Effects of olopatadine hydrochloride on the cutaneous vascular hyperpermeability and the scratching behavior induced by poly-L-arginine in rats.

Intradermal injections of poly-L-arginine induce cutaneous vascular hyperpermeability and scratching behavior in rats. Recently, we elucidated that the plasma extravasation involved both histamine and substance P, while the scratching behavior involved substance P, but not histamine. This study examined the effects of olopatadine hydrochloride (olopatadine), an antiallergic drug with histamine H1-antagonistic action, on the poly-L-arginine-induced responses. Olopatadine (1 mg/kg, p.o.) significantly inhibited both the plasma extravasation and the scratching behavior, suggesting that its inhibitory effects are mediated by the suppression of neuropeptidergic action as well as histaminic action. Olopatadine seems to be a novel-type drug for the treatment of dermatitis.

Occurrence of organo-arsenicals in jellyfishes and their mucus.

Water-soluble arsenic compound fractions were extracted from seven species of jellyfishes and subjected to analysis by high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) for arsenicals. A low content of arsenic was found to be the characteristic of jellyfish. Arsenobetaine (AB) was the major arsenic compound without exception in the tissues of the jellyfish species and mucus-blobs collected from some of them. Although the arsenic content in Beroe cucumis, which preys on Bolinopsis mikado, was more than 13 times that in B. mikado, the chromatograms of these two species were similar in the distribution pattern of arsenicals. The nine species of jellyfishes including two species treated in the previous paper can be classified into arsenocholine (AC)-rich and AC-poor species. Jellyfishes belonging to Semaostamae were classified as AC-rich species.

Roles of mast cells and sensory nerves in cutaneous vascular hyperpermeability and scratching behavior induced by poly-L-arginine in rats.

We investigated whether the polycation poly-L-arginine elicited cutaneous vascular hyperpermeability and scratching behavior and, if so, whether these responses involved mast cells and sensory nerves in rats. Intradermal injections of poly-L-arginine induced vascular hyperpermeability and scratching behavior. Combined treatment with chlorpheniramine and methysergide almost completely suppressed the poly-L-arginine (50 microg/site)-induced plasma leakage. Capsaicin desensitization and the tachykinin NK(1) receptor antagonist LY303870, (R)-1-[N-(2-methoxybenzyl)acetylamino]-3-(1H-indol-3-yl)-2-[N-(2-(4-(piperidin-1-yl)piperidin-1-yl)acetyl)amino]propane, partially inhibited the leakage. In mast cell-deficient rats, poly-L-arginine only minimally induced plasma leakage. On the other hand, capsaicin desensitization and LY303870, but not chlorpheniramine or methysergide, suppressed the poly-L-arginine (200 microg/site)-induced scratching. Moreover, poly-L-arginine elicited the scratching even in mast cell-deficient rats. These results suggest that substance P is at least partly involved in both the cutaneous plasma leakage and the scratching behavior induced by poly-L-arginine. Moreover, mast cell-derived amines are suggested to be involved in the plasma extravasation but scarcely, if any, in the scratching behavior.

Involvement of neuropeptides in the allergic nasal obstruction in guinea pigs.

The purposes of the present study were i) to determine whether neuropeptides induce the nasal obstruction in guinea pigs, and ii) to examine the possible involvement of neuropeptides in allergic nasal obstruction. The decrease in nasal cavity volume was determined by acoustic rhinometry as an index of nasal obstruction. In non-sensitized guinea pigs, substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) caused the nasal obstruction 10 to 30 min after their intranasal application. LY303870 (1 mg/kg), a tachykinin NK1-receptor antagonist; SR48968 (1 mg/kg), a tackykinin NK2-receptor antagonist; and CGRP(8-37) (50 nmol/kg), a CGRP1-receptor antagonist, administered intravenously before the intranasal application of the neuropeptides, inhibited the responses induced by SP, NKA and CGRP, respectively. In the guinea pigs sensitized with dinitrophenyl-coupled Ascaris suum allergenic extract, the intranasal antigen challenge caused nasal obstruction. The response was biphasic and consisted of the early phase response (EPR) and the late phase response (LPR), which developed 30 min and 6 h, respectively, after the antigen challenge. Intravenous administration of LY303870 (1 mg/kg) before the antigen challenge inhibited the EPR, while those of SR48968 (1 mg/kg) and CGRP(8-37) (50 nmol/kg) inhibited the LPR. The present results suggest that neuropeptides are involved in the allergic nasal obstruction.

