Hideko Nishimura

Effects of Y-27632, a Rho/Rho Kinase Inhibitor, on Leukotriene D4- and Histamine-Induced Airflow Obstruction and Airway Microvascular Leakage in Guinea Pigs in vivo

Recent in vitro studies have shown that the Rho/Rho kinase pathway is involved in the mechanism of not only airway smooth muscle contraction but also vascular endothelial permeability caused by certain stimuli. This suggests that Rho/Rho kinase inhibitors may become useful agents against asthma via reduction of increased airway microvascular leakage, one of the main features of this disease. Thus, we wanted to know the in vivo effect of Y-27632, a selective Rho kinase inhibitor, on airway microvascular leakage caused by leukotriene D4 (LTD4) and histamine, potent mediators of allergic airway inflammation, by comparing its effect against airflow obstruction. For comparison, the effects of procaterol, a β2-adrenoceptor agonist, on these responses were also studied. Tracheostomized guinea pigs were given either aerosolized Y-27632 (3 or 15 mmol/l), procaterol (6 µmol/l) or vehicle (0.9% NaCl) for 5 min under spontaneous breathing. After being mechanically ventilated, the animals were given intravenous Evans blue dye 15 min after the end of inhalation. One minute later, either 2 nmol/kg LTD4, 300 nmol/kg histamine or vehicle was administered intravenously. After measurements of lung resistance (RL) for 6 min, the lungs of animals were taken out, and the amount of extravasated Evans blue dye was examined as an index of leakage. Inhaled Y-27632 dose-dependently attenuated increases in RL caused by LTD4 and histamine. The degree of inhibition was almost similar between 15 mmol/l Y-27632 and 6 µmol/l procaterol. By contrast, only 15 mmol/l, but not 3 mmol/l, Y-27632 partially reduced LTD4-induced leakage. Histamine-induced Evans blue dye extravasation was not inhibited by 15 mmol/l Y-27632. Procaterol significantly inhibited the dye extravasation caused by either LTD4 or histamine. These results suggest that Y-27632 is not a useful agent in attenuating airway microvascular leakage which is seen in asthma, although it is potent in inhibiting airflow obstruction.

Acute effects of prostaglandin D2 to induce airflow obstruction and airway microvascular leakage in guinea pigs: role of thromboxane A2 receptors.

BACKGROUND Although prostaglandin D2 (PGD2), a mast cell-derived inflammatory mediator, may trigger allergic airway inflammation, its potency and the mechanism by which it induces airway microvascular leakage, a component of airway inflammation, is not clear. OBJECTIVE We wanted to evaluate the relative potency of PGD2 to cause microvascular leakage as compared to airflow obstruction, because both responses were shown to occur simultaneously in allergic airway diseases such as asthma. The role of thromboxane A2 receptors (TP receptors) in inducing these airway responses was also examined. METHODS Anesthetized and mechanically ventilated guinea pigs were given i.v. Evans blue dye (EB dye) and, 1 min later, PGD2 (30, 100, 300 or 1,000 nmol/kg). For comparison, the effect of 150 nmol/kg histamine or 2 nmol/kg leukotriene D4 (LTD4) was also examined. Lung resistance (R(L)) was measured for 6 min (or 25 min for selected animals) and the lungs were removed to calculate the amount of extravasated EB dye into the airways as a marker of leakage. In some of the animals, specific TP receptor antagonists, S-1452 (10 microg/kg) or ONO-3708 (10 mg/kg), or a thromboxane A2 synthase inhibitor, OKY-046 (30 mg/kg), was pretreated before giving PGD2. RESULTS Injection of PGD2 produced an immediate and dose-dependent increase in RL (peaking within 1 min), which was significant at all doses studied. At 1,000 nmol/kg, PGD2 induced a later increase in R(L), starting at 3 min and reaching a second peak at 8 min. By contrast, only PGD2 at doses of 300 and 1,000 nmol/kg produced a significant increase in EB dye extravasation. The relative potency of 1,000 nmol/kg PGD2 to induce leakage as compared to airflow obstruction was comparable to histamine at most of airway levels, but less than LTD4. Both responses caused by PGD2 were completely abolished by S-1452 and ONO-3708, but not by OKY-046. CONCLUSION PGD2 may induce airway microvascular leakage by directly stimulating TP receptors without generating TXA2 in guinea pigs. Microvascular leakage may play a role in the development of allergic airway inflammation caused by PGD2.

Effect of Disodium Cromoglycate on Airway Mucus Secretion during Antigen-Induced Late Asthmatic Responses in a Murine Model of Asthma

Background: Disodium cromoglycate (DSCG) is known to inhibit both immediate and late asthmatic responses (IAR and LAR). However, its effect on mucus hypersecretion is unknown. Using a murine model of asthma, we aimed to determine whether mucus secretion increased during IAR and LAR. We also studied the potency of DSCG in inhibiting mucus secretion and on airway eosinophilia. Methods: Mice were subjected to initial intraperitoneal sensitizationand airway challenge to ovalbumin (OVA) and then provokedby additional exposure to OVA. Some mice were pretreated with aerosolized DSCG (20 mg/ml) 1 h before the provocation with OVA. After serial measurements of enhanced pause (Penh), an indicator of airflow obstruction, serum samples and bronchoalveolar lavage fluids (BALF) were collected. Then, the lungs were excised and a morphometric analysis for mucus hypersecretion was performed. Results: A biphasic increase in Penh (IAR and LAR) was observed in sensitized animals after provocation with OVA. Airway eosinophilia was observed during both responses. Intraluminal mucus significantly increased during LAR, but not during IAR. DSCG significantly attenuated both IAR and LAR, and significantly inhibited the increase in intraluminal mucus during LAR, but had no effect on eosinophilia in BALF. Conclusion: Our results suggest that airway hypersecretion may be involved as a component of airflow obstruction during LAR, and that this is unlikely during IAR. DSCG may be effective in reducing excessive airway mucus caused by exposure to allergens.

