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

在与 PMN 分离的膜中，U-73122 TFA 有效抑制 PLC 的受体偶联激活 [1]。 U-73122 TFA 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 TFA 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]。

---

1-[6-[[17β-3-甲氧基雌甾-1,3,5（10）-三烯-17基]氨基]己基]-1H-吡咯-2,5-二酮（U-73122）抑制了各种激动剂诱导的人血小板聚集（IC50值1-5微M），但吡咯烷二酮取代吡咯烷二酮类的类似物1-[6-[17β3-甲氧基雄甾-1,3,5-（10）三烯-17-基]氨基]-2,5-吡咯烷二烯（U-73343）没有抑制这种聚集。U-73122的抑制作用不是由细胞内环AMP的增加介导的。相比之下，U-73122抑制了肌醇1,4,5-三磷酸（IP3）的产生，随后凝血酶或血栓素模拟物（5Z，9α，11α，13E，15S）15-羟基-11,9-（环氧甲烷）前列腺素-5,13，-二烯-1-酸（U-46619）诱导的细胞质钙离子的快速增加，但U-73343没有抑制。IP3水平的降低似乎反映了IP3产生的抑制，因为U-73122抑制了血小板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（Ki=9和40微M）。此外，U-73122抑制了胶原蛋白诱导的血栓素B2的产生，但不抑制外源添加的花生四烯酸支持的血栓素B2的产生，这表明U-73122还抑制了花生四烯酸的受体偶联动员。血小板与[3H]花生四烯酸预孵育后，凝血酶诱导的[3H]磷脂酰肌醇的损失和[3H]磷酸的积累被U-73122减弱。U-73122不抑制从猪胰腺或从金刚石龙子和眼镜蛇毒中纯化的磷脂酶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-[（苯胺基）-羰基]-肼基]甲基]-7-氧杂二环[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U-73122通过抑制信号转导中磷脂酶C依赖性成分的机制对血栓素受体激动剂U-46619激活血小板的抑制一致。U-73122，而非U-73343，也抑制了N-甲酰基-甲磺酰基-亮氨酰基-苯丙氨酸诱导的人中性粒细胞（PMN）聚集以及相关的IP3和二甘甘油的产生。N-甲酰基-甲酰基亮酰苯丙氨酸刺激PMN产生的二甲酰甘油可皂化74+/-7%，被U-73122抑制（Ki=2微M）。[2]

---

对于完整和经洋地黄皂苷渗透的人SK-N-SH神经母细胞瘤细胞，已经研究了磷酸肌醇水解的毒蕈碱受体激活与细胞表面毒蕈碱感受器隔离之间的关系。将氨基类固醇1-[6-[[17β-3-甲氧基雌甾-1,3,5（10）-三烯-17-基]氨基]己基]-1H-吡咯-2,5-二酮（U-73122）添加到完整细胞中，导致对氧震颤菌素-M刺激的肌醇磷酸释放和Ca2+信号传导的抑制超过75%。相比之下，当磷脂酶C被钙离子载体离子霉素直接激活时，U-73122的包合几乎没有抑制作用。向完整细胞中添加U-73122还抑制了激动剂诱导的细胞表面毒蕈碱受体的隔离及其随后的下调，IC50值（4.1微M）与抑制磷酸肌醇释放（3.7微M）的观察值相似。相比之下，当氧震颤素-M刺激的磷酸肌醇水解被细胞外Ca2+的耗竭抑制时，没有观察到受体隔离程度的降低。当引入洋地黄皂苷透性细胞时，U-73122比添加Ca2+更显著地抑制了由氧震颤素-M或鸟苷-5'-O-（3-硫代三磷酸）诱导的肌醇磷酸释放。在透化细胞中添加氧曲莫林-M导致毒蕈碱受体隔离和下调。鸟苷-5'-O-（2-硫代二磷酸）的加入抑制了渗透细胞中毒蕈碱乙酰胆碱受体的丧失和磷酸肌醇水解的激活。结果表明，激动剂诱导的SK-N-SH细胞中毒蕈碱乙酰胆碱受体的隔离需要GTP结合蛋白的参与，但不需要产生磷酸肌醇衍生的第二信使分子。[3]

