Chloride ion transport and overexpression of TMEM16A in a guinea-pig asthma model.

BACKGROUND: TMEM16A, a Ca-activated Cl channel, regulates various physiological functions such as mucin secretion. However, the role of TMEM16A in hyper-secretion in asthma is not fully understood. OBJECTIVE: The aim of this study is to evaluate Cl ion transport via TMEM16A and determine the localization of TMEM16A in a guinea-pig asthma model. METHODS: Guinea-pigs were sensitized with ovalbumin (OVA) i.p. on Days 1 and 8. On Day 22, we assessed OVA challenge-induced Cl ion transport in the sensitized tracheas ex vivo in an Ussing chamber, compared with the non-sensitized tracheas. We then examined the effect of T16Ainh-A01, a TMEM16A inhibitor, on the increase in Cl ion transport. The tracheal epithelium was immunostained with an anti-TMEM16A antibody. Epithelial cells from guinea-pig tracheas were cultured at the air-liquid interface in the presence of IL-13 for in vitro study. We studied the effect of TMEM16A inhibitors on Ca-dependent agonist, uridine triphosphate (UTP)-induced increases in Cl ion transport in the cultured cells. The cells were immunostained with an anti-TMEM16A antibody, an anti-MUC5AC antibody and an anti-α-tubulin antibody. RESULTS: OVA challenge induced an increase in short circuit current within 1 min in the OVA-sensitized tracheas but not in the non-sensitized tracheas, which was inhibited by pretreatment of T16Ainh-A01. Sensitized tracheas showed goblet cell metaplasia with more positive TMEM16A immunostaining, particularly in the apical portion compared with the non-sensitized tracheas. The in vitro UTP-induced increase in Cl ion transport was strongly inhibited by pretreatment with T16Ainh-A01, benzbromarone, and niflumic acid. TMEM16A was positively immunostained at the apical portion and in the MUC5AC-positive area in IL-13-induced goblet cell metaplasia. CONCLUSIONS: Antigen challenge and Ca-dependent agonist treatment increased Cl ion transport via the overexpression of TMEM16A in goblet cell metaplasia in a guinea-pig asthma model. TMEM16A inhibitors may be useful for the treatment of hyper-secretion in asthma.

Inhibition of neutrophil elastase-induced goblet cell metaplasia by tiotropium in mice.

Airway occlusion by mucus in chronic obstructive disease (COPD) is associated with a poor prognosis. We hypothesised that tiotropium has the ability to inhibit neutrophil elastase (NE)-induced goblet cell metaplasia in mice and mucin production in vitro. On days 1, 4, and 7, tiotropium or vehicle was administered to C57BL/6 mice by inhalation and they were allowed to intranasally aspirate human NE. Bronchoalveolar lavage fluid (BALF) and lung sections were analysed on days 8, 11 and 14. The effect of late administration of tiotropium on the goblet cell metaplasia by NE aspiration was also assessed. NE-induced MUC5AC production by NCI-H292 cells was measured with ELISA. Repeated NE aspiration induced marked goblet cell metaplasia. The grading of goblet cell metaplasia, neutrophil count and eosinophil count in BALF, keratinocyte-derived chemokine level and leukotriene B(4) level in BALF, and M(3) receptor expression by immunohistochemistry, were lower in the tiotropium group than in the vehicle group. Late administration of tiotropium inhibited the established goblet cell metaplasia. Tiotropium inhibited NE-induced MUC5AC production. Tiotropium inhibited NE-induced goblet cell metaplasia and mucin production, probably mediated by suppression of inflammation and a direct action on epithelial cells. This result suggests that tiotropium may be useful for the treatment of mucus overproduction in COPD.

Role of epidermal growth factor receptor in maintaining airway goblet cell hyperplasia in rats sensitized to allergen.

