Takeo Horie

Receptor-dependent G protein-mediated Ca2+ sensitization in canine airway smooth muscle.

To determine the mechanisms of receptor-dependent Ca2+ sensitization in airway smooth muscle, canine tracheal smooth muscle (CTSM) was permeabilized with alpha-toxin or beta-escin. Although the effects of 5-hydroxytryptamine (100 microM), histamine (100 microM), and the thromboxane A2 analogue U-46619 (100 microM) were negligible, carbachol (100 microM) and endothelin-1 (ET-1, 1 microM) evoked additional contractions of 47.0 +/- 5.90% and 25.0 +/- 5.37% (n = 6) at pCa 6.7 with GTP (3 microM) (normalized to the maximum contraction at pCa 4.5) in alpha-toxin-permeabilized CTSM. GDP-beta-S (1 mM) reversed the carbachol and ET-1 responses completely. GTP-gamma-S (30 microM) and 4 beta-phorbol 12,13-dibutyrate (PDBu, 3 microM) increased the Ca2+ sensitivity (median effective pCa) of contraction by 1.8- and 4.4-fold, respectively (n = 4-11, P < 0.05). The effects of saturating concentrations of GTP-gamma-S and PDBu were additive. A synthetic peptide (T2) corresponding to the actin-binding site of calponin caused a dose-dependent contraction of beta-escin permeabilized CTSM, with the peak effect (25 +/- 4%, n = 4) at 1200 microM, PDBu (3 microM) caused contraction of the T2 peptide-treated CTSM. In conclusion, Ca2+ sensitization of CTSM depends on receptor type and is mediated by G proteins and protein kinase C whose effects are additive, with a partial contribution by calponin.

InsP3, but not novel Ca2+ releasers, contributes to agonist-initiated contraction in rabbit airway smooth muscle

1 To examine the contributions of the putative Ca2+ releasers, inositol 1,4,5‐trisphosphate (InsP3), cyclic ADP ribose (cADPR), and nicotinate adenine dinucleotide phosphate (NAADP), to carbachol (CCh)‐induced contraction in airway smooth muscle, we measured force development of permeabilized rabbit tracheal smooth muscle, human bronchial smooth muscle and guinea‐pig ileum longitudinal smooth muscle. 2 In the presence of 50 μm GTP, CCh and InsP3 contracted α‐toxin‐permeabilized tracheal smooth muscle dose dependently; the EC50 values for CCh and InsP3 were 1.84 μm and 363 μm, and the maximum responses (normalized to the 30 mM caffeine response) to 100 μm CCh and to 800 μm InsP3 were 206 ± 13.4 % (mean ± s.e.m.) and 84.4 ± 5.3 %, respectively. 3 However, cADPR (10‐300 μm), β‐NAD+ (2.5 mM), FK506 (30 μm) and NAADP (100 μm) neither contracted the strip by themselves nor affected the subsequent CCh (1 μm) response. α‐Toxin‐permeabilized bronchial smooth muscle and ileum smooth muscle also responded to caffeine, InsP3 and CCh but not to cADPR. 4 Both 100 μm 8‐amino‐cADPR, a selective cADPR antagonist, and 100 μm thionicotinamide‐NADP, a selective NAADP antagonist, failed to inhibit the CCh response, although procaine abolished the caffeine, InsP3 and CCh responses in the permeabilized tracheal smooth muscle. 5 Although inhibition of the caffeine response by 30 μm ryanodine was nearly complete, approximately 30 % of the InsP3 (300 μm) plus GTP (50 μm) response was retained, and the resultant response disappeared after the caffeine response was evoked in the presence of ryanodine. 6 Heparin (300 μg ml−1) blocked InsP3 (300 μm) and CCh (3 μm) responses in β‐escin‐permeabilized tracheal smooth muscle, while Ruthenium Red (100 μm) partially inhibited the CCh response. 7 Collectively, InsP3 but not cADPR or NAADP plays a key role in CCh‐initiated contraction, and InsP3 utilizes a single compartment of the caffeine/ryanodine‐sensitive stored Ca2+ in airway smooth muscle.

Interferon-γ rescues TNF-α-induced apoptosis mediated by up-regulation of TNFR2 on EoL-1 cells

Recent studies show that apoptosis is important for the resolution of chronic inflammation. Using a human myeloblastic leukemia cell line, EoL-1, we investigated the effect of interferon-gamma (IFN-gamma), which differentiates EoL-1 into monocyte/macrophage-like cells on Fas antigen (Fas)- and tumor necrosis factor-alpha (TNF alpha)-induced apoptosis. Both TNF and anti-Fas monoclonal antibody (CH-11) induced apoptosis of EoL-1 cells. Pretreatment with IFN-gamma for 72 hours enhanced the CH-11-induced apoptosis with up-regulation of Fas. However, the treatment markedly inhibited the TNF-induced apoptosis. In flow cytometric analysis, EoL-1 expressed two types of tumor necrosis factor receptors (TNFR1 and TNFR2), and the expression of TNFR2 but not of TNFR1 was up-regulated significantly after the IFN-gamma treatment. The TNF-induced apoptosis was mimicked by a TNFR1 stimulating antibody (htr-9), and was reversed by a TNFR1 blocking antibody (H398). Although the TNFR1-mediated cytotoxic signal was not affected by IFN-gamma pretreatment, blocking TNFR2 by a specific antagonistic antibody (utr-1) canceled the inhibitory effect of IFN-gamma. In conclusion, TNF-induced apoptosis was mediated preferentially by TNFR1, and the anti-apoptotic effect of IFN-gamma was result from up-regulated TNFR2 in EoL-1 cell line. This cell line is a useful model to provide new insights into crosstalk among Fas/FasL-, TNF-, and IFN-gamma-mediated signaling.

Barium sulphate aspiration

Correspondence to: Dr Kyoichi Kaira k-kaira@maebashi.jrc.or.jp An asymptomatic 70-year-old man had a barium swallow to screen for gastric cancer. During the examination, the patient aspirated high-density barium contrast medium (200% weight/volume) into his lungs. After 1 h, the patient developed fever, so doctors gave him faropenem 600 mg orally. Chest radiographs showed massive barium sulphate depositions in both lower lobes, middle lobe, and lingula (figure). 48 h later, he became dyspnoeic with worsening hypoxaemia (PaO2 57·2 mm Hg, PaCO2 32·1 mm Hg, breathing room air), and was transferred to our hospital. We treated him with intravenous meropenem 1 g/day and clindamycin 1200 mg/day, physical therapy with postural drainage, and oxygen. He recovered, and was discharged 7 days later. Aspiration of large amounts of barium sulphate is a rare incident during radiographic contrast procedures. In spite of the inert character of barium sulphate, the aspiration of highdensity barium is potentially life-threatening. Barium sulphate aspiration

Effects of isoproterenol on IL-2 and cAMP production in peripheral T cells from asthmatic and non-asthmatic subjects sensitive to Candida.

Summry— Immunity to Candida albicans (Candida) is characterized by a Th‐1 type pattern of reactivity. Candida is rarely a cause antigen for bronchial asthma. Beta agonists have been found to inhibit secretion of IL‐2 from T cells through intracellular cAMP elevation. We examined effects of isoproterenol (ISO) on Candida‐stimulated T cells. Peripheral T cells obtained from six Candida‐sensitive asthmatics, six Candida‐sensitive non‐asthmatic subjects, and six normal donors by Ficoll‐Hypaque gradient centrifugation and nylon‐wool column chromatography were incubated with Candida antigen or concanavalin A (Con A) in the absence or presence of ISO. Secretion of IL‐2 and intracellular accumulation of cAMP were assayed by ELISA. Con A induced secretion of IL‐2 in each of the three groups. Candida antigen induced IL‐2 secretion in the normal and the non‐asthmatic subjects, but not in the asthmatics. ISO, which reduced Con A‐induced secretion of IL‐2 in a dose‐dependent manner, had no effect on Candida‐induced secretion of IL‐2. Although ISO increased the intracellular concentration of cAMP in untreated and Con A‐treated T cells from all donors, cells from the normal and the non‐asthmatic subjects, but not from the asthmatics, that were co‐incubated with ISO and Candida had lower levels of cAMP than those treated with ISO alone. It is suggested that Candida antigen induces secretion of IL‐2 and reduces ISO‐inducible accumulation of cAMP in Candida‐responsive IL‐2 secreting cells, which may make Candida‐induced secretion of IL‐2 insensitive to ISO.

Influence of inhaled procaterol on pulmonary rehabilitation in chronic obstructive pulmonary disease

BACKGROUND Chronic obstructive pulmonary disease (COPD) is a progressive condition that classically causes dyspnea during physical activity. Destruction of alveoli and bronchostenosis are thought to lead to shortness of breath and result in decreased physical activity. In this study, we examined the influence of inhaled procaterol on exercise therapy for pulmonary rehabilitation. METHODS Patients with moderate to severe stable COPD were randomly divided into 2 groups those who inhaled procaterol before exercise (n=10) and those who did not (control group) (n=11). For 12 weeks, all patients performed their pulmonary rehabilitation exercises at home. We measured the 6-minute walking distance (6MWD) to assess exercise tolerance and used St. Georges respiratory questionnaire (SGRQ) to assess health-related quality of life (HRQOL) before and after the 12-week exercise program. RESULTS Compared to the control group, the group receiving inhaled procaterol showed significant improvement of 6MWD and SGRQ scores. CONCLUSION Our data suggest that a pulmonary rehabilitation program combined with inhaled procaterol can improve both HRQOL and exercise tolerance in COPD patients.

Effect of β-Agonists on Production of Cytokines by Activated T Cells Obtained from Asthmatic Patients and Normal Subjects

Intracellular levels of cAMP were found to regulate T cell activity. We examined whether beta2-agonists altered cytokine production and cyclic adenosine monophosphate (cAMP) accumulation in concanavalin A (ConA)-activated peripheral T cells from asthmatic patients. Procaterol and isoproterenol weakly decreased the ConA-elicited interleukin (IL)-4 and IL-5 secretion; however, the inhibitory effect of procaterol on the ConA-induced IL-2 secretion was inferior to that of isoproterenol in normal controls and was little in asthmatics. The intracellular accumulation of cAMP by procaterol was not altered compared with that by isoproterenol. Results suggest that there is a qualitative difference between procaterol- and isoproterenol-induced cAMP accumulation in T cells.