Characterization of protein factor(s) in rat bronchoalveolar lavage fluid that enhance insulin transport via transcytosis across primary rat alveolar epithelial cell monolayers.

The aim of this study was to characterize factor(s) in rat bronchoalveolar lavage fluid (BALF) that enhance(s) insulin transport across primary rat alveolar epithelial cell monolayers (RAECM) in primary culture. BALF was concentrated 7.5-fold using the Centricon device and the retentate was used to characterize the factor(s) involved in enhancing apical-to-basolateral transport of intact (125)I-insulin across various epithelial cell monolayers. These factor(s) enhanced transport of intact insulin across type II cell-like RAECM (3-fold increase) and type I cell-like RAECM (2-fold increase), but not across Caco-2 or MDCK cell monolayers. The insulin transport-enhancing factor(s) were temperature- and trypsin-sensitive. The mechanism of enhancement did not seem to involve paracellular transport or fluid-phase endocytosis, since fluxes of sodium fluorescein and FITC-dextran (70kDa) were not affected by the factor(s) in the apical bathing fluid. BALF enhancement of intact (125)I-insulin transport was abolished at 4 degrees C and in the presence of monensin, suggesting involvement of transcellular pathways. Sephacryl S-200 purification of BALF retentate, followed by LC-MS/MS, indicated that the high molecular weight (>100kDa) fractions (which show some homology to alpha-1-inhibitor III, murinoglobulin gamma 2, and pregnancy-zone protein) appear to facilitate transcellular transport of insulin across RAECM.     

Transcytosis of GCSF-Transferrin Across Rat Alveolar Epithelial Cell Monolayers

Purpose. The purpose of this study was to use primary cultured rat alveolar epithelial cell monolayers to examine the potential of using transferrin receptor (TfR)-mediated transcytosis for noninvasive systemic protein drug delivery via the pulmonary route. Methods. Freshly isolated rat type II pneumocytes were plated onto tissue culture-treated polycarbonate 12-mm Transwells. AEC monolayers (G 2500 cm²) were treated with keratinocyte growth factor (10 ng/mL) for maintenance of type II cell-like characteristics. Filgrastim (GCSF)-Tf conjugates were prepared using the linkers SPDP and DPDPB. TfR-specific binding and uptake were determined using ¹²⁵I-Tf and ⁵⁹Fe-Tf treatment, respectively. Apical-to-basolateral (A-to-B) transferrin receptor (TfR)-mediated transcytosis was determined by dosing the apical compartment with 1.5 g/mL of ¹²⁵I-Tf or ¹²⁵I-GCSF-Tf. Nonspecific TfR-independent transport of ¹²⁵I-Tf and ¹²⁵I-GCSF-Tf was determined in parallel by including 150 g/mL of nonradiolabeled Tf. Basolateral samples (500 L) were taken at 2, 4, and 6 h post-dosing, subjected to 15% trichloroacetic acid precipitation, and assayed in a Packard gamma counter. TfR-specific transport was determined as the difference between total and nonspecifc transport. The effects of brefeldin-A (BFA) on TfR distribution and (A-to-B) transport of ¹²⁵I-Tf, ¹²⁵I-GCSF and ¹²⁵I-GCSF-Tf was studied by including the agent in the apical fluid at 1 g/mL.Results. BFA treatment resulted in a small significant reduction in TfR at the basolateral surface of type II cell-like monolayers, while it had no effect on TfR distribution in type I cell-like monolayers. In contrast, BFA treatment significantly altered the endocytosis of TfR, reducing the basolateral uptake of ⁵⁹Fe-Tf while greatly increasing the apical uptake of ⁵⁹Fe-Tf. BFA treatment, however, did not affect the TfR-specific uptake of ⁵⁹Fe-Tf in type I cell-like monolayers. TfR-specific apical-to-basolateral transcytosis of ¹²⁵I-Tf and ¹²⁶I-GCSF-Tf conjugates was significantly enhanced in the presence of BFA in type II cell-like monolayers, whereas it had no effect on apical-to-basolateral transport of ¹²⁵I-GCSF. BFA-enhanced transport of ¹²⁵I-GCSF-Tf was approximately 3-fold higher than that of ¹²⁵I-GCSF in the presence or absence of BFA. Moreover, ¹²⁵I-GCSF transport in the presence of BFA was not significantly different from non-specific ¹²⁵I-GCSF-Tf transport. Chromatographic analyses and bio-assays revealed that GCSF-Tf was not degraded during transport via TfR-specific processes, and that GCSF retained biologic activity when liberated from the conjugate via dithiothreitol reduction. Conclusion. This study suggests the possibility of using TfR-mediated transcytosis for systemic delivery of therapeutic proteins via the alveolar epithelium.     