BACKGROUND: Stimulation of epidermal growth factor receptor (EGFR) induces airway goblet cell hyperplasia, but the role of this molecule in the maintenance of this pathologic change remains uncertain. OBJECTIVE: To determine the mechanisms by which goblet cell hyperplasia is maintained in airway epithelium, we investigated EGFR-induced signalling pathways that lead to both mucin production and antiapoptosis in vitro. We also tested whether the inhibition of EGFR tyrosine kinase speeds reversal of established goblet cell hyperplasia to normal epithelial phenotype in vivo. METHODS: MUC5AC production was measured by immunoassay, and antiapoptotic responses were determined by Bcl-2 expression and terminal deoxynucleotidyl transferase-mediated dUTP-biotin Nick End Labelling staining using NCI-H292 cells. The effect of an inhibitor of EGFR tyrosine kinase (AG1478) on goblet cell hyperplasia was also determined in rats sensitized with ovalbumin (OVA). RESULTS: MUC5AC was constitutively expressed and few apoptotic cells were observed in NCI-H292 cells under non-stimulated condition. TGF-alpha increased MUC5AC and Bcl-2 expression, an effect that was prevented by inhibitors of EGFR tyrosine kinase (AG1478), MEK (PD98059), and NF-kappaB (CAPE). After the addition of TGF-alpha, AG1478 and an inhibitor of phosphatidylinositol 3 kinase/Akt (LY294002), but not PD98059, induced a marked apoptotic response, which was prevented by the caspase inhibitor Z-VAD fmk. Goblet cell hyperplasia and EGFR expression in airway epithelium were noted in the OVA-sensitized rats. Intratracheal instillation of AG1478 induced apoptosis of goblet cells, reverting the airway epithelium to normal epithelial phenotype. CONCLUSION: These findings indicate that EGFR plays an important role in the maintenance of goblet cell hyperplasia. We speculate that inhibitors of the EGFR cascade might be an effective therapy of airway remodelling.

Interleukin-9 and Interleukin-13 augment UTP-induced Cl ion transport via hCLCA1 expression in a human bronchial epithelial cell line.

BACKGROUND: IL-9 and IL-13 induce airway goblet cell metaplasia, which is associated with expression of a Ca(2+)-activated Cl channel, hCLCA1. OBJECTIVE: As UTP stimulates both mucin secretion and Cl ion transport via a Ca(2+)-dependent pathway, the purpose of this study is to determine whether IL-9 and IL-13 affect UTP-induced Cl ion transport in human bronchial epithelial cell line 16HBE cells, and if they do, to elucidate whether such an effect is associated with hCLCA1 expression. METHODS: The increases in short-circuit current (I(sc)) in response to UTP were measured in the presence of amiloride by the Ussing chamber method. The morphology of epithelial cells was assessed by light microscopic findings, and hCLCA1 expression was investigated by immunocytochemistry and immunoblotting. RESULTS: UTP-induced increases in I(sc) in the cells treated with IL-9 or IL-13 for 48 h were greater than those in non-treated cells, and the potency of IL-13 was greater than that of IL-9. Pre-treatment with Ca(2+)-activated Cl channel inhibitors diisothocyanatostilbene-2, 2-disulphonic acid and niflumic acid completely inhibited the augmenting effects of IL-9 and IL-13 on I(sc). The epithelial layer of the cells treated with IL-9 or IL-13 was thicker than that of non-treated cells. The expression of hCLCA1 protein was induced by IL-13 in a concentration-dependent manner. These effects of IL-13 were more potent than those of IL-9. CONCLUSION: IL-9 and IL-13 augmented UTP-induced Cl ion transport, probably via proliferation of the cells with hCLCA1 expression, and IL-13 was more potent than IL-9 in producing such an effect in 16HBE cells.

Beta-adrenergic receptor-mediated growth of human airway epithelial cell lines.

Abnormal growth of airway epithelium and the resultant thickening of airway walls may produce narrowing of airway calibre, thereby contributing to deterioration of bronchoconstriction in chronic obstructive pulmonary disease (COPD). Beta2-adrenergic agonists have been widely used for the treatment of COPD, but their effects on the growth of airway epithelial cells is unknown. Growth of three human airway epithelial cell lines was studied in vitro. Exposure to salbutamol in serum-free medium increased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide reduction and intracellular deoxyribonucleic acid (DNA) contents in 16-human bronchial epithelium (16-HBE) cells and NCI-H292 cells, but not in A549 cells. The growth-promoting effect of salbutamol in 16-HBE cells was equipotent to 10% foetal bovine serum and was inhibited by propranolol and a cyclic adenosine monophosphate (cAMP) antagonist, Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS). Likewise, forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) caused cell growth and DNA synthesis. Western blot analysis showed that salbutamol, forskolin, and 8-Br-cAMP each induced expression of the phosphorylated form of mitogen-activated protein (MAP) kinase, and that the salbutamol-induced phosphorylation was inhibited by propranolol, Rp-cAMPS, and the MAP kinase-kinase inhibitor PD98059. These results suggest that in certain airway epithelial cell lines stimulation of beta2-adrenergic receptors and the consequent production of cyclic adenosine monophosphate may upregulate cell growth, probably through activation of the mitogen-activated protein kinase cascade.

Acute cigarette smoke exposure induces apoptosis of alveolar macrophages.

Alveolar macrophages (AMs) may play a critical role in cigarette smoke (CS)-related pulmonary diseases. This study was designed to determine whether CS induces apoptosis of AMs. In in vitro studies, mouse, rat, and human AMs and human blood monocyte-derived macrophages cultured with aqueous whole CS extracts underwent apoptosis that was detected by light and electron microscopy and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. The gas phase of CSE did not cause apoptosis. The CS-induced apoptosis was associated with increased oxidative stress, Bax protein accumulation, mitochondrial dysfunction, and mitochondrial cytochrome c release but was independent of p53, Fas, and caspase activation. This apoptosis was inhibited by antioxidants such as glutathione, ascorbic acid, and alpha-tocopherol. In in vivo studies where rats were exposed to the smoke from 10 cigarettes over 5 h in an exposure chamber, approximately 3% of AMs obtained by bronchoalveolar lavage after 24 h showed apoptosis. These results suggest that acute CS exposure is capable of inducing apoptosis of AMs.

Differential regulations between adenosine triphosphate (ATP)- and uridine triphosphate-induced Cl(-) secretion in bovine tracheal epithelium. Direct stimulation of P1-like receptor by ATP.

Adenosine 5'-triphosphate (ATP) stimulates airway epithelial Cl(-) secretion in a complicated manner. We examined the difference between ATP- and uridine 5'-triphosphate (UTP)-induced responses of short-circuit current (Isc) in bovine tracheal epithelium treated with amiloride. Each nucleotide caused an increase in Isc composed of the first and second peaks, where the second peak induced by ATP was higher compared with UTP. The ATP-induced second peak was inhibited by the protein kinase (PK) A inhibitor H89, saturation of P1 receptor with adenosine, and the P1 receptor antagonist 8-p-sulfophenyltheophylline, but not by the Ca(2+) chelator ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid plus the endoplasmic reticulum Ca(2+)-pump inhibitor thapsigargin, the adenosine breakdown enzyme adenosine deaminase, the ectonucleotidase inhibitor alpha,beta-methyleneadenosine 5'-diphosphate, or saturation of P2Y2 receptor with UTP. Thus, the response is associated with PKA-dependent pathway via P1-like receptor but not with Ca(2+)-dependent pathway via P2Y2 receptor, and ATP degradation products do not contribute to this response. Further, stimulation of cells with ATP increased PKA activity. In addition, pretreatment with glybenclamide, an inhibitor of cystic fibrosis transmembrane conductance regulator, reduced the second peak of Isc induced by ATP but was without effect on that induced by UTP. Therefore, ATP stimulates glybenclamide-sensitive Cl(-) secretion, and this action is partly mediated by PKA-dependent pathway via P1-like receptor.

Effects of new quinolones on transepithelial electrical potential difference of tracheal mucosa in vivo.

Superfusion of canine tracheal mucosa with 100 microg each of grepafloxacin and ciprofloxacin per ml reduced the electrical transepithelial potential difference in vivo by more than 50%. This effect was dose dependent, specific for new quinolones, and inhibited by Cl channel blockers, indicating that new quinolones attenuate Cl secretion across the airway epithelium.

Validity and safety of sputum induction by inhaled uridine 5'-triphosphate.

UNLABELLED: Inhalation of hypertonic saline during sputum induction causes bronchoconstriction. We studied the validity and safety of sputum induction by uridine 5'-triphosphate (UTP). Sputum was induced by a 5-min inhalation of hypertonic saline (3%) on Day 1 and UTP (5 mg/ml in 0.9% saline) on Days 8 and 15 in 16 healthy subjects and 16 patients with mild-to-moderate asthma. Inhaled UTP produced twofold greater amounts of sputum than did hypertonic saline. There were significant differences in oxygen desaturation and bronchoconstriction during the procedure between the two methods: the maximal fall in Sa(O(2)), the AUC of the Sa(O(2))-time response, and the fall in PEF were less in the subjects who received UTP than in those who received hypertonic saline. Sputum total cell and differential cell counts, with a high proportion of eosinophils in asthmatics, were similar between specimens obtained by hypertonic saline and UTP. When we compared two consecutive measurements on the UTP-induced sputum samples, the reproducibility calculated by the intraclass correlation coefficient was high for the proportion of eosinophils, neutrophils, and macrophages. Therefore, inhalation of UTP aerosols may provide an effective, relatively noninvasive, valid, and reproducible method of sputum induction for the assessment of airway inflammation in asthma. KEYWORDS: uridine triphosphate; induced sputum; airway inflammation; bronchoconstriction; asthma

Serine proteases increase oxidative stress in lung cells.

Several serine proteases are directly cytotoxic. We investigated whether the cytotoxic effects of proteases are associated with increased levels of reactive oxygen species (ROS) in cells. We found that treatment of lung fibroblasts or bronchial epithelial cells with relatively high concentrations (0.1--100 U/ml) of neutrophil elastase, trypsin, and Pronase increased ROS levels in the mitochondria and cytoplasm. The protease-induced increase in ROS was associated with oxidative cellular injury as determined by generation of 8-hydroxy-2'-deoxyguanosine and malonaldehyde plus 4-hydroxyalkenal. The protease-induced increase in ROS was not merely due to cell detachment because the proteases also caused an increase in ROS in suspended cells, which precluded attachment to the extracellular matrix. The protease-induced increase in ROS appears to contribute to cytotoxicity because cell death induced by proteases was attenuated by treatment with catalase, a decomposer of H(2)O(2), and accelerated by treatment with aminotriazole, a catalase inhibitor. These results suggest that several proteases increase oxidative stress, indicating a direct interaction between proteases and ROS in mediating cytotoxicity.

FK506 inhibits Cl- secretion in airway epithelium via calcineurin-independent mechanism.

FK506 (tacrolimus)-binding protein (FKBP) is associated with intracellular Ca2+ release channel and modulates its function. To elucidate the effect of FK506 on Ca2+ dynamics and Ca2+-mediated Cl- secretion in airway epithelium, we studied intracellular Ca2+ ([Ca2+]i) concentration and Cl(-)-dependent short-circuit current (Isc), in cultured bovine tracheal epithelial cells. Addition of ATP induced an increase in [Ca2+]i, and this response was dose dependently inhibited by FK506. Rapamycin, which binds FKBP with high affinity, likewise inhibited the [Ca2+]i rise, but cyclosporin A, a specific calcineurin inhibitor, did not. In Cl- secretion studies using Ussing chamber, ATP increased Ca2+-mediated Isc in amiloride-treated cells, an effect that was inhibited by FK506 and rapamycin but not by cyclosporin A. Therefore, FK506 inhibits Ca2+ mobilization in airway epithelium via FKBP but not calcineurin-dependent mechanism, which may result in the suppression of Ca2+-activated Cl- secretion.

Role of Na(+)-K(+)-ATPase in sodium nitroprusside-induced relaxation of pulmonary artery under hypoxia.

BACKGROUND: The sodium pump (Na(+)-K(+)-ATPase) plays a part in the regulation of smooth muscle contractility, and alterations of enzyme activity by hypoxia could contribute to the mechanism of hypoxic pulmonary vasoconstriction. OBJECTIVE: To determine the role of Na(+)-K(+)-ATPase in the sodium nitroprusside (SNP)-induced relaxation of pulmonary artery in hypoxia. METHODS: Using isolated canine pulmonary arterial rings, we measured the relaxant responses of KCI-contracted tissues to SNP under hyperoxic (95% O2, 5% O2) and hypoxic conditions (5% O2, 5% CO2, 90% N2 in vitro. Na(+)-K(+)-ATPase activity was assessed by measuring ouabain-sensitive (86)Rb uptake. RESULTS: The SNP-induced relaxation was reduced under hypoxia, so that the maximal relaxation decreased from 80.1 +/- 8.6 to 57.8 +/- 6.8% (p < 0.01) and the concentration of SNP required to produce 50% relaxation increased from 1.9 +/- 0.4 x 10(-6) to 2.6 +/- 0.6 x 10(-5) M (p < 0.01). Addition of ouabain, an Na(+)-K(+)-ATPase inhibitor, attenuated the relaxant response to SNP and this inhibition was still observed under hypoxia. Incubation of endothelium-denuded rings with SNP caused dose-dependent increases in intracellular cGMP levels and ouabain-sensitive (86)Rb uptake, and these effects were not significantly altered by hypoxia. CONCLUSION: These results suggest that sarcolemmal Na(+)-K(+)-ATPase activity may be implicated in the mechanism of nitrovasodilator-induced vasodilation of pulmonary artery and may still be functioning under hypoxia.

New quinolone, grepafloxacin, inhibits Cl- secretion across bovine airway epithelium in culture.

OBJECTIVE: Transepithelial ion transport plays an important role in the regulation of the amount and the rheological properties of bronchial secretion. The effect of grepafloxacin (GPFX), a new quinolone agent, on bioelectrical properties of airway epithelium was determined. METHODOLOGY: Electrical properties of bovine tracheal epithelium cultured under an air-liquid interface condition were measured by the short-circuit technique. RESULTS: Addition of GPFX (100 microg/mL) to the mucosal side decreased short-circuit current (Isc) from 14.4 +/- 1.3 to 5.6 +/- 0.6 microA/cm2 (P < 0.001), and the response was accompanied by corresponding decreases in transepithelial potential difference and cell conductance. This effect was concentration dependent, and a similar response was also noted when GPFX was added to the submucosal side. The GPFX-induced decrease in Isc was not altered by the Na+ channel blocker amiloride, but was inhibited by the Cl- channel blocker diphenylamine-2-carboxylate or Cl(-)-free medium (P < 0.001, in each case). Furthermore, GPFX reduced Cl- conductance (P < 0.01) without affecting Na+ conductance of the epithelium. CONCLUSIONS: Grepafloxacin selectively inhibits Cl- secretion across tracheal epithelial cells, which may result in the inhibition of water secretion and, hence, the reduction of airway secretion.

[Effect of endothelin on DNA synthesis in human bronchial epithelium: role of MAP kinase cascade].

To elucidate the effect of endothelin (ET) on airway epithelial cell proliferation, we measured intracellular DNA levels and assessed a possible contribution of mitogen activated protein kinase (MAPK) cascade to the ET action. Incubation of transformed human bronchial epithelial (16 HBE) cells with ET in the serum-free medium caused time-dependent increases in DNA synthesis and MTT reduction, an effect that was attenuated by the MAPK kinase inhibitor PD 98059 in a concentration-dependent manner. Western blot analysis showed that ET induced the expression of phosphorylated MAPK protein, indicating an activation of MAPK, and that this effect was inhibited in the presence of PD 98059 or the ETA receptor antagonist BQ-123. These results suggest that ET may stimulate the proliferation of human airway epithelium via ETA receptors and the concomitant activation of MAPK cascade.

Impairment of airway mucociliary transport in patients with sinobronchial syndrome: role of nitric oxide.

Nitric oxide (NO) produced within the respiratory tract can stimulate ciliary motility of airway epithelial cells and hence mucociliary transport. In this study, to determine the role of NO in mucociliary dysfunction in sinobronchial syndrome (SBS), we measured NO concentrations in the exhaled air by chemiluminescence analyzer and nasal clearance time (NCT) by saccharin test. Exhaled NO concentrations in patients with SBS were 39% of those in healthy nonsmokers and 55% of those in healthy smokers. The patients also showed prolonged NCT compared with healthy subjects, and there was a significant negative correlation between exhaled NO concentrations and NCT. Furthermore, concentrations of chloride (Cl) in the sputum supernatant were higher in SBS patients than in healthy subjects, and there was a significant negative correlation between sputum Cl concentrations and exhaled NO concentrations. These results suggest that airway mucociliary clearance is impaired in patients with SBS and that this impairment might result from the reduced production of NO and the impaired availability of the molecule in the mucociliary apparatus.

[Role of nitric oxide in airway mucociliary dysfunction in diffuse panbronchiolitis].

Nitric oxide (NO) stimulates the ciliary motility of the airway epithelium, thereby assisting in the regulation of mucociliary transport in the respiratory tract. In the present study, to elucidate a possible involvement of NO in mucociliary disturbance in diffuse panbronchiolitis (DPB), we measured nasal clearance time (NCT) using the saccharin test, NO concentration in the exhaled air with a chemiluminescence analyzer, and electrolyte concentration in the sputum. Compared with healthy nonsmokers and smokers, patients with DPB showed a lower NO concentration in the exhaled air (p < 0.05), prolonged NCT (p < 0.01), and a higher Cl concentration in the sputum (p < 0.05). Among these variables, exhaled NO concentration was negatively correlated with NCT (p < 0.01) and sputum Cl concentration (p < 0.05). These results suggest that the inhibition of NO generation associated with an elevated Cl concentration in the airway surface liquid may contribute to airway mucociliary dysfunction in DPB.

Effect of suplatast tosilate, a Th2 cytokine inhibitor, on steroid-dependent asthma: a double-blind randomised study. Tokyo Joshi-Idai Asthma Research Group.

BACKGROUND: Th2 cytokines play an important part in the pathogenesis of asthma. Our aim was to study the effect of suplatast tosilate, a selective Th2 cytokine inhibitor, on asthma control and asthma exacerbations during reduction of inhaled corticosteroid dose in patients with steroid-dependent asthma. METHODS: 85 patients with moderate to severe asthma taking high doses (> or = 1500 microg per day) of inhaled beclometasone dipropionate, were assigned suplatast tosilate (100 mg three times daily) or placebo for 8 weeks in a double-blind, randomised, parallel-group, multicentre trial. During the first 4 weeks, other medications remained unchanged (add-on phase); during the next 4 weeks, the doses of beclometasone were halved (steroid-reduction phase). Main outcome measures were pulmonary function, asthma symptoms, and use of beta2-agonists. FINDINGS: Data were available from 77 patients. During the add-on phase, suplatast tosilate treatment, compared with placebo, was associated with higher forced expiratory volume in 1 s (mean difference between groups for changes from baseline at week 4, 0.20 L [95% CI 0.16-0.24], p=0.043), morning peak expiratory flow (18.6 L/min [14.1-23.1], p=0.037), and less diurnal variation in peak expiratory flow rate, asthma symptom scores (7.1 [6.6-7.6], p=0.029), and serum concentrations of eosinophil cationic protein and IgE. In the steroid-reduction phase, pulmonary function, asthma symptoms, and use of beta2-agonist deteriorated significantly more in the placebo group than in the suplatast group. INTERPRETATION: Treatment with a Th2 cytokine inhibitor in steroid-dependent asthma improves pulmonary function and symptom control, and allows a decrease in dose of inhaled corticosteroid without significant side-effects. Some improvements in pharmacokinetics are, however, needed.

