), or saline vehicle. Thirty minutes after the agonist or vehicle infusions, lordosis and motor behaviour of hamsters is reassessed (post agonist infusion test). All hamsters are assigned to one pretreatment condition, U73122 or vehicle, and are tested once a week for 3 weeks until all infusion conditions (SKF38393, muscimol or vehicle), are received. The order in which hamsters receive SKF38393, muscimol or vehicle infusions is counterbalanced across the group [5].

[1]. Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness. J Pharmacol Exp Ther. 1990 May;253(2):688-97.

[2]. Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils. J Pharmacol Exp Ther. 1990 Nov;255(2):756-68.

[3]. The aminosteroid U-73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. A role for Gp in receptor compartmentation. J Biol Chem. 1991 Dec 15;266(35):23856-62.

[4]. Disruption of phospholipase Cgamma1 signalling attenuates cardiac tumor necrosis factor-alpha expression and improves myocardial function during endotoxemia. Cardiovasc Res. 2008 Apr 1;78(1):90-7. Epub 2007 Dec 12.

[5]. In the ventral tegmental area, the membrane-mediated actions of progestins for lordosis of hormone-primed hamsters involve phospholipase C and protein kinase C. J Neuroendocrinol. 2007 Sep;19(9):717-24.

[6]. Inhibition of 5-lipoxygenase by U73122 is due to covalent binding to cysteine 416. Biochim Biophys Acta. 2012 Feb;1821(2):279-86.

[7]. 3Beta-hydroxy-6-aza-cholestane and related analogues as phosphatidylinositol specific phospholipase C (PI-PLC) inhibitors with antitumor activity. Bioorg Med Chem. 2000 Apr;8(4):699-706.

U-73122 is an aza-steroid that is 3-O-methyl-17beta-estradiol in which the 17beta-hydroxy group is replaced by a 6-(maleimid-1-yl)hexylamino group. An inibitor of phospholipase C. It has a role as an EC 3.1.4.11 (phosphoinositide phospholipase C) inhibitor. It is an aza-steroid, a member of maleimides and an aromatic ether. It is functionally related to a 17beta-estradiol.

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1-[6-[[17 beta-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]- 1H-pyrrole-2,5-dione (U-73122), an inhibitor of phospholipase C (PLC)-dependent processes in human platelets, was found to be a potent inhibitor of human polymorphonuclear neutrophil (PMN) activation by structurally unrelated receptor-specific agonists. U-73122 caused a time- and concentration-dependent (0.1-1 microM) inhibition of myeloperoxidase and vitamin B12-binding protein release from PMNs exposed to N-formyl-methionyl-leucyl-phenylalanine, recombinant human C5a, leukotriene B4 and platelet-activating factor. Activation of the respiratory burst, as measured by superoxide anion production, in PMNs stimulated with these agonists was also suppressed by U-73122. These data suggested that U-73122 inhibited a component of signal transduction that was common to the mechanisms of action of these stimuli. Production of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol and the rise in the cytosolic free calcium concentration, which are early postreceptor events in PMN activation, were all suppressed in U-73122-treated PMNs stimulated with the agonists. These signal transduction events require activation of PLC. Receptor-coupled activation of PLC in membranes isolated from PMNs was potently inhibited by U-73122. U-73122, however, had no direct effect on PMN protein kinase C activity. 1-[6-[[17 beta-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl] -2,5- pyrrolidine-dione (U-73343), a close analog of U-73122 that does not suppress PLC activity, did not inhibit receptor-specific agonist-induced PMN responsiveness. U-73122, therefore, is a novel reagent that is useful in investigating PLC function in receptor-mediated PMN activation.[1]

