Interaction Of Bile Salts Research Articles

Interaction of bile salts with hexadecyltrimethylammonium bromide and sodium dodecyl sulfate

Interaction of bile salts with hexadecyltrimethylammonium bromide and sodium dodecyl sulfate

Taurocholate induces pericanalicular localization of C6-NBD-ceramide in isolated hepatocyte couplets

The mechanisms and pathways involved in hepatocellular transport of lipid destined for biliary excretion remain poorly understood. Using fluorescence microscopy of rat hepatocyte couplets in primary culture, we examined the effects of taurocholate (TC) on the intracellular distribution of 6-N-[7-nitrobenz-2-oxa-1,3-diazol- 4-yl]aminocaproyl-sphingosine (C6-NBD-ceramide), a lipid that accumulates in the Golgi apparatus. Microscopic findings were quantified with morphometric and digital image analysis and were correlated with the metabolism of C6-NBD-ceramide in isolated hepatocyte suspensions and the biliary excretion of fluorescent lipid in the isolated perfused liver. After plasma membrane uptake of C6-NBD-ceramide from albumin at 0 degrees C, the lipid was rapidly internalized at 37 degrees C but exhibited only a modest concentration of fluorescence in intracellular organelles. With 17 microM TC in the medium, there was enhanced localization of fluorescence to organelles and significant recruitment of fluorescent lipid to the pericanalicular region of the couples within 30 min. C6-NBD-ceramide was partially metabolized to C6-NBD-sphingomyelin and -glucosylceramide, indicative of transit through the Golgi apparatus. The generation of C6-NBD-sphingomyelin was significantly increased by TC. After a similar loading protocol in the perfused liver, there was little biliary excretion of fluorescent lipid at 37 degrees C under control conditions. However, infusion of TC markedly enhanced the biliary output of fluorescent lipid over the first 30 min, primarily as C6-NBD-sphingomyelin and -glucosylceramide. We conclude that TC modulates the distribution of C6-NBD-ceramide in hepatocytes by inducing translocation of lipid to a pericanalicular location, most likely the Golgi apparatus, before excretion of its metabolites in bile. Our findings support the concept that bile salt-induced biliary lipid excretion is facilitated by the interaction of bile salts with lipids at the level of intracellular organelles.

Substrate specificity of lysosomal cholesteryl ester hydrolase isolated from rat liver.

The effect of various physicochemical forms of substrate on the activity of acid cholesteryl ester hydrolase isolated from rat liver lysosomes was studied. The amount of sodium taurocholate was varied in the substrate mixture which contained constant amounts of egg phosphatidylcholine (PC) and cholesteryl oleate. The resulting substrate forms produced were PC vesicles, PC vesicles with incorporated sodium taurocholate, mixed micelles, and mixed micelles together with free bile salt micelles. Gradually increasing amounts of sodium taurocholate activated cholesteryl oleate hydrolysis until the molar sodium taurocholate/PC ratio of ca. 0.6; thereafter hydrolytic activity decreased rapidly. The presence of sodium taurocholate micelles clearly inhibits cholesteryl oleate hydrolysis. We therefore propose that the activation observed at low bile salt concentrations depends on bile salt interaction with the substrate vehicle, whereas the inhibition observed at high bile salt concentrations depends on sodium taurocholate interacting with the enzyme. When comparing different phospholipid components in the supersubstrate, the enzyme activity was highest in the presence of dioleoyl PC and decreased when present with dipalmitoyl PC and egg PC. Egg lysoPC completely inhibited the enzyme activity. A net negative charge on the surface of the vesicle substrate increased cholesteryl ester hydrolase activity while a net positive charge on the surface inhibited the enzyme activity. Only part of the product inhibition of cholesteryl oleate hydrolase caused by Na-oleate was reversible when tested with bovine serum albumin present in the incubation mixture.

Hepatic bile flow.