The Fas/Fas-ligand system is not required for bleomycin-induced pulmonary fibrosis in mice.

Recent studies suggest that Fas-Fas-ligand (FasL) interactions play an important role in the development of lung injury and fibrosis. However, evidence to support this concept is still indirect. To determine whether Fas-FasL interaction is required for the development of bleomycin-induced pulmonary fibrosis in mice, we used Fas-deficient (lpr) and FasL-deficient (gld ) mice as animal models. After intratracheal instillation of bleomycin, we examined the lungs of mice through bronchoalveolar lavage, histologic studies, DNA nick-end labeling, and hydroxyproline assay. The development of cellular infiltrates, bronchiolar and alveolar epithelial apoptosis, and fibrosis following bleomycin instillation in the lungs in lpr mice and gld mice was similar to their development in wild-type mice. The results of this study show that bleomycin-induced pulmonary fibrosis does not require Fas-FasL interaction, and that epithelial cell apoptosis after bleomycin exposure is mediated by Fas-independent pathways.

Ginsenoside-induced relaxation of human bronchial smooth muscle via release of nitric oxide.

Ginsenoside, an extract of Panax ginseng, is an essential constituent of anti-asthmatic Chinese herbal medicine. To elucidate whether ginsenoside affects airway smooth muscle tone and, if so, what the mechanism of action is, we studied relaxant responses of human bronchial strips under isometric condition in vitro, and directly measured the release of nitric oxide (NO) by an amperometric sensor for this molecule. Addition of ginsenoside relaxed the tissues precontracted with acetylcholine in a dose-dependent manner, the maximal relaxation and the ginsenoside concentration required to produce 50% relaxation being 67+/-8% and 210+/-29 microg ml(-1), respectively. The relaxant responses to ginsenoside were inhibited by N(G)-nitro-L-arginine methylester (L-NAME) and removal of the epithelium, but not by N(G)-nitro-D-arginine methylester (D-NAME) or tetrodotoxin. This inhibitory effect of L-NAME was reversed by L-arginine but not by D-arginine. Addition of ginsenoside to the medium containing bronchial tissues dose-dependently increased NO-selective electrical current, and this effect was greatly attenuated by the epithelial removal or Ca(2+)-free medium. Ginsenoside also increased tissue cyclic GMP contents, an effect that was abolished in the presence of L-NAME. It is concluded that ginsenoside induces relaxation of human bronchial smooth muscle via stimulation of NO generation predominantly from airway epithelium and cyclic GMP synthesis. This action might account for the anti-asthmatic effect of Panax ginseng.

Effects of reactive oxygen species on intracellular calcium in bovine tracheal epithelium: modulation by nitric oxide.

We studied the effects of reactive oxygen species (ROS) on intracellular Ca2+ concentration ([Ca2+]i) and their possible modulation by nitric oxide (NO) in fura-2-loaded cultured bovine tracheal epithelium. Hypoxanthine (HX) and xanthine oxidase (XO), which generate superoxide anion (O2-) and hydrogen peroxide (H2O2), dose dependently increased [Ca2+]i. The increase in [Ca2+]i was reduced in the presence of superoxide dismutase (SOD, 200 U/mL) and catalase (200 U/mL) by 29% and 43%, respectively. The iron chelator o-phenanthroline and the hydroxyl radical (.OH) scavenger dimethylthiourea (DMTU) more potently inhibited the response of [Ca2+]i. H2O2-derived .OH generated by the Fenton reaction caused a marked [Ca2+]i elevation, but exogenous H2O2 did not. Sodium nitroprusside (100 microM), an NO donor, potentiated HX-XO-induced [Ca2+]i rise by 50%, an effect that was abolished in the presence of SOD or DMTU. These results suggest that .OH formed by interaction of O2- and H2O2 in the presence of iron may play a major role in the HX-XO-induced disruption of airway epithelial Ca2+ homeostasis, and that NO potentiates ROS-induced [Ca2+]i response, presumably by reacting with O2- and producing .OH.