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Insulin Aggregation and Asymmetric Transport Across Human Bronchial Epithelial Cell Monolayers (Calu-3)
Insulin Aggregation and Asymmetric Transport across Human Bronchial Epithelial Cell Monolayers (Calu-3) ISABELLE PEZRON, RANJANA MITRA, DAHNANJAY PAL, ASHIM K. MITRA Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri, 5005 Rockhill Road, Kansas City, Missouri 64110-2499
Received 30 May 2001; revised 28 November 2001; accepted 18 December 2001
ABSTRACT: The purpose of this work was to elucidate the transport pathways of zinc insulin across the Calu-3 cell monolayer, an in vitro model of the human airway epithelium. Calu-3 cells grown in liquid-covered conditions formed a confluent monolayer with a high transepithelial electrical resistance value of 1000 150 O cm2. The cell monolayer was characterized by a low mannitol permeability of 4.7 0.5 107cm/s. Transport of zinc insulin (donor concentration 1 U/mL) in Dulbecco’s modified phosphate buffer saline at 378C was found to be higher in the basolateral (BL) to apical (AP) (Papp ¼ 3.0 0.2 108 cm/s), than in the AP to BL direction (Papp ¼ 0.41 0.02 108 cm/s). P-glycoprotein efflux or specific enzymatic degradation did not appear to contribute toward this asymmetric transport. Insulin receptors, though apparently more abundant on the BL side than on the AP side of Calu-3 cells, did not mediate the direction-dependent transport of insulin. However, transport of a monomeric human insulin analog, Asp(B10)des(B28-30), across the Calu-3 cell monolayer was similar in both directions (BL to AP and AP to BL). The corresponding permeability, Papp ¼ 2.9 0.2 108 cm/s, was not significantly different from the permeability of zinc insulin in the BL to AP direction. The paracellular pathway seems to play a major role in the insulin transport across the Calu-3 cell monolayers. We hypothesize that the transport of zinc insulin oligomers is restricted at the AP surface by the presence of the tight junctional complexes. From the BL side, oligomers may undergo dissociation in the intercellular space and diffuse readily as monomers to the AP surface of the membrane. ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:1135–1146, 2002
Keywords: pulmonary delivery; Calu-3 cells; asymmetric insulin transport; insulin receptor; monomeric insulin; insulin aggregation
INTRODUCTION Pulmonary delivery has resulted in a substantially higher systemic bioavailability of several proteins and peptides relative to oral administration.1 The inhalation route seems to be the leading mode of nonparenteral administration of insulin.2 Previous studies in our laboratory using animal Isabelle Pezron is on leave from the De´partement de Ge´nie Chimique, Universite´ de Technologie de Compie`gne, BP 529, 60205 Compie`gne cedex, France. Correspondence to: Ashim K. Mitra (Telephone: 816-2351615; Fax: 816-235-5190; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 91, 1135–1146 (2002) ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association
models have shown that nasal and pulmonary routes have high potential for systemic insulin absorption.3,4 However, the exact mechanism of pulmonary insulin transport has not so far been delineated because of the anatomical and physiologic complexities of the lung epithelia (e.g., tracheal, bronchial, bronchiolar, alveolar). A drug can be delivered by intratracheal instillation, where it is deposited in the central part of the conducting airway, or by aerosol deposition, where the drug is distributed more uniformly and reaches peripheral alveolar region of the lung.5 The bioavailability of insulin after intratracheal instillation is usually found to be lower than the bioavailability of aerosolized insulin.6 However,
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pulmonary absorption of insulin is enhanced in the presence of absorption promoters such as bile salts7 and/or phospholipids.8 The enhancement mechanisms are not well understood and can involve different factors including dissociation of insulin oligomers, opening of the pulmonary epithelial tight junctions, aminopeptidase inhibition, and a ciliostatic effect.7 Cell monolayer culture of the lung epithelium— the primary barrier to pulmonary drug transport— has been developed to better delineate drug absorption mechanisms.9 According to a recent review, the most promising lung-derived cell lines are Calu-3 and 16HBE14o for transport and absorption studies, and BEAS-2B for drug metabolism studies.10 These are human bronchial cell lines. The only available alveolar cell line is A549, but its ability to form functional tight junctions has been questioned.10,11 Alveolar epithelial culture models have also been developed using primary cell culture,12 but they may be more delicate and prone to extreme variability. Most of the current drugs delivered through inhalation devices are primarily deposited in the lung airways and Calu-3 seems to be the most appropriate currently available cell line to study drug absorption across the airway epithelium.9–11 Calu-3 cells form polarized monolayers and express functional tight junctions, restricting the paracellular transport of hydrophilic solutes.10,11 Until now, Calu-3 cell monolayers were mainly grown on air-interface culture to study the transport of ions through the cystic fibrosis transmembrane conductance regulator.13,14 Only recently, a few studies have reported permeability of drugs across this cell monolayer grown on air-interface conditions10,15–17 and liquid-covered culture.15,18,19 The aim of this study was to elucidate the mechanisms of insulin transport across Calu-3 cell monolayers grown at the liquid-covered interface. In this report, we describe the transport of insulin across Calu-3 cell monolayers grown on permeable filters in the absorptive [apical (AP) to basolateral (BL)] and secretory (BL to AP) directions. The possible involvement of the insulin receptor a-subunit on insulin transport was also examined. A major difficulty in determining insulin absorption mechanisms resides in the complexity of the insulin molecular structure. Insulin molecules have a strong tendency to aggregate into dimers and hexamers in the presence of zinc.20 The absorption of insulin depends on its association state, the monomer being the fastest absorbing species.21 However, the generation of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
monomeric insulin requires modification of the insulin molecule through recombinant DNA technology and site-specific mutagenesis, and optimum systems for its clinical applications are still under investigation.22 To investigate the effect of insulin association on the absorption process, we describe here the transport of zinc insulin and a monomeric insulin analog across the Calu-3 cell monolayer and discuss the differences observed as well as the possible mechanisms involved.
EXPERIMENTAL SECTION Materials Crystalline porcine zinc insulin (potency 26.3 U/mg) and lyophilized monomeric human insulin analog [Asp(B10)des(B28-30); potency 28.85 U/mg] were generously donated by Eli Lilly and Co. (Indianapolis, IN). Human lung carcinoma derived Calu-3 cells were obtained from American Type Culture Collection (ATTC, Rockville, MD). The growth medium was a 1:1 mixture of Dulbecco’s modified Eagle medium and Ham’s F-12 medium (DMEMF12, reference 12500-062) obtained from Life Technologies (Grand Island, NY). MEM nonessential amino acids, penicillin, streptomycin, sodium bicarbonate, and HEPES were obtained from Sigma (St. Louis, MO). Fetal bovine serum was purchased from Mediatech, Inc. (Herndon, VA). The buffer used in transport studies was Dulbecco’s modified phosphate buffer saline (DPBS), containing 129 mM NaCl, 2.5 mM KCl, 7.4 mM Na2HPO4, 1.3 mM KH2PO4, 1 mM CaCl2, 0.7 mM MgSO4, and 5.3 mM glucose at pH 7.4. These chemicals, of analytical grade, were obtained from Sigma. 14C-mannitol (50 mCi/mmol) was supplied by Amersham (Piscataway, NJ) and 14 C-PEG 4000 (11.2 mCi/g) by ICN (Costa Mesa, CA). Anti-human insulin receptor, a-subunit, chicken polyclonal immunoglobulin (Ig)Y, was purchased from Upstate Biotechnology (Lake Placid, NY) and secondary antibodies, peroxidase conjugated goat anti-chicken IgG, from Rockland (Gilbertsville, PA). 3,30 ,5,50 Tetramethylbenzidine (TMB) was obtained from Pierce (Rockland, IL). Coat-A-Count insulin radioimmunoassay kits were supplied by Diagnostic Products Corporation (Los Angeles, CA). Transwell1 filters (sixwell plate, clear polyester membrane, pore size 0.4 mm with an average of 4 106 pores/cm2, diameter 24 mm) and 12-well plates were purchased from Costar (Bedford, MA).
