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  • 1
    Electronic Resource
    Electronic Resource
    Transport and Hydrolysis of Enkephalins in Cultured Alveolar Epithelial Monolayers (1993)
    Springer
    Pharmaceutical research 10 (1993), S. 1662-1667 
    Details
    ISSN: 1573-904X
    Keywords: alveolar epithelium ; calcium ; cell culture ; enkephalins ; epithelial transport ; peptide hydrolysis ; pulmonary absorption
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1018941223967
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  • 2
    Electronic Resource
    Electronic Resource
    Regulation of Tight Junction Permeability by Calcium Mediators and Cell Cytoskeleton in Rabbit Tracheal Epithelium (1993)
    Springer
    Pharmaceutical research 10 (1993), S. 991-997 
    Details
    ISSN: 1573-904X
    Keywords: tracheal epithelium ; paracellular ; tight junction permeability ; calcium ; cytoskeleton
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The present study investigates the mechanisms controlling tight junction permeability of the tracheal epithelium, with an emphasis on the regulatory role of intra- and extracellular calcium as well as the cell cytoskeleton. The tracheas were isolated from rabbits and their junctional permeability barrier was investigated in vitro by means of transepithelial electrical resistance measurements and flux measurements of the radiolabeled paracellular tracer, 14C-mannitol. The effects of intra- and extracellular calcium were studied using the calcium ionophore A 23187 and EGTA, and that of the cytoskeleton was investigated using cytochalasin B. Intracellular calcium of the tracheal epithelium was monitored microfluorometrically using the specific calcium indicator, Fura-2 AM (acetoxymethyl ester). The results indicate that the tight junction permeability of the trachea was significantly increased upon treatment with all three of the test compounds, as evidenced by a substantial decrease in transepithelial electrical resistance and an increase in transepithelial flux of 14C-mannitol. The effects of EGTA and cytochalasin B on the tight junction permeability are fully reversible upon removal of the compounds from the bathing media. On the other hand, tissues treated with the calcium ionophore demonstrate a partial or no recovery in membrane permeability, depending on the intracellular calcium levels. Moderate and transient increases in intracellular calcium caused a partial reversibility of the membrane resistance, while high and sustained intracellular calcium levels induce a complete irreversibility of the membrane resistance. These results suggest that high extracellular calcium levels and low intracellular calcium levels are required for the normal maintenance of the junctional permeability in the tracheal epithelium. Studies using cytochalasin B indicate that there is also a close relationship between the tight junctions and the organization of actin microfilaments. Alterations of these structures as well as cellular calcium levels can result in a substantial change in transepithelial permeability. Therefore compounds that affect tight junction permeability may exert their action through the calcium and cytoskeleton mechanisms.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1018906504944
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  • 3
    Electronic Resource
    Electronic Resource
    Targeted Gene Delivery to Alveolar Macrophages via Fc Receptor-Mediated Endocytosis (1994)
    Springer
    Pharmaceutical research 11 (1994), S. 1731-1736 
    Details
    ISSN: 1573-904X
    Keywords: gene delivery ; receptor-mediated endocytosis ; alveolar macrophages ; IgG ; Fc receptor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Alveolar macrophage (AM) plays important roles in lung homeostasis and pathogenesis of diseases. The study of macrophage gene function and regulation as well as its potential therapeutic intervention will require the development of vectors capable of safe and efficient transfer of DNA to the AM. In the present study, we report a new transfection system that utilizes Fc receptor-mediated endocytosis as a means to target DNA to the AM. This system employs molecular conjugates consisting of a cognate moiety, in this case IgG which recognizes the AM Fc receptor, covalently-linked to a DNA-binding moiety, such as a cationic polyamine. A Complex was formed between immunoglobulin G-polylysine conjugate (IgG-pL) and plasmid DNA carrying the LacZ reporter