Introduction

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P-Glycoprotein (P-gp) is a member of the ATP-binding cassette superfamily of membrane transport proteins that mediates the vectorial movement across cell membranes of a wide range of physicochemically diverse solutes (Stouch and Gudmundsson, 2002). In humans, two P-gp-related genes have been cloned and subsequently termed MDR1 and MDR3 (for review, see Ambudkar et al., 1999). The MDR1/P-gp gene product is recognized in particular to actively efflux from a cell a diverse range of cytotoxic drugs, a characteristic that is an important facet in the multidrug resistant (MDR) cell phenotype. Current evidence suggests that the MDR3/P-gp gene product does not contribute to an MDR phenotype.

In rodents, three P-gp-related genes have been identified and designated mdr-1a, mdr-1b, and mdr-2. Sequencing of the mdr1a and mdr1b gene products has shown them to correspond to the human MDR1, and as expected both mdr-1 genes encode for distinct functional multidrug transporters (Endicott et al., 1991). Cloning of the cognate rodent mdr-2 gene has shown it to be homologous to the human MDR3 (Endicott et al., 1991).

The MDR1 and MDR3 gene products are constitutively expressed in normal tissues, however, the full scope of their biological functions in normal tissues remains to be fully determined. Among other sites the MDR3/P-gp has been localized to the biliary domain of the hepatocyte cell membrane where it functions as a phospholipid transporter, facilitating the selective translocation of phosphatidylcholine into the outer leaflet of the liver canalicular membrane (Deleuze et al., 1996). The MDR1/P-gp is present at a number of anatomical barriers where one of its main functions is to reduce the systemic exposure and specific tissue access of potentially harmful compounds (for review, see Schinkel, 1997).

The lung alveolar septa represent the gaseous exchange region of the lung and comprise mainly the thin cellular barriers of alveolar epithelium and the less restrictive pulmonary capillary endothelium. To facilitate efficient gaseous exchange the alveolar epithelium occupies a total surface area approximately 40-fold greater than that of the epithelium lining the lung conducting airways (from trachea to terminal bronchioles). This large alveolar epithelial surface is comprised essentially of two cell types, the squamous alveolar epithelial (AE) type I cell and the cuboidal AE type II cell. The latter AE type II cell is more numerous and undertakes a range of functions, including, among others, the synthesis and secretion of pulmonary surfactant. Although less abundant, the thin (<0.3 µm in its peripheral attenuated regions) but larger squamous AE type I cell constitutes approximately 95% of the total alveolar epithelial surface area (Crapo et al., 1982). Beyond serving as a cellular conduit for gaseous exchange it is increasingly clear that the AE type I cell, in effectively being the limiting barrier to solute movement between alveolar airspace and capillary blood, possesses the capacity for a wide range of functions, including solute transporter activity, ion transport and fluid homeostasis, and macromolecule uptake (for review, see Crandall and Matthay, 2001).

Given a spatial pattern of constitutive MDR-1/P-gp expression within barriers critical to xenobiotic exposure then on teological grounds the functional expression of MDR-1/P-gp (or its species equivalent) within alveolar epithelium should be anticipated. The aim of this research is to determine whether P-gp is functionally expressed within alveolar epithelium, and in particular within the alveolar type I epithelial cell that forms the limiting barrier to solute transport. The implications for localizing P-gp within this barrier would be significant to studies of fundamental respiratory cell biology as well as to further clarification of the nature of the barrier to xenobiotic transfer from alveolar airspace to pulmonary interstitium and capillary blood. In this study, we report the microanatomical immunolocalization of MDR1/mdr1 P-gp expression to alveolar epithelium within intact normal human and rat lung tissue, with clear localization evident on the luminal membranes of AE type I epithelium. Using a well characterized in vitro approach to study AE differentiation, namely, a rat primary culture model of AE cells (Dobbs et al., 1988; Cheek et al., 1989; Danto et al., 1992; Campbell et al., 1999), we went on to show the increased expression (confirmed by RT-PCR, Western blot, and immunoflow cytometry techniques) of mdr1/P-gp in these primary cultures as the AE culture model progressed toward an AE type I phenotype; freshly isolated rat AE type II cells seemed negative for mdr1/P-gp The functionality of P-gp within the AE primary cultures was established by a flow cytometric accumulation-retention assay using rhodamine-123 as substrate, and also by the polarized transport of vinblastine across confluent AE monolayers grown on semipermeable supports.

