Effect of bisphenol A on drug efflux in BeWo, a human trophoblast-like cell line.

Bisphenol A (BPA) is a monomer of polycarbonate plastics that has estrogenic activities and has been shown to be a substrate for multidrug resistant efflux mechanisms, specifically, P-glycoprotein. Since the natural hormone estrogen reverses multidrug resistance in some cell types, we hypothesized that BPA might have a similar activity in trophoblasts. We have used BeWo cells as an in vitro model for human trophoblasts and calcein AM as a substrate for drug efflux mechanism to characterize BPA interactions with placental P-glycoprotein. We found that chronic exposure of BeWo cells to BPA did not alter intracellular calcein accumulation in a fashion that would be reflective of changes in P-glycoprotein expression. Immunoblots affirmed that BPA had small effects on P-glycoprotein expression. However, BeWo cells acutely exposed to BPA pretreatment were observed to have a significantly decreased calcein accumulation. Addition of cyclosporin A, a P-glycoprotein inhibitor and substrate, completely reversed BPA's effects on calcein accumulation and resulted in a net increase, relative to controls, in calcein accumulation by the BeWo cells. BPA was found not to stimulate P-gp ATPase or alter intracellular esterases mediating calcein release from calcein AM. Therefore, our results suggested that BPA stimulated drug efflux by BeWo cells probably by direct effects on P-glycoprotein.

{beta}-Amyloid-induced neurodegeneration and protection by structurally diverse microtubule-stabilizing agents.

Deposition of beta-amyloid peptide (Abeta) and hyperphosphorylation of the tau protein are associated with neuronal dysfunction and cell death in Alzheimer's disease. Although the relationship between these two processes is not yet understood, studies have shown that both in vitro and in vivo exposure of neurons to Abeta leads to tau hyperphosphorylation and neuronal dystrophy. We previously reported that the microtubule-stabilizing drug paclitaxel (Taxol) protects primary neurons against toxicity induced by the Abeta(25-35) peptide. The studies in this report were undertaken to characterize the actions of paclitaxel more fully, to assess the effectiveness of structurally diverse microtubulestabilizing agents in protecting neurons, and to determine the time course of the protective effects of the drugs. Primary neurons were exposed to Abeta in the presence or absence of several agents shown to interact with microtubules, and neuronal survival was monitored. Paclitaxel protected neurons against Abeta(1-42) toxicity, and paclitaxel-treated cultures exposed to Abeta showed enhanced survival over Abeta-only cultures for several days. Neuronal apoptosis induced by Abeta was blocked by paclitaxel. Other taxanes and three structurally diverse microtubule-stabilizing compounds also significantly increased survival of Abeta-treated cultures. At concentrations below 100 nM, the drugs that protected the neurons did not produce detectable toxicity when added to the cultures alone. Although multiple mechanisms are likely to contribute to the neuronal cell death induced by oligomeric or fibrillar forms of Abeta, low concentrations of drugs that preserve the integrity of the cytoskeletal network may help neurons survive the toxic cascades initiated by these peptides.

Rapid optimization of the post-column fluorogenic ninhydrin reaction for the HPLC-based determination of bradykinin and related fragments.

A flow injection analysis scheme is demonstrated for the rapid optimization of reagent concentrations, flow rates, delay time and temperature using the guanidino moiety specific fluorogenic ninhydrin reaction. Using the amino acid arginine, non-arginine containing peptides, and the arginine-containing peptides, bradykinin and related fragments, specificity is demonstrated. These results serve to extend previous descriptions of the post-column reaction by offering a time efficient approach for the optimization of newly assembled post-column reactors using this chemistry. The reactor is subsequently added to a gradient elution HPLC system with the separation of bradykinin and bradykinin fragments demonstrated. Detection sensitivity in the high femtomole-low picomole mass range was achieved for these substances.

The presence of inducible cytochrome P450 types 1A1 and 1A2 in the BeWo cell line.

