Smooth muscle is a central structure involved in the regulation of airway tone. In addition, it plays an important role in the development of some pathologies generated by alterations in contraction, such as hypercontractility and the airway hyperresponsiveness observed in asthma. The molecular processes associated with smooth muscle contraction are centered around myosin light chain (MLC) phosphorylation, which is controlled by a balance in the activity of myosin light-chain kinase (MLCK) and myosin light-chain phosphatase (MLCP). MLCK activation depends on increasing concentrations of intracellular Ca 2+ , while MLCP activation is independent of Ca 2+. MLCP contains a phosphatase subunit (PP1c) that is regulated through myosin phosphatase target subunit 1 (MYPT1) and other subunits, such as glycogenassociated regulatory subunit and myosin-binding subunit 85 kDa. Interestingly, MLCP inhibition may contribute to exacerbation of smooth muscle contraction by increasing MLC phosphorylation to induce hypercontractility. Many pathways inhibiting MLCP activity in airway smooth muscle have been proposed and are focused on inhibition of PP1c, inhibitory phosphorylation of MYPT1 and dissociation of the PP1c-MYPT1 complex.

Background Airway obstruction (AO) in asthma is driven by airway smooth muscle (ASM) contraction. AO can be induced extrinsically by direct stimulation of ASM with contractile agonists as histamine, or by indirect provocation with antigens as ovalbumin, while the airway tone is dependent on intrinsic mechanisms. The association of the ASM phenotypes involved in different types of AO and airway tone in guinea pigs was evaluated. Methods Guinea pigs were sensitized to ovalbumin and challenged with antigen. In each challenge, the maximum OA response to ovalbumin was determined, and before the challenges, the tone of the airways. At third challenge, airway responsiveness (AR) to histamine was evaluated and ASM cells from trachea were disaggregated to determinate: (a) by flow cytometry, the percentage of cells that express transforming growth factor-β1 (TGF-β1), interleukin-13 (IL-13) and sarco-endoplasmic Ca2+ ATPase-2b (SERCA2b), (b) by RT-PCR, the SERCA2B gene expression, (c) by ELISA, reduced glutathione (GSH) and, (d) Ca2+ sarcoplasmic reticulum refilling rate by microfluorometry. Control guinea pig group received saline instead ovalbumin. Results Antigenic challenges in sensitized guinea pigs induced indirect AO, AR to histamine and increment in airway tone at third challenge. No relationship was observed between AO induced by antigen and AR to histamine with changes in airway tone. The extent of antigen-induced AO was associated with both, TGF-β1 expression in ASM and AR degree. The magnitude of AR and antigen-induced AO showed an inverse correlation with GSH levels in ASM. The airway tone showed an inverse association with SERCA2b expression. Conclusions Our data suggest that each type of AO and airway tone depends on different ASM phenotypes: direct and indirect AO seems to be sensitive to the level of oxidative stress; indirect obstruction induced by antigen appears to be influenced by the expression of TGF-β1 and the SERCA2b expression level plays a role in the airway tone.

Background: Airway obstruction in asthma is driven by airway smooth muscle (ASM) contraction. Airway obstruction can be induced extrinsically by direct stimulation of ASM with contractile agonists or by indirect provocation with antigens, while the airway baseline tone is dependent on intrinsic obstruction. The ASM phenotypes involved in all types of obstruction seem to be related.Methods: To determination the associations of the ASM phenotypes involved in different types of airway obstruction, guinea pigs were sensitized to ovalbumin and repetitively challenged with antigen. At the third challenge, histamine provocation was used to evaluate airway responsiveness (AR), and lung samples were obtained to calculate the airway wall area. ASM cells from the trachea were disaggregated to determine 1) the percentage of cells that expressed transforming growth factor-β1 (TGF-β1), interleukin-13 (IL-13) and sarco-endoplasmic Ca2+ ATPase-2b (SERCA2b) by flow cytometry; 2) SERCA2B gene expression by RT-PCR; 3) the level of reduced glutathione (GSH) by ELISA; and 4) the sarcoplasmic reticulum Ca2+ refilling rate by microfluorometry. The control guinea pig group received only saline instead of ovalbumin. Comparisons were made using t-tests, and the associations were determined using Spearman correlation coefficient analysis.Results: Antigenic challenges induced airway obstruction and progressive incremental changes in airway baseline tone. The AR to histamine and the expression of TGF-β1 in ASM cells was increased in the asthma model. The airway wall mass and expression of IL-13 and SERCA2b in ASM cells were similar between groups. SERCA2B gene expression and GSH levels were reduced in the asthma group. The extent of antigen-induced airway obstruction was directly associated with ASM cell TGF-β1 expression and the degree of AR. The magnitude of AR and antigen-induced airway obstruction showed an inverse correlation with GSH levels. The airway baseline tone showed an inverse association with SERCA2b expression. No relationship was observed between direct or indirect airway obstruction and the airway tone. After caffeine withdrawal, the rate of sarcoplasmic reticulum Ca2+ refilling was similar in both groups.Conclusions: Each type of airway obstruction depends on different ASM phenotypes: 1) direct and indirect airway obstruction seems to be sensitive to the level of ASM oxidative stress; 2) indirect obstruction induced by antigen appears to be influenced by the expression of TGF-β1 in ASM; and 3) the SERCA2b expression level in ASM cells plays a role in the intrinsic airway tone.

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