Arachidonic acid-dependent phosphorylation of troponin I and myosin light chain 2 in cardiac myocytes.
Recent evidence has suggested that arachidonic acid (AA) may be an important signaling molecule in cardiac excitation-contraction coupling. We previously showed that AA and endothelin-1 (ET) inhibit distinct K+ channels via protein kinase C-dependent pathways in rat ventricular myocytes. In addition, we demonstrated that Ca2+ transients in populations of fura 2-loaded myocytes were potentiated by AA and ET via activation of protein kinase C. In this study, we have used suspensions of [32P]orthophosphate (32Pi)-labeled rat ventricular myocytes to study the effects of AA and ET at the level of the myofilaments. After a 10-minute incubation of the labeled cells with phorbol 12-myristate 13-acetate (PMA), AA, or ET in the presence or absence of the protein kinase C inhibitor calphostin C, the myofibrillar proteins were separated by PAGE. Measurement of unloaded cell shortening using video edge detection in single electrically stimulated myocytes was also used to assess the effects of AA and ET on myocyte contractility. Incubation with either PMA, AA, or ET resulted in similar increases in 32Pi incorporation into troponin I (TnI) and myosin light chain 2 (MLC2), which was inhibited by preincubation with the protein kinase C antagonist calphostin C. In addition, the ability of these agonists to stimulate phosphorylation of TnI or MLC2 did not require extracellular Ca2+ or intact intracellular Ca2+ stores. The effects of AA and ET together on phosphorylation of TnI or MLC2 were not additive.(ABSTRACT TRUNCATED AT 250 WORDS)
Damron
D S
DS
Center for Anesthesiology Research, Cleveland Clinic Foundation, OH 44195, USA.
Darvish
A
A
Murphy
L
L
Sweet
W
W
Moravec
C S
CS
Bond
M
M
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism* / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D016232:Endothelins / Q000494:pharmacology; D004789:Enzyme Activation; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects; D066298:In Vitro Techniques; D008387:Marine Toxins; D009200:Myocardial Contraction; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D009210:Myofibrils / Q000187:drug effects / Q000378:metabolism; D009218:Myosins / Q000032:analysis / Q000378:metabolism*; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014336:Troponin / Q000032:analysis / Q000378:metabolism*; D019210:Troponin I
D016232:Endothelins; D008387:Marine Toxins; D010080:Oxazoles; D014336:Troponin; D019210:Troponin I; D016718:Arachidonic Acid; C059041:calyculin A; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D009218:Myosins; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium