The role of phosphoinositide 3-kinase in taurocholate-induced trafficking of ATP-dependent canalicular transporters in rat liver.

Recent studies indicate that wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3-kinase, interferes with bile acid secretion in rat liver; taurocholate induces recruitment of ATP-dependent transporters to the bile canalicular membrane, and PI 3-kinase products are important in intracellular trafficking. We investigated the role of PI 3-kinase in bile acid secretion by studying the in vivo effect of taurocholate, colchicine, and wortmannin on bile acid secretion, kinase activity, and protein levels in canalicular membrane vesicle (CMV) and sinusoidal membrane vesicle (SMV) fractions from rat liver. Treatment of rats or perfusion of isolated liver with taurocholate significantly increased PI 3-kinase activity in both membrane fractions. Taurocholate increased protein content of ATP-dependent transporters, which were detected only in CMVs, whereas increased levels of p85 and a cell adhesion molecule, cCAM 105, were observed in both fractions. Colchicine prevented taurocholate-induced changes in all proteins studied, as well as the increase in PI 3-kinase activity in CMVs, but it resulted in further accumulation of PI 3-kinase activity, p85, and cCAM 105 in SMVs. These results indicate that taurocholate-mediated changes involve a microtubular system. Wortmannin blocked taurocholate-induced bile acid secretion. The effect was more profound when wortmannin was administered prior to treatment with taurocholate. When wortmannin was given after taurocholate, the protein levels of each ATP-dependent transporter were maintained in CMVs, whereas the levels of p85 and cCAM decreased in both membrane fractions. Perfusion of liver with wortmannin before taurocholate administration blocked accumulation of all proteins studied in CMVs and SMVs. These results indicate that PI 3-kinase is required for intracellular trafficking of itself, as well as of ATP-dependent canalicular transporters.

Regulation and translocation of ATP-dependent apical membrane proteins in rat liver.

The bile canalicular membrane contains four specific ATP-dependent transport processes that are involved in secretion of bile acids, non-bile acid organic anions (mrp1), phospholipids (mdr2), and organic cations (mdr3). The aim of this study was to determine how the canalicular presence of these transport proteins is regulated. Canalicular membrane vesicles (CMV) were prepared from livers of rats treated with taurocholate (TC) and/or dibutyryl-adenosine 3',5'-cycle monophosphate (DBcAMP) with and without pretreatment with colchicine. Transport studies were performed with radiolabeled substrates. Changes in the relative amounts of transport proteins were determined by Western blots. Compared with controls, the specific activity of each of the transport processes was enhanced 1.5- and 3-fold with TC and DBcAMP treatments, respectively. Western blots revealed the same increases with mdr2 and mdr3. Pretreatment of rats with colchicine prevented these responses fully with TC treatment, whereas only partial prevention was obtained with DBcAMP treatment. Besides the ATP-dependent transporters, the relative specific activities of the canalicular membrane ectoenzyme markers, leucine aminopeptidase and gamma-glutamyltranspeptidase, were also affected the same way. These results suggest that TC and DBcAMP increase the specific activity of the canalicular ATP-dependent transport proteins and some canalicular membrane ectoenzymes by stimulating an increase in the relative amounts of these proteins in the membrane.

Ectonucleotidases, purine nucleoside transporter, and function of the bile canalicular plasma membrane of the hepatocyte.

