6alpha-hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes.
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The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The Vmax for 6alpha-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent Km was 90 microM. Cytochrome b5 was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6alpha-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r2=0.97) and testosterone 6beta-hydroxylation (r2=0.9). There was also a strong correlation between 6alpha-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6beta-hydroxylation (r2=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6alpha-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 microM concentration. Other inhibitors, such as alpha-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent Km for 6alpha-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 microM). This might give an explanation for the limited formation of 6alpha-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver. (+info)
The role of different P-glycoproteins in hepatobiliary secretion of fluorescently labeled short-chain phospholipids.
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Class III P-glycoproteins (Pgps) mediate biliary phosphatidylcholine (PC) secretion. Recent findings that class I P-glycoproteins are able to transport several short-chain phospholipid analogues raises questions about the role of these Pgps in physiological lipid transport. We investigated the biliary secretion of C6-7-nitro-2,1, 3-benzoxadiazol-4-yl (NBD)-labeled ceramide and its metabolites in Mdr1a/b and Mdr2 knockout mice compared to control mice. Biliary secretion of these NBD-lipids was unaffected in Mdr1a/b -/- mice. Thus neither Mdr1a nor Mdr1b Pgp mediates biliary secretion of these lipids. In contrast, secretion of all three NBD-labeled short-chain phospholipids was significantly reduced in Mdr2 -/- mice. As in vitro studies revealed that Mdr2 Pgp is not able to translocate these lipid analogues, we hypothesized that Mdr2 -/- mice had a reduced PC content of the exoplasmic canalicular membrane leaflet so that extraction of the short-chain lipid probes from this membrane by canalicular bile salts was impaired. To investigate this possibility we studied the bile salt-mediated extraction of natural sphingomyelin (SM) and NBD-labeled short-chain SM from small unilamellar vesicles of different lipid composition. Natural SM could be extracted by the bile salt tauroursodeoxycholate from vesicles containing PC, cholesterol (CHOL), and SM (1:2:2) but not from vesicles containing only SM and CHOL (3:2). NBD-labeled short-chain SM could be extracted from vesicles containing PC while its extraction from pure SM:CHOL vesicles was reduced by 65%. These data confirm that the efficiency of NBD-SM extraction depends on the lipid composition and suggest that the canalicular membrane outer leaflet of Mdr2 -/- mice has a reduced PC content. (+info)
Comparison of the effects of bile acids on cell viability and DNA synthesis by rat hepatocytes in primary culture.
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Bile acid-induced inhibition of DNA synthesis by the regenerating rat liver in the absence of other manifestation of impairment in liver cell viability has been reported. Because in experiments carried out on in vivo models bile acids are rapidly taken up and secreted into bile, it is difficult to establish steady concentrations to which the hepatocytes are exposed. Thus, in this work, a dose-response study was carried out to investigate the in vitro cytotoxic effect of major unconjugated and tauro- (T) or glyco- (G) conjugated bile acids and to compare this as regards their ability to inhibit DNA synthesis. Viability of hepatocytes in primary culture was measured by Neutral red uptake and formazan formation after 6 h exposure of cells to bile acids. The rate of DNA synthesis was determined by radiolabeled thymidine incorporation into DNA. Incubation of hepatocytes with different bile acid species - cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), in the range of 10-1000 microM - revealed that toxicity was stronger for the unconjugated forms of CDCA and DCA than for CA and UDCA. Conjugation markedly reduced the effects of bile acids on cell viability. By contrast, the ability to inhibit radiolabeled thymidine incorporation into DNA was only slightly lower for taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA) than for DCA. When the effect of these bile acids on DNA synthesis and cell viability was compared, a clear dissociation was observed. Radiolabeled thymidine incorporation into DNA was significantly decreased (-50%) at TDCA concentrations at which cell viability was not affected. Lack of a cause-effect relationship between both processes was further supported by the fact that well-known hepatoprotective compounds, such as tauroursodeoxycholic acid (TUDCA) and S-adenosylmethionine (SAMe) failed to prevent the effect of bile acids on DNA synthesis. In summary, our results indicate that bile acid-induced reduction of DNA synthesis does not require previous decreases in hepatocyte viability. This suggests the existence of a high sensitivity to bile acids of cellular mechanisms that may affect the rate of DNA repair and/or proliferation, which is of particular interest regarding the role of bile acids in the etiology of certain types of cancer. (+info)
Critical role of micelles in pancreatic lipase activation revealed by small angle neutron scattering.
