Functional characterization and cloning of amino acid transporter B(0,+) (ATB(0,+)) in primary cultured rat pneumocytes.

Cationic amino acid transport in primary cultured rat pneumocytes exhibiting characteristics of alveolar epithelial type I-like cells are described. Asymmetry and activator ion dependency of (3)H-L-arginine uptake were characterized from the apical or basolateral fluid of pneumocytes grown on permeable support. Substrate specificity of transport was evaluated as a function of (3)H-L-arginine uptake inhibition in the presence of other amino acids. Transepithelial transport studies estimated (3)H-L-arginine flux in the apical-to-basolateral and basolateral-to-apical directions. Full length cDNA of rat amino acid transporter B(0,+) (rATB(0,+)) was cloned and its relative expression level studied. Results indicate that uptake of (3)H-L-arginine from apical fluid is dependent on Na(+) and Cl(-). Zwitterionic and cationic amino acids (excluding L-proline and anionic amino acids) inhibited uptake of (3)H-L-arginine from apical, but not basolateral incubation fluid. Apical-to-basolateral transepithelial flux of (3)H-L-arginine was 20x higher than basolateral-to-apical transport. Kinetic studies of (3)H-L-arginine uptake from apical fluid revealed maximal velocity (V(max)) and Michaelis-Menten constants (K(t)) of 33.32 +/- 2.12 pmol/mg protein/15 min and 0.50 +/- 0.11 mM, respectively, in a cooperative process having a coupling ratio of 1.18 +/- 0.16 with Na(+) and 1.11 +/- 0.13 with Cl(-). Expression of rATB(0,+) mRNA was identified by RT-PCR and Northern analysis. Corresponding cloned 3.2 kb rATB(0,+) cDNA sequence exhibits pronounced homology in deduced amino acid sequence to mouse (95% identity and 97% similarity) and human (89% identity and 95% similarity) ATB(0,+) homologues. We conclude that rat pneumocytes express ATB(0,+), which may partly contribute towards recovering cationic and neutral amino acids from alveolar luminal fluid.

Molecular and functional expression of multidrug resistance-associated protein-1 in primary cultured rat alveolar epithelial cells.

Multidrug resistance-associated protein-1 (MRP1) is an integral membrane efflux protein that is implicated in multidrug resistance in cancer, but it is also expressed in normal tissues. The objective of this study was to determine the expression, localization and functional activity of MRP1 in primary cultured rat alveolar epithelial cells of types I- and II cell-like phenotypes. RT-PCR data showed 550-base pair fragments in both types I- and II-like pneumocytes that exhibited 99% identity to the rat MRP1 isoform. Significant levels of MRP1 protein were detected by western analysis of immunoprecipitates in both cell types, and immunofluorescence combined with confocal laser scanning microscopy indicated basolateral localization of MRP1. Indomethacin (0-100 microM) increased fluorescein basolateral-to-apical transport, and accumulation of fluorescein in the cells, in a dose-dependent manner. We therefore conclude that the MRP1 gene is present in primary cultured rat epithelial cells of both types I- and II-like phenotypes and its corresponding protein (MRP1) is localized in the basolateral membrane of these cells. Primary cultured monolayers of rat type II-like pneumocytes appear to be a useful tool for screening MRP1 substrates designed for pulmonary delivery/targeting.

Glutathione and its transporters in ocular surface defense.

Glutathione (GSH) is an abundant antioxidant ubiquitous in nearly all cell types. Deficiency of GSH has been linked to ocular disease and viral infection. Other established vital roles of GSH include detoxification and immunoprotection. Endogenous GSH plays a protagonist's role in safeguarding active transport processes compartmentalized at the interface between conjunctival mucosa and the tear film. Optimal electrokinetic transport across the conjunctival epithelium requires the mucosal presence of GSH. Glutathione is the most abundant known endogenous antioxidant molecule in tear fluid, mainly derived from conjunctival secretion. Conjunctival GSH transport, a major kinetic component of GSH turnover, occurs through multiple functionally distinct mechanisms. Cell membrane potential regulates conjunctival GSH efflux, while conjunctival GSH uptake requires extracellular Na(+). Significant modulation of GSH, its constituent amino acids, and functions of associated transporters occurs in the conjunctival epithelium with viral inflammatory disease. Topical conjunctival delivery of GSH, its metabolic precursors, or pharmacologic stimulation of endogenous conjunctival GSH secretion carry potential in alleviating viral-inflammatory conjunctivitis.

Cysteine scanning of transmembrane domain three of the human dipeptide transporter: implications for substrate transport.