Kinetic Analysis of Tetraethylammonium Transport in the Kidney Epithelial Cell Line,LLC-PK1

Purpose. The aims of this study were to establish a kinetic means of analyzing the membrane transport of organic cations in renal epithelial cells, and to simultaneously evaluate drug interactions in apical and basolateral membranes. Methods. Tetraethylammonium (TEA) transport was measured using LLC-PK₁, cell monolayers grown on microporous membrane filters. After incubating the cells with unlabeled TEA or other drugs, apical or basolateral medium was changed to that containing labeled TEA, and transcellular transport and cellular accumulation were measured. Clearance from apical medium to cells (CL₁₂), cells to apical medium (CL₂₁), cells to basolateral medium (CL₂₃) and basolateral medium to cells (CL₃₂) were calculated based on a three compartment model. Results. TEA was accumulated progressively in the monolayers from the basolateral side and was transported unidirectionally to the apical side. CL₃₂ was greater than CL₁₂ and CL₂₃ was greater than CL₂₁. Therefore, the rate limiting step of TEA transport from the basolateral to the apical medium was the cell-to-apical step. Co-incubation of TEA with procainamide decreased the transport parameters of TEA, CL₁₂, CL₂₁ and CL₃₂, whereas that with levofloxacin decreased only CL₁₂ and CL₂₁, not affecting the parameters in basolateral membranes. Conclusions. Using a simple model, we analyzed the transport of organic cation in kidney epithelial cell line, LLC-PK₁ This method can be useful for the analysis of cation transport and drug interactions in the apical and basolateral membranes of renal tubules.     

Transport mechanism(s) of poly (amidoamine) dendrimers across Caco-2 cell monolayers

The objective of this research was to investigate the mechanism(s) of transport of generation 2 (G2) poly (amidoamine) dendrimers across Caco-2 cell monolayers. The contribution of an energy-dependent process such as adsorptive endocytosis was investigated by determining G2 permeability at 4 and 37 degrees C. The contribution of P-gp efflux to transport was examined by determining the apical to basolateral (AB) and basolateral to apical (BA) permeability of 14C-paclitaxel in presence of G2, and by determining AB and BA permeability of G2 in presence of paclitaxel. The permeability of G2 and 14C-mannitol was investigated in the presence of palmitoyl carnitine to determine the contribution of the paracellular pathway. Permeability of G2 at 4 degrees C was significantly (P<0.05) lower than that observed at 37 degrees C. AB and BA permeability of 14C-paclitaxel did not change in the presence of G2. AB and BA permeability of G2 did not change in the presence of paclitaxel. The permeability of G2 and 14C-mannitol increased significantly (P<0.05) in the presence of palmitoyl carnitine, and in addition, 14C-mannitol permeability was increased in presence of G2. The permeability of G2 across Caco-2 cell monolayers appears to involve a combination of paracellular transport and an energy-dependent process, possibly adsorptive endocytosis. G2 dendrimers do not appear to be substrates for the P-gp efflux system.     