TRANSPORT PATHWAYS OF ZINC INSULIN
Preparation of Insulin Solutions Zinc insulin or monomeric insulin powder was dissolved in a minimal volume of 0.1 N HCl solution to which sterile DPBS solution was added. The solution pH was adjusted to 7.4 by the addition of 0.1 N NaOH. A stock insulin solution (1 mg/mL) was freshly prepared before each experiment. For conducting transport experiments, insulin solutions were prepared by dilution of the stock solution. Concentrations are expressed in units/milliliter. Zinc insulin has a potency 26.3 U/mg; therefore 1 U/mL corresponds to 0.038 mg/mL (6.6 mM; Mw ¼ 5778). Insulin conformation in the stock solution was analyzed by using circular dichroism (CD) (cell path length 0.02 cm, Spectropolarimeter JASCO J/720; Japan Spectroscopic Co, Japan.). Cell Culture Calu-3 cells of passage number 20–40 were cultured in DMEM-F12 medium containing L-glutamine, supplemented with 10 to 15% fetal bovine serum (heat inactivated), 1% nonessential amino acids, 100 U/mL penicillin, 100 mg/mL streptomycin, 14 mM HEPES, and 29 mM sodium bicarbonate at pH 7.4. The cells were grown at 378C in a tissue culture incubator with 5% CO2 and 95% humidity. The medium was changed every other day. To conduct transport experiments, cells were grown at a density of 1.1 to 1.3 105 cells per cm2 on collagen-coated Transwell filters under liquidcovered culture conditions. The monolayer growth was observed by using optical microscopy (Carl Zeiss Telaval 31, Germany). The integrity of the cell monolayer was monitored by determining the transport of paracellular markers 14C-mannitol, or 14C-PEG 4000 and by measuring the transepithelial electrical resistance (TEER) with an EVOM Epithelial Voltohmmeter (World Precision Instruments, Inc., Sarasota, FL). Microscopy Light, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) were performed to characterize the morphology of Calu3 cells grown under liquid-covered culture conditions. Cells grown at confluency after 9 days were washed twice with cold DPBS, then with sodium cacodylate buffer for 5 min at 48C. The cells were fixed with 2% glutaraldehyde in 0.15 M sodium cacodylate buffer for 1 h at 48C, and then washed several times with cacodylate buffer. The cell sur-
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face was covered with a gelatin layer to protect the surface features, and subsequently fixed with osmium tetroxide for 30 min. Cells were processed for thin sectioning using routine procedures (section thickness 0.5 mm for light microscopy and 90 nm for TEM). The cells used for SEM were dehydrated with graded ethanol and the cell surface was coated with gold/palladium. An optical microscope (Olympus Bmax, Japan), a Jeol Electron Microscope, Japan (model JEM 1200 EX II) at 100 kV, and a Philips Electron Microscope, Holland (model XL-30 ESEM-FEG) were used. Transport Experiments Transport experiments were conducted in six-well Transwell plates both in the AP (1.5 mL), to BL (2.5 mL), and BL to AP direction at 378C. The receiver chamber contained only buffer, DPBS, whereas the donor chamber contained insulin, at various concentrations, or a radiolabeled paracellular marker (14C-mannitol, or 14C-PEG 4000). Samples (100–200 mL) from the receiver side were withdrawn at predetermined times (for up to 2 or 3 h) and were replaced by equal volumes of buffer. Insulin concentrations in the receiver chamber were determined using radioimmunoassay. In this method, 125I-insulin competes with the unlabeled insulin contained in receiver samples for binding to insulin-specific antibodies immobilized on a polypropylene tube. After overnight incubation, the tube bound fraction of labeled insulin is measured with a gamma counter (Beckman 5500, USA). The unlabeled insulin concentration is inversely related to the count obtained, and is determined by calibration with solutions of known insulin concentrations in DPBS. The concentrations of the radiolabeled markers were determined using a scintillation counter (Beckman LS6500, USA). Concentration in the receiver chamber (cR) was calculated by taking into account the loss in the receiver side due to periodic sampling: cR ¼ cn þ
vs n1 ci VR i¼1
ð1Þ
In eq. 1, cn is the concentration measured in sample number n, vs the sampling volume, and VR the volume of the receiver chamber. Transport experiments across Calu-3 cell monolayers as well as across the plain collagen-coated filter were performed. The cumulative amounts of solute transported cR(t)VR were plotted as a function of time. Each experiment was performed at least in JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
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triplicate. Statistical significance ( p < 0.05) of the data was analyzed with Student’s t-test. Transport Data Analysis Passive diffusion driven by a concentration gradient across a membrane was analyzed with the Fickian diffusion model. The kinetics of the solute appearance in the receiver chamber was calculated by integration of the flux equations under apparent steady-state conditions, which generates eq. 223: VD cR ðtÞ ¼ cD ð0Þ VR þ VD 1 1 1 exp A þ Papp t VR VD
ð2Þ
A is the membrane area (4.7 cm2) and Papp (in cm/s) denotes the solute apparent permeability. The indices D and R refer respectively to the donor and the receiver sides. When the amount transported is only a minute fraction of the donor amount, such that the concentration gradient remains constant, this equation was simplified and reduced to eq. 3, which denotes concentration as a linear function of time: cR ðtÞ ¼ cD ð0Þ
A Papp t VR
ð3Þ
The experimental permeability accounts for the permeability of the cell monolayer and also the permeability of the collagen-coated filter and the aqueous boundary layer (ABL) as represented by eq. 424: 1 1 1 1 ¼ þ þ Papp Pmonolayer Pcollagen-coated filter PABL
ð4Þ
The last two terms were determined by measuring the permeability of the solute across the collagen-coated filter containing no cell monolayer. The permeability across the cell monolayer was then calculated from eq. 4. Insulin Receptor Binding Studies Enzyme-linked immunoabsorbent assay (ELISA)25 was used to assay insulin receptors on Calu-3 cells. Antibodies against human insulin receptor molecules (anti-IR), specific for the 135 kDa asubunit (extracellular fraction), were used for ELISA. The cells were grown on six-well inserts or 12-well plates. They were first washed with cold PBS, and then fixed with paraformaldehyde at JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
48C for 20 min. Subsequently, the cells were preincubated with a 3% bovine serum albumin solution at 378C for 1 h to reduce the nonspecific binding. Then, the AP or BL side of the monolayer was incubated with a solution of primary antibodies at a concentration of 10 mg/mL for 2 h at 378C. After washing with PBS, a solution of secondary antibodies (anti-chicken IgG peroxidase conjugate, concentration 0.13 mg/mL) was applied to both sides of the chamber together with 0.1% bovine serum albumin for 2 h at 378C. After several washes with PBS, the cells were incubated with the peroxidase substrate-TMB, for 20 min. The reaction was stopped with 1 M sulfuric acid and the solution color was measured by using UVvisible spectroscopy (Beckman DU 7400) at 450 nm. A control study without primary antibodies was also performed. Insulin was coadministrated at different concentrations to investigate the competitive binding to insulin receptors a-subunits on Calu-3 cells. Each experiment was performed at least in triplicate. The effect of anti-IR molecules, applied to the AP or BL sides, on the insulin transport across Calu-3 cells, was also studied. In these experiments, Calu-3 cells were not fixed, but incubated with the primary antibody solution for 2 h at 378C before conducting any transport experiment.
RESULTS AND DISCUSSION Morphology of Calu-3 Cells The Calu-3 cells grown under liquid-covered conditions on collagen-coated Transwell filters formed a single layer of cells at confluency, as shown by light microscopy (Fig. 1A). TEM analysis revealed that the cells were interconnected by AP tight junctions and displayed morphologic characteristics similar to those observed with cells grown under air-interface conditions13,16 and under liquid-covered conditions on untreated filters19 (Fig. 1B). The presence of abundant cilia, and the production of mucus were evidenced by SEM (Fig. 1C and D). Contrary to what was observed in primary culture of tracheal or bronchial epithelial cells,26,27 it appears that only limited differences in morphology and differentiation are observed in Calu-3 cells grown at the liquid-covered interface compared with the air-interface growth condition. These results are in agreement with previous publications stating that tight junctions and desmosomes, cAMP-dependent chloride secretion,
Figure 1. (A) Light microscopy of a transversal section of confluent Calu-3 cells grown in liquid-covered conditions on collagen-coated filters illustrating monolayer formation (scale 10 mm). (B) TEM picture (scale 1 mm) showing characteristic features of the Calu-3 cells [tight junction (Tj), mucus secretion (ms), and microvilli (mv)]. (C) SEM picture showing abundant cilia (original magnification 15,000). (D) SEM picture showing both mucus secretion and cilia (original magnification 20,000). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
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Figure 1. (Continued)
mucus vesicles, and cilia were evidenced in Calu-3 with both culture conditions.15,18,19 The main difference observed was the shape of the cilia: thick and short in liquid-covered culture conditions compared with long and thin in air-interface culture conditions.15 Therefore, Calu-3 cell monolayers grown under liquid-covered interface seem to be suitable models to study drug transport across the airway epithelium. Calu-3 Cell Monolayer Integrity The growth of the Calu-3 cells was followed by microscopic observations as well as by the measurement of the TEER value associated with the monolayer. The Calu-3 cell monolayer was generally confluent after 6–7 days of growth and the transport experiments were performed after 8– 10 days of growth. The resistance of the monolayer was measurable after 4 days in culture and reached a high plateau value, 1000 150 O cm2 (after subtraction of the filter background), after 7–8 days. This value is in the same order as the ones reported in recent studies, ranging from 800 to 2000 O cm2 in air-interface conditions14 and from 700 to 2500 O cm2 in liquid-covered conditions.15,19 Other studies reported lower values of 100 to 300–600 O cm2.11,13,16–18 Microscopic experiments confirmed that the high value obtained JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
in this study is attributed to the formation of functional tight junctions and not to the formation of multilayers (Fig. 1A). Consistent TEER values, in addition to the examination of confluency and monolayer formation by optical microscopy, are necessary prerequisites to conducting any transport study. The integrity of the cell monolayer during a transport experiment was further investigated by using paracellular markers,28 14C-mannitol ˚ ) and 14C-PEG 4000 (Mw& (Mw ¼ 184, radius 3.9 A ˚ ) with a donor concentration of 4000, radius 15.9 A 0.5 mCi/mL in both cases. The appearance of paracellular markers concentration in the receiver chamber was linear with time and independent of the direction of transport (2-h experiment). Values of the apparent permeability of mannitol and PEG-4000 across Calu-3 cells are Papp ¼ 4.7 0.5 107 cm/s and Papp ¼ 0.96 0.05 107 cm/s, respectively, compared with Papp ¼ 4.3 0.2 105 cm/s and Papp ¼ 0.78 0.06 105 cm/s across the collagen-coated filter. The ratio between the solute permeability across the filter and across the monolayer denotes the ability of the cells to form tight junctions and to restrict solute movement. Apparent permeability values of 14C-mannitol and 14C-PEG 4000 in the presence of the Calu-3 cell monolayer were decreased similarly by a factor 91 14 and 81 13 compared with the
TRANSPORT PATHWAYS OF ZINC INSULIN
collagen-coated filter, suggesting that PEG 4000 ˚ ,28 are molecules, of hydrodynamic radius 15.9 A transported across the Calu-3 monolayer through the same aqueous pore system as mannitol. These results clearly suggest that the cell monolayer was the rate-limiting barrier to the transport and its integrity was maintained under these experimental conditions. The value obtained for 14 C-mannitol permeability across the Calu-3 cell monolayer is compatible with results reported for various pulmonary cell culture model systems, which ranged between 107 and 8.107 cm/s.10,29–31 Transport of Insulin Across Calu-3 Monolayers Polarization of Insulin Transport Figure 2 shows the cumulative amount of insulin transported across the Calu-3 cell monolayer from a donor zinc insulin (1 U/mL). The results indicate that insulin transport across the Calu-3 cell monolayer is direction-dependent. After 2 h, the amount of insulin transported from BL to AP was about 1 mU, whereas only 0.16 mU was transported from the AP to BL direction. The corresponding permeability values, obtained by linear fit of the data, are 3.0 0.2 108 cm/s and 0.41 0.02 108 cm/s in the BL to AP and AP to BL directions, respectively. Direction dependency was even more pronounced at higher donor concentration (12.5 U/mL; see insert in Fig. 2). The measure of the donor insulin concentrations at the end of transport experiments by highpressure liquid chromatography or UV-visible spectroscopy did not indicate any significant loss of donor insulin molecules.