	Materials and Methods

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Immunohistochemical Localization of mdr-1/MDR-1 P-Glycoprotein in Rat and Human Alveolar Epithelium within Intact Normal Lung Tissue. Paraffin wax blocks of in vivo rat lung were prepared as described previously (Newman et al., 1999). Briefly, under terminal anesthesia lung tissue was fixed by a whole-body perfusion method, whereby the pulmonary vasculature was perfused with highly purified 1% monomeric 0.1 M phosphate-buffered glutaraldehyde (pH 7.4) (TAAB Laboratories Equipment Ltd., Berks, UK) for a total of 15 min at normal physiological hydrostatic pressure. After perfusion fixation, small specimens of peripheral lung tissue (approximately 1-5 mm³) were dissected from the whole fixed lungs in situ and immersed in the same fixative for a further 2 h. The tissue blocks were then embedded in paraffin wax using an automated tissue processor (Shanndon, Cheshire, UK).

Isolation and Culture of Primary Rat AE Cells. Male pathogen-free CD rats (120-180 g b.wt.) were used throughout and were bred and maintained at Cardiff University in controlled temperature and lighting under barrier conditions with access to food and water ad libitum. Animal procedures were conducted in compliance with the Animal (Scientific Procedures) Act 1986. Isolation of AE type II cells was undertaken following previously described approaches (Danto et al., 1992; Campbell et al., 1999). Briefly, lung tissue was enzymatically disaggregated using aqueous porcine elastase (2 units/ml) (Worthington Biochemicals, Freehold, NJ). Purification of AE type II cells was undertaken by density centrifugation (250g for 20 min) upon a discontinuous Percoll gradient (1.040 and 1.089 g/ml) (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK). AE type II cells enriched in the opaque band located at the interface of the discontinuous gradient were removed and plated onto a Petri dish containing DMEM (Invitrogen, Paisley, UK) for 1 h at 37°C to aid further purification. After differential attachment the cell suspension containing the isolated AE type II cells (purity >95% AE type II cells) was either plated onto tissue culture-treated plastic or semipermeable polycarbonate membranes (Transwells, 0.4-µm pore size; Corning Costar, High Wycombe, UK) at a seeding density of 0.9 × 10⁶ cells/cm². Cultures were maintained in a humidified atmosphere (5% CO₂, 95% air) with culture medium comprising DMEM supplemented with 10% fetal bovine serum, the antibiotics penicillin G (100 units/ml) and gentamicin (50 µg/ml), and with or without the addition of dexamethasone (0.1 µM) as indicated. Culture medium was replenished every 48 h. After seeding the AE type II cells were allowed to adhere and their purity reconfirmed at 40 h by tannic acid stain to detect the presence of lamellar bodies. Beyond this time in culture, the AE type II cells, when grown on a plastic substratum, acquire (over the subsequent 3 to 6 days) a phenotype more characteristic of the AE type I cell (Dobbs et al., 1988; Cheek et al., 1989; Danto et al., 1992; Campbell et al., 1999). However, parallels between in vivo and in vitro AE cell differentiation remain to be fully defined; hence, the term AE type I-like cell is often adopted to represent the in vitro-derived AE type I phenotype.