Maturational Changes in Airway Remodeling after Chronic Exposure to Ovalbumin in Sensitized Guinea Pigs: Role of Cell Renewal of Airway Resident Cells

We wanted to know whether airway remodeling caused by chronic exposures to antigen differed depending on the degree of maturation of animals. We sensitized guinea pigs at different stages of maturation: juvenile (approximately 200 g in body weight), adult (400 g), and old animals (800 g). Then, animals were repeatedly challenged with inhaled ovalbumin (0.3% or 3%) or vehicle twice a week for 6 wk. After the final challenge, the lungs were excised for the histologic evaluation of changes in the thickness of the inner wall area (Ti), the smooth muscle area (Tm), and the outer wall area (To) in noncartilaginous airway dimensions. To clarify whether or not the observed changes were due to renewal of airway cells, we stained the samples with labeled nucleotide 5′-bromo-2′-deoxyuridine (BrdU), which we injected repeatedly during the challenge periods. Chronic exposures to antigen induced airway wall thickening regardless of their stages of maturation. However, prominent areas of thickening differed between the three groups. Ti increased more remarkably in juvenile and adult animals than in old ones. By contrast, Tm significantly increased only in old animals. BrdU staining revealed more renewal of epithelial cells in juvenile and adult animals than in old ones (juvenile ≧ adult > old), suggesting that increased renewal of epithelial cells contributed to the thickening of Ti in juvenile and adult animals. By contrast, only a slight increase in smooth muscle cell renewal was found even in old animals, indicating that an increase in Tm was due to factors such as hypertrophy. These results show that the development of antigen-induced airway remodeling is partly modified by the degree of maturation of animals in vivo.

Effects of STA2, a Thromboxane A2 Mimetic, in Inducing Airflow Obstruction and Airway Microvascular Leakage in Guinea Pigs

Background: U-46619, a thromboxane A2 (TXA2) mimetic, is shown to cause airway microvascular leakage, although the effects is weak when comparing with that to induce bronchoconstriction in guinea pigs. Objective: In order to know the airway effect of TXA2 more accurately, we have examined the effects of STA2, a TXA2 mimetic with higher affinity to TXA2 (TP) receptors than U-46619, to induce airway microvascular leakage and airflow obstruction. Methods: Anesthetized and ventilated guinea pigs were i.v. given STA2 (3–30 nmol/kg) or U-46619 (3–100 nmol/kg) 1 min after i.v. Evans blue dye. STA2- and U-46619-induced increases in lung resistance (RL) was measured for 6 min. The amount of extravasated Evans blue dye in the lower airways was, then, examined as an index of leakage. In selected animals, specific TP receptor antagonists (10 µg/kg S-1452 or 10 mg/kg ONO-3708) were pretreated i.v. Results: Both STA2 and U-46619 induced significant increases in leakage and airflow obstruction. However, STA2 induced a slow and significantly less increase in RL but caused a significantly greater increase in extravasation of Evans blue dye compared to U-46619. Specific TP receptor antagonists completely abolished both airway effects induced by STA2 and U-46619. Conclusion: Our present results have supported a possibility that TXA2 induces microvascular leakage as well as bronchoconstriction in the airways.

Airway Responsiveness and Airway Remodeling after Chronic Exposure to Procaterol and Fenoterol in Guinea Pigs in vivo

Background: Chronic exposure to fenoterol (FEN), a β2-adrenergic receptor (β2-AR) agonist, was shown to induce both airway hyperresponsiveness and airway remodeling in experimental animals. Objective: We wanted to know the effects of chronic exposure to procaterol (PRO), a β2-AR agonist, on airway function and structure, because this agent is widely used as a bronchodilator in Japan. For comparison, the effects of FEN were also examined. Methods: Aerosolized PRO (0.1 or 1 mg/ml), FEN (1 mg/ml) or vehicle (0.9% NaCl) was given to guinea pigs 3 times a day for 6 weeks. Sublaryngeal deposition of these agents was calculated using radioisotopes. At 72 h after the last inhalation of PRO, FEN or vehicle, the dose-response relationship between lung resistance (RL) and intravenously administered acetylcholine (ACh) was measured. After measuring RL, histological changes in noncartilaginous airway dimensions were evaluated. Results: The amount of sublaryngeal deposition of 0.1 mg/ml PRO in the present study was speculated to be 100 times larger than that of therapeutic dose. ACh concentrations causing 2-fold, 10-fold and maximal increases in RL were not different in 4 groups tested. In the smaller membranous airways (<0.4 mm in diameter), but not the larger ones, thickening of adventitial areas was significantly greater in animals treated with β2-AR agonists than in control animals (23 and 25, and 96% higher in animals treated with 0.1 and 1 mg/ml PRO or 1 mg/ml FEN, respectively). The degree of the increase was significantly less in PRO-treated animals than in FEN-treated animals (p < 0.01). Conclusion: Our results did not provide any evidence that regular inhalation of PRO at the therapeutic dose might induce bronchial hyperresponsiveness. In addition, huge amounts of PRO only caused a mild thickening of the adventitial areas, suggesting that PRO may be a weak inducer of airway remodeling compared with FEN.