Hepatitis D Virus Entry Inhibitors Based on Repurposing Intestinal Bile Acid Reabsorption Inhibitors.

Identification of Na+/taurocholate co-transporting polypeptide (NTCP) as high-affinity hepatic entry receptor for the Hepatitis B and D viruses (HBV/HDV) opened the field for target-based development of cell-entry inhibitors. However, most of the HBV/HDV entry inhibitors identified so far also interfere with the physiological bile acid transporter function of NTCP. The present study aimed to identify more virus-selective inhibitors of NTCP by screening of 87 propanolamine derivatives from the former development of intestinal bile acid reabsorption inhibitors (BARIs), which interact with the NTCP-homologous intestinal apical sodium-dependent bile acid transporter (ASBT). In NTCP-HEK293 cells, the ability of these compounds to block the HBV/HDV-derived preS1-peptide binding to NTCP (virus receptor function) as well as the taurocholic acid transport via NTCP (bile acid transporter function) were analyzed in parallel. Hits were subsequently validated by performing in vitro HDV infection experiments in NTCP-HepG2 cells. The most potent compounds S985852, A000295231, and S973509 showed in vitro anti-HDV activities with IC50 values of 15, 40, and 70 µM, respectively, while the taurocholic acid uptake inhibition occurred at much higher IC50 values of 24, 780, and 490 µM, respectively. In conclusion, repurposing of compounds from the BARI class as novel HBV/HDV entry inhibitors seems possible and even enables certain virus selectivity based on structure-activity relationships.

Effect of a Common Genetic Variant (p.V444A) in the Bile Salt Export Pump on the Inhibition of Bile Acid Transport by Cholestatic Medications.

The bile salt export pump (BSEP) is the primary canalicular transporter responsible for the secretion of bile acids from hepatocytes into bile canaliculi, and inhibition of this transporter has been associated with drug-induced liver injury (DILI). A common variant (rs2287622; p.V444A) in the gene encoding BSEP has been associated with an increased risk of cholestatic DILI. Although p.444V BSEP (reference) and p.444A BSEP (variant) do not differ in their transport kinetics of taurocholic acid (TCA), transport of the more abundant glycocholic acid (GCA) has not been investigated. Importantly, differences in the susceptibility of p.444V and p.444A BSEP to inhibition by drugs causing cholestatic DILI have not been investigated. To address these issues, the transport kinetics of GCA were evaluated by incubating membrane vesicles expressing either p.444V or p.444A BSEP with GCA over a range of concentrations (1, 10, 25, 50, and 100 µM). The abilities of commonly used cholestatic medications to inhibit the transport of TCA and GCA by the reference and variant proteins were compared. Resulting data indicated that GCA transport kinetics for reference and variant BSEP followed Michaelis-Menten kinetics and were not statistically different [ Vmax values of 1132 ± 246 and 959 ± 256 pmol min-1 (mg of protein)-1, respectively, and Km values of 32.7 ± 18.2 and 45.7 ± 25.5 µM, respectively]. There were no statistically significant differences between the reference and variant BSEP in the inhibition of TCA or GCA transport by the cholestatic drugs tested. In conclusion, differential inhibition of TCA or GCA transport cannot account for an association between the variant BSEP and the risk for cholestatic DILI due to the drugs tested.

Effect of substituent pattern and molecular weight of cellulose ethers on interactions with different bile salts.

Some known mechanisms proposed for the reduction of blood cholesterol by dietary fibre are: binding with bile salts in the duodenum and prevention of lipid absorption, which can be partially related with the bile salt binding. In order to gain new insights into the mechanisms of the binding of dietary fibre to bile salts, the goal of this work is to study the main interactions between cellulose derivatives and two types of bile salts. Commercial cellulose ethers: methyl (MC), hydroxypropyl (HPC) and hydroxypropylmethyl cellulose (HPMC), have been chosen as dietary fibre due to their highly functional properties important in manufactured food products. Two types of bile salts: sodium taurocholate (NaTC) and sodium taurodeoxycholate (NaTDC), have been chosen to understand the effect of the bile salt type. Interactions in the bulk have been investigated by means of differential scanning calorimetry (DSC) and linear mechanical spectroscopy. Results show that both bile salts have inhibitory effects on the thermal structuring of cellulose ethers and this depends on the number and type of substitution in the derivatised celluloses, and is not dependent upon molecular weight. Concerning the bile salt type, the more hydrophobic bile salt (NaTDC) has greater effect on these interactions, suggesting more efficient adsorption onto cellulose ethers. These findings may have implications in the digestion of cellulose-stabilised food matrices, providing a springboard to develop new healthy cellulose-based food products with improved functional properties.