The activity and inducibility of cytochrome P450 systems (CYP1A1:1A2) of the human placenta were assessed in a representative human trophoblast-like cell line, BeWo. The activity of CYP1A1 and CYP1A2 in microsome preparations from human liver, placenta, primary cultures of human cytotrophoblast, and BeWo cells was measured by O -dealkylation of 7-ethoxyresorufin (EROD) and 7-methoxyresorufin O -demethylation (MROD), respectively. Results indicated high EROD and MROD activity associated with human liver microsomes, sometimes comparable activities in human placenta microsomes prepared from smokers, and relatively low activities in human placenta microsomes from nonsmokers and in the primary cultures of cytotrophoblasts isolated from nonsmokers. Microsomes from BeWo cell monolayers exhibited the lowest EROD and MROD activities relative to all other microsome preparations. However, compared to primary cultures of normal trophoblasts, the EROD activity of the BeWo cells was far more sensitive to typical inducers, 3-methylcholanthrene, 1,2-benzanthracene, and beta-naphthoflavone. EROD activity in BeWo cells was induced approximately 200-fold by 3-methylcholanthrene. Both EROD and MROD activity in BeWo cells was readily induced by 1,2-benzanthracene, 100-fold and 60-fold, respectively. After induction with 1,2-benzanthracene, the CYP1A1 selective inhibitor, alpha-naphthoflavone, and the CYP1A2 selective inhibitor, furafylline, effectively inhibited enzyme activities with IC(50)s of 2.4 microM and 12.8 microM, respectively, in microsomes from both trophoblasts culture systems. These results show that major cytochrome P450 forms present in human placenta are present and inducible in BeWo cells, a potential model for investigation of drug metabolism mechanisms in the human trophoblast.

Contribution of efflux pump activity to the delivery of pulmonary therapeutics.

To date, there are few in vitro models of the human lung that have been used to characterize multidrug resistant (MDR) efflux pump activity. It is expected that the presence of these protein transporter molecules, such as P-glycoprotein (Pgp) and the multidrug resistance protein associated protein-1 (MRP1), might play a role in limiting drug absorption through the pulmonary epithelium, as has been reported for other epithelial drug delivery barriers such as the intestine and brain. To date, the exact role of the lung resistance related protein (LRP) in MDR is unclear. In this article, we have summarized the biochemistry, function and in vitro/in vivo modulation of Pgp and MRP1. These topics are discussed in light of pulmonary delivery of therapeutic agents, with particular emphasis being placed on the bronchial region of human airways.

Progress and limitations in the use of in vitro cell cultures to serve as a permeability screen for the blood-brain barrier.

A relatively simple, widely applicable, and robust in vitro method of predicting blood-brain barrier (BBB) permeability to central nervous system-acting drugs is an increasing need. A cell-based model offers the potential to account for transcellular and paracellular drug diffusional processes, metabolism, and active transport processes, as well as nondefined interactions between a drug and cellular material that may impact upon a membrane's overall permeability profile. Any in vitro BBB cell model to be utilized for the transendothelial BBB permeability screening of potential central nervous system drugs must display reproducible solute permeability, and a number of other general criteria including: a restrictive paracellular barrier; a physiologically realistic cell architecture; the functional expression of key transporter mechanisms; and allow ease of culture to meet the technical and time constraints of a screening program. This article reviews the range of in vitro cell-based BBB models available, including the primary/low passage bovine and porcine brain endothelial cultures as well as the spectrum of immortalized brain endothelial cell lines that have been established. The article further discusses the benefits and limitations of exploiting such systems as in vitro BBB permeability screens.

Investigation of the metabolism of substance P at the blood-brain barrier using capillary electrophoresis with laser-induced fluorescence detection.

Substance P (SP) metabolism was investigated upon exposure to a monolayer of bovine brain microvessel endothelial cells (BBMECs), a cell culture model of the blood-brain barrier. SP was incubated with the BBMECs and its metabolism was followed as a function of time over a 5-h period. The resulting samples were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA)/cyanide, separated, and detected using cyclodextrin-modified electrokinetic chromatography with laser-induced fluorescence detection (CDMEKC-LIF). Upon exposure to the BBMEC monolayer, SP rapidly degraded to produce the N-terminal (1-9), (1-4) and (1-7) and C-terminal (2-11) and (3-11) fragments. These results were compared with those in an earlier report from our laboratory, where SP metabolism was investigated in vivo by microdialysis sampling in rat striatum.