gene (pSVβ). The conjugate-DNA complex was added directly to the AMs in culture and incubated for 24 h, after which LacZ gene expression was analyzed for β-galactosidase activity by microfluorometry using a fluorogenic β-galactosidase substrate, 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG). The AMs treated with the IgG-pL/DNA complex exhibited galactosidase activity significantly augmented over background levels. Effective gene transfer was shown to require both the DNA-binding moiety and cognate moiety for the cell surface receptor. Specific internalization of the complex by the Fc receptor pathway was verified by competitive inhibition using excess IgG. Under this condition, LacZ gene expression was inhibited, suggesting complex internalization through the Fc mediated endocytosis pathway. The requirement of Fc receptors for complex internalization was further demonstrated using cells that lack Fc receptors, e.g., alveolar epithelial cells. When exposed to the IgG-pL/pSVβ complex, these epithelial cells showed no susceptibility to gene transfer. Thus, the immune conjugate system may be used to accomplish targeted gene delivery to the AMs via the endocytosis pathway. Finally, the conjugate system was found to be nontoxic at concentrations effectively enhancing gene transfer, thereby, suggesting its potential safety in vivo.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1018959231951
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  • 4
    Electronic Resource
    Electronic Resource
    Novel Delivery of Antioxidant Enzyme Catalase to Alveolar Macrophages by Fc Receptor-Mediated Endocytosis (1994)
    Springer
    Pharmaceutical research 11 (1994), S. 1110-1114 
    Details
    ISSN: 1573-904X
    Keywords: macrophages ; immune complex ; catalase ; oxidation ; endocytosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Excessive production of reactive oxygen species by alveolar macrophages (AMs) in response to inhaled toxic substances is a major cause of oxidative lung injury. Therapeutic approaches designed to protect the lungs from oxidative injury by administering native antioxidant enzymes such as catalase and superoxide dismutase have been suggested. However, problems associated with poor penetration of these enzymes to the intracellular target sites have limited their effective use. The present study reports a drug targeting method based on receptor-mediated endocytosis of the antioxidant enzyme catalase to the AMs. This method employs molecular conjugate consisting of a cognate moiety, in this case IgG which recognizes the macrophage Fc receptor, covalently linked to the enzyme catalase via the reversible disulfide linkage. The uptake efficiency of the enzyme conjugate and its protection against oxidative injury were evaluated microfluorometrically using the intracellular oxidative probe dichlorodihydrofluorescein BSA: anti BSA antibody complex (DCHF-IC), and the cell viability indicator propidium iodide. The DCHF-IC-stimulated macrophages exhibited a dose- and time-dependent increase in intracellular fluorescence with a half maximal response dose of approximately 120 µg/ml. Free catalase (50–500 U/ml) failed to inhibit the DCHF-IC-induced oxidative burst and had only a marginal protective effect on AM injury. In contrast, the catalase-IgG conjugate (50–500 U/ml) strongly inhibited both the DCHF-IC-induced oxidation and injury in a dose-dependent manner. Effective inhibition was shown to require both the antioxidant catalase moiety and the cognate moiety for the cell surface receptor. Specific internalization of the conjugate through the Fc receptor was also investigated by competitive inhibition using free IgG. Under this condition, the conjugate showed a much reduced protective effect on intracellular oxidation, indicating conjugate internalization through the Fc endocytosis pathway. Thus, the enzyme-IgG conjugate system may be used as an effective and selective means to deliver antioxidant enzymes to the intracellular oxidative targets of the AMs.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1018976529766
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  • 5
    Electronic Resource
    Electronic Resource
    Alveolar Permeability Enhancement by Oleic Acid and Related Fatty Acids: Evidence for a Calcium-Dependent Mechanism (1994)
    Springer
    Pharmaceutical research 11 (1994), S. 513-517 
    Details
    ISSN: 1573-904X