Synthesis and in vitro evaluation of bile acid prodrugs of floxuridine to target the liver.

Floxuridine is often used to treat metastatic liver disease and is given as an infusion directly into the hepatic artery to increase the amount of intact drug that reaches the liver. The objective of this work was to design and synthesize prodrugs of floxuridine through conjugation to chenodeoxycholic acid (CDCA) to target the liver via the bile acid liver uptake transporter Na(+)/taurocholate cotransporting polypeptide (NTCP, SLC10A1). Two isomeric prodrugs of floxuridine were synthesized: floxuridine 3'glutamic acid-CDCA and floxuridine 5'-glutamic acid-CDCA. Both were potent inhibitors and substrates of NTCP. Floxuridine 3'glutamic acid-CDCA showed Ki=6.86±1.37 µM, Km=10.7±2.1 µM, and passive permeability=0.663(±0.121)×10(-7) cm/s while floxuridine 5'-glutamic acid-CDCA showed Ki=0.397±0.038 µM, Km=40.4±15.2 µM, and passive permeability=1.72(±0.18)×10(-7) cm/s. Floxuridine itself had a higher passively permeability of 7.54(±0.45)×10(-7) cm/s in the same cell line, indicating that both prodrugs have the potential for lower non-specific effects than the drug alone. Prodrugs were stable in rat plasma (t=3 h), but quickly released in rat liver s9 fraction, suggesting future in vivo evaluation.

Nitric oxide-mediated inhibition of taurocholate uptake involves S-nitrosylation of NTCP.

The sodium-taurocholate (TC) cotransporting polypeptide (NTCP) facilitates bile formation by mediating sinusoidal Na(+)-TC cotransport. During sepsis-induced cholestasis, there is a decrease in NTCP-dependent uptake of bile acids and an increase in nitric oxide (NO) levels in hepatocytes. In rat hepatocytes NO inhibits Na(+)-dependent uptake of taurocholate. The aim of this study was to extend these findings to human NTCP and to further investigate the mechanism by which NO inhibits TC uptake. Using a human hepatoma cell line stably expressing NTCP (HuH-NTCP), we performed experiments with the NO donors sodium nitroprusside and S-nitrosocysteine and demonstrated that NO inhibits TC uptake in these cells. Kinetic analyses revealed that NO significantly decreased the V(max) but not the K(m) of TC uptake by NTCP, indicating noncompetitive inhibition. NO decreased the amount of NTCP in the plasma membrane, providing a molecular mechanism for the noncompetitive inhibition of TC uptake. One way that NO can modify protein function is through a posttranslational modification known as S-nitrosylation: the binding of NO to cysteine thiols. Using a biotin switch technique we observed that NTCP is S-nitrosylated under conditions in which NO inhibits TC uptake. Moreover, dithiothreitol reversed NO-mediated inhibition of TC uptake and S-nitrosylation of NTCP, indicating that NO inhibits TC uptake via modification of cysteine thiols. In addition, NO treatment led to a decrease in Ntcp phosphorylation. Taken together these results indicate that the inhibition of TC uptake by NO involves S-nitrosylation of NTCP.

Use of sandwich-cultured hepatocytes to evaluate impaired bile acid transport as a mechanism of drug-induced hepatotoxicity.

