Regular Article

Enhancing Effect of Poly(amino acid)s on Albumin Uptakein Human Lung Epithelial A549 Cells

Ryoko YUMOTO, Sayuri SUZUKA, Saori NISHIMOTO, Junya NAGAI and Mikihisa TAKANO*Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical & Health Sciences,

Hiroshima University, Hiroshima, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: The clearance of albumin from the alveolar space is a critical process in the recovery fromedema. In this study, we investigated the effect of poly(amino acid)s such as poly-L-ornithine (PLO) onalbumin uptake in the cultured lung epithelial cell line A549. FITC-albumin uptake as well as cell surfacebinding was markedly stimulated by co-incubation with PLO, and there was a good correlation betweenthem. After being taken up by A549 cells, FITC-albumin was predominantly targeted to lysosomes.Interestingly, pretreatment of A549 cells with PLO further stimulated FITC-albumin uptake, even in theabsence of PLO in the uptake buffer. FITC-albumin uptake in the presence of PLO was inhibited by ametabolic inhibitor, clathrin-mediated endocytosis inhibitors, and a macropinocytosis inhibitor, indicatingthe involvement of clathrin-mediated endocytosis and/or macropinocytosis. The effect of PLO on FITC-albumin clearance was also examined in an in vivo pulmonary administration method in rats, and co-administration of PLO enhanced fluorescence elimination from the lungs. These findings suggest thatpulmonary administration of poly(amino acid)s such as PLO is a possible strategy for enhancing albuminclearance from the alveolar space, and thereby facilitating the recovery from pulmonary edema.

Keywords: pulmonary edema; alveolar epithelial cells; albumin clearance; poly(amino acid)s;endocytosis; in vivo pulmonary administration

Introduction

Alveolar lining fluid contains various proteins such as albumin,immunoglobulin G, IgA, and transferrin that are important forphysiological and protective functions.1–3) The concentration ofalbumin is normally about 5mg/mL in alveolar fluid and ismuch lower than that in the plasma (40mg/mL). However, theconcentration can increase to 40–65% of the plasma level inhydrostatic pulmonary edema and to 75–95% in lung injurypulmonary edema. Alveolar clearance of proteins is a criticalprocess in the recovery from pulmonary edema as well as inmaintaining the normal alveolar milieu. The inability to clearexcess proteins from the alveolar space results in poor prognosis inpatients suffering from pulmonary diseases, because of inefficientclearance of edema fluid due to increased oncotic pressure.2–4)

It was reported that patients dying with acute lung injury and acuterespiratory distress syndrome (ARDS) had large quantities ofinsoluble protein in their air spaces, and non-survivors of lunginjury after ARDS had three times as much protein in their alveolias survivors.3,5,6) Therefore, understanding protein transport mecha-

nisms in alveolar epithelial cells and the development of strategiesto enhance the protein clearance would be important for the earlyrecovery from the lung injury accompanied by edema.

The alveolar region of the lung is lined with a continuousepithelium comprising two major types of epithelial cells, type Iand type II. Type I epithelial cells have a squamous morphologyand cover 90–95% of the alveolar surface area. Type II cells arecuboidal epithelial cells and cover 5–10% of the surface area,although the number of type II cells in alveolar epithelia is similarto or more than that of type I cells. Type II cells also serve asprogenitors of type I cells, and transdifferentiate into type I cells torepair the alveolar epithelium when it is injured.7–9) We have beenstudying the transport mechanisms of albumin in alveolar epithelialcells, and have shown that albumin transport activity was muchhigher in primary cultured alveolar type II cells than that in type I-like cells.10) In addition, the contribution of type II cells for theclearance of albumin from the alveolar space was supposed to bemuch higher (>75%) than expected from their small surface area inthe alveoli. Thus, type II cells may play a principal role in albuminclearance from the alveolar lining fluid.

