Biomedical subjects

Y Rojanasakul

Publications and source records attributed to Y Rojanasakul.

51 records · Page 3Linked to original sources

PubMed1994

Novel delivery of antioxidant enzyme catalase to alveolar macrophages by Fc receptor-mediated endocytosis.

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 micrograms/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 ant 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Receptor-mediated peptide delivery in pulmonary epithelial monolayers.

The present study investigated the feasibility of utilizing receptor-mediated endocytosis as a means to enhance peptide delivery to the pulmonary epithelium. The strategy employs a molecular conjugate consisting of a cognate moiety, transferrin (TF), covalently-linked to a model polypeptide, horseradish peroxidase (HRP), via a reversible disulfide linkage. A cultured alveolar epithelial monolayer system was used to simulate the conditions of the pulmonary epithelium and to allow accurate quantitation of intra- and transcellular peroxidase transport. The alveolar cells were isolated from rat lungs by enzymatic digestion and grown on microporous tissue culture-treated polycarbonate filters. A significant increase in the uptake of HRP by the cell monolayer was observed upon its conjugation with TF. The effect was found to be concentration-dependent, being more pronounced at low concentrations, i.e., 3.9- and 1.2-fold increase over unconjugated HRP controls at the concentration levels of 0.05 and 1.50 U/ml respectively. Effective peroxidase uptake was shown to require the TF cognate moiety for the cell surface receptor. Specific internalization of the conjugate by the TF endocytic pathway was verified by competition for the TF receptor. Conjugate internalization was not followed by a proportional increase in transcytosis, i.e., at 0.05 U/ml conjugate level, a 1.7-fold increase in transcytosis was observed as compared to 3.9-fold for endocytosis. Effective enhancement of transcytosis was achieved by treating the monolayers with brefeldin A (BFA), a compound known to affect intracellular transport of TF receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Reaction of Cr(VI) with ascorbate and hydrogen peroxide generates hydroxyl radicals and causes DNA damage: role of a Cr(IV)-mediated Fenton-like reaction.

Incubation of Cr(VI) with ascorbate generated Cr(V), Cr(IV) and ascorbate-derived carbon-centered alkyl radicals, as well as formyl radicals. H2O2 caused generation of hydroxyl radicals (OH) and much higher levels of Cr(V), showing that .OH can be generated via a Cr(IV)-mediated Fenton-like reaction (Cr(IV) + H2O2-->Cr(V) + .OH + OH-). 1,10-Phenanthroline and deferoxamine inhibited the formation of both .OH and Cr(V) from the reaction of Cr(VI) with ascorbate in the presence of H2O2. Electrophoretic assays showed that ascorbate-derived free radicals caused DNA double-strand breaks. .OH radicals generated by Cr(V)- and Cr(IV)-mediated Fenton-like reactions also caused DNA double-strand breaks. HPLC measurements showed that .OH radicals generated by Cr(IV) and Cr(V) from H2O2 caused 2'-deoxyguanine hydroxylation to form 8-hydroxy-2'-deoxyguanine.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1994

Fluoromicroscopic studies of bleomycin-induced intracellular oxidation in alveolar macrophages and its inhibition by taurine.