The human intestinal dipeptide transporter (hPepT1) transports dipeptides and pharmacologically active drugs from the intestine to the blood. The role of transmembrane domain 3 (TMD3) of hPepT1 was studied using cysteine-scanning mutagenesis and methane thiosulfonate (MTS) cysteine modification. Each amino acid in TMD3 was individually mutated to a cysteine and Gly-Sar uptake by each mutated and modified transporter was determined relative to wild-type hPepT1. Uptake data for mutated transporters modified with the lipid-insoluble cysteine-modifying reagent MTSET suggested tilting of TMD3 relative to the substrate translocation pathway; the extracellular region of TMD3 showed little MTSET reactivity, indicative of solvent inaccessibility, whereas the intracellular part of TMD3 was relatively solvent accessible. Modification at 10 positions of TMD3 with MTSEA, a lipid-soluble cysteine-modifying reagent, gave unusual and statistically significant increases in Gly-Sar uptake relative to untreated mutants. We interpret these data in terms of the spatial properties of the hPepT1 substrate translocation channel and possible interactions of TMD3 with other transmembrane domains.

Multidrug resistance protein 1 (MRP1) in rabbit conjunctival epithelial cells: its effect on drug efflux and its regulation by adenoviral infection.

PURPOSE: To evaluate the expression, localization, function, and regulation of multidrug resistance protein (MRP1) in rabbit conjunctival epithelial cells (RCEC). MATERIALS AND METHODS: MRP1 gene expression in RCEC was determined by reverse transcription-polymerase chain reaction (RT-PCR), and MRP1 protein expression and its localization were determined by Western blot analysis and immunofluorescence using an anti-MRP1 monoclonal antibody, MRPr1. The effect of MRP1 on the transport and uptake of fluorescein was evaluated in RCEC grown on Transwell filters. Moreover, the effect of adenovirus type 5 (Ad5)-infected RCEC, and cytokines (Interleukin 1 (IL-1), IL-6, and tumor necrosis factor alpha (TNFalpha)) on MRP1 expression and leukotriene C4 (LTC4) uptake were investigated. RESULTS: A 652 bp RT-PCR product from rabbit conjunctiva showed a 87% homology to human MRP1. Immunostaining with MRPr1 revealed a predominant basolateral localization of MRP1 in RCEC. Uptake of fluorescein, a MRP1 substrate, was increased (203-290%) in the presence of uricosuric drug probenecid at 100 microM, anti-inflammatory drug indomethacin at 10 microM and diclofenac, flurbiprofen, and ofloxacin at 1 mM, and by ATP depletion, but not influenced by the depletion of GSH, and the presence of antiviral cidofovir and anti-inflammatory drug cromolyn and prednisolone. Apical-to-basolateral facilitated transport of LTC4 was abolished in the presence of probenecid. Western blot analysis with MRPr1 revealed a distinct band at approximately 190 kDa for freshly isolated and cultured RCEC. Both Ad5 and cytokines (IL-1, IL-6, and TNF-alpha) up-regulated MRP1 expression, thereby reducing LTC4 uptake. CONCLUSIONS: MRP1 appears to be primarily localized in the basolateral membrane of RCEC and function in the efflux of certain organic anions and inflammatory factors out of cells from the basolateral membrane. The upregulation in the expression of MRP1 by Ad5-infection and cytokines suggests a role of MRP1 in the transport of inflammatory factors during ocular inflammation. Supported by NIH grants EY12578, EY10421, and EY12356.

A charge pair interaction between Arg282 in transmembrane segment 7 and Asp341 in transmembrane segment 8 of hPepT1.

PURPOSE: To determine whether R282 in transmembrane segment 7 (TMS7) of hPepT1 forms a salt bridge with D341 in TMS8. METHODS: Mutated hPepT1 transporters containing point mutations at R282 and/or D341 were transiently transfected into HEK293 cells. Their steady state expression and functional activity were measured using immunoprecipitation and 3H-gly-sar uptake, respectively. Gly-sar uptake by cysteine mutants (R282C and D341C) was also measured in the presence and absence of cysteine-modifying MTS reagents. RESULTS: The reverse-charge mutants R282D-hPepT1 and D341R-hPepT1 showed significantly reduced gly-sar uptake, but the double mutant (R282D/D341R-hPepT1) has functionality comparable to that of wild-type hPepT1. Gly-sar uptake by R282C-hPepT1 is reduced, but pre-incubation with 1 mM MTSET, a positively charged cysteine-modifying reagent, restored function to wild-type levels. Similarly, pre-incubation of D341C-hPepT1 with 10 mM MTSES, a negatively charged cysteine-modifying reagent, increased gly-sar uptake compared to unmodified D341C-hPepT1. In contrast, MTSET modification of D341C-hPepT1 (giving a positive charge at position 341) resulted in significant reduction in gly-sar uptake, compared to D341C-hPepT1. CONCLUSION: Our results are consistent with a salt bridge between R282 and D341 in hPepT1, and we use these and other data to propose a role for the R282-D341 charge pair in the hPepT1 translocation mechanism.

Thermodynamic stoichiometry of Na+-coupled glutathione transport.

