Limits...
Nafamostat mesilate promotes endothelium-dependent vasorelaxation via the Akt-eNOS dependent pathway

View Article: PubMed Central - PubMed

ABSTRACT

Nafamostat mesilate (NM), a synthetic serine protease inhibitor, has anticoagulant and anti-inflammatory properties. The intracellular mediator and external anti-inflammatory external signal in the vascular wall have been reported to protect endothelial cells, in part due to nitric oxide (NO) production. This study was designed to examine whether NM exhibit endothelium dependent vascular relaxation through Akt/endothelial nitric oxide synthase (eNOS) activation and generation of NO. NM enhanced Akt/eNOS phosphorylation and NO production in a dose- and time-dependent manner in human umbilical vein endothelial cells (HUVECs) and aorta tissues obtained from rats treated with various concentrations of NM. NM concomitantly decreased arginase activity, which could increase the available arginine substrate for NO production. Moreover, we investigated whether NM increased NO bioavailability and decreased aortic relaxation response to an eNOS inhibitor in the aorta. These results suggest that NM increases NO generation via the Akt/eNOS signaling pathway, leading to endothelium-dependent vascular relaxation. Therefore, the vasorelaxing action of NM may contribute to the regulation of cardiovascular function.

No MeSH data available.


Nafamostat mesilate (NM) stimulates phosphorylation of Akt/endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO) ex vivo.(A) Aorta segments were treated with various concentrations (1~100 µg/ml) of NM for 2 h then harvested for western blot analysis of p-eNOS and p-Akt. Total forms of the proteins were included as a loading control. The levels of p-eNOS and p-Akt were quantified by densitometric analyses (B and C). All western blots are representative of three independent experiments. The data are presented as the means±SEM of three independent experiments. *p<0.05 compared with the control. (D) Aorta segments were treated with various concentrations (1~100 µg/ml) of NM for 2 h, stained with DAF dye, and analyzed by microscopy. Images (a, b, c, d) were captured using 100 × magnification. Images (e, f, g, h) were captured using 200 × magnification. Scale bar=40 µm. Corresponding graphs for the relative fluorescence intensity are represented in (E). (F) Rats were treated intravenously with various concentrations (1~3 µg/ml) of NM for 2 h then measured NO production in the plasma. Bars represent means±standard error (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5015001&req=5

Figure 3: Nafamostat mesilate (NM) stimulates phosphorylation of Akt/endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO) ex vivo.(A) Aorta segments were treated with various concentrations (1~100 µg/ml) of NM for 2 h then harvested for western blot analysis of p-eNOS and p-Akt. Total forms of the proteins were included as a loading control. The levels of p-eNOS and p-Akt were quantified by densitometric analyses (B and C). All western blots are representative of three independent experiments. The data are presented as the means±SEM of three independent experiments. *p<0.05 compared with the control. (D) Aorta segments were treated with various concentrations (1~100 µg/ml) of NM for 2 h, stained with DAF dye, and analyzed by microscopy. Images (a, b, c, d) were captured using 100 × magnification. Images (e, f, g, h) were captured using 200 × magnification. Scale bar=40 µm. Corresponding graphs for the relative fluorescence intensity are represented in (E). (F) Rats were treated intravenously with various concentrations (1~3 µg/ml) of NM for 2 h then measured NO production in the plasma. Bars represent means±standard error (n=3).

Mentions: To identify the physiological relevance of NM on eNOS and vascular function, we first examined whether the phosphorylation of NOS and Akt in aortic rings treated with NM was activated. Aortas from SD rats were obtained, cut into segments and incubated with various doses of NM for 2 h, then analyzed by western blot analysis. As shown in Fig. 3A, phosphorylation of both eNOS and Akt increased with increasing dose of NM, similar to the in vitro results.


Nafamostat mesilate promotes endothelium-dependent vasorelaxation via the Akt-eNOS dependent pathway
Nafamostat mesilate (NM) stimulates phosphorylation of Akt/endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO) ex vivo.(A) Aorta segments were treated with various concentrations (1~100 µg/ml) of NM for 2 h then harvested for western blot analysis of p-eNOS and p-Akt. Total forms of the proteins were included as a loading control. The levels of p-eNOS and p-Akt were quantified by densitometric analyses (B and C). All western blots are representative of three independent experiments. The data are presented as the means±SEM of three independent experiments. *p<0.05 compared with the control. (D) Aorta segments were treated with various concentrations (1~100 µg/ml) of NM for 2 h, stained with DAF dye, and analyzed by microscopy. Images (a, b, c, d) were captured using 100 × magnification. Images (e, f, g, h) were captured using 200 × magnification. Scale bar=40 µm. Corresponding graphs for the relative fluorescence intensity are represented in (E). (F) Rats were treated intravenously with various concentrations (1~3 µg/ml) of NM for 2 h then measured NO production in the plasma. Bars represent means±standard error (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5015001&req=5

Figure 3: Nafamostat mesilate (NM) stimulates phosphorylation of Akt/endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO) ex vivo.(A) Aorta segments were treated with various concentrations (1~100 µg/ml) of NM for 2 h then harvested for western blot analysis of p-eNOS and p-Akt. Total forms of the proteins were included as a loading control. The levels of p-eNOS and p-Akt were quantified by densitometric analyses (B and C). All western blots are representative of three independent experiments. The data are presented as the means±SEM of three independent experiments. *p<0.05 compared with the control. (D) Aorta segments were treated with various concentrations (1~100 µg/ml) of NM for 2 h, stained with DAF dye, and analyzed by microscopy. Images (a, b, c, d) were captured using 100 × magnification. Images (e, f, g, h) were captured using 200 × magnification. Scale bar=40 µm. Corresponding graphs for the relative fluorescence intensity are represented in (E). (F) Rats were treated intravenously with various concentrations (1~3 µg/ml) of NM for 2 h then measured NO production in the plasma. Bars represent means±standard error (n=3).
Mentions: To identify the physiological relevance of NM on eNOS and vascular function, we first examined whether the phosphorylation of NOS and Akt in aortic rings treated with NM was activated. Aortas from SD rats were obtained, cut into segments and incubated with various doses of NM for 2 h, then analyzed by western blot analysis. As shown in Fig. 3A, phosphorylation of both eNOS and Akt increased with increasing dose of NM, similar to the in vitro results.

View Article: PubMed Central - PubMed

ABSTRACT

Nafamostat mesilate (NM), a synthetic serine protease inhibitor, has anticoagulant and anti-inflammatory properties. The intracellular mediator and external anti-inflammatory external signal in the vascular wall have been reported to protect endothelial cells, in part due to nitric oxide (NO) production. This study was designed to examine whether NM exhibit endothelium dependent vascular relaxation through Akt/endothelial nitric oxide synthase (eNOS) activation and generation of NO. NM enhanced Akt/eNOS phosphorylation and NO production in a dose- and time-dependent manner in human umbilical vein endothelial cells (HUVECs) and aorta tissues obtained from rats treated with various concentrations of NM. NM concomitantly decreased arginase activity, which could increase the available arginine substrate for NO production. Moreover, we investigated whether NM increased NO bioavailability and decreased aortic relaxation response to an eNOS inhibitor in the aorta. These results suggest that NM increases NO generation via the Akt/eNOS signaling pathway, leading to endothelium-dependent vascular relaxation. Therefore, the vasorelaxing action of NM may contribute to the regulation of cardiovascular function.

No MeSH data available.