Received March 1, 2013; Accepted May 16, 2013J-STAGE Advance Published Date: May 28, 2013, doi:10.2133/dmpk.DMPK-13-RG-028*To whom correspondence should be addressed: Mikihisa TAKANO, Ph.D., Department of Pharmaceutics and Therapeutics, Graduate School ofBiomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Tel. ©81-82-257-5315, Fax. ©81-82-257-5319, E-mail: takanom@hiroshima-u.ac.jpThis work was supported in part by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (JSPS).

Drug Metab. Pharmacokinet. 28 (6): 497–503 (2013). Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)

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A549 is an epithelial cell line derived from human lung carci-noma, and has been widely used as an in vitro model of humanalveolar type II epithelial cells for biochemical and toxicologicalstudies.11–13) Using this human cell line, we recently reportedthe characteristics of albumin uptake.14) Like albumin uptake inprimary cultured rat alveolar type II cells as well as in the RLE-6TN cell line derived from the rat lung,10,15,16) albumin uptake inA549 cells was mediated predominantly by the clathrin-mediatedendocytic pathway. Therefore, albumin may be transported bya similar mechanism and/or pathway in rat and human alveolarepithelial type II cells.

As for a strategy to enhance peptide and protein transport inalveolar epithelial cells, we recently found that cationic poly(aminoacid)s such as poly-L-ornithine (PLO) and poly-L-lysine (PLL)were effective in stimulating insulin uptake in RLE-6TN cells.17)

The effect of PLO was also observed in in vivo pulmonary admin-istration, and co-administration of PLO with insulin enhancedthe hypoglycemic action of insulin. Such a stimulatory effect ofPLO on insulin uptake may be partly due to the charge inter-action between negatively charged insulin and positively chargedPLO, and between insulin-PLO complex and cell surface negativecharge, though the details for the enhancing mechanisms need tobe explored further.

Because albumin is also a negatively charged molecule,poly(amino acid)s may be effective in enhancing albumin uptakein alveolar epithelial cells. In this study, we aimed to clarify theeffect of poly(amino acid)s such as PLO on FITC-albumin uptakein cultured alveolar epithelial cell line A549. In addition, the effectof PLO on in vivo FITC-albumin clearance from the lungs wasexamined in rats.

Materials and Methods

Materials: Fetal bovine serum (FBS) and Dulbecco’s modi-fied Eagle medium (DMEM) were purchased from MP Bio-medicals (Solon, OH). Trypsin-EDTA, penicillin-streptomycin,LysoTracker Red, and Hoechst 33342 were purchased from LifeTechnologies (Carlsbad, CA). FITC-labeled bovine serum albumin(FITC-albumin), poly-L-ornithine (PLO, MW = 15,000–30,000),and poly-L-lysine (PLL, MW = 15,000–30,000), phenylarsineoxide, indomethacin, nystatin, and 5-(N-ethyl-N-isopropyl) ami-loride were purchased from Sigma-Aldrich (St. Louis, MO).Chlorpromazine and 2,4-dinitrophenol were purchased fromNacalai Tesque (Kyoto, Japan). All other chemicals used for theexperiments were of the highest purity commercially available.

Cell culture: A549 cells were provided by the RIKEN BRCthrough the National Bio-Resource Project of the MEXT, Japan.Cells were cultured in DMEM containing 10% FBS, 100 IU/mLpenicillin, and 100mg/mL streptomycin, in an atmosphere of 5%CO2–95% air at 37°C, and subcultured every 7 days using 0.25%trypsin and 1mM EDTA. A549 cells were used between passages96 and 112. The medium was replaced every 2 or 3 days, and thecells were used for the experiments on the sixth day after seeding.

Animals: Male Wistar rats (seven weeks old) weighingapproximately 210 g were anaesthetized with pentobarbital (30mg/kg) by intraperitoneal injection before the experiments. Theexperiments with animals were performed in accordance withthe Guideline for the Committee of Animal Experimentation,Hiroshima University, and the Committee of Research Facilities forLaboratory Animal Science, Natural Science Center for BasicResearch and Development (N-BARD), Hiroshima University.