Figure 2. Direction-dependent transport of zinc insulin across the Calu-3 monolayer formed on collagen-coated six-well plates for donor concentrations 1 and 12.5 U/mL (insert) at 378C.
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The permeation of a solute across a biological membrane is a complex phenomenon. Simultaneous pathways, i.e., paracellular and transcellular pathways, can contribute to the overall solute transport. The paracellular pathway represents the free diffusion of the solute through the aqueous channels between the cells. Elaborate models have been developed to take into account molecular size, restricted diffusion of large molecules, and electrical effects of small ions.24,32 The transcellular pathway, which represents the transport of the solute across the cell membrane, may be passive or involve pinocytosis and/or active transport mechanisms. The passive transcellular pathway is related to the partitioning of the solute onto the cell lipid membrane and is a predominant mechanism for the transport of lipophilic molecules. Direction-dependent transport of drugs can be observed under various circumstances. P-glycoprotein (P-gp) molecules are present in many tissues and often expressed on the AP side of various cell lines where they function as efflux pumps.33 Consequently, a greater transport of P-gp substrates is observed from the BL to the AP side than in the opposite direction. On the contrary, carrier-mediated transport of amino acids, driven by electrochemical gradients, is favored in the AP to BL direction.34 The presence of enzymes, located specifically on one side of the membrane, can also create transport directionality.35 Specific interaction of peptides with the tight junctional complex was observed in some cases, where tight junction opening occurred only from one side of the membrane.36 An asymmetric paracellular transport resulted from this particular interaction. In another example, receptormediated transcytosis of dimeric immunoglobulin was favored in the BL to AP direction in Calu-3, through receptors present predominantly on the BL surface of the membrane.19 We have examined these possibilities to elucidate the mechanism of insulin transport. The presence of P-gp molecules at the AP surface of Calu-3 cells has been investigated and reported in two recent publications.17,37 Contradictory results show on one hand P-gp efflux of rhodamine 123, a well-known P-gp substrate,17 and on the other hand, a very faint presence of P-gp molecules on Calu-3, which have to be induced by the addition of ouabain.37 P-gp substrates are usually lipophilic molecules with a rather low molecular weight,33 and no study has been conducted with insulin in that regard. To investigate this possibility, we studied the effect of a well-known P-gp JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
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substrate (verapamil, 100 mM) added to the AP chamber, on insulin transport. The presence of verapamil did not change the asymmetric character of the insulin transport, BL to AP directional flux being predominant over the AP to BL flux (data not shown). Consequently, P-gp efflux does not seem to be involved in the asymmetric insulin transport across the Calu-3 cell monolayer, which is consistent with previously published results with other substrates.18 Yahamara et al.35 studied the transport of bovine insulin (zinc-free) across a primary culture of rat alveolar epithelium. The permeability obtained in the BL to AP and AP to BL directions was 0.8 0.1 and 1.2 0.2 108 cm/s, respectively, values which are not significantly different from each other. When using radioactive 125I-insulin in nanomolar donor concentration range, enzymatic degradation of insulin was observed and appeared to be prevalent on the AP side of the membrane. To analyze whether the asymmetric transport observed in our study was due to the specific presence of insulin degrading enzyme on one side of the Calu-3 cell monolayer, we performed the insulin transport experiment in the presence of peptidase inhibitors (bestatin, 1 mM and captopril, 2 mM). The asymmetry and extent of the insulin transport was not affected by the presence of these inhibitors in the donor or the receiver chambers (data not shown). The association of insulin molecules as oligomers may reduce the probability of enzymatic degradation of insulin, as reported previously.21 We also verified that zinc insulin molecules do not have a specific effect on the tight junction complex. Adding insulin to the donor chamber did not affect the 14C-mannitol transport in both directions. Any remaining mechanism that can explain the direction-dependent insulin transport is the receptor-mediated transcytosis of insulin, or an asymmetric transport due to specific interaction between insulin oligomers and the AP side of the membrane. These two possibilities were further investigated in detail and are presented in the sections below.
by an excess of unlabeled insulin.39 Internalization of insulin through the receptor-mediated process was proposed, but direction-dependent transport of unlabeled insulin by transcytosis through the receptor-mediated process has not been proven. In another study, in vivo transendothelial transport of insulin from plasma to interstitial fluid was found to be mainly caused by diffusion with no evidence of the receptor-mediated process.40 Calu-3 cells responded positively to ELISA with anti-human insulin receptor (a-subunit). Figure 3A compares the response of the BL and AP sides of a Calu-3 monolayer after incubation with anti-IR molecules. The percentage of receptor bound antibodies appears to be approximately three times higher on the BL side than the AP side. However, zinc insulin at concentrations of 1 U/mL and 2 U/mL does not appear to bind competitively to the receptors present on the BL side, as shown in Figure 3B.
Insulin Receptor Binding Studies The insulin receptor is widely expressed on mammalian tissues.38 A receptor-mediated transcytosis of insulin in the blood-brain barrier has been suggested.39 Binding of radiolabeled insulin to bovine brain microvessel endothelial cells was evidenced, polarized to the AP side, and inhibited JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
Figure 3. Response of Calu-3 cells to ELISA with anti-human insulin receptors (anti-IR). (A) Comparison of the responses of the AP and BL sides (in a six-well plate). (B) Influence of zinc insulin added on the BL side at concentrations of 1 and 2 U/mL on the receptor binding (12-well plates).
