Human Endogenous Metabolite

At low doses (5 µM), tTaurolithocholic acid (TLCA) tends to raise the membrane-associated fraction of the e-isoform of PKC by 44.1% ± 40.2%[1]. The activation of mobile PKC isoforms requires the selective translocation of PKC's epsilon-isoform to hepatocellular membranes, which is induced by TLCA (10 µM)[1].

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The protein kinase C (PKC) family of isoenzymes plays a key role in the regulation of hepatocellular secretion. The hydrophobic and cholestatic bile acid, Taurolithocholic acid (TLCA), acts as a potent Ca++ agonist in isolated hepatocytes. However, its effect on PKC isoforms has not been elucidated. Here we investigate the effects of TLCA at low micromolar concentrations on the distribution of PKC isoforms and on membrane-associated PKC activity. The distribution of PKC isoforms was determined in isolated rat hepatocytes in short-term culture using Western blotting and immunofluorescence techniques. PKC activity was measured radiochemically. TLCA (10 micromol/L) induced selective translocation of epsilon-PKC by 47.9% +/- 20.5% (P <.02 vs. controls; n = 7), but not of alpha-, delta-, and zeta-PKC to the hepatocellular membranes, whereas the phorbol ester, phorbol 12-myristate 13-acetate (PMA) (1 micromol/L) caused translocation of all mobile isoforms, alpha-, delta-, and epsilon-PKC, as shown by immunoblotting. Immunofluorescence studies demonstrated selective translocation of epsilon-PKC to the canalicular membranes of isolated rat hepatocyte couplets by TLCA (10 micromol/L), but predominant translocation to intracellular and basolateral membranes by PMA (1 micromol/L). Both TLCA (10 micromol/L) and PMA (1 micromol/L) stimulated membrane-bound PKC activity by 60.5% +/- 45. 8% (P <.05 vs. controls; n = 5) and 72.4% +/- 37.2% (P <.05; n = 5), respectively. TLCA at lower concentrations (5 micromol/L) was less effective. Because activation of epsilon-PKC has been associated with impairment of vesicle-mediated targeting and insertion of membrane proteins in secretory cells, it is attractive to speculate that TLCA reduces bile secretory capacity of the liver cell by activation of epsilon-PKC at the canalicular membrane.[1]

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PI3K-dependent PKB (PKB/Akt) Activity in Isolated Rat Hepatocytes [2]
The amount of phospho-PKB(Ser-473), a sensitive read-out of the activation of the PI3K pathway (27, 32), was markedly enhanced by Taurolithocholic acid /TLCA (5 μmol/liter) in hepatocytes in short term culture (Fig. 7) and reached levels up to 194 ± 46% of controls after 60 min (p < 0.005versus control; p < 0.05 versusTUDCA; p < 0.01 versus TCA). In contrast, TUDCA (10 μmol/liter) only transiently increased PKB activity, whereas TCA (10 μmol/liter) had no effect under the experimental conditions chosen (Fig. 7). Thus, TLCA markedly affected PI3K activity in isolated hepatocytes in vitro, whereas TUDCA exerted only minor transient effects on the PI3K pathway when administered at low micromolar concentrations.

Taurolithocholic acid (TLCA) in rat hepatocyte couplets and perfused rat livers exhibits cholestatic effects through PI3K-dependent mechanisms[2].
Taurolithocholic acid (TLCA) is a potent cholestatic agent. Our recent work suggested that TLCA impairs hepatobiliary exocytosis, insertion of transport proteins into apical hepatocyte membranes, and bile flow by protein kinase Cepsilon (PKCepsilon)-dependent mechanisms. Products of phosphatidylinositol 3-kinases (PI3K) stimulate PKCepsilon. We studied the role of PI3K for TLCA-induced cholestasis in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets (IRHC). In IPRL, TLCA (10 micromol/liter) impaired bile flow by 51%, biliary secretion of horseradish peroxidase, a marker of vesicular exocytosis, by 46%, and the Mrp2 substrate, 2,4-dinitrophenyl-S-glutathione, by 95% and stimulated PI3K-dependent protein kinase B, a marker of PI3K activity, by 154% and PKCepsilon membrane binding by 23%. In IRHC, TLCA (2.5 micromol/liter) impaired canalicular secretion of the fluorescent bile acid, cholylglycylamido fluorescein, by 50%. The selective PI3K inhibitor, wortmannin (100 nmol/liter), and the anticholestatic bile acid tauroursodeoxycholic acid (TUDCA, 25 micromol/liter) independently and additively reversed the effects of TLCA on bile flow, exocytosis, organic anion secretion, PI3K-dependent protein kinase B activity, and PKCepsilon membrane binding in IPRL. Wortmannin also reversed impaired bile acid secretion in IRHC. These data strongly suggest that TLCA exerts cholestatic effects by PI3K- and PKCepsilon-dependent mechanisms that are reversed by tauroursodeoxycholic acid in a PI3K-independent way [2].

PKB/Akt activity in isolated rat hepatocytes was determined by an immunoblotting technique. In brief, 4 h after plating (see above) cells were incubated for 5, 15, 30, and 60 min with Taurolithocholic acid /TLCA (5 μmol/liter; at concentrations >5 μmol/liter, TLCA caused visible damage in isolated hepatocytes in short term culture), TUDCA (10 μmol/liter), TCA (10 μmol/liter), or the carrier Me2SO only (control, 0.1%, v/v). Culture dishes were then placed on ice, and cells were scraped and immediately shock-frozen (−80 °C). Shock-frozen cells were homogenized in ice-cold lysis buffer (1 ml/100 mg) and processed as described above [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

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

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.11 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 20.8 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: ≥ 2.08 mg/mL (4.11 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 20.8 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: ≥ 2.08 mg/mL (4.11 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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