Nanoparticle effects on rat alveolar epithelial cell monolayer barrier properties

Inhaled nanoparticles have been reported to contribute to deleterious effects on human health. In this study, we investigated the effects of ultrafine ambient particulate suspensions (UAPS), polystyrene nanoparticles (PNP; positively and negatively charged; 20, 100, 120 nm), quantum dots (QD; positively and negatively charged; 30 nm) and single-wall carbon nanotubes (SWCNT) on alveolar epithelial cell barrier properties. Transmonolayer resistance (R_t) and equivalent short-circuit current (I_eq) of primary rat alveolar epithelial monolayers were measured in the presence and absence of varying concentrations of apical nanoparticles. In some experiments, apical-to-basolateral fluxes of radiolabeled mannitol or inulin were determined with or without apical UAPS exposure and lactate dehydrogenase (LDH) release was analyzed after UAPS or SWCNT exposure. Results revealed that exposure to UAPS decreased R_t and I_eq significantly over 24 h, although neither mannitol nor inulin fluxes changed. Positively charged QD decreased R_t significantly (with subsequent recovery), while negatively charged QD did not. R_t decreased significantly after SWCNT exposure (with subsequent recovery). On the other hand, PNP exposure had no effects on R_t or I_eq. No significant increases in LDH release were observed after UAPS or SWCNT exposure. These data indicate that disruption of alveolar epithelial barrier properties due to apical nanoparticle exposure likely involves alteration of cellular transport pathways and is dependent on specific nanoparticle composition, shape and/or surface charge.     

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.     

Feasibility of a 3D human airway epithelial model to study respiratory absorption

The respiratory route is an important portal for human exposure to a large variety of substances. Consequently, there is an urgent need for realistic in vitro strategies for evaluation of the absorption of airborne substances with regard to safety and efficacy assessment. The present study investigated feasibility of a 3D human airway epithelial model to study respiratory absorption, in particular to differentiate between low and high absorption of substances. Bronchial epithelial models (MucilAir™), cultured at the air–liquid interface, were exposed to eight radiolabeled model substances via the apical epithelial surface. Absorption was evaluated by measuring radioactivity in the apical compartment, the epithelial cells and the basolateral culture medium. Antipyrine, caffeine, naproxen and propranolol were highly transported across the epithelial cell layer (>5%), whereas atenolol, mannitol, PEG-400 and insulin were limitedly transported (<5%). Results indicate that the 3D human airway epithelial model used in this study is able to differentiate between substances with low and high absorption. The intra-experimental reproducibility of the results was considered adequate based on an average coefficient of variation (CV) of 15%. The inter-experimental reproducibility of highly absorbed compounds was in a similar range (CV of 15%), but this value was considerably higher for those compounds that were limitedly absorbed. No statistical significant differences between different donors and experiments were observed. The present study provides a simple method transposable in any lab, which can be used to rank the absorption of chemicals and pharmaceuticals, and is ready for further validation with respect to reproducibility and capacity of the method to predict respiratory transport in humans.     