The hepatocyte is a polar cell that can remove a variety of molecules from blood and excrete them into bile. This review is primarily concerned with the mechanism of transport of the principal anions--the bile salts--across the sinusoidal membrane, their passage through the cell, and excretion across the canalicular membrane. Clearly much of this process is poorly understood, but the study of the membrane stages should be facilitated by the ability to prepare purified sinusoidal and canalicular membrane vesicles. For example, the relative importance of albumin-binding sites as well as the putative bile salt receptor proteins can be better assessed. It seems likely that although the interaction of bile salts with receptor proteins is important, it is an initial event that puts the bile salt in the correct place for uptake to occur. The driving force for uptake is the Na+ gradient created across the basolateral membrane by the activity of the Na+-K+-ATPase. Within the cell, various modes of transport have been suggested. Several authors emphasize the importance of protein binding of bile salts, either because of their presumed ability to maintain the concentration of these anions in the hepatocyte below their critical micellar concentration or because of their putative role in transport. It is important to understand these aspects of the role of cytosolic proteins for several reasons. Knowledge of the true concentration of free bile salt within the cell should allow estimation of whether the electrochemical gradient is sufficient for bile salts to accumulate in bile without the need for active transport of molecules from the cell into the canaliculus. The compartmental model described by Strange et al. (153) offers one theoretical way of determining the concentration of free bile salt, although the problems inherent in studying amphipath binding to the membranes of subcellular organelles (31) require that the model be reevaluated by the hygroscopic-desorption method. The second role suggested for the cytosolic bile salt-binding proteins is as transport proteins. As discussed in section VI, I think it is unlikely that the proteins identified so far act in this way, and it is more likely that movement occurs by diffusion in free solution. It is also important to determine the possible involvement of subcellular organelles such as Golgi bodies. Little is known of their role in the transport of bile salts or indeed where bile salt micelles are formed.(ABSTRACT TRUNCATED AT 400 WORDS)

360-MHz nuclear magnetic resonance and laser photochemically induced dynamic nuclear polarization studies of bile salt interaction with porcine colipase A.

Porcine pancreatic colipase possesses a particular three‐dimensional surface domain containing six out of the seven aromatic residues of the molecule in a highly hydrophobic environment [P. Canioni and P. Cozzone (1979) Biochimie (Paris) 61, 343–354; J. Wieloch, B. Borgström, K. E. Falk and S. Forsen (1979) Biochemistry, 18, 1622–1628]. The domain corresponds to the 49–57 and 77–86 β‐sheet fragments brought into spatial proximity by protein folding [P. Canioni, P. Cozzone and L. Sarda (1980) Biochim. Biophys. Acta, 621, 29–42]. The identification of this specific domain as being the lipid binding site on colipase is proposed on the basis of the NMR and photochemically induced dynamic nuclear polarization (photo‐CIDNP) studies of the complexes of colipase with organized bile salt micelles.The binding of taurodeoxycholate micelles specifically perturbs the proton NMR ring resonances of Tyr‐I and Tyr‐II (Tyr‐56 and Tyr‐57) and His‐II (His‐86) together with several aliphatic resonances, reflecting the involvement of the hydrophobic aromatic domain in micelle fixation. In the presence of colipase, specific shifts and broadening of the methyl groups at position 18 and 21 of the sterane ring of taurodeoxycholate are observed and suggest that the hydrophobic side of the bile salt is primarily involved. The strong photo‐CIDNP effects of Tyr‐I and Tyr‐II in the presence of lumiflavin dye, which have been described on free colipase [P. Canioni, P. Cozzone and R. Kaptein (1980) FFBS Lett. 111, 219–222] are totally suppressed in the presence of taurodeoxycholate or chenocholamine micelles, indicating that due to the protection by the bile salt, the aromatic surface residues are no longer accessible. The pH dependence of the NMR perturbations and photo‐CIDNP effects observed in the colipase‐micelle complexes confirms that (1) the hydrophobic aromatic domain is directly involved and (2) the driving force of the primary micelle binding is essentially hydrophobic.A general model for micelle binding to colipase is proposed and involves a two‐step mechanism. Initially, the lipid hydrophobic binding site participates in the building of the bound taurodeoxycholate aggregate to form a mixed micelle through hydrophobic surface interactions (stoichiometric complex). At higher bile salt concentrations, polar forces might account for further growth of the micellar structure bound on colipase.