Uptake studies: Uptake experiments were performed asdescribed previously.14–17) A549 cells grown on 12-well cultureplates were used. After removal of the culture medium, each wellwas washed and preincubated with 1mL of phosphate-bufferedsaline (PBS buffer; 137mM NaCl, 3mM KCl, 8mM Na2HPO4,1.5mM KH2PO4, 0.1mM CaCl2, and 0.5mM MgCl2, pH 7.4)supplemented with 5mM D-glucose (PBS-G buffer) at 37°Cor 4°C for 10min. Then, 0.5mL of PBS-G buffer containing FITC-albumin (20 µg/mL or 20mg/mL) with or without various con-centrations of PLO or PLL was added to each well, and the cellswere incubated at 37°C or 4°C for a specified period.

To evaluate the effect of pretreatment of PLO on albuminuptake, A549 cells were preincubated for 10min with PBS-Gbuffer in the absence or presence of PLO (80 µg/mL), and thenincubated with FITC-albumin (20mg/mL) for 60min in theabsence or presence of PLO (80µg/mL) at 37°C or 4°C. Toevaluate the effect of PLO addition in the middle of incubation,A549 cells were incubated with FITC-albumin (20mg/mL) for30min after preincubation with PBS-G buffer for 10min, andthen PLO (80 µg/mL) or PBS-G buffer was added to the incubationbuffer followed by incubation for 30min at 37°C or 4°C. Forinhibition studies, A549 cells were preincubated for 10min withPBS or PBS-G buffer with or without the inhibitor as follows:1mM 2,4-dinitrophenol in PBS buffer, chlorpromazine (35–140 µM) and indomethacin (100–300 µM) in PBS-G buffer, phen-ylarsine oxide (5–30µM), 5-(N-ethyl-N-isopropyl) amiloride (25–75µM) and nystatin (15, 30 µM) in PBS-G buffer containing 0.5%DMSO. The same vehicles were used for each control experiment.After removal of the preincubation buffer, the cells were incubatedwith 0.5mL of the uptake buffer containing FITC-albumin (20mg/mL) and PLO (80µg/mL) in the absence or presence of theinhibitors at 37°C or 4°C for 60min. Phenylarsine oxide was usedonly in the preincubation buffer.

At the end of the incubation, the uptake buffer was aspiratedand the cells were washed rapidly three times with 1mL of ice-coldPBS buffer. The cells were scraped with a rubber policeman into0.75mL of ice-cold PBS buffer and the wells were rinsed againwith 0.75mL of ice-cold PBS buffer to improve the recovery ofthe cells. The cells were further washed by centrifugation at 4°Cfor 3min at 9,838©g twice. After the supernatant was aspirated,the pellet was solubilized in 1mL of 0.1% Triton X-100 in PBSbuffer without CaCl2 or MgCl2 at room temperature for 30min, andcentrifuged for 3min at 5,600©g. The supernatant was used forfluorescence and protein assays.

The amount of FITC-albumin taken up by A549 cells wasmeasured using a Hitachi fluorescence spectrophotometer F-2700(Tokyo, Japan) at an excitation wavelength of 500 nm and anemission wavelength of 520 nm. Protein was determined by theLowry method with bovine serum albumin as the standard.

Confocal laser scanning microscopy: A549 cells weregrown on 35-mm glass bottom culture dishes for 6 days. Thecells were incubated with FITC-albumin (20mg/mL), LysoTrackerRed (75 nM), and Hoechst 33342 (10 µM) in the presence orabsence of PLO (80 µg/mL) for 60min at 37°C, and after washingthe cells with ice-cold PBS buffer three times for 5min each,florescence in the cells was visualized by confocal laser scanningmicroscopy (LSM5 Pascal, Carl Zeiss, Germany).