Ectonucleotidases are enzymes that degrade extracellular nucleotides. Extracellular nucleotides (especially ATP) and their degradation products (particularly adenosine) have multiple effects on cell functions by acting through purinergic receptors. Adenosine nucleotides are present in bile, which suggests that hepatocytes may release nucleotides into the canaliculus where they are promptly degraded into adenosine by ecto-ATPase and 5'-nucleotidase, which have been identified in the canalicular plasma membrane. Adenosine is then transported into hepatocytes by a Na+-dependent nucleoside transporter that is present in the canalicular plasma membrane. Purification and molecular cloning of ecto-ATPase and other canalicular proteins are complicated by an abundant canalicular plasma membrane protein, cCAM 105. However, the recent cloning of an ecto-ATPase (apyrase) from potato tubers provides a new opportunity to identify the canalicular ecto-ATPase. The canalicular Na+-dependent purine nucleoside transporter has been cloned from rat liver. Study of its expression during development and other physiological circumstances suggests that the transporter may play an important role in maintaining hepatic purine levels that are essential for the liver to serve as a major source of purines for tissues (i.e., brain, muscle) that lack pathways for de novo purine biosynthesis.

Studies on fenestral contraction in rat liver endothelial cells in culture.

Liver endothelial cells possess fenestrae, which are pores supported by a cytoskeleton ring composed of actin and myosin. Fenestrae are dynamic structures that can contract or dilate, although the mechanism for this phenomenon remains to be elucidated. Staining of actin and/or of myosin permitted measurement of fenestral diameter and area in cultured rat liver endothelial cells using digitized video-intensified fluorescence microscopy with image analysis. Within 1 minute of incubation with 0.1 micromol/L serotonin, fenestral diameter and area decreased by 24 +/- 5% and 56 +/- 7%, respectively. Contraction of fenestrae by serotonin was inhibited by chelation of extracellular Ca2+ with EGTA and by addition of Ca2+ channel blockers, such as dilthiazem and verapamil. The response of fenestrae to serotonin was mimicked by addition of a Ca2+ ionophore, A23187. Serotonin inhibited cAMP production, had no effect on inositol phosphate production, and activated phospholipase A2, causing release of arachidonic acid. These results suggest that contraction of fenestrae is associated with Ca2+ influx. In response to 0.1 micromol/serotonin, intracellular Ca2+ levels increased within 3 to 5 seconds from 150 nmol/L to >400 nmol/l followed by rapid phosphorylation of the 20-kd subunit of myosin light chain; both events dependent on extracellular Ca2+.

ATP-dependent phosphatidylcholine translocation in rat liver canalicular plasma membrane vesicles.

Phosphatidylcholine (PC) translocation was studied in rat liver canalicular plasma membrane vesicles using a fluorescent PC analogue that permitted the quantitation of asymmetric PC distribution in the outer and inner leaflet of the vesicles. PC translocation to the outer leaflet of the canalicular membrane was stimulated by ATP and an ATP-regenerating system in a time and temperature-dependent manner resulting in 200 pmol PC translocated/mg protein per 30 min. A non-hydrolyzable ATP analogue did not support translocation. Translocating activity was observed with PC but not with phosphatidylethanolamine and was specific for inside-out oriented canalicular membrane vesicles. Addition of taurocholate (10 microM), a micelle-forming bile acid, enhanced ATP-dependent PC translocation 1.5 +/- 0.1-fold, whereas addition of taurodehydrocholate (10 microM), a non-micelle-forming bile acid, did not. These results indicate the presence of an ATP-dependent transporter that "flips" phosphatidylcholine from the inner to the outer leaflet of the rat bile canalicular plasma membrane from where it can become associated with bile acids in the canalicular lumen, thereby enhancing ATP-dependent flipping activity. Several lines of evidence suggest that the transporter is Mdr2 P-glycoprotein.

The role of thiols in ATP-dependent transport of S-(2,4-dinitrophenyl)glutathione by rat liver plasma membrane vesicles.