Drug-induced liver toxicity is a significant problem in drug development and clinical practice, yet its mechanisms are not well understood. Growing evidence suggests that inhibition of bile acid transport may be one mechanism of hepatotoxicity. A number of hepatic transporters work in concert to transport bile acids and xenobiotics from blood to bile, and many drugs have been shown to perturb this process with detrimental consequences. Hepatocytes cultured in a sandwich configuration maintain transporter activity and liver-specific metabolic functions; thus, the sandwich-cultured hepatocyte model represents a useful tool for evaluating hepatotoxicity caused by interference with hepatic transporters. As an example, the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist troglitazone is one such drug that has been shown to inhibit bile acid transport in vitro. Data presented in this manuscript indicate that troglitazone inhibits both basolateral uptake and canalicular excretion of taurocholate in a concentration-dependent manner in both sandwich-cultured and suspended human and rat hepatocytes. These data confirm both the interaction of troglitazone with bile acid transporters in hepatocytes and the utility of the sandwich-cultured hepatocyte model to study such interactions.

Gastroprotective and ulcer-healing activity of oleanolic acid derivatives: in vitro-in vivo relationships.

The triterpene oleanolic acid 1 and its semisynthetic derivatives 2-7 were assessed for gastroprotective and ulcer-healing effect using human epithelial gastric cells (AGS) and human lung fibroblasts (MRC-5). The ability of the compounds to protect the AGS cells against the damage induced by sodium taurocholate (NaT), to stimulate the cellular reduced glutathione (GSH) and prostaglandin E(2) content, to enhance AGS and MRC-5 cell proliferation and to scavenge superoxide anion in vitro was studied. The cytotoxicity of the compounds was assessed towards MRC-5 and AGS cells. In addition, the gastroprotective activity of the compounds was assessed in vivo using the HCl/EtOH-induced ulcer model in mice. All the assayed compounds displayed a significant reduction of AGS cells damage after incubation with NaT. None of the studied compounds was active as a superoxide anion scavenger nor stimulated the GSH content in AGS cell cultures. Compounds 1, 2, 4 and 6 were able to increase the prostaglandin content in AGS cell cultures. Concerning the proliferation assays, a significant stimulating effect was observed for compounds 3 and 7 on AGS cells and for 1 and 7 on MRC-5 fibroblasts. Regarding cytotoxicity, derivatives 2, 4, 6 and 7 were less toxic than the parent compound oleanolic acid. Our results strongly support the predictive capacity of the in vitro assessment of gastroprotective activity allowing the reduction of experimental animals.

Novel non-systemic inhibitor of ileal apical Na+-dependent bile acid transporter reduces serum cholesterol levels in hamsters and monkeys.

1-{7-[(1-(3,5-Diethoxyphenyl)-3-{[(3,5-difluorophenyl)(ethyl)amino]carbonyl}-4-oxo-1,4-dihydroquinolin-7-yl)oxy]heptyl}-1-methylpiperidinium bromide, R-146224, is a potent, specific ileum apical sodium-dependent bile acid transporter (ASBT) inhibitor; concentrations required for 50% inhibition of [3H]taurocholate uptake in human ASBT-expressing HEK-293 cells and hamster ileum tissues were 0.023 and 0.73 microM, respectively. In bile-fistula rats, biliary and urinary excretion 48 h after 10 mg/kg [14C]R-146224, were 1.49+/-1.75% and 0.14+/-0.05%, respectively, demonstrating extremely low absorption. In hamsters, R-146224 dose-dependently reduced gallbladder bile [3H]taurocholate uptake (ED50: 2.8 mg/kg). In basal diet-fed hamsters, 14-day 30-100 mg/kg R-146224 dose-dependently reduced serum total cholesterol (approximately 40%), high density lipoprotein (HDL) cholesterol (approximately 37%), non-HDL cholesterols (approximately 20%), and phospholipids (approximately 20%), without affecting serum triglycerides, associated with reduced free and esterified liver cholesterol contents. In normocholesterolemic cynomolgus monkeys, R-146224 specifically reduced non-HDL cholesterol. In human ileum specimens, R-146224 dose-dependently inhibited [3H]taurocholate uptake. Potent non-systemic ASBT inhibitor R-146224 decreases bile acid reabsorption by inhibiting the ileal bile acid active transport system, resulting in hypolipidemic activity.

