Carbon monoxide-mediated alterations in paracellular permeability and vesicular transport in acetaminophen-treated perfused rat liver.

This study aimed to examine whether acetaminophen (AAP), an anti-inflammatory agent producing hepatocellular damages with its overdose, evokes hepatocellular dysfunction through mechanisms involving carbon monoxide (CO) generated by heme oxygenase (HO). In perfused rat livers, CO and bilirubin were determined in venous perfusate and bile samples as indices of heme degradation. Biliary excretion of transportally injected horseradish peroxidase was also determined to assess paracellular junctional permeability and vesicular transport across hepatocytes. AAP at 20 mmol/L induced a transient choleresis, followed by a reduction of bile output. Under these circumstances, the release of CO and bilirubin IXalpha, terminal products of the HO-mediated heme degradation, became 2. 5-fold greater than the control. The rate of CO production appeared stoichiometric to the degradation rate of microsomal cytochrome P-450. Mechanisms for the AAP-induced cholestasis involved an increase in the junctional permeability that coincided with a reduction of vesicular transport across hepatocytes. Clotrimazole, a cytochrome P-450 inhibitor, or zinc protoporphyrin IX, an HO inhibitor, but not copper protoporphyrin IX, which did not inhibit HO, attenuated these AAP-induced changes. Furthermore, administration of CO at concentrations comparable with those induced by AAP elicited a marked elevation of the paracellular junctional permeability concurrent with a reduction of transcellular vesicular transport, mimicking effects of the AAP administration. Thus, CO serves as a putative regulator of hepatocellular function that is overproduced through acute heme degradation during xenobiotic transformation.     

Functional reconstitution of the canalicular bile salt transport system of rat liver.

Recent studies have suggested that the canalicular bile salt transport system of rat liver corresponds to a 100-kDa membrane glycoprotein. In the present study we attempted to functionally reconstitute the 100-kDa protein into artificial proteoliposomes. Canalicular membrane proteins were solubilized with octyl glucoside in the presence of asolectin phospholipids. The extracts were treated with preimmune serum or the 100-kDa protein selectively immunoprecipitated with a polyclonal antiserum. Proteins remaining in the supernatant were then incorporated into proteoliposomes by gel-filtration chromatography. Canalicular proteoliposomes containing the 100-kDa protein exhibited transstimulatable taurocholate uptake that could be inhibited by 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS). In contrast, no DIDS-sensitive transstimulatable taurocholate uptake was found in 100-kDa protein-free canalicular proteoliposomes. However, when the immunoprecipitated 100-kDa protein was dissociated from the antibodies and exclusively incorporated into liposomes, reconstitution of DIDS-sensitive transstimulatable and electrogenic taurocholate anion transport was again positive. Although incorporation of solubilized basolateral membrane proteins into liposomes also resulted in a prompt reconstitution of Na+ gradient-driven taurocholate uptake, the anti-100-kDa antibodies had no effects on the reconstituted transport activity of basolateral proteins. Thus, the findings establish that the previously characterized canalicular-specific 100-kDa protein is directly involved in the transcanalicular secretion of bile salts.     

Uptake of bile acids by perfused rat liver: evidence of a structure-activity relationship

The hepatic extraction of unconjugated and taurine-conjugated bile acids, provided with different hydrophilicity values, has been measured in the perfused rat liver, in order to evaluate the role of the bile acid structure and bile acid hydrophilicity on their uptake by the liver. Ursocholic, cholic, ursodeoxycholic and chenodeoxycholic acids, free and taurine-conjugated, were injected into the portal vein in dose response studies, using a nonrecirculating perfusion system. For all of the bile acids, the uptake process showed saturation. In addition, a nonsaturable component was apparent in bile acids provided with the lowest hydrophilicity values, as expressed by the lowest values of the water to octanol partition coefficient. The maximum uptake velocity increased with increasing values of the partition coefficient, which in turn were associated with 7-OH alpha to beta epimerization, the presence of 12-OH in alpha position and taurine conjugation. The ratio of maximum uptake velocity to Km (Km being the half-saturation constant) appeared to be markedly increased by taurine conjugation and by 7-OH alpha to beta epimerization, whereas it was reduced by the presence of 12-OH in alpha position.     

Papaverine inhibits transcytotic vesicle transport and lipid excretion into bile in isolated perfused rat liver.