In-vitro transfer of nitazoxanide across the intestinal epithelial barrier

Nitazoxanide is a thiazolide compound that exhibits antimicrobial properties against helminths, protozoa, anaerobic bacteria and also Helicobacter pylori. The mucosal diffusion of this new drug has not been studied. The aim of this study was to examine the transport of radiolabelled nitazoxanide across the epithelial barrier according to the mode (mucosal or serosal) of drug administration. HT29-19A intestinal epithelial cells, grown as monolayers on microporous filters, were used as an epithelial model. In a short-term (100 min) transport study, the apical to basal and the basal to apical transport of nitazoxanide across the monolayers was studied in an Ussing chamber. In a long-term (24 h) study, the transport of the drug and its intracellular accumulation were studied in filter-grown epithelial monolayers kept in culture plates. In the short-term transport study, both the apical and basal fluxes achieved a steady state after 70 min and there was no significant difference between the apical to basal (3991+/-1001 ng h(-1) cm(-2)) and the basal to apical (4246+/-856 ng h(-1) cm(-2)) nitazoxanide fluxes. In the long-term transport study, after apical or basal drug application, a gradual increase in the drug concentration in the opposite compartment was noted, which reached similar values for apical and basal fluxes (2497+/-125 and 2309+/-81 ng mL(-1), respectively) after 24 h. Moreover, a rapid, although transitory, intracellular accumulation of nitazoxanide was observed at 10 min after apical (299+/-25 ng/10(6) cells) and basal (124+/-10 ng/10(6) cells) drug application, but decreased thereafter. There is an important transepithelial transport of nitazoxanide across the digestive epithelial monolayer with a rapid intracellular accumulation of the drug. No difference between the apical to basal and basal to apical fluxes of the drug was observed, suggesting that both topical and systemic modes of action of this antibiotic are successful.     

The prevention of cyclosporin A adsorption to Transwell surfaces by human plasma

Prevention of cyclosporin A (CsA) adsorption onto the inner surface of Transwell during transport experiments, by the addition of human plasma to the receiver compartment (basolateral side), was investigated. The addition of plasma to a level of 50% (v/v) of the transport medium led to a reduction in the adsorption of CsA (0.1 microM) down to a level of 5%. As a result, the apical to basolateral flux of CsA across the Caco-2 cell monolayer in the presence of 50% (v/v) plasma was estimated to be 2.7-fold higher than that obtained in the absence of plasma. Thus, the adsorption problem can be overcome simply by the addition of an appropriate volume of human plasma to the transport medium. This method appears to be applicable to the routine estimation of CsA flux across epithelial cell monolayers using Transwell.     

Carrier-mediated uptake of cisplatin by the OK renal epithelial cell line

The purpose of this study was to investigate whether transport of cis-diamminedichloroplatinum II (cisplatin) across renal epithelial cell line OK cells is mediated by the organic cation transport system. OK cell monolayers cultured on permeable membranes were incubated with 100 microM cisplatin on the apical or basolateral side, and the cellular accumulation and the transport of cisplatin across the monolayer were measured. The accumulation from the basolateral medium and the basolateral-to-apical transport of cisplatin were higher than the accumulation from the apical medium and the apical-to-basolateral transport, respectively. The cell monolayers were incubated with different concentrations of cisplatin (0.02 approximately 3 mM) in the basolateral medium. The relationship between the cisplatin concentrations in the medium and in the cells revealed that cisplatin accumulation tended to be saturable. The basolateral-to-apical transport of cisplatin was increased when the pH of the apical medium was decreased, with a concomitant decrease in the accumulation of cisplatin. Coincubation of cisplatin with tetraethylammonium (TEA), a typical substrate for the organic cation transporter, significantly decreased the accumulation and transport of cisplatin from the basolateral medium. These results suggest that the uptake and basolateral-to-apical transport of cisplatin are mediated by not only simple diffusion but also by the organic cation transport system.     

Use of alveolar cell monolayers of varying electrical resistance to measure pulmonary peptide transport

The apparent permeability coefficient (P(app)) of two fluorescently tagged model hydrophilic peptides, acXASNH(2) and acXAS(GAS)(7)NH(2), and (14)C-mannitol across monolayers of cultured rat alveolar epithelial cells of varying transepithelial electrical resistance (TER) has been examined. In line with their design features, the peptides were not degraded under the conditions of the test. Furthermore, no concentration dependence of transport of the tripeptide acXASNH(2) was observed over the concentration range studied, nor was any directional transport seen for either of the model peptides, indicating that under the conditions of the test they were not substrates for any transporters or efflux pumps. From the hydrophilic nature of the peptides (as assessed by their log P), and their inverse dependence of transport with molecular weight and TER, it was assumed that the peptides were transported across the cell monolayer passively via the paracellular route. The observed P(app) for the transport of (14)C-mannitol and the peptides across rat alveolar epithelial cell monolayers were found to be inversely (though not linearly) related to the measured TER and could be well-modeled assuming the presence of two populations of "pores" in the cell monolayer, namely, cylindrical pores of diameter 1.5 nm and large pores of diameter 20 nm. The relative populations of the two types of pores varied with the TER of the monolayer, with the number of large pores decreasing with an increase in TER (and the number of small pores taken as fixed). These results suggest that if the cell monolayer is well characterized with respect to the passage of a range of probe molecules across monolayers of varying electrical resistance, it should be possible to predict the P(app) of any hydrophilic peptide or drug crossing the membrane by the paracellular route at any desired TER using a monolayer of any electrical resistance, above a minimum value.     