Inhibitory effect of olopatadine hydrochloride on the sneezing response induced by intranasal capsaicin challenge in guinea pigs.

To investigate the possible inhibitory effect of olopatadine hydrochloride (olopatadine), an antiallergic drug, on the tachykinin-mediated nasal responses, we examined the effect of olopatadine on the sneezing and the nasal rubbing responses induced by intranasal capsaicin challenge in guinea pigs. Olopatadine (10 mg/kg, p.o.) inhibited the sneezing response by 57% without affecting the nasal rubbing one. The antihistamines chlorpheniramine and clemastine did not affect the responses. Morphine caused the inhibition of both responses, which was antagonized by naloxone. These results suggest that olopatadine inhibits the sneezing response by the inhibition of the tachykinin release and not by its antihistaminic action.

Isolation and characterization of arsenate-sensitive and resistant mutants of Chlamydomonas reinhardtii.

Arsenate-sensitive and resistant mutants of Chlamydomonas reinhardtii were obtained by screening mutants generated by random insertional mutagenesis for growth in the presence of various concentrations of arsenate. The intracellular concentrations of arsenic in the mutants kept in the arsenate-containing medium were determined with an atomic absorption spectrophotometer. The intracellular levels of arsenic in the arsenate-resistant mutants were all lower than that of the parent strain CC425. Some of the arsenate-sensitive mutants, AS1 and AS3, showed obviously higher levels of arsenic than that of CC425, while other sensitive mutant, AS2, did not accumulate arsenic so much. Analysis of the chemical species of arsenic suggested that inorganic arsenic was converted to dimethylarsinic acid (DMAA) in CC425. However, DMAA was hardly detected in AS2. The mechanisms of the resistance to arsenate are discussed on its uptake and detoxification.

Protective effect of erdosteine against hypochlorous acid-induced acute lung injury and lipopolysaccharide-induced neutrophilic lung inflammation in mice.

The effect of erdosteine, a mucoactive drug, on hypochlorous acid (HOCl)-induced lung injury, and the lipopolysaccharide (LPS)-induced increase in tumour necrosis factor-alpha (TNF-alpha) production and neutrophil recruitment into the airway, was investigated. Male BALB/c mice were orally administered erdosteine (3-100 mgkg(-1)), ambroxol hydrochloride (ambroxol) (3-30 mgkg(-1)), S-carboxymethyl-L-cysteine (S-CMC) (100-600 mgkg(-1)) or prednisolone (10 mgkg(-1)), 1 h before intratracheal injection of HOCl or LPS. In the HOCl-injected mice, erdosteine markedly suppressed increases in the ratios of lung wet weight to bodyweight and lung dry weight to bodyweight, whereas the other mucoactive drugs ambroxol and S-CMC had little effect. Erdosteine also inhibited the LPS-induced neutrophil influx, although it did not affect the increased level of TNF-alpha in the bronchoalveolar lavage fluid. The results suggest that attenuation of reactive oxygen species and neutrophil recruitment is involved in the clinical efficacy of erdosteine in the treatment of chronic bronchitis.

Mucolytic and antitussive effects of erdosteine.

To investigate the influence of erdosteine, a new homocysteine-derived expectorant, on airway clearance we studied the effects of the drug on the viscosity of mucin, on the mucociliary transport rate in quails, on airway secretion in rats and on the cough reflex in guinea-pigs. The active metabolite of erdosteine, M1 (10 microM to 1 mM), significantly reduced the viscosity of porcine stomach mucin. Erdosteine by itself did not reduce viscosity. Erdosteine significantly promoted mucociliary transport in quails and increased airway secretion in rats. The effect was still apparent 24h after administration. Erdosteine significantly suppressed citric acid-induced cough reflexes in guinea-pigs but did not suppress mechanical stimuli-induced cough reflexes. Erdosteine suppressed the reduction of the recovery volume of bronchoalveolar lavage fluid and albumin leakage into the fluid in citric acid-exposed guinea-pigs. These results indicate that erdosteine removes sputum by reducing its viscosity, and by promoting mucociliary transport and sustained enhancement of airway secretion. It also suppressed the chemical stimulation-induced cough reflex and plasma leakage into the airway. These results suggest that erdosteine is an excellent expectorant with several modes of action.