Ambiguity exists with respect to mechanisms of glutathione (GSH) transport and the molecular identity of GSH transporters. Empirical and theoretical limitations have hindered functional and molecular characterizations. Published literature referring to the isolation and molecular identification of Na+-coupled GSH transporters that mediate the cellular uptake of GSH is highly debated. Whereas a number of functional and kinetic reports of this putative symport mechanism exist, the hypothetical transmembrane Na+-coupled GSH transporter protein or the genetic message encoding it has not been isolated. Theoretical thermodynamic calculations to support the concept of secondary active GSH transport and to rationalize accounts of physical-kinetic measurements describing Na+-coupled cellular GSH uptake were performed. The adequacy of requisite energy and stoichiometric conservation of the separate electrical and chemical components of a Na+ gradient in maintaining a high cellular accumulation gradient for GSH was examined through a purely phenomenological perspective. Dependent on the biological context, the energetic coupling between Na+ and GSH cotransport may occur at ratios from 1:1 to 3:1. Molecular identification of specific transporters responsible for cellular Na+-coupled GSH uptake will facilitate determination of their relative contribution to the overall plasma membrane resting potential. In tissues with a high GSH concentration relative to their extracellular milieu, particularly in pathologies of cystic fibrosis and dry eye syndromes, large energy coupling ratios in cotransport of Na+ and GSH may be expected. Na+-coupled GSH transport may play an important role in disease onset and (or) progression, or treatment modalities thereof.

Nucleoside transport in primary cultured rabbit tracheal epithelial cells.

The present study aimed at elucidating the mechanisms of nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) grown on a permeable filter support. Uptake of (3)H-uridine, the model nucleoside substrate, from the apical fluid of primary cultured RTEC was examined with respect to its dependence on Na(+), substrate concentration, temperature and its sensitivity to inhibitors, other nucleosides and antiviral nucleoside analogs. Apical (3)H-uridine uptake in primary cultured RTEC was strongly dependent on an inward Na(+) gradient and temperature. Ten micromolar nitro-benzyl-mercapto-purine-ribose (NBMPR) (an inhibitor of es-type nucleoside transport in the nanomolar range) did not further inhibit this process. (3)H-uridine uptake from apical fluid was inhibited by basolateral ouabain (10 microM) and apical phloridzin (100 microM), indicating that uptake may involve a secondary active transport process. Uridine uptake was saturable with a K(m) of 3.4 +/- 1.8 microM and the V(max) of 24.3 +/- 5.2 pmoles/mg protein/30 s. Inhibition studies indicated that nucleoside analogs that have a substitution on the nucleobase competed with uridine uptake from apical fluid, but those with modifications on the ribose sugar including acyclic analogs were ineffective. The pattern of inhibition of apical (3)H-uridine, (3)H-inosine and (3)H-thymidine uptake into RTEC cells by physiological nucleosides was consistent with multiple systems: A pyrimidine-selective transport system (CNT1); a broad nucleoside substrate transport system that excludes inosine (CNT4) and an equilibrative NBMPR-insensitive nucleoside transport system (ei type). These results indicate that the presence of apically located nucleoside transporters in the epithelial cells lining the upper respiratory tract can lead to a high accumulation of nucleosides in the trachea. At least one Na(+)-dependent, secondary, active transport process may mediate the apical absorption of nucleosides or analogous molecules.

Fine tuning of rabbit equilibrative nucleoside transporter activity by an alternatively spliced variant.

The full-length cDNA encoding an equilibrative nucleoside transporter (rbENT2) and its novel C-terminal variant, rbENT2A, were isolated from rabbit trachea. Rabbit ENT2 protein consists of 456 amino acid residues; rbENT2A is shorter by 41 residues. Both rbENT2 and rbENT2A transcripts are found in rabbit tissues including intestine, kidney cortex, kidney, and trachea, at varying levels of expression. When transfected in a heterologous expression system-Madin Darby canine kidney (MDCK) epithelial cell line-both rbENT2 and rbENT2A were expressed. rbENT2 had a molecular mass of 49 kDa; rbENT2A had a molecular mass of 44 kDa. Clones of both transporters yielded functional proteins that were capable of mediating uridine uptake and efflux without the needing to be coupled to a secondary ion (e.g. Na(+)). Remarkably, rbENT2A displayed a higher affinity (K(m) = 41 microM) and a lower capacity (V(max) = 0.6 nmol/mg protein/5 min) towards substrates than rbENT2 (K(m) = 272.8 microM, V(max) = 1.26 nmol/mg protein/5 min). Pharmacological profiles showed that nitro-benzyl-mercapto-purine-ribose (NBMPR) potently inhibited (3)H-uridine uptake mediated by rbENT2A, but not uptake mediated by rbENT2. The constitutive splicing, broad expression, markedly different kinetics, and distinct pharmacological characteristics of rbENT2A appear to act in conjunction with the wild type, rbENT2, to fine-tune basolateral nucleoside transport function in rabbit trachea.