The effect of thiol/disulfide exchange on ATP-dependent S-(2,4-dinitrophenyl)glutathione (GS-DNP) transport was studied in sodium nitrate treated rat liver plasma membrane vesicles. Transport followed Michaelis-Menten kinetics with an apparent Km of 9.6 microM for GS-DNP and 124 microM for ATP. 5,5'-Dithiobis(2-nitrobenzoate) (DTNB) and N-ethylmaleimide (NEM) efficiently inactivated GS-DNP transport activity in a dose- and time-dependent manner. Half-maximal inactivation occurred in 10 min at 40 microM for DTNB and 550 microM for NEM. Inactivation by DTNB was reversed by dithiothreitol. S-(N-Ethyl)maleimyl glutathione and/or ATP-Mg2+, but neither S-(N-ethyl)maleimyl cysteinylglycine nor oxidized glutathione could protect transport activity from inactivation by NEM or cystamine. These results suggest that reactive thiols are located near the active site of the transporter and that S-alkyl and the gamma-glutamyl residues of glutathione are important for protection. Biological disulfides which were tested included cystine, oxidized glutathione, oxidized Coenzyme-A, oxidized lipoic acid, and oxidized lipoamide; cystamine was the most potent reversible inactivator. Molecular oxygen also inactivated transport activity, which was recovered on addition of dithiothreitol, suggesting intramolecular disulfide formation by vicinal thiols. We interpret these results to indicate that the ATP-dependent GS-DNP transporter contains two or more thiols which are necessary for the maintenance of transport activity. The reversible inactivation of the activity by biological disulfides suggests that the transporter may be regulated by thiol/disulfide exchange in vivo.

Impaired biliary excretion and whole body elimination of methylmercury in rats with congenital defect in biliary glutathione excretion.

Biliary excretion of methylmercury, a major route of elimination of this toxic compound, was less than 2% of control in Eisai hyperbilirubinemic (EHBR) rats, a mutant Sprague-Dawley strain with a defect in biliary excretion of a variety of organic anions, including glutathione S-conjugates and reduced glutathione (GSH). Biliary GSH excretion in EHBR rats was also < 2% of controls, confirming previous findings. Impaired biliary methylmercury and GSH excretion was not explained by decreased hepatic content of these compounds. Indeed, hepatic methylmercury and GSH concentrations in EHBR rats were actually double those of controls. To assess the significance of the impaired biliary excretion in the whole body elimination of the toxicant, 203Hg excretion was measured over a 17-day period after intraperitoneal administration of either 0.5 or 5 mumol/kg of 203Hg-methylmercury chloride. The results for the two doses were similar. Methylmercury was eliminated by a first order process; however, the biological half-line was significantly longer in the EHBR rats, 46 to 54 days versus 18 to 22 days. Fecal excretion was the main route of elimination in both control and mutant animals. At necropsy (17 days), 16% to 25% of the 203Hg dose was recovered in the liver of the EHBR rats, whereas livers of control animals contained less than 2%of the administered dose. These findings demonstrate the biliary excretion of methylmercury is markedly impaired in EHBR rats and is associated with a low biliary GSH excretion, providing support for the hypothesis that methylmercury is normally transported across the canalicular membrane by a GSH-dependent mechanism, and presumably as a GSH mercaptide (CH3Hg-SG).(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-specific inhibition by bile acids of adenosine triphosphate-dependent taurocholate transport in rat canalicular membrane vesicles.

The adenosine triphosphate (ATP)-dependent transport system is a major determinant of canalicular bile acid secretion. The system transports bile acids and neither organic cations nor non-bile acid organic anions, such as glucuronides or glutathione adducts. To define the structural specificity of the ATP-dependent system, the authors examined the ability of various bile acids to inhibit ATP-dependent taurocholate transport by rat liver canalicular membrane vesicles. Only bile acids with a negative charge inhibited transport, which was unaffected by side chain length. Conjugated, but not unconjugated, mono- and di-hydroxy bile acids inhibited transport. The presence of 7 alpha- and 12 alpha-hydroxylation also influenced inhibition of ATP-dependent taurocholate transport. Inhibition of transport by bile acids was kinetically competitive. These results suggest that the canalicular ATP-dependent bile acid transport system depends on bile acid side chain charge, conjugation, and hydroxylation.

Bile acid inhibition of P-glycoprotein-mediated transport in multidrug-resistant cells and rat liver canalicular membrane vesicles.