TRANSPORT PATHWAYS OF ZINC INSULIN
Anti-IR molecules added to the AP or BL donor chamber did not affect the insulin transport. Moreover, preliminary results involving the passage of 125I-insulin across Calu-3 cells also showed no inhibition of 125I-insulin transport when zinc insulin was added to the donor side (data not shown). Experiments at 48C were also attempted to confirm the absence of the receptor-mediated process. However, a decrease in TEER value of the Calu-3 cell monolayer together with an increased permeability of paracellular marker indicated a loss of integrity of the monolayer maintained at 48C (data not shown). Even though our results suggest that insulin receptors are present on the surface of Calu-3 cells and are more abundant on the BL side, the reason for a higher transport from BL to AP does not seem to be due to receptor-mediated transport. An aggregation of zinc insulin molecules in solution may hinder insulin for binding to the active sites of the receptors. Additional experiments will be necessary to confirm and quantitate the presence of insulin receptors on Calu-3 cells. Transport of a Monomeric Insulin Analog Asp(B10)des(B28-30) Zinc insulin exists predominantly in the mono˚ ) only at meric form (hydrodynamic radius 12.8 A 7 a very low concentration (< 10 M &0.015 U/mL) under neutral conditions.20,41 At higher concentrations, insulin self-associates into dimers, which in the presence of zinc, further assemble into ˚ .20 stable hexamers of hydrodynamic radius 28 A 20 Hvidt has characterized the formation of zinc insulin oligomers by light scattering and sizeexclusion chromatography. The equilibrium association constants obtained are K1,2 ¼ 14 104 M1 for the monomer-dimer equilibrium and K2,6 ¼ 15 1010 M2 or the dimer-hexamer equilibrium.20 Calculation of the corresponding molar oligomer concentrations yields to 2.9, 1.16, and 0.23 106 M for the monomers, dimers, and hexamers, respectively, which represent 44, 35, and 21% in weight of the total insulin concentration (1 U/mL, 6.6 106 M).42 It clearly shows that even if the monomers remain the dominant species, the formation of dimers and hexamers, including respectively 2 and 6 insulin molecules, results in a very significant reduction of the monomer concentration. These association properties have been shown to affect insulin absorption in vivo.21 Many insulin monomeric analogs have been developed, as reported recently, in order to better control biological
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action of insulin in diabetes mellitus.22 Modifications in the insulin B chain, particularly on the B10 and B26–30 positions, have a dramatic effect on insulin association.43 Monomers may still form dimers at high concentrations, but are unable to form zinc-coordinated hexamers.43 The insulin analog used in this study is Asp(B10)des(B28-30), in which the histidine residue in B10 has been replaced by an aspartine residue, and the B28–30 residues have been removed. The CD spectra of zinc insulin and the human insulin analog are shown in Figure 4 at a concentration of 1 mg/mL (about 25 U/mL). The far-UV region of the CD spectrum of zinc insulin is characterized by two negative maxima at 209 and 222 nm. The 222-nm band is attributed to the b-structure, characteristic of the dimer conformation, whereas the 209-nm band, assigned to the a-helix structure, is a characteristic feature of the monomer.44 The attenuation of the 222 nm maximum and the increase in intensity of the 209 nm maximum in the insulin analog signal a predominance of monomeric structures.44,45 No data on the transport of this monomeric insulin analog across cell monolayer model have been reported so far. The results of the transport studies with the monomeric insulin analog are displayed in Figure 5. Contrary to zinc insulin, no directiondependence of the transport was observed. The amount of monomeric insulin transported in 2 h in both directions was around 1.2 mU, which is not significantly different from what was obtained with zinc insulin in the BL to AP direction. The corresponding permeability (calculated by linear
Figure 4. CD spectra showing mean residue ellipticity of porcine zinc insulin and monomeric human insulin analog Asp(B10)des(B28–30) in the far-UV region. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
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Figure 5. Unidirectional transport of human monomeric insulin analog Asp(B10)des(B28–30) across the Calu-3 monolayer formed on collagen-coated six-well plates (donor concentration 1 U/mL, 378C).
fit of the data between 20 and 120 min) is 2.9 0.2 108 cm/s. The unidirectional permeability of monomeric insulin across the collagen-coated filter was determined to be 2.7 0.2 106 cm/s. Therefore, the permeability of monomeric insulin across the Calu-3 cell monolayer is decreased by a factor 93 14 compared with the collagen-coated membrane, which is not significantly different from the ratios obtained for the paracellular markers 14C-mannitol and 14C-PEG 4000. Concentration dependence studies of the permeability of monomeric insulin across Calu-3 cell monolayers revealed no saturation effect. Moreover, monomeric insulin molecules did not compete with anti-IR molecules for binding to insulin receptors, which was observed for zinc insulin (data not shown). Consequently, paracellular permeability appears to be the major pathway for monomeric insulin transport across Calu-3 cell monolayers. The modification of the insulin chain chemical composition also affects the isoelectric point and the charges present in the chain. The replacement of the histidine residue by an aspartine residue in B10 leads to a decrease in isoelectric point from 5.6 to 4.55.43 The monomeric insulin chain is expected to bear more fractional negative charges at pH ¼ 7.4 than the corresponding porcine zinc insulin chain. However, electric field effects on paracellular transport of solutes are only significant for small molecules.32 In the case of macromolecules, size effects are expected to be predominant, and in the Caco-2 model, charge effects were undetectable for molecule sizes ˚ .32 Therefore, the charge effects are above 6 A not likely to explain the difference in transport patterns of zinc and monomeric insulin. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
We can deduce from these interesting results that the directionality of zinc insulin transport is likely to be caused by the presence of insulin oligomers. According to literature data, pulmonary epithelial membrane contains intercellular junctions with a large distribution of pore size. A majority of small pore size (< 1 nm) is present together with a few numbers of large pores (4– 8 nm, sometimes up to 25 nm), mediating the transport of macromolecular solutes.9 A solute with a radius close to the junctional size will undergo increasing restricted diffusion because of steric hindrance at proximity of the junction, and will experience increasing frictional resistance with the pore wall during transport.9 Although the size distribution of the intercellular junctions in the Calu-3 cell monolayer is unknown, it is reasonable to assume that the insulin oligomers may be subjected to restricted diffusion. The strongest restriction occurs through the tight junctional complexes, located at the uppermost AP region of the membrane.46 We suggest that the asymmetric transport arises from restricted diffusion of zinc insulin oligomers at the AP surface because of the presence of the tight junctional complexes. In addition, insulin oligomers may interact with muco-proteins secreted on the AP side of the membrane. On the BL side, the insulin oligomers may readily enter the intercellular space. The insulin concentration inside the intercellular aqueous pores decreases and reaches a very low value near the AP surface, because of the concentration gradient across the membrane. Dissociation of insulin oligomers can then take place and the monomers diffuse freely to the AP side of the membrane. This hypothesis is supported by the similarity of permeability values for zinc insulin in the BL to AP direction and the permeability value obtained for monomeric insulin.
CONCLUSION The results reported herein indicate that the Calu-3 cell monolayer grown under liquid-covered conditions, like the air-interface model, is also a suitable cell culture model for conducting transport studies. High TEER value and low mannitol permeability demonstrate that the confluent monolayer is the rate-limiting barrier to solute transport. Restricted insulin transport across the Calu-3 cell line is consistent with the previous in vivo results obtained from intratracheal
TRANSPORT PATHWAYS OF ZINC INSULIN
instillation experiments in the absence of any absorption enhancer.8 P-gp efflux or specific enzymatic degradation did not seem to be major contributing factors for higher insulin transport in the BL to AP direction. Insulin receptors, more abundant on the BL side than on the AP side of Calu-3 cells, did not mediate the direction-dependent transport of insulin. Experiments conducted with a monomeric insulin analog revealed that the paracellular pathway plays a major role in insulin transport. Also, molecular aggregation and size restriction effects are likely to hinder the diffusion of the insulin oligomers, particularly on the AP side of the membrane where the tight junction complexes are located. From the BL side, oligomers may undergo dissociation in the intercellular space and diffuse freely as monomers to the AP region of the membrane.
ACKNOWLEDGMENTS We thank Dr. Douglas Law and Jessica Kueger, from the Department of Biological Sciences, UMKC, and Dr. Vladimir Dusevich, from the Dental School, UMKC, for sharing their expertise in electron microscopy. This work was supported by a grant from Hoechst-Marion-Roussel Pharmaceuticals, Kansas City, MO.
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7. Li Y, Shao Z, DeNicola DB, Mitra AK. 1993. Effect of a conjugated bile salt on the pulmonary absorption of insulin in rats. Eur J Pharm Biopharm 39:216–221. 8. Mitra R, Pezron I, Li Y, Mitra AK. 2001. Enhanced pulmonary delivery of insulin by lung lavage fluid and phospholipids. Int J Pharm 217:25–31. 9. Mathias NR, Yamashita F, Lee VHL. 1996. Respiratory epithelial cell culture models for evaluation of ion and drug transport. Adv Drug Del Rev 22: 215–249. 10. Forbes B. 2000. Human airway epithelial cell lines for in vitro drug transport and metabolism studies. Pharm Sci Technol Today 3:18–27. 11. Winton HL, Wan H, Cannell MB, Gruenert DC, Thompson PJ, Garrod DR, Stewart GA, Robinson C. 1998. Cell lines of pulmonary and non-pulmonary origin as tools to study the effects of house dust mite proteinases on the regulation of epithelial permeability. Clin Exp Allergy 28:1273–1285. 12. Elbert KJ, Scha¨fer UF, Scha¨fers HJ, Kim KJ, Lee VHL, Lehr CM. 1999. Monolayers of human alveolar epithelial cells in primary culture for pulmonary absorption and transport studies. Pharm Res 16:601–608. 13. Shen BQ, Finkbeiner WE, Wine JJ, Mrsny RJ, Widdicombe JH. 1994. Calu-3: A human airway epithelial cell line that shows cAMP-dependent Cl secretion. Am J Physiol 266:L493–L501. 14. Illek B, Tam AWK, Fisher H, Machen TE. 1999. Anion selectivity of apical membrane conductance of Calu-3 human airway epithelium. Pflugers Arch 437:812–822. 15. Meaney C, Florea BI, Borchard G, Juninger HE. 1999. Characterization of a human submucosal gland cell line (Calu-3) as an in vitro model of the airway epithelium. Proc Int Symp Control Rel Bioact Mater 26:198–199. 16. Foster KA, Avery ML, Yazdanian MA, Audus KL. 2000. Characterization of the Calu-3 cell line as a tool to screen pulmonary drug delivery. Int J Pharm 208:1–11. 17. Hamilton KO, Backstrom G, Yazdanian MA, Audus KL. 2001. P-glycoprotein efflux pump expression and activity in Calu-3 cells. J Pharm Sci 90:647–658. 18. Cavet ME, West M, Simmons NL. 1997. Transepithelial transport of fluoroquinolone ciprofloxacin by human airway epithelial Calu-3 cells. Antimicrob Agents Chemother 41:2693–2698. 19. Loman S, Radl J, Jansen HM, Out TA, Lutter R. 1997. Vectorial transcytosis of dimeric IgA by the Calu-3 human lung epithelial cell line: Upregulation by IFN-g. Am J Physiol 272:L951– L958. 20. Hvidt S. 1991. Insulin association in neutral solutions studied by light scattering. Biophys Chem 39:205–213.