In vivo pulmonary administration of FITC-albumin: Pul-monary administration of FITC-albumin with or without PLO wasperformed as reported previously.17) Briefly, after being anesthe-

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tized, the trachea was exposed and incised transversely betweenthe fourth and fifth tracheal rings. Cannulation (2.5 cm longpolyethylene tubing, O.D. 3mm) was done through the trachealincision. Then, 50 µL of PBS buffer containing FITC-albumin(200µg/mL) with or without PLO (240 µg/mL) was injected intothe lung over a period of 1–2 s through the tracheal cannula using a100µL syringe. The inferior aorta and vena cava were cut 2 h afteradministration of the drug solution, and the lungs were perfusedwith 0.15M NaCl through the right ventricle until the lungs turnedwhite. After the lungs were extirpated, all steps were performedon ice or at 4°C. The lungs were homogenized with Ultra Turrax(IKA Japan K.K., Osaka) in 9mL of PBS buffer at 19,000 rpm for2min (approximately 10% homogenate), and then homogenized ina glass/Teflon Potter homogenizer with 10 strokes at 1,000 rpm. Thehomogenate was centrifuged at 3,000©g for 15min in an Avanti 30Compact Centrifuge (Beckman Instruments, Inc., Fullerton, CA).The supernatant was centrifuged at 100,000©g for 60min in aHimac CP80WX Preparative Ultracentrifuge (Hitachi, Ltd., Tokyo,Japan). NaOH was added to the supernatant (final concentration,125mM), and was used for fluorescence measurement.

Statistical analysis: Data are expressed as means « S.E.Statistical analysis was performed by Student’s t-test or one-wayANOVA followed by Tukey’s test for multiple comparisons. Thelevel of significance was set at *p < 0.05 or **p < 0.01.

Results

Effect of various concentrations of poly(amino acid)s onFITC-albumin uptake: As we previously reported,14) FITC-albumin uptake by A549 cells was temperature-sensitive, andtherefore, intracellular uptake was estimated by subtracting thecell surface binding at 4°C from the total cell association at 37°Cin the present study. First, the effects of two positively chargedpoly(amino acid)s, PLO and PLL, on FITC-albumin uptake byA549 cells were examined. As shown in Figure 1A, the uptakeof FITC-albumin (20 µg/mL) was stimulated by co-incubationwith PLO and PLL, and maximum effects were observed at 20µg/mL (PLO) and 10µg/mL (PLL). At higher concentrations, thestimulatory effects of these poly(amino acid)s decreased. Becausethe albumin concentration in alveolar fluid would increase to 40–65% of the plasma level (about 40–50mg/mL) in hydrostaticpulmonary edema, the effects of poly(amino acid)s on 20mg/mLFITC-albumin uptake were examined (Fig. 1B). PLO stimulatedFITC-albumin uptake in a concentration-dependent manner. PLLalso stimulated FITC-albumin uptake, though the stimulatoryeffect reached the maximum at PLL concentrations higher than 30µg/mL. Similar enhancing effects of PLO and PLL on the uptakewere observed even when a higher concentration of FITC-albumin(40mg/mL) was used (data not shown).

Relationship between the intracellular uptake and cellsurface binding of FITC-albumin in the presence of PLO andPLL: The relationship between the intracellular uptake and cellsurface binding of FITC-albumin (20mg/mL) in the presence ofvarious concentrations of PLO and PLL is shown in Figure 2.Poly(amino acid)s enhanced not only the intracellular uptake ofFITC-albumin but also its cell surface binding, and a significantcorrelation (p < 0.01) was observed between FITC-albumin uptakeand binding in both cases.

Intracellular localization of FITC-albumin taken up byA549 cells: Intracellular localization of FITC-albumin wasexamined by confocal laser scanning microscopy. When A549

cells were incubated with FITC-albumin, punctate localization ofgreen fluorescence was observed both in the absence and presenceof PLO, and the intensity of the fluorescence was much strongerin the cells incubated with PLO (Figs. 3A and 3D). Figures 3Band 3E show the fluorescence of LysoTracker Red, a lysosomalmarker, simultaneously added to the uptake buffer. As shownin Figures 3C and 3F, colocalization of FITC-albumin andLysoTracker Red was observed both in the absence and presenceof PLO, indicating that FITC-albumin taken up by the cells wastargeted to lysosomes.