To study the effect of bile acids on P-glycoprotein-mediated drug transport, we performed experiments using multidrug resistant cells and rat canalicular membrane vesicles. Cellular accumulation and efflux of rhodamine 123 were measured in drug-resistant cells by means of computerized quantitative image analysis and fluorescence microscopy. ATP-dependent [3H]daunomycin transport was studied by means of rapid filtration in canalicular membrane vesicles prepared from normal rats. Doxorubicin-sensitive (PSI-2) and -resistant (PN1A) 3T3 cells and human-derived hepatocellular carcinoma doxorubicin-sensitive and -resistant cells were used. Taurochenodeoxycholate and glycochenodeoxycholate, taurolithocholate and ursodeoxycholate (50 to 200 mumol/L) inhibited rhodamine 123 and [3H]daunomycin transport in multidrug-resistant cells and canalicular membrane vesicles, respectively, whereas taurocholate, taurodeoxycholate and tauroursodeoxycholate did not. Primary and secondary unconjugated bile acids had no effect. These results reveal that taurolithocholate, taurochenodeoxycholate and glycochenodeoxycholate and ursodeoxycholate inhibit P-glycoprotein-mediated drug transport function in multidrug resistant cell lines and in canalicular membrane vesicles. These results suggest possible interaction between P-glycoprotein function and bile acids in cholestasis and after treatment of patients with ursodeoxycholic or chenodeoxycholic acid.

Dubin-Johnson-like syndrome in golden lion tamarins (Leontopithecus rosalia rosalia).

On routine blood screens, persistent conjugated hyperbilirubinemia was discovered in two groups of closely related adult female golden lion tamarins (Leontopithecus rosalia rosalia, n = 8). Bromosulfophthalein (BSP) retention tests were performed on four hyperbilirubinemic and three control tamarins. BSP excretion was delayed in hyperbilirubinemic tamarins as compared with controls. Grossly, liver of affected tamarins was dark brown to black, with a prominent reticulated pattern. Histologic examination revealed abundant intrahepatic pigment, primarily in a centrilobular and midzonal distribution. Most of the pigment did not react with Perls' Prussian blue method for iron, Hall's method for bilirubin, or the Armed Forces Institute of Pathology acid-fast method for lipofuscin but was positive with Fontana and lipofuscin-ferric ferricyanide reduction techniques. Liver from control golden lion tamarins had intrahepatocellular Perls' iron-positive pigment diffusely throughout the lobule with a small amount of Fontana method-positive pigment. Ultrastructurally, hepatocytes from a hyperbilirubinemic tamarin contained pleomorphic electron-dense structures within lysosomes. Transport studies demonstrated secretion of fluorescein isothiocyanate-labeled glycocholic acid, a fluorescent bile acid analog, into bile canaliculi and no secretion of carboxydichlorofluorescein diacetate, a non-bile acid organic anion, by liver from a hyperbilirubinemic tamarin. In contrast, control liver secreted carboxydichlorofluorescein diacetate readily into bile canaliculi. The clinicopathologic presentation of this syndrome in golden lion tamarins is similar to that described for Dubin-Johnson syndrome of human beings.

Two distinct mechanisms for bilirubin glucuronide transport by rat bile canalicular membrane vesicles. Demonstration of defective ATP-dependent transport in rats (TR-) with inherited conjugated hyperbilirubinemia.

Bilirubin is conjugated with glucuronic acid in hepatocytes and subsequently secreted in bile. The major conjugate is bilirubin diglucuronide. Using sealed vesicles which are primarily derived from the canalicular (CMV) and sinusoidal (SMV) membrane vesicle domains of the plasma membrane of hepatocytes, we demonstrated that bilirubin glucuronides are transported by CMV by both ATP- and membrane potential-dependent transport systems. In CMV from normal rats, these processes are additive. In CMV from TR- rats, which have an autosomal recessively inherited defect in biliary secretion of nonbile acid organic anions, ATP-dependent transport of bilirubin diglucuronide was absent whereas the membrane potential driven system was retained. Other canalicular ATP-dependent transport systems, which were previously described for organic cations and bile acids, are functionally retained in TR- rats. Our study indicates that bilirubin glucuronides are primarily secreted into the bile canaliculus by an ATP-dependent mechanism which is defective in an animal model of the human Dubin-Johnson syndrome.