---

U-73122最初被鉴定为磷脂酶C抑制剂，是纯化5-脂氧合酶（5-LO）的有效直接抑制剂，IC50值为30 nM。5-LO催化花生四烯酸（AA）转化为白三烯，白三烯是参与炎症和过敏反应以及宿主对微生物防御反应的介质。由于人类5-LO酶的有效抑制取决于U73122的马来酰亚胺基团的巯基反应性，我们利用这一特性鉴定了5-LO蛋白中的半胱氨酸残基，这些残基对U-73122的5-LO抑制很重要。我们通过MALDI-MS发现U73122与半胱氨酸残基99、159、248、264、416和449共价结合。Cys416突变为丝氨酸显著降低了U73122对5-LO的抑制作用，而靠近Cys416的三个半胱氨酸的额外突变进一步削弱了该化合物对5-LO抑制作用。U73122和5-LO的洗脱实验表明U73122的结合是不可逆的。总之，我们的数据表明，Cys416周围靠近活性位点拟议AA进入通道的区域是开发新5-LO抑制剂的一个有趣靶点[6]。

在内毒素血症小鼠中，U73122 TFA 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 TFA (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 TFA 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]。

---

在实验1中，用雌二醇（10微克；h 0）+孕酮（100微克；h 45）预处理卵巢切除的仓鼠，进行前凸和运动行为的预测试（h 48），然后输注PLC抑制剂U-73122（400 nM/侧）或赋形剂。30分钟后，对仓鼠进行重新测试，然后向VTA输注SKF38393（100ng/侧）、麝香莫醇（100ng/方）或赋形剂。30分钟后，对仓鼠进行前凸和运动行为的后测。在实验2中，使用了类似的方案，除了用PKC抑制剂双吲哚基马来酰亚胺（75 nM/侧）代替PLC抑制剂输注仓鼠。与VTA的载体相比，通过输注PLC抑制剂U73122或PKC抑制剂双吲哚基马来酰亚胺，全身孕酮、SKF38393-和肌松醇促进的前凸得以减轻。因此，VTA中孕激素通过D（1）和/或GABA（A）增强前凸的作用可能包括PLC和PKC的下游活性。[5]

---

实验1：U-73122预处理对SKF38393或肌氨醇介导的仓鼠孕酮促前凸增加的影响[5]
现场分析显示，39只仓鼠接受了双侧VTA输注，3只仓鼠接受双侧黑质输注。接受黑质双侧输注的三只仓鼠的数据被排除在统计分析之外（数据未显示）。接受黑质注射的仓鼠没有表现出与接受VTA注射的仓鼠相似的行为模式。

与向VTA输注赋形剂相比，向VTA注射PLC抑制剂U-73122显著缩短了经雌二醇和孕酮预处理的仓鼠的总前凸持续时间[F（1111）=146.44，P < 0.01]. 与向VTA输注赋形剂相比，向VTA中输注D1激动剂SKF38393或GABAA激动剂蝇蕈醇显著增加了总前凸持续时间[F（2111）=25.53，P < 0.01]. 这些变量之间存在显著的相互作用[F（2111）=25.40，P < 0.01] 由于U-73122而非赋形剂，输注可减轻孕酮促进的前凸，并阻断SKF38393或麝香醇对雌二醇引发的仓鼠孕酮介导的前凸的增强作用（图2）。

输注U-73122的VTA不会改变活动监测器中仓鼠的运动行为，但与施用SKF38393的仓鼠相比，麝香莫醇对减少束流断裂总数有显著作用[F（2111）=4.13，P < 0.02]（表1）。

