title: Ca2+-dependent rapid Ca2+ sensitization of contraction in arterial smooth muscle.
abstract: Ca(2+) ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca(2+) with calmodulin activates myosin light chain (MLC) kinase to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase is involved in the rapid development of MLC phosphorylation and contraction during Ca(2+) transient, we compared Ca(2+) signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during alpha(1) agonist-induced contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from a negligible amount to a peak value of 0.38+/-0.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca(2+) rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca(2+) release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca(2+)-dependent protein kinase C. This was followed by a slow Ca(2+)-independent and Rho-kinase/protein kinase C-dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.29+/-0.09 at rest to the peak of 0.68+/-0.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca(2+) sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca(2+) rise induces a rapid inhibition of MLC phosphatase coincident with the Ca(2+)-induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content.
authors.affiliation: Boston Biomedical Research Institute, 64 Grove St, Watertown, MA 02472, USA.
authors.forename: George J
authors.initials: GJ
authors.lastname: Dimopoulos
authors.forename: Shingo
authors.initials: S
authors.lastname: Semba
authors.forename: Kazuyo
authors.initials: K
authors.lastname: Kitazawa
authors.forename: Masumi
authors.initials: M
authors.lastname: Eto
authors.forename: Toshio
authors.initials: T
authors.lastname: Kitazawa
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling; D005263:Femoral Artery / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors; D009124:Muscle Proteins / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000502:physiology*; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D010656:Phenylephrine / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D021122:Protein Subunits / Q000378:metabolism; D011817:Rabbits; D013997:Time Factors; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology*; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D054460:rho-Associated Kinases
chemical_list: D000316:Adrenergic alpha-Agonists; C491521:CPI-17 protein, Oryctolagus cuniculus; D002121:Calcium Channel Blockers; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D018994:Myosin Light Chains; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D021122:Protein Subunits; D014662:Vasoconstrictor Agents; D010656:Phenylephrine; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; D002118:Calcium