Carrier-mediated transport of folic acid in BeWo cell monolayers as a model of the human trophoblast.

Using cultured BeWo cells as a model of human trophoblast, we investigated whether carrier-mediated transport of folic acid occurs. BeWo cells, which were derived from human choriocarcinoma, were cultured on a tissue culture plate or in a permeation chamber. When the cells reached confluence, drug uptake or transport experiments were performed. The uptake of [(3)H]folic acid by BeWo cells occurred at a much lower rate at 4 degrees C than at 37 degrees C. The uptake of [(3)H]folic acid was saturable at higher concentrations and inhibited by typical metabolic inhibitors, sodium azide and 2,4-dinitrophenol. The uptake of [(3)H]folic acid was significantly increased with decreasing pH of the incubation buffer and markedly inhibited by 4,4'-diidothiocyanostilbene-2,2'-disulfonic acid (DIDS). Analogs of folic acid, methotrexate and 5-methyltetrahydrofolate, inhibited the uptake of [(3)H]folic acid by BeWo cells. Kinetic analysis using Lineweaver-Burk plots revealed that methotrexate competitively inhibited the uptake of [(3)H]folic acid and folic acid competitively inhibited the uptake of [(3)H]methotrexate. In transport experiments, the permeation of [(3)H]folic acid from the apical-to-basal side was greater than that from the basal-to-apical side, and the transport of [(3)H]folic acid from the apical-to-basal side was inhibited by an excess of folic acid. The findings obtained in the present study confirm the existence of an asymmetric, carrier-mediated transport system for folic acid and its analog, methotrexate, across BeWo cells, a representative of the human trophoblast.

Modulation of P-glycoprotein activity in Calu-3 cells using steroids and beta-ligands.

The purpose of this work was to investigate if P-glycoprotein (Pgp) efflux pump activity could be inhibited in the sub-bronchial epithelial cell line, Calu-3, by glucocorticosteroids and beta-ligands. The Pgp modulation efficiency of each compound was determined by its ability to increase the accumulation of the Pgp substrate rhodamine 123 (Rh123) accumulation in these cells. Pgp inhibition was observed at > or =100 microM steroids and beta-ligand. The modulation effectiveness of the beta-ligands increased with increasing hydrophobicity (logP(octanol/aqueous)) whereas an obvious correlation was not obtained with the complete set of steroids tested. Steroidal Pgp substrates did not affect Rh123 accumulation (e.g. aldosterone, dexamethasone, 11beta,17alpha,21-OH progesterone). In contrast, two hydrophobic non-Pgp steroidal substrates (testosterone and progesterone) displayed different effects on Rh123 accumulation, with progesterone being the more potent modulator. The most hydrophobic beta-ligand, propranolol, a known Pgp substrate, gave the largest increase in Rh123 accumulation in this therapeutic class. The beta-ligand modulation efficiency could also be correlated to Pgp structural recognition elements such as hydrogen bonding potential, the presence of a basic nitrogen and planar aromatic ring. No effect on Rh123 accumulation was observed with the formulation additives tested (ethanol, glycerol and palmitoyl carnitine) at concentrations previously reported to be non-toxic to Calu-3 cells.

Multidrug resistance-associated protein-1 functional activity in Calu-3 cells.

