Investigation of signaling pathways that mediate the inotropic effect of urotensin-II in human heart.
OBJECTIVE
This study investigated signaling pathways that may contribute to the potent positive inotropic effect of human urotensin-II (hU-II) in human isolated right atrial trabeculae obtained from patients with coronary artery disease.


METHODS
Trabeculae were set up in tissue baths and stimulated to contract at 1 Hz. Tissues were incubated with 20 nM hU-II with or without phorbol 12-myristate 13-acetate (PMA, 10 microM) to desensitize PKC, the PKC inhibitor chelerythrine (10 microM), 10 microM 4alpha-phorbol that does not desensitize PKC, the myosin light chain kinase inhibitor wortmannin (50 nM, 10 microM), or the Rho kinase inhibitor Y-27632 (0.1-10 microM). Activated RhoA was determined by affinity immunoprecipitation, and phosphorylation of signaling proteins was determined by SDS-PAGE.


RESULTS
hU-II caused a potent positive inotropic response in atrial trabeculae, and this was concomitant with increased phosphorylation of regulatory myosin light chain (MLC-2, 1.8+/-0.4-fold, P<0.05, n=6) and PKCalpha/betaII (1.4+/-0.2-fold compared to non-stimulated controls, P<0.05, n=7). Pretreatment of tissues with PMA caused a marked reduction in the inotropic effect of hU-II, but did not affect hU-II-mediated phosphorylation of MLC-2. The inotropic response was inhibited by chelerythrine, but not 4alpha-phorbol or wortmannin. Although Y-27632 also reduced the positive inotropic response to hU-II, this was associated with a marked reduction in basal force of contraction. RhoA.GTP was immunoprecipitated in tissues pretreated with or without hU-II, with findings showing no detectable activation of RhoA in the agonist stimulated tissues.


CONCLUSIONS
The findings indicated that hU-II increased force of contraction in human heart via a PKC-dependent mechanism and increased phosphorylation of MLC-2, although this was independent of PKC. The positive inotropic effect was independent of myosin light chain kinase and RhoA-Rho kinase signaling pathways.
Department of Medicine, University of Queensland, The Prince Charles Hospital, Rode Road Chermside, 4032 Queensland, Australia. russell@medicine.uq.edu.au
Fraser D
FD
Russell
Peter
P
Molenaar
D000470:Alkaloids; D000577:Amides / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D053119:Benzophenanthridines; D019332:Endothelin-1 / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D009200:Myocardial Contraction / Q000187:drug effects*; D009206:Myocardium / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors; D010617:Phenanthridines / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology*; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014579:Urotensins / Q000494:pharmacology*; D000077191:Wortmannin; D054460:rho-Associated Kinases
D000470:Alkaloids; D000577:Amides; D000730:Androstadienes; D053119:Benzophenanthridines; D019332:Endothelin-1; D047908:Intracellular Signaling Peptides and Proteins; D010617:Phenanthridines; D011725:Pyridines; D014579:Urotensins; C108830:Y 27632; C029677:urotensin II; C016299:chelerythrine; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D009219:Myosin-Light-Chain Kinase; D013755:Tetradecanoylphorbol Acetate; D000077191:Wortmannin