The effect of the interdependence of protein and bile salt concentrations on the measurement of cerebroside sulphate sulphatase activity in human leucocyte and fibroblast extracts

The previously observed differences in properties of human leucocyte and fibroblast cerebroside sulphate sulphatase (cerebroside-3-sulphate 3-sulphohydrolase, EC 3.1.6.8) measured in vitro have been found to be due to subtle differences in incubation conditions. Maximum enzyme activity was observed with either crude sodium taurocholate or with pure sodium taurodeoxycholate. The optimum bile salt concentration of the enzyme in leucocyte or fibroblast extracts, but not the pure ox liver enzyme, was critically dependent on protein concentration. At low concentrations of the latter (less than 0.1 mg/ml), maximum activity was observed at taurocholate concentrations less than 0.5 mg/ml; at protein concentrations greater than 0.20 mg/ml substantially more bile acid (more than 1.3 mg/ml) was required to stimulate maximum activity. Addition of Triton X-100 or bovine serum albumin to the incubation mixtures increased the optimum taurocholate concentration. The dependence of the bile salt optimum on protein concentration appears to be related to the binding of the lipid substrate to membranous protein present in the tissue extracts. Release of the bound lipid is effected either by increasing the bile salt human leucocyte, fibroblast and liver X-100. In the presence of excess bile salt human leucocyte, fibroblast and liver cerebroside sulphate sulphatase activity is stimulated by Triton at low protein concentrations; under identical conditions the pure or crude ox-liver enzyme is substantially inhibited. Our data also show that cerebroside sulphate sulphatase activity measured in extracts from leucocytes and fibroblasts, the tissues normally used to effect a diagnosis of metachromatic leucodystrophy, is the result of a complex interaction of bile salt, protein, Triton X-100 and probably the substrate itself. Any slight alteration in any of those factors, without a corresponding change in any or all of the others, can have a marked effect on the measured enzyme activity, and may lead to errors in the diagnosis of metachromatic leucodystrophy.

Properties and biological significance of the ileal bile salt transport system.

The properties of a specific transport system for bile salts, which is located in the ileum of the small intestine are described. The system operates by a sodium ion cotransport mechanism, and it functions in maintaining a normal enterohepatic circulation of bile salts. Analysis of structure-activity data allows us to depict our hypothesis for the interaction of bile salt and Na with the membranal recognition site of this transport system. The sequellae of metabolic disorders which can arise following disease or surgical ablation of the ileal region of the intestine which result in an interrupted bile salt enterohepatic circulation are described. We suggest that these findings hold interest to toxicologists, since it is not beyond reason that toxic agents might exist which impair the function of this transport system specifically or which could poison the ileal mucosal cell. Such agents might be detected by the presence of some of the described metabolic disorders. Finally, we discuss the ileal transport of the sulfated esters of bile salts and the possibility that this might relate to that aspect of detoxification pertaining to their enhanced excretion.

Properties and Biological Significance of the Ileal Bile Salt Transport System

The properties of a specific transport system for bile salts, which is located in the ileum of the small intestine are described. The system operates by a sodium ion cotransport mechanism, and it functions in maintaining a normal enterohepatic circulation of bile salts. Analysis of structure-activity data allows us to depict our hypothesis for the interaction of bile salt and Na with the membranal recognition site of this transport system. The sequellae of metabolic disorders which can arise following disease or surgical ablation of the ileal region of the intestine which result in an interrupted bile salt enterohepatic circulation are described. We suggest that these findings hold interest to toxicologists, since it is not beyond reason that toxic agents might exist which impair the function of this transport system specifically or which could poison the ileal mucosal cell. Such agents might be detected by the presence of some of the described metabolic disorders. Finally, we discuss the ileal transport of the sulfated esters of bile salts and the possibility that this might relate to that aspect of detoxification pertaining to their enhanced excretion.