The purpose of this work was to determine whether the in vitro bronchiolar epithelial cell model, Calu-3, possesses efflux pump activity by the multidrug resistance-associated protein-1 (MRP1). Reverse transcription-polymerase chain reaction demonstrated MRP1 gene expression in Calu-3 cells. Indirect fluorescence studies showed a basolateral membrane localization of MRP1 compared with P-glycoprotein (Pgp) that was found on the apical side of these cells. An increase in the rate of accumulation of the MRP1 substrate calcein was observed following treatment with the organic anion/MRP1 inhibitor indomethacin, the Pgp inhibitors cyclosporin A (CsA) and vinblastine, as well as conditions of energy depletion. Total calcein efflux was significantly decreased with the MRP1 inhibitors probenecid and indomethacin, while total efflux was unchanged following treatment with CsA. In the latter case, however, intracellular calcein levels postefflux were significantly greater. Probenecid and indomethacin increased calcein net secretion 2.4- and 3.5-fold, respectively. The efflux of etoposide, a known substrate for both Pgp and MRP1, was shown to be mainly Pgp-mediated by using the multidrug-resistant inhibitors quinidine (mixed Pgp/MRP1), CsA (Pgp), and MK571 (MRP1). Together, these data suggest that Calu-3 cells possess MRP1 functional activity that is subordinate to Pgp efflux. We present here kinetic analysis of calcein efflux from Calu-3 cells to support our findings.

Improving the selectivity of HAV-peptides in modulating E-cadherin-E-cadherin interactions in the intercellular junction of MDCK cell monolayers.

PURPOSE: The objective of this work is to understand the sequence specificity of HAV peptides and to improve their selectivity in regulating E-cadherin-E-cadherin interactions in the intercellular junctions. METHODS: Peptide 1 was modified using an alanine scanning method to give peptides 2-6. The ability of these peptides to modulate intercellular junctions was evaluated using Madin-Darby Canine Kidney (MDCK) cell monolayers on Transwell membranes from either the apical (AP) or the basolateral (BL) side. Modulation of the intercellular junctions was measured by the ability to lower the transepithelial electrical resistance (TEER) of MDCK monolayers and by the increase in mannitol flux. Molecular docking experiments were performed to model the binding properties of these peptides to the EC1 domain of E-cadherin. RESULTS: Peptides 5 (Ac-SHAVAS-NH2) and 6 (Ac-SHAVSA-NH2) were found to be more effective than the parent peptide 1 in decreasing the resistance of the cell monolayer. Furthermore, comparative studies with the control and the weak inhibitor peptide 2 indicate that peptide 5 displayed a significant increase in mannitol flux. Molecular docking of peptides 1, 2 and 5 to the EC1 domain suggests that peptide 5 has the lowest binding energy. CONCLUSIONS: HAV peptides have the ability to modulate E-cadherin-E-cadherin interactions in the intercellular junctions of the MDCK cell monolayer, thus indirectly increasing the permeability of the tight junctions. This observation indicates that residues flanking the HAV sequence are important in the binding selectivity of HAV peptides to E-cadherin. Molecular docking can further aid in the design of peptides with better selectivity to the EC1 domain of E-cadherin.

Nitric oxide and blood-brain barrier integrity.

The blood-brain barrier (BBB) is comprised of the endothelial cells that line the capillaries of the brain. The unique characteristics of this barrier include tight intercellular junctions, a complex glycocalyx, a paucity of pinocytic vesicles, and an absence of fenestra. These properties allow for the selective exchange of substances between the systemic circulation and the extracellular fluid compartment of the brain. It is well established that there are many conditions, including those mediated by nitric oxide (NO), that can lead to an opening of the BBB, eventually leading to vasogenic edema and secondary brain damage. The precise molecular mechanisms mediating NO-induced tissue injury and the breakdown of the BBB are complex and not completely understood. NO is a soluble, easily diffusible gas that is generated by NO synthase. Two of the isoforms of NO synthase are constitutive, calcium-dependent enzymes that modulate many physiological functions, including the regulation of smooth muscle contraction and blood flow. The third isoform is calcium-independent and inducible and can be stimulated by stress, inflammation, and infection. Under these conditions, NO can be generated in large quantities and has detrimental effects on the CNS. NO has been shown to increase permeability of the BBB, allowing substances to enter into the brain passively. This review considers the role of NO and BBB integrity.

Conjugation with L-Glutamate for in vivo brain drug delivery.