The mechanism of bleomycin-induced pulmonary fibrosis is not yet clear. Recent studies have shown that alveolar macrophages (AM) can be stimulated by bleomycin in vitro releasing inflammatory cytokines, suggesting that the interaction of bleomycin with AM is an important step in the drug-induced fibrotic process. Bleomycin is known to bind DNA and generate oxygen radicals through complexation with Fe2+ and oxygen. To provide more insight into the cellular oxidative property of bleomycin, we have developed a fluoromicroscopic method using 2',7'-dichlorofluorescin diacetate (DCFHDA) as an oxidative fluorescence probe to study the bleomycin-induced intracellular oxidation in rat AM and the inhibition of the oxidation by taurine, a compound known to inhibit the bleomycin-induced fibrosis. Bleomycin at 5 to 20 micrograms/ml has a moderate stimulatory effect (1.87- to 2.66-fold) on the secretion of superoxide anion. A high concentration of bleomycin (20 micrograms/ml), however, inhibits cell response to zymosan-induced secretion of superoxide anion. At 4 micrograms/ml, bleomycin has no effect on cell membrane integrity or morphology but results in a significant increase in intracellular oxidation. This oxidative process is Fe(2+)-dependent and is accompanied by an increase in intracellular calcium (35 nM). Both the intracellular oxidation and calcium rise induced by internalized bleomycin are inhibited by pretreatment of cells with varying concentrations of taurine (25, 125, and 187.5 microM). The inhibitory effect on intracellular oxidation was found to be 36, 57, and 60%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Altered calcium homeostasis and cell injury in silica-exposed alveolar macrophages.

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 microns 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 (approximately 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 preservation 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 Ca(2+)-ATPase 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).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Inhibition of proliferative activity of pulmonary fibroblasts by tetrandrine.

Tetrandrine, an herbal drug, has been employed in China to treat pulmonary fibrosis. To date, the mechanisms governing the antifibrotic action of tetrandrine are unknown. The present study employs a fibroblast mitogenic assay to determine whether tetrandrine directly inhibits the ability of fibroblasts to respond to stimulation by growth factors. The data indicate that tetrandrine blocks proliferation and the incorporation of tritiated thymidine into DNA by fibroblasts stimulated with human serum, PDGF plus plasma, FGF plus plasma, or TNF plus plasma. Since tetrandrine inhibits the response to a variety of growth factors, its action does not appear to involve the blockade of a specific stimulatory receptor. Tetrandrine is effective in inhibiting thymidine incorporation when added up to 6 hr after stimulation of quiescent cells, suggesting either that tetrandrine does not block the attainment of competence by fibroblasts or that its activity is not limited to blocking the attainment of competence by these cells. Growth factor-induced mitogenesis is also inhibited by nitrendipine, a calcium channel blocker, and by cytochalasin B, a microfilament blocker. However, tetrandrine treatment of fibroblasts neither results in the changes of morphology seen with cytochalasin B nor is limited to the early events of stimulus-response coupling. Therefore, the mechanism of action for tetrandrine is not identical to that for either cytochalasin B or nitrendipine. In summary, these results suggest that the antifibrotic action of tetrandrine may be mediated in part by direct inhibition of fibroblast proliferation normally associated with the development and progression of silicosis.

Alkaloids↗

PubMed1993

Regulation of tight junction permeability by calcium mediators and cell cytoskeleton in rabbit tracheal epithelium.

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Transport and hydrolysis of enkephalins in cultured alveolar epithelial monolayers.

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

Animals↗

PubMed1993

Mechanisms of hydroxyl free radical-induced cellular injury and calcium overloading in alveolar macrophages.

Excessive production of reactive oxygen radicals by alveolar macrophages is proposed to play an important role in oxidative lung injury. A major product oxygen radical formation is the highly reactive hydroxyl radical (.OH) generated via a biologic Fenton reaction. In addition to its known ability to induce lipid peroxidation, recent studies have suggested that the .OH may exert its cytotoxic effect through the alteration of [Ca2+]i homeostasis. To test this potential mechanism as well as to investigate the relationship between .OH and Ca2+ overloading in cytotoxic injury, isolated rat alveolar macrophages were exposed to externally generated radical system, H2O2 (0.01 to 1 mM) and Fe2+ (1 mM) and their [Ca2+]i levels and cell injury were monitored using quantitative fluorescence microscopy with the aid of the specific Ca2+ indicator, Fura-2, and membrane integrity indicator, propidium iodide. Electron spin resonance measurements using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) confirmed the production of the .OH radical by this system. Upon the addition of the radicals, the macrophages displayed a rapid initial rise in [Ca2+]i which was followed by a slower but more pronounced [Ca2+]i elevation that reached a level 3 to 5 times higher than the basal level. This process preceded cell death as evident by nuclear propidium iodide fluorescence. Depletion of extracellular Ca2+ inhibited both the [Ca2+]i response and cell injury. Preincubation of the cells with the Ca2+ channel blocker verapamil or .OH radical scavenger mannitol similarly inhibited the [Ca2+]i rise and loss of viability. Firefly luciferase assay of cellular ATP content demonstrated that the alterations in [Ca2+]i following .OH treatment preceded the depletion of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

The transport barrier of epithelia: a comparative study on membrane permeability and charge selectivity in the rabbit.