Cell Lines. Investigations also made use of various cell lines, including the human colon adenocarcinoma cell line Caco-2 (passage 35) and the human lung type II alveolar adenocarcinoma cell line A549 (passage 98-101), both obtained from European Collection of Animal Cell Cultures (Porton Down, UK). The Madin-Darby canine kidney (MDCK) epithelial cell line and its recombinant clone containing the human MDR-1/P-gp gene (MDCK-MDR1) were both kind gifts from Piet Borst (The Netherlands Cancer Institute, Amsterdam, The Netherlands). The human breast carcinoma cell line MCF-7 and the human nasopharyngeal carcinoma cell line KB3-1, both P-gp negative, and their respective P-gp-induced sublines MCF-7/ADR and KBV1 were all obtained as kind gifts from the Imperial Cancer Research Fund (London, UK). All cell lines were routinely cultured in DMEM supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin, and 100 U/ml penicillin G. The culture media for KBV1 and MCF-7/ADR cell lines were supplemented with vinblastine (1 µg/ml) and doxorubicin (0.1 µg/ml), respectively, to maintain the MDR phenotype. However, before experimentation these cells were taken through one round of subculture in the absence of cytotoxic agents.

RT-PCR Analysis for the Detection of mdr-1/mdr-2 P-gp mRNA in Rat Alveolar Epithelial Cells. Total RNA was harvested from freshly isolated rat AE type II cells, denoted as AE(0). Total RNA was also extracted from the isolated AE cells grown in primary culture to 60 h, AE(60); 120 h, AE(120); and 192 h, AE(192) postseeding. By the 120 and 192 h time points postseeding, the cells had adopted a characteristic AE type I-like phenotype. Total RNA was harvested using a commercial kit (Ultraspec RNA reagent; Biogenesis, Dorset, UK) following the manufacturer's protocol. For each variable, three or four separate samples from different isolations were collected. Quantification and purity assessment of the extracted RNA was carried using a GeneQuant pro RNA/DNA calculator (Pharmacia Biotech, Cambridge, UK). First-strand cDNA was synthesized using 500 ng of RNA that was initially reverse transcribed using 200 U of Moloney murine leukemia virus reverse transcriptase (Invitrogen). The reverse transcription reaction consisted of 10 pmol of random hexamers (pdN6; Amersham Biosciences UK, Ltd.), 10 mM dithiothreitol, 1 mM dNTPs, and 1 U/µl RNasin (20-µl total volume). Samples were heat denatured at 80°C for 4 min before the addition of reverse transcriptase, followed further by incubations at 25°C for 10 min, 42°C for 50 min to complete the reverse transcription process, and finally at 99°C for 2 min to inactivate the reverse transcriptase enzyme to terminate the whole reaction. The cDNA reaction product was either held at 4°C for immediate use or stored at 80°C until required.

P-gp Immunoblot Analysis in Rat Alveolar Epithelial Cells. Confluent cell monolayers of AE(192), A549, KBV-1, KB3-1, and MDCK-MDR1 were harvested in lysis buffer containing 50 mM Tris (pH 7.5), 1% Triton, 5 mM EGTA, 150 mM NaCl, and protease/phosphatase inhibitors and incubated on ice for 20 min. After this, the crude cell lysates were centrifuged in a microcentrifuge at 10,000g for 15 min and the cell supernatants subjected to immunoblot analysis. Total protein (equivalent to 30 µg for each sample) was loaded and resolved by 7.5% SDS-PAGE and then electroblotted to nitrocellulose (0.2-µm pore size) membrane (Schleicher & Schuell, Dassel, Germany). Rainbow prestained molecular weight markers (Amersham Biosciences UK, Ltd.) were concurrently run. For signal generation the membrane was incubated at room temperature first for 2 h with 5% nonfat-dried milk in Tris-buffered saline with 0.1% Tween 20 (TBST, 0.1 M, pH 7.4) and then for 16 h at 4°C with the monoclonal P-gp C219 antibody (ID Labs, Inc.) diluted 1:100 in TBST. After this, the membrane was washed three times in TBST and further incubated for 1 h at room temperature with HRP-conjugated anti-mouse IgG rabbit antibody (Dako, Cambridge, UK) diluted 1:6000 in TBST. The membrane was finally washed six times with TBST and the chemiluminescence signal generated (Super Signal Ultra; Pierce, Chester, UK) and recorded onto Hyperfilm ECL (Amersham Biosciences UK, Ltd.). Image acquisition and band quantitation were undertaken on GS-700 densitometer with Molecular Analyst software (Bio-Rad).