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In the duodenum, pancreatic lipase (PL) develops its activity on triglycerides by binding to the bile-emulsified oil droplets in the presence of its protein cofactor pancreatic colipase (PC). The neutron crystal structure of a PC-PL-micelle complex (Hermoso, J., Pignol, D., Penel, S., Roth, M., Chapus, C., and Fontecilla-Camps, J. C. (1997) EMBO J. 16, 5531-5536) has suggested that the stabilization of the enzyme in its active conformation and its adsorption to the emulsified oil droplets are mediated by a preformed lipase-colipase-micelle complex. Here, we correlate the ability of different amphypathic compounds to activate PL, with their association with PC-PL in solution. The method of small angle neutron scattering with D(2)O/H(2)O contrast variation was used to characterize a solution containing PC-PL complex and taurodeoxycholate micelles. The resulting radius of gyration (56 A) and the match point of the solution indicate the formation of a ternary complex that is similar to the one observed in the neutron crystal structure. In addition, we show that either bile salts, lysophospholipids, or nonionic detergents that form micelles with radii of gyration ranging from 13 to 26 A are able to bind to the PC-PL complex, whereas smaller micelles or nonmicellar compounds are not. This further supports the notion of a micelle size-dependent affinity process for lipase activation in vivo. (+info)
Hydrophilic bile salts protect bile duct epithelium during cold preservation: a scanning electron microscopy study.
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Prolonged graft preservation is associated with postoperative bile duct strictures after liver transplantation. We previously showed that hydrophilic bile salts mitigate bile duct preservation injury in a pig model. Because this injury occurs at the epithelial level, scanning electron microscopy was performed to further characterize this effect in vitro. Swine livers were harvested after the intravenous infusion of 1 of 3 solutions: saline (n = 7), tauroursodeoxycholate ([TUDC] hydrophilic; n = 4), or taurodeoxycholate ([TDC] hydrophobic; n = 4). Livers were perfused with University of Wisconsin solution. The bile ducts were flushed retrograde, and the liver was stored at 0 degrees C to 1 degrees C for 20 hours. Bile duct samples were obtained at the time of harvest and 8, 12, 16, and 20 hours thereafter. In saline-infused controls at time 0, the epithelium was intact and composed of uniform cuboidal cells covered with fine regular microvilli. There were no spaces between individual cells. After 8 to 12 hours of preservation, cells were more irregular in shape, with loss of cell-cell contact. The cell surfaces showed fewer microvilli. Surface erosions suggested loss of cell-wall integrity. TUDC was protective, evidenced by normal-appearing cells with uniform microvilli after 16 hours. In contrast, TDC accelerated the injury process, causing cell-surface erosions, blebs, and loss of microvilli as early as time 0. Scanning electron microscopy is an excellent tool to study injury to bile duct epithelium. This study supports the hypothesis that hydrophilic bile salts protect bile ducts during preservation. To determine whether treatment with hydrophilic bile salts can prevent postoperative stricture, in vivo transplantation studies are needed. (+info)
Interactions of bile salt micelles and colipase studied through intermolecular nOes.