The transport barrier of the epithelial 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 approximately nasal greater than or equal to bronchial greater than or equal to tracheal greater than vaginal greater than or equal to rectal greater than corneal greater than buccal greater than 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Immunohistochemical detection of steroid binding in rabbit cornea.

An indirect immunoperoxidase technique was used to demonstrate progesterone binding in rabbit cornea as a measure of steroid absorption into corneal tissues. Tissue preparation prior to immune staining was prepared by both a pre-fixed, paraffin-embedding method and a post-fixed, cryostat sectioning method. Rabbit antiprogesterone antiserum and peroxidase conjugated goat antirabbit antiserum were used as a primary and secondary antibody respectively. The immune staining of the antibody-treated sections was then developed in a solution of 3-3' diaminobenzidine-hydrogen peroxide system. The resulting, brown colored, end-products at the site of reaction indicate the location of bound steroid in the sections. Paraffin-embedded sections showed negative staining in both steroid treated and untreated control corneas while fixed frozen sections showed only positive staining for the treated corneas. The results suggest the preservation of the immunogenicity in the treated frozen sections. The negative result observed in the paraffin-embedded sections may be attributed to the loss of steroid from the tissues during the dehydration and paraffin embedding processes. It was found that the immunoreactive progesterone localized mainly in the epithelial cells and the bottom portion of the stroma. Prior application with an excess of triamcinolone acetonide, to the treated corneas gave negative results indicating the reversible and competitive nature of the binding.

Animals↗

PubMedDate not supplied

A cell-based drug delivery system for lung targeting: I. Preparation and pharmacokinetics.

A drug-loaded tumor cell (DLTC) system has been developed for lung metastasis-targeting drug delivery. Doxorubicin was loaded into B16-F10 murine melanoma cells (96 microg/10(6) cells). The loading process led to the death of all the carrier cells. The diameter of DLTC was 15.03+/-2.36 microm (mean +/- SD). The amount and rate of doxorubicin being released from the DLTC mainly depended on the drug loading and carrier cell concentration. Over a 6-month storage in phosphate buffered saline (PBS) at 4 degrees C, the decrease in intracellular drug concentration and the carrier cell number were less than 25% and 5%, respectively. After a bolus injection of 30 microg doxorubicin in either DLTC form or free solution into the mice tail veins, drug deposit in the lung from DLTC was 3.6-fold of that achieved by free drug solution. The latter resulted in higher drug content in liver and spleen. Extensive trypsinization of DLTC reduced its lung targeting effect by 30%, and the density of surface adhesion molecule GM3 on DLTC surface by 25%. In conclusion, this DLTC system demonstrated a lung-targeting activity that may be partially attributed to its specific surface characteristics.

Animals↗

PubMedDate not supplied

A cell-based drug delivery system for lung targeting: II. Therapeutic activities on B16-F10 melanoma in mouse lungs.