Troglitazone-induced intrahepatic cholestasis by an interference with the hepatobiliary export of bile acids in male and female rats. Correlation with the gender difference in troglitazone sulfate formation and the inhibition of the canalicular bile salt export pump (Bsep) by troglitazone and troglitazone sulfate.

Troglitazone is a thiazolidinedione insulin sensitizer drug for the treatment of type 2 non-insulin-dependent diabetes mellitus (NIDDM). Based on an increasing number of reports on troglitazone-associated liver toxicity, the cholestatic potential of troglitazone and its major metabolite troglitazone sulfate has been investigated. In isolated perfused rat livers troglitazone (10 microM) reduced the bile flow by 25% (female) to 50% (male) within 60 min. After single intravenous administrations of troglitazone to rats of both genders, rapid and dose-dependent increases in the plasma bile acid concentrations were observed, with male rats being more sensitive than female rats. In male rat liver tissue fivefold higher troglitazone sulfate levels were measured as compared to female rat liver tissue. This difference was due to the formation rate of troglitazone sulfate, which was four times faster in cytosolic fractions of male rat liver as compared to female rat liver (Clint=132 and 35 microl min(-1) mg(-1), respectively). Troglitazone sulfate strongly inhibited the ATP-dependent taurocholate transport mediated by the canalicular bile salt export pump (Bsep) in isolated canalicular rat liver plasma membrane preparations of both genders (IC(50) value of 0.4-0.6 microM), while troglitazone was 10 times less potent (IC(50) values of 3.9 microM). This high Bsep inhibition potential and the efficient formation and accumulation of troglitazone sulfate in liver tissue, suggested that troglitazone sulfate was mainly responsible for the interaction with the hepatobiliary export of bile acids at the level of the canalicular Bsep in rats. Such an interaction might lead potentially also in man to a troglitazone-induced intrahepatic cholestasis, potentially contributing to the formation of troglitazone-induced liver injuries.

Cholestan-3beta,5alpha,6beta-triol, but not 7-ketocholesterol, suppresses taurocholate-induced mucin secretion by cultured dog gallbladder epithelial cells.

In order to investigate oxysterol-mediated effects on the biliary system, we studied the effects of cholestan-3beta,5alpha,6beta-triol (TriolC) and 7-ketocholesterol (7KC) on gallbladder epithelial cells. We compared their cell proliferation effects in cultured dog gallbladder epithelial cells (DGBE) to their effects in cultured human pulmonary artery endothelial cells (HPAE). Oxysterols inhibited cell proliferation in a dose-dependent fashion. Oxysterols inhibited cell growth to 50% of control at a higher dose for DGBE cells than for HPAE cells. TriolC was more cytotoxic than 7KC. We also investigated the effect of oxysterols on bile salt-induced mucin secretion by DGBE cells. TriolC suppressed mucin secretion by DGBE cells, whereas 7KC did not. These findings support the hypothesis that biliary oxysterols affect gallbladder mucosal function.

Bile salt excretion in skate liver is mediated by a functional analog of Bsep/Spgp, the bile salt export pump.

