Dipeptide Transport Across Rat Alveolar Epithelial Cell Monolayers

The transepithelial transport and metabolism of two model peptides, glycyl-D-phenylalanine (Gly-D-Phe) and glycyl-L-phenylalanine (Gly-L-Phe), across primary cultured monolayers of rat alveolar epithelial cells were studied. These tight monolayers (>2000 -cm²) exhibited type I pneumocyte morphological and phenotypic characteristics. A reverse-phase HPLC was used to monitor the appearance of parent dipeptides and their metabolites (D- or L-Phe) in the receiver fluid. The apparent permeability coefficient (P_app) for Gly-D-Phe was about 1.6 × 10^–7 cm/sec at both 1 and 10 mM and in both the apical-to-basolateral (AB) and the basolateral-to-apical (BA) directions. In contrast, the P _app of Gly-L-Phe at 1 mM was about two times higher than that at 10 mM in the AB direction. The P _app of Gly-L-Phe in the BA direction at either concentration was about the same (about 1.4 × 10^–7 cm/sec). Whereas no metabolite was detected during Gly-D-Phe transport, the proportions of a metabolite, L-Phe, observed at 4 hr in the basolateral receiver fluid for 1 and 10 mM apical donor Gly-L-Phe accounted for 83 and 77% of the estimated total Gly-L-Phe (i.e., L-Phe + Gly-L-Phe), respectively. The corresponding values in the BA direction were 40 and 19% of the estimated total Gly-L-Phe in the apical receiver reservoir. Metabolism of Gly-L-Phe was significantly reduced in the presence of 3 µM actinonin (an inhibitor relatively specific for aminopeptidase M) in the apical but not the basolateral fluid. Under all experimental conditions, the monolayers remained intact, as indicated by no appreciable changes in the bioelectric parameters of transepithelial potential difference and electrical resistance. The above data provide evidence for cellular metabolism of Gly-L-Phe as well as paracellular restricted diffusional transport of intact Gly-D-Phe and Gly-L-Phe and comparatively lower transcellular transport of Gly-L-Phe across the rat alveolar epithelial cell monolayer.     

Horseradish Peroxidase Transport Across Rat Alveolar Epithelial Cell Monolayers

Purpose. To evaluate the transport characteristics of horseradish peroxidase (HRP, a nonspecific fluid-phase endocytosis marker) across an in vitro model of tight (> 2,000 ohm-cm²) rat alveolar epithelial cell monolayers grown on tissue culture-treated polycarbonate filters. Methods. Unidirectional HRP fluxes were estimated from the appearance rate of HRP in the receiver fluid following instillation in the donor fluid as a function of donor [HRP] and temperature. Molecular species present in either bathing fluid were determined at the end of flux experiments using fluorescein isothiocyanate (FITC)-labeled HRP by gel permeation chromatography. Cell-associated HRP activity at the end of the transport experiment was determined, as were the rates of recycling and transcellular movement of HRP. An enzymatic assay was used to quantify HRP activity in the bathing fluid and cells. Results. Unidirectional HRP fluxes were symmetric and increased linearly with up to 50 µM donor [HRP]. The apparent permeability coefficient of HRP was reduced by 3.5 times upon lowering the temperature from 37 to 4°C. About 50% of the FITC-labeled species present in either receiver fluid was intact HRP. Cell-associated HRP estimated from apical HRP incubation was about 4 times greater than that from basolateral incubation. Recycling into apical fluid of cell-associated HRP following apical incubation occurred rapidly with a half-time (T_1/2) of ~5 min, reaching a plateau at ~67% of the initial cell-associated HRP, while transcellular movement of HRP (into basolateral fluid) took place with a T_1/2 of ~20 min, attaining a steady-state at ~13% of the initial cell-associated HRP. Basolateral recycling of HRP was also rapid (T_1/2 = ~5 min) reaching a steady-state at ~35% of the initial basolaterally-bound HRP. Transcellular movement of HRP following basolateral incubation was slower (T_1/2 = ~70 min), leveling off at 50% of the initial cell-associated HRP. Conclusions. HRP appears to be transported relatively intact (~50%) across rat alveolar epithelial barrier via nonspecific fluid-phase endocytosis. The transepithelial pinocytotic rate of alveolar epithelial cells is estimated to be about 25 nL/cm²/h.     

