Hideo Tsukagoshi

A major role for the Rho‐associated coiled coil forming protein kinase in G‐protein‐mediated Ca2+ sensitization through inhibition of myosin phosphatase in rabbit trachea

G protein‐mediated Ca2+ sensitization of airway smooth muscle contraction was investigated with respect to the relative importance of Rho‐associated coiled coil forming protein kinase (ROCK) and protein kinase C (PKC). We examined the effects of Y‐27632, a ROCK inhibitor, and GF 109203X, a PKC inhibitor, on guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS)‐induced contraction in α‐toxin‐ or β‐escin‐permeabilized rabbit trachea. Although pre‐treatment with Y‐27632 dose‐dependently inhibited GTPγS (10 μM)‐induced Ca2+ sensitization of α‐toxin‐permeabilized trachea, a Y‐27632‐insensitive component (approximately 16% of the maximum contraction) was retained during the early phase of the GTPγS response in the presence of Y‐27632 (100 μM). GF 109203X (5 μM) abolished 1 μM 4β‐phorbol 12, 13‐dibutyrate (PDBu)‐induced, but only partially inhibited the GTPγS‐induced Ca2+ sensitization. A combination of Y‐27632 (100 μM) and GF 109203X (5 μM) totally abolished the GTPγS response. GTPγS caused only a small contraction in the absence of Ca2+. Wortmannin (30 μM), a myosin light chain kinase (MLCK) inhibitor, completely inhibited Ca2+‐induced contraction. ATP‐triggered contraction of the strip which had been treated with calyculin A (1 μM), a phosphatase inhibitor, in rigor solutions was markedly slowed by worthmannin (30 μM), but not by Y‐27632 (100 μM), in the presence of GTPγS and Ca2+. GTPγS, but not PDBu, contracted the β‐escin‐permeabilized trachea in the absence of Ca2+, but the presence of Ca2+‐independent MLCK. We conclude that ROCK plays a primary role in G‐protein‐mediated Ca2+ sensitization, which requires MLCK activity, with minor contribution of PKC to the early phase of contraction, and PDBu utilizes conventional PKC(s) in airway smooth muscle.

Evaluation of Y-27632, a Rho-kinase inhibitor, as a bronchodilator in guinea pigs

To evaluate (+)-(R)-trans-4-(l-Aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride, monohydrate (Y-27632), a selective Rho-kinase inhibitor, as a novel bronchodilator in vivo and in vitro, we investigated the effect of Y-27632 on the acetylcholine- or ovalbumin-induced increase in lung resistance (R(L)) in non-sensitized or passively sensitized guinea pigs, and the relaxant effects of salbutamol, Y-27632 and theophylline on acetylcholine- or ovalbumin-induced contraction of isolated trachea. Y-27632 inhalation (1 mM, 2 min) inhibited acetylcholine- or ovalbumin-induced increase in R(L) without changes in mean blood pressure, and the effect persisted for at least 3 h. Salbutamol, Y-27632 and theophylline each completely reversed the acetylcholine- or ovalbumin-induced contraction of isolated trachea with rank order of potency, salbutamol>Y-27632>theophylline. The relaxant effect of Y-27632 was not affected by propranolol. We conclude that, although Y-27632 is not as potent as a beta-adrenoceptor agonist, Y-27632 may become an alternative inhaled bronchodilator, because Y-27632 is more potent than theophylline, and the relaxant effect is independent of beta-adrenoceptors.

Sensitized Mast Cells Migrate Toward the Agen: A Response Regulated by p38 Mitogen-Activated Protein Kinase and Rho-Associated Coiled-Coil-Forming Protein Kinase

Although mast cells accumulate within the mucosal epithelial layer of patients with allergic rhinitis and bronchial asthma, the responsible chemotactic factors are undefined. We investigated whether mast cells sensitized with Ag-specific IgE migrate toward the Ag. MC/9 mast cells sensitized with anti-DNP IgE migrated toward DNP-conjugated human serum albumin. This migration was directional, and the degree was stronger than that induced by stem cell factor. IL-3 and stem cell factor-dependent cultured mast cells derived from mouse bone marrow also migrated toward the Ag. Subsequent migration mediated by the FcεRI was significantly inhibited by incubating the cells with Y-27632, a Rho-associated coiled-coil-forming protein kinase inhibitor, or with SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor. Both p38 MAPK and MAPK-activated protein kinase (MAPKAPK)2 were activated following FcεRI aggregation, and activation of MAPKAPK2 was almost completely inhibited by 10μM SB203580. Wortmannin or a low concentration of SB203580 partially inhibited MAPKAPK2, but did not block mast cell migration. In contrast, Y-27632 did not affect the activation of MAPKAPK2. These results indicate that Ag works not only as a stimulant for allergic mediators from IgE-sensitized mast cells, but also as a chemotactic factor for mast cells. Both p38 MAPK activation and Rho-dependent activation of Rho-associated coiled-coil-forming protein kinase may be required for FcεRI-mediated cell migration.

Atrial natriuretic peptide inhibits tumor necrosis factor-alpha production by interferon-gamma-activated macrophages via suppression of p38 mitogen-activated protein kinase and nuclear factor-kappa B activation.

We investigated whether the atrial natriuretic peptide (ANP) might have an inhibitory effect on inflammatory cells. Treatment of RAW264.7 macrophages with interferon-gamma (IFN- gamma) caused a significant increase in tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) production. Activation of p38 mitogen-activated protein (MAP) kinase was observed 30 to 120 min after IFN-gamma, and transcription factor nuclear factor-kappa B (NF-kappaB) was activated about 7 to 9 times of the basal activity. Human ANP(99-126) and a specific p38 MAP kinase inhibitor SB203580 inhibited the IFN-gamma-induced TNF-alpha production in a dose-dependent manner without affecting NO production. ANP inhibited the IFN-gamma-induced p38 MAP kinase activation, and ANP and SB203580 inhibited NF-kappaB activation. To study the involvement of oxidative stress in this system, the effects of allopurinol and acetovanillone, inhibitors of xanthine oxidase and NADPH oxidase, respectively, were studied. Allopurinol or acetovanillone did not inhibit the IFN-gamma-induced production of TNF-alpha or NO, suggesting little involvement of oxidative stress in this system. This is the first evidence in vitro that ANP has an anti-inflammatory activity on IFN-gamma-activated macrophages by suppressing signal transduction pathway leading to p38 MAP kinase and NF-kappaB activation.

Elevated levels of peripheral-blood, naturally occurring aliphatic polyamines in bronchial asthmatic patients with active symptoms

The levels of peripheral‐blood, naturally occurring aliphatic polyamines, such as putrescine, spermidine, and spermine, from 21 bronchial asthmatic patients (11 atopics and 10 nonatopics) were measured by postcolumn derivatization high‐performance liquid chromatography analysis. None of the patients, except the 44‐year‐old woman in the case report below, were given prednisolone, and they were instructed to take only regular medication during the tests. Blood was drawn from the patients in a fasting state, and the polyamine levels were compared between the times when they were free of asthmatic symptoms and when they had mild spontaneous attacks. Nine (5 atopics and 4 nonatopics), 6 (3 atopics and 3 nonatopics), and 4 (3 atopics and 1 nonatopic) out of 20 patients, when they had relatively mild asthmatic attacks, showed higher putrescine, spermidine, and spermine levels, respectively, than those of normal healthy control subjects. The levels of peripheral blood polyamines from a 44–year‐old atopic bronchial asthmatic woman, who was admitted to the hospital with severe asthmatic attacks, were measured serially, and the putrescine and spermidine levels were found to be elevated during the asthmatic attacks, returning to normal levels in parallel with the clinical course. These data may suggest a role for naturally occurring aliphatic polyamines in bronchial asthma.

Inhibition by a novel peptide leukotriene receptor antagonist ONO‐1078 of airway wall thickening and airway hyperresponsiveness to histamine induced by leukotriene C4 or leukotriene D4 in guinea‐pigs

We studied the effect of intravenous administration of leukotriene (LT) C4 or LTD., on airway responsiveness to histamine and airway wall thickening in guinea‐pigs. Guinea‐pigs were killed and the lungs were fixed in formalin. Slides from paraffin‐embedded section of the lungs were stained and the airways that were cut in transverse section were measured by tracing enlarged images using a digitizer, Moreover, airway resistance (Raw) was determined by a pulmonary mechanics analyser and we calculated two indices, an index of airway wall thickening and the one of airway hyperresponsiveness to histamine, from changes of baseline‐Raw and peak‐Raw following intravenous administration of histamine before and after the intravenous administration of LTC4 or LTD4. The infusion of LTC4 or LTD4 induced an increase of the relative thickness of the airway wall in peripheral bronchi demonstrable by the histological examination. In analysis of airway function, intravenous administration of LTC4 or LTD4 induced airway hyperresponsiveness to histamine with airway wall thickening. The LTC4 and LTD4 receptor antagonist ONO‐1078 inhibited these effects of LTC4 and LTD4, suggesting LTC4 and LTD4 may induce airway wall thickening and airway hyperresponsiveness through LTC4 and LTD4 receptors in the airways.

Detection of tryptase-, chymase+ cells in human CD34 bone marrow progenitors.

Background Mast cells (MCs) arise from haematopoietic stem cells. We have recently reported that CD34+ progenitors derived from human bone marrow (BM) develop into tryptase+, chymase+ MCs when cultured in the presence of recombinant human stem cell factor (rhSCF) and recombinant human IL‐6 (rhIL‐6). In an experiment for the expression of chymase during differentiation, chymase+ cells were detected in human BM, but tryptase+ cells were not found.

Recurrent bronchiolitis obliterans organizing pneumonia in a patient with limited cutaneous systemic sclerosis

Abstract. Systemic sclerosis (SSc) is a generalized disorder characterized by fibrosis and vascular obliteration in the skin, lung, gastrointestinal tract, and kidney. One of its two subsets is a stable, limited cutaneous group (lSSc). Pulmonary involvement in scleroderma is common, and several types of pulmonary disorders are associated with SSc. Bronchiolitis obliterans organizing pneumonia (BOOP) is a rare finding in lung disorders associated with SSc. We describe a case of lSSc with BOOP that was responsive to steroid therapy. Of interest is that the lung disorders appeared in different periods and areas. It might be important to diagnose abnormal shadows in lung fields before treatment of patients with SSc.

Functional expression of high‐affinity receptor for immunoglobulin E on mast cells precedes that of tryptase during differentiation from human bone marrow‐derived CD34 progenitors cultured in the presence of stem cell factor and interleukin‐6

Background CD34+ progenitor cells develop into tryptase+, CD117+ mast cells when cultured in the presence of recombinant human stem cell factor (rhSCF). However, spontaneous IgE receptor (FcɛRI) expression during human mast cell development is not well examined.

Effects of the calcium ionophore A23187 on airway responsiveness to histamine and substance P in guinea pigs

Abstract.Objective: We evaluated the mechanism of the airway hyperresponsiveness (AHR) induced by a calcium ionophore in guinea pigs. ¶Materials and Methods: Airway responsiveness to intravenous histamine (HS) and substance P (SP) was measured 24 h after a 1-h exposure to aerosolized A23187 (0.03 or 0.1 mg/ml) or its vehicle (10% DMSO). Changes were assessed by calculating – logPC350HS and – logPC350SP. Neutral endopeptidase (NEP) activity in the airway tissues, as well as the nitrite (NO2−) levels and the cell population in bronchoalveolar lavage fluid (BALF) was determined after measurement of pulmonary function. Changes in SP-induced vascular permeability 24 h after exposure to A23187 were measured by the Evans Blue dye extravasation technique. ¶Results: Exposure to A23187 caused a significant AHR to SP, along with a significant increase in the number of neutrophils and epithelial cells in the BALF. While there was no significant change in NEP activity in the airway tissues, the levels of nitrite in the BALF were significantly decreased in A23187-exposed animals. Significant correlations were found between the number of epithelial cells in the BALF and – logPC350SP (r = 0.477, p < 0.05) and between nitrite levels in the BALF and – logPC350SP (r = −0.491, p < 0.05). A23187 exposure did not significantly change the SP-induced airway microvascular leakage. ¶Conclusions: These data suggest that A23187 exposure induced AHR to SP possibly by reducing NO levels in the airway tissues. This may be due to damaged airway epithelium and/or NO breakdown by activated inflammatory cells in the airways of these guinea pigs.