Biliary secretion of bile salts in mammals is mediated in part by the liver-specific ATP-dependent canalicular membrane protein Bsep/Spgp, a member of the ATP-binding cassette superfamily. We examined whether a similar transport activity exists in the liver of the evolutionarily primitive marine fish Raja erinacea, the little skate, which synthesizes mainly sulfated bile alcohols rather than bile salts. Western blot analysis of skate liver plasma membranes using antiserum raised against rat liver Bsep/Spgp demonstrated a dominant protein band with an apparent molecular mass of 210 kDa, a size larger than that in rat liver canalicular membranes, approximately 160 kDa. Immunofluorescent localization with anti-Bsep/Spgp in isolated, polarized skate hepatocyte clusters revealed positive staining of the bile canaliculi, consistent with its selective apical localization in mammalian liver. Functional characterization of putative ATP-dependent canalicular bile salt transport activity was assessed in skate liver plasma membrane vesicles, with [(3)H]taurocholate as the substrate. [(3)H]taurocholate uptake into the vesicles was mediated by ATP-dependent and -independent mechanisms. The ATP-dependent component was saturable, with a Michaelis-Menten constant (K(m)) for taurocholate of 40+/-7 microM and a K(m) for ATP of 0.6+/-0.1 mM, and was competitively inhibited by scymnol sulfate (inhibition constant of 23 microM), the major bile salt in skate bile. ATP-dependent uptake of taurocholate into vesicles was inhibited by known substrates and inhibitors of Bsep/Spgp, including other bile salts and bile salt derivatives, but not by inhibitors of the multidrug resistance protein-1 or the canalicular multidrug resistance-associated protein, indicating a distinct transport mechanism. These findings provide functional and structural evidence for a Bsep/Spgp-like protein in the canalicular membrane of the skate liver. This transporter is expressed early in vertebrate evolution and transports both bile salts and bile alcohols.

Substrate specificity of the ileal and the hepatic Na(+)/bile acid cotransporters of the rabbit. II. A reliable 3D QSAR pharmacophore model for the ileal Na(+)/bile acid cotransporter.

To design a reliable 3D QSAR model of the intestinal Na(+)/bile acid cotransporter, we have used a training set of 17 inhibitors of the rabbit ileal Na(+)/bile acid cotransporter. The IC(50) values of the training set of compounds covered a range of four orders of magnitude for inhibition of [(3)H]cholyltaurine uptake by CHO cells expressing the rabbit ileal Na(+)/bile acid cotransporter allowing the generation of a pharmacophore using the CATALYST algorithm. After thorough conformational analysis of each molecule, CATALYST generated a pharmacophore model characterized by five chemical features: one hydrogen bond donor, one hydrogen bond acceptor, and three hydrophobic features. The 3D pharmacophore was enantiospecific and correctly estimated the activities of the members of the training set. The predicted interactions of natural bile acids with the pharmacophore model of the ileal Na(+)/bile acid cotransporter explain exactly the experimentally found structure;-activity relationships for the interaction of bile acids with the ileal Na(+)/bile acid cotransporter (Kramer et al. 1999. J. Lipid. Res. 40: 1604;-1617). The natural bile acid analogues cholyltaurine, chenodeoxycholyltaurine, or deoxycholyltaurine were able to map four of the five features of the pharmacophore model: a) the five-membered ring D and the methyl group at position 18 map one hydrophobic site and the 21-methyl group of the side chain maps a second hydrophobic site; b) one of the alpha-oriented hydroxyl groups at position 7 or 12 fits the hydrogen bond donor feature; c) the negatively charged side chain acts as hydrogen bond acceptor; and d) the hydroxy group at position 3 does not specifically map any of the five binding features of the pharmacophore model. The 3-hydroxy group of natural bile acids is not essential for interactions with ileal or hepatic Na(+)/bile acid cotransporters. A modification of the 3-position of a natural bile acid molecule is therefore the preferred position for drug targeting strategies using bile acid transport pathways.

Enhanced Na+-dependent bile salt uptake by WIF-B cells, a rat hepatoma hybrid cell line, following growth in the presence of a physiological bile salt.