Serotonin stimulates a Ca2+ permeant nonspecific cation channel in hepatic endothelial cells.

The contraction of hepatic endothelial cell fenestrae after exposure to serotonin is associated with an increase in intracellular Ca2+ which is derived from extracellular Ca2+, is inhibited by pertussis toxin and is not associated with activation of phosphoinositol turnover or cAMP. Using cell-attached and excised patches in primary cultures of rat hepatic endothelial cells, we identified a serotonin-activated channel with conductance of 26.4+/-2.3 pS. The channel was also permeant to Na+, K+ and Ca++ but not to anions. In cell-attached patch recordings, addition of serotonin to the bath significantly increased channel activity with Ca2+ or Na+ as the charge-carrying ions. This channel provides a mechanism whereby serotonin can raise the cytosolic Ca2+ concentration in hepatic endothelial cells.

Defective biliary excretion of epinephrine metabolites in mutant (TR-) rats: relation to the pathogenesis of black liver in the Dubin-Johnson syndrome and Corriedale sheep with an analogous excretory defect.

Dubin-Johnson patients, mutant Corriedale sheep and TR- and EHBR mutant rats have recessively inherited defective bile canalicular secretion of many nonbile acid organic anions. The human and ovine mutants have black livers and lysosomal pigment accumulation. The livers in TR- and EHBR mutant rats are not black, and sparse lysosomal pigment accumulation is seen. Previously, we postulated that the unidentified pigment in the Dubin-Johnson syndrome results from the accumulation of tyrosine, phenylalanine and tryptophan metabolites, such as metanephrine, which are normally secreted in bile as organic anions. We tested this hypothesis in TR- rats. 3H-epinephrine was injected intravenously; control rats secreted 2.80% +/- 0.52% of the injected dose in bile as compared with 0.19% +/- 0.07% in TR- rats. From 82% to 90% of biliary radioactivity was due to polar conjugates in control rats and mutant rats. TR- rats retained more of the injected dose in the liver, particularly in lysosomes, and secreted more in urine than did control rats. After feeding control and TR- rats for 4 mo with a rat chow diet supplemented with 4% tyrosine, tryptophan and phenylalanine, the liver did not become grossly black; however, histological and electron microscopic study revealed dense lysosomal pigment accumulation in TR- rats. Intraportal injection of metanephrine resulted in the appearance of black liver in TR- rats that persisted for at least 2 hr and was not associated with pigment accumulation by light or electron microscopic examination.(ABSTRACT TRUNCATED AT 250 WORDS)

A nucleoside transporter is functionally linked to ectonucleotidases in rat liver canalicular membrane.

Prevention of nucleoside loss in bile is physiologically desirable because hepatocytes are the main source of nucleosides for animal cells which lack de novo nucleoside biosynthesis. We have demonstrated a Na+ gradient-energized, concentrative nucleoside transport system in canalicular membrane vesicles (CMV) from rat liver by studying [3H]adenosine uptake using a rapid filtration technique. The Na(+)-dependent nucleoside transporter accepts purine, analogues of purine nucleosides and uridine; exhibits high affinity for adenosine (apparent Km, 14 microM); is not inhibited by nitrobenzylthioinosine or dipyridamole, and is present in CMV but not in rat liver sinusoidal membrane vesicles. Adenosine transport in right side-out CMV was substantially greater than with inside-out CMV. CMV also contain abundant ecto-ATPase and ecto-AMPase (5'-nucleotidase). These ectoenzymes were shown to degrade nucleotides into nucleosides which were conserved by the Na(+)-dependent nucleoside transport system.