Flow Cytometric Immunofluorescence Assay for P-gp Expression in Rat Alveolar Epithelial Cells. Freshly isolated AE(0) cells and confluent monolayers of AE(192) cells and of select continuous cell lines were harvested by trypsin/EDTA disaggregation and washed with phosphate-buffered saline (PBS, 0.1 M, pH 7.4) containing 1% BSA (Sigma-Aldrich, Poole, Dorset, UK).

Retention and Accumulation of Rhodamine-123 in Rat Alveolar Epithelial Cells. To examine the functional expression of P-gp, the intracellular accumulation and efflux of the highly selective fluorescent P-gp substrate, rhodamine-123, were studied using a previously described method (Lee et al., 1994). In this particular assay, the inhibition of P-gp-mediated cell efflux leads to the increased cellular accumulation and retention of rhodamine-123. Briefly, confluent monolayers of AE(192) cells and of select continuous cell lines (harvested at 120-192 h postseeding) were rinsed with Ca²⁺- and Mg²⁺-free PBS, and the cells harvested by treatment with 0.05% trypsin and 0.02% EDTA for 2 min at 37°C. Cells were then suspended (5 × 10⁵ cells/ml) in DMEM (minus serum) containing rhodamine-123 (0.2 µg/ml) in the presence or absence of the P-gp competitive inhibitor verapamil (40 M). Cells were then incubated in the dark for 1 h at 37°C. After this, the cells were pelleted (100g centrifugation for 5 min), washed in PBS, and 50% of the cells then aliquoted for flow cytometric analysis representative of the accumulation phase of rhodamine-123. For the examination of rhodamine-123 efflux the remaining cells were resuspended in fresh DMEM (minus serum) either with or without verapamil (40 µM) but devoid of rhodamine-123. The cell efflux of the accumulated rhodamine-123 was then conducted over 2 h at 37°C, after which the cells were pelleted, washed in ice-cold PBS, and analyzed by FACScan flow cytometry. The fluorescence of rhodamine-123 was collected in a FL1 band pass filter. Each treatment was represented by at least six replicates, minimum of 4000 events was collected for each sample and each experiment repeated at least twice. Sample analysis gated the cell population to exclude cell debris.

Polarized Transepithelial Transport of Vinblastine across Monolayer Cultures of Rat Alveolar Epithelial Cells. Freshly isolated AE type II cells were seeded upon Transwell polycarbonate inserts (0.9 × 10⁶ cells/cm²) and cultured to 192 h postseeding AE(192) in the presence of dexamethasone. The MDR-1/P-gp-positive cells, Caco-2, and MDCK-MDR1 were also seeded onto Transwell inserts (both at a seeding density of 0.04 × 10⁶/cm²) and cultured to days 21 and 4 postseeding, respectively. The formation of restrictive monolayers was monitored for all cell types by microscopical examination and measurement of transepithelial electrical resistance (TEER) using an EVOM epithelial voltammeter (WPI, Sarasota, FL).

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Statistics. Results are presented as mean ± S.D unless otherwise stated. Statistical analysis was undertaken using either nonpaired Student's t test or analysis of variance with post hoc analysis by Duncan's multiple range test. Details are provided in figure legends. Statistical significance was at P < 0.05 unless otherwise stated.

	Results

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Immunohistochemical Localization of mdr-1/MDR-1 P-gp within Alveolar Epithelium of Normal Intact Lung Tissue. Paraffin wax sections (5-10 µm) of rat and human normal lung tissue were prepared and immunostained for P-gp using JSB-1 and C494 antibodies, both of which react specifically with the MDR-1 protein and not the MDR-3 protein but also have been shown previously to react with both rat and human species (Jette et al., 1993). Immunocytochemistry with C219 failed to provide any reproducible staining pattern even within positive control tissue.

