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To study the regulation of fenestrations by vascular endothelial growth factor in liver sinusoidal endothelial cells, SK Hepl cells were transfected with green fluorescence protein (GFP)-actin and GFP-caveolin-1. SK Hep 1 cells had pores; some of which appeared to be fenestrations (diameter 55 [+ or -] 28 nm, porosity 2.0 [+ or -] 1.4%), rudimentary sieve plates, bristlecoated micropinocytotic vesicles and expressed caveolin-1, yon Willebrand factor, vascular endothelial growth factor receptor-2, endothelial nitric oxide synthase and clathrin, but not CD31. There was avid uptake of formaldehyde serum albumin, consistent with endocytosis. Vascular endothelial growth factor caused an increase in porosity to 4.8 [+ or -] 2.6% (P < 0.01) and pore diameter to 104 [+ or -] 59 nm (P < 0.001). GFP-actin was expressed throughout the cells, whereas GFPcaveolin-1 had a punctate appearance; both responded to vascular endothelial growth factor by contraction toward the nucleus over hours in parallel with the formation of fenestrations. SK Hepl cells resemble liver sinusoidal endothelial cells, and the vascular endothelial growth factor-induced formation of fenestration-like pores is preceded by contraction of actin cytoskeleton and attached caveolin-1 toward the nucleus. sinusoidal endothelial cell; fenestration; liver

Progressive familial cholestasis (PFIC) 2 and benign recurrent intrahepatic cholestasis (BRIC) 2 are caused by mutations in the bile salt export pump (BSEP, ABCB 11) gene; however, their prognosis differs. PFIC2 progresses to cirrhosis and requires liver transplantation, whereas BRIC2 is clinically benign. To identify the molecular mechanism(s) responsible for the phenotypic differences, eight PFIC2 and two BRIC2 mutations were introduced in rat Bsep, which was transfected in MDCK II cells. Taurocholate transport activity, protein expression, and subcellular distribution of these mutant proteins were studied in a polarized MDCK II monolayer. The taurocholate transport activity was approximately half of the wild-type (WT) in BRIC2 mutants (A570T and R1050C), was substantially less in two PFIC2 mutants (D482G and E297G), and was almost abolished in six other PFIC2 mutants (K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X). Bsep protein expression levels correlated closely with transport activity, except for R1057X. The half-life of the D482G mutant was shorter than that of the WT (1.35 h vs. 3.49 h in the mature form). BRIC2 mutants and three PFIC mutants (D482G, E297G, and R1057X) were predominantly distributed in the apical membrane. The other PFIC2 mutants remained intracellular. The R1057X mutant protein was stably expressed and trafficked to the apical membrane, suggesting that the COOH-terminal tail is required for transport activity but not for correct targeting. In conclusion, taurocholate transport function was impaired in proportion to rapid degradation of Bsep protein in the mutants, which were aligned in the following order: A570T and R1050C > D482G > E297G > K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X. These results may explain the phenotypic difference between BRIC2 and PFIC2. bile salt export pump; bile secretion; cholestasis; ABCB 11; mutation

The aim of this study was to determine the role of N-linked glycosylation in protein stability, intracellular trafficking, and bile acid transport activity of the bile salt export pump [Bsep (ATP-binding cassette B11)]. Rat Bsep was fused with yellow fluorescent protein, and the following mutants, in which Asn residues of putative glycosylation sites ([Asn.sup.109], [Asn.sup.116], [Asn.sup.122], and [Asn.sup.125]) were sequentially replaced with Gln, were constructed by site-directed mutagenesis: single N109Q, double N109Q + N116Q, triple N109Q + N116Q + N122Q, and quadruple N109Q + N116Q + N122Q + N125Q. Immunoblot and glycosidase cleavage analysis demonstrated that each site was glycosylated. Removal of glycans decreased taurocholate transport activity as determined in polarized MDCK II cells. This decrease resulted from rapid decay of the mutant Bsep protein; biochemical half-lives were 3.76, 3.65, 3.24, 1.35, and 0.52 h in wild-type, single-mutant, double-mutant, triple-mutant, and quadruple-mutant cells, respectively. Wild-type and single-and double-mutant proteins were distributed exclusively along the apical membranes, whereas triple- and quadruple-mutant proteins remained intracellular. MG-132 but not bafilomycin [A.sub.1] extended the half-life, suggesting a role for the proteasome in Bsep degradation. To determine whether a specific glycosylation site or the number of glycans was critical for protein stability, we studied the protein expression of combinations of N-glycan-deficient mutants and observed that Bsep with one glycan was considerably unstable compared with Bsep harboring two or more glycans. In conclusion, at least two N-linked glycans are required for Bsep protein stability, intracellular trafficking, and function in the apical membrane. cholestasis; bile acid; bile secretion