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21. Liu FY, Kildsig DO, Mitra AK. 1991. Insulin aggregation in aqueous media and its effect on alpha-chymotrypsin-mediated proteolytic degradation. Pharm Res 8:925–929. 22. Vajo Z, Duckworth WC. 2000. Genetically engineered insulin analogs: Diabetes in the new millenium. Pharmacol Rev 52:1–9. 23. Kobayashi S, Kondo S, Juni K. 1995. Permeability of peptides and proteins in human cultured alveolar A549 cell monolayer. Pharm Res 12:1115–1119. 24. Knipp GT, Ho NFH, Barsuhn CL, Borchardt RT. 1997. Paracellular diffusion in Caco-2 cell monolayers: Effect of perturbation on the transport of hydrophilic compounds that vary in charge and size. J Pharm Sci 86:1105–1110. 25. Pal D, Audus KL, Siahaan TJ. 1997. Modulation of cellular adhesion in bovine brain microvessel endothelial cells by a decapeptide. Brain Res 747:103– 113. 26. Mathias NR, Kim KJ, Robinson TW, Lee VHL. 1995. Development and characterization of rabbit tracheal epithelial cell monolayer models for drug transport studies. Pharm Res 12:1499–1505. 27. de Jong P, van Sterkenburg AJA, Hesseling SC, Kempenaar JA, Mulder AA, Mommaas AM, Dijkman JH, Ponec M. 1994. Ciliogenesis in the human bronchial epithelial cells cultured at the air–liquid interface. Am J Respir Cell Mol Biol 10:271–277. 28. Ghandehari H, Smith PL, Ellens H, Yeh PY, Kopecek J. 1997. Size-dependent permeability of hydrophilic probes across rabbit colonic epithelium. J Pharmacol Exp Ther 280:747–753. 29. Willumsen NJ, Davis CW, Boucher RC. 1994. Selective response of human airway epithelia to luminal but not serosal solution hypertonicity: Possible role for proximal airway epithelia as an osmolality transducer. J Clin Invest 94:779–787. 30. Kim KJ, Cheek JM, Crandall ED. 1991. Contribution of active Naþ and Cl fluxes to net ion transport by alveolar epithelium. Respir Physiol 85:245–256. 31. Dodoo ANO, Bansal SS, Barlow DJ, Bennet F, Hider RC, Lansley AB, Lawrence MJ, Marriott C. 2000. Use of alveolar cell monolayers of varying electrical resistance to measure pulmonary peptide transport. J Pharm Sci 89:223–231. 32. Adson A, Raub TJ, Burton PS, Barsuhn CL, Hilgers AR, Audus KL, Ho NFH. 1994. Quantitative approaches to delineate paracellular diffusion in cultured epithelial cell monolayers. J Pharm Sci 83:1529– 1536. 33. Kim RB, Wandel C, Leake B, Cvetkovic M, Fromm MF, Dempsey PJ, Roden MM, Belas F, Chaudhary AK, Roden DM, Wood AJJ, Wilkinson G. 1999. Interrelationship between substrates and inhibi-
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Received 30 May 2001; revised 28 November 2001; accepted 18 December 2001
ABSTRACT: The purpose of this work was to elucidate the transport pathways of zinc insulin across the Calu-3 cell monolayer, an in vitro model of the human airway epithelium. Calu-3 cells grown in liquid-covered conditions formed a confluent monolayer with a high transepithelial electrical resistance value of 1000 150 O cm2. The cell monolayer was characterized by a low mannitol permeability of 4.7 0.5 107cm/s. Transport of zinc insulin (donor concentration 1 U/mL) in Dulbecco’s modified phosphate buffer saline at 378C was found to be higher in the basolateral (BL) to apical (AP) (Papp ¼ 3.0 0.2 108 cm/s), than in the AP to BL direction (Papp ¼ 0.41 0.02 108 cm/s). P-glycoprotein efflux or specific enzymatic degradation did not appear to contribute toward this asymmetric transport. Insulin receptors, though apparently more abundant on the BL side than on the AP side of Calu-3 cells, did not mediate the direction-dependent transport of insulin. However, transport of a monomeric human insulin analog, Asp(B10)des(B28-30), across the Calu-3 cell monolayer was similar in both directions (BL to AP and AP to BL). The corresponding permeability, Papp ¼ 2.9 0.2 108 cm/s, was not significantly different from the permeability of zinc insulin in the BL to AP direction. The paracellular pathway seems to play a major role in the insulin transport across the Calu-3 cell monolayers. We hypothesize that the transport of zinc insulin oligomers is restricted at the AP surface by the presence of the tight junctional complexes. From the BL side, oligomers may undergo dissociation in the intercellular space and diffuse readily as monomers to the AP surface of the membrane. ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:1135–1146, 2002
Keywords: pulmonary delivery; Calu-3 cells; asymmetric insulin transport; insulin receptor; monomeric insulin; insulin aggregation
INTRODUCTION Pulmonary delivery has resulted in a substantially higher systemic bioavailability of several proteins and peptides relative to oral administration.1 The inhalation route seems to be the leading mode of nonparenteral administration of insulin.2 Previous studies in our laboratory using animal Isabelle Pezron is on leave from the De´partement de Ge´nie Chimique, Universite´ de Technologie de Compie`gne, BP 529, 60205 Compie`gne cedex, France. Correspondence to: Ashim K. Mitra (Telephone: 816-2351615; Fax: 816-235-5190; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 91, 1135–1146 (2002) ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association
models have shown that nasal and pulmonary routes have high potential for systemic insulin absorption.3,4 However, the exact mechanism of pulmonary insulin transport has not so far been delineated because of the anatomical and physiologic complexities of the lung epithelia (e.g., tracheal, bronchial, bronchiolar, alveolar). A drug can be delivered by intratracheal instillation, where it is deposited in the central part of the conducting airway, or by aerosol deposition, where the drug is distributed more uniformly and reaches peripheral alveolar region of the lung.5 The bioavailability of insulin after intratracheal instillation is usually found to be lower than the bioavailability of aerosolized insulin.6 However,
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pulmonary absorption of insulin is enhanced in the presence of absorption promoters such as bile salts7 and/or phospholipids.8 The enhancement mechanisms are not well understood and can involve different factors including dissociation of insulin oligomers, opening of the pulmonary epithelial tight junctions, aminopeptidase inhibition, and a ciliostatic effect.7 Cell monolayer culture of the lung epithelium— the primary barrier to pulmonary drug transport— has been developed to better delineate drug absorption mechanisms.9 According to a recent review, the most promising lung-derived cell lines are Calu-3 and 16HBE14o for transport and absorption studies, and BEAS-2B for drug metabolism studies.10 These are human bronchial cell lines. The only available alveolar cell line is A549, but its ability to form functional tight junctions has been questioned.10,11 Alveolar epithelial culture models have also been developed using primary cell culture,12 but they may be more delicate and prone to extreme variability. Most of the current drugs delivered through inhalation devices are primarily deposited in the lung airways and Calu-3 seems to be the most appropriate currently available cell line to study drug absorption across the airway epithelium.9–11 Calu-3 cells form polarized monolayers and express functional tight junctions, restricting the paracellular transport of hydrophilic solutes.10,11 Until now, Calu-3 cell monolayers were mainly grown on air-interface culture to study the transport of ions through the cystic fibrosis transmembrane conductance regulator.13,14 Only recently, a few studies have reported permeability of drugs across this cell monolayer grown on air-interface conditions10,15–17 and liquid-covered culture.15,18,19 The aim of this study was to elucidate the mechanisms of insulin transport across Calu-3 cell monolayers grown at the liquid-covered interface. In this report, we describe the transport of insulin across Calu-3 cell monolayers grown on permeable filters in the absorptive [apical (AP) to basolateral (BL)] and secretory (BL to AP) directions. The possible involvement of the insulin receptor a-subunit on insulin transport was also examined. A major difficulty in determining insulin absorption mechanisms resides in the complexity of the insulin molecular structure. Insulin molecules have a strong tendency to aggregate into dimers and hexamers in the presence of zinc.20 The absorption of insulin depends on its association state, the monomer being the fastest absorbing species.21 However, the generation of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
monomeric insulin requires modification of the insulin molecule through recombinant DNA technology and site-specific mutagenesis, and optimum systems for its clinical applications are still under investigation.22 To investigate the effect of insulin association on the absorption process, we describe here the transport of zinc insulin and a monomeric insulin analog across the Calu-3 cell monolayer and discuss the differences observed as well as the possible mechanisms involved.