Effects of Protease Inhibitors on Vasopressin Transport Across Rat Alveolar Epithelial Cell Monolayers

The transepithelial transport of arginine vasopressin (AVP) across cultured rat alveolar epithelial cell monolayers was studied. At 0.1 nM donor [¹²⁵I]AVP, the radiolabel flux measured in the apical-to-basolateral (AB) direction was about 10 times greater than that in the reverse (BA) direction. HPLC analyses of the basolateral receiver fluid collected at the end of these flux measurements showed that about 97% of total [¹²⁵I]label represented subspecies of AVP, whereas the apical receiver fluid contained largely intact AVP (-85% of total [¹²⁵I]label). Both donor fluids contained virtually no degradation products of AVP (>99%). In the presence of an excess 0.1 mM unlabeled AVP in the apical donor fluid, the P_app for radiolabeled AVP in the AB direction was decreased by ~68%, while the fraction of intact AVP in the basolateral receiver fluid was increased six-fold as compared to that observed at 0.1 nM [¹²⁵I]AVP alone. Under this condition, the flux of intact AVP was approximately the same in both directions. When the concentration of apical camostat mesylate, an aminopeptidase inhibitor, was varied from 0 to 2 mM, the radiolabeled flux in the AB direction (with 0.1 nM [¹²⁵I]AVP in the donor fluid) was significantly decreased in a dose-dependent manner, yielding commensurably elevated concentrations of intact AVP in the basolateral receiver fluid. In contrast, leupeptin (0.5 mM), a serine protease inhibitor, was without effect. These data, taken together, suggest that apically-presented AVP undergoes proteolysis (most likely by peptidases localized at apical cell membranes of alveolar epithelium). It does not appear that intact AVP traverses the alveolar epithelium by saturable processes but primarily via passive diffusional pathways. Thus, the high bioavailability reported in previous studies on the pulmonary instillation and/or delivery via aerosolization of AVP is likely due to passive diffusion of the peptide utilizing the large surface area available in the distal respiratory tract of the mammalian lung. Furthermore, inclusion of appropriate protease inhibitor may increase the overall transport of intact AVP across the alveolar epithelial barrier.     

Enhancement of Insulin Transport Across Primary Rat Alveolar Epithelial Cell Monolayers by Endogenous Cellular Factor(s)

Purpose To characterize factor(s) contained in apical medium of primary cultured rat alveolar epithelial type II cell-like monolayers (RAECM-II) that enhance insulin absorption across alveolar epithelial cells. Materials and Methods Primary rat alveolar epithelial cell monolayers cultured on Transwells in the presence and absence of 10 ng/ml keratinocyte growth factor for 6 days were dosed from the apical compartment with radiolabeled insulin in: newborn bovine serum-containing medium (SM), conditioned medium from apical compartment of rat alveolar epithelial type I cell-like monolayers (RAECM-I) (CMI), or conditioned medium from apical compartment of RAECM-II (CMII). At the end of 2 h incubation, basolateral medium was collected and amounts of transported radiolabeled insulin were determined using a gamma counter. In order to determine the molecular size range of the enhancing factor(s), CMII was centrifuged in 50 kDa molecular weight cut-off Centricon tubes, and both retentate and filtrate were used as separate dosing solutions. Heat denaturation and ammonium sulphate precipitation were used to determine if the involved factor(s) represent proteins or other smaller soluble factors. Transalveolar transport rates of a paracellular marker, ¹⁴C-mannitol, and fluid-phase marker, horseradish peroxidase, were determined in the presence and absence of the factors. Effects of temperature (4, 16 and 37°C) on radiolabeled insulin fluxes were also measured. Results Conditioned medium obtained from the apical compartment of RAECM-II, CMII, increased transport of insulin across the monolayers when compared to SM or CMI. The enhancing effect of CMII was retained in the precipitate following ammonium sulfate treatment and in the retentate after Centricon filtration. The enhancing effect of CMII was significantly decreased when heated at 80°C for 15 min. CMII did not affect the transport of ¹⁴C-mannitol or HRP, while its effect on insulin transport was decreased by 87% when temperature was lowered to 4°C from 37°C. Conclusions Conditioned medium from type II cell-like monolayer cultures appears to contain protein factor(s) which seem to be involved in facilitating active transcellular transport of insulin across primary cultured RAECM-II.     

