|Tongxinluo reduces myocardial no-reflow and ischemia-reperfusion injury by stimulating the phosphorylation of eNOS via the PKA pathway. |
Li, XD; Yang, YJ; Geng, YJ; Jin, C; Hu, FH; Zhao, JL; Zhang, HT; Cheng, YT; Qian, HY; Wang, LL; Zhang, BJ; Wu, YL
American journal of physiology. Heart and circulatory physiology
The objective of the present study was to investigate whether pretreatment with single low loading dose of tongxinluo (TXL), a traditional Chinese medicine, 1 h before myocardial ischemia could attenuate no-reflow and ischemia-reperfusion injury by regulating endothelial nitric oxide synthase (eNOS) via the PKA pathway. In a 90-min ischemia and 3-h reperfusion model, minipigs were randomly assigned to the following groups: sham, control, TXL (0.05 g/kg, gavaged 1 h before ischemia), TXL + H-89 (a PKA inhibitor, intravenously infused at a dose of 1.0 μg·kg(-1)·min(-1) 30 min before ischemia), and TXL + N(ω)-nitro-L-arginine (L-NNA; an eNOS inhibitor, intravenously administered at a dose of 10 mg/kg 30 min before ischemia). TXL decreased creatine kinase (CK) activity (P less than 0.05) and reduced the no-reflow area from 48.6% to 9.5% and infarct size from 78.5% to 59.2% (P less than 0.05), whereas these effects of TXL were partially abolished by H-89 and completely reversed by L-NNA. TXL elevated PKA activity and the expression of PKA, Thr(198) phosphorylated PKA, Ser(1179) phosphorylated eNOS, and Ser(635) phosphorylated eNOS in the ischemic myocardium. H-89 repressed the TXL-induced enhancement of PKA activity and phosphorylation of eNOS at Ser(635), and L-NNA counteracted the phosphorylation of eNOS at Ser(1179) and Ser(635) without an apparent influence on PKA activity. In conclusion, pretreatment with a single low loading dose of TXL 1 h before ischemia reduces myocardial no-reflow and ischemia-reperfusion injury by upregulating the phosphorylation of eNOS at Ser(1179) and Ser(635), and this effect is partially mediated by the PKA pathway.
|Src kinase activates endothelial nitric-oxide synthase by phosphorylating Tyr-83. |
Fulton, D; Church, JE; Ruan, L; Li, C; Sood, SG; Kemp, BE; Jennings, IG; Venema, RC
The Journal of biological chemistry
The endothelial nitric-oxide synthase (eNOS) is regulated in part by serine/threonine phosphorylation, but eNOS tyrosine phosphorylation is less well understood. In the present study we have examined the tyrosine phosphorylation of eNOS in bovine aortic endothelial cells (BAECs) exposed to oxidant stress. Hydrogen peroxide and pervanadate (PV) treatment stimulates eNOS tyrosine phosphorylation in BAECs. Phosphorylation is blocked by the Src kinase family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Moreover, eNOS and c-Src can be coimmunoprecipitated from BAEC lysates by antibodies directed against either protein. Domain mapping and site-directed mutagenesis studies in COS-7 cells transfected with either eNOS alone and then treated with PV or cotransfected with eNOS and constitutively active v-Src identified Tyr-83 (bovine sequence) as the major eNOS tyrosine phosphorylation site. Tyr-83 phosphorylation is associated with a 3-fold increase in basal NO release from cotransfected cells. Furthermore, the Y83F eNOS mutation attenuated thapsigargin-stimulated NO production. Taken together, these data indicate that Src-mediated tyrosine phosphorylation of eNOS at Tyr-83 modulates eNOS activity in endothelial cells.
|Identification of regulatory sites of phosphorylation of the bovine endothelial nitric-oxide synthase at serine 617 and serine 635. |
Michell, Belinda J, et al.
J. Biol. Chem., 277: 42344-51 (2002)
Endothelial nitric-oxide synthase (eNOS) is regulated by signaling pathways involving multiple sites of phosphorylation. The coordinated phosphorylation of eNOS at Ser(1179) and dephosphorylation at Thr(497) activates the enzyme, whereas inhibition results when Thr(497) is phosphorylated and Ser(1179) is dephosphorylated. We have identified two further phosphorylation sites, at Ser(617) and Ser(635), by phosphopeptide mapping and matrix-assisted laser desorption ionization time of flight mass spectrometry. Purified protein kinase A (PKA) phosphorylates both sites in purified eNOS, whereas purified Akt phosphorylates only Ser(617). In bovine aortic endothelial cells, bradykinin (BK), ATP, and vascular endothelial growth factor stimulate phosphorylation of both sites. BK-stimulated phosphorylation of Ser(617) is Ca(2+)-dependent and is partially inhibited by LY294002 and wortmannin, phosphatidylinositol 3-kinase inhibitors, suggesting signaling via Akt. BK-stimulated phosphorylation of Ser(635) is Ca(2+)-independent and is completely abolished by the PKA inhibitor, KT5720, suggesting signaling via PKA. Activation of PKA with isobutylmethylxanthine also causes Ser(635), but not Ser(617), phosphorylation. Mimicking phosphorylation at Ser(635) by Ser to Asp mutation results in a greater than 2-fold increase in activity of the purified protein, whereas mimicking phosphorylation at Ser(617) does not alter maximal activity but significantly increases Ca(2+)-calmodulin sensitivity. These data show that phosphorylation of both Ser(617) and Ser(635) regulates eNOS activity and contributes to the agonist-stimulated eNOS activation process.