ATP-dependent organic anion transport system in normal and TR- rat liver canalicular membranes.

Many non-bile acid organic anions enter hepatocytes, where they are converted into polar conjugates and preferentially secreted into bile. ATP-dependent and membrane potential-dependent transport of non-bile acid organic anions across the plasma membrane of rat hepatocytes was studied in canalicular (CMV) or sinusoidal membrane vesicles (SMV) using sulfobromophthalein (BSP), a representative non-bile acid organic anion. Temperature-dependent, osmotically sensitive, saturable, ATP-dependent, and unidirectional (inside to outside) BSP transport was observed in CMV but not in SMV. Apparent Michaelis constants for ATP and BSP were 0.26 mM and 31 microM, respectively. ATP-dependent BSP transport was inhibited by oxidized glutathione, dinitrophenyl-glutathione (GSDNP), BSP glutathione, and bilirubin diglucuronide but not by daunomycin, taurocholate, and reduced glutathione. Inhibition by GSDNP and bilirubin diglucuronide was competitive, with apparent inhibitor constants of 41 and 4.2 microM, respectively. CMV from normal rats demonstrated ATP-dependent and membrane potential-dependent BSP transport that was additive. CMV from TR- rats lacked ATP-dependent BSP transport but retained membrane potential-dependent transport of non-bile acid organic anions as well as ATP-dependent transport mechanisms for organic cations (i.e., daunomycin) and bile acids (i.e., taurocholate).

Erythrocyte membrane transport of glutathione conjugates and oxidized glutathione in the Dubin-Johnson syndrome and in rats with hereditary hyperbilirubinemia.

The Dubin-Johnson syndrome is manifested by conjugated hyperbilirubinemia and pigment accumulation in hepatocellular lysosomes. The TR-rat model is a phenotypic model of the Dubin-Johnson syndrome and is characterized by defective ATP-dependent transport of a group of nonbile acid organic anions, including glutathione-S-conjugates and oxidized glutathione, across the bile canaliculus. Similar ATP-dependent transport mechanisms have been described in erythrocytes. Intact erythrocytes and inverted erythrocyte membrane vesicles from Dubin-Johnson patients, TR-rats and appropriate controls were studied with regard to ATP-dependent transport of dinitrophenyl glutathione and oxidized glutathione. No significant differences were observed, indicating that the erythrocyte and canalicular ATP-dependent transporters for these substrates are functionally and potentially genetically distinct.

The function of Gp170, the multidrug-resistance gene product, in the brush border of rat intestinal mucosa.

Gp170 is a transmembrane glycoprotein that is overexpressed in multidrug-resistant tumor cells and is present in the apical plasma membrane domain of small intestinal mucosal cells. The function of Gp170 was studied in the small intestine of the rat. Jejunal and ileal brush border membrane vesicles, but not basolateral membrane vesicles, manifested adenosine triphosphate (ATP)-dependent transport of daunomycin, a substrate for Gp170, and contained a approximately 170-kilodalton protein that reacts with anti-Gp170 monoclonal antibody. Whereas ATP supported daunomycin transport, nonhydrolyzable ATP analogues were ineffective. ATP-dependent daunomycin transport by brush border vesicles was unidirectional (inside to outside) and temperature dependent and was blocked by Gp170 inhibitors but not by taurocholate or bromsulphalein glutathione. Studies using everted small intestine revealed transport of rhodamine 123, a Gp170 substrate, from the serosal surface through the mucosa and inhibition by Gp170 inhibitors. These results suggest that Gp170 in rat small intestinal brush border membrane vesicles is an ATP-dependent efflux pump responsible for the transport of Gp170 substrates into the small intestinal lumen. Gp170 may protect against exogenously derived potentially damaging hydrophobic cations and contribute to the rarity of small intestinal cancer in humans and many animals.