AIM: Urocortin II (UcnII) exerts beneficial effects in heart failure. In cardiac myocytes, UcnII exerts positive inotropic and lusitropic effects through a PKA-dependent pathway. We tested the hypothesis that, in addition, UcnII stimulates endothelial NO synthase (eNOS) and evaluated the underlying signaling pathways and mechanisms.METHODS: UcnII-induced phosphorylation of Akt and eNOS was measured using phospho-specific antibodies. Isolated cardiac myocytes were loaded with 5x10-6M DAF-FM and UcnII-induced changes in NO production were assessed by changes in DAF-FM fluorescence in electrically paced myocytes (0.5 Hz, room temperature) by means of confocal microscopy.RESULTS: In rabbit ventricular myocytes, UcnII caused increases in phosphorylation of Akt at Ser473 (+89.4±21.4%) and Thr308 (+60.4±39.7%) and phosphorylation of eNOS at Ser1177 (+49.6±25.9%; n=6-11, all P<0.05 vs untreated controls). Wortmannin (300nM) and LY294002 (10-5M), inhibitors of PI3K, largely reduced UcnII-induced phosphorylation of Akt and eNOS, respectively (n=11-20, P<0.05). Cellular NO production increased by 41.1±5.5% (n=20, P<0.01), which was inhibited by eNOS inhibitors, L-NIO (10-5M) and L-NAME (1mM) by ∼30% (n=4-8, P<0.05). Inhibition of PKA by H89 (5x10-6M) also reduced eNOS phosphorylation (n=11, P<0.05) but did not affect Akt phosphorylation. Direct stimulation of cAMP/PKA signaling via forskolin (10-5M) increased phosphorylation of eNOS (n=5, P<0.05) but not of Akt. When both PI3K/Akt (LY294002 10-5M) and cAMP/PKA (H89 10-6M) signaling were inhibited, the UcnII-induced increase of [NO]i was attenuated by ∼20% (n=10, P<0.01). UcnII also increased [NO]i in mouse, rat, and human ventricular myocytes as well as in rabbit atrial myocytes (n=4-12).SUMMARY: We conclude that, in cardiac myocytes, UcnII causes eNOS phosphorylation to stimulate cellular NO production via both cAMP/PKA and PI3K/Akt signaling.