Organic Cation Transport in Rabbit Alveolar Epithelial Cell Monolayers

Purpose. To characterize organic cation (OC) transport in primary cultured rabbit alveolar epithelial cell monolayers, using [^l4C]-guanidine as a model substrate. Methods. Type II alveolar epithelial cells from the rabbit lung were isolated by elastase digestion and cultured on permeable filters pre-coated with fibronectin and collagen. Uptake and transport studies of [¹⁴C]-guanidine were conducted in cell monolayers of 5 to 6 days in culture. Results. The cultured alveolar epithelial cell monolayers exhibited the characteristics of a tight barrier. [¹⁴C]-Guanidine uptake was temperature dependent, saturable, and inhibited by OC compounds such as amiloride, cimetidine, clonidine, procainamide, propranolol, tetraethyl-ammonium, and verapamil. Apical guanidine uptake (K_m = 129 ± 41 M, V_max = 718 ± 72 pmol/mg protein/5 min) was kinetically different from basolateral uptake (K_m = 580 ± 125 (M, V_max = 1,600 ± 160 pmol/mg protein/5 min). [¹⁴C]-Guanidine transport across the alveolar epithelial cell monolayer in the apical to basolateral direction revealed a permeability coefficient (P_app) of (7.3 ± 0.4) × 10^-7 cm/sec, about seven times higher than that for the paracellular marker [¹⁴C]-mannitol. Conclusions. Our findings are consistent with the existence of carrier-mediated OC transport in cultured rabbit alveolar epithelial cells.     

Transport and Hydrolysis of Enkephalins in Cultured Alveolar Epithelial Monolayers

An in vitro cultured monolayer system of alveolar epithelial cells was used as a model to investigate transport and hydrolysis of two enkephalin peptides, Met-enkephalin (TGGPM) and [D-Ala²]Met-enkephalinamide (TAGPM), in pulmonary epithelium. Isolated alveolar type II cells formed continuous monolayers when grown on microporous tissue culture-treated polycarbonate filters in serum-free, hormonally defined medium. Transport and hydrolysis studies of enkephalins in the monolayer system obtained after 6 days in culture, using fluorescence reversed-phase HPLC, indicate a reduced but significant degradation of enkephalins in the alveolar epithelium compared to most other epithelia previously reported. Aminopeptidases and dipeptidyl carboxypeptidase represent two major hydrolytic enzymes for TGGPM, as indicated by the formation of the degradative products Tyr and Tyr-Gly-Gly, while dipeptidyl peptidase, which is responsible for the formation of Tyr-Gly, contributes much less. The enkephalinase inhibitor thiorphan failed to prevent the hydrolysis of TGGPM whereas the enkephalin analog TAGPM was relatively resistant to enzymatic cleavage. The rate of enkephalin transport across the alveolar epithelium was directly proportional to drug concentration and occurred irrespective of transport direction, suggesting passive diffusion as the major mechanism for transepithelial transport. Agents that affect paracellular transport pathways, e.g., EGTA and the calcium ionophore A-23187, greatly promoted the transport rate. The ionophore at high doses, in addition to promoting tight junction permeability, also caused cellular damage associated with a sustained rise in intracellular calcium levels, as indicated by nuclear propidium iodide fluorescence. The cultured monolayer of alveolar epithelium may be used to study pulmonary drug absorption, degradation, and toxicity.     

Gly-L-Phe Transport and Metabolism Across Primary Cultured Rabbit Tracheal Epithelial Cell Monolayers

Pharmaceutical Research -     

Systematic Investigations of the Influence of Molecular Structure on the Transport of Peptides Across Cultured Alveolar Cell Monolayers

Purpose. To determine how the structures of peptides influence theiralveolar permeability. Methods. The studies were performed using 14 synthetic modelpeptides, labelled with a novel, non-intrusive amino acid fluorophore, andtheir transport studied using rat alveolar cell monolayers cultured onpermeable supports. Results. The passage of the peptides across the epithelial cellmonolayers is shown to be primarily paracellular, with an inverse dependenceon molecular size, and an enhanced flux observed for cationic peptides.The apparent permeability coefficients (P _app ) for the peptides(together with those for other organic solutes, taken from the literature) areshown to be well-modelled assuming two populations of pores in themonolayers, modelled as cylindrical channels of radii 15 Å and 22nm. The former pores are shown to be numerically equatable withthe monolayer tri-junctional complexes, and the latter are taken asmonolayer defects. Conclusions. The various monolayer P _app values correlatewell with the results from in vivo transport experiments, and the conclusion isdrawn that the pulmonary delivery of peptide drugs is perfectlyexploitable.     