In vitro studies have shown that conjugation of a model compound [p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD)] with L-Glu can improve D-MOD permeation through the bovine brain microvessel endothelial cell monolayers (Sakaeda et al., 2000). The transport of this D-MOD-L-Glu conjugate is facilitated by the L-Glu transport system. In this paper, we evaluate the in vivo brain delivery of model compounds (i.e. D-MOD, p-nitro-D-phenylalanine (p-nitro-D-Phe), 5,7-dichlorokynurenic acid (DCKA) and D-kyotorphin) and their L-Glu conjugates. DCKA was also conjugated with L-Asp and L-Gln amino acids. The analgesic activities of D-kyotorphin and its L-Glu conjugate were also evaluated. The results showed that the brain-to-plasma concentration ratio of D-MOD-L-Glu was higher than the D-MOD alone; however, the plasma concentration of both compounds were the same. The plasma concentration of p-nitro-D-Phe-L-Glu conjugate was higher than the parent p-nitro-D-Phe; however, the brain-to-plasma concentration ratio of p-nitro-D-Phe was higher than its conjugate. On the other hand, both DCKA and DCKA conjugates have a low brain-to-plasma concentration ratio due to their inability to cross the blood-brain barrier (BBB). The L-Asp and L-Glu conjugates of DCKA have elevated plasma concentrations relative to DCKA; however, the DCKA-L-Gln conjugate has the same plasma concentration as DCKA. For D-kyotorphin, both the parent and the L-Glu conjugate showed similar analgesic activity. In conclusion, conjugation of a non-permeable drug with L-Glu may improve the drug's brain delivery; however, this improvement may depend on the physicochemical and receptor binding properties of the conjugate.

P-glycoprotein efflux pump expression and activity in Calu-3 cells.

The purpose of this work was to determine if the sub-bronchial epithelial cell model, Calu-3, expresses the functionally active P-glycoprotein (Pgp) efflux pump. Calu-3 cells express lower levels of Pgp than both Caco-2 and A549 cells as determined by Western Blot analysis. In Calu-3 cells, accumulation of the Pgp substrates rhodamine 123 (Rh123) and calcein acetoxymethyl ester (calcein-AM) was increased in the presence of the specific Pgp inhibitors cyclosporin A (CsA), vinblastine, and taxol. Significant inhibition of Pgp activity was not observed until after 2 h in both cell lines. The organic anion/multidrug resistance associated protein-1 (MRP1) inhibitors, probenecid and indomethacin, did not affect Rh123 accumulation, whereas an increase in calcein accumulation was observed by both agents. The metabolic inhibitor sodium azide decreased the efflux of Rh123 out of Calu-3 cells to the same degree as CsA, supporting inhibition of an active, efflux pathway. The basolateral-to-apical transport of Rh123 was significantly higher than that in the reverse direction, indicating a secretory pathway of efflux that was inhibited 25-fold by CsA. Basolateral-to-apical transport of Rh123 was inhibited slightly with both MRP1 inhibitors; however, no significant effect of Rh123 net secretion was observed. Mixed inhibitor studies demonstrated that Rh123 efflux was mainly Pgp mediated. These results support an energy-dependent Pgp efflux pump pathway that is sensitive to inhibition with CsA in Calu-3 cells.

Microparticulate uptake mechanisms of in-vitro cell culture models of the respiratory epithelium.

The objective of this study was to examine the uptake mechanisms of fluorescent polystyrene microspheres of various diameters and surface chemistry by two human cell lines derived from the respiratory epithelium, A549 and Calu-3. Briefly, A549 and Calu-3 cells were grown to confluence in 12-well cluster plates and the uptake of fluorescent microspheres by the cells was determined at various time points. The amount of microspheres internalized by the cells was determined by correcting for non-specific binding to the cell surface. The data showed that A549 cells appeared to have more phagocytic activity than Calu-3 cells. Albumin-coated microspheres as large as 3 microm diameter can be internalized by A549 cells. The amount of internalization by A549 cells observed for 0.5-microm diameter albumin-coated microspheres was approximately 10-times greater than that observed for 1-microm diameter spheres and approximately 100-times greater than values observed for 2- and 3-microm diameter beads. Transmission electron micrographs confirmed that the microspheres were internalized by the cells. Uptake experiments conducted with Calu-3 cells indicated that albumin-coated microspheres were neither bound nor internalized by the cells. The effect of microsphere surface chemistry on the uptake mechanism indicated that amidine microspheres were internalized more rapidly and to a greater extent by both A549 and Calu-3 cells than carboxylate microspheres and non-coated microspheres. This phenomenon is thought to be attributed to masking of the negative polystyrene core by the positive amidine functional group; this effect was less marked for the carboxylate microspheres. These results suggest that A549 and Calu-3 cells can internalize microspheres and that size and effective charge played an important role in the uptake process.