View larger version (117K): [in this window] [in a new window]   Fig. 1.   Immunohistochemical staining for P-gp (JSB-1 antibody) in normal rat (a-c) and human (d and e) lung tissue. Paraffin wax sections (5 µm in thickness) were prepared and labeled with JSB-1 antibody, as described under Materials and Methods. In rat tissue, immunostaining for P-gp was observed across what appeared as the entire alveolar epithelial surface membrane, with staining clearly present at the apical surface of the type I alveolar epithelial cell (a, magnification, 67×; c, higher magnification, 100× image of the boxed region in a). b, serves as a positive control showing labeling for P-gp within bronchial epithelium (BE) (magnification, 67×). Similarly, in human tissue the squamous alveolar type I epithelium clearly showed P-gp immunostaining across its apical membrane surfaces (d, magnification, 67×; g and f, a higher magnification (100×) image of the boxed region in d). Specific staining for P-gp was observed in the ciliated cells of the lower bronchial epithelium (e). Negative control sections were run in parallel consisting of antibody omission and replacement of primary antibody with an isotypic IgG2a antibody; no staining was observed in any of these control sections. CP, capillary; AS, alveolar airspace.

mdr-1/P-gp mRNA in Primary Rat Alveolar Epithelial Cell Cultures. Total RNA was isolated from primary cultures of rat alveolar epithelial cells and analyzed by RT-PCR using previously published mdr-1a and mdr-1b and mdr-2 rat gene-specific primers (Table 1). The alveolar cultures were grown from seeding in either the presence or absence of dexamethasone (0.1 µM); the extensive literature base reporting the use of this primary cell system to study alveolar epithelial cell differentiation or alveolar epithelial solute transport varies in either the inclusion or exclusion of the above-mentioned glucocorticoid.

View larger version (53K): [in this window] [in a new window]   Fig. 2.   Agarose gel (loading of cDNA equivalent to 100 ng of RNA) of the RT-PCR analysis of total RNA obtained from primary rat AE cell culture using oligonucleotide primers shown in Table 1 for rat mdr1a and mdr1b and the control housekeeper gene GAPDH. Lanes 1 and 2, freshly isolated rat AE type II cells [AE(0)]. Lanes 3 and 4, AE(60) grown in the absence or presence of glucocorticoid, respectively. Lanes 5 and 6, AE(120) grown in the absence or presence of glucocorticoid, respectively. Lanes 7 and 8, AE(192) grown in the absence or presence of glucocorticoid, respectively. GAPDH levels are consistent throughout the samples. Transcript for mdr-2 was not detected in any of the AE samples. The vertical arrow shows direction of electrophoresis. Histogram shows band quantitation, where for each variable three or four separate samples from different isolations were collected. Statistical analyses by analysis of variance and Duncan's multiple range test, where + represents a statistical difference (P < 0.05) from all other mdr-1a signals, and where * represents a statistical difference (P < 0.05) from all other mdr-1b signals.

P-gp Protein Expression by Western Blot in Primary Rat Alveolar Epithelial Cell Cultures. Immunoblot analysis (Fig. 3) with the antibody C219 was used to confirm Pgp protein expression in the late AE cultures grown to 192 h postseeding in the absence (lane 1) or presence (lane 2) of 0.1 µM dexamethasone; little morphological difference was observed between cells grown in the presence or absence of the glucocorticoid.

View larger version (42K): [in this window] [in a new window]   Fig. 3.   Western blot showing expression of P-gp in cultured AE(192) cells and control cell lines. Expression of P-gp in the AE(192) cells is evident by a single band at 170 to 190 kDa, the appropriate molecular mass range for P-gp in both AE(192) cells cultured with glucocorticoid (lane 1) or without glucocorticoid (lane 2). Lanes 3 to 6, select cell lines: KB3-1 cells (lane 3); A549 cells (lane 4); KBV-1 cells (lane 5), and MDCK-MDR (lane 6). Each lane was loaded with 25 µg, separated by SDS-polyacrylamide gel electrophoresis (7.5%), blotted onto nitrocellulose membrane, and P-gp detected by C219 monoclonal antibody. Signal bleed is evident in lane 4 arising from the adjacent lane 5. The vertical arrow shows direction of electrophoresis.