EXPERIMENTAL SECTION Materials Crystalline porcine zinc insulin (potency 26.3 U/mg) and lyophilized monomeric human insulin analog [Asp(B10)des(B28-30); potency 28.85 U/mg] were generously donated by Eli Lilly and Co. (Indianapolis, IN). Human lung carcinoma derived Calu-3 cells were obtained from American Type Culture Collection (ATTC, Rockville, MD). The growth medium was a 1:1 mixture of Dulbecco’s modified Eagle medium and Ham’s F-12 medium (DMEMF12, reference 12500-062) obtained from Life Technologies (Grand Island, NY). MEM nonessential amino acids, penicillin, streptomycin, sodium bicarbonate, and HEPES were obtained from Sigma (St. Louis, MO). Fetal bovine serum was purchased from Mediatech, Inc. (Herndon, VA). The buffer used in transport studies was Dulbecco’s modified phosphate buffer saline (DPBS), containing 129 mM NaCl, 2.5 mM KCl, 7.4 mM Na2HPO4, 1.3 mM KH2PO4, 1 mM CaCl2, 0.7 mM MgSO4, and 5.3 mM glucose at pH 7.4. These chemicals, of analytical grade, were obtained from Sigma. 14C-mannitol (50 mCi/mmol) was supplied by Amersham (Piscataway, NJ) and 14 C-PEG 4000 (11.2 mCi/g) by ICN (Costa Mesa, CA). Anti-human insulin receptor, a-subunit, chicken polyclonal immunoglobulin (Ig)Y, was purchased from Upstate Biotechnology (Lake Placid, NY) and secondary antibodies, peroxidase conjugated goat anti-chicken IgG, from Rockland (Gilbertsville, PA). 3,30 ,5,50 Tetramethylbenzidine (TMB) was obtained from Pierce (Rockland, IL). Coat-A-Count insulin radioimmunoassay kits were supplied by Diagnostic Products Corporation (Los Angeles, CA). Transwell1 filters (sixwell plate, clear polyester membrane, pore size 0.4 mm with an average of 4 106 pores/cm2, diameter 24 mm) and 12-well plates were purchased from Costar (Bedford, MA).
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Preparation of Insulin Solutions Zinc insulin or monomeric insulin powder was dissolved in a minimal volume of 0.1 N HCl solution to which sterile DPBS solution was added. The solution pH was adjusted to 7.4 by the addition of 0.1 N NaOH. A stock insulin solution (1 mg/mL) was freshly prepared before each experiment. For conducting transport experiments, insulin solutions were prepared by dilution of the stock solution. Concentrations are expressed in units/milliliter. Zinc insulin has a potency 26.3 U/mg; therefore 1 U/mL corresponds to 0.038 mg/mL (6.6 mM; Mw ¼ 5778). Insulin conformation in the stock solution was analyzed by using circular dichroism (CD) (cell path length 0.02 cm, Spectropolarimeter JASCO J/720; Japan Spectroscopic Co, Japan.). Cell Culture Calu-3 cells of passage number 20–40 were cultured in DMEM-F12 medium containing L-glutamine, supplemented with 10 to 15% fetal bovine serum (heat inactivated), 1% nonessential amino acids, 100 U/mL penicillin, 100 mg/mL streptomycin, 14 mM HEPES, and 29 mM sodium bicarbonate at pH 7.4. The cells were grown at 378C in a tissue culture incubator with 5% CO2 and 95% humidity. The medium was changed every other day. To conduct transport experiments, cells were grown at a density of 1.1 to 1.3 105 cells per cm2 on collagen-coated Transwell filters under liquidcovered culture conditions. The monolayer growth was observed by using optical microscopy (Carl Zeiss Telaval 31, Germany). The integrity of the cell monolayer was monitored by determining the transport of paracellular markers 14C-mannitol, or 14C-PEG 4000 and by measuring the transepithelial electrical resistance (TEER) with an EVOM Epithelial Voltohmmeter (World Precision Instruments, Inc., Sarasota, FL). Microscopy Light, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) were performed to characterize the morphology of Calu3 cells grown under liquid-covered culture conditions. Cells grown at confluency after 9 days were washed twice with cold DPBS, then with sodium cacodylate buffer for 5 min at 48C. The cells were fixed with 2% glutaraldehyde in 0.15 M sodium cacodylate buffer for 1 h at 48C, and then washed several times with cacodylate buffer. The cell sur-
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face was covered with a gelatin layer to protect the surface features, and subsequently fixed with osmium tetroxide for 30 min. Cells were processed for thin sectioning using routine procedures (section thickness 0.5 mm for light microscopy and 90 nm for TEM). The cells used for SEM were dehydrated with graded ethanol and the cell surface was coated with gold/palladium. An optical microscope (Olympus Bmax, Japan), a Jeol Electron Microscope, Japan (model JEM 1200 EX II) at 100 kV, and a Philips Electron Microscope, Holland (model XL-30 ESEM-FEG) were used. Transport Experiments Transport experiments were conducted in six-well Transwell plates both in the AP (1.5 mL), to BL (2.5 mL), and BL to AP direction at 378C. The receiver chamber contained only buffer, DPBS, whereas the donor chamber contained insulin, at various concentrations, or a radiolabeled paracellular marker (14C-mannitol, or 14C-PEG 4000). Samples (100–200 mL) from the receiver side were withdrawn at predetermined times (for up to 2 or 3 h) and were replaced by equal volumes of buffer. Insulin concentrations in the receiver chamber were determined using radioimmunoassay. In this method, 125I-insulin competes with the unlabeled insulin contained in receiver samples for binding to insulin-specific antibodies immobilized on a polypropylene tube. After overnight incubation, the tube bound fraction of labeled insulin is measured with a gamma counter (Beckman 5500, USA). The unlabeled insulin concentration is inversely related to the count obtained, and is determined by calibration with solutions of known insulin concentrations in DPBS. The concentrations of the radiolabeled markers were determined using a scintillation counter (Beckman LS6500, USA). Concentration in the receiver chamber (cR) was calculated by taking into account the loss in the receiver side due to periodic sampling: cR ¼ cn þ
vs n1 ci VR i¼1
ð1Þ
In eq. 1, cn is the concentration measured in sample number n, vs the sampling volume, and VR the volume of the receiver chamber. Transport experiments across Calu-3 cell monolayers as well as across the plain collagen-coated filter were performed. The cumulative amounts of solute transported cR(t)VR were plotted as a function of time. Each experiment was performed at least in JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
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triplicate. Statistical significance ( p < 0.05) of the data was analyzed with Student’s t-test. Transport Data Analysis Passive diffusion driven by a concentration gradient across a membrane was analyzed with the Fickian diffusion model. The kinetics of the solute appearance in the receiver chamber was calculated by integration of the flux equations under apparent steady-state conditions, which generates eq. 223: VD cR ðtÞ ¼ cD ð0Þ VR þ VD 1 1 1 exp A þ Papp t VR VD
ð2Þ
A is the membrane area (4.7 cm2) and Papp (in cm/s) denotes the solute apparent permeability. The indices D and R refer respectively to the donor and the receiver sides. When the amount transported is only a minute fraction of the donor amount, such that the concentration gradient remains constant, this equation was simplified and reduced to eq. 3, which denotes concentration as a linear function of time: cR ðtÞ ¼ cD ð0Þ
A Papp t VR
ð3Þ
The experimental permeability accounts for the permeability of the cell monolayer and also the permeability of the collagen-coated filter and the aqueous boundary layer (ABL) as represented by eq. 424: 1 1 1 1 ¼ þ þ Papp Pmonolayer Pcollagen-coated filter PABL
ð4Þ
The last two terms were determined by measuring the permeability of the solute across the collagen-coated filter containing no cell monolayer. The permeability across the cell monolayer was then calculated from eq. 4. Insulin Receptor Binding Studies Enzyme-linked immunoabsorbent assay (ELISA)25 was used to assay insulin receptors on Calu-3 cells. Antibodies against human insulin receptor molecules (anti-IR), specific for the 135 kDa asubunit (extracellular fraction), were used for ELISA. The cells were grown on six-well inserts or 12-well plates. They were first washed with cold PBS, and then fixed with paraformaldehyde at JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
48C for 20 min. Subsequently, the cells were preincubated with a 3% bovine serum albumin solution at 378C for 1 h to reduce the nonspecific binding. Then, the AP or BL side of the monolayer was incubated with a solution of primary antibodies at a concentration of 10 mg/mL for 2 h at 378C. After washing with PBS, a solution of secondary antibodies (anti-chicken IgG peroxidase conjugate, concentration 0.13 mg/mL) was applied to both sides of the chamber together with 0.1% bovine serum albumin for 2 h at 378C. After several washes with PBS, the cells were incubated with the peroxidase substrate-TMB, for 20 min. The reaction was stopped with 1 M sulfuric acid and the solution color was measured by using UVvisible spectroscopy (Beckman DU 7400) at 450 nm. A control study without primary antibodies was also performed. Insulin was coadministrated at different concentrations to investigate the competitive binding to insulin receptors a-subunits on Calu-3 cells. Each experiment was performed at least in triplicate. The effect of anti-IR molecules, applied to the AP or BL sides, on the insulin transport across Calu-3 cells, was also studied. In these experiments, Calu-3 cells were not fixed, but incubated with the primary antibody solution for 2 h at 378C before conducting any transport experiment.
