Transfer of fatty acids between intracellular membranes: roles of soluble binding proteins, distance, and time.

Soluble fatty acid binding proteins (FABPs) are thought to facilitate exchange of fatty acids between intracellular membranes. Although many FABP variants have been described, they fall into two general classes. "Membrane-active" FABPs exchange fatty acids with membranes during transient collisions with the membrane surface, whereas "membrane-inactive" FABPs do not. We used modeling of fatty acid transport between two planar membranes to examine the hypothesis that these two classes catalyze different steps in intracellular fatty acid transport. In the absence of FABP, the steady-state flux of fatty acid from the donor to the acceptor membrane depends on membrane separation distance (d) approaching a maximum value (J(max)) as d approaches zero. J(max) is one-half the rate of dissociation of fatty acid from the donor membrane, indicating that newly dissociated fatty acid has a 50% chance of successfully reaching the acceptor membrane before rebinding to the donor membrane. For larger membrane separations, successful transfer becomes less likely as diffusional concentration gradients develop. The mean diffusional excursion of the fatty acid into the water phase (d(m)) defines this transition. For d<>d(m), aqueous diffusion is rate limiting. All forms of FABP increase d(m) by reducing the rate of rebinding to the donor membrane, thus maintaining J(max) over larger membrane separations. Membrane-active FABPs further increase J(max) by catalyzing the rate of dissociation of fatty acids from the donor membrane, although frequent membrane interactions would be expected to reduce their diffusional mobility through a membrane-rich cytoplasm. Individual FABPs may have evolved to match the membrane separations and densities found in specific cell lines.

Mechanism of indinavir-induced hyperbilirubinemia.

Indinavir is a viral protease inhibitor used for the treatment of HIV infection. Unconjugated hyperbilirubinemia develops in up to 25% of patients receiving indinavir, prompting drug discontinuation and further clinical evaluation in some instances. We postulated that this side-effect is due to indinavir-mediated impairment of bilirubin UDP-glucuronosyltransferase (UGT) activity and would be most pronounced in individuals with reduced hepatic enzyme levels, as occurs in approximately 10% of the population manifesting Gilbert's syndrome. This hypothesis was tested in vitro, in the Gunn rat model of UGT deficiency, and in HIV-infected patients with and without the Gilbert's polymorphism. Indinavir was found to competitively inhibit UGT enzymatic activity (K(I) = 183 microM) while concomitantly inducing hepatic bilirubin UGT mRNA and protein expression. Although oral indinavir increased plasma bilirubin levels in wild-type and heterozygous Gunn rats, the mean rise was significantly greater in the latter group of animals. Similarly, serum bilirubin increased by a mean of 0.34 mg/dl in indinavir-treated HIV patients lacking the Gilbert's polymorphism versus 1.45 mg/dl in those who were either heterozygous or homozygous for the mutant allele. Whereas saquinavir also competitively inhibits UGT activity, this drug has not been associated with hyperbilirubinemia, most likely because of the higher K(I) (360 microM) and substantially lower therapeutic levels as compared with indinavir. Taken together, these findings indicate that elevations in serum-unconjugated bilirubin associated with indinavir treatment result from direct inhibition of bilirubin-conjugating activity.

Localization of bilirubin in phospholipid bilayers by parallax analysis of fluorescence quenching.

It has been proposed that the neurotoxicity observed in severely jaundiced infants results from the binding of unconjugated bilirubin to nerve cell membranes. However, despite potentially important clinical ramifications, there remains significant controversy regarding the physical nature of bilirubin-membrane interactions. We used the technique of parallax analysis of fluorescence quenching (Chattopadhyay, A., and E. London. 1987. Biochemistry. 26: 39;-45) to measure the depth of penetration of bilirubin in model phospholipid bilayers. The localization of unconjugated bilirubin and ditaurobilirubin within small unilamellar vesicles composed of dioleoylphosphatidylcholine was determined through an analysis of the quenching of bilirubin fluorescence by spin-labeled phospholipids, and by bilirubin-mediated quenching of a series of anthroyloxy fatty acid probes at various depths within the membrane bilayer. Findings were further verified with potassium iodide as an aqueous quencher. Our results indicate that, at pH 10, unconjugated bilirubin localizes approximately 20 A from the bilayer center, in the region of the polar head groups. Further analyses suggest a modest influence of pH, membrane cholesterol content, and vesicle diameter on the bilirubin penetration depth. Taken together, these data support that, under physiologic conditions, bilirubin localizes to the polar region of phospholipid bilayers, near the membrane-water interface.

