Abstract P220: UrocortinII Increases eNOS-mediated NO Production in Rabbit Ventricular Myocytes via Both PKA- and Akt-dependent Pathways
AIM: Urocortin II (UcnII) exerts beneficial and cardioprotective effects in heart failure. The cellular signaling pathways underlying these effects are unclear. We tested the hypothesis that part of the UcnII-induced effects are mediated via stimulation of eNOS and increased cellular NO signaling.
METHODS: Isolated rabbit ventricular myocytes were loaded with the NO dye DAF-FM (5μM, 30min). UcnII-induced changes in NO production were measured as changes in DAF-FM fluorescence in electrically paced myocytes (0.5Hz, RT) by means of confocal microscopy. Unloaded cell shortening was measured simultaneously as fractional shortening (FS) of resting cell length. Phosphorylation of Akt and eNOS was determined as the ratio of phosphorylated to total protein using Western Blotting.
RESULTS: UcnII (100nM) increased DAF-FM fluorescence by 41±5% and FS by 42±6% (n=9–20, P<0.01). The inhibitors of eNOS, L-NIO (10μM) and L-NAME (1mM), decreased the NO production and the FS by ~31% and ~33% and by ~33% and ~43% (n=4–8, P<0.05), respectively. UcnII increased Akt phosphorylation at Ser473 by 79±19% and at Thr308 by 60±19% (n=9–13, both P<0.05) and phosphorylation of eNOS at Ser1177 by 89±34% (n=20, P<0.05). Wortmannin (300nM) or LY294002 (10μM), both inhibitors of PI3K, almost completely reduced UcnII-induced phosphorylation of Akt and eNOS, respectively (n=9–16, P<0.05). UcnII-induced Akt phosphorylation was not affected by H89 (5μM, PKA inhibitor, n=15–16). However, UcnII-induced phosphorylation of eNOS was reduced by H89 by ~83% (n=14, P<0.05). Elevation of cAMP by forskolin (10μM) also increased eNOS phosphorylation at Ser1177 (n=5, P<0.05), but did not affect Akt phosphorylation (n=8). These data suggest that both PKA and Akt may phosphorylate eNOS at Ser1177 and thereby stimulate NO production. In line with this notion, the combined use of H89 (1μM) and LY294002 (10μM) reduced the UcnII-induced NO production and FS by ~20% and ~51%, respectively (n=10, P<0.05).
SUMMARY: In isolated rabbit ventricular myocytes UcnII stimulates eNOS-mediated NO production via both PKA- and PI3K-Akt-dependent phosphorylation of eNOS. Increased cardiomyocyte NO signaling may contribute to the beneficial effects of UcnII in heart failure.