    Keywords: oleic acid ; alveolar epithelium ; permeability ; cellular damage ; calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Pulmonary exposure to oleic acid (OA) is associated with permeability alterations and cellular damage; however, the exact relationship between these two effects has not been clearly established. Using cultured alveolar epithelial monolayers, we demonstrated that OA and some other fatty acids (≤50 µM) can induce permeability changes without detectable cellular damage. At higher concentrations, however, OA caused severe membrane damage and leakage to solute flux. The permeability enhancing effect of OA was observed with both the paracellular marker 3H-mannitol and the lipophilic transcellular indicator 14C-progesterone. While the effect of OA on transcellular permeability may be attributed to its known effect on membrane fluidity, the paracellular promoting effect of OA and its mechanism are not well established. We postulated that OA may increase paracellular permeability through a Ca2+-dependent tight junction mechanism. Using dual-excitation fluorescence microscopy, we demonstrated that OA can increase intracellular calcium, [Ca2+]i , in a dose-dependent manner. This effect was transient at low OA concentrations (≤50 µM) but became more pronounced and sustained at higher concentrations. Free hydroxyl and unsaturated groups were required for this activation since esterified OA (oleic methyl ester) and stearic acid (a saturated fatty acid with equal chain length) had much reduced effects on both the [Ca2+]i and the permeability alterations. Degree of unsaturation was unimportant since linolenic acid (18:3), linoleic acid (18:2), and OA (18:1) had similar and comparable effects on the two parameters. When the alveolar epithelium was bathed with Ca2+ -free medium, OA failed to elevate [Ca2+ ]i , suggesting that Ca2+ influx from the extracellular medium is responsible for the observed [Ca2+]i rise. This effect of OA was not due to nonspecific membrane damage since the monolayer maintained its integrity and the [Ca2+]ireturned to pretreatment levels after an initial transient rise. Moreover, the permeability alteration was fully reversible upon removal of OA. These results suggest that the alveolar permeability may be reversibly enhanced by sublethal concentrations of oleic acid.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1018906330308
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  • 6
    Electronic Resource
    Electronic Resource
    Receptor-Mediated Peptide Delivery in Pulmonary Epithelial Monolayers (1994)
    Springer
    Pharmaceutical research 11 (1994), S. 1121-1126 
    Details
    ISSN: 1573-904X
    Keywords: pulmonary absorption ; receptor-mediated endocytosis ; transcytosis ; alveolar epithelium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1018980630675
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  • 7
    Electronic Resource
    Electronic Resource
    The Transport Barrier of Epithelia: A Comparative Study on Membrane Permeability and Charge Selectivity in the Rabbit (1992)
    Springer
    Pharmaceutical research 9 (1992), S. 1029-1034 
    Details
    ISSN: 1573-904X
    Keywords: epithelial transport ; permeability ; permselectivity ; absorption ; electrical resistance ; electrical conductance ; diffusion potential
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The transport barrier of the epithelia presents one of the major problems limiting the effective use of these tissues as alternate delivery routes for macromolecules such as peptides and proteins. In the present study, two membrane transport properties, namely, the permeability and permselectivity of the shunt pathway, were investigated and compared in various tissues including the nasal, tracheal, bronchial, buccal, rectal, vaginal, corneal, epidermal, duodenal, jejunal, ileal, and colonic epithelia. Membrane permeability was evaluated using a combined method based on electrical conductance and flux measurements of a hydrophilic fluorescent probe, 6-carboxy fluorescein (CF). Membrane permselectivity or the charge discriminating ability of the membrane was evaluated by KCl diffusion potential measurements. The results indicate that all epithelia under investigation possess a relatively high degree of permeation barrier and are highly selective for the absorption of positively charged solutes. Shunt path permeability was found to vary greatly among tissues from different epithelia, whereas membrane charge selectivity was relatively constant in these tissues. A good correlation was observed