Monolayers of Human Alveolar Epithelial Cells in Primary Culture for Pulmonary Absorption and Transport Studies

Purpose. To develop a cell culture model of human alveolar epithelial cells in primary culture for the in vitro study of pulmonary absorption and transport. Methods. Type II pneumocytes isolated from normal human distal lung tissue by enzyme treatment and subsequent purification were plated on fibronectin/collagen coated polyester filter inserts, and cultured using a low-serum growth medium. Characterization of the cell culture was achieved by bioelectric measurements, cell-specific lectin binding, immunohistochemical detection of cell junctions, and by assessment of transepithelial transport of dextrans of varying molecular weights. Results. In culture, the isolated cells spread into confluent monolayers, exhibiting peak transepithelial resistance of 2,180 ± 62 X cm² and potential difference of 13.5 ± 1.0 mV (n = 30–48), and developing tight junctions as well as desmosomes. As assessed by lectin-binding, the cell monolayers consisted of mainly type I cells with some interspersed type II cells, thus well mimicking the situation in vivo. The permeability of hydrophilic macromolecular FITC-dextrans across the cell monolayer was found to be inversely related to their molecular size, with P_app values ranging from 1.7 to 0.2 X 10^–8 cm/sec. Conclusions. A primary cell culture model of human alveolar epithelial cells has been established, which appears to be a valuable in vitro model for pulmonary drug delivery and transport studies.     

Receptor-Mediated Peptide Delivery in Pulmonary Epithelial Monolayers

The present study investigated the feasibility of utilizing receptor-mediated endocytosis as a means to enhance peptide delivery to the pulmonary epithelium. The strategy employs a molecular conjugate consisting of a cognate moiety, transferrin (TF), covalently-linked to a model polypeptide, horseradish peroxidase (HRP), via a reversible disulfide linkage. A cultured alveolar epithelial monolayer system was used to simulate the conditions of the pulmonary epithelium and to allow accurate quantitation of intra- and transcellular peroxidase transport. The alveolar cells were isolated from rat lungs by enzymatic digestion and grown on microporous tissue culture-treated polycarbonate filters. A significant increase in the uptake of HRP by the cell monolayer was observed upon its conjugation with TF. The effect was found to be concentration-dependent, being more pronounced at low concentrations, i.e., 3.9- and 1.2-fold increase over unconjugated HRP controls at the concentration levels of 0.05 and 1.50 U/ml respectively. Effective peroxidase uptake was shown to require the TF cognate moiety for the cell surface receptor. Specific internalization of the conjugate by the TF endocytic pathway was verified by competition for the TF receptor. Conjugate internalization was not followed by a proportional increase in transcytosis, i.e., at 0.05 U/ml conjugate level, a 1.7-fold increase in transcytosis was observed as compared to 3.9-fold for endocytosis. Effective enhancement of transcytosis was achieved by treating the monolayers with brefeldin A (BFA), a compound known to affect intracellular transport of TF receptor complexes. At 1.6 µ/ml concentration level, BFA promoted a >20-fold increase in the rate of transcytosis of the conjugate in both the apical-to-basal and basal-to-apical directions. This effect was not associated with membrane leakage since BFA-treated monolayers maintained tight barrier to transport of the paracellular permeability solute ¹⁴C mannitol. In addition, BFA had no significant effect on the transport of free HRP. Instead, the effect of BFA on conjugate transport was mediated by TF receptors since excess free TF competitively inhibited transcytosis of the conjugate. Thus, our results are consistent with the TF receptor-mediated transport of the conjugate and its enhancement through the intracellular rerouting of the conjugate by BFA. The findings in this study may potentially be relevant to the design of drug delivery systems that can enhance intra- or transcellular uptake of therapeutic peptides in the pulmonary epithelium.     