Functional and pharmacological mechanisms of nucleoside transport across the basolateral membrane of rabbit tracheal epithelial cells.

The role of basolateral membrane nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) was studied. Primary cultured RTEC were grown on permeable support at an air-interface. Transport studies were conducted in the uptake, efflux, and transepithelial transport configurations using (3)H-uridine as a model substrate. Time, temperature and concentration dependency of (3)H-uridine transport were evaluated in parallel to the metabolism of this substrate using scintillation counting and thin layer chromatography. Inhibition of (3)H-uridine uptake from basolateral fluid was estimated in presence of all unlabeled natural nucleosides as well as analogs and nucleobases. Functional modulation pathways of (3)H-uridine uptake were studied after treatment of RTEC with pharmacological levels of A23187, forskolin, tamoxifen, H89 and colchicine. The basolateral aspect has a low-affinity and high-capacity transport system that exhibits characteristics of bi-directionality, temperature/concentration dependency, and broad specificity towards purines and pyrimidines without requiring Na(+). Basolateral equilibrative-sensitive/insensitive (es/ei) type transport machinery manifested as a biphasic dose response to nitro-benzyl-mercapto-purine-ribose (NBMPR) inhibition. In addition, a number of therapeutically relevant nucleoside analogs appeared to compete with the uptake of uridine from basolateral fluid. Short-term pre-incubation of primary cultured RTEC with the calcium ionophore A23187 inhibited basolateral uridine uptake without affecting the J(max) and K(m). The inhibitory effect was not reversible with a protein kinase C (PKC) antagonist, tamoxifen. In contrast, basolateral uridine uptake was increased by adenylyl cyclase activator forskolin (reversible with protein kinase A (PKA) inhibitor H89), resulting in a decreased K(m), but a lower J(max). Uridine exit across the basolateral membrane of primary cultured RTEC occurs via a facilitative diffusion carrier, which can be modulated by intracellular Ca(2+) levels and PKA. Information about these carriers will help improve the transportability of antitumor and antiviral nucleoside analogs in the pulmonary setting.

Roles of the conjunctiva in ocular drug delivery: a review of conjunctival transport mechanisms and their regulation.

Conjunctiva plays many roles including protection of ocular surface, production of tear film, and a conduit for drug clearance (depending on drug properties) into the systemic circulation or for drug transport to the deep tissues of the eye. The conjunctiva, which is a moderately tight epithelium, endowed with various transport processes for the homeostasis of ions, solutes, and water in the conjunctival surface and tear film. Modulation of ion transport in the conjunctiva leads to alterations in transconjunctival fluid flow that may become useful for treatment of dry-eye state in the eye. As a possible drug delivery route to the posterior portion of the eye, conjunctiva is an attractive route due to both larger surface area than that of cornea and expression of several key transport processes. Tear contains D-glucose and many amino acids, in addition to the usual ions in the body fluids. Several ion-coupled solute transport processes for absorption of amino acids, D-glucose, monocarboxylate, nucleosides, and dipeptides are expressed in the conjunctiva. Thanks to the rich endowment of these transport processes, drug transport across the conjunctiva into the intraocular tissues may become quite feasible. Subconjunctival injection of microparticles and matrix materials (which allows sustained release of drugs) is shown to maintain reasonable levels of various drugs in the vitreous, perhaps attesting to the fact that conjunctiva per se may contribute as a part of multiple transport barrier(s) in ocular drug delivery. In addition, several conjunctival approaches have been investigated to optimize treatment of dry-eye syndrome and intraocular diseases, and more can be accomplished in the coming years.

Tissue distribution of moxaverine-hydrochloride in the rabbit eye and plasma.

OBJECTIVE: The aim of this study was to determine the tissue distribution and epithelial penetration of moxaverine-hydrochloride (MOX) in the rabbit eye. METHODS: For systemic application, a radioactively labeled MOX solution was injected into the ear vein of Dutch-belted pigmented male rabbits. For topical dosing, an identical solution was administered. At predetermined time points, rabbits were sacrificed, the eyes dissected, and the amount of MOX in the ocular tissues measured. To examine the MOX permeability across the corneal epithelium, transport studies using rabbit corneal epithelial cell culture were conducted and the respective apparent permeability coefficient in absorptive (a to b) or secretive (b to a) direction was calculated. RESULTS: Topical delivery resulted in high concentrations of MOX in the cornea and conjunctiva, although other tissues of the anterior part yielded lower MOX concentrations. In the tissues of the posterior part, high amounts were detected in the retina. Plasma levels were low. The apparent permeability coefficient across corneal epithelial cell layers was in the range of 10(5) cm/s, exhibiting no apparent directionality. CONCLUSION: A topical dosing of MOX to posterior regions of the eye seems feasible. MOX levels in the posterior part of the eye were remarkably high, without causing stringent plasma levels. The high apparent permeability coefficient of MOX across the corneal epithelial cell layers might be caused by the lipophilic nature of the drug and was in the range of other compounds with comparable physicochemical properties.

