br Results and discussion The effects of hypoxia a
Results and discussion The effects of hypoxia, a combination of shear stress and hyperoxia, and a combination of shear stress and hypoxia in BAECs were assessed at 0, 15, 30, 60, 120, and 180 min. Changes in phosphorylation levels of eNOS at Ser635 were investigated using immunoblotting, and representative immunoblotting images are shown for all treatment conditions and times in Fig. 1. These data show differing band intensities of Ser635-phosphorylated eNOS between treatment groups and variations with exposure times. Fig. 2 shows phosphorylation levels of eNOS at Ser635 under hypoxia (A), a combination of shear stress and hyperoxia (B), and a combination of shear stress and hypoxia (C). Relative to baseline conditions, phosphorylated eNOS protein lactone levels increased after 180 min under all conditions. Following exposures to hypoxia (Fig. 2A), phosphorylation of eNOS at Ser635 was not increased significantly after 15 min compared with baseline, and was increased significantly after 30 min. As the previous study  speculated, exposures to a combination of shear stress and hyperoxia (Fig. 2B) led to increase in eNOS phosphorylation at Ser635, which was detected after only 15 min (2.15-fold), and significant 2.23- and 2.75-fold increases were observed at 30 and 60 min, respectively. Phosphorylation were still 2.64 and 2.46 times higher at 120 and 180 min than those at baseline conditions, respectively. Under a combination of shear stress and hypoxia (Fig. 2C), phosphorylation at Ser635 tended to increase with time, and the highest Ser635 phosphorylation levels were observed at 60 min and were significantly greater (2.44-fold) than those under baseline conditions. At 15, 30, 120, and 180 min, phosphorylation levels were 1.71-, 1.87-, 1.91-, and 2.29-fold greater than at baseline. The phosphorylation at Ser635 was significantly greater at 30, 60, 180 min than at 15 min under hypoxia (Fig. 2A), however the significant increase was only found compared with baseline under a combination of shear stress and hyperoxia (Fig. 2B) and a combination of shear stress and hypoxia (Fig. 2C). The maximum increase was 1.94-fold at 180 min under hypoxia, on the other hand was 2.75- and 2.44- fold at 60 min under a combination of shear stress and hyperoxia and a combination of shear stress and hypoxia, respectively. Phosphorylation at Ser635 may play an important chronic regulation , and we speculate the differential mechanisms by hypoxia and shear stress may contribute the choric regulation of phosphorylation at Ser635. Additionally, taken with these observations, the data described above indicates that increases in phosphorylation levels are greater after 15-min shear stress and hyperoxia than after exposures to hypoxia only or a combination of shear stress and hypoxia. Combination treatment of shear stress and hypoxia induced phosphorylation within 15 min. However, although there were no significant differences in eNOS phosphorylation at Ser635 between a combination of shear stress and hyperoxia and a combination of shear stress and hypoxia all time points, the resulting phosphorylation levels tended to be lower under a combination of shear stress and hypoxia than those under a combination of shear stress and hyperoxia, indicating that hypoxia inhibits shear stress-mediated phosphorylation of eNOS at Ser635. Boo et al. indicated that phosphorylation of eNOS at Ser635 and Ser1179 in response to shear stress is regulated by the following two mechanisms, 1) protein kinase A (PKA)-dependent mechanisms but also follows phosphosinositide-3-kinase (PI3K)-independent pathways, and 2) PKA- and PI3K-dependent pathways . Similarly, Chen et al. reported that AMP-activated protein kinase (AMPK) phosphorylates Ser635 . Additionally, extracellular signal-regulated kinase (ERK) 1/2 was also proposed [19,20], and this signaling pathway was also activated by shear stress [21,22]. On the other hand, hypoxia activates Pim kinase 1 (Pim1) , which was reported to be a novel kinase that mediates the phosphorylation of eNOS at Ser635 . Considering our results, the aforementioned studies warrant speculation that Pim1 inhibits PKA, AMPK, and/or ERK1/2. This hypothesis is further supported by data showing that AMPK and ERK1/2 were activated by a Pim1 inhibitor, suggesting that Pim1 negatively regulates AMPK and ERK1/2 [24,25]. On the other hand, phosphorylation at Ser635 by PKA was not influenced by the Pim1 inhibitor , indicating that PKA may be independent of Pim1. Therefore, simultaneous exposures to shear stress and hypoxia likely induce phosphorylation of eNOS at Ser635 and lead to increase NO production, and additive effects may be abolished due to hypoxic inhibition of AMPK and ERK1/2 signaling pathways by Pim1.