Comparison of Albumin Uptake in Rat Alveolar Type II and Type I-like Epithelial Cells in Primary Culture

Purpose To elucidate and compare the activity and mechanism of albumin uptake in primary cultured alveolar type II and type I-like epithelial cells. Materials and methods Type II epithelial cells isolated from rat lungs were cultured for 2 days at 5 × 10⁶ cells/35-mm dish or for 6 days at 2 × 10⁶ cells/35-mm dish. The mRNA expression of marker genes and FITC-albumin uptake were examined. Results The cells cultured for 2 days exhibited cuboidal type II epithelial morphology with lamellar bodies inside the cells, while the cells cultured for 6 days exhibited squamous type I epithelial morphology. These morphological characteristics were consistent with the changes in mRNA expression pattern of marker genes. FITC-albumin uptake in both cells was temperature-dependent and was inhibited by metabolic inhibitors and bafilomycin A₁. The rate of uptake was much higher in type II cells than type I-like cells. In both cells, FITC-albumin uptake was inhibited by clathrin mediated-endocytosis inhibitors, but not by caveolae mediated-endocytosis inhibitors. Conclusions These findings indicate that albumin in alveolar lining fluid is internalized into type II and type I epithelial cells via clathrin-mediated endocytosis, and the rate of albumin uptake is higher in type II cells than type I cells.     

Permeability of Peptides and Proteins in Human Cultured Alveolar A549 Cell Monolayer

Purpose. The transport of peptides or proteins across the alveolar cell monolayer was studied in vitro in order to elucidate their transport pathway. Methods. The permeability of 14 peptides or proteins and 6 dextrans with MW 1,000~150,000 was measured in cultured human lung adenocarcinoma A549 cell monolayers at 37°C or 4°C. The stability of the tested peptides and proteins was also evaluated. Results. The permeability coefficients of these macromolecules across the A549 cell monolayer at 37°C ranged from 10 ^–5 to 10 ^–7 (cm/sec), and exhibited a good inverse correlation with molecular weight. All macromolecules were stable throughout the transport experiment, and degradation by proteases was minimal. Permeability at 4°C did not differ from that at 37°C. Clear selectivity for direction of transport was not observed. Conclusions. These results suggested that the tested peptides and proteins appeared to penetrate the A549 cell monolayer via a paracellular route by passive diffusion.     

Transepithelial Transport of Diphenhydramine Across Monolayers of the Human Intestinal Epithelial Cell Line Caco-2

Purpose. The transepithelial transport characteristics of theantihistamine, diphenhydramine, were studied in human intestinal Caco-2 cellmonolayers to elucidate the mechanisms of its intestinal absorption. Methods. The transepithelial transport and the cellular accumulationof diphenhydramine were measured using Caco-2 cell monolayersgrown in Transwell chambers. Results. The transepithelial transport of diphenhydramine from theapical to basolateral side was saturable, and the flux and cellularaccumulation of diphenhydramine were dependent on the apicalextracellular pH (pH 7.4 > 6.5 > 5.5). Transport and accumulation ofdiphenhydramine from the apical side were inhibited by anotherantihistamine, chlorpheniramine, while typical substrates for the renal organiccation transport system such as tetraethylammonium, cimetidine andguanidine had no effect. The transepithelial transport and cellularaccumulation of diphenhydramine from the basolateral side were alsopH-dependent and inhibited by chlorpheniramine. In addition, intracellulardiphenhydramine preloaded was preferentially effluxed to the apicalside, suggesting the involvement of the secretory pathway indiphenhydramine transport. Furthermore, diphenhydramine uptake from boththe apical and basolateral sides was stimulated by preloadingmonolayers with chlorpheniramine (trans-stimulation effect). Conclusions. Transepithelial transport of diphenhydramine acrossCaco-2 cells is mediated by pH-dependent, specific transport systemsthat exist in both the apical and basolateral membranes.     

Melittin as an Epithelial Permeability Enhancer I: Investigation of Its Mechanism of Action in Caco-2 Monolayers

Purpose Melittin is an amphipathic antimicrobial peptide which has been shown to enhance the permeability of mannitol and reduce transepithelial electrical resistance (TER) across Caco-2 monolayers. The aim of this work was to further examine the potential of melittin as a paracellular permeability enhancer and to investigate the mechanism of interaction with tight junction proteins in Caco-2. Materials and Methods The permeability of a range of fluorescent markers of differing molecular weights across monolayers was examined and immunofluorescence and western blotting analysis of tight junction proteins were also carried out. The mechanism of TER reduction was also examined using cell signalling inhibitors. Results Apical but not basolateral addition of melittin increased the permeability of a range FITC-dextrans (4–70 kDa) across monolayers. Melittin effects were reversible and no cytotoxicity was evident in polarized Caco-2 epithelia at the concentrations used. Altered expression of ZO-1, E-cadherin and F-actin was also detected. The phospholipase A2 inhibitors, aristolochic acid and indomethacin and the cyclooxygenase inhibitor, piroxicam, partially attenuated melittin-induced TER reduction, suggesting that part of the mechanism by which melittin opens tight junctions involves prostaglandin signalling. Conclusions Apically-added melittin opens tight junctions, causing dramatic TER reductions with significant increases in flux of dextrans. These effects appear mediated in part via PLA2 and involve alterations in specific tight junction proteins.     