Biliary excretion is the rate-limiting step in transfer of bilirubin, other organic anions, and xenobiotics across the liver. Multidrug resistance-associated protein 2 (Mrp2, Abcc2) is the major transporter for conjugated endo- and xenobiotic-conjugated compounds into bile. Hormones regulate bilirubin and xenobiotic secretion into bile, which have dimorphic differences. Therefore, we examined the possible role of sex steroids and growth hormone in the regulation of Mrp2. In ~8-wk-old rats, mRNA, transcriptional activity, and hepatic content of Mrp2 were selectively increased fourfold (P < 0.001) in females compared with males. In males, estrogens increased and testosterone decreased Mrp2 mRNA and protein, whereas no significant effect was measured in females, suggesting either a direct effect on the liver or an alteration in growth hormone secretory pattern. After hypophysectomy, Mrp2 mRNA was markedly reduced and the effects of estrogens and testosterone on Mrp2 were prevented, supporting the role of pituitary hormones in controlling Mrp2 expression. Mrp2 increased following growth hormone infusion in males. Mrp2 mRNA was decreased in growth hormone-deficient 'Little' mice. Growth hormone infusions in hypophysectomized rats partially restored Mrp2 levels, whereas thyroxine addition returned Mrp2 mRNA and protein to basal levels. Morphology as well as biochemical measurements demonstrated that Mrp2 was localized to the bile canaliculus in equal density in both genders, whereas hormone replacements increased Mrp2 in hypophysectomized animals. In cultured hepatocytes, thyroxine did not have an effect, but growth hormone alone and combined with thyroxine increased Mrp2 mRNA levels. In conclusion, Mrp2 levels are regulated by the combination of thyroxine and different growth hormone secretory patterns. estrogen; thyroid; glucocorticoids; bilirubin

Hepatocytes polarize by forming functionally distinct sinusoidal (basolateral) and canalicular (apical) plasma membrane domains. Two distinct routes are used for delivery of membrane proteins to the canaliculus. Proteins having glycosylphosphatidylinositol anchors or single transmembrane domains are targeted to the sinusoidal plasma membrane from where they transcytose to the canalicular domain. In contrast, apical ATP-binding-cassette (ABC) transporters, which are required for energy-dependent biliary secretion of bile acids (ABCB11), phospholipids (ABCB4), and nonbile acid organic anions (ABCC2), lack initial residence in the basolateral plasma membrane and traffic directly from Golgi membranes to the canalicular membrane. While investigating mechanisms of apical targeting in WIF-B9 cells, a polarized hepatic epithelial cell line, we observed that rab11a is required for canalicular formation. Knockdown of rab11a or overexpression of the rab11a-GDP locked form prevented canalicular formation as did overexpression of the myosin Vb motorless tail domain. In WIF-B9 cells, which lack bile canaliculi, apical ABC transporters colocalized with transcytotic membrane proteins in rab11a-containing endosomes and, unlike the transcytotic markers, did not distribute to the plasma membrane. We propose that polarization of hepatocytes (i.e., canalicular biogenesis) requires recruitment of rab11a and myosin Vb to intracellular membranes that contain apical ABC transporters and transcytoticf markers, permitting their targeting to the plasma membrane. In this model, polarization is initiated upon delivery of rab11a-myosin Vb-containing membranes to the surface, which causes plasma membrane at the site of delivery to differentiate into apical domain (bile canaliculus). hepatocyte polarization | plasma membrane segregation