Characterization of active ion transport across primary rabbit corneal epithelial cell layers (RCrECL) cultured at an air-interface.

Previously, we reported the development of a primary culture model of tight rabbit corneal epithelial cell layers (RCrECL) characterizing bioelectric parameters, morphology, cytokeratin, and passive permeability. In the present study, we specifically evaluated the active ion transport processes of RCrECL cultured from either pigmented or albino rabbits. Primary cultured RCrECL were grown at an air-interface on Clear-Snapwells precoated with collagen/fibronectin/laminin and mounted in a modified Ussing-type chamber for the evaluation of their active ion transport processes under short-circuited conditions. Contribution of active Na(+) and Cl(-) transport to overall short-circuit current (I(sc)) was evaluated by removing Na(+) and Cl(-), respectively, from bathing fluids of RCrECL and measurements of net fluxes of Na(+) and Cl(-) using (22)Na and (36)Cl, respectively. Amiloride and benzamil were used to determine the role of apical Na(+)-channel activities to net Na(+) fluxes. N-phenylanthranilic acid (NPAA), ouabain, BaCl(2) and bumetanide were used to determine the role of basolateral Na,K-ATPase, apical Cl(-)-channel, and basolateral K(+)-channel and Na(+)(K(+))2Cl(-)-cotransporter activities, respectively, in active ion transport across RCrECL. I(sc) of RCrECL derived from pigmented rabbits was comprised of 64+/-2% and 44+/-5% for active Na(+) and Cl(-) transport, respectively, consistent with net Na(+) absorption and Cl(-) secretion of 0.062+/-0.006 and 0.046+/-0.008 muEq/cm(2)/hr estimated from radionuclide fluxes. Apical amiloride and benzamil inhibited I(sc) by up to approximately 50% with an IC(50) of 1 and 0.1 microm, respectively, consistent with participation of apical epithelial Na(+)-channels to net Na(+) absorption across RCrECL cultured from pigmented rabbits. Addition of ouabain to the basolateral, NPAA to the apical, BaCl(2) to the basolateral and bumetanide to basolateral fluid decreased I(sc) by 86+/-1.5%, 53+/-3%, 18+/-1.8% and 13+/-1.9% in RCrECL cultured from pigmented rabbits, while 85+/-0.7%, 36+/-1.6%, 38+/-1.8% and 15+/-3.5% decreases are observed for RCrECL from albino rabbits, respectively. Air-interface cultured RCrECL from either pigmented or albino rabbits exhibited active ion transport properties similar to those present in excised tissues. This primary culture system may be a reliable in-vitro model for mechanistic characterization of corneal epithelial function and regulation of transport properties.

Lectins as endocytic ligands: an assessment of lectin binding and uptake to rabbit conjunctival epithelial cells.

PURPOSE: To investigate the binding and uptake pattern of three plant lectins in rabbit conjunctival epithelial cells (RCECs) with respect to their potential for enhancing cellular macromolecular uptake. METHODS: Three fluorescein-labeled plant lectins (Lycoperison esculentum, TL; Solanum tuberosum, STL; and Ulex europaeus 1, UEA-1) were screened with respect to time-, concentration-, and temperature-dependent binding and uptake. Chitin (30 mg/ml) and L-alpha-fucose (10 mM) were used as inhibitory sugars to correct for nonspecific binding of TL or STL and UEA-1, respectively. Confocal microscopy was used to confirm internalization of STL. RESULTS: The binding and uptake of all three lectins in RCECs was time-dependent (reaching a plateau at 1-2 h period) and saturable at 1-h period. The rank order of affinity constants (km) was STL>TL>UEA-1 with values of 0.39>0.48>4.81 microM, respectively. However, maximal, specific binding/uptake potential was in the order UEA-1>STL>TL with values of 53.7, 52.3, and 15.0 nM/mg of cell protein, respectively. Lectins showed temperature dependence in their uptake, with STL exhibiting the highest endocytic capacity. Internalized STL was visualized by confocal microscopy to be localized to the cell membrane and cytoplasm. CONCLUSION: Based on favorable binding and uptake characteristics, potato lectin appears to be a useful candidate for further investigation as an ocular drug delivery system.

Impairment of conjunctival glutathione secretion and ion transport by oxidative stress in an adenovirus type 5 ocular infection model of pigmented rabbits.