Effect of Restricted Conformational Flexibility on the Permeation of Model Hexapeptides Across Caco-2 Cell Monolayers

Purpose. To determine how restricted conformational flexibility of hexapeptides influences their cellular permeation characteristics. Methods. Linear (Ac-Trp-Ala-Gly-Gly-X-Ala-NH₂; X = Asp, Asn, Lys) and cyclic (cyclo[Trp-Ala-Gly-Gly-X-Ala]; X = Asp, Asn, Lys) hexapeptides were synthesized, and their transport characteristics were assessed using the Caco-2 cell culture model. The lipophilicities of the hexapeptides were determined using an immobilized artificial membrane. Diffusion coefficients used to calculate molecular radii were determined by NMR. Two-dimensional NMR spectroscopy, circular dichroism, and molecular dynamic simulations were used to elucidate the most favorable solution structure of the cyclic Asp-containing peptide. Results. The cyclic hexapeptides used in this study were 2–3 times more able to permeate (e.g., P_app = 9.3 ± 0.3 × 10^–8 cm/sec, X = Asp) the Caco-2 cell monolayer than were their linear analogs (e.g., P_app = 3.2 ± 0.3 × 10_–8 cm/sec, X = Asp). In contrast to the linear hexapeptides, the flux of the cyclic hexapeptides was independent of charge. The cyclic hexapeptides were shown to be more lipophilic than the linear hexapeptides as determined by their retention times on an immobilized phospholipid column. Determination of molecular radii by two different techniques suggests little or no difference in size between the linear and cyclic hexapeptides. Spectroscopic data indicate that the Asp-containing linear hexapeptide exists in a dynamic equilibrium between random coil and -turn structures while the cyclic Asp-containing hexapeptide exists in a well-defined compact amphophilic structure containing two -turns. Conclusions. Cyclization of the linear hexapeptides increased their lipophilicities. The increased permeation characteristics of the cyclic hexapeptides as compared to their linear analogs appears to be due to an increase in their flux via the transcellular route because of these increased lipophilicities. Structural analyses of the cyclic Asp-containing hexapeptide suggest that its well-defined solution structure and, specifically the existence of two -turns, explain its greater lipophilicity.     

Neutrophil-Mediated Transport of Liposomes Across the Madin Darby Canine Kidney Epithelial Cell Monolayer

Targeted drug delivery to peripheral blood neutrophils (PMNs) should be of therapeutic potential in various disease states. In addition, substances taken up by PMNs in the circulation may be delivered to an extravascular site via the naturally occurring cell infiltration. The present study employs an in vitro chemotaxis model to test whether particulate drug carriers such as liposomes can be transported across a cellular barrier by migrating PMNs. The system contained 10⁷ human PMNs/ml, 0.3-µm liposomes at a total lipid concentration of 2.5 mM, and 10% autologous human serum in the apical side of a confluent Madin Darby canine kidney (MDCK) epithelial cell monolayer of 4.71 cm². The MDCK cells were grown on a polycarbonate membrane with 3-µm pores without any extracellular matrix, and 10^–7 M f-Met-Leu-Phe was added to the basolateral side as a trigger of chemotaxis. The aqueous phase of the reverse-phase evaporation vesicles (REVs) contained lucifer yellow CH (LY) and [¹⁴C]sucrose. The lipid bilayer of the REVs was spiked with [³H]dipalmitoylphosphatidylcholine (DPPC). Transmission electron micrographs showed that, in response to the formyl peptide, PMNs adhered to the apical surface of MDCK cells, emigrated across the MDCK cell layer, passed through the 3-µm pores in the polycarbonate membrane, and finally, appeared in the bottom well. Epifluorescence micrographs showed that most, if not all, of the migrated PMNs contained punctate fluorescence derived from LY. Transport data over a 3.5-hr period indicated that those markers that appeared in the basal side were indeed transported by phagocytosis of REVs by PMNs and that intact serum was an essential component in the process. The PMN-mediated transport of REVs may serve as a possible targeted drug delivery to an extravascular site in vivo in various inflammatory diseases.     