Transcellular transport of creatinine in renal tubular epithelial cell line LLC-PK1

BACKGROUND/AIM: Creatinine is excreted into urine via tubular secretion in addition to glomerular filtration. In the present study, characteristics of the creatinine transport in renal epithelial cells were investigated. METHODS: The transcellular transport and accumulation of [14C]creatinine and [14C]tetraethylammonium (TEA) were assessed using LLC-PK1 cell monolayers cultured on porous membrane filters. RESULTS: [14C]Creatinine was transported directionally from the basolateral to apical side of LLC-PK1 cell monolayers. Basolateral uptake of [14C]creatinine was dependent on membrane potential, and was saturable with apparent K(m) and V(max) values of 13.2+/-2.8 mM and 13.1+/-3.1 nmol/mg protein/5 min, respectively. Concomitant administration of organic cations (1 mM) such as cimetidine, quinidine and trimethoprim inhibited both the transcellular transport and accumulation of [14C]creatinine. Furthermore, apical excretion of [14C]creatinine was not dependent on acidification of the apical medium. CONCLUSIONS: Creatinine was subjected to directional transport across renal epithelial cells from the basolateral to apical side. The organic cation transporter should be involved in the basolateral uptake of creatinine.     

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.     

Fingerprinting rat liver microsomal cytochromes P-450 as a means of delineating sexually distinctive forms

The cytochrome P-450 fraction of microsomes separated on lauric acid AH-Sepharose 4B columns contains about 75% of the microsomal P-450. This was finger-printed by means of two dimensional isoelectric focusing/ SDS-PAGE. Separation of the fraction by highly reproducible, standard procedures on carboxymethyl Sepharose CL6B into four fractions allowed ready isolation and purification of seven forms of P-450, RLM2, 2b, 3, f4, 5, 5a and f5a. Comparison of the four fractions CMI, CMII, CMIII and CMIV revealed qualitative differences in the proteins contained in CMI and CMII of male and female rats. Identification of these proteins revealed RLM2, present in the CMI fraction of adult male rats, is not present in detectable levels in the comparable fraction from females. Similarly, RLM3 and 5 were present in the CMII fraction of male rats but could not be detected in the corresponding fraction of females. Instead, another protein, fRLM4, was found in the females. RLM5a, found in the CMII fraction of males, was also present in females. Examination of the physical properties of these P-450 proteins revealed those isolated in the CMI and CMII fractions to have fairly neutral isoelectric points (7.1–7.6). Based upon the NH₂-terminal amino acid sequence, three classes of constitutive forms of P-450 can be recognized. All of the constitutive forms have methionine in position one and leucine in position seven. By comparing sequence homologies, RLM2 and 2b form one sub-class, RLM3, f4 and 5 form a second sub-class, and P-450f and RLM5a form a third sub-class.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthdayThis work was supported by United States Public Health Service Grant GM26114 from the National Institutes of Health     

Oligopeptide Transport in Rat Lung Alveolar Epithelial Cells is Mediated by Pept2

Purpose

Studies were conducted in primary cultured rat alveolar epithelial cell monolayers to characterize peptide transporter expression and function.