The effect of erdosteine and its active metabolite on reactive oxygen species production by inflammatory cells.

OBJECTIVE: We examined the effect of erdosteine (KW-9144), an expectorant, and related compounds on inflammatory cell-derived reactive oxygen species which are involved in airway inflammation. METHODS: Neutrophils were isolated from peritoneal lavages of casein-injected rats and from peripheral blood of healthy human donors. Eosinophils were isolated from peritoneal lavages of horse serum-injected guinea pigs. These cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and the production of reactive oxygen species was measured with luminol-dependent chemiluminescence (LDCL). RESULTS: M1, an active metabolite of erdosteine, significantly inhibited PMA-induced LDCL of the all cell populations with treatment before stimulation. The effects of S-carboxymethylcysteine (S-CMC), ambroxol and N-acetylcysteine (NAC) on the LDCL response were weaker than those of M1. Furthermore, PMA-induced LDCL was decreased by posttreatment with M1. CONCLUSION: These results suggest that M (an active metabolite of erdosteine) may exert an antiinflammatory effect by scavenging inflammatory cells-derived reactive oxygen species.

Accuracy of measurement of acoustic rhinometry applied to small experimental animals.

Nasal obstruction is one of the major symptoms of allergic rhinitis. In the study of the mechanism of nasal obstruction, experiments on animal are useful. In adult humans, acoustic rhinometry has been used to evaluate nasal obstruction by determining nasal cavity dimensions in terms of cross-sectional areas as a function of the distance from the nostril. We modified the equipment used on humans to assess dimensions of nasal airway geometry of small experimental animals. The purpose of this study was to investigate the accuracy of measurement of the modified acoustic rhinometry applied to small experimental animals using nasal cavity models and guinea pigs. Measurement of the nasal cavity models (made of cylindrical silicone tubes) showed that the acoustic rhinometry estimated 85.5% of actual area and 79.0% of actual volume. In guinea pigs, nasal cavity volume determined by the acoustic rhinometry was 73.7 +/- 20.0% of actual volume. The actual volume was estimated by impression material instilled into the nasal cavity of the animals (IM volume), and volume determined by acoustic rhinometry significantly correlated with IM volume. Furthermore, there was a significant negative correlation between the volume and nasal airway resistance in guinea pigs. Measurement of the nasal airway resistance is the method frequently used in the evaluation of the nasal obstruction in guinea pigs. These results suggest that acoustic rhinometry is useful in evaluating nasal obstruction in small experimental animals.

Inorganic and methylated arsenic compounds induce cell death in murine macrophages via different mechanisms.