P-gp Protein Expression by Immunoflow Cytometry in Primary Rat Alveolar Epithelial Cell Cultures. Further verification of P-gp protein expression in the late (192-h) primary cultures of AE cells was provided by immunoflow cytometry. Figure 4 shows some examples of the fluorescent distributions obtained by immunoflow cytometry for P-gp protein expression in the primary rat alveolar epithelial cells together with other select cell lines. The tabulated data in Fig. 4 show for each cell culture the ratio of the pooled median fluorescence intensities obtained with P-gp antibody compared with that obtained with an isotypic control antibody; ratios >1.0 indicate increasing evidence of P-gp protein expression.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Expression of P-glycoprotein by immunoflow cytometry. Figure shows some example fluorescence distributions for AE(192) cells grown in the presence of dexamethasone (+dex), and some other select cell lines. Each fluorescence distribution was obtained from collecting 10,000 events per sample (n = 4 samples for each cell) using a FACScan flow cytometer, and for each cell line compares the P-gp antibody (either C219 or MRK16) to that of an isotypic control antibody. The tabulated data in Fig. 4 shows for each cell culture the ratio of the pooled median fluorescence intensities obtained with P-gp antibody compared with that obtained with an isotypic control antibody; ratios >1.0 indicate increasing evidence of P-gp protein expression. Statistical analysis by nonpaired Student's t test.

Functional Expression of P-gp in Primary Rat Alveolar Epithelial Cell Cultures: Retention and Accumulation Assay of Rhodamine-123. A fluorescent based flow cytometry assay using the P-gp substrate rhodamine-123 was used as one of the techniques to confirm P-gp functionality in the AE cells.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Intracellular accumulation and retention of rhodamine-123. a and b, accumulation. Cells were incubated for 60 min at 37°C in media containing rhodamine-123 in the absence (untreated control) or presence of P-glycoprotein inhibitor verapamil at 40 µM. c and d, retention. Cells were then washed and rhodamine-123 efflux carried out over 120 min at 37°C in media not containing rhodamine-123 and in the absence (untreated control) or presence of 40 µM verapamil. Data are mean ± S.D. (n = 6) and presented as a percentage of untreated control cells. Statistical analysis by nonpaired Student's t test. *, indicates statistical difference (P < 0.05) compared with the respective untreated control.

Functional Expression of P-gp in Primary Rat Alveolar Epithelial Cell Cultures: Polarized Transport of Vinblastine. To provide a more ready assessment of the potential transepithelial transport barrier provided by P-gp within the cultured AE cells we examined the polarized (A-B and B-A) cell monolayer permeability to vinblastine in the presence of P-gp inhibitors verapamil or cyclosporin. For comparative purposes, in addition to monolayers of AE(192) cells we examined the polarized permeability of MDCK-MDR1 and Caco-2 cell monolayers to vinblastine.

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View larger version (18K): [in this window] [in a new window]   Fig. 6.   Polarized transport of vinblastine. Permeability of vinblastine across monolayers of AE(192) + dexamethasone (dex) cells (a), Caco-2 (b), and MDCK-MDR1 (c). Transport studies were undertaken in the A-B and B-A direction in the absence or presence of verapamil (40 µM) or cyclosporin (10 µM) added to both the apical and basal compartments. *, indicates statistical (P < 0.05) difference in the B-A direction compared with the respective A-B direction.

	Discussion

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mdr-1/MDR1 P-gp is constitutively expressed at a number of anatomical barriers separating "environment" from systemic blood, e.g., luminal membranes of intestinal enterocytes or of proximal tubule epithelial cells of the kidney, or systemic blood from certain tissues, e.g., luminal membranes of endothelial cells that form the blood-brain barrier (Schinkel et al., 1994), the blood-retinal barrier (Holash and Stewart 1993), and the blood-testes barrier (Holash et al., 1993). Such a spatial distribution of this efflux transporter has defined it as a functionally important element in reducing the systemic exposure and specific tissue access of potentially harmful xenobiotics (for review, see Schinkel, 1997). The lung is constantly exposed to inhaled xenobiotics from the immediate atmosphere. With expression of P-gp within the epithelium of the lung conducting airways already recognized (Lechapt-Zalcman et al., 1997), we sought in this investigation to use several different but complimentary experimental approaches to examine for functional P-gp expression within normal mammalian alveolar epithelium, which comprises the majority of the total lung epithelial surface area (~120-m² surface area for alveolar epithelium versus ~3 m² for the conducting airways). In particular, we sought to define whether P-gp expression was evident within the terminally differentiated squamous AE type I cell that constitutes the major part (>95%) of the total alveolar epithelial surface area, and the loss of which to chemical injury would be highly detrimental to the maintenance of the alveolar-capillary blood barrier.