Although bile salts are toxic to the liver at high plasma concentrations, the effects of physiological concentrations of bile salts on normal hepatic function are poorly understood. We examined the effect of taurocholate (TC) on the basolateral uptake of [3H]TC in WIF-B cells, a hybrid cell line stably exhibiting in vitro the structural and functional polarity of hepatocytes. Cells were grown in the absence or presence of TC (50 micromol/L) over 12 days, and then incubated with [3H]TC concentrations ranging from 1 to 250 micromol/L. For both control and TC-grown cells, uptake of [3H]TC was linear over 2 minutes. In control cells, the Km for [3H]TC Na+-dependent uptake over 1 minute was 6 +/- 5 micromol/L, and the Vmax was 45 +/- 6 pmol TC/mg protein/min (+/- SEM). TC-grown cells exhibited no significant change in Km but showed a doubling of Vmax to 87 +/- 6 pmol TC/mg protein/min (P < .005). In both control and TC-grown cells, maximal uptake of [3H]TC occurred following 10 to 12 days in culture, with TC-grown cells consistently showing greater rates of [3H]TC uptake from 4 to 14 days in culture. Western blots immunostained for the basolateral Na+-dependent plasma membrane protein, ntcp, revealed the appropriate approximately 50-kd band in control and TC-grown cells, and confocal immunofluorescence microscopy demonstrated staining along the basolateral plasma membrane. Northern blots hybridized with a cDNA probe directed against ntcp indicated a modest TC-induced increase in mRNA levels. Reverse-transcriptase polymerase chain reaction (RT-PCR) using RNA isolated from WIF-B cells and oligonucleotide primers specific for rat ntcp or human NTCP transcripts revealed only the presence of the rat ntcp transcript. We conclude that bile salts, at concentrations normally found in mammalian portal blood, may be capable of promoting enhanced hepatocellular bile salt uptake via an increase in basolateral Na+-dependent plasma membrane transport capacity.

Secretin prevents taurocholate-induced intrahepatic cholestasis in the rat.

Secretin is known to stimulate the flow of bicarbonate-rich bile from the bile-duct epithelium, but has no effect on hepatocytes. To investigate the effects of secretin on bile production during intrahepatic cholestasis, we infused secretin into rats with taurocholate-induced cholestasis. Secretin was given at 0.25 and 0.50 units.min-1.kg-1 to Wistar rats that simultaneously received a continuous infusion of taurocholic acid at above its maximum hepatic transport capacity to produce cholestasis. When taurocholic acid was infused at doses of 1.4 and 1.6 mumol.min-1.100 g b.w.-1, bile volume decreased in control rats. In contrast, the simultaneous infusion of secretin significantly increased bile flow and the biliary excretion of bile acids and bicarbonate. The serum taurocholic acid level at the end of the experiment was significantly lower in the secretin-treated groups than in the control group. These findings indicate that secretin prevents taurocholate-induced cholestasis and may enhance the biliary excretion of bile acids.

Transepithelial transport of cholyltaurine by Caco-2 cell monolayers is sodium dependent.

Bile acids are efficiently recovered from the intestinal lumen by a Na(+)-dependent transport process that is localized in the ileal enterocyte brush-border membrane. To establish a cell culture model for this process, we examined the Na+ dependence of cholyltaurine (C-tau; taurocholate) transport across monolayers of differentiated Caco-2 cells grown on permeable filter inserts. Transport of [3H]C-tau was Na+ dependent (> 20-fold stimulation), saturable, and time linear for at least 60 min. The apparent Michaelis constant of [3H]C-tau transport was approximately 65 microM, and the maximal transport rate was approximately 800 pmol.min-1.mg protein-1. Transport of [3H]C-tau in the apical-to-basolateral direction was 17-fold greater than transport in the reverse direction. Lowered incubation temperature, various metabolic inhibitors, and various unlabeled bile acids inhibited [3H]C-tau transport. Caco-2 cells thus transport bile acids in a manner similar to that described for ileal brush-border membrane vesicles and isolated ileal enterocytes and are therefore an appropriate model for studying the molecular basis of ileal bile acid transport.

Effect of lysosomotropic agents on the taurocholate-stimulated biliary excretion of horseradish peroxidase.

The effects of the lysosomotropic agents chloroquine and leupeptin on the taurocholate-stimulated biliary excretion of horseradish peroxidase (HRP) was studied in bile fistula rats. HRP (0.5 mg/100 g body wt) was injected into the portal vein during taurocholate (0.4 mumol/min/100 g body wt) or saline infusion. HRP appeared in bile showing both an early (approx. 5 min) and a late (approx. 25 min) excretion peak. The late peak, which represented about 95% of the total HRP excreted, is due to transcellular vesicular transport. The early peak is mainly due to paracellular leakage although a rapid vesicular transport also contributes. Taurocholate infusion significantly increased the biliary output of HRP (both peaks) and of the endogenous lysosomal enzyme acid phosphatase. Pretreatment with chloroquine or leupeptin inhibited the taurocholate-stimulated late excretion of HRP into bile, without affecting its early excretion. The lysosomotropic agents did not affect the biliary excretion of bile salts but significantly inhibited the taurocholate-stimulated biliary excretion of acid phosphatase. The results are consistent with a role of lysosomes in the taurocholate-stimulated major transcellular vesicular transport of HRP into bile.