Characterization of the Calu-3 cell line as a tool to screen pulmonary drug delivery.

The objective of this research was to examine the human sub-bronchial gland cell line, Calu-3, and assess its potential as a metabolic and transport model to study drug delivery to the respiratory epithelium. The present studies were conducted using Calu-3 cells grown in Transwells(R) or in multiwell cluster plates. TEER values for Calu-3 monolayers were determined using the World Precision Instrument Voltohmmeter and STX-2 electrode. The results confirmed that Calu-3 cells form tight monolayers and give appreciable TEER values in culture when grown under air-interface conditions. Permeability data for small lipophilic molecules across Calu-3 monolayers suggested that the cell line is a suitable model to examine the transport of low molecular weight substances and xenobiotics. Calu-3 cells were also found to efflux FITC-transferrin (MW 80000) in a polarized manner. The metabolic capacity of Calu-3 cells was also examined. The P4501A1 and P4502B isozymes were determined to be functional, but not inducible, with fluorescent resorufin assays. The data indicated that the Calu-3 cell line may be useful for studying the contributions of bronchial epithelial cells to mechanisms of drug delivery at the respiratory epithelium.

Enhancement of transport of D-melphalan analogue by conjugation with L-glutamate across bovine brain microvessel endothelial cell monolayers.

In this paper, the L-glutamate (L-Glu) transport system was targeted to improve the delivery of a model compound, p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD), through the blood-brain barrier (BBB) in vitro cell culture model. D-MOD is an analogue of an antitumor agent D-melphalan. To target the L-Glu transport system, D-MOD was conjugated to L-Glu to give D-MOD-L-Glu conjugate. D-MOD and D-MOD-L-Glu transport properties were evaluated using the bovine brain microvessel endothelial cell (BBMEC) monolayers. The results suggest that D-MOD-L-Glu conjugate permeates through the BBMEC monolayers more readily than the parent D-MOD. The improvement of transport may be due to the recognition of D-MOD-L-Glu by the L-Glu transport system. The transport mechanism was evaluated using several different experiments including: (a) concentration-dependent studies; (b) temperature-dependent studies; (c) substrate inhibition studies; and (d) metabolic inhibitor studies. The D-MOD-L-Glu transport was inhibited by the change of temperature from 37 degrees C to 4 degrees C. At higher concentrations, the transport of D-MOD-L-Glu reached plateau due to saturation. Furthermore, some amino acids (i.e., L-Glu, L-Asp, D-Asp, and L-Gln) inhibited the transport of D-MOD-L-Glu; presumably the conjugate was competing with these amino acids for the same transport system. Metabolic inhibitors (i.e., 2,4-dinitrophenol and sodium azide) suppressed the transport of the conjugate. However, the conjugate was not transported by monocarboxylic acid, dipeptide and neutral amino acid transporters. In conclusion, the L-Glu transport system can be utilized to facilitate a non-permeable drug across the BBB by conjugating the drug with L-Glu amino acid.

Functional expression of P-glycoprotein in primary cultures of human cytotrophoblasts and BeWo cells.

