title: Key role of myosin light chain (MLC) kinase-mediated MLC2a phosphorylation in the alpha 1-adrenergic positive inotropic effect in human atrium.
abstract: OBJECTIVE
  Mechanisms of the positive inotropic response to alpha(1)-adrenergic stimulation in the heart remain poorly understood, but recent evidence in rat papillary muscle suggests an important role of regulatory myosin light chain (MLC2) phosphorylation. This study investigated alpha(1)-adrenergic contractile effects and the role of MLC kinase (MLCK)-dependent phosphorylation of MLC2 in human atrial muscle strips.
  
  
  METHODS
  Force measurement was performed on electrically stimulated atrial muscle strips (n=140; 20 hearts) in the presence of the beta-blocker nadolol. MLC2a phosphorylation was determined by 2D-polyacrylamide gel electrophoresis and Western blotting of muscle strips that were immediately freeze-clamped following force measurements.
  
  
  RESULTS
  The alpha(1)-agonist phenylephrine (PE; 0.3-100 microM) exerted a concentration-dependent, monophasic, sustained positive inotropic effect (86% above basal) that was accompanied by an 80% increase in MLC2a phosphorylation. Desinhibition of MLC phosphatase by the Rho kinase inhibitor Y-27632 (10 microM) reduced the effect of PE by 16%. The MLCK inhibitor wortmannin (10 microM) completely abolished both the PE-induced increase in force and MLC2a phosphorylation. The structurally unrelated MLCK inhibitor ML-7 (10 microM) had similar effects. Neither Y-27632 nor wortmannin or ML-7 affected beta-adrenergic force stimulation. In contrast to our findings in atrial muscle strips, we observed no increase in MLC2v phosphorylation after PE in human ventricular muscle strips and wortmannin failed to inhibit PE-induced force of contraction.
  
  
  CONCLUSIONS
  alpha(1)-Adrenergic receptors mediate a prominent increase in contractile force in human atria that depends on MLCK activity and is accompanied by an increase in MLC2 phosphorylation.
authors.affiliation: Institute of Experimental and Clinical Pharmacology, University Hospital Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Grimm
authors.forename: Pascal
authors.initials: P
authors.lastname: Haas
authors.forename: Birthe
authors.initials: B
authors.lastname: Willipinski-Stapelfeldt
authors.forename: Wolfram-Hubertus
authors.initials: WH
authors.lastname: Zimmermann
authors.forename: Thomas
authors.initials: T
authors.lastname: Rau
authors.forename: Klaus
authors.initials: K
authors.lastname: Pantel
authors.forename: Michael
authors.initials: M
authors.lastname: Weyand
authors.forename: Thomas
authors.initials: T
authors.lastname: Eschenhagen
mesh_terms: D000293:Adolescent; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology*; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000328:Adult; D000368:Aged; D000704:Analysis of Variance; D000730:Androstadienes / Q000494:pharmacology; D001381:Azepines / Q000494:pharmacology; D024722:Cardiac Myosins / Q000378:metabolism*; D002648:Child; D002675:Child, Preschool; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006325:Heart Atria; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D066298:In Vitro Techniques; D008297:Male; D018613:Microscopy, Confocal; D008875:Middle Aged; D009200:Myocardial Contraction / Q000187:drug effects; D009206:Myocardium / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009248:Nadolol / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology; D010656:Phenylephrine / Q000494:pharmacology; D010766:Phosphorylation; D011224:Prazosin / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013268:Stimulation, Chemical; D000077191:Wortmannin
chemical_list: D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D000319:Adrenergic beta-Antagonists; D000730:Androstadienes; D001381:Azepines; D004791:Enzyme Inhibitors; D018994:Myosin Light Chains; D009281:Naphthalenes; D047428:Protein Kinase Inhibitors; C107184:myosin light chain 2; C070571:ML 7; D010656:Phenylephrine; D009248:Nadolol; D009219:Myosin-Light-Chain Kinase; D024722:Cardiac Myosins; D011224:Prazosin; D000077191:Wortmannin