Papaverine is a nonspecific smooth muscle relaxant and a phosphodiesterase inhibitor. Its effects on biliary excretion of lipids and horseradish peroxidase were investigated in a single-pass isolated perfused rat liver model. A constant infusion of papaverine (1.6 mumol/min; 40 mumol/L) significantly increased bile flow (microliters per minute per gram of liver) before (2.03 +/- 0.09 vs. 1.0 +/- 0.06) and after sodium taurocholate infusion (2.77 +/- 0.10 vs. 1.88 +/- 0.11). However, papaverine significantly and reversibly reduced biliary excretion of phospholipids and cholesterol (nanomoles per minute per gram of liver) after a 1.0 mumol/min sodium taurocholate infusion, from 7.45 +/- 0.83 and 1.42 +/- 0.15 to 1.75 +/- 0.18 and 0.39 +/- 0.06, respectively (p less than 0.01), whereas secretion of bile acids was unaffected. When a 1-min pulse of horseradish peroxidase (25 mg) was infused in isolated perfused rat liver after a continuous infusion of N6,O-2'-dibutyryladenosine 3',5'-cyclic monophosphate (0.25 mumol/min; 6.25 mumol/L), horseradish peroxidase appeared in bile in an early (4 to 6 min) and late (20 to 25 min) peak. Papaverine significantly reduced the late peak, from 1.211 +/- 0.264 to 0.498 +/- 0.107 (p less than 0.01). Papaverine had no significant effects on either cyclic AMP or cyclic GMP in the liver and bile, although it has been reported that papaverine is a phosphodiesterase inhibitor. These findings indicate that papaverine inhibits biliary excretion of lipids but not bile acids, and they suggest that papaverine has an inhibitory effect on transcytotic vesicle transport independent of an increase of cyclic nucleotides in hepatocytes.     

Adaptive regulation of hepatic bile salt transport. Effect of prolonged bile salt depletion in the rat

Exposure of the liver to increased bile salt flux can increase the bile salt maximum secretory rate (SRm), presumably through the induction of new transport sites. The converse, i.e., the down-regulation of SRm upon bile salt deprivation, has not been demonstrated. We examined the effects of bile salt depletion for 24 h and 48 h on taurocholate SRm and bromsulphalein (BSP) SRm, and on [14C]taurocholate binding to isolated liver surface membranes in unrestrained external biliary fistula rats. Taurocholate SRm was significantly decreased by 35% and 51% in 24-h-depleted and 48-h-depleted rats, respectively, compared with control, sham-operated rats. Maximal taurocholate concentration in bile was also significantly lower in bile salt-deprived rats. In contrast, BSP SRm was not significantly different between depleted animals and controls. Bile salt depletion for 24 h and 48 h did not significantly alter liver surface membrane protein recovery and membrane enzyme specific activity, including Na+ + K+-ATPase. Specific [14C]taurocholate binding to liver surface membranes was significantly decreased by 25% in 24-h-depleted rats compared with control rats. In contrast to taurocholate SRm, bile salt depletion for 48 h did not result in further reduction of specific taurocholate binding sites. This study demonstrates that taurocholate SRm progressively decreased in 24-h- and 48-h-bile salt-depleted rats, this being consistent with adaptive down-regulation of hepatic bile salt transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of brefeldin a on transcytotic vesicular pathway and bile secretion: A study on the isolated perfused rat liver and isolated rat hepatocyte couplets

This study investigated the effect of Brefeldin A (BFA) on the transcytotic vesicular pathway labeled with horseradish peroxidase (HRP) in both isolated rat hepatocyte couplets (IRHC) and the isolated perfused rat liver (IPRL). To evaluate the role of the transcytotic vesicular pathway on bile secretion, the effect of BFA on bile secretion in the IPRL was then investigated. In the basolateral area of IRHC, BFA showed no effect on the density and percentage of area of HRP-labeled vesicles. However, HRP-labeled vesicles tended to accumulate in the juxtanuclear area of BFA-treated hepatocytes (P < .001 vs. controls). In the pericanalicular area, on the other hand, HRP-labeled vesicles were depleted compared with controls (P < .001). In keeping with these findings, although the early peak remained unchanged, BFA inhibited as much as 50% of the late peak of HRP excretion in bile, after a pulse load of HRP in the IPRL. Bile flow and the biliary secretion of bile salts (BS) and phospholipids were not modified by BFA in isolated livers perfused without BS in the perfusate or with 1 μmol min taurocholate (TCA). In BFA-treated livers, peak bile flow and BS output decreased by 20% (P < .05 vs. controls) only when a 5 μmol TCA bolus was administered. In conclusion, this study demonstrates that BFA inhibits the transcytotic vesicular pathway in the liver. However, BFA has no significant effect on bile secretion either in basal conditions or during perfusion with physiological amounts of BS. BFA slightly decreases bile flow and BS output only after an overload of BS, providing evidence against the physiological relevance of the transcystotic vesicular pathway in the process of bile formation.459.)     

Effects of amino acids on bile acid-dependent and independent bile flow in the isolated perfused rat liver.

BACKGROUND/AIMS: Conflicting data on the effects of amino acids on biliary function led us to investigate their interaction with taurocholate in the perfused rat liver model. METHODS: To investigate the influence of amino acids on the bile acid-independent component of bile flow, 12 livers were perfused with (n = 6) and without (n = 6) amino acid addition from t30 min. For the study of bile acid-dependent bile flow, 24 livers were perfused under 8 experimental conditions according to the perfusate taurocholate concentration (12.5, 25, 37.5 or 50 microM) and whether amino acids were or were not added from t30 min. RESULTS: In the absence of taurocholate, amino acids induced a 40% (p<0.01) decrease in bile flow together with an increase in hepatic water content (17.8%, p< 0.05). Thus, amino acids exert an inhibitory effect on bile acid-independent bile flow despite the postulated cell swelling-dependent increase in bile flow. When livers were perfused at various taurocholate concentrations, amino acids induced, in addition to their inhibitory effect on bile acid-independent bile flow, a significant increase in taurocholate apparent choleretic activity (13.2 microl/micromol vs. 10.6 microl/micromol; p = 0.05), while taurocholate intrinsic clearance was significantly decreased (4.5+/-1.2 ml x min(-1) x g(-1) vs. 6.1+/-1.3 ml x min(-1) x g(-1); p<0.01). CONCLUSIONS: These data suggest that at physiological bile acid concentrations amino acids exert an inhibitory effect on both bile acid-dependent and- independent bile flow, whereas at higher taurocholate concentrations this inhibitory effect disappears, probably because of cell swelling-dependent mechanisms.     

Effect of bile acids on hepatic protoporphyrin metabolism in perfused rat liver

To determine the effect of bile acids on hepatic protoporphyrin metabolism, balance studies were performed in isolated perfused rat livers. Hepatic protoporphyrin metabolism was found to increase linearly as a function of protoporphyrin dose in livers infused with and without taurocholate (0.7 mumol/min), but their rates differed significantly. Employing a standard 1500-nmol protoporphyrin bolus dose, infusions (0.7 mumol/min) of taurocholate, glycocholate, deoxycholate, and chenodeoxycholate, but not tauroursodeoxycholate or ursodeoxycholate, increased protoporphyrin metabolism 1.7- to 2.7-fold over control (0 bile acid) values. Bile acid infusion ranging from 0.175 to 1.4 mumol/min confirmed that both taurocholate and chenodeoxycholate increased protoporphyrin disposal significantly more than ursodeoxycholate. For all bile acids, the increase of protoporphyrin metabolism was most pronounced between biliary bile acid excretion rates of 10-50 nmol/min.g liver. These data indicate that (a) bile acids facilitated protoporphyrin metabolism, (b) bile acid structure influenced the effect, and (c) ursodeoxycholate may not be a prime candidate to study the role of bile acids in the treatment of protoporphyria.     

Mechanisms of secretion of proteins into bile: studies in the perfused rat liver

Employing the in situ perfused rat liver, we examined the origins and mechanisms of transport of proteins into bile. First, utilizing polyacrylamide gels, we noted that many biliary proteins co-migrated with dominant serum proteins. Upon liver perfusion with serum-free medium, most proteins disappeared from the biliary profile; one major biliary protein that was not present in serum, identified as secretory component, remained. Kinetic analysis of the disappearance half-lives of the biliary proteins suggested that some serum proteins enter bile by a slow (20 to 30 min; transcellular) route, while others utilize both slow and rapid (5 min; paracellular) routes. In biosynthetic labeling experiments, secretion of newly synthesized proteins into bile was delayed about 20 min when compared with secretion of proteins into the perfusion medium and comprised less than 1% of the total secreted proteins. When a new liver was inserted into the perfusion medium containing newly synthesized secreted proteins, only two proteins, hemopexin and an unidentified protein, were transported into the bile from the perfusion medium; other biliary proteins were presumed to come directly from the hepatocyte. This latter group included some proteins that were secreted into the perfusion medium as well as into bile, and others, e.g., secretory component, that were secreted only into bile. Based on our results we have defined six pathways for entry of proteins into bile.     

