The MLCK-mediated alpha1-adrenergic inotropic effect in atrial myocardium is negatively modulated by PKCepsilon signaling.
The present study examined the role of myosin light chain kinase (MLCK), PKC isozymes, and inositol 1,4,5-trisphosphate (IP(3)) receptor in the positive inotropic effect of alpha(1)-adrenergic stimulation in atrial myocardium. We measured inotropic effects of phenylephrine (0.3-300 microM) in isolated left atrial preparations (1 Hz, 37 degrees C, 1.8 mM Ca(2+), 0.3 microM nadolol) from male 8-week FVB mice (n=200). Phenylephrine concentration-dependently increased force of contraction from 1.5+/-0.1 to 2.8+/-0.1 mN (mean+/-s.e.m., n=42), which was associated with increased MLC-2a phosphorylation at serine 21 and 22 by 67% and translocation of PKCepsilon but not PKCalpha to membrane (+30%) and myofilament (+50%) fractions.MLCK inhibition using ML-7 or wortmannin right-shifted the concentration-response curve of phenylephrine, reducing its inotropic effect at 10 microM by 73% and 81%, respectively. The compound KIE1-1 (500 nM), an intracellularly acting PKCepsilon translocation inhibitor peptide, prevented PKCepsilon translocation and augmented the maximal inotropic effect of phenylephrine by 40%. In contrast, inhibition of Ca(2+)-dependent PKC translocation (KIC1-1, 500 nM) had no effect. Chelerythrine, a PKC inhibitor, decreased basal force without changing the inotropic effect of phenylephrine. The IP(3) receptor blocker 2-APB (2 and 20 microM) concentration-dependently decreased basal force, but did not affect the concentration-response curve of phenylephrine. These results indicate that activation of MLCK is required for the positive inotropic effect of alpha(1)-adrenergic stimulation, that the Ca(2+)-independent PKCepsilon negatively modulates this effect, and that PKCalpha and IP(3) receptor activation is not involved.
Grimm
Michael
M
Institute of Experimental and Clinical Pharmacology and Toxicology, University Medical Center, Hamburg, Germany.
Mahnecke
Nina
N
Soja
Friederike
F
El-Armouche
Ali
A
Haas
Pascal
P
Treede
Hendrik
H
Reichenspurner
Hermann
H
Eschenhagen
Thomas
T
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002316:Cardiotonic Agents / Q000494:pharmacology; D006321:Heart / Q000187:drug effects*; D006325:Heart Atria; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D051379:Mice; D009200:Myocardial Contraction / Q000187:drug effects*; D009219:Myosin-Light-Chain Kinase / Q000502:physiology*; D010656:Phenylephrine / Q000494:pharmacology; D010766:Phosphorylation; D051744:Protein Kinase C-epsilon / Q000502:physiology*; D018340:Receptors, Adrenergic, alpha-1 / Q000502:physiology*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013347:Subcellular Fractions / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000318:Adrenergic beta-Agonists; D002316:Cardiotonic Agents; D018340:Receptors, Adrenergic, alpha-1; D010656:Phenylephrine; D015544:Inositol 1,4,5-Trisphosphate; D051744:Protein Kinase C-epsilon; D009219:Myosin-Light-Chain Kinase; D007545:Isoproterenol; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium