Airway mechanics and methods used to visualize smooth muscle dynamics in vitro.

Contraction of airway smooth muscle (ASM) is regulated by the physiological, structural and mechanical environment in the lung. We review two in vitro techniques, lung slices and airway segment preparations, that enable in situ ASM contraction and airway narrowing to be visualized. Lung slices and airway segment approaches bridge a gap between cell culture and isolated ASM, and whole animal studies. Imaging techniques enable key upstream events involved in airway narrowing, such as ASM cell signalling and structural and mechanical events impinging on ASM, to be investigated.

Ex vivo lung function measurements in precision-cut lung slices (PCLS) from chemical allergen-sensitized mice represent a suitable alternative to in vivo studies.

A wide range of industrial chemicals can induce respiratory allergic reactions. Hence, there is an urgent need for methods identifying and characterizing the biological action of chemicals in the lung. Here, we present an easy, reliable alternative method to measure lung function changes ex vivo after exposure to chemical allergens and compare this to invasive in vivo measurements after sensitization with the industrial chemicals trimellitic anhydride (TMA) and 2,4-dinitrochlorobenzene (DNCB). Female BALB/c mice were sensitized epicutaneously with the respiratory allergen TMA and the contact sensitizer DNCB. The early allergic response to TMA and DNCB was registered in vivo and ex vivo on day 21 after inhalational challenge with dry standardized aerosols or after exposure of precision-cut lung slices (PCLS) to dissolved allergen. Airway hyperresponsiveness (AHR) to increasing doses of methacholine (MCh) was measured on the next day in vivo and ex vivo. Bronchoalveolar lavage (BAL) was performed for immunological characterization of local inflammation. TMA-sensitized mice showed AHR to MCh in vivo (ED(50): 0.06 microg MCh vs. 0.21 microg MCh in controls) and in PCLS (EC(50): 0.24 microM MCh vs. 0.4 microM MCh). TMA-treated animals showed increased numbers of eosinophils (12.8 x 10(4) vs. 0.7 x 10(4)) and elevated eotaxin-2 concentrations (994 pg/ml vs. 167 pg/ml) in BAL fluid 24 h after allergen challenge. In contrast, none of these parameters differed after sensitization with DNCB. The present study suggests that the effects of low molecular weight allergens, like TMA and DNCB, on ex vivo lung functions tested in PCLS reflect the in vivo situation.

Characterisation of guinea pig precision-cut lung slices: comparison with human tissues.

Precision-cut lung slices (PCLS) allow comparison of the airway responses of different species under identical experimental conditions. The aim of this study was to establish and characterise PCLS from guinea pigs (GPs) and to compare them with human PCLS. GP PCLS were prepared according to previously published procedures with the exception that the agarose solution and the initial incubation medium contained isoproterenol to avoid post mortem airway contraction. The median effective concentrations (EC50, expressed as nM) for agonist-induced bronchoconstriction in GP and human PCLS, respectively, were: leukotriene D4 (1.8, 5.0); thromboxane (16, 1.3); serotonin (69, unresponsive); histamine (217, 2,170); and methacholine (231, 234). Allergen-induced bronchoconstriction of passively sensitised PCLS was attenuated by histamine or thromboxane-prostanoid receptor antagonists and was almost completely prevented by their combination with leukotriene receptor antagonists. Airways pre-contracted with methacholine were relaxed by the beta-agonist salbutamol or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Simultaneous studies of airways and vessels are possible with, for example, EC50 values for endothelin-1 of 37 nM (pulmonary arteries), 10 nM (pulmonary veins) and 9.6 nM (airway). When compared with previous findings in rat and mouse, these data show that guinea pig lungs are a more appropriate model for human airway pharmacology than lungs from rats or mice.

Pressor responses to platelet-activating factor and thromboxane are mediated by Rho-kinase.

Platelet-activating factor (PAF) contracts smooth muscle of airways and vessels primarily via release of thromboxane. Contraction of smooth muscle is thought to be mediated either by calcium and inositol trisphosphate (IP(3))-dependent activation of the myosin light chain kinase or, alternatively, via the recently discovered Rho-kinase pathway. Here we investigated the contribution of these two pathways to PAF and thromboxane receptor-mediated broncho- and vasoconstriction in two different rat models: the isolated perfused lung (IPL) and precision-cut lung slices. Inhibition of the IP(3) receptor (1-10 microM xestospongin C) or inhibition of phosphatidylinositol-specific PLC (30 microM L-108) did not affect bronchoconstriction but attenuated the sustained vasoconstriction by PAF. Inhibition of myosin light chain kinase (35 microM ML-7) or of calmodulin kinase kinase (26 microM STO609), which regulates the phosphorylation of the myosin light chain, had only a small effect on PAF- or thromboxane-induced pressor responses. Similarly, calmidazolium (10 microM), which inhibits calmodulin-dependent proteins, only weakly reduced the airway responses. In contrast, Y-27632 (10 microM), a Rho-kinase inhibitor, attenuated the thromboxane release triggered by PAF and provided partial or complete inhibition against PAF- and thromboxane-induced pressor responses, respectively. Together, our data indicate that PAF- and thus thromboxane receptor-mediated smooth muscle contraction depends largely on the Rho-kinase pathway.