Conjunctival epithelial cells of pigmented rabbits secrete reduced glutathione (GSH) into the apical (mucosal) fluid. The aim of the current study was to determine the effect of oxidative stress resulting from viral infection and that of GSH supplementation on redox status, GSH, and ion transport in freshly excised conjunctival tissues and epithelial cell layers in primary culture (RCEC) of adenovirus type 5 (Ad5)-infected rabbits. Lipid peroxidation (LPO) products, nitric oxide (NO), and expression of nitric oxide synthase (NOS2) were quantitated as a function of time after viral inoculation. Unidirectional fluxes of [3H]GSH and changes in short-circuit current (Isc) from mucosal supplementation of Ad5-inoculated conjunctival tissues with GSH and glutathione monoethyl ester (GSH-MEE) were also measured. Ad5 inoculation significantly decreased conjunctival GSH level by 19, 45, 48, and 50% at 8, 24, 48, and 72 h postinfection, respectively. LPO product and NO levels increased significantly (2- and 100-fold, respectively) above that of uninfected controls on Day 3 post-Ad5 inoculation, and co-treatment with GSH-MEE and tocopherol succinate abolished this effect. NO levels showed a progressive increase post-Ad5 inoculation, reaching 0.22 +/- 0.06, 8.12 +/- 0.91, and 2.05 +/- 0.65 microM on Days 1, 3, and 5, respectively, and the highest level was observed on the day of maximal viral replication (Day 3). A very significant induction of the expression of NOS2 on Days 1, 3, and 5 post-Ad5 inoculation was observed. Uninfected control conjunctival tissues displayed a net serosal-to-mucosal GSH flux (Jsm), where the mucosal-to-serosal flux (Jms) was approximately 14 pmol h(-1) cm(-2) and the Jsm was approximately 22 pmol h(-1) cm(-2). In Ad5-inoculated rabbits similar GSH flux was observed in both the sm and ms directions, and the net GSH flux was negligible. Isc and potential difference (PD) across conjunctival tissues of Ad5-inoculated rabbits decreased by > or = 50% compared with control, while the transepithelial electrical resistance (TEER) remained unchanged. Mucosal, but not serosal, superfusion of GSH or GSH-MEE in Ad5-inoculated conjunctival tissues increased the Isc by up to 40% in approximately 100 min. Our results show that net secretion of GSH across rabbit conjunctiva is totally blocked after Ad5 inoculation and active ion transport rate decreased by approximately 50%. Decreased net GSH secretion into mucosal fluid after Ad5 infection may have resulted from a decreased intracellular GSH pool due to oxyradical-induced changes in redox status and lower active ion transport. Mucosal treatment of Ad5-infected conjunctival tissues with pharmacological levels of GSH appears to transstimulate mucosal GSH secretion and restore active ion transport activity, suggesting a potentially useful therapeutic regimen for ocular infections.

The characteristics and mechanisms of uptake of PLGA nanoparticles in rabbit conjunctival epithelial cell layers.

PURPOSE: To delineate the characteristics and mechanisms of uptake of biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles in primary cultured rabbit conjunctival epithelial cells (RCECs). METHODS: Poly(D,L-lactide-co-glycolide) nanoparticles (PLGA 50:50. 101 nm in diameter) containing 6-coumarin (as a fluorescent marker) were used. The effect of size was studied using various particle sizes (100 nm, 800 nm, and 10 microm). The effect of cytochalasin D, nocodazole, and metabolic inhibitors on nanoparticle uptake was investigated. The capability of nanoparticles to enhance the uptake of an encapsulated protein. BSA bound to Texas red (TR-BSA), was evaluated. RESULTS: Maximal uptake of nanoparticles at 37 degrees C occurred at 2 h, and 100-nm particles had the highest uptake in RCECs in comparison with 800-nm and 10-microm particles. Nanoparticle uptake was saturable over the 0.1-4 mg/ml concentration range. Nanoparticle uptake was confirmed by confocal microscopy and was inhibited significantly by coumarin-free nanoparticles (of similar size), by lower incubation temperature, and by the presence of metabolic inhibitors and cytochalasin D. The uptake of encapsulated TR-BSA in RCECs at 4 h was 28% higher than free BSA application. CONCLUSION: Our findings suggest that PLGA nanoparticle uptake in primary cultured rabbit conjunctival epithelial cells occurs most likely by adsorptive-type endocytosis.

Clathrin and caveolin-1 expression in primary pigmented rabbit conjunctival epithelial cells: role in PLGA nanoparticle endocytosis.