Kinetic model of protein-mediated ligand transport: influence of soluble binding proteins on the intermembrane diffusion of a fluorescent fatty acid.

The mechanism (or mechanisms) whereby fatty acids and other amphipathic compounds are transported from the plasma membrane to intracellular sites of biotransformation remains poorly defined. In an attempt to better characterize the role of cytosolic binding proteins in this process, a kinetic model of intermembrane ligand transport was developed in which diffusional transfer of ligand between membrane and protein is assumed. The model was tested by utilizing stopped-flow techniques to monitor the transfer of the fluorescent fatty acid analogue, 12-anthroyloxy stearate (12-AS), between model membrane vesicles. Studies were conducted in the presence or absence of bovine serum albumin (BSA), liver fatty acid-binding protein (L-FABP), and intestinal fatty acid-binding protein (I-FABP) in order to determine the effect of soluble proteins on the rate of intermembrane ligand transfer. As predicted by the model, the initial velocity of 12-AS arrival at the acceptor membrane increases in an asymptotic manner with the acceptor concentration. Furthermore, probe transfer velocity was found to decline asymptotically with increasing concentrations of BSA or L-FABP, proteins that exhibit diffusional transfer kinetics. This observation was found to hold true independent of whether donor or acceptor vesicles were preequilibrated with the protein. In contrast, 12-AS transfer velocity exhibited a linear correlation with the concentration of I-FABP, a protein that is thought to transport fatty acids, at least in part, via a collisional mechanism. Taken together, these findings validate the derived kinetic model of protein-mediated ligand transport and further suggest that the mechanism of ligand-protein interaction is a key determinant of the effect of cytosolic proteins on intracellular ligand diffusion.

Role of apolipoprotein D in the transport of bilirubin in plasma.

Apolipoprotein D (apo D) is a 30-kDa glycoprotein of unknown function that is associated with high-density lipoproteins (HDL). Because unconjugated bilirubin has been shown to bind apo D with a 0. 8:1 stoichiometry, we examined the contribution of this protein to transport of bilirubin in human plasma. Density gradient centrifugation analysis using physiological concentrations of [(14)C]bilirubin reveals that 9% of unconjugated bilirubin is associated with HDL, with the remaining pigment bound primarily to serum proteins (i.e., albumin). The percentage of total plasma bilirubin bound to HDL was found to increase proportionally with bilirubin concentration. Affinity of human apo D for bilirubin was determined by steady-state fluorescence quenching, with Scatchard analysis demonstrating a single binding site for unconjugated bilirubin with an affinity constant (K(a)) of approximately 3 x 10(7) M(-1). Incorporation of apo D into phosphatidylcholine vesicles had no effect on K(a), suggesting that a lipid environment does not alter the affinity of the protein for bilirubin. Using stopped-flow techniques, the first-order rate constant for bilirubin dissociation from apo D was measured at 5.4 s(-1) (half-time = 129 ms). Our findings indicate that HDL is the principal nonalbumin carrier of bilirubin in human plasma and further support the proposition that the affinity of HDL for bilirubin is primarily the result of binding to apo D.

Mechanism of hepatocellular uptake of albumin-bound bilirubin.