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The relationship between muscarinic receptor activation of phosphoinositide hydrolysis and the sequestration of cell surface muscarinic receptors has been examined for both intact and digitonin-permeabilized human SK-N-SH neuroblastoma cells. Addition of the aminosteroid 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U-73122) to intact cells resulted in the inhibition of oxotremorine-M-stimulated inositol phosphate release and of Ca2+ signaling by greater than 75%. In contrast, when phospholipase C was directly activated by the addition of the calcium ionophore ionomycin, inclusion of U-73122 had little inhibitory effect. Addition of U-73122 to intact cells also inhibited the agonist-induced sequestration of cell surface muscarinic receptors and their subsequent down-regulation with an IC50 value (4.1 microM) similar to that observed for inhibition of inositol phosphate release (3.7 microM). In contrast, when oxotremorine-M-stimulated phosphoinositide hydrolysis was inhibited by depletion of extracellular Ca2+, no reduction in the extent of receptor sequestration was observed. When introduced into digitonin-permeabilized cells, U-73122 more markedly inhibited inositol phosphate release elicited by either oxotremorine-M or guanosine-5'-O-(3-thiotriphosphate) than that induced by added Ca2+. Addition of oxotremorine-M to permeabilized cells resulted in muscarinic receptor sequestration and down-regulation. Both the loss of muscarinic acetylcholine receptors and activation of phosphoinositide hydrolysis in permeabilized cells were inhibited by the inclusion of guanosine-5'-O-(2-thiodiphosphate). The results indicate that the agonist-induced sequestration of muscarinic acetylcholine receptor in SK-N-SH cells requires the involvement of a GTP-binding protein but not the production of phosphoinositide-derived second messenger molecules.[3]

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U73122 which was originally identified as a phospholipase C inhibitor represents a potent direct inhibitor of purified 5-lipoxygenase (5-LO) with an IC50 value of 30 nM. 5-LO catalyzes the conversion of arachidonic acid (AA) into leukotrienes which represent mediators involved in inflammatory and allergic reactions and in host defense reactions against microorganisms. Since the efficient inhibition of the human 5-LO enzyme depended on the thiol reactivity of the maleinimide group of U73122, we used this property to identify cysteine residues in the 5-LO protein that are important for 5-LO inhibition by U73122. We found by MALDI-MS that U73122 covalently binds to cysteine residues 99, 159, 248, 264, 416 and 449. Mutation of Cys416 to serine strongly reduces inhibition of 5-LO by U73122 and the additional mutation of three cysteines close to Cys416 further impairs 5-LO inhibition by the compound. Wash out experiments with U73122 and 5-LO indicated an irreversible binding of U73122. Together, our data suggest that the area around Cys416 which is close to the proposed AA entry channel to the active site is an interesting target for the development of new 5-LO inhibitors.[6]

C29H40N2O3

464.64

464.303

C, 74.96; H, 8.68; N, 6.03; O, 10.33

112648-68-7

104794

Off-white to yellow solid

1.2±0.1 g/cm3

617.1±55.0 °C at 760 mmHg

327.0±31.5 °C

0.0±1.8 mmHg at 25°C

1.589

6.59

58.64

1

4

9

34

763

5

O(C([H])([H])[H])C1C([H])=C([H])C2=C(C=1[H])C([H])([H])C([H])([H])[C@]1([H])[C@]2([H])C([H])([H])C([H])([H])[C@]2(C([H])([H])[H])[C@]([H])(C([H])([H])C([H])([H])[C@]21[H])N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])N1C(C([H])=C([H])C1=O)=O

LUFAORPFSVMJIW-ZRJUGLEFSA-N

InChI=1S/C29H40N2O3/c1-29-16-15-23-22-10-8-21(34-2)19-20(22)7-9-24(23)25(29)11-12-26(29)30-17-5-3-4-6-18-31-27(32)13-14-28(31)33/h8,10,13-14,19,23-26,30H,3-7,9,11-12,15-18H2,1-2H3/t23-,24-,25+,26+,29+/m1/s1

1-(6-(((8R,9S,13S,14S,17S)-3-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione

U 73122; U73122; 112648-68-7; U-73122; U73122; U 73122; 1-(6-((3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; U-73,122; U-73122 hydrate; 1-[6-[[(8R,9S,13S,14S,17S)-3-methoxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl]amino]hexyl]pyrrole-2,5-dione; U73122.

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: ≥ 0.62 mg/mL (1.33 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 6.2 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: ≥ 0.62 mg/mL (1.33 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 6.2 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% Tween 80+saline: 5mg/mL

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配方 4 中的溶解度: 10 mg/mL (21.52 mM) in 0.5% CMC-Na/saline water (这些助溶剂从左到右依次添加，逐一添加), 悬浮液； 超声助溶 (<60°C).
*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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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;
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