RESULTS AND DISCUSSION Morphology of Calu-3 Cells The Calu-3 cells grown under liquid-covered conditions on collagen-coated Transwell filters formed a single layer of cells at confluency, as shown by light microscopy (Fig. 1A). TEM analysis revealed that the cells were interconnected by AP tight junctions and displayed morphologic characteristics similar to those observed with cells grown under air-interface conditions13,16 and under liquid-covered conditions on untreated filters19 (Fig. 1B). The presence of abundant cilia, and the production of mucus were evidenced by SEM (Fig. 1C and D). Contrary to what was observed in primary culture of tracheal or bronchial epithelial cells,26,27 it appears that only limited differences in morphology and differentiation are observed in Calu-3 cells grown at the liquid-covered interface compared with the air-interface growth condition. These results are in agreement with previous publications stating that tight junctions and desmosomes, cAMP-dependent chloride secretion,
Figure 1. (A) Light microscopy of a transversal section of confluent Calu-3 cells grown in liquid-covered conditions on collagen-coated filters illustrating monolayer formation (scale 10 mm). (B) TEM picture (scale 1 mm) showing characteristic features of the Calu-3 cells [tight junction (Tj), mucus secretion (ms), and microvilli (mv)]. (C) SEM picture showing abundant cilia (original magnification 15,000). (D) SEM picture showing both mucus secretion and cilia (original magnification 20,000). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
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Figure 1. (Continued)
mucus vesicles, and cilia were evidenced in Calu-3 with both culture conditions.15,18,19 The main difference observed was the shape of the cilia: thick and short in liquid-covered culture conditions compared with long and thin in air-interface culture conditions.15 Therefore, Calu-3 cell monolayers grown under liquid-covered interface seem to be suitable models to study drug transport across the airway epithelium. Calu-3 Cell Monolayer Integrity The growth of the Calu-3 cells was followed by microscopic observations as well as by the measurement of the TEER value associated with the monolayer. The Calu-3 cell monolayer was generally confluent after 6–7 days of growth and the transport experiments were performed after 8– 10 days of growth. The resistance of the monolayer was measurable after 4 days in culture and reached a high plateau value, 1000 150 O cm2 (after subtraction of the filter background), after 7–8 days. This value is in the same order as the ones reported in recent studies, ranging from 800 to 2000 O cm2 in air-interface conditions14 and from 700 to 2500 O cm2 in liquid-covered conditions.15,19 Other studies reported lower values of 100 to 300–600 O cm2.11,13,16–18 Microscopic experiments confirmed that the high value obtained JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
in this study is attributed to the formation of functional tight junctions and not to the formation of multilayers (Fig. 1A). Consistent TEER values, in addition to the examination of confluency and monolayer formation by optical microscopy, are necessary prerequisites to conducting any transport study. The integrity of the cell monolayer during a transport experiment was further investigated by using paracellular markers,28 14C-mannitol ˚ ) and 14C-PEG 4000 (Mw& (Mw ¼ 184, radius 3.9 A ˚ ) with a donor concentration of 4000, radius 15.9 A 0.5 mCi/mL in both cases. The appearance of paracellular markers concentration in the receiver chamber was linear with time and independent of the direction of transport (2-h experiment). Values of the apparent permeability of mannitol and PEG-4000 across Calu-3 cells are Papp ¼ 4.7 0.5 107 cm/s and Papp ¼ 0.96 0.05 107 cm/s, respectively, compared with Papp ¼ 4.3 0.2 105 cm/s and Papp ¼ 0.78 0.06 105 cm/s across the collagen-coated filter. The ratio between the solute permeability across the filter and across the monolayer denotes the ability of the cells to form tight junctions and to restrict solute movement. Apparent permeability values of 14C-mannitol and 14C-PEG 4000 in the presence of the Calu-3 cell monolayer were decreased similarly by a factor 91 14 and 81 13 compared with the
TRANSPORT PATHWAYS OF ZINC INSULIN
collagen-coated filter, suggesting that PEG 4000 ˚ ,28 are molecules, of hydrodynamic radius 15.9 A transported across the Calu-3 monolayer through the same aqueous pore system as mannitol. These results clearly suggest that the cell monolayer was the rate-limiting barrier to the transport and its integrity was maintained under these experimental conditions. The value obtained for 14 C-mannitol permeability across the Calu-3 cell monolayer is compatible with results reported for various pulmonary cell culture model systems, which ranged between 107 and 8.107 cm/s.10,29–31 Transport of Insulin Across Calu-3 Monolayers Polarization of Insulin Transport Figure 2 shows the cumulative amount of insulin transported across the Calu-3 cell monolayer from a donor zinc insulin (1 U/mL). The results indicate that insulin transport across the Calu-3 cell monolayer is direction-dependent. After 2 h, the amount of insulin transported from BL to AP was about 1 mU, whereas only 0.16 mU was transported from the AP to BL direction. The corresponding permeability values, obtained by linear fit of the data, are 3.0 0.2 108 cm/s and 0.41 0.02 108 cm/s in the BL to AP and AP to BL directions, respectively. Direction dependency was even more pronounced at higher donor concentration (12.5 U/mL; see insert in Fig. 2). The measure of the donor insulin concentrations at the end of transport experiments by highpressure liquid chromatography or UV-visible spectroscopy did not indicate any significant loss of donor insulin molecules.
Figure 2. Direction-dependent transport of zinc insulin across the Calu-3 monolayer formed on collagen-coated six-well plates for donor concentrations 1 and 12.5 U/mL (insert) at 378C.
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The permeation of a solute across a biological membrane is a complex phenomenon. Simultaneous pathways, i.e., paracellular and transcellular pathways, can contribute to the overall solute transport. The paracellular pathway represents the free diffusion of the solute through the aqueous channels between the cells. Elaborate models have been developed to take into account molecular size, restricted diffusion of large molecules, and electrical effects of small ions.24,32 The transcellular pathway, which represents the transport of the solute across the cell membrane, may be passive or involve pinocytosis and/or active transport mechanisms. The passive transcellular pathway is related to the partitioning of the solute onto the cell lipid membrane and is a predominant mechanism for the transport of lipophilic molecules. Direction-dependent transport of drugs can be observed under various circumstances. P-glycoprotein (P-gp) molecules are present in many tissues and often expressed on the AP side of various cell lines where they function as efflux pumps.33 Consequently, a greater transport of P-gp substrates is observed from the BL to the AP side than in the opposite direction. On the contrary, carrier-mediated transport of amino acids, driven by electrochemical gradients, is favored in the AP to BL direction.34 The presence of enzymes, located specifically on one side of the membrane, can also create transport directionality.35 Specific interaction of peptides with the tight junctional complex was observed in some cases, where tight junction opening occurred only from one side of the membrane.36 An asymmetric paracellular transport resulted from this particular interaction. In another example, receptormediated transcytosis of dimeric immunoglobulin was favored in the BL to AP direction in Calu-3, through receptors present predominantly on the BL surface of the membrane.19 We have examined these possibilities to elucidate the mechanism of insulin transport. The presence of P-gp molecules at the AP surface of Calu-3 cells has been investigated and reported in two recent publications.17,37 Contradictory results show on one hand P-gp efflux of rhodamine 123, a well-known P-gp substrate,17 and on the other hand, a very faint presence of P-gp molecules on Calu-3, which have to be induced by the addition of ouabain.37 P-gp substrates are usually lipophilic molecules with a rather low molecular weight,33 and no study has been conducted with insulin in that regard. To investigate this possibility, we studied the effect of a well-known P-gp JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
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substrate (verapamil, 100 mM) added to the AP chamber, on insulin transport. The presence of verapamil did not change the asymmetric character of the insulin transport, BL to AP directional flux being predominant over the AP to BL flux (data not shown). Consequently, P-gp efflux does not seem to be involved in the asymmetric insulin transport across the Calu-3 cell monolayer, which is consistent with previously published results with other substrates.18 Yahamara et al.35 studied the transport of bovine insulin (zinc-free) across a primary culture of rat alveolar epithelium. The permeability obtained in the BL to AP and AP to BL directions was 0.8 0.1 and 1.2 0.2 108 cm/s, respectively, values which are not significantly different from each other. When using radioactive 125I-insulin in nanomolar donor concentration range, enzymatic degradation of insulin was observed and appeared to be prevalent on the AP side of the membrane. To analyze whether the asymmetric transport observed in our study was due to the specific presence of insulin degrading enzyme on one side of the Calu-3 cell monolayer, we performed the insulin transport experiment in the presence of peptidase inhibitors (bestatin, 1 mM and captopril, 2 mM). The asymmetry and extent of the insulin transport was not affected by the presence of these inhibitors in the donor or the receiver chambers (data not shown). The association of insulin molecules as oligomers may reduce the probability of enzymatic degradation of insulin, as reported previously.21 We also verified that zinc insulin molecules do not have a specific effect on the tight junction complex. Adding insulin to the donor chamber did not affect the 14C-mannitol transport in both directions. Any remaining mechanism that can explain the direction-dependent insulin transport is the receptor-mediated transcytosis of insulin, or an asymmetric transport due to specific interaction between insulin oligomers and the AP side of the membrane. These two possibilities were further investigated in detail and are presented in the sections below.
by an excess of unlabeled insulin.39 Internalization of insulin through the receptor-mediated process was proposed, but direction-dependent transport of unlabeled insulin by transcytosis through the receptor-mediated process has not been proven. In another study, in vivo transendothelial transport of insulin from plasma to interstitial fluid was found to be mainly caused by diffusion with no evidence of the receptor-mediated process.40 Calu-3 cells responded positively to ELISA with anti-human insulin receptor (a-subunit). Figure 3A compares the response of the BL and AP sides of a Calu-3 monolayer after incubation with anti-IR molecules. The percentage of receptor bound antibodies appears to be approximately three times higher on the BL side than the AP side. However, zinc insulin at concentrations of 1 U/mL and 2 U/mL does not appear to bind competitively to the receptors present on the BL side, as shown in Figure 3B.