We previously demonstrated that unconjugated bilirubin spontaneously diffuses through phospholipid bilayers at a rate which exceeds albumin dissociation, suggesting that solvation from albumin represents the rate-limiting step in hepatic bilirubin clearance. To further examine this hypothesis, we studied the uptake of bovine serum albumin (BSA)-bound bilirubin by cultured hepatoblastoma (HepG2) cells. Uptake of bilirubin was saturable, with a K(m) and V(max) of 4.2+/-0.5 microM (+/-S.E.M.) and 469+/-41 pmol min(-1) mg(-1) at 25 degrees C. Substantial bilirubin uptake also was observed at 4 degrees C (K(m)=7.0+/-0.8 microM, V(max)=282+/-26 pmol min(-1) mg(-1)), supporting a diffusional transport mechanism. Consistent with reported solvation rates, the cellular uptake of bilirubin bound to human serum albumin was more rapid than for BSA-bound bilirubin, indicative of dissociation-limited uptake. Counterintuitively, an inverse correlation between pH and the rate of bilirubin flip-flop was observed, due to pH effects on the rate of dissociation of bilirubin from albumin and from the membrane bilayer. The identification of an inflection point at pH 8.1 is indicative of a pK(a) value for bilirubin in this range. Taken together, our data suggest that hepatocellular uptake of bilirubin is dissociation-limited and occurs principally by a mechanism involving spontaneous transmembrane diffusion.

Unconjugated bilirubin exhibits spontaneous diffusion through model lipid bilayers and native hepatocyte membranes.

The liver is responsible for the clearance and metabolism of unconjugated bilirubin, the hydrophobic end-product of heme catabolism. Although several putative bilirubin transporters have been described, it has been alternatively proposed that bilirubin enters the hepatocyte by passive diffusion through the plasma membrane. In order to elucidate the mechanism of bilirubin uptake, we measured the rate of bilirubin transmembrane diffusion (flip-flop) using stopped-flow fluorescence techniques. Unconjugated bilirubin rapidly diffuses through model phosphatidylcholine vesicles, with a first-order rate constant of 5.3 s-1 (t(1)/(2) = 130 ms). The flip-flop rate is independent of membrane cholesterol content, phospholipid acyl saturation, and lipid packing, consistent with thermodynamic analyses demonstrating minimal steric constraint to bilirubin transmembrane diffusion. The coincident decrease in pH of the entrapped vesicle volume supports a mechanism whereby the bilirubin molecule crosses the lipid bilayer as the uncharged diacid. Transport of bilirubin by native rat hepatocyte membranes exhibits kinetics comparable with that in model vesicles, suggesting that unconjugated bilirubin crosses cellular membranes by passive diffusion through the hydrophobic lipid core. In contrast, there is no demonstrable flip-flop of bilirubin diglucuronide or bilirubin ditaurate in phospholipid vesicles, yet these compounds rapidly traverse isolated rat hepatocyte membranes, confirming the presence of a facilitated uptake system(s) for hydrophilic bilirubin conjugates.

Effects of submicellar bile salt concentrations on biological membrane permeability to low molecular weight non-ionic solutes.

Bile salts have been hypothesized to mediate cytotoxicity by increasing membrane permeability to aqueous solutes. We examined whether submicellar bile salt concentrations affect model and native membrane permeability to small uncharged molecules such as water, urea, and ammonia. Osmotic water permeability (Pf) and urea permeability were measured in large unilamellar vesicles composed with egg yolk phosphatidylcholine (EYPC) +/- cholesterol (Ch) or rat liver microsomal membranes by monitoring self-quenching of entrapped carboxyfluorescein (CF). Ammonia permeability was determined utilizing the pH dependence of CF fluorescence. Submicellar bile salt concentrations did not significantly alter Pf of EYPC +/- Ch or rat liver microsomal membranes. At taurodeoxycholate (TDC) or tauroursodeoxycholate concentrations approaching those that solubilized membrane lipids, CF leakage occurred from vesicles, but Pf remained unchanged. Higher bile salt concentrations (0.5-2 mM TDC) did not alter Pf of equimolar EYPC/Ch membranes. The activation energy for transmembrane water flux was unchanged (12.1 +/- 1.2 kcal/mol for EYPC) despite the presence of bile salts in one or both membrane hemileaflets, suggesting strongly that bile salts do not form transmembrane pores that facilitate water flux. Furthermore, submicellar bile salt concentrations did not increase membrane permeability to urea or ammonia. We conclude that at submicellar concentrations, bile salts do not form nonselective convective channels that facilitate transmembrane transport of small uncharged molecules. These results suggest that bile salt-mediated transport of specific substrates, rather than nonselective enhancement of membrane permeability, underlies bile salt cytotoxicity for enterocytes and hepatocytes.