Protective action of amiodarone against ventricular fibrillation in the isolated perfused rat heart.

The pretreatment of rats with amiodarone for 2 minutes to 3 weeks before the excision of their hearts caused a dose-related decrease in heart rate and an increase in the ventricular fibrillation threshold both before and after coronary arterial ligation. Similarly, amiodarone decreased the incidence of ventricular premature extrasystoles, ventricular tachycardia and fibrillation during the period of regional ischemia after coronary arterial ligation and also after reperfusion of the ischemic myocardium. There was no evidence of a metabolic protective effect on ischemic myocardium because tissue high energy phosphate content decreased to a similar extent in ischemic myocardium from control and amiodarone-treated rats. Instead, the protective effect of amiodarone against fibrillation was accompanied by attenuation of the increase in tissue cyclic adenosine monophosphate in ischemic myocardium after coronary arterial ligation. It is proposed that amiodarone exerts a potent antifibrillatory effect by decreasing tissue cyclic adenosine monophosphate in ischemic myocardium.     

Taurocholate stimulates transcytotic vesicular pathways labeled by horseradish peroxidase in the isolated perfused rat liver

The effect of taurocholate on transcytotic vesicular pathways labeled with horseradish peroxidase was assessed in isolated perfused rat liver preparations. Forty-five minutes after a horseradish peroxidase load in a recirculating system, continuous infusion of taurocholate but not taurodehydrocholate significantly increased horseradish peroxidase excretion in bile by 50% compared with controls. When horseradish peroxidase (25 mg) was pulse loaded for 1 minute in control perfusions, it appeared in bile in early (4-6 minutes) and late (20-25 minutes) peaks, the latter accounting for 90% of total horseradish peroxidase output. Taurocholate infusion significantly increased horseradish peroxidase output in both early and late peaks, whereas only a small increase in the early peak was observed with taurodehydrocholate. Colchicine pretreatment increased the early peak in bile but abolished the second peak. Electron micrographs from control livers revealed the accumulation of horseradish peroxidase-containing vesicles in pericanalicular regions at early (2 minutes) as well as late (18 minutes) periods. When a morphometric analysis of electron micrographs was performed from pericanalicular regions 2 minutes after a 1-minute pulse of horseradish peroxidase (500 mg), taurocholate but not taurodehydrocholate increased both the density and percent area of horseradish peroxidase-containing vesicles compared with controls. In contrast, colchicine pretreatment had no effect on the density of the early-appearing vesicles, although their individual sizes were reduced. Taurocholate but not taurodehydrocholate also increased the percent of tubular structures in the pericanalicular region. These findings indicate that taurocholate stimulates both early and late transcytotic vesicle pathways and therefore probably microtubule-independent vesicle pathway is present in hepatocytes that must be distinguished from paracellular routes.     

Communication via gap junctions modulates bile secretion in the isolated perfused rat liver

BACKGROUND & AIMS: Bile secretion is regulated in part by adenosine 3',5'-cyclic monophosphate (cAMP) and cytosolic Ca2+ (Ca2+i). Hormone receptors that link to these second messengers are not uniformly distributed across the hepatic lobule, but both cAMP and Ca2+i cross gap junctions, so we tested whether gap junctional communication plays a role in changes in bile flow induced by the activation of these receptors. METHODS: cAMP levels in isolated perfused rat livers were increased by using glucagon, because glucagon receptors are predominantly on pericentral hepatocytes, or by using dibutyryl cAMP, which acts on hepatocytes throughout the hepatic lobule. Ca2+i concentration was increased by using vasopressin, because V1a receptors are most heavily expressed on pericentral hepatocytes, or by using 2,5-di(tert-butyl)-1, 4-benzo-hydroquinone (t-BuBHQ), which increases the Ca2+i concentration in hepatocytes throughout the hepatic lobule. We used 18alpha-glycyrrhetinic acid (alphaGA) to block gap junction conductance, which was assessed by fluorescence recovery after photobleaching. RESULTS: alphaGA blocked fluorescence recovery after photobleaching without altering the basal rate of bile flow. Glucagon and dibutyryl cAMP increased bile flow; alphaGA blocked the glucagon-induced increase but not that induced by dibutyryl cAMP. Vasopressin and t-BuBHQ decreased bile flow; alphaGA exacerbated the decrease induced by vasopressin but not by t-BuBHQ. CONCLUSIONS: Glucagon and vasopressin modulate bile flow in a manner that depends in part on gap junctional communication, even though the two hormones activate second messengers with opposing effects on bile flow. The organization of second messenger signals across the hepatic lobule may be an important component of hormonal regulation of bile secretion.     