We demonstrate in this study the cytotoxic effects of inorganic arsenicals, arsenite and arsenate, and organic arsenic compounds, monomethylarsonic acid (MAA), dimethylarsinic acid (DMAA), and trimethylarsine oxide (TMAO), which are metabolites of inorganic arsenicals in human bodies, using murine macrophages in vitro. Inorganic arsenicals, both arsenite and arsenate, are strongly toxic to macrophages, and the concentration that decreased the number of surviving cells to 50% of that in untreated controls (IC50) was 5 or 500 microM, respectively. These inorganic arsenicals mainly caused necrotic cell death with partially apoptotic cell death; about 80% of dead cells were necrotic, and 20% were apoptotic. The inorganic arsenicals also induced marked release of an inflammatory cytokine, tumor necrosis factor alpha (TNF alpha), at cytotoxic doses. This strong cytotoxicity of an inorganic arsenical, arsenite, might be mediated via active oxygen and protease activation because it was inhibited by the addition of some antioxidant reagents, such as superoxide dismutase (SOD), catalase, and GSH, or by a peptide inhibitor of interleukin-1 beta-converting enzyme (ICE). It is likely that these immunotoxic effects of inorganic arsenicals may evoke both immunosuppression and inflammation, and they may be central factors causing carcinogenesis and severe inflammatory responses, such as hepatomegaly and splenomegaly, in chronic arsenicosis patients who daily ingested arsenic-contaminated well water. In contrast, the cytotoxic effects of methylated arsenic compounds were lower than those of inorganic arsenicals. The IC50 value of DMAA was about 5 mM, and MAA and TMAO had no toxicity even at concentrations over 10 mM. Additionally, these methylated chemicals suppressed the TNFalpha release from macrophages. DMAA induced mainly apoptotic cell death in macrophages as indicated by cellular morphological changes, condensed nuclei, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), and DNA fragmentation. However, the cytotoxicity of DMAA might be induced via a different mechanism from that of inorganic arsenicals because it was not abolished by the additions of SOD, catalase, or ICE inhibitor. Conversely, GSH enhanced the toxicity of DMAA. These data suggest that methylation of inorganic arsenicals in mammals plays an important role in suppression of both severe immunosuppression and inflammatory responses caused by inorganic arsenicals.

Effect of oxatomide on experimental allergic rhinitis in guinea pigs.

We have investigated the effect of oxatomide, an antiallergic agent, on experimental allergic rhinitis in sensitized guinea pigs. Oxatomide (1 and 10 mg/kg, p.o.) significantly inhibited the sneeze response and nasal rubbing after antigen challenge. Oxatomide (10 and 30 mg/kg) reduced the increase in nasal vascular permeability induced by the antigen-antibody reaction. The decreases in nasal cavity volume caused by nasal mucosal swelling 10 min, 30 min and 6 hr after antigen challenge were significantly inhibited by oxatomide (30 mg/kg). These results indicate that oxatomide inhibits the experimental allergic rhinitis in guinea pigs.

Erdosteine enhances mucociliary clearance in rats with and without airway inflammation.

Erdosteine is a new homocysteine-derived expectorant and has been reported to have many mucolytic effects. In this report, we studied the activities of erdosteine on mucociliary clearance in normal and airway-inflammation-induced rats. In normal rats, erdosteine at doses of 100-600 mg/kg significantly promoted mucociliary clearance. However, erdosteine did not change the concentrations of mucopolysaccharides in bronchoalveolar lavage fluid (BALF). In the LPS-instillated rats, the mucociliary clearance was inhibited and the number of inflammatory cells, albumin concentration, and mucopolysaccharides concentration in BALF were increased. Erdosteine at doses of 100-600 mg/kg significantly attenuated the inhibition of mucociliary clearance and the increase of inflammatory cells, however, it did not prevent the increase of albumin and mucopolysaccharides. Other mucolytic drugs which are ambroxol and S-carboxymethylcysteine, had no effect. These results indicate that erdosteine promotes the mucociliary clearance in normal and airway-inflammation-induced rats.

Polymer-mediated extraction of 8-quinolinolato metal chelates for the determination of metal ions in water with graphite furnace atomic absorption spectrometry.

Water-insoluble 8-quinolinolato metal chelates were formed and were stably solubilized in the aqueous solution of a water-soluble polymer, poly (N-isopropylacrylamide)(PNIPAAm), at room temperature. When the solution was heated at 50 degrees C, PNIPAAm precipitated and then formed a gum-like aggregate (polymer phase) having a very small volume. Accompanying the polymer precipitation, hydrophobic 8-quinolinolato chelates with cobalt(II), iron(III), nickel(II), and copper(II) ions were efficiently incorporated into the polymer phase. At 0.5% (w/v) of PNIPAAm and 8.0 mM of 8-quinolinol, the recoveries in the incorporation of four metal chelates were quantitative. The fluorescence spectra of a probe suggests that the hydrated polymer in the aqueous solution provides hydrophobic portions which can incorporate hydrophobic metal chelates. The polymer phase was easily taken out from the solution and was dissolved with a small amount of acetonitrile. The resulting solution could be directly introduced into a graphite furnace of atomic absorption spectrometry. The signal intensities for the absorbance of cobalt after concentrating the chelate were 100-fold greater than those before the concentration.

Different role of serum components and cytokines on alveolar macrophage activation by soluble fungal (1-->3)-beta-D-glucan.

In this study, we investigated the mechanism of alveolar macrophage activation by systemic administration of SSG, a soluble highly branched (1-->3)-beta-D-glucan obtained from a fungus Sclerotinia sclerotiorum IFO 9395. Multiple i.v. administration (10 mg/kg; once daily for 10 consecutive days) of SSG enhanced some functions of alveolar macrophages, such as lysosomal enzyme activity and nitric oxide secretion, on day 1 after the last administration, and it also elevated the concentrations of serum protein, interferon gamma and SSG in bronchoalveolar lavage fluid on the same day. On the in vitro assay system, stimulation by SSG alone (500 microg/ml) slightly augmented the lysosomal enzyme activity of alveolar macrophages, but it had no effect on nitric oxide production of cells. Stimulation by serum (1 or 10% mouse serum) or serum components, such as fibronectin (25 microg/ml) and albumin (500 microg/ml), alone strongly augmented only the lysosomal enzyme activity of alveolar macrophages, but it had no effect on nitric oxide secretion from cells, and no synergism or additive-like effect was observed between serum components and SSG. In contrast, stimulation by crude lymphokine (5%) or recombinant murine interferon gamma (100 U/ml) alone did not induce augmentation of lysosomal enzyme activity and nitric oxide production of alveolar macrophages in vitro, but when cells were incubated together with crude lymphokine or recombinant murine interferon gamma and SSG (500 microg/ml), a significant combined effect was observed on both functions of alveolar macrophages. In addition, pretreatment of crude lymphokine or recombinant murine interferon gamma enhanced the expression of beta-D-glucan specific binding sites on the alveolar macrophage surface in vitro though pretreatment by serum components had no effect. Based on these findings, the enhancement of alveolar macrophage functions by systemic administration of SSG appears to be mediated, at least in part, by both the simple effect of serum components including fibronectin and albumin leaked from pulmonary peripheral blood into the alveoli and the synergistic effect between lymphokines released from activated pulmonary T cells and SSG itself entering the alveoli after SSG injection via the priming effect of lymphokines which enhances the expression of beta-D-glucan specific binding sites on the alveolar macrophage surface.

Study of in vitro cytotoxicity of a water soluble organic arsenic compound, arsenosugar, in seaweed.

In the present study, we demonstrated the cytotoxic effect of a dimethylarsenic compound in seaweed, (R)-(2',3'-dihydroxypropyl) 5- deoxy-5-dimethylarsinoyl-beta-D-riboside, namely arsenosugar (AsSug), on mammalian cells, murine macrophages, in comparison with that of an inorganic arsenical, arsenite, in vitro. More than 99.5% pure AsSug was synthesized. Arsenite was strongly and equally toxic to both peritoneal macrophages (PMs) and alveolar macrophages (AMs), and the concentration of arsenite that inhibited the viability of cells by 50% compared to the viability of control cells (50% inhibitory concentration; IC50) was 5 microM. In contrast, AsSug showed no cytotoxicity to both PMs and AMs at the microM concentration level; however, it induced different and interesting cellular responses in both macrophages at high concentrations, 1-10 mM. AsSug enhanced the viability of PMs at an optimal dose of 5 mM; conversely, it showed weak but significant cytotoxicity to AMs (IC50 = 8 mM).

Induction of chromosomal aberrations in cultured human fibroblasts by inorganic and organic arsenic compounds and the different roles of glutathione in such induction.