Intracellular transport of small hydrophobic compounds by the hepatocyte.

The liver is responsible for the detoxification and biliary excretion of a variety of endogenous and xenobiotic compounds and, therefore, is capable of responding to rapid fluctuations in metabolic demand. In order to accomplish this function, the hepatocyte must efficiently transport a host of substrates with wide-ranging physical-chemical properties to intracellular sites for biotransformation and subsequent secretion into the bile. The trafficking of substrates and metabolites within the liver cell is a complex process, involving the coordinated action of cellular proteins, organellar membranes, the cytoskeleton, vesicular transport pathways, and bulk convective cytoplasmic flow. This review summarizes recent developments in the field of intracellular transport, with particular reference to the metabolism of small hydrophobic and amphipathic molecular species (e.g., bilirubin, bile salts, fatty acids) by the hepatocyte.

A new voyage of discovery: transport through the hepatocyte.

In summary, hepatocellular membranes likely play an essential role in the binding and directed trafficking of unconjugated bilirubin, and potentially of a variety of other small hydrophobic molecules. Targeting of these substrates to the endoplasmic reticulum is determined by membrane cholesterol content, surface area and integral protein binding and enzyme activity. The rate of intracellular transport potentially may be modulated by the concentration of cytosolic binding proteins, but, at least for ligandin, this protein does not appear to function primarily as an intracellular bilirubin transporter.

Influence of glutathione S-transferase B (ligandin) on the intermembrane transfer of bilirubin. Implications for the intracellular transport of nonsubstrate ligands in hepatocytes.

To examine the hypothesis that glutathione S-transferases (GST) play an important role in the hepatocellular transport of hydrophobic organic anions, the kinetics of the spontaneous transfer of unconjugated bilirubin between membrane vesicles and rat liver glutathione S-transferase B (ligandin) was studied, using stopped-flow fluorometry. Bilirubin transfer from glutathione S-transferase B to phosphatidylcholine vesicles was best described by a single exponential function, with a rate constant of 8.0 +/- 0.7 s-1 (+/- SD) at 25 degrees C. The variations in transfer rate with respect to acceptor phospholipid concentration provide strong evidence for aqueous diffusion of free bilirubin. This finding was verified using rhodamine-labeled microsomal membranes as acceptors. Bilirubin transfer from phospholipid vesicles to GST also exhibited diffusional kinetics. Thermodynamic parameters for bilirubin dissociation from GST were similar to those for human serum albumin. The rate of bilirubin transfer from rat liver basolateral plasma membranes to acceptor vesicles in the presence of glutathione S-transferase B declined asymptotically with increasing GST concentration. These data suggest that glutathione S-transferase B does not function as an intracellular bilirubin transporter, although expression of this protein may serve to regulate the delivery of bilirubin, and other nonsubstrate ligands, to sites of metabolism within the cell.

Kinetics of bilirubin transfer between serum albumin and membrane vesicles. Insight into the mechanism of organic anion delivery to the hepatocyte plasma membrane.

Unconjugated bilirubin is transported in the plasma bound primarily to serum albumin, from which it is taken up and metabolized by the liver. To better characterize the mechanism of bilirubin delivery to the hepatocyte, stopped-flow techniques were utilized to study the kinetics of bilirubin transfer between serum albumin and both model phospholipid and native hepatocyte plasma membrane vesicles. The transfer process was best described by a single exponential function, with rate constants of 0.93 +/- 0.04, 0.61 +/- 0.03, and 0.10 +/- 0.01 s-1 (+/- S.D.) at 25 degrees C for human, rat, and bovine serum albumins, respectively. The observed variations in rate with respect to donor and acceptor concentrations provide strong evidence for the diffusional transfer of free bilirubin. Thermodynamic analysis suggests that the binding site on bovine serum albumin demonstrates higher specificity for the bilirubin molecule than that on human or rat serum albumin, which exhibit similar binding characteristics. Kinetic analysis of bilirubin transfer from rat serum albumin to isolated rat basolateral liver plasma membranes indicates that the delivery of albumin-bound bilirubin to the hepatocyte surface occurs via aqueous diffusion, rather than a collisional process, thereby mitigating against the presence of an "albumin receptor."