Glucose transport and hypoxia–reoxygenation injury in the perfused rat liver

Abstract During liver transplantation, oxidative stress occurs during hypoxia and reoxygenation of the donor organ. Chemical oxidative stress impairs cell membrane transport. Therefore, in this study the influence of hypoxia and reoxygenation on hepatocellular membrane transport was investigated. Specifically, glucose transport was studied in the perfused rat liver using the multiple indicator-dilution technique. First, it was observed that in normal rat livers, glucose transport was rapid but saturable ( K _m48 ± 10 mmol/L and V _max9.4 ± 0.9 μmol/s per g of liver). To simulate hypoxia and reoxygenation, livers were perfused for 30 min with nitrogen-saturated buffer and then with oxygen-saturated buffer for 20 min. The livers from fed rats were protected from hypoxia-reoxygenation injury whereas those from fasted rats were highly susceptible to injury as determined by lactate dehydrogenase release. After reoxygenation, the rate of glucose influx decreased significantly by ∼50% in the fasted livers ( P < 0.001) but was unaffected in the fed livers. This impairment of the hepatocellular transport of glucose, which could be secondary to oxidative injury to the hepatocyte membrane, has implications for the function of donor livers that have sustained hypoxia-reoxygenation ('preservation') injury during transplantation.     

Inhibition by colchicine of biliary secretion of diethylmaleate in the rat: evidence for microtubule-dependent vesicular transport

It has been proposed that a microtubule-dependent transport of vesicles derived from the Golgi apparatus may play a role in biliary secretion of bile salts and other cholephilic anions. To test this hypothesis, we examined the influence of colchicine and vinblastine, two microtubule inhibitors, on diethylmaleate-induced bile flow and on the biliary secretion of diethylmaleate, an organic anion whose glutathione conjugates may be secreted into bile through the Golgi apparatus and Golgi-derived vesicles. Rats were pretreated with colchicine or vinblastine, and diethylmaleate was injected intraperitoneally at doses of 28 to 400 mumol/100 gm body wt. Basal bile flow was unaffected by colchicine or vinblastine. In contrast, diethylmaleate-induced bile flow and the secretion into bile of diethylmaleate conjugates (estimated by the cation-anion gap in bile) were significantly lower in colchicine-treated and vinblastine-treated animals than in controls. Diethylmaleate-induced bile flow was reduced in proportion to diethylmaleate conjugate secretion. A linear relationship was seen between bile flow and biliary output of diethylmaleate conjugates: this relationship was similar in colchicine-treated or vinblastine-treated animals and in controls. At electron microscopy, diethylmaleate had induced distension of the Golgi saccules of the hepatocytes. In conclusion, colchicine and vinblastine inhibited the secretion into bile of diethylmaleate conjugates and diethylmaleate-induced bile flow. These results support the view that microtubule-dependent transport of Golgi-derived vesicles is involved in the biliary secretion of diethylmaleate and, perhaps, other cholephilic organic anions.     

Adaptive regulation of bile salt transporters in kidney and liver in obstructive cholestasis in the rat.

BACKGROUND & AIMS: Cholestasis results in adaptive regulation of bile salt transport proteins in hepatocytes that may limit liver injury. However, it is not known if changes also occur in the expression of bile salt transporters that reside in extrahepatic tissues, particularly the kidney, which might facilitate bile salt excretion during obstructive cholestasis. METHODS: RNA and protein were isolated from liver and kidney 14 days after common bile duct ligation in rats and assessed by RNA protection assays, Western analysis, and tissue immunofluorescence. Sodium-dependent bile salt transport was also measured in brush border membrane vesicles from the kidney. RESULTS: After common bile duct ligation, serum bile salts initially rose and then declined to lower levels after 3 days. In contrast, urinary bile salt excretion rose progressively over the 2-week period. By that time, the ileal sodium-dependent bile salt transporter messenger RNA and protein expression in total liver had increased to 300% and 200% of controls, respectively, while falling to 46% and 37% of controls, respectively, in the kidney. Sodium-dependent uptake of (3)H-taurocholate in renal brush border membrane vesicles was decreased. In contrast, the multidrug resistance-associated protein 2 expression in the kidney was increased 2-fold, even 1 day after ligation. Immunofluorescent studies confirmed the changes in the expression of these transporters in liver and kidney. CONCLUSIONS: These studies show that the molecular expression of bile salt transporters in the kidney and cholangiocytes undergo adaptive regulation after common bile duct obstruction in the rat. These responses may facilitate extrahepatic pathways for bile salt excretion during cholestasis.     