The in vitro and in vivo anticancer activities of doxorubicin-loaded B16-F10 murine melanoma cells (DLTC) were evaluated. DLTC showed similar growth-inhibitory effects against live B16-F10 cells with doxorubicin solution in cell culture system, with the IC50 of 0.11 microM and 0.17 microM, respectively. However, DLTC demonstrated higher effectiveness than the free solution in treating mouse lung cancer caused by live B16-F10 cells. Syngeneic C57BL mice were inoculated intravenously with live B16-F10 cells first, and then received daily treatment of intravenous injections of doxorubicin in either DLTC or free solution form. Compared with the control group treated with phosphate-buffered saline, DLTC eradicated almost all the lung cancer colonies (>99%), while the free solution form reduced the colonies by 61%, when the treatment was given at an early stage. If the treatment started after the establishment of micrometastatic colonies in the mouse lungs, DLTC and free solution treatment resulted in 85% and 30% cancer reduction, respectively. Additional experiments demonstrated that the reduction of lung cancer colonies by DLTC was related to the initial treatment time: the earlier the treatment, the greater the effect. In conclusion, DLTC showed better therapeutic outcomes than free solution form in treating lung cancer of our animal model.

Animals↗

PubMedDate not supplied

Inhibitory action of tetrandrine on macrophage production of interleukin-1 (IL-1)-like activity and thymocyte proliferation.

Tetrandrine is a bisbenzylisoquinoline alkaloid which has been shown to exhibit antifibrotic activity against silicosis. Tetrandrine is characterized by its strong binding to alveolar macrophages and inhibition of particle-induced respiratory burst activity in these phagocytes. In contrast, tubocurine and tubocurarine are structurally similar to tetrandrine but exhibit little effect on fibrosis or activation of alveolar macrophages. The objective of the present study was to test the effect of tetrandrine on macrophage production of monokines in response to occupational dusts, and to determine tetrandrine's effect on monokine-medicated cell growth using a mouse thymocyte proliferation assay and lipopolysaccharide (LPS) as a positive control. Stimulation of alveolar macrophages by respirable silica dust resulted in a release of monokines which caused a fourfold increase in thymocyte proliferation. Coal dust, on the other hand, had no effect on macrophage production of this cytokine. Tetrandrine was found to exhibit a dose-dependent inhibition of monokine release from both silica and LPS-stimulated alveolar macrophages. In experiments where thymocytes were directly treated with tetrandrine, a dose-dependent inhibition of thymocyte proliferation was noted with both interleukin-1-(IL-1) specific and nonspecific mitogenic (concanavalin A) actions. In contrast to the inhibitory potency of tetrandrine, tubocurarine was found to have no effect on either the production of monokines by LPS-stimulated alveolar macrophages or IL-1-mediated thymocyte proliferation. These results provide a correlation between the antifibrotic effect of tetrandrine and inhibition of macrophage activation.

Alkaloids↗

PubMedDate not supplied

Role of hydroxyl radical in silica-induced NF-kappa B activation in macrophages.

Nuclear transcription factor kappa B (NF-kappa B) is a multiprotein complex that regulates a variety of genes important for immunity and inflammation. The present study investigates the silica-induced activation of this transcription factor in mouse macrophage cell line RAW 264.7 cells, the role of free radical reactions in the mechanism of the activation, and its possible inhibition. Tetrandrine, a benzylisoquinoline alkaloid, which has been used as an antifibrotic drug to treat the lesions of silicosis and has been characterized as a hydroxyl radical (.OH) scavenger, inhibited the NF-kappa B activation induced by silica, lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA). Catalase, metal chelator, deferoxamine, and the silanol group (SiOH) blocker, poly(2-vinylpyridine-N-oxide) (PVPNO), also inhibited silica-induced NF-kappa B activation. Electron spin resonance (ESR) spin trapping measurements show that both deferoxamine and PVPNO decreased silica-mediated .OH radical generation from H2O2. It is shown that Fe(II) and not Fe(III) is able to cause NF-kappa B activation. The antioxidant, ascorbate, attenuated the NF-kappa B activation induced by silica but not by LPS. The .OH radical scavenger, sodium formate, inhibited NF-kappa B activation induced by silica but had only a minor effect on NF-kappa B activation induced by LPS. The results indicate that silica-mediated free radical generation via the Fenton or Fenton-like reaction (M(n)+ + H2O2-->M(n + 1)+ + OH- + .OH) and silanol groups on the silica surface play an important role in silica-induced NF-kappa B activation.

Alkaloids↗