Pulmonary, Gastrointestinal and Urogenital Pharmacology
Enhanced airway smooth muscle cell thromboxane receptor signaling via activation of JNK MAPK and extracellular calcium influx

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Abstract

Thromboxane is a key inflammatory mediator and potent airway constrictor. It acts on thromboxane A2 (TP) receptors and contributes to airway inflammation and airway hyperresponsiveness that is the characteristic feature of asthma. The present study was designed to study TP receptor signaling in airway smooth muscle cells by using an organ culture model and a set of selective pharmacological inhibitors for mitogen-activated protein kinase (MAPK) and calcium signal pathways. Western-blot, immunohistochemistry, myograph and a selective TP receptor agonist U46619 were used for examining TP receptor signal proteins and function. Organ culture of rat bronchial segments for up to 48 h induces a time-dependently increased airway contractile response to U46619. This indicates that organ culture increases TP receptor signaling in the airway smooth muscle cells. The enhanced bronchial contraction was attenuated by the inhibition of c-Jun N-terminal kinase (JNK) MAPK activity, chelation of extracellular calcium and calcium channel blocker nifedipine, suggesting that JNK MAPK activity and elevated intracellular calcium level are required for the TP receptor signaling. In conclusion, airway smooth muscle cell TP receptor signaling occurs via JNK MAPK activity and the elevation of extracellular calcium influx, which may provide knowledge for understanding the signaling pathway responsible for the modulation of TP receptor mediated airway hyperresponsiveness to thromboxane.

Introduction

Thromboxane A2 (TXA2) was identified in extracts of human platelets and shown to be capable of initiating the contraction of both vascular and airway smooth muscle (Armour et al., 1989, Hamberg et al., 1975). TXA2 and other prostanoids are elevated in a number of lung diseases including asthma and chronic obstructive pulmonary disease (Barnes, 2001, Pratico et al., 1998, Robinson et al., 1985). It is involved in acute bronchoconstriction after antigen inhalation in asthmatic patients, as demonstrated by a markedly elevated level of thromboxane B2 (Davi et al., 1997), a stable metabolite of TXA2 (Iwamoto et al., 1988, Manning et al., 1991, Wenzel et al., 1989). A wealth of studies has shown the role of TXA2 in the pathogenesis and pathophysiology of asthma (Devillier and Bessard, 1997, Dogne et al., 2002a, Wenzel, 1997). TXA2 is generated in greater amounts in asthmatics than in normal subjects, and may participate in thickening and remodeling of the airway wall, subsequently contributing to airway hyperresponsiveness (Davi et al., 1997, Robinson et al., 1985).

TXA2 mediates a number of cellular responses through binding to a specific receptor, the prostanoid thromboxane (TP) receptor (Alm et al., 2002), which signals through the activation of the Gq/11 family of G proteins (Kinsella, 2001). The TP receptor is activated by its natural agonist as well as by high levels of other eicosanoids such as prostaglandin H2, prostaglandin F2α (PGF), and isoprostanes, all of which may play a role in asthma (Antczak et al., 2002, Dogne et al., 2002b). Many aspects of bronchial hyperresponsiveness are potentially mediated by the TP receptor, which associates the TP receptor with the pathophysiology of asthma. TXA2 synthase inhibitors and TP receptor antagonists have been developed as anti-asthma drugs, and demonstrated to improve TP receptor-induced airflow limitation and bronchial hyperresponsiveness (Hanson et al., 2005, Ishimura et al., 2008, McKenniff et al., 1991). However, the signaling pathway responsible for the modulation of TP receptor mediated airway hyperresponsiveness to TXA2 is not clear.

The mitogen-activated protein kinase (MAPK) pathway activation has been suggested to contribute to airway inflammation and airway hyperresponsiveness (Duan and Wong, 2006). The best-characterized of the mammalian MAPKs are 1) the 42- and 44-kDa extracellular signal-regulated kinases (ERKs) ERK2 and ERK1; 2) the c-Jun N-terminal kinase (JNK) or stress-activated protein kinase (SAPK); and 3) p38 MAPK. Among them, the JNK signaling is demonstrated to tightly regulate the TP receptor related inflammation (Bayat et al., 2008, Kumar et al., 2005) in vasculature and the JNK inhibitor SP600125 exerts effects on allergen-induced airway inflammation and remodeling (Eynott et al., 2004, Nath et al., 2005). A common feature associated with the regulation of airway smooth muscle contraction is a change in intracellular Ca2+ concentration, the modification of calcium channel activity may predispose airway smooth muscle to hyperresponsiveness (Perez-Zoghbi et al., 2009).

Previously, we have demonstrated that organ culture induced airway hyperresponsiveness to bradykinin occurs via the up-regulated bradykinin receptors (Lei et al., 2010). The present study was designed to investigate if an organ culture affects TP receptor mediated airway contractile responses focusing on the roles of transcription and MAPK activity and calcium channel activities.

Section snippets

Tissue preparation

Male Sprague Dawley rats (body weight 250–300 g, M&B, Denmark) were acclimatized for a week under standardized temperature (21–22 °C), humidity (50–60%) and light (12:12 light–dark) conditions in the Animal Department of Wallenberg Center in Lund. The rats were anesthetized by CO2 inhalation and were exsanguinated. The lungs were immersed in cold buffer solution (NaCl 119 mM; NaHCO3 15 mM; KCl 4.6 mM; MgCl2 1.2 mM; NaH2PO4 1.2 mM; CaCl2 1.5 mM and glucose 5.5 mM) and the bronchi were freed of adhering

Enhancement of TP agonist U46619-induced contraction

Basal contractile responses to U46619 (TXA2 analog) on isolated rat secondary bronchial ring segments were studied in fresh isolated segments. A weak contraction was elicited by U46619 in the fresh segments. The bronchial segments were cultured in serum-free medium for 12, 24 and 48 h in order to study the time course effects. Compared with control (fresh segments), organ culture time-dependently enhanced U46619-induced contraction. The Emax was reached at 24 h and the concentration-effect curves

Discussion

Airway hyperresponsiveness is characterized by an increased sensitivity of airway smooth muscle cells to constrictor agents, which can be demonstrated in almost all patients with current symptomatic asthma (Cockcroft and Davis, 2006). The increased sensitivity of the airways to constrictor agonists results in a steeper slope of the dose–response relationship and a greater maximal response to the agonists (O'Byrne and Inman, 2003). The present studies have for the first time demonstrated that

Acknowledgements

This study was supported by the Heart-Lung Foundation (grant no. 20070273), the Swedish Research Council (5958), and the Flight Attendant Medical Research Institute (FAMRI, USA).

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