Kazuo Isono

Vasoactive intestinal peptide stimulates ciliary motility in rabbit tracheal epithelium: modulation by neutral endopeptidase

We studied the effect of vasoactive intestinal peptide (VIP) on ciliary activity in rabbit cultured tracheal epithelium by a photoelectric method in vitro. Administration of VIP (10(-7) M) elicited an increase in ciliary beat frequency (CBF) from the baseline values of 970 +/- 52 to 1139 +/- 75 beats/min (mean +/- S.E., P less than 0.01). This ciliostimulatory effect was dose-dependent, with the maximal increase and EC50 value being 17.4 +/- 1.0% (P less than 0.05) and 6.10(-11) M, respectively. The VIP-induced increase in CBF was abolished by pretreatment of cells with [4-Cl-D-Phe6, Leu17]-VIP, a VIP receptor antagonist. The neutral endopeptidase inhibitor phosphoramidon (10(-5) M) potentiated the effect of VIP, so that the CBF dose-response curve for VIP was shifted to lower concentrations by 0.5 log U. The administration of VIP increased cyclic AMP levels in epithelial cells, an effect that was also potentiated by phosphoramidon. These results suggest that VIP may interact with its specific receptors and stimulate airway ciliary activity probably through the activation of adenylate cyclase, and that neutral endopeptidase may play a role in modulating this effect of VIP.

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.

Effects of platelet-activating factor on bioelectric properties of cultured tracheal and bronchial epithelia

To elucidate the effect of platelet-activating factor (PAF) on ion transport function of airway epithelial cells, we studied bioelectric properties of cultured tracheal and bronchial epithelia from dogs under short-circuit conditions in vitro. Addition of PAF (10(-5) mol/L) to mucosal solution of Ussing chamber increased short-circuit current of tracheal epithelium from 3.3 +/- 0.7 to 8.5 +/- 1.2 microA/cm2 (p less than 0.001). This effect was dose dependent, and there was a corresponding increase in transepithelial potential difference. In contrast, PAF was without effect when it was added to the submucosal side. Electrical properties of bronchial epithelium remained unchanged by PAF. The PAF-induced increase in short-circuit current was not affected by amiloride but abolished by diphenylamine-2-carboxylate, bumetanide, or Cl-free medium. The effects of PAF were not altered by AA-861 or U-60257, but attenuated by indomethacin and piroxicam, and dose-dependently blocked by CV 6209 and WEB 2086. Mucosal, but not submucosal, addition of PAF increased the rate of prostaglandin release from tracheal epithelium. These results suggest that PAF selectively stimulates Cl secretion across tracheal epithelium, probably through activation of its specific receptors and the subsequent production of prostaglandins.

β‐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). β2‐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 β2‐adrenergic receptors and the consequent production of cyclic adenosine monophosphate may upregulate cell growth, probably through activation of the mitogen-activated protein kinase cascade.

Role of regular treatment with inhaled corticosteroid or leukotriene receptor antagonist in mild intermittent asthma.

Current guidelines for asthma treatment do not recommend daily maintenance therapy in patients with mild intermittent (step 1) asthma. However, because there is increasing evidence that airway inflammation is present even in this patient group, maintenance anti-inflammatory therapy may be considered. We investigated the clinical impact of regular treatment with the inhaled corticosteroid beclomethasone dipropionate and the leukotriene receptor antagonist pranlukast in the patients concerned. The study was a randomized, controlled, parallel-group, multicenter trial. Eighty-five symptomatic patients with newly diagnosed mild intermittent asthma having normal pulmonary function were assigned beclomethasone or pranlukast for 8 weeks. Then, these medications were stopped for the next 16 weeks. Main outcome measures were asthma symptoms, pulmonary function, and airway inflammation. Treatment with beclomethasone and pranlukast significantly increased forced expiratory volume in 1 second and peak expiratory flow from baseline and decreased asthma symptom scores and sputum eosinophil counts and eosinophil cationic protein contents. After discontinuation of the treatment, symptom scores remained unchanged, but pulmonary function and airway inflammation were aggravated and then returned to the baseline levels. Therefore, maintenance therapy with inhaled corticosteroid or leukotriene receptor antagonist can provide further improvements in asthma symptoms, pulmonary function, and airway inflammation, and discontinuation of the therapy causes worsening of asthma, indicating that stopping or interrupting anti-inflammatory therapy may not be advisable in patients with symptomatic mild intermittent asthma.

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 Ca2+‐activated Cl channel, hCLCA1.