PURPOSE: The internalization of poly (dl-lactide-co-glycolide, PLGA) nanoparticles in rabbit conjunctival epithelial cells (RCEC) was previously shown to occur by an endocytic process, as evidenced its energy-dependence, inhibition by the vesicle formation blocker cytochalasin D, and by the characteristic display of punctate distribution under confocal microscopy. In addition, clathrin protein was implicated in the endocytosis of these nanoparticles in vascular smooth muscle cells. We sought to examine the expression of clathrin and caveolin-1 in RCECs and to determine whether they play a role in PLGA nanoparticle endocytosis. METHODS: PLGA (50:50) nanoparticles (100 nm in diameter) containing 6-coumarin (fluorescent marker, 0.05% w/v) were used in this study. The effect of pharmacological treatments aimed at disrupting formation of clathrin-coated vesicles (hypertonic challenge and intracellular K+ depletion) and caveolae (nystatin and filipin) on apical uptake of nanoparticles in primary cultured RCEC was investigated. Transferrin was chosen as a marker for clathrin-dependent endocytosis from the basolateral aspect, whereas cholera toxin B subunit was chosen as a marker for caveolae-mediated endocytosis. The staining pattern of nanoparticles in RCECs was compared with that of clathrin heavy chain (HC) and caveolin-1 under fluorescent confocal microscopy to examine possible colocalization using clathrin HC and caveolin-1 mouse monoclonal antibodies (mAb). Two pairs of primers were designed (based on conserved regions of clathrin and caveolin-1 gene in different species) to amplify a 744-bp and 152-bp fragment of clathrin HC and caveolin-1 gene, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) to detect the message for clathrin HC and caveolin-1 was performed using total RNA prepared from freshly isolated RCECs. HEK293 cells were used as positive control for clathrin gene expression, whereas rabbit heart muscle and HEK cells were used as positive control for caveolin-1 gene expression. The RT-PCR products were separated using 2% agarose gel electrophoresis. Western blot analysis was performed to detect the expression of both clathrin and caveolin-1 proteins in RCECs using mouse mAbs. HeLa cells and A431 epidermoid cells were used as positive controls. The effect of transfection of RCECs (using Lipofectamine 2000TM reagent) with specific antisense oligonucleotides designed against the rabbit clathrin isoform on clathrin protein expression and PLGA nanoparticle uptake was investigated. RESULTS: Apical uptake of nanoparticles in primary cultured RCECs was decreased by 45% and 35%, respectively, as a result of K+ depletion and hypertonic media treatments. Likewise, the same treatments significantly decreased the basolateral uptake of FITC-transferrin by 50%. In contrast, nystatin and filipin had no effect on apical uptake of nanoparticles and cholera toxin B subunit in RCECs, suggesting a lack of the involvement of caveolae in the internalization of these two agents. Confocal microscopy showed fluorescent staining of cell membrane in the presence clathrin mAb, but not in the presence of caveolin-1 mAb, with partial overlap with a nanoparticle staining pattern. RT-PCR confirmed the presence of the clathrin HC gene, but not the caveolin-1 gene, in RCECs as indicated by a 744-bp fragment of the gene. However, caveolin-1 gene was detected in other rabbit tissues such as the epithelium of the cornea and trachea, and heart muscle, as indicated by a 152-bp fragment of the gene. Western blot analysis revealed a clathrin HC band (180 kDa) in RCEC culture and HeLa cells. However, caveolin-1 protein (22 kDa) was not detected in RCEC culture, but was detected in A431 cells. Transfection of RCECs with antisense oligonuceotide directed against clathrin HC resulted in knockdown of the clathrin HC protein in a concentration dependent manner. However, clathrin HC protein knockdown had no effect on apical uptake of nanoparticles in RCECs. CONCLUSIONS: Our findings indicate that endocytosis of nanoparticles in primary cultured RCECs occurs mostly independently of clathrin- and caveolin-1-mediated pathways. In addition, the gene and protein expression of clathrin HC, but not caveolin-1, was identified in rabbit conjunctival epithelial cells.

Analysis of transmembrane segment 7 of the dipeptide transporter hPepT1 by cysteine-scanning mutagenesis.

To investigate the involvement of transmembrane segment 7 (TMS7) of hPepT1 in forming the putative central aqueous channel through which the substrate traverses, we individually mutated each of the 21 amino acids in TMS7 to a cysteine and analyzed the mutated transporters using the scanning cysteine accessibility method. Y287C- and M292C-hPepT1 did not express at the plasma membrane. Out of the remaining 19 transporters, three (F293C-, L296C-, and F297C-hPepT1) showed negligible glycyl-sarcosine (gly-sar) uptake activity and may play an important role in defining the overall hPepT1 structure. K278C-hPepT1 showed approximately 40% activity and the 15 other transporters exhibited more than 50% gly-sar uptake when compared with wild type (WT)-hPepT1. Gly-sar uptake for the 16 active transporters containing cysteine mutations was then measured in the presence of 2.5 mM 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) or 1 mM [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET). Gly-sar uptake was significantly inhibited for each of the 16 single cysteine mutants in the presence of 2.5 mM MTSEA. In contrast, significant inhibition of uptake was only observed for K278C-, M279C-, V280C-, T281C-, M284C-, L286C-, P291C-, and D298C-hPepT1 in the presence of 1 mM MTSET. MTSET modification of R282C-hPepT1 resulted in a significant increase in gly-sar uptake. To investigate this further, we mutated WT-hPepT1 to R282A-, R282E-, and R282K-hPepT1. R282E-hPepT1 showed a 43% reduction in uptake activity, whereas R282A- and R282K-hPepT1 had activities comparable with WT-hPepT1, suggesting a role for the Arg-282 positive charge in substrate translocation. Most of the amino acids that were MTSET-sensitive upon cysteine mutation, including R282C, are located toward the intracellular end of TMS7. Hence, our results suggest that TMS7 of hPepT1 is relatively solvent-accessible along most of its length but that the intracellular end of the transmembrane domain is particularly so. From a structure-function perspective, we speculate that the extracellular end of TMS7 may shift following substrate binding, providing the basis for channel opening and substrate translocation.

