Human Endogenous Metabolite

在低剂量 (5 µM) 下，Taurolithocholic acid /牛磺石胆酸 (TLCA) 往往会使 PKC e 亚型的膜相关部分提高 44.1% ± 40.2%[1]。移动 PKC 同工型的激活需要 PKC 的 epsilon 同工型选择性易位至肝细胞膜，这是由 TLCA (10 µM) 诱导的[1]。

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蛋白激酶C（PKC）同工酶家族在肝细胞分泌的调节中起着关键作用。疏水性和胆汁淤积性胆汁酸，牛磺酸胆酸（TLCA），在分离的肝细胞中充当强效的钙离子激动剂。然而，其对PKC亚型的影响尚未阐明。在这里，我们研究了低微摩尔浓度的TLCA对PKC亚型分布和膜相关PKC活性的影响。使用蛋白质印迹和免疫荧光技术在短期培养的分离大鼠肝细胞中确定PKC亚型的分布。放射化学法测定PKC活性。Taurolithocholic acid /TLCA（10微摩尔/升）诱导εPKC选择性易位47.9%+/-20.5%（与对照组相比，P<.02；n=7），但α-、δ-和ζPKC未选择性易位至肝细胞膜，而佛波醇酯佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）（1微摩尔/L）导致所有可移动异构体α-、Δ-和εPKC易位，如免疫印迹所示。免疫荧光研究表明，TLCA（10微摩尔/升）选择性地将ε-PKC转运到分离的大鼠肝细胞联的小管膜，但PMA（1微摩尔/L）主要转运到细胞内和基底外侧膜。TLCA（10μmol/L）和PMA（1μmol/L）均能刺激膜结合PKC活性60.5%+/-45。分别为8%（与对照组相比P<0.05；n=5）和72.4%+/-37.2%（P<0.05；n=5）。较低浓度（5微摩尔/升）的TLCA效果较差。由于εPKC的激活与囊泡介导的分泌细胞膜蛋白靶向和插入的损伤有关，因此推测TLCA通过激活小管膜上的εPKC来降低肝细胞的胆汁分泌能力是有吸引力的。[1]

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分离的大鼠肝细胞中PI3K依赖性PKB（PKB/Akt）活性[2]
磷酸化PKB（Ser-473）的量是PI3K通路激活的敏感读数（27，32），在短期培养中，肝细胞中的Taurolithocholic acid /牛磺酸/TLCA（5μmol/l）显著增强（图7），60分钟后达到对照组的194±46%（与对照组相比p<0.005；与TUDCA相比p<0.05；与TCA相比p<0.01）。相比之下，TUDCA（10μmol/升）仅短暂增加PKB活性，而TCA（10µmol/升，在所选实验条件下没有影响（图7）。因此，TLCA在体外显著影响了分离肝细胞中的PI3K活性，而TUDCA在低微摩尔浓度下给药时仅对PI3K通路产生轻微的短暂影响。

大鼠肝细胞对和灌注大鼠肝脏中的Taurolithocholic acid /牛磺石胆酸 (TLCA) 通过 PI3K 依赖性机制表现出胆汁淤积作用 [2]。
Taurolithocholic acid /牛磺酸胆酸（TLCA）是一种强效的胆汁淤积剂。我们最近的工作表明，TLCA通过蛋白激酶Cepsilon（PKCepsilon）依赖性机制损害肝胆分泌、运输蛋白插入肝细胞顶膜和胆汁流动。磷脂酰肌醇3-激酶（PI3K）的产物刺激PKCε。我们研究了PI3K在离体灌流大鼠肝脏（IPRL）和离体大鼠肝细胞偶联物（IRHC）中对TLCA诱导的胆汁淤积的作用。在IPRL中，TLCA（10微摩尔/升）使胆汁流量受损51%，使辣根过氧化物酶（囊泡分泌的标志物）的胆汁分泌受损46%，Mrp2底物2,4-二硝基苯基-S-谷胱甘肽受损95%，并刺激PI3K依赖性蛋白激酶B（PI3K活性的标志）受损154%，使PKEpsilon膜结合受损23%。在IRHC中，TLCA（2.5微摩尔/升）使荧光胆汁酸（胆酰甘氨酰荧光素）的小管分泌受损50%。选择性PI3K抑制剂渥曼青霉素（100 nmol/升）和抗胆固醇胆汁酸牛磺脱氧胆酸（TUDCA，25微摩尔/升）独立和相加地逆转了TLCA对IPRL中胆汁流量、胞吐、有机阴离子分泌、PI3K依赖性蛋白激酶B活性和PKCε膜结合的影响。渥曼青霉素还逆转了IRHC中受损的胆汁酸分泌。这些数据强烈表明，TLCA通过PI3K和PKCε依赖的机制发挥胆汁淤积作用，这些机制被牛磺脱氧胆酸以PI3K非依赖的方式逆转[2]。

通过免疫印迹技术测定分离的大鼠肝细胞中PKB/Akt的活性。简而言之，在接种后4小时（见上文），细胞与Taurolithocholic acid /牛磺胆酸/TLCA（5μmol/l；浓度>5μmol/l时，TLCA在短期培养中对分离的肝细胞造成可见损伤）、TUDCA（10μmol/l）、TCA（10µmol/l）或仅使用载体Me2SO（对照，0.1%，v/v）孵育5、15、30和60分钟。然后将培养皿放在冰上，刮取细胞并立即冷冻（-80°C）。将休克冷冻细胞在冰冷的裂解缓冲液（1ml/100mg）中均质化，并如上所述进行处理[2]。

Bile acid secretion by IRHC was assessed by measuring the hepatocellular uptake and secretion of 1 μmol/liter cholylglycylamido fluorescein (CGamF) into the canalicular space as previously described. CGamF was synthesized according to Schteingart et al. and was kindly provided by Dr. Alan Hofmann. Four hours after isolation, hepatocytes (on coverslips) were briefly transferred to HEPES buffer. Then, cells were pretreated for 15 min at 37 °C with (i) Me2SO (0.1%, v/v), (ii) 100 nmol/liter wortmannin and Me2SO, (iii) Me2SO for 5 min, and 2.5 μm Taurolithocholic acid /TLCA (in Me2SO, 0.1%, v/v) for 10 min, (iv) 100 nmol/liter wortmannin and Me2SO for 5 min, and 100 nmol/liter wortmannin and 2.5 μm Taurolithocholic acid /TLCA (in Me2SO, 0.1%, v/v) for 10 min, (v) Me2SO for 5 min and 5 μmol/liter Taurolithocholic acid /TLCA (in Me2SO, 0.1%, v/v) for 10 min, and (vi) 100 nmol/liter wortmannin and Me2SO for 5 min, and 100 nmol/liter wortmannin and 5 μmol/liter Taurolithocholic acid /TLCA for 10 min. Cells were then transferred for 5 min to HEPES buffer containing 1 μmol/liter fluorescent CGamF at 37 °C to allow adequate loading of the fluorescent bile acid and transferred back for 10 min to their previous dishes (i-vi). Hepatocyte secretion was stopped by placing coverslips in ice-cold HEPES buffer on ice, and cells were viewed immediately on a Zeiss LSM 510 microscope (Thornwood, NY). Laser settings were optimized for a dynamic range to avoid saturation of the fluorescence. The same settings were used for all conditions. Cells were analyzed on the confocal laser scanning microscope by one investigator (C. J. Soroka) who was blinded to the experimental conditions. Couplets were selected based upon the presence of a well defined canalicular space as determined under bright field optics. Images were then acquired with rapid scanning to avoid quenching of the fluorescence. Quantitation of uptake (uptake = (F° cell + F° can)/μm2) and secretion (% secretion = [F° can/(F° cell + F° can)] × 100) of CGamF was performed as previously published, except that NIH Image software was used.[2]

[1]. Modulation of protein kinase C by taurolithocholic acid in isolated rat hepatocytes. Hepatology. 1999 Feb;29(2):477-82.

[2]. Taurolithocholic acid exerts cholestatic effects via phosphatidylinositol 3-kinase-dependent mechanisms in perfused rat livers and rat hepatocyte couplets. J Biol Chem. 2003 May 16;278(20):17810-8.

Sodium taurolithocholate is a bile acid. It is functionally related to a taurolithocholic acid.
See also: Sodium taurolithocholate (annotation moved to).

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In the present study, co-administration of a PI3K inhibitor not only reversed TLCA-induced impairment of bile secretion but also cellular damage as determined by lactate dehydrogenase release (Table I). The improvement in bile flow alone could not account for this effect since TUDCA also improved secretion in TLCA-treated livers but failed to abolish the cell damage induced by TLCA in IPRL. Future studies will be necessary to elucidate the role of PI3K in TLCA-induced acute liver cell damage.
The present data suggest that PI3K represents a potential target of future anticholestatic treatment strategies. It should be mentioned, however, that PI3K may activate a survival pathway in rat hepatocytes treated with the hydrophobic bile acid, taurochenodeoxycholic acid (TCDCA) which protects liver cells from TCDCA-induced damage in vitro as well as in vivo (Rust C, unpublished observation). Interestingly, the taurochenodeoxycholic acid-induced survival pathway did not involve PKB activation in vitro. Thus, different bile acids may exert differential effects on PI3K- and PKB-mediated processes in liver cells. It remains to be clarified whether involvement of different PI3K isoforms or action in different subcellular compartments may contribute to these diverse effects of bile acids on PI3K and PKB.
In summary, the present study demonstrates that TLCA-induced impairment of bile flow, hepatobiliary exocytosis, secretion of bile acids, and other organic anions as well as liver cell damage is mediated by PI3K- and putatively PKCε-dependent mechanisms. TUDCA reversed the inhibitory effects of TLCA on bile secretion by a PI3K-independent mechanism.[2]

C26H44NNAO5S

505.69

505.284

6042-32-6

Taurolithocholic Acid-d5 sodium;1265476-97-8;Taurolithocholic acid-d4 sodium;2410279-97-7;Taurolithocholic acid-d4-1 sodium; 6042-32-6; 516-90-5

23662757

White to off-white solid powder

5.555

114.91

2

5

7

34

832

9

C[C@H](CCC(=O)NCCS(=O)(=O)[O-])[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2CC[C@H]4[C@@]3(CC[C@H](C4)O)C)C.[Na+]

YAERYJYXPRIDTO-HRHHVWJRSA-M

InChI=1S/C26H45NO5S.Na/c1-17(4-9-24(29)27-14-15-33(30,31)32)21-7-8-22-20-6-5-18-16-19(28)10-12-25(18,2)23(20)11-13-26(21,22)3;/h17-23,28H,4-16H2,1-3H3,(H,27,29)(H,30,31,32);/q;+1/p-1/t17-,18-,19-,20+,21-,22+,23+,25+,26-;/m1./s1

sodium;2-[[(4R)-4-[(3R,5R,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethanesulfonate

Taurolithocholic acid sodium salt; 6042-32-6; Sodium taurolithocholate; Taurolithocholic Acid (Sodium Salt); 2-((R)-4-((3R,5R,8R,9S,10S,13R,14S,17R)-3-Hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)ethanesulfonic acid, sodium salt; MFCD00036746; CHEMBL2028239; CHEBI:229584;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮和光照。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 100 mg/mL (197.75 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.11 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 20.8 mg/mL澄清DMSO储备液加入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: ≥ 2.08 mg/mL (4.11 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 20.8 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: ≥ 2.08 mg/mL (4.11 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 20.8 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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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：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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