Peripheral Blood Th1 and Th2 Profile in Patients with Moderate Asthma: Effect of Inhaled Corticosteroid

The peripheral blood from healthy subjects and asthma patients was stimulated with phorbol 12-myristate 13-acetate and ionomycin, and the cells were stained with anti-CD4 antibody, permeabilized, stained with anti-IFN-γ and anti-IL-4 antibodies, and analyzed by flow cytometry. Compared with healthy subjects, asthma patients showed a greater percentage of both IL-4(+) IFN-γ (–) CD4 cells (Th2 cells) and IFN-γ(+) IL-4(–) CD4 cells (Th1 cells). The percentage of Th2 cells was correlated with serum IgE level. After treatment with inhaled corticosteroid, Th2 cells decreased at week 24, but not week 4. Long-term therapy with inhaled steroid may thus be required for improvement in lymphocytic inflammation.

Airway hyperresponsiveness to histamine in mycoplasmal infection: role of histamine N-methyltransferase.

To elucidate the modulatory role of histamine-degrading enzymes in airway constrictor responses in mycoplasmal infection, we studied hamster tracheal segments under isometric conditions in vitro. Nasal inoculation with Mycoplasma pneumoniae potentiated the contractile responses to histamine but not to methacholine. Pretreatment of tissues with the histamine N-methyltransferase inhibitor SKF 91488 abolished the infection-induced potentiation, whereas, the diamine oxidase inhibitor aminoguanidine had no effect. The histamine N-methyltransferase but not diamine oxidase activity in tracheal tissues was decreased in infected animals. These results suggest that M. pneumoniae causes airway hyperresponsiveness to histamine probably through a reduction of endogenous histamine N-methyltransferase activity.

Effect of Neutral Endopeptidase Inhibition on Substance-P-Induced Increase in Short-Circuit Current of Canine Cultured Tracheal Epithelium

We studied the effect of substance P (SP) on the electric properties of cultured canine tracheal epithelium and its possible modulation by neutral endopeptidase (NEP) by Ussings short-circuited technique in vitro. Addition of SP (5 x 10(-6) M) to the mucosal side increased short-circuit current (SCC) from 5.1 +/- 0.9 to 10.3 +/- 2.2 microA/cm2 (mean +/- SE; p less than 0.01), which was accompanied by increases in transepithelial potential difference and conductance. The effect of the mucosal SP on SCC was dose-dependent, with the maximal increase from the baseline value being 5.8 +/- 1.0 microA/cm2 observed at 5 x 10(-5) M. The NEP inhibitor phosphoramidon (10(-5) M) did not affect these responses. On the other hand, SCC was not altered by the addition of SP to the submucosal side. However, it was increased dose-dependently in the presence of phosphoramidon (10(-5) M) but not in the presence of captopril, bestatin or leupeptin. This stimulatory effect of submucosal SP was abolished by furosemide, diphenylamine-2-carboxylate and Cl-free medium, but not by amiloride. These results suggest that SP may selectively stimulate Cl secretion across the airway epithelium and that this effect may be modulated by submucosal NEP.

Budesonide/formoterol maintenance and reliever therapy in moderate-to-severe asthma: effects on eosinophilic airway inflammation.

Although the budesonide and formoterol in a single inhaler for maintenance and reliever therapy has been evaluated in recent studies, the effects on eosinophilic airway inflammation remain uncertain. The purpose of this study was to compare the efficacy, including anti-inflammatory effects, of as-needed budesonide/formoterol with salbutamol in Japanese patients with moderate-to-severe asthma. Patients with asthma using an inhaled corticosteroid plus a long-acting beta2-agonist as a controller and at least one asthma exacerbation in the previous 12 months were randomized to budesonide/formoterol maintenance therapy (160/4.5 micrograms, 2 inhalations twice daily) plus either as-needed budesonide/formoterol (160/4.5 micrograms; n = 32) or salbutamol (100 micrograms; n = 31) up to 4 inhalation daily for 48 weeks. The time to first asthma exacerbation was significantly prolonged with as-needed budesonide/formoterol compared with salbutamol (log-rank test; p = 0.0342). There was a 66% reduction in the hazard ratio for a first exacerbation with as-needed budesonide/formoterol (p = 0.0334). The frequencies of both mild and severe exacerbations and reliever use were consistently less with budesonide/formoterol than salbutamol. As-needed budesonide/formoterol significantly improved in lung function and symptom scores compared with salbutamol. In addition, the contents of eosinophil cationic protein and B12 tryptase, as well as number of eosinophils and mast cells in induced sputum, decreased to a greater extent with budesonide/formoterol compared with salbutamol. In conclusion, the budesonide and formoterol for maintenance and reliever therapy seems more effective in controlling persistent asthma with a significant reduction of airway inflammation. Clinical trial 121104, www.clinicaltrials.gov.