title: Quantitative determination of α(2B)-adrenoceptor-evoked myosin light chain phosphorylation in vascular smooth muscle cells.
abstract: BACKGROUND
  Phosphorylation of myosin light chains is a biochemical readout of smooth muscle cell contraction. α2-Adrenoceptor agonists and antagonists may have important applications in cardiovascular drug development. To assess α2-adrenoceptor-mediated drug effects on vascular smooth muscle contraction, we developed a cell-based assay for the quantitative determination of myosin light chain phosphorylation (pMLC20) in cultured A7r5 smooth muscle cells from rat aorta, transfected to express the human α2B-adrenoceptor (A7r5-α2B cell line).
  
  
  METHODS
  In a 96-well format, confluent and serum-starved cells (+/- inhibitor preincubation) were treated with receptor ligands for 5-120 s and the evoked pMLC20 response was monitored with a quantitative in-cell immunoassay, employing time-resolved fluorescence technology. Western blotting, immunofluorescent labelling and intracellular calcium concentration measurements were used for assay validation.
  
  
  RESULTS
  The α2-adrenoceptor agonist dexmedetomidine induced rapid, transient and dose-dependent (EC50 30-65 nM) myosin light chain phosphorylation, peaking at 20-45 s with an Emax value of approximately 60% over vehicle control. The endogenous agonist arginine vasopressin produced responses that were comparable to those evoked by dexmedetomidine. Blockers of α2-adrenoceptors, myosin light chain kinase, Gi-proteins, Gβγ subunits, L-type calcium channels and phospholipase C antagonized the dexmedetomidine-evoked myosin light chain phosphorylation, whereas blockers of protein kinase C and protein kinase A potentiated the response to dexmedetomidine.
  
  
  CONCLUSIONS
  The novel method is suitable as a ligand profiling tool to assess the capacity of ligands to evoke or inhibit vascular smooth muscle cell contraction and for investigating the intracellular pathways involved in this process. The assay now allows the quantitative determination of pMLC20 signal induction or inhibition in vascular smooth muscle cells and is superior to conventional Western blotting due to the reduced number of cells required and the potential for measurement of detailed time curves, multiple treatments and replicates on each plate.
authors.affiliation: Department of Pharmacology, Drug Development and Therapeutics, Institute of Biomedicine, University of Turku, Turku, Finland.
authors.forename: Susann
authors.initials: S
authors.lastname: Björk
authors.affiliation: Department of Pharmacology, Drug Development and Therapeutics, Institute of Biomedicine, University of Turku, Turku, Finland. Electronic address: anna.huhtinen@utu.fi.
authors.forename: Anna
authors.initials: A
authors.lastname: Huhtinen
authors.affiliation: Department of Pharmacology, Drug Development and Therapeutics, Institute of Biomedicine, University of Turku, Turku, Finland; Department of Biosciences, Åbo Akademi University, Turku, Finland.
authors.forename: Anne
authors.initials: A
authors.lastname: Vuorenpää
authors.affiliation: Department of Pharmacology, Drug Development and Therapeutics, Institute of Biomedicine, University of Turku, Turku, Finland; Unit of Clinical Pharmacology, Turku University Hospital, Turku, Finland.
authors.forename: Mika
authors.initials: M
authors.lastname: Scheinin
mesh_terms: D002478:Cells, Cultured; D020927:Dexmedetomidine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018994:Myosin Light Chains / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D018341:Receptors, Adrenergic, alpha-2 / Q000032:analysis* / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D018994:Myosin Light Chains; D018341:Receptors, Adrenergic, alpha-2; D020927:Dexmedetomidine
keywords: 96-well plate; Contraction; Functional assay; Methods; Myosin light chain phosphorylation; Quantitative immunostaining; Vascular smooth muscle cell; alpha2-Adrenoceptor