Effect of pretreatment of A549 cells with PLO on FITC-albumin uptake: In the above experiments, poly(aminoacid)s were added to the uptake buffer with FITC-albumin (co-incubation). So, we next examined the effect of pretreatment ofA549 cells with PLO on FITC-albumin uptake (Fig. 4). Interest-ingly, pretreatment of A549 cells with PLO enhanced FITC-albumin uptake more potently than co-incubation, even thoughPLO was not added to the uptake buffer (pretreatment alone).When PLO was added to the uptake buffer in addition to thepretreatment, FITC-albumin uptake was rather decreased comparedwith pretreatment alone (pretreatment plus co-incubation).

Effect of PLO addition in the middle of incubation: For theenhancement of albumin clearance to facilitate the recovery frompulmonary edema, the enhancer should be administered to thelungs, where albumin concentration in the alveolar lining fluid isalready high. Therefore, we examined the effect of PLO addition inthe middle of the incubation. As shown in Figure 5, addition ofPLO at 30min after starting incubation of A549 cells with FITC-

Fig. 1. Effect of various concentrations of poly(amino acid)s on FITC-albumin (A: 20 µg/mL, B: 20mg/mL) uptake by A549 cellsConfluent monolayers were incubated with FITC-albumin for 60min in thepresence (closed symbol) or absence (open symbol) of various concentrations ofPLO (circle) or PLL (triangle). Each point represents the mean « S.E. of threemonolayers.

Effect of Poly¤amino acid¥s on Albumin Uptake by A549 Cells 499

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Fig. 2. Relationship between intracellular uptake of FITC-albumin andbinding in the presence or absence of poly(amino acid)s in A549 cellsIntracellular uptake and cell surface binding of FITC-albumin in the presence(closed symbol) or absence (open symbol) of PLO (A) or PLL (B) were plotted(data from the experiments in Fig. 1B were used). The correlation coefficient(R2) was calculated to be 0.86 for PLO and 0.90 for PLL.

Fig. 3. (Color online) Confocal laser scanning micrographs of A549 cellsAfter incubation with FITC-albumin, LysoTracker Red, and Hoechst 33342 for 60min in the absence (A, B, C) or presence of PLO (D, E, F), cells were observed byconfocal laser scanning microscopy. A, D: FITC-albumin (green), B, E: LysoTracker Red (red; lysosome), and Hoechst 33342 (blue; nucleus). Colocalization offluorescence derived from FITC-albumin and LysoTracker Red is shown in yellow in C and F (merged). (For interpretation of the references to color in this figurelegend, the reader is referred to the web version of this article.)

Fig. 4. Effect of pretreatment of A549 cells with PLO on FITC-albuminuptakeA549 cells were preincubated in the absence (Pretreat; PBS(G)) or presence(Pretreat; PLO) of 80 µg/mL PLO, and FITC-albumin (20mg/mL) uptake for60min with (Co-PLO; (+)) or without (Co-PLO; (¹)) co-incubation with 80µg/mL PLO was measured. Each value represents the mean « S.E. of threemonolayers. **p < 0.01, significantly different from the value for co-incubation(PBS(G), (+)).

Fig. 5. Effect of PLO addition in the middle of incubationThe time-course of FITC-albumin (20mg/mL) uptake in A549 cells wasmeasured with ( ) or without ( ) the addition of PLO (80 µg/mL) at 30minafter starting incubation. Each value represents the mean « S.E. of threemonolayers. **p < 0.01, significantly different from the value without theaddition of PLO ( ).

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albumin markedly enhanced the following uptake. The uptake ratesof FITC-albumin with and without PLO addition were 10.3 µg/mgprotein/min and 1.2 µg/mg protein/min (estimated from the uptakerate from 30 to 60min), respectively.

Effect of various inhibitors on FITC-albumin uptake inthe presence of PLO: In order to understand the mechanism ofalbumin uptake in the presence of PLO in A549 cells, the effectsof various inhibitors on FITC-albumin uptake were elucidated(Table 1). Pretreatment of A549 cells with a metabolic inhibitor, 2,4-dinitrophenol, significantly inhibited FITC-albumin uptake in thepresence of PLO. The uptake of FITC-albumin was inhibited byclathrin-mediated endocytosis inhibitors, phenylarsine oxide andchlorpromazine, in a concentration-dependent manner. In addition,FITC-albumin uptake was also inhibited by a macropinocytosisinhibitor, 5-(N-ethyl-N-isopropyl) amiloride. On the other hand,FITC-albumin uptake was not affected by the caveolae-mediatedendocytosis inhibitors indomethacin and nystatin.

In vivo pulmonary administration of FITC-albumin with orwithout PLO: The effect of co-administration of PLO on FITC-albumin clearance from the lung was examined in rats underin vivo conditions. A solution containing FITC-albumin without(control) or with PLO was administered to the lungs, and after 2 h,the fluorescence remaining in the lungs was estimated. When PLOwas co-administered, the fluorescence derived from FITC-albuminsignificantly decreased to 48.6 « 3.0% of control (p < 0.01,n = 3), suggesting that PLO would enhance albumin clearancefrom the lungs.

Discussion

In pulmonary edema, the concentration of albumin in alveolarlining fluid can increase to about 4- to 8-fold of normal con-centration, and for the restoration of the normal alveolar milieu, theexcess amount of albumin needs to be cleared from the alveolarspace. We have previously shown that, after being taken up bycultured RLE-6TN cells, part of albumin is targeted to lysosomes,where it is gradually degraded.15) Another fate of albumin taken upby the epithelial cells might be transcytosis to the basal side. Buret al.18) showed that albumin transport across the monolayers wasstrongly direction-dependent, and apical-to-basal permeability wasmuch higher than in the basal-to-apical direction. More recently,

Buchäckert et al.19) observed the transepithelial transport of 125I-albumin in both type II and type I-like cell monolayers. We alsoconfirmed the transcytosis of albumin from the apical-to-basal side(data not shown), using primary cultured rat alveolar epithelialcells grown on permeable Transwell filters in the presence ofkeratinocyte growth factor, which would be effective to preservethe type II phenotype of primary cultured alveolar epithelialcells.20) Thus, the uptake of albumin across the apical membraneinto the alveolar epithelial cells is the first step of albuminclearance from the alveolar space.

Recently, several researchers have used cationic peptides forenhancing the uptake of high-molecular weight compounds intothe cells.21–23) PLO and PLL are cationic poly(amino acid)s havingpositive charges at physiological pH. In addition, it was reportedthat covalent binding was not needed to enhance the deliveryof high-molecular weight compounds like plasmid DNA into thecells.24) Based on these backgrounds, we previously examined theeffects of these cationic poly(amino acid)s on insulin transport, andfound that they successfully enhanced in vitro uptake in alveolarepithelial cells as well as in vivo pulmonary absorption of insulinto the systemic circulation.17) In addition, the cytotoxicity of PLOin RLE-6TN cells was fairly low, even after repeated treatmentof the cells with PLO. In this study, we investigated the effect ofpoly(amino acid)s on albumin uptake in A549 cells.

The uptake of FITC-albumin (20 µg/mL) was stimulated by co-incubation with PLO and PLL, and the maximum stimulatoryeffects on FITC-albumin uptake were observed at 20 µg/mL PLOand 10µg/mL PLL (Fig. 1A). We previously examined the effectsof PLO and PLL on FITC-insulin (20 µg/mL) uptake by RLE-6TNcells under the same experimental conditions.17) Though the cellsand substrates were different, a similar bell-shaped pattern wasobserved. Therefore, the concentration of poly(amino acid)s and/orsubstrate:poly(amino acid)s ratio would be an important factor toobtain an efficient stimulatory effect of poly(amino acid)s on theuptake of macromolecules.