表1。实验1：在雌二醇和孕酮刺激的仓鼠的水平交叉室中，接受U-73122或赋形剂和SKF38393、肌肉醇或赋形剂注入腹侧肌区的布雷姆骨折总数。

---

为了研究PLCgamma1在体内内毒素血症中的作用，将野生型和杂合型PLCgamma 1敲除（PLCgamma2（+/-））小鼠用U-73122或其非活性类似物U73343或载体预处理15分钟，然后用LPS处理4小时。U73122或PLCgamma基因杂合缺失抑制PLCgamma可降低心脏TNF-α表达。更重要的是，PLCgamma1（+/-）小鼠或U73122治疗也减轻了LPS诱导的心肌功能障碍。[4]
PLCγ1在内毒素血症诱导的心肌抑制中的作用也通过使用药理学抑制剂U-73122进行了研究。野生型小鼠用U73122或其非活性类似物U73343（9mg/kg，i.p.）预处理15分钟，然后用赋形剂或LPS（4mg/kg，i.p.4）预处理4小时。如上所述评估心脏功能。U73122对假手术动物没有影响，但与U73343相比，在不影响内毒素血症小鼠心率的情况下，显著增加了心脏功和收缩和舒张速率（图6）。这些数据进一步支持PLCγ1对内毒素血症心肌功能障碍的贡献[4]。

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 TFA）（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 TFA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 TFA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 TFA（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 TFA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 TFA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 TFA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 TFA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 TFA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 TFA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 TFA，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 TFA（Ki=2 microM）抑制[2]。

---

质谱分析的样品制备[6]
使用离心浓缩器浓缩和脱盐纯化的5-LO。将蛋白质在pH 7.4的PBS中稀释至终浓度为150μg·mL-1，并加入1 mMU-73122（乙醇溶液）以获得30μM的终浓度。在37°C下孵育30分钟后，用50 mM NH4HCO3 pH 7.8作为洗涤缓冲液的离心浓缩器将蛋白质溶液与未结合的U-73122分离。在5 mM二硫苏糖醇的存在下，蛋白质内的二硫键在57°C下还原1小时。游离半胱氨酸的烷基化是通过在室温下在黑暗中与35 mM碘乙酰胺一起孵育1小时来实现的。通过加入终浓度为35 mM的二硫苏糖醇来淬灭过量的碘乙酰胺。5-LO的消化是在37°C下用胰蛋白酶以1:50（w:w）的胰蛋白酶/5-LO比进行的，持续12小时。对于野生型5-LO，消化后需要进行第二步还原和烷基化，以增强含有两个紧邻半胱氨酸的肽的信号。

---

MALDI-MS[6]
将1μL消化样品（~3pmol 5-LO）与1μL 2.5 mg·mL-1 CHCA溶液在70%乙腈和0.1%三氟乙酸中的不锈钢MALDI靶上混合。结晶后，使用Ultraflex I MALDI-TOF/TOF仪器获取MS光谱）。光谱经过内部校准。峰值检测的最小信噪比设置为3，峰值分配的误差容限高达30ppm。蛋氨酸氧化、谷氨酰胺形成焦谷氨酸以及半胱氨酸被U-73122、脲甲基化和焦脲甲基化烷基化作为峰分配的可选修饰。

---

放射性配体结合分析[2]
如前所述，测量[3H]NMS或[3H]东莨菪碱与完整细胞（重新悬浮在缓冲液A中）或地高辛渗透细胞（重新悬在KGEH缓冲液中）的结合。

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 TFA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]。

---

细胞培养条件[2]
在先前描述的条件下培养人SK-N-SH神经母细胞瘤细胞（传代数未知）。所有实验均使用检测后10-20天的细胞。在吸取培养基后，通过加入Puck的Dl溶液将细胞从组织培养瓶中分离出来，通过离心（300 X g，1分钟）收集，除非另有说明，否则将其重新悬浮在缓冲液A中（142 mM NaC1、5.6 mM KCl、2.2 mM CaC12、3.6 mM NaHC03、1 mM MgCl、5.6 mM D-glUCOSe和30 mM Na+-HPES缓冲液，pH 7.4）。