Facilitation of hepatic uptake of phenol 3,6-dibromphthalein disulfonate by taurocholate.

The influence of sodium taurocholate (TC) on the hepatic uptake of phenol 3,6-dibromphthalein disulfonate (dibromosulphophthalein, DBSP) was examined in the dog. Single injections of DBSP (7.5-83.3 mg-kg-1) were given intravenously and the initial velocity of uptake (V) of the dye was calculated from the plasma disappearance rate measured during the 1st 5 min. It was observed that: a) the initial velocity of uptake of DBSP increased with the dose (D) in a nonlinear way, a finding consistent with Michaelis-Menten kinetics; the maximal initial velocity of uptake (Vmax) was 7.5+/-1.0 (SD) mg-min-1-kg body wt-1, and the half-saturation constant (Kd) was 27.7+/-7.0 (SD) mg-kg-1; b) when a TC infusion was given prior to the DBSP injection, the initial disappearance of the dye was more rapid than in the absence of the bile acid; Vmax increased to 12.3+/-2.0 (SD) mg-min-1-kg-1 (P less than 0.001) and Kd increased to 50.3+/-12.8 (SD) mg-kg-1 (P less than 0.001). These results indicate that: 1) the uptake of DBSP by the liver cell is a saturable process; 2) TC increases both the Vmax, suggesting a facilitation of the uptake process, and the Kd, suggesting competition for a common site. This effect of TC on DBSP uptake is similar to the previously described effect of this bile salt on BSP excretion into bile and suggests an interaction of bile salts with the uptake process of dyes.

Bile salts and bioelectric properties of rat jejunum.

Addition of conjugated bile salts increased transmural- and transserosal-potential differences of sheets of rat jejunum. Removal of Cl- from buffer solutions minimized the bile-sale induced bioelectric changes. Bile-salt induced doubling of tissue resistance was not explained by an observed increase in net Cl- serosa leads to mucosa flux. Electrical effects were unrelated to concentration and were observed only when bile-salt solutions perfused the jejunal mucosa. The molecular events associated with bile-salt interactions with the plasma membrane affecting ion fluxes may relate to their unique effects on sterol absorption.

Studies on simple and mixed bile salt micelles by nuclear magnetic resonance spectroscopy

High resolution nuclear magnetic resonance (NMR) spectroscopy has been used to study (a) the spectra of the common bile acids in organic solvents, (b) the structure of the bile-salt micelle and (c) the interaction of bile salt with lecithin in mixed micelles. The following is concluded: 1. 1. The common bile acids may be identified by their NMR spectra in organic solvents. 2. 2. When bile salt micelle formation takes place in 'HaO, a differential broadening occurs of the signals from the hydrophobic side of the bile salt molecule compared with those of the hydrophilic side. This observation suggests that the bile salt micelle associates hydrophobically with the hydrocarbon parts of the bile salt on the inside of the micelle and the hydroxyls and ionic parts of the molecule on the outside in contact with the aqueous environment. 3. 3. Relatively small amounts of bile salt convert the broad spectrum of lecithin in 2H 2O to a high resolution NMR spectrum. As the ratio of lecithin to bile salt in the mixed micelle is increased, the signals from the hydrophobic side of the bilesalt molecule are broadened indicating decreased molecular motion of these protons. Further, the percent of lecithin alkyl chains present as a high resolution peak increases as the amount of bile salt is decreased. These facts are consistent with the hypothesis that the lecithin-bile salt micelle exists as a bilayer of lecithin surrounded on its hydrophobic surfaces by bile salt molecules. The hydrocarbon side of the bile salt molecules associate with some of the alkyl chains of the lecithin thus hiding them from the aqueous environment. The entire exterior of the micelle is hydrophilic, the ends being covered with the polar groups of the lecithin and the sides with the polar groups and hydroxyls of the bile salts.