The uptake of FITC-albumin in A549 cells was saturable, andwas mediated by a high-affinity (Km value: 1.3mg/mL), lowcapacity system and by a low-affinity (Km value: 45.6mg/mL),high capacity system.14) At the concentration of 20 µg/mL of FITC-albumin, the high-affinity uptake system is the predominant path-way. On the other hand, the concentration of albumin in alveolarlining fluid is much higher, especially in pulmonary edema. There-fore, we next examined the effects of PLO and PLL on the uptakeof 20mg/mL FITC-albumin. At this concentration, the low-affinityuptake system is the predominant pathway for the entry of FITC-albumin into the cells. Though the concentration of albumin was1,000-fold higher than that employed in Figure 1A, PLO and PLLin the same concentration range stimulated FITC-albumin uptake(Fig. 1B). In contrast to the effect on 20µg/mL FITC-albuminuptake, a bell-shaped stimulation pattern was not observed andthe uptake increased with increased poly(amino acid)s concentra-tion, though the stimulatory effect of PLL seemed to be maximal atconcentrations higher than 30 µg/mL. When intracellular uptakewas plotted against cell surface binding, a good correlation wasobserved between FITC-albumin uptake and binding in both thePLO and PLL experiments (Fig. 2). Therefore, the enhancement ofalbumin uptake by these poly(amino acid)s may be due to increasedbinding of albumin to the cell surface at least in part.

It has been suggested that the additional -CH2- group containedwithin lysine may make the ¡-helix conformation of PLL more

Table 1. Effect of various inhibitors on FITC-albumin uptake in thepresence of PLO

InhibitorConcentration

(µM)Albumin uptake(% of control)

2,4-Dinitrophenol 1,000 52.6 « 8.2*Phenylarsine oxide 5 74.1 « 2.5

15 51.6 « 6.7**30 57.1 « 10.3**

Chlorpromazine 35 71.2 « 4.8*70 67.7 « 6.1*

140 52.0 « 2.9**5-(N-ethyl-N-isopropyl)-amiloride 25 60.6 « 2.6*

50 45.4 « 9.9**75 45.3 « 7.5**

Indomethacin 100 103.1 « 1.9200 99.2 « 4.6300 105.7 « 2.2

Nystatin 15 108.8 « 2.530 99.7 « 10.1

Each value represents the mean « S.E. of three monolayers.*p < 0.05, **p < 0.01, significantly different from each control.

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stable than PLO.24,25) On the other hand, pKa values of the primaryamine groups for lysine and ornithine are comparable (about 10.5–10.7). Therefore, though the precise mechanism is not known, thedifference in FITC-albumin uptake enhancing activity betweenPLO and PLL may be due to their conformational differences. Thestimulation effect of PLO on FITC-insulin uptake in RLE-6TNcells was also stronger than that of PLL.17)

As described above, Ramsay et al.24) examined the biophysicalinteraction between poly(amino acid)s, PLO and PLL, and anegatively charged plasmid DNA. They found that the poly(aminoacid)s:plasmid DNA complexes showed positive zeta potentials(+20 to +30mV) over a wide range of poly(amino acid)s:plasmidDNA ratios. Like plasmid DNA, albumin is also a negativelycharged macromolecule (the pI value is about 4.5). Therefore, it islikely that poly(amino acid)s and albumin would form a complexhaving positive zeta potential, resulting in increased interactionbetween the complex and the negatively charged cell surface andsubsequent uptake of FITC-albumin.

The intracellular localization of FITC-albumin after being takenup by A549 cells was evaluated by confocal laser scanning micros-copy (Fig. 3). The enhancing effect of PLO on FITC-albuminuptake was also supported by these experiments, as shown by theincreased fluorescence intensity of FITC-albumin in the presenceof PLO. In addition, using LysoTracker Red as a lysosomal marker,it was shown that FITC-albumin was predominantly localized inlysosomes either in the absence or presence of PLO, where FITC-albumin may be degraded.

