Diverse actions of cadmium on the smooth muscle myosin phosphorylation system

AbstractThe effects of cadmium (Cd) on smooth muscle myosin phosphorylation have been investigated using an in vitro system comprising myosin filaments containing endogenous calmodulin (CM) and myosin light chain kinase (MLCKase). In the absence of calcium (Ca), Cd as well as some other divalent cations caused no activation of phosphorylation. However, when at least one (or possibly two) Ca2+ were bound per CM, the addition of 10 μM to 40 μM Cd2+ resulted in a 2 to 3 fold acceleration of the phosphorylation rate. Higher Cd concentrations caused inhibition of the system independent of Ca2+ concentration through the formation of Cd-ATP complexes. These results explain some previously controversial data on the complex effects of Cd in intact smooth muscles

Purification of Myosin from Bovine Tracheal Smooth Muscle, Filament Formation and Endogenous Association of Its Regulatory Complex

Dynamic regulation of myosin filaments is a crucial factor in the ability of airway smooth muscle (ASM) to adapt to a wide length range. Increased stability or robustness of myosin filaments may play a role in the pathophysiology of asthmatic airways. Biochemical techniques for the purification of myosin and associated regulatory proteins could help elucidate potential alterations in myosin filament properties of asthmatic ASM. An effective myosin purification approach was originally developed for chicken gizzard smooth muscle myosin. More recently, we successfully adapted the procedure to bovine tracheal smooth muscle. This method yields purified myosin with or without the endogenous regulatory complex of myosin light chain kinase and myosin light chain phosphatase. The tight association of the regulatory complex with the assembled myosin filaments can be valuable in functional experiments. The purification protocol discussed here allows for enzymatic comparisons of myosin regulatory proteins. Furthermore, we detail the methodology for quantification and removal of the co-purified regulatory enzymes as a tool for exploring potentially altered phenotypes of the contractile apparatus in diseases such as asthma

Purification of Myosin from Bovine Tracheal Smooth Muscle, Filament Formation and Endogenous Association of Its Regulatory Complex

Dynamic regulation of myosin filaments is a crucial factor in the ability of airway smooth muscle (ASM) to adapt to a wide length range. Increased stability or robustness of myosin filaments may play a role in the pathophysiology of asthmatic airways. Biochemical techniques for the purification of myosin and associated regulatory proteins could help elucidate potential alterations in myosin filament properties of asthmatic ASM. An effective myosin purification approach was originally developed for chicken gizzard smooth muscle myosin. More recently, we successfully adapted the procedure to bovine tracheal smooth muscle. This method yields purified myosin with or without the endogenous regulatory complex of myosin light chain kinase and myosin light chain phosphatase. The tight association of the regulatory complex with the assembled myosin filaments can be valuable in functional experiments. The purification protocol discussed here allows for enzymatic comparisons of myosin regulatory proteins. Furthermore, we detail the methodology for quantification and removal of the co-purified regulatory enzymes as a tool for exploring potentially altered phenotypes of the contractile apparatus in diseases such as asthma.Medicine, Faculty ofNon UBCMedicine, Department ofPathology and Laboratory Medicine, Department ofReviewedFacultyResearcherOthe

Binding of phosphorylated and dephosphorylated heavy meromyosin to F-actin

The effect of myosin light chain phosphorylation in skeletal muscle was investigated with respect to the binding affinity of phosphorylated and dephosphorylated heavy meromyosin (HMM) for F-actin in the absence of ATP. For phosphorylated HMM the affinity was 2.5-times weaker in the presence of Ca 2+ as in its absence (HMM divalent binding sites saturated only with Mg). For dephosphorylated HMM the reverse was true, the binding being 2.4-times higher in the presence of Ca 2+

Length adaptation of airway smooth muscle

Many types of smooth muscle, including airway smooth muscle (ASM), are capable of generating maximal force over a large length range due to length adaptation - a relatively rapid process in which smooth muscle regains contractility after experiencing a force decrease induced by length fluctuation. Although the underlying mechanism is unclear, it is believed that structural malleability of smooth muscle cells is essential for the adaptation to occur. The process is triggered by strain on the cell cytoskeleton that results in a series of yet-to-be-defined biochemical and biophysical events leading to restructuring of the cytoskeleton and contractile apparatus, and consequently optimization of the overlap between the myosin and actin filaments. Although length adaptability is an intrinsic property of smooth muscle, maladaptation of ASM could result in excessive constriction of the airways and inability of deep inspirations to dilate them. In this review, we describe the phenomenon of length adaptation in ASM and some possible underlying mechanisms that involve the myosin filament assembly and disassembly. We also discuss a possible role of maladaptation of ASM in the pathogenesis of asthma. We believe that length adaptation in ASM is mediated by specific proteins and their post-translational regulations involving covalent modifications, such as phosphorylation. The discovery of these molecules and the processes that regulate their activity will greatly enhance our understanding of the basic mechanisms of ASM contraction and will also suggest molecular targets to alleviate asthma exacerbation related to excessive constriction of the airways