Compartmental analysis of steady-state taurocholate transport kinetics by isolated rat hepatocytes.

We used compartmental modeling to describe taurocholate transport by isolated rat liver cells in suspension. Cells are preincubated in the presence of unlabeled taurocholate. When a steady-state for taurocholate is reached, radiolabeled taurocholate is added to the medium and its exchange kinetics between the medium and the cells are followed over time. Because the studies are performed under steady-state conditions, the kinetics can be described by linear compartmental models. We found a closed two-compartment model sufficient to describe the steady-state transport data. Simulations reveal that if the pools of free and bound intracellular taurocholate exchange rapidly, the cells will behave as a single, kinetically homogeneous compartment and intracellular events will not influence the exchange kinetics of taurocholate between the medium and the cells. The two-compartment model was used to study the concentration dependence of taurocholate transport by isolated cells. Steady-state transport rates and taurocholate concentrations in the medium and the cells were calculated using the model equations. Taurocholate influx, accumulation and efflux processes were studied simultaneously by examining the relationship between appropriate combinations of these variables. Application of this approach to study the inhibition of taurocholate transport by taurochenodeoxycholate is illustrated. In conclusion, this method provides a complementary approach to initial rate studies, which are generally used to investigate bile acid transport by isolated cells.

Preventive effect of cimetidine on chronic erosive gastritis induced by taurocholate in rats.

We studied the preventive effect of cimetidine at the microscopic level on chronic erosive gastritis induced experimentally by 6-months of administration of drinking water containing 5 mmol/l of the sodium salt of taurocholic acid (TCA) in rats. The chronic erosive gastritis was characterized by mucosal erosions, reduction of mucosal thickness and reduction in the number of parietal cells per unit area, infiltration of inflammatory cells which were mainly lymphocytes and plasmocytes, and proliferation of collagenous fibers in the gastric mucosa. A standard meal including cimetidine 0.4 and 0.8%, which was administered ad libitum with TCA, reduced the total length of erosions, normalized the mucosal thickness and the number of parietal cells, and reduced inflammatory cell infiltration in the gastric mucosa. However, cimetidine did not show any effect on the proliferation of collagenous fibers in the interstitial space of the mucosa. The doses administered were 400 mg/kg/day and 800 mg/kg/day for 6 months. Cimetidine, thus, had a preventive effect on experimental chronic erosive gastritis in rats.

Cytoprotective effect of acetaminophen against taurocholate-induced damage to rat gastric monolayer cultures.

Acetaminophen has recently been reported to protect against drug damage to gastric mucosa in vivo. The present study tested acetaminophen protection in cultured rat gastric mucous cells against sodium taurocholate-induced damage and assessed the role of endogenous prostaglandins. Cell damage was assessed by phase-contrast microscopy and quantitated by Chromium-51 release assay which positively correlated with the trypan blue dye exclusion test (r = 0.98). The effect of acetaminophen on the production of PGE2 and 6-keto-prostaglandin F1a (6KF) was also studied. Sodium taurocholate caused cell death in a dose-dependent manner as indicated by increased 51Cr release. Preincubation with 5 mM acetaminophen significantly reduced 51Cr release caused by 5 mM sodium taurocholate, producing a 40% increase in cell survival. This cytoprotection was not blocked by indomethacin. PGE2 and 6KF of the media did not change after preincubation with nondamaging concentrations of acetaminophen or taurocholate. These results indicate that: (1) acetaminophen exerts a direct protective effect on gastric mucous cells cultured in vitro independent of indirect factors such as blood flow and (2) this protection is not associated with increased prostaglandin production.