Korean J Physiol Pharmacol.  2016 Sep;20(5):539-545. 10.4196/kjpp.2016.20.5.539.

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

• Choi S
• Kwon HJ
• Song HJ
• Choi SW
• Nagar H
• Piao S
• Jung SB
• Jeon BH
• Kim DW
• Kim CS

• KMID: 2350511
• DOI: http://doi.org/10.4196/kjpp.2016.20.5.539

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.

Keyword

Endothelial nitric oxide synthase; Nafamostat mesilate; Nitric oxide; Vascular relaxation

MeSH Terms

Animals
Aorta
Arginase
Arginine
Biological Availability
Endothelial Cells
Endothelium
Human Umbilical Vein Endothelial Cells
Mesylates*
Nitric Oxide
Nitric Oxide Synthase
Nitric Oxide Synthase Type III
Phosphorylation
Rats
Relaxation
Serine Proteases
Vasodilation*
Arginase
Arginine
Mesylates
Nitric Oxide
Nitric Oxide Synthase
Nitric Oxide Synthase Type III
Serine Proteases

Figure

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  Fig. 1 Nafamostat mesilate (NM) dose- and time-dependently stimulates phosphorylation of endothelial nitric oxide synthase (eNOS) and Akt in human umbilical vein endothelial cells (HUVECs).(A) Dose dependent effect of NM on eNOS and Akt phosphorylation in HUVECs. Cells were treated with various concentrations (10~1000 ng/ml) of NM for 2 h then harvested for western blot analysis of p-eNOS and p-Akt. The levels of p-eNOS and p-Akt were quantified by densitometric analyses (B and C). (D) Time dependent effect of NM on eNOS and Akt phosphorylation in HUVECs. Cells were treated with 300 ng/mL of NM for various time points starting from 5 min up to 2 h followed by harvesting the cells for western blot analysis. Total forms of the proteins were included as a loading control. The levels of p-eNOS and p-Akt were quantified by densitometric analyses (E and F). 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 control cells.

• 

  Fig. 2 Nafamostat mesilate (NM) stimulates the production of nitric oxide and inhibits arginase activity in human umbilical vein endothelial cells (HUVECs).(A) NO production in HUVECs treated with various concentrations (10~1000 ng/ml) of NM for 2 h. (B) Arginase activity measured in HUVECs treated with various concentrations (30~1000 ng/mL) of NM for 2 h. Bars represent means±standard error (n=3).

• 

  Fig. 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).

• 

  Fig. 4 Nafamostat mesilate (NM) improves impaired endothelial-dependent vascular relaxation ex vivo.(A) Aortic rings incubated with various concentrations (10~30 µg/ml) of NM were used to measure vascular reactivity. Increasing aortic relaxation induced by increasing dose of NM in aortic rings preconstricted with phenylephrine (10–5 M) compared with NM plus L-NAME treated aortic rings. *p<0.05compared with the control. (B) Increased bioavailable NO with NM treatment compared to saline control. Bars represent means±standard error (n=3). (C) Graphical representation of NM activity and pathway.

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