Review

Regulated vesicle trafficking of membrane transporters in hepatic epithelia

Understanding the mechanisms of drug transport in the human lung is an important issue in pulmonary drug discovery and development. For this purpose, there is an increasing interest in immortalized lung cell lines as alternatives to primary cultured lung cells. We recently reported the protein expression in human lung tissues and pulmonary epithelial cells in primary culture, (Sakamoto A, Matsumaru T, Yamamura N, Uchida Y, Tachikawa M, Ohtsuki S, Terasaki T. 2013. J Pharm Sci 102(9):3395–3406) whereas comprehensive quantification of protein expressions in immortalized lung cell lines is sparse. Therefore, the aim of the present study was to clarify the drug transporter protein expression of five commercially available immortalized lung cell lines derived from tracheobronchial cells (Calu-3 and BEAS2-B), bronchiolar–alveolar cells (NCI-H292 and NCI-H441), and alveolar type II cells (A549), by liquid chromatography–tandem mass spectrometry-based approaches. Among transporters detected, breast cancer-resistance protein in Calu-3, NCI-H292, NCI-H441, and A549 and OCTN2 in BEAS2-B showed the highest protein expression. Compared with data from our previous study,(Sakamoto A, Matsumaru T, Yamamura N, Uchida Y, Tachikawa M, Ohtsuki S, Terasaki T. 2013. J Pharm Sci 102(9):3395–3406) NCI-H441 was the most similar with primary lung cells from all regions in terms of protein expression of organic cation/carnitine transporter 1 (OCTN1). In conclusion, the protein expression profiles of transporters in five immortalized lung cell lines were determined, and these findings may contribute to a better understanding of drug transport in immortalized lung cell lines. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Las enfermedades fibropoliquísticas congénitas del hígado son un conjunto de enfermedades que se caracterizan por un defecto en el proceso de crecimiento y maduración de las células que constituyen los conductos biliares (placa ductal). La expresión clínica de estas enfermedades es muy variada, y depende del nivel en el que se produce la malformación de la placa ductal. Así, la fibrosis hepática congénita, la enfermedad poliquística hepática, la enfermedad de Caroli y los quistes de colédoco representan formas diferentes de la alteración en el desarrollo de la placa ductal con manifestaciones clínicas muy diferentes entre sí. El diagnóstico y el tratamiento es variable en función de la forma de presentación. Es importante la evaluación de la presencia de quistes renales y del desarrollo de insuficiencia renal.

Congenital hepatic fibropolycystic diseases are a combination of diseases characterized by a defect in the growth and maturation process of the cells constituting the bile ducts (ductal plate). The clinical expression of these diseases varies greatly, and depends on the leve on which malformation of the ductal plate occurs. Thus, congenital hepatic fibrosis, hepatic polytheistic disease, Caroli disease and choledoch cysts represent different forms of the alteration in the development of the ductal plate, with very different clinical manifestations. Diagnoses and treatment varies based on the presentation form. Evaluation of the presence of retinal cysts and development of bread of failure is important.

Formation of bile ducts in culture is important for reconstructing hepatic organoids with bile drainage systems. However, morphogenic factors of biliary epithelial cells (BECs) have been poorly understood because of the lack of experimental models. Here, we demonstrated that rat BECs formed bile ductular networks in dynamic culture, when culture conditions were sequentially controlled. BEC morphogenesis was achieved through two-dimensional culture on collagen gel, collagen gel sandwich configuration, and 1% dimethylsulfoxide stimulation. In this culture system, BECs developed into large bile duct structures (LBDs) that formed interconnected networks of continuous lumens. LBD luminal surfaces possessed well developed microvilli, consisted of 7 to 10 BECs, and their inner diameters measured 20 to 50 μm. Quantitative PCR analysis revealed that the cells in LBDs expressed apical and basal domain markers of BECs. Immunofluorescent staining identified apical domain markers such as Cl⁻/HCO₃⁻ anion exchanger 2 and cystic fibrosis transmembrane regulator on the luminal surface of LBDs, responding to secretin stimulation as well as laminin protein surrounding LBDs. Furthermore, the cells in LBDs transported metabolized fluorescein from the basal side to the luminal space, further demonstrating that the reconstructed LBDs were functionally and morphologically similar to the bile ducts in vivo. The culture model described here will be useful in reconstructing hepatic tissues as well as in understanding the mechanism of bile duct development and its disruption in disease.

Background & Aims: In polycystic liver diseases (PCLDs), increased cholangiocyte proliferation and fluid secretion are key features and cholangiocyte adenosine 3′,5′-cyclic monophosphate (cAMP) is an important regulator of these processes. Thus, we assessed cAMP levels and evaluated octreotide (an analogue of somatostatin known to inhibit cAMP) in hepatic cyst growth using an in vitro model of cystogenesis and an in vivo animal model of autosomal recessive polycystic kidney disease (ARPKD), one of the PCLDs. Methods: Expression of somatostatin receptors (SSTRs) was assessed by reverse-transcription polymerase chain reaction and confocal microscopy in cholangiocytes from normal and polycystic kidney (PCK) rats, the ARPKD model of autosomal recessive polycystic kidney disease. Effects of octreotide on cAMP levels and cyst expansion were studied in vitro using PCK bile ducts grown in 3-dimensional culture. The effects of octreotide on hepatic and renal cystogenesis were investigated in PCK rats in vivo. Results: In cholangiocytes and serum of PCK rats, cAMP concentrations were ∼2 times higher than in normal rats. SSTR subtypes that bind octreotide (ie, SSTR2, SSTR3, and SSTR5) were expressed in both normal and PCK cholangiocytes. In vitro, octreotide inhibited cAMP levels by 35% and reduced cyst growth by 44%. In vivo, octreotide lowered cAMP content in cholangiocytes and serum by 32%–39% and inhibited hepatic disease progression, leading to 22%–60% reductions in liver weight, cyst volume, hepatic fibrosis, and mitotic indices. Similar effects were observed in kidneys of PCK rats. Conclusions: This preclinical study provides a strong rationale for assessing the potential value of octreotide in the treatment of PCLDs.

The apical membrane of the hepatocyte fulfils a unique function in the formation of primary bile. For all important biliary constituents a primary active transporter is present that extrudes or translocates its substrate toward the canalicular lumen. Most of these transporters are ATP-binding cassette (ABC) transporters. Two types of transporters can be recognized: those having endogenous metabolites as substrates (which could be referred to as “physiologic” transporters) and those involved in the elimination of drugs, toxins, and waste products. It should be emphasized that this distinction cannot be strictly made as some endogenous metabolites can be regarded as toxins as well. The importance of the canalicular transporters has been recognized by the pathologic consequence of their genetic defects. For each of the physiologic transporter genes an inherited disease has now been identified and most of these diseases have a quite serious clinical phenotype. Strikingly, complete defects in drug transporter function have not been recognized (yet) or only cause a mild phenotype. In this review we only briefly discuss the inherited defects in transporter function, and we focus on the pathophysiologic concepts that these diseases have generated.