Arginase Inhibition by Ethylacetate Extract of Caesalpinia sappan Lignum Contributes to Activation of Endothelial Nitric Oxide Synthase

Woosung Shin; To Dao Cuong; Jeong Hyung Lee; Byungsun Min; Byeong Hwa Jeon; Hyun Kyo Lim; Sungwoo Ryoo

doi:10.4196/kjpp.2011.15.3.123

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Shin, Cuong, Lee, Min, Jeon, Lim, and Ryoo: Arginase Inhibition by Ethylacetate Extract of Caesalpinia sappan Lignum Contributes to Activation of Endothelial Nitric Oxide Synthase

Original Article

Korean J Physiol Pharmacol 2011;15(3):123-128.

Published online: 18 February 2011

DOI: https://doi.org/10.4196/kjpp.2011.15.3.123

Arginase Inhibition by Ethylacetate Extract of Caesalpinia sappan Lignum Contributes to Activation of Endothelial Nitric Oxide Synthase

Woosung Shin¹, To Dao Cuong³, Jeong Hyung Lee², Byungsun Min³, Byeong Hwa Jeon⁴, Hyun Kyo Lim⁵, Sungwoo Ryoo^1,

¹Department of Biology, Kangwon National University, Chuncheon 200-701, Korea

²Department of Biochemistry, Kangwon National University, Chuncheon 200-701, Korea

³Department of Pharmacy, Catholic University, Daegu 712-702, Korea

⁴Infectious Signaling Network Research Center, Department of Physiology, School of Medicine, Chungnam National University, Daejeon 301-747, Korea

⁵Department of Anesthesiology and Pain Medicine, Yonsei University Wonju College of Medicine, Wonju 220-701, Korea

Corresponding to: Sungwoo Ryoo, Department of Biology, Kangwon National University, Kangwondae-gil 1, Chuncheon 200-701, Korea. (Tel) 82-33-250-8534, (Fax) 82-33-251-3990, (E-mail) ryoosw08@kangwon.ac.kr

Received 5 April 2011 Revised 16 May 2011 Accepted 30 May 2011

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Caesalpinia sappan (C. sappan) is a medicinal plant used for promoting blood circulation and removing stasis. During a screening procedure on medicinal plants, the ethylacetate extract of the lignum of C. sappan (CLE) showed inhibitory activity on arginase which has recently been reported as a novel therapeutic target for the treatment of cardiovascular diseases such as atherosclerosis. CLE inhibited arginase II activity prepared from kidney lysate in a dose-dependent manner. In HUVECs, inhibition of arginase activity by CLE reciprocally increased NOx production through enhancement of eNOS dimer stability without any significant changes in the protein levels of eNOS and arginase II expression. Furthermore, CLE-dependent arginase inhibition resulted in increase of NO generation and decrease of superoxide production on endothelium of isolated mice aorta. These results indicate that CLE augments NO production on endothelium through inhibition of arginase activity, and may imply their usefulness for the treatment of cardiovascular diseases associated with endothelial dysfunction.

Keywords: Caesalpinia sappan lignum, Arginase, Endothelial nitric oxide synthase, Nitric oxide, Superoxide

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Fig. 1.

CLE inhibits arginase activity in a dose-dependent manner. Arginase II solution was prepared from kidney lysate. Arginase activities were measured in the presence of different concentrations of CLE as described in Methods. Incubation of CLE significantly decreased arginase II activity (n=12 from 4 different experiments; 1-way ANOVA, p<0.01). DMSO (10 μM) was used as a control.

Fig. 2.

CLE-dependent arginase inhibition results in increased NOx production. HUVECs were incubated with 20 μg/ml of CLE for 18 hours. CLE significantly inhibited arginase activity (A, ^∗ vs. untreated, p<0.01, n=4) and reciprocally increased NOx production in a dose-dependent manner (B, ^∗ vs. untreated, p<0.05; ^# vs. untreated, p<0.01, n=4).

Fig. 3.

CLE enhances the formation of eNOS dimer without altering expression levels of arginase II and eNOS. Protein levels of arginase II and eNOS were analyzed after incubation with CLE (18 hours, 20 μg/ml). Arginase II and eNOS protein levels were not significantly changed by CLE treatment (A, n=3). CLE incubation (20 μg/ml, 6 hours), however, induced eNOS dimerization, as detected by low-temperature SDS-PAGE and Western blot analysis (B). The dimer to monomer ratio of eNOS was shown in the bar graph from 4 independent experiments (^∗ vs. untreated, p<0.01, n=4). Boiled samples were used as a control.

Fig. 4.

Arginase inhibition results in increased NO production and decreased superoxide generation in isolated mice aorta. Incubation of mice aortic rings with CLE (20 μg/ml, 16 hours) resulted in a significant decrease in arginase activity (A, ^∗ vs. untreated, p<0.01, n=4). (B) Pretreated aorta were loaded with DAF (5 μM) followed by measurement of fluorescence (endothelial side up). The graph shows representative traces of DAF fluorescence in CLE- and CLE plus L-NAME (10 μM)-treated aorta. (C) The slope of DAF fluorescence was monitored and then determined (^∗ vs. untreated, p<0.01; ^# vs. CLE, p<0.01; n=4 mice). (D) ROS production in the aortic endothelium was traced at different time points after preloading with DHE (5 μM). (E) The slope of DHE fluorescence was determined based on cumulative data (^∗ vs. untreated, p<0.01; ^# vs. CLE, p<0.01; n=4 mice).

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