---

PPI的测量[2]
水解-SK-N-SH细胞在37“C下在含有10 pCi/ml[3H]肌醇的Dulbecco改良Eagle培养基/胎牛血清中在90%空气、10%COZ的气氛中预标记2或3天。在这些条件下，肌醇脂质的标记达到同位素平衡。对于完整细胞，在Li+存在下监测总[3H]磷酸肌醇部分的积累，如前所述。之前已经报道了这些部分中存在的单个磷酸肌醇异构体的鉴定。为了测量渗透细胞中的PPI水解，用Puck的Dl溶液洗涤预标记的细胞一次，然后重新悬浮。置于含有20p~洋地黄皂苷的KGEH缓冲液（139mM K+-谷氨酸盐、2mM ATP、4mM MgCl、10mM LiCl、10mM-EGTA和30mM Na+-HPES缓冲液，pH 7.4）中。细胞在37°C下以约3-4mg/ml的蛋白质浓度渗透5分钟。然后将可渗透的细胞离心，用等体积的KGEH缓冲液（减去洋地黄皂苷）洗涤，并重新悬浮在相同的缓冲液中。在终止tlme反应并如前所述定量肌醇磷酸盐释放后，常规允许孵育（游离[Ca2+]=60 nM）进行30分钟。

---

细胞质Ca2+浓度的测量[2]
SK-N-SH细胞中的细胞质Caz+浓度（[Ca2+Ii）是通过在岛津RF-5000荧光分光光度计中监测fura-2荧光来测定的，如前所述，但Grynkiewicz等人的双波长方法是常规使用的。在这些条件下，[Caz+]i=（R-R，i，/Rm..-R）B.Kd，其中R、Rmi和Rmax是在340和380 nm激发波长下获得的荧光的比率（Xernisaion=505 nm）。B是380nm处无Ca2+/Ca'+饱和信号的荧光比，Kd是fura-2对Ca2+的亲和力常数（224nM）。在任一激发波长下，组织自发荧光都可以忽略不计（<6%）。

---

细胞内CAMP的测量[2]
在1 mM 3-异丁基-1-甲基黄嘌呤的存在下，将完整细胞（0.3 mg蛋白质/ml）与各种试剂在37°C下孵育30分钟。终止反应，提取CAMP，并通过放射免疫测定法进行定量，如前所述。

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 TFA (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 TFA 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]. he PLC inhibitor, U73122, was dissolved in saline at a concentration of 400 nM/µl. The concentration of PLC utilised was based upon published reports and our pilot data [5].

---

Effect of U-73122 on myocardial function in endotoxemia. Wild-type mice were pre-treated with either U-73122 or U73343 (9 mg/kg, i.p.) for 15 min, followed by vehicle or lipopolysaccharide (4 mg/kg, i.p.) for 4 h. Changes in heart rate (A), heart work (B), rate of contraction (+dF/dtmax, C) and relaxation (−dF/dtmin, D) are presented. Heart work and rate of contraction and relaxation were significantly improved in U73122 + lipopolysaccharide compared with U73343 + lipopolysaccharide. Data are mean ± SEM, n = 7–9 per group, *P < 0.05 vs. Sham; †P < 0.05 vs. lipopolysaccharide + U73122. [4]

[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.

[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.

---

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]

---

In conclusion, the results obtained demonstrate that inclusion of the aminosteroid U-73122 leads to an inhibition of both mAChR-stimulated PPI hydrolysis and receptor sequestration in SK-N-SH cells. Because the primary site of action of U-73122 appears to be an inhibition of G, regulation of phospholipase C, we conclude that activation of this G-protein either directly or indirectly is required for mAChR sequestration. [2]

---

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]

---

U-73122 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]

---

We could show that U-73122 is a 5-LO inhibitor that covalently and irreversibly binds to Cys416 and that this interaction is essential for inhibition of the human 5-LO enzyme by this compound. The data suggest that the surface area close to Cys416 could be an interesting target for the development of novel 5-LO inhibitors. [6]

C29H40N2O3.CF3COOH

578.66

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 TFA; 112648-68-7; U-73122 TFA; U73122 TFA; 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 TFA 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 TFA.

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。

---

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。

---

View More

配方 3 中的溶解度: 2% Tween 80+saline: 5mg/mL

---

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。

---

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。