Specialized protective role of mucosal glutathione in pigmented rabbit conjunctiva.

PURPOSE: To investigate mechanisms of H(2)O(2)-induced reduction in rates of active ion transport (I(sc)) across the pigmented rabbit conjunctival tissue and the protective role afforded by mucosal glutathione (GSH). METHODS: Changes in I(sc) and specific binding properties of ouabain were evaluated in a modified Ussing chamber setup, using conjunctival tissues freshly excised from pigmented rabbits. Effective concentrations of H(2)O(2) at which 50% of I(sc) was inhibited (IC(50)) were determined for the mucosal and serosal instillation of the agent. The rate of exogenous H(2)O(2) consumption in the mucosal and serosal bathing fluids was estimated. Mucosal 8-Br cAMP at 3 mM, serosal bumetanide at 0.5 mM, and both mucosal and serosal bathing of the conjunctiva with Na(+)-free bicarbonated Ringer's solution (BRS) were used to estimate contributions of conjunctival ion transport mechanisms in I(sc) changes elicited by mucosal H(2)O(2) at IC(50). Specific binding of (3)H-ouabain to the serosal side of the conjunctiva was estimated in the presence of mucosal or serosal H(2)O(2) to assess the role of functional Na(+)/K(+)-ATPase pumps in H(2)O(2) injury. The effect of mucosally instilled GSH and other reductive and nonreductive agents on possible restoration of oxidant-induced decrease in conjunctival I(sc) was also determined. RESULTS: Mucosal and serosal H(2)O(2) decreased conjunctival I(sc) gradually in a dose-dependent manner. The mucosal IC(50) of H(2)O(2)was 1.49 +/- 0.20 mM, whereas the serosal IC(50) was estimated at 10.6 +/- 2.0 micro M. The rate of H(2)O(2) consumption from mucosal fluid was six times faster than that from serosal fluid. Conjunctival tissues pretreated with mucosal H(2)O(2) at IC(50) retained approximately 50% of their maximum 8-Br cAMP-dependent increases in I(sc). Serosal bumetanide did not further reduce the I(sc) beyond the initial 70% decrease caused by mucosal H(2)O(2). When conjunctiva was bathed with Na(+)-free BRS on both the mucosal and serosal sides, before or after addition of mucosal H(2)O(2), the combined effects were additive, decreasing I(sc) by up to 95% to 99%. Mucosal, but not serosal, GSH or reduced L-glutathione mono-ethyl ester (GSH-MEE) superfusion of conjunctival tissues pre-exposed to mucosal H(2)O(2) at IC(50) recovered to 60% to 80% of the initial pre-H(2)O(2) I(sc) after approximately 100 minutes. The specific binding of (3)H-ouabain to the serosal side of the tissue was inhibited by 85% in the presence of mucosal or serosal treatment with H(2)O(2) at their respective IC(50) values. Pretreatment for 60 minutes with either 5 mM GSH, 2 mM GSH-MEE, or 0.1 mM ebselen, when instilled into the mucosal fluid, resulted in 30%, 45%, or 55% reductions, respectively, in ouabain binding after exposure to mucosal H(2)O(2) at IC(50). Furthermore, mucosal posttreatment with 10 mM GSH or 5 mM GSH-MEE of conjunctival tissues pre-exposed to mucosal H(2)O(2) resulted in a 30% recovery of the ouabain-binding level above that observed in tissues exposed to 1.5 mM H(2)O(2) alone on the mucosal side. By contrast, the decrease in conjunctival I(sc) or in the ouabain-binding level elicited by serosal H(2)O(2) at IC(50) was irreversible. CONCLUSIONS: A higher mucosal IC(50) of [H(2)O(2)] on conjunctival I(sc) corresponds to the faster consumption of exogenous H(2)O(2) from mucosal bathing fluid. In addition, actively secreted GSH by conjunctival epithelial cells may help reduce the injury by mucosally applied H(2)O(2). Injury by H(2)O(2) may directly affect vital membrane components (e.g., Na(+),K(+)-ATPase) involved in active ion transport across conjunctiva. Mucosal protection by GSH (or its analogues) of active conjunctival ion transport may be useful in maintaining the physiological functions of conjunctiva under oxidative stress.