Clastogenic effects of a variety of arsenic compounds were examined on cultured human fibroblasts. The following compounds were tested: inorganic arsenicals (arsenite and arsenate), the major metabolites of inorganic arsenicals in human and experimental animals [methylarsonic acid (MAA), dimethylarsinic acid (DMAA) and trimethylarsine oxide (TMAO)], and water-soluble organoarsenic derivatives [2', 3'-dihydroxypropyl-5-deoxy-5-dimethylarsinoyl-beta-D-riboside (arsenosugar), arsenocholine, arsenobetaine and tetramethylarsonium iodide] found in marine organisms. Arsenic compounds induced mainly chromatid gaps and chromatid breaks. The rank order of compounds in terms of clastogenic potency was arsenite > arsenate > DMAA > MAA > TMAO. DMAA was very potent and caused chromosome pulverizations in most metaphases when present at doses higher than 7 x 10(-3) M. Arsenosugar, arsenocholine, arsenobetaine and tetramethylarsonium iodide were less effective. Depletion of cellular glutathione (GSH) with L-buthionine-SR-sulfoximine (BSO), increased the incidence of chromosomal aberrations induced by arsenite, arsenate and MAA, and markedly suppressed the clastogenic effects of DMAA. DMAA was highly clastogenic even in GSH-depleted cells when the cells were incubated with DMAA in the presence of GSH (5 and 10 mM). These results suggest that GSH might play a role in protecting cells against the clastogenic effects of arsenite, arsenate and MAA. GSH might be involved in the expression of clastogenic actions of DMAA.

Dimethylarsinic acid causes apoptosis in HL-60 cells via interaction with glutathione.

Inducibility of apoptosis in cultured human HL-60 cells by arsenic compounds, such as arsenite, arsenate, methylarsonic acid (MAA), and dimethylarsinic acid (DMAA), was investigated, together with the role of glutathione (GSH) in the induction. Among the arsenic compounds DMAA was the most potent in terms of the ability to cause the morphological changes (formation of nuclear fragmentation and apoptotic bodies) characteristic of apoptosis. Furthermore, fragmentation of internucleosomal DNA was also induced by DMAA. Depletion of cell GSH by L-buthionine-SR-sulfoximine, a selective inhibitor of gamma-glutamylcysteine synthetase, enhanced the cytotoxicity of arsenite, arsenate, and MAA, while such depletion suppressed the cytotoxicity of DMAA. The depletion of GSH also suppressed the morphological changes and the fragmentation of internucleosomal DNA caused by DMAA, both of which are characteristic features of apoptosis. The results suggest that the death of cells caused by DMAA is due to apoptosis and that GSH is involved in the induction of apoptosis by this arsenic compound.

Ability of histamine to increase nasal mucosal permeability to macromolecules in guinea pigs.

The effect of histamine on nasal mucosal permeability against an antigen was investigated by using modified passive cutaneous anaphylaxis (PCA) reactions in normal and actively sensitized guinea pigs. The administration of a dinitrophenyl-coupled Ascaris (DNP-Ascaris) solution as an antigen into the nasal cavity caused PCA reactions in the dorsal skin of normal guinea pigs. The administration of histamine into the nasal cavity before the antigen treatment significantly enhanced the anaphylactic responses. The PCA reactions did not occur when ovalbumin (OA) was administered intranasally in normal guinea pigs. In guinea pigs sensitized against DNP-Ascaris, however, PCA reactions to anti-OA antiserum were elicited by the intranasal administration of OA. The intranasal administration of histamine before the antigen treatment also enhanced anaphylactic responses in sensitized guinea pigs. These results indicate that histamine increases nasal mucosal permeability and that this may be one of the causes of nasal hypersensitivity in nasal allergy.

The effect of KW-4679, an antiallergic drug, on experimental allergic rhinitis in guinea pigs: effects on nasal blockage.

We investigated the effect of KW-4679 (Z-11-(dimethylaminopropyliden)-6,11-dihydrodibenzoxepin-2-a cetic acid hydrochloride), an antiallergic agent, on the nasal blockage induced by antigen challenge into the nostrils of actively sensitized guinea pigs. The change of the nasal cavity volume caused by nasal mucosal swelling after antigen challenge was measured by acoustic rhinometry. Oral administration of KW-4679 (0.01-10 mg/kg) significantly inhibited the decrease in the nasal cavity volume at 10 min, 30 min and 6 hr after antigen challenge. Ketotifen (1-10 mg/kg, p.o.) also inhibited the decrease in the nasal cavity volume after antigen challenge. These results indicate that KW-4679 may be useful for the treatment of allergic rhinitis.