Phosphoinositide 3-kinase (PI3K)/Akt plays a pivotal role in the vascular response. The present study is to determine whether PI3K/Akt pathway in vascular smooth muscle cells is involved in nitroglycerin (NTG) tolerance and the underlying mechanism. Nitrate tolerance of porcine coronary arteries in vitro was induced by incubation of NTG (10(-5) M) for 24 h. Nitrate tolerance in vivo was obtained by subcutaneous injection of mice with NTG (20 mg kg(-1), tid, 3 days) and the aortas were used. Protein levels of total and phosphorylated Akt, forkhead box protein O1 (FoxO1), and cGMP-dependent protein kinase (PKG) were determined by western blot analysis. Isometric vessel tension was recorded by organ chamber technique. PKG mRNA was determined by real-time PCR. The cellular translocation of FoxO1 was observed by immunofluorescence. Reactive oxygen species (ROS) level was measured by DHE staining. The vascular relaxation to NTG was significantly inhibited in in vivo and in vitro NTG tolerant arteries. Meanwhile, the protein level of phosphorylated Akt at Ser473 was increased in the tolerant arteries. The attenuated relaxation and the augmented Akt-p were ameliorated by LY294002, a specific inhibitor of P13K. The protein and mRNA expression of PKG were significantly down-regulated in NTG tolerant arteries, which were reversed by LY294002. The level of phosphorylated FoxO1 at Ser256 and its translocation from the nucleus to the cytosol were both increased in NTG tolerance and were also inhibited by LY294002. ROS production was significantly increased in NTG tolerant arteries, which was not be affected by LY294002 but inhibited by N-acetyl-L-cysteine. In conclusion, the present study suggests that PI3K/Akt in vascular smooth muscle is involved in the development of NTG tolerance via inhibiting PKG transcription and the effect is mediated by FoxO1. (C) 2018 Elsevier Inc. All rights reserved.