Insulin Receptor Binding Studies The insulin receptor is widely expressed on mammalian tissues.38 A receptor-mediated transcytosis of insulin in the blood-brain barrier has been suggested.39 Binding of radiolabeled insulin to bovine brain microvessel endothelial cells was evidenced, polarized to the AP side, and inhibited JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
Figure 3. Response of Calu-3 cells to ELISA with anti-human insulin receptors (anti-IR). (A) Comparison of the responses of the AP and BL sides (in a six-well plate). (B) Influence of zinc insulin added on the BL side at concentrations of 1 and 2 U/mL on the receptor binding (12-well plates).
TRANSPORT PATHWAYS OF ZINC INSULIN
Anti-IR molecules added to the AP or BL donor chamber did not affect the insulin transport. Moreover, preliminary results involving the passage of 125I-insulin across Calu-3 cells also showed no inhibition of 125I-insulin transport when zinc insulin was added to the donor side (data not shown). Experiments at 48C were also attempted to confirm the absence of the receptor-mediated process. However, a decrease in TEER value of the Calu-3 cell monolayer together with an increased permeability of paracellular marker indicated a loss of integrity of the monolayer maintained at 48C (data not shown). Even though our results suggest that insulin receptors are present on the surface of Calu-3 cells and are more abundant on the BL side, the reason for a higher transport from BL to AP does not seem to be due to receptor-mediated transport. An aggregation of zinc insulin molecules in solution may hinder insulin for binding to the active sites of the receptors. Additional experiments will be necessary to confirm and quantitate the presence of insulin receptors on Calu-3 cells. Transport of a Monomeric Insulin Analog Asp(B10)des(B28-30) Zinc insulin exists predominantly in the mono˚ ) only at meric form (hydrodynamic radius 12.8 A 7 a very low concentration (< 10 M &0.015 U/mL) under neutral conditions.20,41 At higher concentrations, insulin self-associates into dimers, which in the presence of zinc, further assemble into ˚ .20 stable hexamers of hydrodynamic radius 28 A 20 Hvidt has characterized the formation of zinc insulin oligomers by light scattering and sizeexclusion chromatography. The equilibrium association constants obtained are K1,2 ¼ 14 104 M1 for the monomer-dimer equilibrium and K2,6 ¼ 15 1010 M2 or the dimer-hexamer equilibrium.20 Calculation of the corresponding molar oligomer concentrations yields to 2.9, 1.16, and 0.23 106 M for the monomers, dimers, and hexamers, respectively, which represent 44, 35, and 21% in weight of the total insulin concentration (1 U/mL, 6.6 106 M).42 It clearly shows that even if the monomers remain the dominant species, the formation of dimers and hexamers, including respectively 2 and 6 insulin molecules, results in a very significant reduction of the monomer concentration. These association properties have been shown to affect insulin absorption in vivo.21 Many insulin monomeric analogs have been developed, as reported recently, in order to better control biological
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action of insulin in diabetes mellitus.22 Modifications in the insulin B chain, particularly on the B10 and B26–30 positions, have a dramatic effect on insulin association.43 Monomers may still form dimers at high concentrations, but are unable to form zinc-coordinated hexamers.43 The insulin analog used in this study is Asp(B10)des(B28-30), in which the histidine residue in B10 has been replaced by an aspartine residue, and the B28–30 residues have been removed. The CD spectra of zinc insulin and the human insulin analog are shown in Figure 4 at a concentration of 1 mg/mL (about 25 U/mL). The far-UV region of the CD spectrum of zinc insulin is characterized by two negative maxima at 209 and 222 nm. The 222-nm band is attributed to the b-structure, characteristic of the dimer conformation, whereas the 209-nm band, assigned to the a-helix structure, is a characteristic feature of the monomer.44 The attenuation of the 222 nm maximum and the increase in intensity of the 209 nm maximum in the insulin analog signal a predominance of monomeric structures.44,45 No data on the transport of this monomeric insulin analog across cell monolayer model have been reported so far. The results of the transport studies with the monomeric insulin analog are displayed in Figure 5. Contrary to zinc insulin, no directiondependence of the transport was observed. The amount of monomeric insulin transported in 2 h in both directions was around 1.2 mU, which is not significantly different from what was obtained with zinc insulin in the BL to AP direction. The corresponding permeability (calculated by linear
Figure 4. CD spectra showing mean residue ellipticity of porcine zinc insulin and monomeric human insulin analog Asp(B10)des(B28–30) in the far-UV region. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
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Figure 5. Unidirectional transport of human monomeric insulin analog Asp(B10)des(B28–30) across the Calu-3 monolayer formed on collagen-coated six-well plates (donor concentration 1 U/mL, 378C).
fit of the data between 20 and 120 min) is 2.9 0.2 108 cm/s. The unidirectional permeability of monomeric insulin across the collagen-coated filter was determined to be 2.7 0.2 106 cm/s. Therefore, the permeability of monomeric insulin across the Calu-3 cell monolayer is decreased by a factor 93 14 compared with the collagen-coated membrane, which is not significantly different from the ratios obtained for the paracellular markers 14C-mannitol and 14C-PEG 4000. Concentration dependence studies of the permeability of monomeric insulin across Calu-3 cell monolayers revealed no saturation effect. Moreover, monomeric insulin molecules did not compete with anti-IR molecules for binding to insulin receptors, which was observed for zinc insulin (data not shown). Consequently, paracellular permeability appears to be the major pathway for monomeric insulin transport across Calu-3 cell monolayers. The modification of the insulin chain chemical composition also affects the isoelectric point and the charges present in the chain. The replacement of the histidine residue by an aspartine residue in B10 leads to a decrease in isoelectric point from 5.6 to 4.55.43 The monomeric insulin chain is expected to bear more fractional negative charges at pH ¼ 7.4 than the corresponding porcine zinc insulin chain. However, electric field effects on paracellular transport of solutes are only significant for small molecules.32 In the case of macromolecules, size effects are expected to be predominant, and in the Caco-2 model, charge effects were undetectable for molecule sizes ˚ .32 Therefore, the charge effects are above 6 A not likely to explain the difference in transport patterns of zinc and monomeric insulin. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002
We can deduce from these interesting results that the directionality of zinc insulin transport is likely to be caused by the presence of insulin oligomers. According to literature data, pulmonary epithelial membrane contains intercellular junctions with a large distribution of pore size. A majority of small pore size (< 1 nm) is present together with a few numbers of large pores (4– 8 nm, sometimes up to 25 nm), mediating the transport of macromolecular solutes.9 A solute with a radius close to the junctional size will undergo increasing restricted diffusion because of steric hindrance at proximity of the junction, and will experience increasing frictional resistance with the pore wall during transport.9 Although the size distribution of the intercellular junctions in the Calu-3 cell monolayer is unknown, it is reasonable to assume that the insulin oligomers may be subjected to restricted diffusion. The strongest restriction occurs through the tight junctional complexes, located at the uppermost AP region of the membrane.46 We suggest that the asymmetric transport arises from restricted diffusion of zinc insulin oligomers at the AP surface because of the presence of the tight junctional complexes. In addition, insulin oligomers may interact with muco-proteins secreted on the AP side of the membrane. On the BL side, the insulin oligomers may readily enter the intercellular space. The insulin concentration inside the intercellular aqueous pores decreases and reaches a very low value near the AP surface, because of the concentration gradient across the membrane. Dissociation of insulin oligomers can then take place and the monomers diffuse freely to the AP side of the membrane. This hypothesis is supported by the similarity of permeability values for zinc insulin in the BL to AP direction and the permeability value obtained for monomeric insulin.
CONCLUSION The results reported herein indicate that the Calu-3 cell monolayer grown under liquid-covered conditions, like the air-interface model, is also a suitable cell culture model for conducting transport studies. High TEER value and low mannitol permeability demonstrate that the confluent monolayer is the rate-limiting barrier to solute transport. Restricted insulin transport across the Calu-3 cell line is consistent with the previous in vivo results obtained from intratracheal
TRANSPORT PATHWAYS OF ZINC INSULIN
instillation experiments in the absence of any absorption enhancer.8 P-gp efflux or specific enzymatic degradation did not seem to be major contributing factors for higher insulin transport in the BL to AP direction. Insulin receptors, more abundant on the BL side than on the AP side of Calu-3 cells, did not mediate the direction-dependent transport of insulin. Experiments conducted with a monomeric insulin analog revealed that the paracellular pathway plays a major role in insulin transport. Also, molecular aggregation and size restriction effects are likely to hinder the diffusion of the insulin oligomers, particularly on the AP side of the membrane where the tight junction complexes are located. From the BL side, oligomers may undergo dissociation in the intercellular space and diffuse freely as monomers to the AP region of the membrane.
ACKNOWLEDGMENTS We thank Dr. Douglas Law and Jessica Kueger, from the Department of Biological Sciences, UMKC, and Dr. Vladimir Dusevich, from the Dental School, UMKC, for sharing their expertise in electron microscopy. This work was supported by a grant from Hoechst-Marion-Roussel Pharmaceuticals, Kansas City, MO.
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