Membrane lipid composition and vesicle size modulate bilirubin intermembrane transfer. Evidence for membrane-directed trafficking of bilirubin in the hepatocyte.

To characterize the mechanisms underlying intracellular bilirubin transport, stopped-flow fluorometry was utilized to study the effects of membrane vesicle size and lipid composition on the kinetics of unconjugated bilirubin movement between model and native hepatocyte membranes. Bilirubin transfer rates declined asymptotically with increasing donor vesicle diameter, due primarily to a 1.4 kcal.mol-1 decrease in the entropy of activation for the larger vesicles. The incorporation of phosphatidylethanolamine and phosphatidylserine significantly enhanced the dissociation of bilirubin from phosphatidylcholine vesicles. Cholesterol induced a biphasic effect on the transfer rate constant; an initial decrease in rate from 248 to 217 s-1 associated with cholesterol:phospholipid ratios up to 20% was followed by a dramatic rise to 312 s-1 as the cholesterol concentration was increased to 70 mol %. The bilirubin dissociation rate from isolated rat liver endoplasmic reticulum (9.1 s-1) was significantly slower than for both basolateral and canalicular plasma membranes, which exhibited rate constants of 11.7 and 25.8 s-1, respectively. Collectively, these data suggest that the cholesterol: phospholipid ratio is the principal determinant of bilirubin dissociation from membranes. We postulate that the inherent cellular membrane cholesterol gradient in the hepatocyte creates a directed flux of bilirubin from the plasma membrane to teh endoplasmic reticulum and represents a potential driving force for intracellular bilirubin transport.

Mechanism of the spontaneous transfer of unconjugated bilirubin between small unilamellar phosphatidylcholine vesicles.

Unconjugated bilirubin (bilirubin-IX alpha), the hydrophobic end product of heme degradation, is esterified in the hepatocyte endoplasmic reticulum to water-soluble conjugates prior to excretion in bile. To characterize the process of intracellular bilirubin transport, the kinetic and thermodynamic activation parameters for the spontaneous transfer of bilirubin between small unilamellar egg lecithin vesicles were determined. Bilirubin-IX alpha was added to donor vesicles labeled with the fluorescent phospholipid probe, (5-(dimethylamino)naphthalene-1-sulfonyl) dipalmitoyl-L-alpha-phosphatidylethanolamine (dansyl-PE). When bound to the donor vesicles, bilirubin quenches the dansyl probe fluorescence through resonance energy transfer. The movement of bilirubin from dansyl-labeled donor vesicles to unlabeled acceptor vesicles was monitored directly by the reemergence of dansyl fluorescence over time. Vesicle fusion and intervesicle transfer of the dansyl-PE probe were excluded by quasielastic light scattering and fluorescence resonance energy transfer studies. Stopped-flow analysis demonstrated that the transfer of bilirubin was described by a single-exponential function with a mean half-time of 2.0 +/- 0.1 ms (+/- SD) at 37 degrees C. The rate of bilirubin transfer was independent of acceptor vesicle concentration and decreased with increasing buffer ionic strength, indicating that intermembrane transfer occurred via aqueous diffusion, rather than vesicle collisions. The free energy of activation (delta G++) for the dissociation of bilirubin from donor vesicles was 14.2 kcal.mol-1. These studies suggest that bilirubin is associated with phospholipid bilayers at the membrane-water interface. We postulate that the movement of unconjugated bilirubin between intracellular membranes occurs via spontaneous transfer through the aqueous phase.