The immunohistochemical studies we present in both rat and human lung tissue show clear staining for mdr-1/MDR-1 P-gp, respectively, along the alveolar type I epithelial membranes lining the alveolar airspace. Previous studies that have commented upon P-gp expression within the alveolar region are limited with a lack of definitive investigations examining the alveolar epithelium specifically. Using an in situ hybridization technique with probes against mdr-1b, Johannesson et al. (1997) noted positive staining in rat lung parenchyma, although they were unable to identify the cell type expressing the mdr-1b transcript. Using RT-PCR analysis of primary cultures of human lung epithelial cells (separated on the basis of size into ciliated epithelial cells >40 µm and alveolar epithelial cells <40 µm), Bagru et al. (1998) reported the presence of P-gp transcript in primary 7 day cultures of the <40-µm cell population. Using immunohistochemistry techniques with C219 antibody (that cross-reacts with both MDR-1 and MDR-3 P-gp), Cordon-Cardo et al. (1990) found P-gp to be undetectable within normal human lung alveolar tissue, while observing luminal staining in the bronchial epithelium, the latter finding in bronchial epithelium in agreement with the subsequent work of Lechapt-Zalcman et al. (1997).

Extending our investigations to examine P-gp functional expression within the cultured alveolar epithelial cell, we exploited a well characterized in vitro model system using rat primary culture of AE cells (Dobbs et al., 1988; Cheek et al., 1989; Danto et al., 1992; Campbell et al., 1999). Paralleling an in vivo process whereby the AE type II cell serves as a progenitor for, and differentiates into, the AE type I cell (for review, see Uhal, 1997), the isolation and primary culture of AE type II cells over a 5- to 8-day period on a substratum of tissue culture-treated plastic leads to the loss of the AE type II phenotype and acquisition of the morphology, and expression of biochemical markers, characteristic of the in vivo AE type I cell phenotype. When grown in the presence of low glucocorticoid concentrations (0.1 µM dexamethasone) such cultures not only adopt with time the well characterized squamous morphology but also generate a highly restrictive solute barrier with TEERs of between 1000 and 2000  · cm² (Cheek et al., 1989; Campbell et al., 1999).

With the above-described model system, we confirmed the expression of both mdr-1a and mdr-1b mRNA transcripts in the cultured cells as they develop toward the AE type-I-like phenotype, i.e., transcripts became evident between 60 and 120 h postseeding with an absence of both mdr-1a or -1b in the freshly isolated AE type II cells. The lack of P-gp protein in the freshly isolated AE type II cells was also observed by immunoflow cytometry (C219 antibody) and leads to the tentative conclusion that the absence of mdr-1 transcript or P-gp protein in fresh isolates of pure (>95% AE type II) AE type II cells is a true reflection of the in vivo cell phenotype in this species. Of more note with respect to the aim of the investigation was confirmation of expression of mdr-1 transcript and P-gp protein (immunoflow cytometry and Western blot) within the cultured AE cells as they progress in culture toward an AE type I-like phenotype at 120 to 192 h postseeding. The temporal nature of this change was apparent for both mdr-1a and mdr-1b transcripts and in both the  and + dexamethasone-treated cultures. Beyond this temporal pattern (a function of the differentiation status of the culture) a direct comparison between the  and + dexamethasone-treated cells revealed a glucocorticoid mdr-1-inductive effect (for the mdr-1b notable at 60 h, and for both mdr-1a and mdr-1b notable at 120 h postseeding). This inductive effect was not evident by the time the cells had reached 192 h postseeding. A number of reports exist noting dexamethasone induction of P-gp levels in liver, brain, and intestinal tissue and also in lung tissue (Demeule et al., 1999), an effect which seems to be glucocorticoid concentration-dependent. The above-mentioned RT-PCR data would not be inconsistent with low-level glucocorticoid induction of cellular mdr-1 transcript levels against a background where the changing phenotype of the cell itself is also leading to increased mdr-1 transcript. Nevertheless, whether the alveolar epithelial cells were grown in the presence or absence of glucocorticoid the AE type I-like cells at 120 and 192 h express both mdr-1a and 1b and P-gp protein, whereas the freshly isolated AE type II progenitor cells, and the AE cells in early primary culture at 60 h, lack both the protein and transcript.

The functionality of P-gp within the AE type I-like cells (192) cells was established by a flow cytometric accumulation-retention assay using rhodamine-123 as substrate, and also by the polarized transport of vinblastine across confluent AE monolayers grown on semipermeable supports. The studies confirmed a basal to apical efflux mechanism present within the AE type I-like (192) cells that presents a transport barrier that can be considered, within the limits of this study design, to be comparable with that observed in the well characterized Caco-2 cell model.

The functional studies provide further support for the supposition that P-gp expressed in alveolar type I epithelium can serve as an efflux pump available to extrude potentially harmful inhaled xenobiotics and environmental pollutants from the alveolar epithelial cell back into the alveolar airspace away from the systemic circulation. Beyond this fundamental protective role, however, other speculative physiological functions of P-gp within the alveolar airspace may be considered. For example, MDR-1 or mdr-1a P-gp seems able to positively regulate cell volume-dependent chloride ion channel activity (for review, see Higgins, 1995), although P-gp itself lacks intrinsic chloride ion transport function. This modulatory role could subserve homeostatic volume regulation within the alveolar epithelium and help to maintain efficient respiratory gaseous exchange. Some evidence suggests that phosphatidylcholine, among other phospholipids, behaves as substrate for MDR1/P-gp (Bosch et al., 1997; Abulrob et al., 1999). The alveolar epithelial type I cell through P-gp functionality may fulfill a role in alveolar surfactant phospholipid homeostasis.

From a pharmacological perspective, the presence of P-gp within alveolar epithelium is of note given the current intense interest in exploiting the inhaled pulmonary route of drug delivery to gain increased systemic absorption for a range of therapeutic entities, from proteins and peptides (for review, see Patton, 1998) to, for example, inhaled opiate analgesics (Ward et al., 1997). With anatomical determinants and pharmacokinetic data supporting the view that deep penetration of therapeutic aerosols leads to improved systemic bioavailability, particularly for biotechnology products, it is natural that the alveolar epithelium is considered an appropriate absorption surface to target. In the knowledge that peptides and peptide-like drugs (Sharom et al., 1996), opiates (Wandel et al., 2002), and a wide range of structurally diverse molecules can serve as P-gp substrates, the finding of functional P-gp expression with alveolar epithelium provides a further possible mechanism that may limit the systemic absorption of inhaled products or indeed lead to nonlinear absorption pharmacokinetics for pulmonary-administered products.

In summary, we have demonstrated expression of P-gp within alveolar type I epithelium from normal human and rat lung tissue. Using a well characterized rat primary alveolar epithelial cell culture model, we observed mdr-1 transcript and P-gp protein expression within the cultured alveolar type I cell phenotype. The P-gp expressed was functional and supports a role for MDR-1/mdr-1 as an element in mediating the reduced alveolar airspace to epithelium, and alveolar airspace-to-blood exposure of potentially harmful xenobiotics. Given the number of possible physiological functions that P-gp may serve within normal alveolar epithelium a critical evaluation of lung physiology and biochemistry within P-gp knockout mice is warranted and would extend to the examination of alveolar fluid composition and determination of possible underlying pathology compared with their wild type counterparts. Such studies would ultimately improve our knowledge of the exact physiological function of P-gp in normal alveolar epithelium and how these may change under pathological conditions.