The objective of this study was to investigate the functional expression of the efflux transporter, P-glycoprotein (P-gp), in primary cultures of human cytotrophoblasts and BeWo cell monolayers. Uptake studies with primary cultures of human cytotrophoblasts or BeWo cells were conducted with calcein-AM and vinblastine (P-gp markers) or fluorescein (MRP marker) in the presence of specific P-gp or MRP inhibitors. Results showed that the accumulation of P-gp substrates calcein-AM and vinblastine by BeWo cells or primary cultures of human cytotrophoblasts was significantly enhanced in the presence of a typical P-gp inhibitor, cyclosporin-A, or other inhibitors such as quinidine, verapamil, and dipyridamole. MRP inhibitors had no effect on the accumulation of calcein or fluorescein by BeWo cells. Western blots confirmed the presence of multidrug resistant gene product 1 (MDR1) in both primary cultures of human cytotrophoblasts and BeWo cells. This study demonstrates functional P-gp in term human trophoblasts and further supports the use of primary cultures of human cytotrophoblasts and BeWo cells as in vitro models of the trophoblast to investigate mechanisms regulating drug distribution across the placenta.

Fatty acid transport regulatory proteins in the developing rat placenta and in trophoblast cell culture models.

The placenta forms a selective barrier that is able to transport nutrients that are of critical use to the fetus. Delivery of essential fatty acids to the fetus is dependent upon transplacental transport and provides the backbone for the biosynthesis of biological membranes, myelin and various signalling molecules. The primary objective of this research was to elucidate the expression patterns of genes that regulate fatty acid transport across the placenta. Several fatty acid transport regulatory genes have been identified in the rat including; cytoplasmic heart fatty acid binding protein (hFABP), plasma membrane fatty acid binding protein (FABPpm), fatty acid translocase (FAT) and fatty acid transport protein (FATP). In this study, we have elucidated temporal and spatial expression patterns for these genes in the rat placenta and in cell culture models of the rat placenta by Northern blot, RT-PCR, Western blot and/or by in situ hybridization analyses. Expression of hFABP was specific to the labyrinth zone, the main barrier and site of transplacental transport in the rat placenta. In addition, the levels of hFABP expression increased with gestational age, suggesting a growing requirement for fatty acid transport with advancing stages of pregnancy. FABPpm, FAT and FATP are expressed in both the junctional and labyrinth zones of the rat placenta. FAT was predominantly localized to the labyrinth zone by in situ hybridization analysis. The placental cell expression patterns of the genes involved in fatty acid transport were supported by our observations of HRP-1 (labyrinth zone) and Rcho-1 (junctional zone) trophoblast cell culture models. Given their cell surface location, we predict that FABPpm, FAT and FATP potentially participate in placental fatty acid uptake. The predominant expression of hFABP and FAT in the labyrinth zone of the chorioallantoic placenta implicates hFABP and FAT in the transplacental movement of fatty acids from maternal to fetal compartments.

Regeneration and maintenance of bile canalicular networks in collagen-sandwiched hepatocytes.

The morphological and cytoskeletal reorganization of collagen-sandwiched rat hepatocytes during the de novo formation of complete canalicular networks was examined by phase, fluorescence and electron microscopy. During the initial stages of membrane repolarization, there was a marked accumulation of both microfilaments and microtubules at the sites of canalicular generation. Microtubule-disrupting agents (colchicine, nocodazole) inhibited the localization of actin filaments at cell margins and the initiation and branching of canalicular networks. After removal of microtubule-disrupting agents, microfilaments relocalized to the canalicular borders and microtubules nucleated along the margins of the bile canaliculi at sites distinct from the peri-canalicular actin networks. Microfilament-perturbing agents (cytochalasin D, phalloidin) did not affect the de novo initiation of bile canaliculi and only slightly impaired the development of canalicular lumina into networks. In established cultures with complete canalicular networks, subsequent treatment with microtubule-disrupting agents did not acutely affect the integrity of preformed canalicular networks. In contrast, treatment with microfilament-perturbing agents caused a marked dilation of most canaliculi. These results illustrate the differential role of the cytoskeleton in the regeneration and maintenance of bile canalicular networks by collagen-sandwiched hepatocytes. Moreover, this study shows the utility of this system as an in vitro model for examining the regulation of cell and membrane polarity.