Bile acid secretion induced by cAMP and taurocholate is associated with recruitment of several ATP binding cassette (ABC) transporters to the canalicular membrane. Taurocholate-mediated bile acid secretion and recruitment of ABC transporters are phosphatidylinositol 3-kinase (PI3K) dependent and require an intact microtubular apparatus. We examined mechanisms involved in cAMP-mediated bile acid secretion. Bile acid secretion induced by perfusion of rat liver with dibutyryl cAMP was blocked by colchicine and wortmannin, a PI3K inhibitor. Canalicular membrane vesicles isolated from cAMP-treated rats manifested increased ATP-dependent transport of taurocholate and PI3K activity that were reduced by prior in vivo administration of colchicine or wortmannin. Addition of a PI3K lipid product, phosphoinositide 3,4-bisphosphate, but not its isomer, phosphoinositide 4,5-bisphosphate, restored ATP-dependent taurocholate in these vesicles. Addition of a decapeptide that activates PI3K to canalicular membrane vesicles increased ATP-dependent transport above baseline activity. In contrast to effects induced by taurocholate, cAMP-stimulated intracellular trafficking of the canalicular ABC transporters was unaffected by wortmannin, and recruitment of multidrug resistance protein 2, but not bile salt excretory protein (bsep), was partially decreased by colchicine. These studies indicate that trafficking of bsep and other canalicular ABC transporters to the canalicular membrane in response to cAMP is independent of PI3K activity. In addition, PI3K lipid products are required for activation of bsep in the canalicular membrane. These observations prompt revision of current concepts regarding the role of cAMP and PI3K in intracellular trafficking, regulation of canalicular bsep, and bile acid secretion. ATP binding cassette transporters; bile acid secretion; bile salt export protein; multidrug resistance protein 2; phosphatidylinositol 3-kinase

Previous studies in rat bile canalicular membrane vesicles and WIF-B9 cells revealed that cAMP-induced trafficking of ATP-binding cassette (ABC) transporters to the canalicular membrane and their activation require phosphoinositide 3-kinase (PI3-K) products. In the present studies, canalicular secretion of fluorescein isothiocyanate-glycocholate in WIF-B9 cells was increased by cAMP and a decapeptide that enhances PI3-K activity; these effects were inhibited by wortmannin. To determine the mechanism(s) whereby cAMP activates PI3-K, we examined signal transduction pathways in WIF-B9 and COS-7 cells, cAMP activated PI3-K in both cell lines in a phosphotyrosine-independent manner. PI3-K activity increased in association with p110[beta] in both cell lines. The effect of cAMP was KT-5720 sensitive, suggesting involvement of protein kinase A. Expression of a dominant-negative [beta]-adrenergic receptor kinase COOH terminus ([beta]-ARKct), which blocks G[beta][gamma]/ signaling, decreased PI3-K activation in both cell lines, cAMP increased GTP-bound Ras in COS-7 but not WIF-B9 cells. Expression of dominant-negative Ras abolished cAMP-mediated PI3-K, which suggests that the effect is downstream of Ras and G[beta][gamma]. These data indicate that cAMP activates PI3-K in a cell type-specific manner and provide insight regarding mechanisms of PI3-K activation required for bile acid secretion. bile secretion; heterotrimeric G protein; G[beta][gamma]; protein kinase A; Ras

The rMRP3 gene was isolated from rat liver, which codes for a protein 1522 amino acids in length that shows extensive sequence similarity with MRP1 and MRP2. Northern blot analyses indicated that rMRP3 is expressed in lung and intestine of Sprague-Dawley rats as well as in liver of Eisai hyperbilirubinemic rats and TR- mutant rats, which are deficient in MRP2 expression. Immunoblot analysis and immunoconfocal microscopy indicate that rMRP3 is found in the canalicular membrane, suggesting that it may play a role in bile formation.

Crude vesicular fractions isolated from rat liver total microsomes were analyzed to determine the presence of adenosine triphosphate (ATP)-dependent canalicular transporters in transcytotic vessels. Analysis of the crude vesicular fractions that were isolated by sucrose gradient flotation indicated the concentration-dependent regulation of the specific activities of bile canalicular ATP-dependent transport proteins. Furthermore, the ATP-dependent transporters were localized in a distinct population of transport vesicles with different traffic patterns.

I. INTRODUCTION Plasma membranes from hepatocytes have three separate domains that maintain polarity of the cell and can be recognized morphologically (56, 222). They consist of a microvillous-rich sinusoidal membrane [...]