Abstract
Different interacting signaling modules involving Ca(2+)/calmodulin-dependent myosin light chain kinase, Ca(2+)-independent regulatory light chain phosphorylation, myosin phosphatase inhibition, and actin filament-based proteins are proposed as specific cellular mechanisms involved in the regulation of smooth muscle contraction. However, the relative importance of specific modules is not well defined. By using tamoxifen-activated and smooth muscle-specific knock-out of myosin light chain kinase in mice, we analyzed its role in tonic airway smooth muscle contraction. Knock-out of the kinase in both tracheal and bronchial smooth muscle significantly reduced contraction and myosin phosphorylation responses to K(+)-depolarization and acetylcholine. Kinase-deficient mice lacked bronchial constrictions in normal and asthmatic airways, whereas the asthmatic inflammation response was not affected. These results indicate that myosin light chain kinase acts as a central participant in the contractile signaling module of tonic smooth muscle. Importantly, contractile airway smooth muscles are necessary for physiological and asthmatic airway resistance.
Highlights
Smooth muscles line the walls of hollow organs such as the airways of the respiratory tract
We conclude that MLCKSMKO mice exhibit an efficient deletion of MLCK in airway smooth muscles
We find that deletion of Ca2ϩ/calmodulin-dependent MLCK in tonic airway smooth muscle greatly attenuated RLC phosphorylation as well as force development initially and during the sustained contraction phase with both depolarization and muscarinic receptor-induced coninflammation of the asthmatic airway is similar in control and traction
Summary
Generation of Floxed Mlck Mice and Tissue-specific Knock-out Mice (MLCKSMKO)—To generate MLCKSMKO mice, Mlckflox/flox mice were crossed with SM-CreERT2 (ki) mice expressing a tamoxifen-activated Cre recombinase under control of the SM22 promoter as previously described [17, 27]. Immunization and Airway Challenge—Six- to eight-week-old female mice were sensitized to ovalbumin by intraperitoneal injection of 80 g of OVA (Grade VI; Sigma) adsorbed to 4 mg of aluminum hydroxide (Pierce) in a total volume of 0.2 ml of sterile saline on days 0 and 14. These mice were challenged for 60 min to aerosolized 1% ovalbumin by ultrasonic nebulization on days 24, 25, and 26. Fresh tracheal and lung tissues from control (CTR) and MLCKSMKO knock-out mice were fixed with 4% formalin and dehydrated in a graded series of ethanol solutions followed by standard paraffin section and HE staining. Differences between groups were determined by Student’s t test with significance at p Ͻ 0.05
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