between membrane electrical conductance and steady-state flux of CF, indicating a paracellular transport of the compound. The rank order of the intrinsic membrane permeability was as follows: intestinal≈ nasal ≥ bronchial ≥ tracheal 〉 vaginal ≥ rectal 〉 corneal 〉 buccal 〉 skin. Membrane permselectivity, expressed as the ratio of transport number (positive over negative), ranges from 1.78 for the buccal to 1.33 for the rectal epithelium. These results suggest that, for effective delivery purposes, permeation enhancing methods, by either increasing tissue permeability or modifying drug-membrane charge selectivity, are generally required. The permeation data also suggest that the respiratory epithelia represent good alternate routes for drug delivery, particularly for those that are orally ineffective, i.e., due to extensive gastrointestinal tract degradation or first-pass metabolism.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1015802427428
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  • 8
    Electronic Resource
    Electronic Resource
    Altered calcium homeostasis and cell injury in silica-exposed alveolar macrophages (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 154 (1993), S. 310-316 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: There is evidence to suggest that cell injury induced in alveolar macrophages (AM) following phagocytic activation by silica particles may be mediated through changes in intracellular free calcium [Ca2+]i. However, the mechanism of silica- induced cytotoxicity relative to [Ca2+]i overloading is not yet clear. To provide a better insight into this mechanism, isolated rat AMs were exposed to varying concentrations of crystalline silica (particle size 〈 5 μm in diameter) and the fluctuation in their [Ca2+]i and cell integrity were quantitatively monitored with the fluorescent calcium probe, Fura-2 AM, and the membrane integrity indicator, propidium iodide (PI). Results from this study indicate that silica can rapidly increase [Ca2+]i in a dose-dependent manner with a characteristic transient calcium rise at low doses (〈0.1 mg/ml) and an elevated and sustained rise at high doses (〉0.1 mg/ml). Depletion of extracellular calcium [Ca2+]o markedly inhibited the [Ca2+]i rise (≈90%), suggesting that Ca2+ influx from extracellular source is a major mechanism for silica-induced [Ca2+]i rise. When used at low doses but sufficient to cause a transient [Ca2+]i rise, silica did not cause significant increase in cellular PI uptake during the time of study, suggesting the presevation of membrane integrity of AMs under these conditions. At high doses of silica, however, a marked increase in PI nuclear fluorescence was observed. Depletion of [Ca2+]o greatly inhibited cellular PI uptake, induced by 0.1 mg/ml or higher doses of silica. This suggests that Ca2+ influx, as a result of silica activation, is associated with cell injury. Indeed, our results further demonstrated that the low dose effect of silica on Ca2+ influx is inhibited by the Ca2+ channel blocker nifedipine. At high doses of silica (〉0.1 mg/ml), cell injury was not prevented by nifedipine or extracellular Ca2+ depletion, suggesting that other cytotoxic mechanisms, i.e., nonspecific membrane damage due to lipid peroxidation, are also responsible for the silica-induced cell injury. Silica had no significant effect on cellular ATP content during the time course of the study, indicating that the observed silica-induced [Ca2+]i rise was not due to the impairment of Ca2+-pumps, which restricts Ca2+ efflux. Pretreatment of the cells with cytochalasin B to block phagocytosis failed to prevent the effect of silica on [Ca2+]i rise. Taken together, these results suggest that the elevation of [Ca2+]i caused by silica is due mainly to Ca2+ influx through plasma membrane Ca2+ channels and nonspecific membrane damage (at high doses). Neither ATP depletion nor Ca2+ leakage during phagocytosis was attributed to the silica-induced [Ca2+]i rise. © 1993 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041540214
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  • 9
    Book
    Book
    Biopharmaceutical drug design and development (1999)
    Totowa, NJ :Humana Pr.,
    Details
    Title: Biopharmaceutical drug design and development
    Contributer: Wu-Pong, Susanna , Rojanasakul, Yongyut
    Publisher: Totowa, NJ :Humana Pr.,
    Year of publication: 1999
    Pages: 435 S.
    Type of Medium: Book
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