Methods

Freshly isolated rat lung alveolar epithelial cells were purified and cultured on permeable support with and without keratinocyte growth factor (KGF). Messenger RNA and protein expression of Pept1 and Pept2 in alveolar epithelial type I- and type II-like cell monolayers (±KGF, resp.) were examined by RT-PCR and Western blotting. ³H–Glycyl-sarcosine (³H–gly-sar) transmonolayer flux and intracellular accumulation were evaluated in both cell types.

Results

RT-PCR showed expression of Pept2, but not Pept1, mRNA in both cell types. Western blot analysis revealed presence of Pept2 protein in type II-like cells, and less in type I-like cells. Bi-directional transmonolayer ³H–gly-sar flux lacked asymmetry in transport in both types of cells. Uptake of ³H–gly-sar from apical fluid of type II-like cells was 7-fold greater than that from basolateral fluid, while no significant differences were observed from apical vs. basolateral fluid of type I-like cells.

Conclusions

This study confirms the absence of Pept1 from rat lung alveolar epithelium in vitro. Functional Pept2 expression in type II-like cell monolayers suggests its involvement in oligopeptide lung disposition, and offers rationale for therapeutic development of di/tripeptides, peptidomimetics employing pulmonary drug delivery.

     

Benzimidazolones enhance the function of epithelial Na+ transport

Background and Purpose

Pharmacological enhancement of vectorial Na⁺ transport may be useful to increase alveolar fluid clearance. Herein, we investigated the influence of the benzimidazolones 1-ethyl-1,3-dihydro-2-benzimidazolone (1-EBIO), 5,6-dichloro-1-EBIO (DC-EBIO) and chlorzoxazone on vectorial epithelial Na⁺ transport.

Experimental Approach

Effects on vectorial Na⁺ transport and amiloride-sensitive apical membrane Na⁺ permeability were determined by measuring short-circuit currents (I_SC) in rat fetal distal lung epithelial (FDLE) monolayers. Furthermore, amiloride-sensitive membrane conductance and the open probability of epithelial Na⁺ channels (ENaC) were determined by patch clamp experiments using A549 cells.

Key Results

I_SC was increased by approximately 50% after addition of 1-EBIO, DC-EBIO and chlorzoxazone. With permeabilized basolateral membranes in the presence of a 145:5 apical to basolateral Na⁺ gradient, the benzimidazolones markedly increased amiloride-sensitive I_SC. 5-(N-Ethyl-N-isopropyl)amiloride-induced inhibition of I_SC was not affected. The benzamil-sensitive I_SC was increased in benzimidazolone-stimulated monolayers. Pretreating the apical membrane with amiloride, which inhibits ENaC, completely prevented the stimulating effects of benzimidazolones on I_SC. Furthermore, 1-EBIO (1 mM) and DC-EBIO (0.1 mM) significantly increased (threefold) the open probability of ENaC without influencing current amplitude. Whole cell measurements showed that DC-EBIO (0.1 mM) induced an amiloride-sensitive increase in membrane conductance.

Conclusion and Implications

Benzimidazolones have a stimulating effect on vectorial Na⁺ transport. The antagonist sensitivity of this effect suggests the benzimidazolones elicit this action by activating the highly selective ENaC currents. Thus, the results demonstrate a possible new strategy for directly enhancing epithelial Na⁺ transport.     