Studies on transmural potentials in vitro in relation to intestinal absorption : V. Kinetic characteristics of lipid interactions with rat gut

The in vitro studies reported here are concerned with the interactions of bile salt alone and of bile salt, fatty acid, monoglyceride and triglyceride in various combinations, with the mucosal surface of paired everted sacs prepared from the rat small intestine. Lipid-induced changes were noted in transmural potential (ΔPD) and in short-circuit current ( ΔI sc ) during incubation in buffered media. Increments in PD and I sc appeared to be saturable functions of graded concentrations of taurocholate and of glyceryl monooleate in the mucosal medium. This was confirmed by Line-weaver-Burk plots of the data and apparent K m values for the interaction of taurocholate and of monooleate with the ileal mucosal epithelium were estimated by extrapolation to be 3.6 and 6.0 mM, respectively. The lipid-membrane interactions are Na +-dependent. For example, apparent K m values increased as the Na + concentration of the medium was decreased by isosmolar replacement with Tris + or K +. Moreover, K +, compared with Tris +, seemed to inhibit markedly the bile salt interaction, but had little effect on the interaction of monoglyceride other than deprivation of Na +. Furthermore, this Na + dependency is reflected in a mutual competitive inhibition between glucose and these lipids as well as between the lipids themselves. A comparison of ΔPD and ΔI sc data, obtained with mixed lipids containing oleic acid or triolein, suggests that 3-component preparations (with taurocholate and monoglyceride) are more effective in interacting with the mucosal surface of the sac than are the corresponsing 2-component preparations (without monoglyceride). The studies with [ 14C]triolein indicate that (1) a small but significant fraction of the lipid may actually enter the mucosal tissues and (2) uptake, determined under open-circuit and short-circuit conditions, appears to be an active, Na +-dependent process.

Surface chemistry of the monoglyceride-bile salt system: Its relationship to the function of bile salts in fat absorption

Ingested triglyceride is hydrolyzed by pancreatic lipase to yield monoglycerides and fatty acids, which are dispersed by bile salts into aggregates of micellar size. To determine the molecular arrangement of these aggregates, the surface interaction of appropriate fatty acids, glycerides, and their analogs with synthetically prepared, pure bile salts varying in conjugation, number, and position of hydroxyl groups and type of A/B ring juncture has been characterized. Monoolein was readily penetrated by bile salts. The degree of penetration was proportional to the intrinsic surface activity of the bile salt, and this correlated with the number and position of hydroxyl substituents: taurocholate <tauroallodeoxycholate (5α) < taurodeoxycholate ≦ taurochenodeoxycholate < taurolithocholate. Curves of π A and Δ V A indicated that at high areas the bile salt nucleus lies parallel to the interface; as the area was reduced, the nucleus became perpendicular and the ionic head of the bile salt was forced into the aqueous phase. With further compression, dihydroxy and trihydroxy bile salts separated from the monolayer and passed into the bulk phase; monohydroxy bile salts remained associated with the film, reflecting their greater surface activity. Penetration of monoglycerides, monoglyceride analogs, diglycerides, and triglycerides of oleyl and stearyl homologs indicated that the interaction of bile salts with monoglycerides was much stronger than that with higher glycerides, fatty acids, or propylene glycol monoesters. At 25°C, penetration did not occur unless spacing between the polar heads of monoglycerides was present. The mechanism of penetration was considered to be hydrogen bonding between the hydroxyl groups of the bile salt nucleus and those of the glycerol moiety of the monoglyceride. Monoglycerides form a mesomophic phase in water. Bile salts adsorb to the presumably lamellar or cylindrical arrays and round them off into smaller aggregates; a spherical model of the bile salt-monoglyceride-fatty acid aggregate is proposed. In this model, bile salts function as wetting agents, rather than as detergents, since they adsorb to the interface and do not penetrate the hydrocarbon chains. The ability of bile salts to penetrate films of monoglycerides plus the existence of a mesomorphic phase in the water-poor region of the ternary composition phase diagram of the bile salt-water monoglyceride system suggests that a mesomorphic phase of bile salt-water-monoglyceride and fatty acid may occur during pancreatic lipolysis at the oil/water interface. Thus the mechanism proposed by Lawrence for spontaneous detergence may well occur in biological systems.