Canalicular bile flow and bile salt secretion are maintained in rats with liver cirrhosis. Further evidence for the intact cell hypothesis

Different aspects of biliary physiology were studied in rat model of liver cirrhosis induced by CCl4/phenobarbitone. We measured bile flow, bile salt secretion, biliary secretion pressure and bile-to-plasma ratios of inert solutes under basal conditions and during infusion of taurocholate (0.4 and 0.8 mumol.min-1.100 g body wt.-1) in 11 cirrhotic and 10 control male Sprague-Dawley rats. Bile flow and biliary bile salt secretion did not differ between the two groups. Analyzing the relationship between bile salt secretion and bile flow, however, we found an increased slope (P less than 0.02) in the cirrhotic animals, suggesting a higher apparent osmotic activity of the bile salts secreted. Maximal biliary secretion pressure was maintained in cirrhotic animals (22.5 +/- 2.5 vs. 25.0 +/- 2.9 cmH2O) in the absence of exogenous bile salt. During taurocholate infusion it decreased to a lesser extent (P less than 0.001) in cirrhotic animals (13.5 +/- 3.4 vs. 19.3 +/- 3.8 cmH2O). Bile-to-plasma ratios of [3H]sucrose and [14C]ferrocyanide were markedly increased in cirrhotic rats. Biliary [14C]erythritol clearance was equal to bile flow in both groups. In the cirrhotic group, the [3H]sucrose bile/plasma ratio was positively correlated with spleen weight (r = 0.744, P less than 0.01), serum concentration of alkaline phosphatase (r = 0.583, P less than 0.05) and basal maximum biliary secretion pressure (r = 0.801, P less than 0.001). We conclude that chronic portal hypertension is associated with increased permeability of the blood/bile barrier. Nevertheless, bile flow and bile salt secretion are maintained in cirrhotic rats, giving support to the intact cell hypothesis for this important hepatocellular function.     

Human and rat bile acid-CoA:amino acid N-acyltransferase are liver-specific peroxisomal enzymes: implications for intracellular bile salt transport

Bile acid-coenzyme A:amino acid N-acyltransferase (BAAT) is the sole enzyme responsible for conjugation of primary and secondary bile acids to taurine and glycine. Previous studies indicate a peroxisomal location of BAAT in peroxisomes with variable amounts up to 95% detected in cytosolic fractions. The absence or presence of a cytosolic pool of BAAT has important implications for the intracellular transport of unconjugated/deconjugated bile salts. We used immunofluorescence microscopy and digitonin permeabilization assays to determine the subcellular location of endogenous BAAT in primary human and rat hepatocytes. In addition, green fluorescent protein (GFP)-tagged rat Baat (rBaat) and human BAAT (hBAAT) were transiently expressed in primary rat hepatocytes and human fibroblasts. Catalase and recombinant GFP-SKL and DsRed-SKL were used as peroxisomal markers. Endogenous hBAAT and rBaat were found to specifically localize to peroxisomes in human and rat hepatocytes, respectively. No significant cytosolic fraction was detected for either protein. GFP-tagged hBAAT and rBaat were efficiently sorted to peroxisomes of primary rat hepatocytes. Significant amounts of GFP-tagged hBAAT or rBaat were detected in the cytosol only when coexpressed with DsRed-SKL, suggesting that hBAAT/rBaat and DsRed-SKL compete for the same peroxisomal import machinery. When expressed in fibroblasts, GFP-tagged hBAAT localized to the cytosol, confirming earlier observations. Conclusion: hBAAT and rBaat are peroxisomal enzymes present in undetectable amounts in the cytosol. Unconjugated or deconjugated bile salts returning to the liver need to shuttle through the peroxisome before reentering the enterohepatic circulation.