Distribution characteristics of clarithromycin and azithromycin, macrolide antimicrobial agents used for treatment of respiratory infections, in lung epithelial lining fluid and alveolar macrophages

The distribution characteristics of clarithromycin (CAM) and azithromycin (AZM), macrolide antimicrobial agents, in lung epithelial lining fluid (ELF) and alveolar macrophages (AMs) were evaluated. In the in vivo animal experiments, the time‐courses of the concentrations of CAM and AZM in ELF and AMs following oral administration (50 mg/kg) to rats were markedly higher than those in plasma, and the area under the drug concentration–time curve (AUC) ratios of ELF/plasma of CAM and AZM were 12 and 2.2, and the AUC ratios of AMs/ELF were 37 and 291, respectively. In the in vitro transport experiments, the basolateral‐to‐apical transport of CAM and AZM through model lung epithelial cell (Calu‐3) monolayers were greater than the apical‐to‐basolateral transport. MDR1 substrates reduced the basolateral‐to‐apical transport of CAM and AZM. In the in vitro uptake experiments, the intracellular concentrations of CAM and AZM in cultured AMs (NR8383) were greater than the extracellular concentrations. The uptake of CAM and AZM by NR8383 was inhibited by ATP depletors. These data suggest that the high distribution of CAM and AZM to AMs is due to the sustained distribution to ELF via MDR1 as well as the high uptake by the AMs themselves via active transport mechanisms. Copyright © 2011 John Wiley & Sons, Ltd.     

Evaluation of creatine transport using Caco-2 monolayers as an in vitro model for intestinal absorption.

Creatine is a nutraceutical that has gained popularity in both well-trained and casual athletes for its performance-enhancing or ergogenic properties. The major disadvantages of creatine monohydrate formulations are poor solubility and oral bioavailability. In the present study, creatine transport was examined using Caco-2 monolayers as an in vitro model for intestinal absorption. Confluent monolayers of Caco-2 cells (passage 25-35) were used for the permeability studies. Monolayers were placed in side-by-side diffusion chambers. (14)C-Creatine (0.1-0.5 microCi/mL) was added to either the apical or basolateral side, and the transport of the creatine across the Caco-2 monolayer was measured over a 90-min period. The apical to basolateral transport of (14)C-creatine was small, ranging from 0.2-3% of the original amount appearing on the receiver side in a 90-min period. Interestingly, the basolateral to apical permeability of radiolabeled creatine was substantially greater than that observed in the apical to basolateral direction. Studies with drug efflux transport inhibitors indicate that neither the P-glycoprotein nor multidrug resistance-associated protein is involved in the enhanced basolateral to apical transport of creatine.     

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.     

Increasing Paracellular Porosity by E-Cadherin Peptides: Discovery of Bulge and Groove Regions in the EC1-Domain of E-Cadherin

Purpose. The objective of this work is to evaluate the ability of peptides derived from the bulge (HAV-peptides) and groove (ADT-peptides) regions of E-cadherin EC1-domain to increase the paracellular porosity of the intercellular junctions of Madin-Darby canine kidney (MDCK) cell monolayers. Methods. Peptides were synthesized using a solid-phase method and were purified using semi-preparative HPLC. MDCK monolayers were used to evaluate the ability of cadherin peptides to modulate cadherin-cadherin interactions in the intercellular junctions. The increase in intercellular junction porosity was determined by the change in transepithelial electrical resistance (TEER) values and the paracellular transport of ¹⁴C-mannitol. Results. HAV- and ADT-peptides can lower the TEER value of MDCK cell monolayers and enhance the paracellular permeation of ¹⁴C-mannitol. HAV- and ADT-decapeptides can modulate the intercellular junctions when they are added from the basolateral side but not from the apical side; on the other hand, HAV- and ADT-hexapeptides increase the paracellular porosity of the monolayers when added from either side. Conjugation of HAV- and ADT-peptides using -aminocaproic acid can only work to modulate the paracellular porosity when ADT-peptide is at the N-terminus and HAV-peptide is at the C-terminus; because of its size, the conjugate can only modulate the intercellular junction when added from the basolateral side. Conclusions. Peptides from the bulge and groove regions of the EC1 domain of E-cadherin can inhibit cadherin-cadherin interactions, resulting in the opening of the paracellular junctions. These peptides may be used to improve paracellular permeation of peptides and proteins. Furthermore, this work suggests that both groove and bulge regions of EC-domain are important for cadherin-cadherin interactions.