Effect of Size and Charge on the Passive Diffusion of Peptides Across Caco-2 Cell Monolayers via the Paracellular Pathway

Purpose. To evaluate the effect of size and charge on the permeation characteristics of peptides across the intestinal mucosa. Methods. The lipophilicities of neutral, positively and negatively charged capped amino acids (Asn, Lys, Asp), tripeptides (Ac-Gly-X-Ala-NH₂; X = Asn, Lys, Asp) and hexapeptides (Ac-Tip-Ala-Gly-Gly-X-Ala-NH₂; X = Asn, Lys, Asp) were estimated using an immobilized artificial membrane. The diffusion coefficients used to calculate the molecular radii were measured by NMR. The transport characteristics of the model peptides were determined across Caco-2 cell monolayers. Results. When model compounds having the same charge were compared, permeation was highly size-dependent (capped amino acids > tripeptides > hexapeptides), suggesting transport predominantly via the paracellular route. For example, the flux of the negatively charged Asp amino acid (P_app = 10.04 ± 0.43 × 10^–8 cm/s) was 3 times greater than that observed for the Asp-containing hexapeptide (P_app = 3.19 ± 0.27 × 10^–8 cm/s). When model compounds of the same size were compared, permeation across the cell monolayer was charge-dependent (negative < positive neutral). For example, the neutral, Asn-containing tripeptide (P_app = 25.79 ± 4.86 × 10^–8 cm/s) was substantially more able to permeate the Caco-2 cell monolayer than the negatively charged Asp-containing tripeptide (P_app = 7.95 ± 1.03 × 10^–8 cm/s) and the positively charged Lys-containing tripeptide (P_app = 9.86 ± 0.18 × 10^–8 cm/s). The permeability of the cell monolayer to peptides became less sensitive to net charge as the size of the peptides increased. Conclusions. A positive net charge of hydrophilic peptides enhances their permeation across the intestinal mucosa via the paracellular pathway. With increasing molecular size, molecular sieving of the epithelial barrier dominates the transport of peptides, and the effect of the net charge becomes less significant.     

Caco-2 Cell Monolayers as a Model for Drug Transport Across the Intestinal Mucosa

Human colon adenocarcinoma (Caco-2) cells, when grown on semipermeable filters, spontaneously differentiate in culture to form confluent monolayers which both structurally and functionally resemble the small intestinal epithelium. Because of this property they show promise as a simple, in vitro model for the study of drug absorption and metabolism during absorption in the intestinal mucosa. In the present study, the transport of several model solutes across Caco-2 cell monolayers grown in the Transwell diffusion cell system was examined. Maximum transport rates were found for the actively transported substance glucose and the lipophilic solutes testosterone and salicylic acid. Slower rates were observed for urea, hippurate, and salicylate anions and were correlated with the apparent partition coefficient of the solute. These results are similar to what is found with the same compounds in other, in vivo absorption model systems. It is concluded that the Caco-2 cell system may give useful predictions concerning the oral absorption potential of new drug substances.     

Stimulation of Phagocytic Activity of Alveolar Macrophages Toward Artificial Microspheres by Infection with Mycobacteria

Purpose The purpose of this study is to know the effect of uptake of mycobacteria on the phagocytic activity of alveolar macrophage (Mφ) cells toward poly(lactic-co-glycolic) acid (PLGA) microspheres (MS) loaded with the anti-tuberculosis agent rifampicin (RFP-PLGA MS). Materials and Methods Biological functions such as phagocytic activity toward PLGA MS loaded with fluorescent coumarin (cPLGA MS) and toward polystyrene latex MS (PSL MS), and generation of tumor necrosis factor-α (TNF-α) and nitric oxide (NO) were examined using alveolar Mφ cell NR8383 after they had phagocytosed Mycobacterium bovis Calmette-Guérin (BCG), heat-killed BCG (h-kBCG) or Escherichia coli. Results The ingestion of BCG, h-kBCG, and E. coli did not affect the viability of the Mφ cells within 2 days. The phagocytosis caused generation of TNF-α and NO, being more significant with E. coli than with both types of BCGs. The phagocytosis of both types of BCGs stimulated the phagocytic uptake of cPLGA and PSL MS’s, which took place prior to the generation of TNF-α or NO, but that of E. coli suppressed the uptake of both MS’s. Conclusion Mycobacterial infection stimulated the phagocytic uptake toward cPLGA MS. These results suggest that RFP-PLGA MS is favorable for overcoming tuberculosis.