Allergic sensitization enhances the contribution of Rho-kinase to airway smooth muscle contraction

Abstract

1. Repeated allergen challenge has been shown to increase the role of Rho-kinase in airway smooth muscle (ASM) contraction. We considered the possibility that active allergic sensitization by itself, that is, without subsequent allergen exposure, could be sufficient to enhance Rho-kinase-mediated ASM contraction. 2. Guinea pigs were actively IgE-sensitized to ovalbumin (OA), using Al(OH)(3) as adjuvant. Contractile responsiveness to G(q)-coupled receptor agonists (methacholine, histamine or PGF(2alpha)) was investigated in tracheal rings. No effect of sensitization was observed on basal- and methacholine-induced myogenic tone. In contrast, potency of histamine and PGF(2alpha) increased, that is, EC(50) decreased, after OA-sensitization by 2.6- and 4.7-fold, respectively, without effect on maximal contraction (E(max)). 3. Basal tone in preparations from both control and OA-sensitized animals was strongly decreased in the presence of the Rho-kinase inhibitor (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide (Y-27632) (1 microm). In control preparations, the E(max) and potency of histamine were unaffected by Y-27632, but were decreased for PGF(2alpha) (by 38.2% and 2.0-fold, respectively). However, in preparations from OA-sensitized animals, Y-27632 induced a significant reduction in E(max) (33.5%) and potency (2.3-fold) of histamine and of PGF(2alpha) (48.3% and 6.6-fold, respectively), normalizing the OA-sensitization-induced increase in sensitivity toward these agonists. 4. We also investigated the contribution of Rho-kinase in vivo by measuring airway responsiveness toward inhaled histamine in permanently instrumented, unanaesthetized control and OA-sensitized guinea pigs. Treatment with Y-27632 by inhalation (5 mm, nebulizer concentration) decreased airway responsiveness toward histamine both in control and OA-sensitized animals. However, the histamine PC(100) ratio pre/post Y-27632 inhalation was significantly smaller in OA-sensitized animals as compared to control animals, indicating an enhanced contribution of Rho-kinase. 5. Expression of RhoA, an upstream activator of Rho-kinase, was significantly increased (2.6-fold) in lung homogenates of OA-sensitized guinea pigs compared to control animals, as determined by Western analysis. 6. In conclusion, the results show a receptor-dependent role of Rho-kinase in agonist-induced ASM contraction. The contribution of Rho-kinase to contractile airway responsiveness, both in vivo and ex vivo, is augmented after active allergic sensitization, as a consequence of increased expression of RhoA presumably. Inhibition of the RhoA/Rho-kinase pathway may be considered a useful pharmacotherapeutical target in allergy and asthma.

Figure 1

Effect of OA-sensitization on basal myogenic tone of open-ring tracheal smooth muscle preparations from control and OA-sensitized guinea pigs in the absence (white bars) and presence (black bars) of 1 μM Y-27632. Data represent means±s.e.m. of 10 to 14 experiments each performed in duplicate. ^***P<0.001 compared to vehicle.

Figure 2

Effects of OA-sensitization on agonist-induced contraction. (a) Histamine- and (b) PGF_2α-induced contraction of guinea pig open-ring tracheal smooth muscle preparations from control (open circles) and OA-sensitized (closed circles) guinea pigs. Data represent means±s.e.m. of five to six experiments each performed in duplicate.

Figure 3

Effects of Rho-kinase inhibition on agonist-induced contraction. Histamine (a, b)- and PGF_2α (c, d)-induced contraction in the absence (open circles) and presence (black circles) of 1 μM Y-27632 of open-ring tracheal smooth muscle preparations of control (a, c) and OA-sensitized (b, d) guinea pigs. Data represent means±s.e.m. of five to six experiments each performed in duplicate.

Figure 4

Effects of basal tone on histamine-induced contraction. Histamine-induced contraction of open-ring tracheal smooth muscle preparations in the absence (circles) or presence of 3 μM indomethacin (triangles) or 3 μM indomethacin plus PGF_2α (squares). Data represent means±s.e.m. of four experiments each performed in duplicate.

Figure 5

Effects of Y-27632 inhalation (5 mM) on airway responsiveness toward histamine in control and actively OA-sensitized guinea pigs. Effects were expressed as the histamine PC₁₀₀ ratio pre/post saline (white bars) or pre/post Y-27632 (black bars) inhalation. Data represent means±s.e.m. of five animals. ^***P<0.001 compared to saline treated; ^#P<0.01 compared to Y-27632-treated controls.

Figure 6

Western analysis of RhoA expression in lung homogenates of control (n=4) and OA-sensitized (n=3) guinea pigs. Upper panel: representative Western blot. Lane 1: control; lane 2: OA-sensitized. Lower panel: Densitometric analysis. Data represent means±s.e.m. ^**P<0.01 compared to controls.