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Colipase is a small protein (10 kDa), which acts as a protein cofactor for the pancreatic lipase. Various models of the activated ternary complex (lipase-colipase-bile salt micelles) have been proposed using detergent micelles, but no structural information has been established with bile salt micelles. We have investigated the organization of sodium taurodeoxycholate (NaTDC) micelles and their interactions with pig and horse colipases by homonuclear nuclear magnetic resonance (NMR) spectroscopy. The NMR data supply evidence that the folding of horse colipase is similar to that already described for pig colipase. Intermolecular nuclear Overhauser effects have shown that two conserved aromatic residues interact with NaTDC micelles. (+info)
Differences in Ca(2+) signaling underlie age-specific effects of secretagogues on colonic Cl(-) transport.
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Taurodeoxycholic acid (TDC) stimulates Cl(-) transport in adult (AD), but not weanling (WN) and newborn (NB), rabbit colonic epithelial cells (colonocytes). The present study demonstrates that stimuli like neurotensin (NT) are also age specific and identifies the age-dependent signaling step. Bile acid actions are segment and bile acid specific. Thus although TDC and taurochenodeoxycholate stimulate Cl(-) transport in AD distal but not proximal colon, taurocholate has no effect in either segment. TDC increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in AD, but not in WN and NB, colonocytes. In AD cells, TDC (5 min) action on Cl(-) transport needs intra- but not extracellular Ca(2+). NT, histamine, and bethanechol increase Cl(-) transport and [Ca(2+)](i) in AD, but not WN, distal colonocytes. However, A-23187 increased [Ca(2+)](i) and Cl(-) transport in all age groups, suggesting that Ca(2+)-sensitive Cl(-) transport is present from birth. Study of the proximal steps in Ca(2+) signaling revealed that NT, but not TDC, activates a GTP-binding protein, Galpha(q), in AD and WN cells. In addition, although WN and AD colonocytes had similar levels of phosphatidylinositol 4,5-bisphosphate, NT and TDC increased 1,4,5-inositol trisphosphate content only in AD cells. Nonresponsiveness of WN cells to Ca(2+)-dependent stimuli, therefore, is due to the absence of measurable phospholipase C activity. Thus delays in Ca(2+) signaling afford a crucial protective mechanism to meet the changing demands of the developing colon. (+info)
Colipase residues Glu64 and Arg65 are essential for normal lipase-mediated fat digestion in the presence of bile salt micelles.
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Pancreatic triglyceride lipase (PTL) requires colipase for activity. Various constituents in meals and in bile, particularly bile acids, inhibit PTL. Colipase restores activity to lipase in the presence of inhibitory substances like bile acids. Presumably, colipase functions by anchoring and orienting PTL at the oil-water interface. The x-ray structure of the colipase.PTL complex supports this model. In the x-ray structure, colipase has a hydrophobic surface positioned to bind substrate and a hydrophilic surface, lying opposite the hydrophobic surface, with two putative lipase-binding domains, Glu(45)/Asp(89) and Glu(64)/Arg(65). To determine whether the hydrophilic surface interacts with PTL in solution, we introduced mutations into the putative PTL binding domains of human colipase. Each mutant was expressed, purified, and assessed for activity against various substrates. Most of the mutants showed impaired ability to reactivate PTL, with mutations in the Glu(64)/Arg(65) binding site causing the greatest effect. Analysis indicated that the mutations decreased the affinity of the colipase mutants for PTL and prevented the formation of PTL.colipase complexes. The impaired function of the mutants was most apparent when assayed in micellar bile salt solutions. Most mutants stimulated PTL activity normally in monomeric bile salt solutions. We also tested the mutants for their ability to bind substrate and anchor lipase to tributyrin. Even though the ability of the mutants to anchor PTL to an interface decreased in proportion to their activity, each mutant colipase bound to tributyrin to the same extent as wild type colipase. These results demonstrate that the hydrophilic surface of colipase interacts with PTL in solution to form active colipase.PTL complexes, that bile salt micelles influence that binding, and that the proper interaction of colipase with PTL requires the Glu(64)/Arg(65) binding site. (+info)