Next, the effect of PLO pretreatment on FITC-albumin uptakewas examined in A549 cells. For this purpose, A549 cells weretreated with 80µg/mL PLO for 10min, and after removing thePLO solution, the cells were incubated in uptake buffer containingFITC-albumin but not PLO for 60min. Surprisingly, pretreatmentalone enhanced FITC-albumin uptake more potently than co-incubation with PLO, even though the amount of PLO remainingin the uptake buffer after pretreatment should be much lower thanunder co-incubation conditions. In addition, when PLO was addedto the uptake buffer in addition to the pretreatment, FITC-albuminuptake rather decreased compared with the uptake after pretreat-ment alone. These results may indicate that cationic poly(aminoacid)s like PLO would bind quite tightly to negatively charged cellsurface containing proteoglycan and sialic acid. Several reportsindicated that cationic peptides would bind to negatively chargedheparan sulfate proteoglycan.26,27) So, PLO bound to the cellsurface during preincubation may remain there and bind FITC-albumin even in the absence of further PLO in the uptake buffer,resulting in stimulation of FITC-albumin uptake. On the otherhand, when PLO was added to the uptake buffer in addition tothe pretreatment, PLO may bind to both the cell surface and FITC-albumin, and electrostatic repulsion may occur between PLObound to the cell surface and PLO-albumin complex, resultingin the decreased stimulatory effect on FITC-albumin uptake. Thishypothesis may also explain the reason why bell-shaped effects ofPLO and PLL on FITC-albumin (20 µg/mL) uptake were observed(Fig. 1A). At lower concentrations of poly(amino acid)s, it maybind to either FITC-albumin or the cell surface. In such a case,increased interaction between poly(amino acid)s:FITC-albumincomplex and the negatively charged cell surface, or negativelycharged FITC-albumin and the cell surface bound with positivelycharged poly(amino acid)s can be expected. On the other hand, athigher concentrations of poly(amino acid)s, it may bind to both

FITC-albumin and the cell surface, resulting in the decreasedstimulatory effect of FITC-albumin as described above.

On one hand, in pulmonary edema, the enhancer of albuminclearance should be administered to the lung, where albuminconcentration in alveolar lining fluid is already high. Consideringsuch a condition, the effect of PLO added to the uptake bufferin the middle of the incubation was examined. Addition of PLOat 30min after starting incubation with FITC-albumin markedlyenhanced the uptake (Fig. 5). Therefore, PLO may be effective instimulating albumin clearance from the alveolar space by pulmo-nary administration.

The endocytic pathway involved in albumin uptake in thepresence of PLO was examined in A549 cells. Based on the sen-sitivity to various endocytosis inhibitors, it was suggested that thepredominant pathway for FITC-albumin uptake would be clathrin-mediated endocytosis and/or macropinocytosis. Therefore, theendocytic pathway involved in albumin uptake in A549 cells maybe the same in the absence and presence of poly(amino acid)s.14)

However, each endocytosis inhibitor employed is not necessarilyspecific for a single pathway. For example, 5-(N-ethyl-N-iso-propyl) amiloride used as a macropinocytosis inhibitor may alsoaffect clathrin-mediated endocytosis.28) Thus, the contribution ofthese two endocytic pathways for the uptake of albumin in A549cells needs to be clarified further.

Recently, Buchäckert et al.19) examined the transepithelialalbumin clearance from the alveolar space of intact rabbit lungs,which was assessed by real-time measurement of 125I-albuminelimination from the alveolar space. They showed that albuminclearance from the alveolar space was decreased by loweringtemperatures compared with that at 37°C, by the addition of anexcess amount of unlabeled albumin into the alveolar space, andby treatment with clathrin-mediated endocytosis inhibitors. Theseresults support the idea that the uptake of albumin into the alveolarepithelial cells is the first step and an important determinant foralbumin clearance from the alveolar space of intact lungs. Further-more, in the present study, we examined the effect of PLO onFITC-albumin clearance from the lungs in rats under in vivoconditions. PLO facilitated the elimination of fluorescence derivedfrom FITC-albumin, suggesting that PLO would enhance albuminclearance from the alveolar space of the lungs.

In conclusion, the present results show that poly(amino acid)ssuch as PLO could effectively stimulate endocytic uptake ofalbumin in cultured alveolar epithelial cells, and enhance in vivoalbumin clearance from the lungs in rats. These findings provideimportant information for the development of novel strategies tofacilitate the recovery from pulmonary edema.

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